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• Published: 23 August 2019

History and progress of hypotheses and clinical trials for Alzheimer’s disease

• Pei-Pei Liu 1 ,
• Xiao-Yan Meng 1 &
• Jian-Sheng Kang   ORCID: orcid.org/0000-0002-2603-9718 1  

Signal Transduction and Targeted Therapy volume  4 , Article number:  29 ( 2019 ) Cite this article

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• Neurological disorders
• Neuroscience

An Author Correction to this article was published on 23 September 2019

This article has been updated

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive memory loss along with neuropsychiatric symptoms and a decline in activities of daily life. Its main pathological features are cerebral atrophy, amyloid plaques, and neurofibrillary tangles in the brains of patients. There are various descriptive hypotheses regarding the causes of AD, including the cholinergic hypothesis, amyloid hypothesis, tau propagation hypothesis, mitochondrial cascade hypothesis, calcium homeostasis hypothesis, neurovascular hypothesis, inflammatory hypothesis, metal ion hypothesis, and lymphatic system hypothesis. However, the ultimate etiology of AD remains obscure. In this review, we discuss the main hypotheses of AD and related clinical trials. Wealthy puzzles and lessons have made it possible to develop explanatory theories and identify potential strategies for therapeutic interventions for AD. The combination of hypometabolism and autophagy deficiency is likely to be a causative factor for AD. We further propose that fluoxetine, a selective serotonin reuptake inhibitor, has the potential to treat AD.

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Introduction

Alzheimer’s disease (AD) is an irreversible progressive neurological disorder that is characterized by memory loss, the retardation of thinking and reasoning, and changes in personality and behaviors. 1 , 2 AD seriously endangers the physical and mental health of the elderly. Aging is the biggest risk factor for the disease, the incidence of which doubles every 5 years after the age of 65. 3 Approximately 40 million people over the age of 60 worldwide suffer from AD, and the number of patients is increasing, doubling every 20 years. 4 , 5 , 6 , 7

In 1906, Alois Alzheimer presented his first signature case and the pathological features of the disease at the 37th convention of Southwestern German Psychiatrists. Later, in 1910, his coworker Emil Kraepelin named the disease in honor of his achievements. In the following years (from 1910 to 1963), researchers and physicians did not pay much attention to the disease until Robert Terry and Michael Kidd revived interest by performing electron microscopy of neuropathological lesions in 1963. Electron microscopy analysis showed that neurofibrillary tangles (NFTs) were present in brain biopsies from two patients with advanced AD. 8 , 9 Since then, studies on the pathological features and mechanisms of AD and drug treatments for the disease have been conducted for more than half a century (from 1963 to present). 10

Clinically, AD is divided into sporadic AD (SAD) and familial AD (FD). FD accounts for 1–5% of all AD cases. 11 , 12 , 13 , 14 , 15 In the early 1990s, linkage analyses of early-onset FD determined that mutations in three genes, namely, amyloid-beta A4 precursor protein ( APP ), presenilin 1 ( PSEN1 ), and presenilin 2 ( PSEN2 ), are involved in FD. PSEN1 mutations account for ~81% of FD cases, APP accounts for ~14%, and PSEN2 accounts for ~6%. 11 In addition to these three genes ( APP , PSEN1 , and PSEN2 ), more than 20 genetic risk loci for AD have been identified. 16 , 17 The strongest genetic risk factor for AD is the ε4 allele of apolipoprotein E ( APOE ). 18 , 19 , 20 , 21 APOE is a class of proteins involved in lipid metabolism and is immunochemically colocalized to senile plaques, vascular amyloid deposits, and NFTs in AD. The APOE gene is located on chromosome 19q13.2 and is associated with late-onset FD. The APOE gene has three alleles, namely, ε2 , ε3 , and ε4 , with frequencies of 8.4%, 77.9%, and 13.7%, respectively. The differences in APOE2 (Cys112, Cys158), APOE3 (Cys112, Arg158), and APOE4 (Arg112, Arg158) are limited to amino acid residues 112 and 158. 22 , 23 , 24 , 25 Analyses of the frequencies of these APOE alleles among human populations have revealed that there is a significant association between APOE4 and late-onset FD (with an ε4 allele frequency of ~40% in AD), suggesting that ApoE4 may be an important susceptibility factor for the etiopathology of AD. 25 , 26 , 27 Moreover, APOE4 can increase the neurotoxicity of β-amyloid (Aβ) and promote filament formation. 28 The APOE4 genotype influences the timing and amount of amyloid deposition in the human brain. 29 Reelin signaling protects synapses against toxic Aβ through APOE receptors, which suggests that APOE is a potential target for AD therapy. 30

The incidence of SAD accounts for more than 95% of all AD cases. Therefore, in this review, we focus our attention on recent SAD research and clinical trials. There are various descriptive hypotheses regarding the causes of SAD, including the cholinergic hypothesis, 31 amyloid hypothesis, 32 , 33 tau propagation hypothesis, 34 mitochondrial cascade hypothesis, 35 calcium homeostasis hypothesis, 36 inflammatory hypothesis, 37 neurovascular hypothesis, 38 metal ion hypothesis, 39 and lymphatic system hypothesis. 40 In addition, there are many other factors that increase the risk for SAD, including family history, 41 midlife hypertension, 42 sleep disorders, 43 midlife obesity, 44 and oxidative stress. 45 , 46 Interestingly, according to the latest evaluation of single-nucleotide polymorphisms (SNPs), Mukherjee et al. found 33 SNPs associated with AD and assigned people to six cognitively defined subgroups. 47

At present, clinical drug treatments are mainly divided into two categories: acetylcholinesterase inhibitors (AChEIs), represented by donepezil, and the antagonist of N-methyl-D-aspartic acid (NMDA) receptor, represented by memantine (Table 1 ). 48 As neurotransmitter regulators, these drugs can only relieve symptoms for a short time but cannot delay the progression of AD. Recent failures and the limited progress of therapeutics in phase III clinical trials suggest that it is time to consider alternative strategies for AD treatment. 49

In this review, we discuss the hypotheses of the molecular mechanisms of AD and related clinical trials (Fig. 1 ) and hope that these discussions will be helpful for developing explanatory theories and potential effective strategies for AD treatment.

Percentage of clinical trials in which each of the various hypotheses for AD were tested up to 2019. The amyloid hypothesis was the most heavily tested (22.3% of trials); the neurotransmitter hypothesis was the second most tested (19.0% of trials); the percentage of trials that tested the tau propagation hypothesis was ~12.7%; 17.0% of trials tested the mitochondrial cascade hypothesis and related hypotheses; 7.9% of trials tested the neurovascular hypothesis; 6.6% of trials tested the exercise hypothesis; 4.6% of trials tested the inflammatory hypothesis; 0.5% of trials tested the virus hypothesis; and the other uncatalogued trials made up approximately 8.4% of all trials

Cholinergic hypothesis

The cholinergic hypothesis was proposed by Peter Davies and A. J. F. Maloney in 1976 31 . They studied and compared the activities of the key enzymes involved in the synthesis of neurotransmitters, including acetylcholine, γ-aminobutyric acid, dopamine, noradrenaline, and 5-hydroxytryptamine, in 20 regions of AD and control brains. The activity of choline acetyltransferase in the AD brains was greatly reduced in the amygdala, hippocampus, and cortex, in which the concentration of acetylcholine was decreased at synapses. 50 , 51 , 52 The activity of glutamic acid decarboxylase, tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine-β-hydroxylase, and monoamine oxidase in all the areas of the AD brains studied appeared to be well within the normal range. Choline acetyltransferase is a key enzyme in the synthesis of acetylcholine, and its catalytic activity requires these substrates: choline, acetyl-CoA, and adenosine triphosphate (ATP). This was the first time that the concept of AD was noted as a cholinergic system failure. 31 , 53 This finding has also been reported in other neurological and psychiatric disorders, such as Parkinson’s disease (PD) and depression. 54 , 55

AChEIs can alleviate cognitive impairment in AD patients by inhibiting the degradation of acetylcholine. 56 , 57 , 58 , 59 Therefore, AChEIs have been used for more than 20 years since the FDA approved tacrine, the first drug for the treatment of AD, in 1995. 60 Tacrine is a reversible AChEI. Because of its liver toxicity, the number of tacrine prescriptions dropped after other AChEIs were introduced, and the usage of tacrine has been largely discontinued. The second generations of AChEI drugs that are widely used at present include donepezil, rivastigmine, and galantamine. 61 , 62 These drugs show fewer side effects and higher central selectivity and improve the cognition level of patients with mild to moderate AD. The daily living ability and overall function of patients treated with rivastigmine and galantamine are better than those treated with donepezil. 61 , 62 , 63 According to the latest meta-analysis on the efficacy of AChEIs for treating the cognitive symptoms of dementia, AChEIs have modest effects on dementia in AD, 64 but the effect is not continuous. 65 , 66

In conclusion, the current clinical drugs used for the treatment of AD improve the quality of life of AD patients, but have no significant effect on the occurrence or progression of AD. In 2012, the French Pharmacoeconomic Committee assessed the medical benefit of these drugs and downgraded its rating of the medical benefit provided by AChEIs in AD from "major" to "low." 67

Amyloid hypothesis

The amyloid hypothesis was first proposed in 1991 by John Hardy and David Allsop. 32 , 33 They found a pathogenic mutation in the Aβ precursor protein (APP) gene on chromosome 21, which suggested that APP mismetabolism and Aβ deposition were the primary events in AD. They thought that the pathological cascades in AD were Aβ deposition, tau phosphorylation, NFT formation, and neuronal death. The presence of Aβ deposits in an APP mutant (APP751) transgenic model supported the hypothesis and further contributed to shifting the amyloid hypothesis from a descriptive to a mechanistic hypothesis. 68 , 69 Positron emission tomography (PET) imaging studies have suggested that ~30% of clinically normal older individuals have signs of Aβ accumulation. 70 , 71 , 72 , 73

Aβ was first isolated by Glenner and Wong in 1984. 74 Aβ may provide a strategy for diagnostic testing for AD and for understanding its pathogenesis. 74 APP was first cloned and sequenced in 1987; APP consists of 695 amino acid residues and a glycosylated receptor located on the cell surface. 75 , 76 Aβ is composed of 39–43 residues derived from multiple proteolytic cleavages of APP. APP is cleaved in two ways (Fig. 2 ). The first method is through the α pathway. APP is hydrolyzed by α-secretase and then by γ-secretase; this process does not produce insoluble Aβ. The second method is through the β pathway. APP is hydrolyzed by β-secretase (BACE1) and then by γ-secretase to produce insoluble Aβ. Under normal conditions, the Aβ protein is not produced since APP hydrolysis is mainly based on the α pathway. A small amount of APP is hydrolyzed via the second method, and the Aβ that produced is eliminated by the immune system. However, when some mutations, such as the Lys670Asn/Met671Leu (Swedish) and Ala673Val mutations near the BACE1 cleavage site, are present, 77 , 78 APP is prone to hydrolysis by the β pathway, resulting in an excessive accumulation of insoluble Aβ and eventually the development of AD. 79 , 80 However, the Ala673Thr mutation has been suggested to be protective. 81

Schematic of the amyloid hypothesis and tau hypothesis. Upper: The transmembrane APP protein can be cleaved by two pathways. Under normal processing, APP is hydrolyzed by α-secretase and then by γ-secretase, which does not produce insoluble Aβ; under abnormal processing, APP is hydrolyzed by β secretase (BACE1) and then by γ secretase, which produces insoluble Aβ. Phase III clinical trials of solanezumab (Eli Lilly), crenezumab (Roche/Genentech/AC Immune), aducanumab (Biogen Idec), and umibecestat (Novartis/Amgen), which target the amyloid hypothesis, have all been terminated thus far. Lower: The tau protein can be hyperphosphorylated at amino residues Ser202, Thr205, Ser396, and Ser404 (which are responsible for tubulin binding), thereby leading to the release of tau from microtubules and the destabilization of microtubules. Hyperphosphorylated tau monomers aggregate to form complex oligomers and eventually neurofibrillary tangles, which may cause cell death

High concentrations of Aβ protein are neurotoxic to mature neurons because they cause dendritic and axonal atrophy followed by neuronal death. 82 The levels of insoluble Aβ are correlated with the decline of cognition. 83 In addition, Aβ inhibits hippocampal long-term potentiation (LTP) in vivo. 84 Neurofibrillary degeneration is enhanced in tau and APP mutant transgenic mice. 85 Transgenic mice that highly express human APP in the brain exhibit spontaneous seizures, which may be due to enhanced synaptic GABAergic inhibition and deficits in synaptic plasticity. 86 Individuals with Aβ are prone to cognitive decline 87 , 88 , 89 and symptomatic AD phenotypes. 90 , 91

The current strategies for AD treatment based on the Aβ hypothesis are mainly divided into the following categories: β- and γ-secretase inhibitors, which are used to inhibit Aβ production; antiaggregation drugs (including metal chelators), which are used to inhibit Aβ aggregation; protease activity-regulating drugs, which are used to clear Aβ; and immunotherapy. 92 We will discuss recent progress regarding immunotherapy and BACE1 inhibitors.

Aβ-targeting monoclonal antibodies (mAbs) are the major passive immunotherapy treatments for AD. For example, solanezumab (Eli Lilly), which can bind monomeric and soluble Aβ, failed to show curative effects in AD patients in phase III, although solanezumab effectively reduced free plasma Aβ concentrations by more than 90%. 93 Gantenerumab (Roche/Genentech) is a mAb that binds oligomeric and fibrillar Aβ and can activate the microglia-mediated phagocytic clearance of plaques. However, it also failed in phase III. 94 Crenezumab (Roche/Genentech/AC Immune) is a mAb that can bind to various Aβ, including monomers, oligomers, and fibrils. On January 30, 2019, Roche announced the termination of two phase III trials of crenezumab in AD patients. Aducanumab (Biogen Idec) is a mAb that targets aggregated forms of Aβ. Although aducanumab can significantly reduce Aβ deposition, Biogen and Eisai announced the discontinuation of trials of aducanumab on March 21, 2019. Together, the failure of these trials strongly suggests that it is better to treat Aβ deposits as a pathological feature rather than as part of a major mechanistic hypothesis.

BACE1 inhibitors aim to reduce Aβ and have been tested for years. However, no BACE1 inhibitors have passed clinical trials. Verubecestat (MK-8931, Merck & Co.) reduced Aβ levels by up to 90% in the cerebrospinal fluid (CSF) in AD. However, Merck no longer listed verubecestat in its research pipeline since verubecestat did not improve cognitive decline in AD patients and was associated with unfavorable side effects. 95 Lanabecestat (AZD3293, AstraZeneca/Eli Lilly) is another BACE1 inhibitor that can lower CSF Aβ levels by up to 75%. However, on June 12, 2018, phase II/III trials of lanabecestat were discontinued due to a lack of efficacy. The BACE1 inhibitor atabecestat (JNJ-54861911, Janssen) induced a robust reduction in Aβ levels by up to 95% in a phase I trial. However, Janssen announced the discontinuation of this program on May 17, 2018. The latest news regarding the BACE inhibitor umibecestat (Novartis/Amgen) was released on July 11, 2019; it was announced that the evaluation of umibecestat was discontinued in phase II/III trials since an assessment demonstrated a worsening of cognitive function. Elenbecestat (E2609, Eisai) is another BACE1 inhibitor that can reduce CSF Aβ levels by up to 80% 96 , 97 and is now in phase III trials (shown in Table 2 ). Although all BACE1 inhibitors seem to reduce CSF Aβ levels, the failure of trials of solanezumab, which can reduce free plasma Aβ concentrations by more than 90%, 93 may be sufficient to lead us to pessimistic expectations, especially considering that the treatment worsened cognition and induced side effects.

Tau propagation hypothesis

Intracellular tau-containing NFTs are an important pathological feature of AD. 98 , 99 NFTs are mainly formed by the aggregation of paired helical filaments (Fig. 2 ). Pathological NFTs are mainly composed of tau proteins, which are hyperphosphorylated. 100 , 101 , 102 , 103 Tau proteins belong to a family of microtubule-binding proteins, and are heterogeneous in molecular weight. A main function of tau is to stabilize microtubules, which is particularly important for neurons since microtubules serve as highways for transporting cargo in dendrites and axons. 34 , 104 Tau cDNA, which encodes a protein of 352 residues, was cloned and sequenced in 1988. RNA blot analysis has identified two major transcripts that are 6 and 2 kilobases long and are widely distributed in the brain. 105 , 106 The alternative splicing of exons 2, 3, and 10 of the tau gene produces six tau isoforms in humans; the differential splicing of exon 10 leads to tau species that contain various microtubule-binding carboxyl terminals with repeats of three arginines (3R) or four arginines (4R). 107 , 108 An equimolar ratio of 3R and 4R may be important for preventing tau from forming aggregates. 109

The tau propagation hypothesis was introduced in 2009. 34 The pathology of tau usually first appears in discrete and specific areas and later spreads to more regions of the brain. Aggregates of fibrillar and misfolded tau may propagate in a prion-like way through cells, eventually spreading through the brains of AD patients (Fig. 2 ). Clavaguera et al. demonstrated that tau can act as an endopathogen in vivo and in culture studies in vitro with a tau fragment. 104 In their study, brain extracts isolated from P301S tau transgenic mice 110 were injected into the brains (the hippocampus and cortical areas) of young ALZ17 mice, a tau transgenic mouse line that only develops late tau pathology. 111 After the injection, the ALZ17 mice developed tau pathology quickly, whereas the brain extracts from wild-type mice or immunodepleted P301S mice, which were used as controls, had no effect. The causes of tau aggregation in sporadic tauopathies are not fully understood. Tau can be phosphorylated at multiple serine and threonine residues (Fig. 2 ). 112 , 113 The gain- and loss-of-function of tau phosphorylation may be due to alterations in the activities of kinases or phosphatases that target tau, and thus, the toxicity of tau can be augmented as a result. Other posttranslational modifications can decrease tau phosphorylation or enhance the harmful states of tau. For example, serine–threonine modifications by O-glycosylation can reduce the extent of tau phosphorylation. 114 , 115 Thus, tau hyperphosphorylation may partially result from a decrease in tau O-glycosylation. In addition, tau can also be phosphorylated at tyrosine residues, 116 sumoylated and nitrated, 117 but the exact roles of these tau modifications remain elusive.

According to the tau propagation hypothesis, abnormally phosphorylated tau proteins depolymerize microtubules and affect signal transmission within and between neurons. 101 , 103 , 118 In addition, mutant forms of human tau cause enhanced neurotoxicity in Drosophila melanogaster . 119 There may be cross-talk between the tau propagation hypothesis and the amyloid hypothesis. As mentioned earlier, among the risk loci for AD, APOE is the most robust factor for AD pathogenesis. 120 Unlike other isoforms, APOE4 may increase Aβ by decreasing its clearance 121 , 122 , 123 and enhancing tau hyperphosphorylation. 124 , 125 , 126 GSK3 is one of the upstream factors that jointly regulates Aβ and tau. Increased GSK3 activity leads to the hyperphosphorylation of the tau protein. 126 GSK3 overactivity may also affect the enzymatic processing of APP and thus increase the Aβ level. 127 , 128 In addition, tau is essential for Aβ-induced neurotoxicity, and dendritic tau can mediate Aβ-induced synaptic toxicity and circuit abnormalities. 129 Moreover, APP and tau act together to regulate iron homeostasis. APP can interact with ferroportin-1 to regulate the efflux of ferrous ions. 130 , 131 As an intracellular microtubule-associated protein, tau can increase iron output by enhancing the transport of APP to the cell surface. 132 Decreased APP trafficking to the cell surface accounts for iron accumulation in tau knockout neurons. 133 , 134

As one of the most important hypotheses of AD, the tau propagation hypothesis has a wide range of impacts. Drugs that target the tau protein are divided into the following categories: tau assembly inhibitors, tau kinase inhibitors, O-GlcNAcase inhibitors, microtubule stabilizers, and immunotherapy drugs. 92 Only a few agents have undergone proof-of-principle tests as tau kinase inhibitors, microtubule-stabilizing agents, and inhibitors of heat shock protein 90 (Hsp90), which stabilize GSK3β. 135 , 136 In addition, some inhibitors of tau aggregation, such as TRx0237 (TauRx Therapeutics), are in clinical trials. The results of TRx 237–005 phase III clinical trials showed that the agent may be effective as a monotherapy since the brain atrophy rate of AD patients declined after 9 months of treatment. 137 ACI-35 (AC Immune/Janssen) and AADvac1 (Axon Neuroscience SE) are vaccines that target the hyperphosphorylated tau protein, and the vaccines are still being evaluated in clinical trials 138 (Table 2 ). Tau-directed therapies will inevitably face challenges similar to those presently encountered in Aβ-targeted trials. Overall, the effectiveness of tau-directed therapies remains to be tested in the future.

Mitochondrial cascade hypothesis and related hypotheses (Fig. 3 )

In 2004, Swerdlow and Khan first introduced the mitochondrial cascade hypothesis 35 and stated that mitochondrial function may affect the expression and processing of APP and the accumulation of Aβ in SAD. The hypothesis includes three main parts. First, an individual's baseline mitochondrial function is defined by genetic inheritance. Second, the rate of age-associated mitochondrial changes is determined by inherited and environmental factors. Moreover, a decline in mitochondrial function or efficiency drives aging phenotypes. 139 , 140 , 141 Third, the rate of change of mitochondrial function in individuals influences AD chronology.

Mitochondrial cascade and related hypotheses. Mitochondria are the main contributors to ROS production, which is significantly increased in AD. The metabolites of mitochondrial TCA, such as pyruvate, fumarate, malate, OAA, and α-KG, not only directly regulate energy production but also play an important role in the epigenetic regulation of neurons and longevity. 164 , 173 , 187 , 188 , 189 For example, SAM provides methyl groups for histone and DNA methyltransferases (HMTs and DNMTs). 165 , 166 α-KG is a necessary cofactor for TET DNA methylases, histone demethylases (HDMs), and lysine demethylases KDMs/JMJDs. 167 , 168 Mitochondria also regulate the levels and redox state of FAD, a cofactor of the histone demethylase LSD1. 175 Dysfunctional mitochondria can be removed by mitophagy, which is also very important in the progression of AD. BNIP3L interacts with LC3 or GABARAP and regulates the recruitment of damaged mitochondria to phagophores. In addition, Beclin 1 is released from its interaction with Bcl-2 to activate autophagy after BNIP3L competes with it. PINK1 promotes autophagy by recruiting the E3 ligase PARK2. Then, VDAC1 is ubiquitinated and then binds to SQSTM1. SQSTM1 can interact with LC3 and target this complex to the autophagosome. 445 L. monocytogenes can promote the aggregation of NLRX1 and the binding of LC3, thus activating mitophagy. 446 The MARCH5-FUNDC1 axis mediates hypoxia-induced mitophagy. 447 The mitochondrial proteins NIPSNAP1 and NIPSNAP2 can recruit autophagy receptors and bind to autophagy-related proteins. 448 ROS: reactive oxygen species; TCA: tricarboxylic acid cycle; OAA: oxaloacetate; α-KG: α-ketoglutarate; SAM: S-adenosyl methionine; TET: ten–eleven translocation methylcytosine dioxygenase; FAD: flavin adenine dinucleotide

Oxidative stress is defined as “an imbalance in pro-oxidants and antioxidants with associated disruption of redox circuitry and macromolecular damage.” 142 Oxidative stress is mainly caused by increased levels of reactive oxygen species (ROS) and/or reactive nitrogen species, including superoxide radical anions (O 2− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (HO − ), nitric oxide (NO), and peroxynitrite (ONOO − ). In intact cells, ROS can be produced from multiple sources, including mitochondria, ER, peroxisomes, NADPH oxidases, and monoamine oxidases. 143 , 144 In AD, neurons exhibit significantly increased oxidative damage and a reduced number of mitochondria, 145 which are the main contributors to ROS generation among these ROS sources. 146 , 147 The overproduction of ROS and/or an insufficient antioxidant defense can lead to oxidative stress. 148 Before the onset of the clinical symptoms of AD and the appearance of Aβ pathology, there is evidence that the production of ROS increases due to mitochondrial damage. 148 Both mtDNA and cytochrome oxidase levels increase in AD, and the number of intact mitochondria is significantly reduced in AD. 145 Several key enzymes involved in oxidative metabolism, including dehydrogenase complexes for α-ketoglutarate (α-KG) and pyruvate, and cytochrome oxidase also show reduced expression or activity in AD. 149 , 150 , 151 , 152 , 153 , 154 In addition, there is evidence in vitro and in vivo for a direct relationship between oxidative stress and neuronal dysfunction in AD. 155 , 156 Aβ-dependent endocytosis is involved in reducing the number of NMDA receptors on the cell surface and synaptic plasticity in neurons and brain tissue in AD mice. 157 Excessive Aβ may also trigger excitotoxicity and stress-related signaling pathways by increasing Ca 2+ influx, increasing oxidative stress, and impairing energy metabolism. 158

Although the majority of efforts have been focused on genetic variations and their roles in disease etiology, it has been postulated that epigenetic dysfunction may also be involved in AD. 159 , 160 Indeed, there is growing evidence that epigenetic dysregulation is linked to AD. 161 , 162 , 163 Mitochondrial metabolites are required for epigenetic modifications, such as the methylation of DNA and the methylation and acetylation of histones. 164 AD brains exhibited a global reduction in DNA modifications, including 5-methylcytosine and 5-hydroxymethylcytosine. 165 , 166 , 167 , 168 S-adenosyl methionine (SAM) provides a methyl group for histones and DNA methyltransferases in the nucleus. SAM is generated and maintained by coupling one-carbon metabolism and mitochondrial energy metabolism. 169 , 170 α-KG, which is generated by the tricarboxylic acid cycle (TCA) cycle in mitochondria and the cytosol, is a cofactor of ten–eleven translocation methylcytosine dioxygenase DNA methylases, histone demethylases (HDMs) and the lysine demethylases KDMs/JMJDs. 171 , 172 However, the activities of KDMs/JMJDs and TETs can be inhibited by fumarate, succinate, and 2-hydroxyglutarate. 173 Mutations that affect the succinate dehydrogenase complex and fumarate hydratase can induce the accumulation of succinate and fumarate, respectively. 174 Oxidized flavin adenine dinucleotide (FAD) is an essential cofactor of the HDM LSD1, a member of the KDM family. 175 In addition, acetyl-CoA, the source of acetyl groups that are consumed by histone acetyltransferases, is generated by ATP citrate lyase and pyruvate dehydrogenase in the cytosol and mitochondria, respectively. 176 In addition, oxidized nicotinamide adenine dinucleotide (NAD + ) is a cofactor for sirtuins (SIRTs), a family of deacetylases that includes nuclear-localized SIRT1, SIRT6, and SIRT7, cytosolic SIRT2, and three mitochondrial SIRTs (SIRT3, SIRT4, and SIRT5) (Fig. 3 ). Therefore, the activities of SIRTs are sensitive and are regulated by cellular NAD + pools. 177 As summarized by Fang, NAD + replenishment can enhance autophagy/mitophagy mainly through SIRT1 or SIRT3; meanwhile, SIRT6 and SIRT7 induce autophagy through the inhibition of mTOR; NAD + may also inhibit autophagy/mitophagy through SIRT2, SIRT4, SIRT5, and poly(ADP-ribose) polymerases. 178 In short, mitochondrial dysfunction can partially explain the epigenetic dysregulation in aging and AD.

Dysfunctional mitochondria can be removed by mitophagy, a term that was first coined by Dr Lemasters in 2005. 179 Since then, mitophagy has been linked to various diseases, including neurodegenerative disorders such as PD 180 and Huntington's disease (HD), 181 as well as normal physiological aging. 182 Mitophagosomes can effectively degrade their internalized cargo by fusing with lysosomes during axonal retrotransport. 183 Fang et al. demonstrated that neuronal mitophagy is impaired in AD. 184 Mitophagy stimulation can reverse memory impairment, diminish insoluble Aβ 1–42 and Aβ 1–40 through the microglial phagocytosis of extracellular Aβ plaques, and abolish AD-related tau hyperphosphorylation. 184 Therefore, deficiencies in mitophagy may have a pivotal role in AD etiology and may be a potential therapeutic target. 178 , 184 , 185 , 186

The metabolites of mitochondrial TCA, such as pyruvate, fumarate, malate, oxaloacetate (OAA), and α-KG, have been demonstrated to extend lifespan when fed to C. elegans . 173 , 187 , 188 , 189 Wilkins et al. found that OAA enhances the energy metabolism of neuronal cells. 190 Moreover, OAA can also activate mitochondrial biogenesis in the brain, reduce inflammation, and stimulate neurogenesis. 191 The application of OAA in AD was also investigated by Swerdlow et al., and the results showed that 100-mg OAA capsules did not result in an elevation of OAA in the blood 192 ; higher doses up to 2 g per day were also evaluated in clinical studies, but no results have been posted or published yet.

Clinical trials related to the mitochondrial cascade hypothesis and related hypotheses account for 17.0% of all clinical trials (Fig. 1 ). Based on the above, the mitochondrial cascade hypothesis and related hypotheses (Fig. 3 ) may link other hypotheses, including the cholinergic hypothesis, amyloid hypothesis, and tau propagation hypothesis.

Calcium homeostasis and NMDA hypotheses

The calcium homeostasis hypothesis was proposed in 1992 by Mattson et al. They found that Aβ can elevate intracellular calcium levels and render neurons more vulnerable to environmental stimuli. 36 The involvement of calcium in AD was first suggested long ago by Khachaturian, 193 and since then, there are many efforts to clarify this hypothesis. 194 , 195 , 196 Calcineurin can trigger reactive/inflammatory processes in astrocytes, which are upregulated in AD models. 197 In addition, calcium homeostasis is closely related to learning and memory. Rapid autopsies of the postmortem human brain have suggested that calcineurin/nuclear factor of activated T-cells signaling is selectively altered in AD and is involved in driving Aβ-mediated cognitive decline. 198 The evidence indicates that calcium homeostasis may be associated with the development of AD. 193 , 199

Memantine, a noncompetitive antagonist of NMDA glutamate receptors in the brain was approved for marketing in Europe in 2002 and received US FDA approval in 2003. 200 , 201 Memantine is not an AChEI. The functional mechanism of memantine likely involves blocking current flow (especial calcium currents) through NMDA receptors and reducing the excitotoxic effects of glutamate. 202 Memantine is also an antagonist of type 3 serotonergic (5-HT 3 ) receptors and nicotinic acetylcholine receptors, but it does not bind other receptors, such as adrenergic, dopamine, and GABA receptors. The inhibition of NMDA receptors can also reduce the inhibition of α-secretase and thus inhibit the production of Aβ. 203 However, the French Pharmacoeconomic Committee downgraded its rating of the medical benefit provided by memantine in AD from "major" to "low," 67 which was also supported by a recent meta-analysis. 64

Neurovascular hypothesis

The homeostasis of the microenvironment and metabolism in the brain relies on substrate delivery and the drainage of waste through the blood; neurons, astrocytes, and vascular cells form a delicate functional unit that supports the integrity of brain structure and function. 204 , 205 , 206 Vascular dysregulation leads to brain dysfunction and disease. Alterations in cerebrovascular function are features of both cerebrovascular pathologies and neurodegenerative diseases, including AD. 38 In 1994, it was demonstrated that the cerebral microvasculature is damaged in AD. 207 Aβ can induce the constriction of the cerebral arteries. 208 In an AD mouse model, neocortical microcirculation is impaired before Aβ accumulation. 209 , 210 Neuroimaging studies in AD patients have demonstrated that neurovascular dysfunction is found before the onset of neurodegeneration. 211 , 212 , 213 , 214 In addition to aberrant angiogenesis and the senescence of the cerebrovascular system, the faulty clearance of Aβ across the blood–brain barrier (BBB) can initiate neurovascular uncoupling and vessel regression and consequently cause brain hypoperfusion, brain hypoxia, and neurovascular inflammation. Eventually, BBB compromise and a chemical imbalance in the neuronal environment lead to neuronal dysfunction and loss. 215 In mice that overexpress APP, impairment in the neocortical microcirculation is observed. The cerebrovascular effects of Aβ in dementia may involve alterations in cerebral blood flow and neuronal dysfunction. 209 Moreover, neurovascular dysfunction may also play a role in the etiology of AD.

Many factors can lead to changes in the neurovasculature, which in turn affect the occurrence and progression of AD. Of these factors, hyperlipidemia is one of the most important. During the last two decades, growing evidence has shown that a high cholesterol level may increase the risk of AD. In one test, higher levels of low-density lipoprotein (LDL) or total cholesterol were correlated with lower scores on the MMSE (modified mini mental state exam) in nondemented patients. High total cholesterol levels in midlife increase the risk of AD nearly threefold: the odds ratio (OR) is 2.8 (95% confidence interval, CI: 1.2–6.7). 216 Midlife obesity is also a risk factor for AD, 217 and midlife adiposity may predict an earlier onset of dementia and Aβ accumulation. 218 Adipose tissue secretes some inflammation factors, such as tumor necrosis factor (TNF-α), interleukin-1 (IL-1), and interleukin-6, in obesity, 219 and these factors may induce insulin resistance, produce Aβ deposits, and stimulate excessive tau phosphorylation. 220

A hyperglycemic state is another risk factor. Type 2 diabetic patients (T2D) have an increased risk of dementia, 221 both vascular dementia (VD) and AD. In the largest and latest meta-analysis of T2D and dementia risk, data from 6184 individuals with diabetes and 38,350 without diabetes were pooled and analyzed. 222 The relative risk (RR) for dementia was 1.51 (95%CI: 1.31–1.74). The results of the analyses further suggested that there are two common subtypes of dementia: AD and VD. The results suggested that T2D conferred an RR of 2.48 (95%CI: 2.08–2.96) for VD and 1.46 (95%CI: 1.20–1.77) for AD. 222 Insulin resistance is a common feature of T2D and SAD. Accumulating evidence supports the involvement of impaired insulin signaling in AD progression. Insulin levels and insulin receptor expression are reduced in AD brains. 223 However, plasma insulin and Aβ levels are both increased in AD patients, suggesting that a decrease in insulin clearance may increase plasma Aβ levels. Blocking insulin signaling in the brain through the intracerebroventricular administration of STZ (the diabetogenic drug streptozotocin) resulted in various pathological features that resemble those found in human SAD, while the administration of insulin and glucose enhances learning and memory in AD patients. 224 , 225

Many institutions have conducted clinical trials of statins, drugs that are used to lower blood cholesterol, for the treatment of AD. However, in a phase IV clinical trial, simvastatin failed to reduce Aβ-42 and tau levels in the CSF. The results suggested that the use of statins for the treatment of AD requires more evidence. 226 To test the hyperglycemic hypothesis, rosiglitazone (RSG), a drug used for the treatment for type II diabetes mellitus, was evaluated. RSG XR had no effect in a phase III trial. 227 In addition, hypertension has also been linked to worse cognition and hypometabolism in AD. AD patients with hypertension exhibit worse cognitive function (on the AD assessment scale-cognitive subscale, P  = 0.038) and a higher burden of neuropsychiatric symptoms (on the neuropsychiatric inventory questionnaire, P  = 0.016) than those without hypertension. 228 As an antihypertensive medication, ramipril is a specific angiotensin-converting enzyme inhibitor; however, ramipril was tested and failed in a pilot clinical trial. 229

Therefore, trial failures of treatments related to the neurovascular hypothesis and related hypotheses suggest that these hypotheses alone may not be sufficient to explain the etiology of AD.

Inflammatory hypothesis

The inflammatory responses of microglia and astrocytes in the central nervous system (CNS) also play important roles in the development of AD. 230 , 231 , 232 Microglial cells are brain-specific macrophages in the CNS, and they make up 10–15% all brain cells. 233 Microglia cells exhibit higher activity in AD patients than in the control group. 234 The concentration of aggregated microglial cells near senile plaques and neurons with NFTs in AD patients is usually 2–5 times higher than that in normal individuals. Inflammatory factors that are expressed by microglia and histocompatibility complexes also cause inflammation. 235 In vitro studies have linked Aβ pathology in AD to neuroinflammation. It has been shown that Aβ possesses a synergistic effect on the cytokine-induced activation of microglia. 236 Two studies have confirmed that Aβ can induce glial activation in vivo. 237 , 238 The fibrillar conformation of Aβ seems to be crucial for such activation. 239 In AD patients, Aβ can bind to microglia cells through the CD36-TLR4-TLR6 receptor complex and the NLRP3 inflammatory complex, destroy cells, release inflammation-inducing factors, such as TNF-α, and cause immune responses. In addition to increased levels of TNF-α, increased levels of the inflammatory cytokines IL-1β, TGF-β, IL-12, and IL-18 in the CNS are also correlated with AD progression and increase damage in the brains of AD patients. 240 Interestingly, CD22 is a B-cell receptor that functions as a negative regulator of phagocytosis. The functional decline of aged microglia may result from the upregulation of CD22; thus, the inhibition of CD22 can enhance the clearance of debris and fibrils, including Aβ oligomers, in vivo, and this process may be potentially beneficial for the treatment of AD. 241

Considerable evidence suggests that the use of anti-inflammatory drugs may be linked with a reduced occurrence of AD. The ability of naproxen and celecoxib to delay or prevent the onset of AD and cognitive decline was evaluated in phase III clinical trials. However, therapeutic efficacy analysis indicated that naproxen and celecoxib do not exert a greater benefit compared with that of placebo. In addition, the naproxen and celecoxib groups experienced more adverse events, including hypertension, gastrointestinal, and vascular or cardiac problems, so these phase III clinical trials were discontinued. 242 A clinical trial of lornoxicam in AD patients was also terminated due to a lack of efficacy. These failures suggest that the clinical application of anti-inflammatory drugs for AD treatment needs to be further validated (Table 2 ).

Metal ion hypothesis

Metal ions that play functional roles in organisms are classified as biometals, while other metal ions are inert or toxic. 243 , 244 The dyshomeostasis of any metal ion in the body usually leads to disease. In the CNS, biometals, such as copper, zinc, and iron, are required to act as cofactors for enzymatic activity, mitochondrial function, and neuronal function. 245 , 246 In healthy brains, free metal ions are stringently regulated and kept at a very low level. 247

Biometal ions are involved in Aβ aggregation and toxicity. In the first study that evaluated biometals and Aβ, which was published by Bush et al. in 1994, zinc was linked to Aβ. The potential link between biometals and AD has been intensively studied. 39 , 248 , 249 , 250 There is evidence of the dyshomeostasis of biometals in AD brains. Biometals, especially zinc and copper, are directly coordinated by Aβ, and biometals such as iron can reach a high concentration (~1 mM) in plaques. 251 , 252 In the serum, the levels of copper, which are not associated with ceruloplasmin, are elevated in AD patients. Moreover, a higher copper content in the serum is associated with lower MMSE scores. 253 , 254 In the serum of AD patients, the levels of Zn 2+ ions are decreased compared with those in age-matched controls, whereas the concentration of Zn 2+ is elevated in the CSF. 255

The important role of biometals in Aβ formation has been reported in various animal models. For example, the role of Cu 2+ in Aβ formation was demonstrated in a cholesterol-fed rabbit model of AD. 256 Administering trace amounts of Cu 2+ in drinking water was sufficient to induce Aβ accumulation, the consequent formation of plaques, and deficits in learning. 256 On the other hand, Cu 2+ also plays a beneficial role. For example, transgenic mice that overexpress mutant human APP and are treated with Cu 2+ show a reduction in Aβ and do not exhibit a lethal phenotype. 257 In contrast, in Drosophila that specifically express human Aβ in the eye, dietary zinc and copper increase Aβ-associated damage, while different chelators of biometals demonstrate favorable effects. 258

During normal aging, the gradual accumulation of iron is observed in some brain areas, such as the substantia nigra, putamen, globus pallidus, and caudate nucleus. 259 , 260 , 261 , 262 , 263 An increase in the level of iron in AD brains was first demonstrated in 1953. 264 More recently, through the use of magnetic resonance imaging (MRI), iron accumulation was found in AD and was shown to be mainly localized to certain brain areas, such as the parietal cortex, motor cortex, and hippocampus. 265 , 266 , 267 , 268 , 269 , 270 , 271 , 272 Studies of gene mutations that affect the metabolism of iron have suggested that the dyshomeostasis of iron plays a role in neuronal death, such as the neuronal death that occurs in neurodegenerative disorders such as AD. 273 , 274 , 275 , 276 , 277 Iron overload accelerates neuronal Aβ production and consequently worsens cognitive decline in a transgenic AD mice. 278 There is evidence that the levels of labile iron can directly affect APP production via iron regulatory element. 279 As a potent source of highly toxic hydroxyl radicals, redox-active iron is actively associated with senile plaques and NFTs. 280

As the most common nutrient deficiency in the world, iron deficiency is also frequently observed and reported in AD. 281 Iron is present in polynuclear iron–sulfur (Fe/S) centers and hemoproteins. Mitochondrial complexes I–III require Fe/S clusters, and complexes II–IV need hemoproteins for electron transfer and the oxidative phosphorylation of the respiratory chain. 282 Thus, iron deficiency may partially account for hypometabolism in AD since women with iron deficiency anemia have a higher prevalence of dementia. 283 Interestingly, iron deficiency and iron accumulation in AD seem paradoxical. One potential explanation is that tau differentially regulates the motor proteins dynein and kinesin; specifically, tau may preferentially inhibit kinesin, which transports cargo toward the cell periphery. 284 Tau is distributed in a proximal-to-distal gradient with a low concentration in the cell body. 284 , 285 , 286 , 287 When tau is hyperphosphorylated, it is released from the distal microtubules into the neuronal axon and soma, and thus inhibits kinesin activity and prevents the transport of iron-containing cargo and other cargo (including mitochondria) to the neuronal periphery; this may result in the accumulation of mtDNA and iron accumulations in the soma of neurons in AD 145 , 280 and deficiencies in mitochondria and iron homeostasis in the white matter of the brain. Iron-targeted therapies were recently updated and reviewed. 288 Similar to the amyloid hypothesis, the conjecture that the therapeutic chelation of iron ions is an effective approach for treating AD remains widespread despite a lack of evidence of any clinical benefits. 288

Aluminum (Al), the most abundant metal in the earth’s crust, is a nonessential metal ion in organisms. The role of Al in AD needs to be further elucidated. Exley et al. hypothesized that Al is associated with Aβ in AD brains and Al can precipitate Aβ in vitro into fibrillar structures; in addition, Al is known to increase the Aβ burden in the brains of treated animals, which may be due to a direct or indirect effect on Aβ anabolism and catabolism. 289 , 290

Biometals may play various roles in AD and may influence the pathogenesis directly or indirectly. For example, biometals indirectly influence energy metabolism and APP processing, 249 while cellular iron levels can directly regulate APP through IREs identified in the 5′ -UTR of mRNA. 291 , 292

Lymphatic system hypothesis

The lymphatic network and the blood vasculature are essential for fluid balance in the body. 293 , 294 Below the human skull, the meninges, a three-layer membrane that envelopes the brain, contains a network of lymphatic vessels. This meningeal lymphatic system was first discovered in 1787, and interest in this system has been revived recently. 295 , 296 , 297 Proteins, metabolites, and waste produced by the brain flow through the interstitial fluid (ISF) and reach the CSF, which circulates through the ventricles and brain meninges. 298 In the classical form of transvascular removal, metabolic waste and other molecules in these fluids are drained from the brain, are transported across capillary walls, and cross the BBB. 298 , 299 Thrane et al.’s found that, in addition to transvascular removal, perivascular removal, in which the blood vasculature allows the CSF to flow into or exit the brain along the para-arterial space or via paravenous routes, occurs and that aquaporin-4 water channels that are expressed in astrocytes are essential for CSF–ISF exchange along the perivascular pathway. 300 , 301 This perivascular route is called the glymphatic system. 302 , 303

During aging, impairments in the transvascular/perivascular removal of waste may result in Aβ accumulation in the brain. 40 , 304 Animals that lack aquaporin-4 channels show a 70% decrease in the ability to remove large solutes, such as Aβ. 305 , 306 Da Masquita et al.’s investigated the importance of meningeal lymphatics for Aβ production in AD mouse models. They found that ablating meningeal lymphatics leads to Aβ accumulation in the meninges, accelerates Aβ deposition, and induces cognitive deficits. These findings are consistent with Aβ accumulation observed in the meninges of AD patients. Strategies for promoting the growth of meningeal lymphatic vessels may have the potential to enhance the clearance of Aβ and lessen the deposition of Aβ, 307 , 308 but this remains to be further validated.

Other hypotheses

In addition to the above hypotheses, there are many other factors that can affect the occurrence of AD. For a long time (at least 60 years), investigators have suspected that microbes may be involved in the onset and progression of AD, this was hypothesized by Sjogren et al. beginning in 1952. 309 In addition to McLachlan et al.’s proposal in 1980, 310 several investigators have proposed that AD may be caused by a viral form of herpes simplex. 311 , 312 , 313 , 314 There have been intensive reports suggesting that AD may be associated with various bacterial and viral pathogens, 315 , 316 , 317 especially herpesviridae (including HSV-1, 318 , 319 EBV, HCMV, HHV-6A, and HHV-7 314 , 320 ). However, these studies did not determine the underlying mechanisms or identify a robust association with a specific viral species. Recent reports have suggested that Aβ aggregation and deposition may be stimulated by different classes of microbes as a part of the innate immune response. Microbes trigger amyloidosis, and newly generated Aβ acts as an antimicrobial peptide to coat microbial particles to fight the infection. 321 , 322 , 323 Valaciclovir, an antiviral drug that is used for the management of herpes simplex and herpes zoster, is now in a phase II trial for AD (Table 2 ).

MicroRNAs (miRNAs) are involved in posttranscriptional gene regulation. 324 , 325 , 326 , 327 The decreased expression of miRNA-107 (miR-107) in AD may accelerate disease progression by regulating the expression of BACE1. 328 In SAD patients, the expression of miR-29a/b-1 is inversely correlated with BACE1 expression. 329 Only one clinical trial related to miRNAs is underway. Gregory Jicha launched a phase I trial to assess the safety and efficacy of gemfibrozil in modulating miR-107 levels for the prevention of AD in subjects with cognitive impairment (Table 2 ).

Mannose oligosaccharide diacid (GV-971) was developed by researchers at the Shanghai Institute of Medicine, the Chinese Academy of Sciences, the Ocean University of China, and the Shanghai Green Valley Pharmaceutical Co., Ltd. GV-971 is an oceanic oligosaccharide molecule extracted from seaweed. GV-971 may capture multiple fragments of Aβ in multiple sites and multiple states, inhibit the formation of Aβ filaments, and depolymerize filaments into nontoxic monomers 330 , 331 ; however, an understanding of the exact mechanism is still lacking. GV-971 has been reported to improve learning and memory in Aβ-treated mice. 332 In phase II trials, GV-971 improved cognition in AD patients. 333 In addition, a phase III clinical trial of GV-971 finished with positive results, and it is on its way to the market in China (Table 2 ).

Interestingly, a pilot clinical trial that included 120 nondemented elderly Chinese individuals (ages 60–79) living in Shanghai compared the effects of interventions (such as walking, Tai Chi, and social interaction) on cognition and whole brain volume, as determined by a neuropsychological battery and MRI scans. 334 The results showed that Tai Chi and social interaction were beneficial, but walking had no effect. Therefore, in addition to promising drugs, a healthy lifestyle can delay the progression of AD.

The whole brain atrophy rate is −0.67 to −0.8% per year in adulthood. 335 Freeman et al.’s results demonstrated that, although the frontal and temporal regions of the cortex undergoing thinning, the total number of neurons remains relatively constant from age 56 to age 103. However, there is a reduction in the number of hippocampal neurons in AD but not in normal aging. The loss of neuronal structural complexity may contribute to the thinning that occurs with aging. 336 The integrity of neurons and dendritic structures is the basis for maintaining the normal function of neurons. 337 , 338 , 339 Brain atrophy affects the function of neurons, which in turn impairs signal transmission and causes movement disorders, cognitive disorders etc. 340 , 341 , 342 , 343 Brain atrophy has been shown to be a key pathological change in AD. 344 , 345 , 346 , 347 In particular, the annual atrophy rate of the hippocampus in AD patients (−3.98 ± 1.92%) is two to four times that of the atrophy rate in healthy individuals (−1.55 ± 1.38%). At the same time, the annual increase in the temporal lobe volume of the lateral ventricle in AD patients (14.16 ± 8.47%) is significantly greater than that in healthy individuals (6.15 ± 7.69%). 348 The ratio of the volume of the lateral ventricle to the volume of the hippocampus may be a reliable measurement for evaluating AD since the ratio can minimize variances and fluctuations in clinical data and may be a more objective and sensitive method for diagnosis and evaluating AD. In 1975, brain atrophy and a reduction in perfusion were detected in AD patients. 349 In 1980, atrophy of hippocampal neurons and abnormal brain metabolism were first discovered in AD patients with PET. 350 Brain volume reduction in patients with AD is significantly associated with dementia severity and cognitive disturbances as well as neuropsychiatric symptoms. 351 The development of broad-spectrum drugs that target brain atrophy, a common feature of neurodegenerative diseases, is still ongoing. In our previous work, RAS–RAF–MEK signaling was demonstrated to protect hippocampal neurons from atrophy caused by dynein dysfunction and mitochondrial hypometabolism (tetramethylrhodamine ethyl ester mediated mitochondrial inhibition), suggesting the feasibility of interventions for neuronal atrophy. 352

The MAPK pathway protects neurons against dendritic atrophy and relies on MEK-dependent autophagy. 352 Autophagy is the principal cellular pathway by which degraded proteins and organelles are recycled, and it plays an essential role in cell fate in response to stress. 353 , 354 , 355 , 356 , 357 Aged organelles and protein aggregates are cleared by the autophagosome–lysosome pathway, which is particularly important in neurons. 358 , 359 , 360 Growing evidence has implicated defective autophagy in neurodegenerative diseases, including AD, PD, amyotrophic lateral sclerosis and HD. 358 , 361 , 362 , 363 , 364 Recent work using live-cell imaging determined that autophagosomes preferentially form at the axon tip and undergo retrograde transport to the cell body. 365 As a key protein in autophagy, Beclin 1 is decreased in the early stage of AD. 357 , 366 , 367 Moreover, a decrease in autophagy induced by the genetic ablation of Beclin 1 increases intracellular Aβ accumulation, extracellular Aβ deposition, and neurodegeneration. 368 Autophagy decline also causes microglial impairments and neuronal ultrastructural abnormalities. 368 On the other hand, transcriptome evidence has revealed enhanced autophagy–lysosome function in centenarians. 369 PPARA-mediated autophagy can reduce AD-like pathology and cognitive decline. 370 These results suggest that autophagy is a potential therapeutic target for AD. MEK-dependent autophagy is protective in neuronal cells. 352 The activation of the MEK–ERK signaling pathway can reduce the production of toxic amyloid Aβ by inhibiting γ-secretase activity. 371 , 372 , 373 , 374 , 375 Thus, MEK-dependent autophagy may provide a potential way to enhance Aβ and NFT clearance and may also be a new potential target for AD therapy (Fig. 4 ).

Schematic representation of autophagy. Yellow box: mTOR-dependent autophagy pathways. Growth factors can inhibit autophagy via activating the PI3K/Akt/mTORC1 pathway; under nutrient-rich conditions, mTORC1 is activated, whereas under starvation and oxidative stress, mTORC1 is inhibited. AMPK-dependent autophagy activation can be induced by starvation and hypoxia. 449 Ras can also activate autophagy via activating PI3K, 352 while p300 can inhibit autophagy. 450 p38 promotes autophagy by phosphorylating and inactivating Rheb and then inhibiting mTOR under stress. 451 Green boxes: mTOR-independent autophagy pathways. The PI3KCIII complex (also called the Beclin 1–Vps34–Vps15 complex) is essential for the induction of autophagy and is regulated by interacting proteins, such as the negative regulators Rubicon, Mcl-1, and Bcl-XL/Bcl-2, while proteins including UVRAG, Atg14, Bif-1, VMP-1, and Ambra-1 induce autophagy by binding Beclin 1 and Vps34 and promoting the activity of the PI3KCIII complex. 357 In addition, various kinases also regulate autophagy. ERK and JNK-1 can phosphorylate Bcl-2, release its inhibition, and consequently induce autophagy; the phosphorylation of Beclin 1 by Akt inhibits autophagy, whereas the phosphorylation of Beclin 1 by DAPK promotes autophagy. 452 Autophagy can be inhibited by the action of PKA and PKC on LC3. Finally, Atg4, Atg3, Atg7, and Atg10 are autophagy-related proteins that mediate the formation of the Atg12–Atg5–Atg16L1 complex and LC3-II. 453 RAS and p300 can also regulate autophagy via the mTOR-independent pathway 454

Hypometabolism is sufficient to cause neuronal atrophy in vitro and in vivo. 352 , 376 , 377 Hypometabolism may be a potential therapeutic target for AD. 378 Regional hypometabolism is another characteristic of AD brains (Fig. 5 ). The human brain makes up ~2% of the body weight but consumes up to ~20% of the oxygen supply; the brain is energy demanding and relies on the efficiency of the mitochondrial TCA cycle and oxidative phosphorylation for ATP generation. 379 , 380 , 381 , 382 However, glucose metabolism in the brain in AD and mild cognitive impairment is significantly impaired compared with that in the brain upon normal aging, and the decline in cerebral glucose metabolism occurs before pathology and symptoms manifest and gradually worsens as symptoms progress. 383 , 384 , 385 In 1983, de Leon et al. examined aged patients with senile dementia and found a 17–24% decline in the cerebral glucose metabolic rate. 386 Inefficient glucose utilization, impaired ATP production, and oxidative damage are closely correlated, and these deficiencies have profound consequences in AD. 387 , 388 For example, ATP deficiency causes the loss of the neuronal membrane potential since Na + /K + ATPase fails to maintain proper intracellular and extracellular gradients of Na + and K + ions. In addition, the propagation of action potentials and the production of neurotransmission is hindered by energy insufficiency. Moreover, after membrane depolarization (mainly due to the dissipation of Na + and K + ion gradients), Ca 2+ flows down the steep gradient (~1.2 mM of extracellular Ca 2+ to ~0.1 μM of intracellular Ca 2+ ) into the cell to raise intracellular Ca 2+ levels and stimulates the activities of various Ca 2+ -dependent enzymes (including endonucleases, phospholipases, and proteinases), eventually contributing to neuronal dysfunction and death. 158 Mitochondria are the most energetically and metabolically active organelles in the cell. 389 , 390 Mitochondria are also dynamic organelles; they experiences changes in their functional capacities, morphologies, and positions 391 , 392 , 393 so that they can be transported, and they respond to physiological signals to meet the energy and metabolic demands of cellular activities. 394 , 395 , 396 In addition to neuronal atrophy, mitochondrial dysfunction leads to hypometabolism, which in turn contributes to the progression of AD. 397 , 398 , 399 Indeed, there is evidence that hypometabolism and neuronal atrophy coexists in patients with amyloid-negative AD. 400 In addition to mitochondrial dysfunction, hypoperfusion and hypoxia in vascular diseases may also cause hypometabolism in the brain and thus contribute to the progression of AD (Fig. 5 ). Meanwhile, as the synthesis of acetylcholine requires the involvement of acetyl-CoA and ATP, hypometabolism leads to a decrease in acetylcholine synthesis in neurons, which suggests that hypometabolism may be an underlying explanation for the acetylcholine hypothesis (Fig. 5 ).

In addition to mitochondrial dysfunction, hypometabolism may underlie the cholinergic hypothesis, metal ion hypothesis, and neurovascular hypothesis. a Glucose is enzymatically catalyzed to produce pyruvate. Pyruvate is converted to acetyl-CoA and then enters the TCA cycle or is used in the cytoplasm to synthesize acetylcholine. However, in AD patients, because of hypometabolism, the production of acetyl-CoA and ATP is insufficient, which leads to a reduction in acetylcholine synthesis. b Mitochondrial complexes I–III require Fe/S clusters, and complexes II–IV need hemoproteins for electron transfer and the oxidative phosphorylation of the respiratory chain. When iron deficiency occurs, the production of Fe/S and hemoproteins decreases, thereby affecting mitochondrial function and resulting in hypometabolism. In addition, copper is essential for the function of complex IV. Clearly, Cu–Zn superoxide dismutase (SOD1) requires copper and zinc. 455 , 456 c Hypoperfusion and hypoxia in vascular diseases leads to insufficient oxygen supply, which in turn leads to insufficient ATP synthesis, resulting in hypometabolism in AD patients. TCA: tricarboxylic acid cycle; SOD1: superoxide dismutase 1

The relationship between hypometabolism and autophagy in neurons is still unknown, 352 but calorie restriction (CR) is known to enhance autophagy. CR-induced autophagy can recycle intracellular degraded components and aggregates to maintain mitochondrial function. 401 Hypometabolism and a simultaneous decrease in autophagy can worsen the situation and lead to the dysfunction and atrophy of neurons. Hypometabolism and a simultaneous decrease in autophagy may be causative factors of brain atrophy and AD (Fig. 6 ).

Hypometabolism and autophagy decline are likely to be causative factors of neuronal atrophy. Normal neurons vs. atrophic neurons. Upper: Normal levels of autophagy and metabolism exist in neurons to maintain their morphology and function. Lower: Hypometabolism and a reduction in autophagy are found in atrophic neurons

Perspective

AD, like the aging population, has increasingly become a medical and social concern. There are currently four clinically used drugs (a total of five therapies, the fifth one of which is a combination of two drugs) that have been approved by the FDA, but they only treat the symptoms and have no significant effect on the progression of AD. Based on this retrospective review of AD and the lessons learned, we propose that fluoxetine, 402 a selective serotonin reuptake inhibitor (SSRI), may have strong potential for the treatment of AD (Fig. 7 ).

The potential mechanisms of fluoxetine in the remission of AD. As a selective 5-HT reuptake inhibitor, fluoxetine can increase the extraneuronal concentration of 5-HT. 5-HT binds to the 5-HT 4 A receptor to promote neuronal dematuration through a Gs-mediated pathway. 5-HT binds to the 5-HT 1 A receptor, which is involved in BDNF-dependent neurogenesis through the Gi-mediated signaling pathway. After 5-HT stimulation, MeCP2 is phosphorylated at Ser421 through CaMKII-dependent signaling, and this promotes the dissociation of CREB from HDAC and then increases the expression of BDNF. BDNF activates downstream signaling pathways, including the MEK-ERK pathway, which might promote the activity of α-secretase, inhibit γ-secretase, and reduce the production of toxic amyloid Aβ. Moreover, the serotonylation of histone H3 at glutamine 5 (Q5) enhances the binding of H3K4me3 and TFIID and allows gene expression. Fluoxetine has been reported to bind and inhibit NMDA receptors directly, which can reduce the inhibition of α-secretase and thus prevent the production of Aβ. In addition, fluoxetine can bind to the endoplasmic reticulum protein sigma-1 receptor, which induces the dissociation of Bip from the sigma-1 receptor and promotes neuroprotection. 5-HT: serotonin; ER: endoplasmic reticulum

Based on functional brain imaging with PET, there is evidence that serotonin plays an important role in aging, late-life depression, and AD. 403 Short-term treatment with the antidepressant fluoxetine can trigger pyramidal dendritic spine synapse formation in the rat hippocampus. 404 In an MRI study of fluoxetine for the treatment of major depression, Vakili et al. found that female responders had a statistically significant higher mean right hippocampal volume than that of nonresponders. 405 Long-term treatment with fluoxetine can promote the neurogenesis and proliferation of hippocampal neurons in mice through the 5-HT 1 A receptor, and this can relieve anxiety phenotypes in mice 406 and enhance mitochondrial motility. 407 5-HT 4 A receptors that are expressed by mature neurons in the hippocampal dentate gyrus are also important for promoting neurogenesis and dematuration. 408 , 409 , 410 Fluoxetine can promote neurogenesis not only in the hippocampus but also in the anterior cortex and hypothalamus. 411 This action depends on BDNF, as fluoxetine can enhance the phosphorylation of methyl-CpG binding protein 2 (MeCP2) at serine 421 to relieve its transcriptional inhibition and thereby promote the expression of BDNF. 412 , 413 In addition to promoting neurite outgrowth and neurogenesis, enhanced BDNF signaling can rearrange the subcellular distribution of α-secretase, which increases its binding to APP peptides; in addition, the activity of β-secretase is inhibited after BDNF treatment. 414 Moreover, the serotonylation of glutamine (at position 5) in histone H3 by a transglutaminase 2-mediated manner is a sign of permissive gene expression. 415

Furthermore, fluoxetine has been reported to bind and inhibit NMDA receptors directly in the CNS, 416 and this can reduce the inhibition of α-secretase and thus prevent the production of Aβ. 203 , 417 Fluoxetine also inhibits γ-secretase activity and reduces the production of toxic amyloid Aβ by activating MEK-ERK signaling. 371 , 372 In addition, fluoxetine can bind to the endoplasmic reticulum protein sigma-1 receptor. 418 Sigma-1 receptor ligands can enhance acetylcholine secretion. 419 , 420 The sigma-1 receptor activator Anavex 2–73 has entered a phase III clinical trial after it was granted fast-track status by the FDA because of the promising results in phase II. The sigma-1 receptor is located in the mitochondrion-associated ER membrane so that the activation of the sigma-1 receptor can prolong Ca 2+ signaling in mitochondria. 421 Consequently, the local and specific elevation of [Ca 2+ ] in the mitochondrial matrix can enhance ATP synthesis, 422 , 423 which ameliorates hypometabolism.

In addition, our group examined the effect of SSRIs on cognitive function in AD by conducting a meta-analysis of randomized controlled studies. Of the 854 articles identified, 14 articles that involved 1091 participants were eligible for inclusion. We compared changes in MMSE scores between SSRI treatment groups and the placebo group, and we found that SSRIs may contribute to improved cognitive function, with a mean difference (MD) of 0.84 (95%CI: 0.32–1.37, P   =  0.002) compared with the control. Further subgroup analysis exploring the effect of fluoxetine and other SSRIs revealed a beneficial effect of fluoxetine (MD = 1.16, 95%CI: 0.41–1.90, P   =  0.002) but no benefit of other SSRIs (MD = 0.58, 95%CI: −0.17–1.33, P   =  0.13) on cognitive function. 424 Consequently, all of the above evidence indicates that fluoxetine has strong potential for the treatment of AD. In addition, because of above wealthy supporting documentation and the weak role of other SSRIs such as escitalopram in promoting BDNF release, 425 fluoxetine was singled out as a potential therapy for the treatment of AD, not just as a complementary treatment. 426 As summarized and illustrated in Fig. 7 , the exact mechanisms of the effects of fluoxetine remain to be further clarified.

Finally, to summarize this review of the history and progress of hypotheses and clinical trials for AD, the most perplexing question is in regards to amyloid hypothesis and its failed clinical trials, which account for 22.3% of all clinical trials (Fig. 1 ). Although mutations in APP , PSEN1 , or PSEN2 only account for ~0.5% of all AD cases, 11 mutations in PSEN1, which is the most common known genetic cause of FD and functions as the catalytic subunit of γ-secretase, 427 , 428 may cast light upon Aβ and its paradox. In 2017, Sun et al. analyzed the effect of 138 pathogenic mutations in PSEN1 on the production of Aβ−42 and Aβ−40 peptides by γ-secretase in vitro; they found that 90% of these mutations led to a decrease in the production of Aβ−42 and Aβ−40 and that 10% of these mutations result in decreased Aβ−42/Aβ−40 ratios. 429 This comprehensive assessment of the impact of FD mutations on γ-secretase activity and Aβ production does not support the amyloid hypothesis and suggests an alternative therapeutic strategy aimed at restoring γ-secretase activity 430 ; this is also supported by the fact that the functional loss of both PSEN1 and PSEN2 in the mouse postnatal forebrain causes memory impairment in an age-dependent manner. 431 Considering that the activation of Notch signaling by the cleavage of γ-secretase 432 is not involved in age-related neurodegeneration, 433 other signaling pathways mediated by Aβ and/or other products of γ-secretase substrates, such as ErbB4, 434 E-cadherin, 435 N-cadherin, 436 ephrin-B2, 437 CD44, 438 and LDL receptor-related protein, 439 may play active roles in neuronal survival in the adult brain.

The most interesting and challenging phenomena regarding fluoxetine is that fluoxetine is clinically more effective in women than in men 440 and that the prevalence of AD and other dementias is higher in women than in men 441 ; meanwhile, women live significantly longer than men. 442 These phenomena suggest that there are interplays or trade-offs between AD and longevity. In particular, APOE is the strongest genetic risk factor for AD 18 , 19 , 20 , 21 and is the only gene associated with longevity that achieves genome-wide significance ( P  < 5 × 10 –8 ). 443 APOE4 is associated with a risk of AD that declines after the age of 70; the OR for APOE4 heterozygotes remains above unity at almost all ages; surprisingly, however, the OR for APOE4 homozygotes dips below unity after the age of 89. 444 There may be genetic and nongenetic factors that interact with APOE4 , lead to shorter survival in more aggressive form of AD, or promote longevity in an age-dependent manner. 11 Uncovering the puzzle of APOE4 and the mystery of longevity may provide insights for AD prevention.

Change history

23 september 2019.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank all researchers for their work in the AD field, as well as all institutes and companies for their efforts in clinical trials. We are also grateful to the many authors for their papers that were uncited due to limited space and time. We are grateful for Wei Liu’s discussion about autophagy. We appreciate funding by the National Natural Science Foundation of China (Grant No. 31171369), the National Basic Research Program (973 Program) (Nos 2011CB910903 and 2010CB912001), the Chinese Academy of Sciences (Hundred Talents Program and 2009OHTP10), the Joint Construction Project of Henan Province (No. 2018020088 and No. 2018020114), and the First Affiliated Hospital of Zhengzhou University.

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Liu, PP., Xie, Y., Meng, XY. et al. History and progress of hypotheses and clinical trials for Alzheimer’s disease. Sig Transduct Target Ther 4 , 29 (2019). https://doi.org/10.1038/s41392-019-0063-8

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DOI : https://doi.org/10.1038/s41392-019-0063-8

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The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: recent challenges and their implications for novel drug development

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• 1 Program in Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA 30912-2450, USA. [email protected]
• PMID: 12805474
• DOI: 10.1124/jpet.102.041616

The cholinergic hypothesis was initially presented over 20 years ago and suggests that a dysfunction of acetylcholine containing neurons in the brain contributes substantially to the cognitive decline observed in those with advanced age and Alzheimer's disease (AD). This premise has since served as the basis for the majority of treatment strategies and drug development approaches for AD to date. Recent studies of the brains of patients who had mild cognitive impairment or early stage AD in which choline acetyltransferase and/or acetylcholinesterase activity was unaffected (or even up-regulated) have, however, led some to challenge the validity of the hypothesis as well as the rationale for using cholinomimetics to treat the disorder, particularly in the earlier stages. These challenges, primarily based on assays of post mortem enzyme activity, should be taken in perspective and evaluated within the wide range of cholinergic abnormalities known to exist in both aging and AD. The results of both post mortem and antemortem studies in aged humans and AD patients, as well as animal experiments suggest that a host of cholinergic abnormalities including alterations in choline transport, acetylcholine release, nicotinic and muscarinic receptor expression, neurotrophin support, and perhaps axonal transport may all contribute to cognitive abnormalities in aging and AD. Cholinergic abnormalities may also contribute to noncognitive behavioral abnormalities as well as the deposition of toxic neuritic plaques in AD. Therefore, cholinergic-based strategies will likely remain valid as one approach to rational drug development for the treatment of AD other forms of dementia.

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• Acetylcholine / metabolism*
• Aging / physiology*
• Alzheimer Disease / metabolism*
• Alzheimer Disease / physiopathology
• Alzheimer Disease / prevention & control
• Cholinergic Antagonists / therapeutic use
• Cognition Disorders / etiology*
• Cognition Disorders / prevention & control
• Cholinergic Antagonists
• Acetylcholine

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• Paul T Francis a ,
• Alan M Palmer b ,
• Michael Snape b ,
• Gordon K Wilcock c
• a Dementia Research Laboratory, Neuroscience Research Centre, Guy’s, King’s and St Thomas’ Schools of Biomedical Sciences, King’s College, London, SE1 9RT, UK, b Cerebrus, Oakdene Court, 613 Reading Road, Winnersh, Wokingham, RG41 5UA, UK, c Department of Care of the Elderly, Frenchay Hospital, Bristol, BS16 2EW, UK
• Dr Paul T Francis, Dementia Research Laboratory, Division of Biomolecular Sciences, Guy’s, King’s and St Thomas’ Schools of Biomedical Sciences, King’s College, St Thomas Street, London SE1 9RT, UK. Telephone 0044 171 955 2611; fax and answer phone 0044 171 955 2600; email p.francis{at}umds.ac.uk

Alzheimer’s disease is one of the most common causes of mental deterioration in elderly people, accounting for around 50%-60% of the overall cases of dementia among persons over 65 years of age. The past two decades have witnessed a considerable research effort directed towards discovering the cause of Alzheimer’s disease with the ultimate hope of developing safe and effective pharmacological treatments. This article examines the existing scientific applicability of the original cholinergic hypothesis of Alzheimer’s disease by describing the biochemical and histopathological changes of neurotransmitter markers that occur in the brains of patients with Alzheimer’s disease both at postmortem and neurosurgical cerebral biopsy and the behavioural consequences of cholinomimetic drugs and cholinergic lesions. Such studies have resulted in the discovery of an association between a decline in learning and memory, and a deficit in excitatory amino acid (EAA) neurotransmission, together with important roles for the cholinergic system in attentional processing and as a modulator of EAA neurotransmission. Accordingly, although there is presently no “cure” for Alzheimer’s disease, a large number of potential therapeutic interventions have emerged that are designed to correct loss of presynaptic cholinergic function. A few of these compounds have confirmed efficacy in delaying the deterioration of symptoms of Alzheimer’s disease, a valuable treatment target considering the progressive nature of the disease. Indeed, three compounds have received European approval for the treatment of the cognitive symptoms of Alzheimer’s disease, first tacrine and more recently, donepezil and rivastigmine, all of which are cholinesterase inhibitors.

https://doi.org/10.1136/jnnp.66.2.137

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Alzheimer’s disease affects an estimated 15 million people worldwide and is the leading cause of dementia in elderly people. With the proportion of elderly people in the population increasing steadily, the burden of the disease, both to carers and national economies, is expected to become substantially greater over the next 2 to 3 decades.

Alzheimer’s disease is a progressive neurodegenerative disorder with a mean duration of around 8.5 years between onset of clinical symptoms and death. Brain regions that are associated with higher mental functions, particularly the neocortex and hippocampus, are those most affected by the characteristic pathology of Alzheimer’s disease. This includes the extracellular deposits of β-amyloid (derived from amyloid precursor protein; APP) in senile plaques, intracellular formation of neurofibrillary tangles (containing an abnormally phosphorylated form of a microtubule associated protein, tau), and the loss of neuronal synapses and pyramidal neurons. These changes result in the development of the typical symptomology of Alzheimer’s disease characterised by gross and progressive impairments of cognitive function and often accompanied by behavioural disturbances such as aggression, depression, and wandering. Carers find these features the most difficult to cope with and they often lead to the need for institutionalisation of the patient. 1

The systematic biochemical investigation of the brains of patients with Alzheimer’s disease began in the late 1960s and early 1970s. The hope was that a clearly defined neurochemical abnormality would be identified, providing the basis for the development of rational therapeutic interventions analogous to levodopa treatment of Parkinson’s disease. Support for this perspective came in the mid-1970s with reports of substantial neocortical deficits in the enzyme responsible for the synthesis of acetylcholine (ACh), choline acetyltransferase (ChAT). 2-4 Subsequent discoveries of reduced choline uptake, 5 ACh release 6 and loss of cholinergic perikarya from the nucleus basalis of Meynert 7 confirmed a substantial presynaptic cholinergic deficit.

These studies, together with the emerging role of ACh in learning and memory, 8 led to the “cholinergic hypothesis of Alzheimers disease” (figure A). Thus it was proposed that degeneration of cholinergic neurons in the basal forebrain and the associated loss of cholinergic neurotransmission in the cerebral cortex and other areas contributed significantly to the deterioration in cognitive function seen in patients with Alzheimer’s disease. 9

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Schematic diagram of a neuron representing (A) alterations in neurotransmission in Alzheimer’s disease and (B) the hypothetical mode of action of AChE inhibitors. Key to figure (A): (1) reduced cortical cholinergic innervation; (2) reduced corticocortical glutamatergic neurotransmission due to neuron or synapse loss; (3) reduced coupling of muscarinic M1 receptors to second messenger system?; (4) shift of tau to the hyperphosphoryalted state—precursor of neurofibrillary tangles; (5) reduced secretion of soluble APP; (6) increased production of β-amyloid protein; (7) decreased glutamate production. *It is hypothesised that these changes give rise to the clinical symptoms of Alzheimer’s disease and contribute to the spread of pathology. 12 49 54 Key to figure B: (1) AChE inhibitors reduce the breakdown of endogenously released ACh, resulting in greater activation of postsynaptic ACh receptors; hypothesised consequences: (2) reduced phosphorylation of tau; (3) secretion of sAPP returned towards normal; (4) reduced β-amyloid production; (5) glutamatergic neurotransmission returns towards normal, possibly due to activation of muscarinic and nicotinic receptors. ACh=acetylcholine; mAChR=ACh muscarinic receptor; APP=a myloid precursor protein; AChE=acetylcholinesterase; nAChR=ACh nicotinic receptor; Glu=glutamate.

Over the 20 years since the origins of the cholinergic hypothesis, data from numerous studies have challenged its veracity as an explanation for the syndrome of dementia in Alzheimer’s disease. Thus, this review attempts to re-evaluate the cholinergic hypothesis in the following ways:

(1) Setting the original findings of reduced cholinergic neurotransmission in the context of changes in other neurotransmitter systems, a clear understanding of the behavioural role of the cholinergic system, and a more detailed understanding of the molecular pathology of the disease.

(2) Charting the preclinical and clinical development of cholinomimetic drugs for the symptomatic treatment of Alzheimer’s disease, focusing on the first generation and second generation cholinesterase inhibitors currently available.

Neurochemical and histopathological changes in cholinergic and non-cholinergic neurons in Alzheimer’s disease

At postmortem, Alzheimer’s disease is characterised by neuronal loss and neurofibrillary tangle formation in circumscribed regions of the neocortex and hippocampus, primarily affecting pyramidal neurons and their synapses. 10 11 Neurotransmitter specific subcortical nuclei that project to the cortex are also affected by neurodegenerative processes, including the cholinergic nucleus basalis of Meynert and medial septum, the serotonergic raphe nuclei, and the noradrenergic locus coeruleus.

Biochemical investigations of biopsy tissue taken from patients with Alzheimer’s disease 3.5 years (on average) after the onset of symptoms indicate that a selective neurotransmitter pathology occurs early in the course of the disease. 12 Specifically, presynaptic markers of the cholinergic system appear uniformly reduced. This is exemplified by reductions in ChAT activity and ACh synthesis which are strongly correlated with the degree of cognitive impairment in patients with Alzheimer’s disease. 12-15 Whereas serotonergic and some noradrenergic markers are affected, markers for dopamine, γ-aminobutyric acid (GABA), or somatostatin are not altered. 12 When postmortem studies of Alzheimer’s disease brain are considered (typically representing a later stage of the disease) many more neurotransmitter systems are involved or are affected to a greater extent. These include GABA 16 17 and somatostatin 18 19 and may indicate that cortical interneurons, for which these are neurochemical markers, are affected later in the disease process. Based on postmortem studies, however, changes in serotonergic neurotransmission may be linked to the behavioural disturbances of Alzheimer’s disease such as depression, rather than cognitive dysfunction. 1 20 21

On the basis of the above evidence, neocortical cholinergic innervation is probably lost at an early stage of the disease, a conclusion substantiated by evidence for similar changes in patients that have displayed clinical symptoms for less than 1 year. 22 However, although the loss of cholinergic function is correlated with the cognitive impairment in Alzheimer’s disease, an association between two such indices does not necessarily indicate a causal relation. Other indices also correlate with measures of cognitive decline in Alzheimer’s disease, such as loss of synapses and pyramidal cell perikarya. 23 Moreover, a few patients with Alzheimer’s disease do not show large decreases in ChAT activity, albeit that a small reduction is found in the amygdala. 24 In addition, patients with inherited olivopontocerebellar atrophy have diminished ChAT activity of a magnitude similar to that seen in Alzheimer’s disease in the absence of cognitive deficits. 25 Thus, although diminished ChAT activity is a necessary correlate of Alzheimer’s disease, additional factors other than impaired cholinergic function are likely to participate in the decline in cognitive function. Other studies have demonstrated a reduction in the number of nicotinic 26 and muscarinic (M2) ACh receptors in Alzheimer’s disease brains, most of which are considered to be located on presynaptic cholinergic terminals, but a relative preservation of postsynaptic muscarinic (M1, M3) receptors. 27 However, there is some evidence for a disruption of the coupling between the muscarinic M1 receptors, their G-proteins, and second messenger systems. 28

In addition to cholinergic dysfunction, other strong correlates of dementia are the chemical and histopathological markers of excitatory amino acid (EAA) releasing cortical pyramidal neurons. These neurons, considered to contribute to normal cognitive function in their own right, also seem to have a pivotal role in cholinergic function as they are cholinoceptive. 29-32 Although neurochemical studies of EAA neurotransmission have failed to show profound or extensive alterations in EAA neuronal indices, 12 this may be related to the difficulty in distinguishing the transmitter pool of aspartate and glutamate from the metabolic pool. Nevertheless, glutamate concentration was reduced by 14% in temporal lobe biopsy samples of patients with Alzheimer’s disease. Greater reductions were evident at postmortem in regions enriched with EAA nerve terminals. 33 Uptake of d -aspartate, a putative marker of EAA nerve endings, is also reduced in many cortical areas in Alzheimer’s disease brains. 34-36

Arguably, in vivo imaging studies of patients with Alzheimer’s disease also support the involvement of pyramidal neurons in the disease as the pattern of regional hypometabolism parallels neuronal loss/atrophy, tangle formation, and synapse loss. 10 37-39 Loss of cortical pyramidal neurons, 23 40 41 synapse loss, 40 and reduced glutamate concentration, 17 together with the formation of neurofibrillary tangles, 42 all correlate with the severity of dementia. These findings indicate that pyramidal neurons and their transmitter glutamate (and/or aspartate) play a part in the cognitive symptoms of Alzheimer’s disease and may therefore represent an additional therapeutic target. However, these neurons are cholinoceptive and it is reasonable to propose that one of the actions of cholinomimetic drugs for the treatment of Alzheimer’s disease is to increase the activity of EAA neurons through muscarinic and nicotinic receptors that are present on such cells. 29 This is supported by electrophysiological studies showing the excitatory actions of cholinomimetic drugs in cortical pyramidal neurons from both rats and humans 30 31 and microdialysis studies in rats. 32 Clearly, as a result of cholinergic and other pyramidal neuronal loss, the profound reduction in EAA neurotransmission will lead to pyramidal hypoactivity compounded by maintained levels of inhibition by GABAergic neurons. Consequently, it may be hypothesised that in addition to the deleterious effects of neuronal loss and tangle formation, there is a change in the balance of neurotransmission in the Alzheimer’s disease brain favouring lower neuronal activity. 12 This may be reflected in the hypometabolism in patients with Alzheimer’s disease seen with imaging techniques, although a component of this is also likely to be due to neuronal atrophy. 43 Likewise, it is of interest that regional cerebral blood flow may be increased in patients with Alzheimer’s disease by cholinesterase (ChE) inhibitors such as physostigmine. 44 45

CHOLINERGIC AND NON-CHOLINERGIC NEURONS AND ALZHEIMER’S DISEASE NEUROPATHOLOGY

The discovery that rare mutations in the gene encoding for APP always led to Alzheimer’s disease in family members carrying the defect resulted in the proposal of the “amyloid cascade hypothesis” of Alzheimer’s disease. 46 Thus, the mismetabolism of APP leading to increased production of β-amyloid was proposed as the critical event in both familial and sporadic Alzheimer’s disease with other changes, tangles, neuron loss, synapse loss, and neurotransmission dysfunction, following as a consequence. Cholinergic neurotransmission may be a specific target for β-amyloid, as it has been shown to reduce both choline uptake and ACh release in vitro. 47 48 It is of interest here that disease related changes in the Alzheimer’s disease brain are focused on pyramidal neurons in that these cells are lost in the disease, subject to tangle formation, represent a major source of APP (and hence, a site for its mismetabolism leading to increased β-amyloid production) and are regulated by a neurotransmitter (ACh), affected early in the disease. These neurons therefore seem to have a central role in the clinical symptoms as well as in the pathophysiology of the disease. Observations in cell lines and primary neuronal cultures that the activation of muscarinic, metabotropic glutamate, and other phospholipase C-linked receptors favours the non-amyloidogenic processing of APP 49 suggests that compounds being developed for symptomatic treatment may have a serendipitous effect on the continuing emergence of pathology by reducing the production of β-amyloid. Furthermore, β-amyloid neurotoxicity is attenuated by muscarinic agonists. 50 No data have yet been reported regarding the potential beneficial effects of cholinomimetic drugs on either increasing APP or reducing β-amyloid production in patients with Alzheimer’s disease. There is, however, some evidence for reductions in CSF fluid APP in depressed patients receiving drugs with anticholinergic side effects. 51 Clearly, long term studies are called for to test this hypothesis in the patient population. However, this may raise ethical problems—for example, the need for serial lumbar puncture and the justification for groups of patients to act as placebo controls.

Other studies have shown that the phosphorylation of tau, thought to be an important step in the formation of tangles (which occur predominately in EAA cortical pyramidal neurons), may also be influenced by the phospholipase C second messenger system. 52 Thus, after muscarinic cholinergic receptor stimulation, activation of protein kinase C may lead to the inactivation of a protein kinase (GSK-3) which phosphorylates tau, in vitro, in a similar manner to that found in Alzheimer’s disease. 52 In support of this tenet, neuronal cells in culture transfected with M1 muscarinic receptors show reduced phosphorylation of tau after treatment with cholinergic agonists. 53 Therefore, as a consequence of reduced cholinergic activity, reduced activation of protein kinase C may lead to a higher level of activity of GSK-3 and hence hyperphosphorylation of tau. Thus, if these neurotransmitter-protein interactions occur in the Alzheimer’s disease brain, it is not inconceivable that the changes in the balance of neurotransmission in the Alzheimer’s disease brain may contribute to increased tau phosphorylation and β-amyloid production and hence neurodegeneration in selectively vulnerable regions. Furthermore, it is possible that ChE inhibitors may reduce the histopathological features of disease progression.

On the basis of recent studies of Alzheimer’s disease, a glutamatergic hypothesis of Alzheimer’s disease has been proposed as an auxiliary to the cholinergic hypothesis. 12 54 Thus, the cholinergic hypothesis may be refined to include the idea that a major target of cholinomimetic action is EAA pyramidal neurons, and that cholinergic hypofunction compounds the loss of EAA function. Together these systems may be largely responsible for the neuropsychological deficits and may contribute to the continuing emergence of pathology in patients with Alzheimer’s disease. This revised cholinergic hypothesis provides a stronger case for the continued development of cholinomimetic drugs for the symptomatic treatment of Alzheimer’s disease.

Behavioural consequences of cholinomimetic drugs and cholinergic lesions

Many pharmacological studies have examined the effect of cholinomimetic drugs and cholinergic receptor antagonists on learning and memory tasks. The most commonly used model is based on the finding that scopolamine, a muscarinic receptor antagonist, induces amnesia in young healthy subjects comparable with that in old, untreated subjects. 8 These deficits may be reversed by ChE inhibitors. Compounds that reverse these scopolamine induced deficits in experimental animals may be considered as potential drugs to treat cognitive impairment.

It is, however, difficult to separate reliably the effects on learning and memory processes from effects on other behavioural domains. For example, methylscopolamine (which does not cross the blood-brain barrier) is as active as scopolamine in several models of cognitive function, 55 56 indicating that peripheral changes induced by these compounds indirectly influence performance in cognitive tasks. It is, therefore, very important to distinguish central versus peripheral effects of cholinminetic agents. Scopolamine induced impairment of performance may also be mediated by direct effects on sensorimotor function or motivation deficits. 56 57 Further, it is likely that the scopolamine induced impairment in the performance of both experimental animals and humans in the delayed matching to position task (a commonly used test of cognitive function) is secondary to attentional deficits that are induced by the drug. 58 59

Both hippocampal and cortical areas of the brain receive major cholinergic input from basal forebrain nuclei. Thus, the lesioning of these nuclei has been used to model cholinergic denervation in Alzheimer’s disease and to establish the behavioural consequences of cholinergic deafferentation. The most significant and consistent effects of such cholinergic lesioning on learning and memory follow lesioning of cholinergic pathways that lead to the hippocampus. 60 61 Initial studies used stereotaxic injection of ibotenic acid to lesion cholinergic nuclei, and caused profound deficits in discrimination learning and memory. However, injection of the toxins quisqualic acid and α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) into the same site causes a greater loss of ChAT activity than ibotenate but only marginal impairments in the same range of cognitive tasks. 62 Thus, in addition to the established role for ACh in learning and memory, there are data to suggest that ACh also plays a critical part in attentional processing. 63-65 This is supported by a study showing that both tacrine and nicotine improve attentional functions in patients with Alzheimer’s disease. 66

Cholinomimetic therapy in Alzheimer’s disease

A prediction of the cholinergic hypothesis is that drugs that potentiate central cholinergic function should improve cognition and perhaps even some of the behavioural problems experienced with Alzheimer’s disease. There are a number of approaches to the treatment of the cholinergic deficit in Alzheimer’s disease, most of which have initially focused on the replacement of ACh precursors (choline or lecithin) but these agents failed to increase central cholinergic activity. Other studies have investigated the use of ChE inhibitors that reduce the hydrolysis of ACh (figure, B)—for example, physostigmine. More recent investigational compounds include specific M1 muscarinic or nicotinic agonists, M2 muscarinic antagonists, or improved “second generation” ChE inhibitors (table).

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Cholinomimetic drugs in clinical development for Alzheimer’s disease including European registrations

Additional potential symptomatic therapeutic avenues relevant to the cholinergic hypothesis of Alzheimer’s disease have resulted from the rapid development in the understanding of the molecular pathology of the disease. For example, during the development of cholinergic neurons in the basal forebrain, they express functional nerve growth factor (NGF) receptors. In adult life, these neurons seem to remain responsive to NGF. Consequently, intraventricular administration of NGF has been shown to prevent the lesion induced loss of cholinergic neuronal cell bodies and to accelerate the recovery of behavioural deficits in learning. 67 Another approach is the transplantation of ACh rich foetal tissue grafts, which has been shown to improve the cognitive performance of primates after excitotoxic lesions of cholinergic nuclei. 68 Thus, although such approaches may provide additional future possibilities for the palliative treatment for Alzheimer’s disease, the use of ChE inhibitors is the most well developed approach to treatment to date.

PRECLINICAL STUDIES OF CHOLINESTERASE INHIBITORS

Although a variety of ChE inhibitors have been developed as potential treatments for Alzheimer’s disease, their pharmacological activities differ. One of the most fundamental differences between them is in the mechanism of ChE inhibition. For example, enzyme kinetic studies have shown that tacrine, an acridine compound, and donepezil, a novel piperidine class agent, are “mixed type” reversible inhibitors of ChE. That is, these compounds inhibit ChE via both non-competitive (by blockade of the deacetylation process) and ACh competitive mechanisms. 69 Thus, these compounds reversibly bind to the hydrophobic region of the enzyme to “allosterically” modulate catalytic activity. Further, the inhibition produced by these compounds is mutually exclusive, suggesting that both compounds act at similar sites within the enzyme, although donepezil is more potent and selective. 70 71

This type of inhibition differs from that produced by the carbamates— for example, rivastigmine and physostigmine derivatives such as heptylphysostigmine. This class of compounds have been termed “pseudoirreversible” ChE inhibitors, in that they are actually cleaved by the enzyme, resulting in a covalent modification of the enzyme. Such inhibition is non-competitive with ACh and is irreversible. However, the association of the carbamate with the esteric site is transient (taking several minutes) due to both rapid metabolism and the relative rapid rate of decarbamylation which regenerates ChE. 72-74 A further compound, metrifonate, inhibits ChE irreversibly. Metrifonate is a prodrug that is converted into dichlorvos, an organophosphorus ChE inhibitor with a very long duration of inhibition (the half life is 52 days). 75

The principal influence of the mechanism of action of enzyme inhibitors in the clinic relates to their duration of action. A more theoretical issue is the effect of pronounced non-competitive inhibition on the rate of enzyme synthesis. Non-competitive inhibitors may produce only slowly reversible ChE inhibition. The rate at which this inhibition is reversed may be of the same order as the rate of enzyme synthesis. 76 Thus, the long term effects of administration of slowly reversible, or irreversible, inhibitors on the overall cholinergic function are difficult to predict.

The selectivity of enzyme inhibition also plays a crucial part in determining the therapeutic profile of any ChE inhibitor. In this regard, several factors should be taken into account. All compounds will possess a greater or lesser degree of selectivity, and many of the differences between compounds may be influenced by the actions of the compound other than its intended ChE inhibition. Not surprisingly, therapeutic agents developed as inhibitors of AChE, which is found primarily in neural tissue, may also inhibit butyrylcholinesterase (BuChE), which acts mainly in the periphery. Although the function of BuChE remains unknown, 77 clinical data with selective and non-selective AChE inhibitors suggest the BuChE inhibition may be associated with unwanted peripheral side effects, 78 79 although to date, this remains an unproved empirical finding. However, compared with tacrine, less peripheral cholinergic-related side effects have been found with donepezil, as it is over 1000-fold more selective for AChE than BuChE. 70 74 79 80 Thus, greater brain AChE inhibition may be achieved with donepezil at the therapeutically effective dose compared with tacrine, increasing donepezil’s potential clinical efficacy. 71

A further factor associated with the in vivo pharmacology of mixed type ChE inhibitors is that such compounds may interact with the site at which ACh is “captured” within the AChE enzyme, and may also act at other sites that bind or recognise Ach. 81 82 Both tacrine and donepezil displace the binding of selective ligands from muscarinic and nicotinic ACh receptors, 57 71 83-85 although neither compound has significant activity at other neurotransmitter receptors. At muscarinic receptors, both compounds act as antagonists. 71 However, these effects only occur at concentrations of the compounds significantly greater than those needed to produce the required degree of ChE inhibition and are not therefore likely to have relevance in the clinic. 86

Donepezil, like tacrine, has been reported to have effects on other neurotransmitter systems other than via receptors. For instance, donepezil is only 10-fold less potent than imipramine at inhibiting the uptake of serotonin. 71 However, unlike tacrine, some second generation ChE inhibitors have been shown, using in vivo microdialysis techniques to measure the extracellular concentration of neurotransmitters and their metabolites, to increase monoamine concentrations in the cortex after administration of therapeutic doses. 87 88 These type of effects might be expected to influence affective states—for example, mood—in a positive manner. Given that depression and aggression are important determinants of quality of life for patients with Alzheimer’s disease and their carers, such effects may have clinical relevance. 1

CLINICAL TRIALS

First generation cholinesterase inhibitors.

During the late 1980s and early 1990s, the first cholinomimetic compound, tacrine, underwent a large number of clinical studies using various doses and treatment periods ranging from a few days to 30 weeks. Tacrine was subsequently approved for use in some, but not all, countries. Evidence from three pivotal studies of tacrine has established clearly the benefits of ChE treatment in patients with a diagnosis of probable Alzheimer’s disease. 89-91 Statistically significant, dose related improvements on objective performance based tests of cognition, clinician and caregiver rated global evaluations of patient wellbeing, and also quality of life measures have been reported. 89-92

Unfortunately, potentially serious adverse side effects have limited the use of this compound. Both tacrine, and the carbamate physostigmine, possess detrimental effects on hepatic and cardiovascular function. Indeed, perhaps the most often documented reason for withdrawal of tacrine is its potential hepatotoxicity. However, this effect seems to be unrelated to dose, and alanine aminotransferase (ALT) concentrations usually return to normal after drug withdrawal. In addition, many patients can be successfully rechallenged with tacrine after the enzymes return to normal. 93

Among the other unwanted side effects of tacrine, the most often occurring are those caused by overstimulation of the peripheral cholinergic system at or below 30% ChE inhibition reflecting its dose related tolerability. 94 These side effects are manifested predominantly by gastrointestinal tract discomfort and overactivity, resulting in nausea, vomiting, abdominal pain, and diarrhoea. Doses of tacrine within the therapeutic range elicit such side effects in about 20% of tacrine treated patients. 95 In one 30 week clinical trial, over 50% of patients treated with tacrine discontinued treatment because of cholinergic related side effects. In addition, over 70% of patients titrated to the highest dosage of tacrine (160 mg/day) failed to complete this 30 week study. 91 These incidental effects limit the compound’s maximum tolerated dose that may be administered to patients, and therefore the extent of brain AChE inhibition that can be achieved.

Despite these limitations, a substantial number of patients, some 250 000–300 000 worldwide, have been exposed to tacrine. Consequently, although tacrine produces a meaningful benefit in a significant proportion of patients with Alzheimer’s disease, the question has been raised as to whether this approach represents a fair test of the cholinergic hypothesis. This issue has been considered in the development of “second generation” ChE inhibitors. Such ChE inhibitors have been designed to limit side effect problems, and the maximum tolerated dose that can be achieved may be determined more by the effects of ChE inhibition itself.

Second generation cholinesterase inhibitors

At least an equivalent level of benefit is likely to be produced by the newer second generation ChE inhibitors including donepezil, 96-98 rivastigmine, 99 metrifonate, 100-102 galantamine 103 and several other compounds. Such compounds show an effect and magnitude of benefit of at least that reported for tacrine, but with a more favourable clinical profile. For example, donepezil has a once daily dosage schedule and produces dose related significant improvements in cognition and global function, with over 80% of patients experiencing an improvement or no deterioration in cognition. Such responses should be viewed positively, considering the progressive, degenerative nature of the disease. In one 30 week randomised, double blind study of donepezil (5 or 10 mg/day) versus placebo (n=150/group, 450 total), statistically significant improvements were obtained with both 5 and 10 mg/day of donepezil for the intent to treat analysis of Alzheimer’s disease assessment scale (ADAS-cog 104 ; p⩽0.001) and the clinician’s interview based impression of change (CIBIC plus 105 ; p⩽0.005). 97 This clinical improvement (as determined by the ADAS-cog) was correlated with both donepezil plasma concentrations and AChE inhibition. 96 Further, a retrospective subanalysis of the 30 week trial clinical dementia rating scale domains that reflect activities of daily living (ADLs): community affairs, home and hobbies, and personal care, suggests that donepezil (10 mg/day) delays the loss of ADLs by about 1 year. 106 Preliminary evidence from open label studies showed that the treatment effect of donepezil is maintained over long periods (at least 2 years). 107 This general thesis that ChE inhibitors will delay the progression of symptoms of Alzheimer’s disease and improve patients, on average, by the equivalent of 6–12 months deterioration, is now receiving further support with the publication of results from the trials of rivastigmine and metrifonate. 99-102

Substantially more patients were able to tolerate and achieve therapeutic concentrations of donepezil than was possible with tacrine. Donepezil (5 and 10 mg/day) is well tolerated with no evidence of hepatotoxicity. 70 96-98 108 and an incidence of side effects (5 mg/day) similar to that of placebo. 96-98 The mainly cholinergic side effects that do occur are usually mild, transient, and resolve with continued treatment. As with the other available ChE inhibitors including tacrine, the incidence of side effects has been reported to be slightly increased in patients treated with the higher dosages of ChE inhibitors. This is likely due to the rapid, forced dose titration schedule used in clinical trials. Indeed, a lower incidence of side effects was found when a longer titration schedule was employed; for example, escalation to 10 mg/day donepezil after 4–6 weeks allowing achievement of a steady state at 5 mg/day donepezil. 97

Diagnostic inaccuracy, which may be as high as 20%, can produce a strong bias in favour of non-response when the results in different treatment arms are “averaged out”. The heterogeneity of Alzheimer’s disease at a genetic, clinical, neurochemical, and neuropathological level may also contribute to differing response rates. Thus, whereas some patients, of course, respond considerably more than the mean reported in clinical trials, others similarly, will respond less. It may prove difficult to develop one therapy with an equivalent effect across the disease range of stage and severity. However, it may be possible to define patients by genotype, or by other markers, and tailor treatment to specific clinical subtypes, and work is underway to explore this possibility.

Response rates of ChE inhibitors do not detract from their clinical importance, as ChE inhibitors provide meaningful and important benefits for some patients with Alzheimer’s disease and their families. There is no doubt now that significant proportions of patients with probable mild to moderate Alzheimer’s disease gain some benefit from ChE inhibitors. In qualitative terms, a delay in symptomatic decline by about 6–12 months is valuable to those with Alzheimer’s disease, and also to those that care for and about them. At an anecdotal level, individual patients that have been treated successfully, and their relatives have reported improved awareness and attention, greater motivation and independence, improved language and communication abilities, and an improvement in the ability to undertake previously impaired or abandoned ADLs and hobbies. For example, a good response can include such features as the patient being able, once again, to manage their own day to day activities, being able to make and take telephone calls spontaneously, undertake their hobbies and pastimes and, in some cases, even to go shopping and successfully return with the required goods without having become lost.

Finally, evidence is emerging from clinical trials of cholinomimetic drugs that such drugs may improve the abnormal non-cognitive, behavioural symptoms of Alzheimer’s disease. Thus, ChE inhibitors have been reported to significantly improve many manifestations of behavioural disturbance including agitation, apathy, hallucinations, and aberrant motor behaviour 101 109 and xanomeline, a selective muscarinic agonist, has been shown to improve vocal outbursts and psychotic symptoms. 110 Further, such long term treatment with ChE inhibitors may delay or reduce the need for nursing home placement, and even reduce mortality (a trend for reduced mortality has been noted with tacrine). 111 These findings require confirmation, and it will be important to establish whether this reduction in mortality increases the duration of the subsequent, more dependent stages of the disease. In addition, although nursing home placement may be delayed, the effect on the actual duration of nursing home care should be ascertained.

CLINICAL USE

Choosing the right patient.

Cholinomimetic treatment is targeted specifically at patients with Alzheimer’s disease, albeit that such treatment may be beneficial in other dementias where a cholinergic deficit also exists—for example, Lewy body disease. Trials are currently underway to explore this possibility. The severity of the dementia is another important factor to be considered as currently these drugs have been assessed adequately in patients with mild to moderately severe Alzheimer’s disease only, but again this is subject to further evaluation and current practice may change as clinical experience increases. In addition, it is essential to make a careful assessment of the patients’ illness to ensure that they are likely to have Alzheimer’s disease. Primary care physicians may screen for and recognise patients with suspected Alzheimer’s disease within the community, but often referral to a specialist service is required. As there is no definitive diagnostic test for Alzheimer’s disease, it is important to base a diagnosis of “probable Alzheimer’s disease” on careful consideration of the patients’ symptoms and signs, preferably using the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM IV) 112 or National Institute of Neurological and Communicative Disorders and Stroke - Alzheimer’s Disease and Related Disorders Association work group (NINCDS-ADRDA) criteria, 113 or an equivalent protocol. If properly applied, the accuracy rate of diagnosis using such criteria, confirmed at necropsy, probably varies between 85% and 95%, 114 115 depending on the experience of the centre in which the patient is assessed.

Decisions on continuing long term cholinesterase inhibitor treatment

Less than half of the patients receiving ChE inhibitors achieve a clinically significant response, although no further deterioration or even a slowing of deterioration are desirable outcomes, given the progressive, degenerative nature of the disease. Nevertheless, all patients with Alzheimer’s disease should have the opportunity of a treatment trial of at least 3 months in duration. Unfortunately, however, it has not yet been possible to predict or distinguish responders from non-responders.

In clinical trials, various psychometric outcome measures are used to assess the efficacy of drug treatment for Alzheimer’s disease. The cognitive subscale of the ADAS-cog and the CIBIC are used often to determine the efficacy of pharmacological agents. The ADAS-cog measures memory, orientation, attention, language, function, and praxis. CIBIC measures patients’ global function in terms of general, cognitive, behavioural, and ADL domains (for example, personal care and hobbies), and is determined by experienced clinicians, but may incorporate input from the primary caregiver of the patient with Alzheimer’s disease (for example, the CIBIC plus). A further assessment, the mini mental state examination (MMSE), 116 is a short collection of cognitive tests that examines several areas of cognition. It is widely used to measure the onset, progression, and severity of Alzheimer’s disease in the clinical setting. The test is easy to administer and score, and can be used readily in a primary care setting, both at the office and in the patient’s home.

In clinical practice, it is rarely possible to undertake such a comprehensive assessment, but some degree of objectivity concerning treatment effect is, nevertheless, essential. Many physicians will rely on a simple general test such as the MMSE, coupled with a global measure formed from the relative or other carer’s opinion about the response, and their own assessment based on notes made at the first assessment. Those patients that clearly benefit from treatment should continue. However, treatment should be terminated in those patients who show deterioration, albeit that such patients must be closely monitored during such periods due to reports of precipitous decline after abrupt discontinuation. In addition, drug free periods, between 3–6 months for example, may be useful in evaluating the response to ChE inhibitors; deterioration during such periods would indicate that continued treatment is appropriate. 117

The cholinergic hypothesis of Alzheimer’s disease is based on the presynaptic deficits found in the brains of patients with Alzheimer’s disease and studies of the role of ACh in animal and human behaviour. Although it is now clear that cholinergic dysfunction may not cause cognitive impairment directly, but rather indirectly, by interfering with attentional processing, the hypothesis predicted that cholinomimetic drugs would improve cognitive function. This prediction was not fully realised with compounds such as physostigmine and tacrine, probably because the emergence of side effects that may have constrained the dosing regimen to sub-efficacious doses. Poor tolerability seems to be less of an issue for the second generation compounds of the type now being licensed for the treatment of Alzheimer’s disease. With improved diagnosis, careful patient selection, and fewer side effects, such compounds will establish if cholinomimetic therapy provides effective and long lasting palliative therapy. Moreover, the emerging relation between neurotransmission and metabolism of two key proteins involved in Alzheimer’s disease, APP and tau, raises the possibility that second generation ChE inhibitors may alter disease pathology and progression.

Acknowledgments

Work on this manuscript was supported by an educational grant from Eisai Inc and Pfizer Pharmaceuticals Group, Pfizer Inc. We thank PPS International, Worthing, UK, for their assistance in the development of this manuscript and Drs K Stanhope and M Sheardown for helpful discussion.

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Article Contents

Introduction, interactions between the cholinergic system and the other pathophysiological hallmarks of alzheimer’s disease, conflicts of interest, supplementary material.

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The cholinergic system in the pathophysiology and treatment of Alzheimer’s disease

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Harald Hampel, M.-Marsel Mesulam, A Claudio Cuello, Martin R Farlow, Ezio Giacobini, George T Grossberg, Ara S Khachaturian, Andrea Vergallo, Enrica Cavedo, Peter J Snyder, Zaven S Khachaturian, The cholinergic system in the pathophysiology and treatment of Alzheimer’s disease, Brain , Volume 141, Issue 7, July 2018, Pages 1917–1933, https://doi.org/10.1093/brain/awy132

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Cholinergic synapses are ubiquitous in the human central nervous system. Their high density in the thalamus, striatum, limbic system, and neocortex suggest that cholinergic transmission is likely to be critically important for memory, learning, attention and other higher brain functions. Several lines of research suggest additional roles for cholinergic systems in overall brain homeostasis and plasticity. As such, the brain’s cholinergic system occupies a central role in ongoing research related to normal cognition and age-related cognitive decline, including dementias such as Alzheimer’s disease. The cholinergic hypothesis of Alzheimer’s disease centres on the progressive loss of limbic and neocortical cholinergic innervation. Neurofibrillary degeneration in the basal forebrain is believed to be the primary cause for the dysfunction and death of forebrain cholinergic neurons, giving rise to a widespread presynaptic cholinergic denervation. Cholinesterase inhibitors increase the availability of acetylcholine at synapses in the brain and are one of the few drug therapies that have been proven clinically useful in the treatment of Alzheimer’s disease dementia, thus validating the cholinergic system as an important therapeutic target in the disease. This review includes an overview of the role of the cholinergic system in cognition and an updated understanding of how cholinergic deficits in Alzheimer’s disease interact with other aspects of disease pathophysiology, including plaques composed of amyloid-β proteins. This review also documents the benefits of cholinergic therapies at various stages of Alzheimer’s disease and during long-term follow-up as visualized in novel imaging studies. The weight of the evidence supports the continued value of cholinergic drugs as a standard, cornerstone pharmacological approach in Alzheimer’s disease, particularly as we look ahead to future combination therapies that address symptoms as well as disease progression.

Late-onset Alzheimer’s disease dementia, the most prevalent age-related neurodegenerative disease, is clinically characterized by a progressive loss of memory and other cognitive functions. In contrast to early-onset autosomal dominant forms of Alzheimer’s disease, which are directly linked to abnormalities of amyloid-β, the cascade of pathophysiological events that leads to late-onset Alzheimer’s disease is not yet fully understood. Contemporary evidence suggests that late-onset Alzheimer’s disease is a complex polygenic disease that involves aberrant interaction among several molecular pathways. By definition, age is the strongest risk factor ( Hebert et al. , 1995 ) followed by the ɛ4 allele of apolipoprotein E ( APOE ɛ4) ( Liu et al. , 2013 ; Shi et al. , 2017 ), and probably also cardiovascular and lifestyle risk factors ( de Bruijn and Ikram, 2014 ). The neuropathological features of Alzheimer’s disease include the accumulation of several abnormal proteins such as amyloid-β in plaques and hyperphosphorylated-tau in neurofibrillary tangles, leading to massive loss of synapses, dendrites, and eventually neurons. Clinical expression of the disease reflects the dysfunction and eventual failure of both neurochemical and structural neural networks, including the ‘cholinergic system’. Although the pivotal events in the pathogenesis of Alzheimer’s disease are not fully understood, several competing theories on the underlying biology of the neurodegeneration have guided research into interventions to modify, arrest, or delay the progression of the disease and its clinical manifestations. In recent years, however, failure of clinical trials in Alzheimer’s disease has been the rule rather than the exception, and no new drugs for Alzheimer’s disease have been approved by the US Food and Drug Administration (FDA) since 2003. The multifaceted, heterogeneous, progressive, and interactive pathophysiology of Alzheimer’s disease also suggests a likely need for individualized combination treatments that may need to be varied from one stage of the disease to another, and perhaps also from one patient to another.

The cholinergic hypothesis revolutionized the field of Alzheimer’s disease research by transporting it from the realm of descriptive neuropathology to the modern concept of synaptic neurotransmission. It is based on three milestones: the discovery of depleted presynaptic cholinergic markers in the cerebral cortex ( Bowen et al. , 1976 ; Davies and Maloney, 1976 ); the discovery that the nucleus basalis of Meynert (NBM) in the basal forebrain is the source of cortical cholinergic innervation that undergoes severe neurodegeneration in Alzheimer’s disease ( Mesulam, 1976 ; Whitehouse et al. , 1981 ); and the demonstration that cholinergic antagonists impair memory whereas agonists have the opposite effect ( Drachman and Leavitt, 1974 ). The hypothesis received compelling validation when cholinesterase inhibitor therapies were shown to induce significant symptomatic improvement in patients with Alzheimer’s disease ( Summers et al. , 1986 ). Although other relevant pathophysiological mechanisms have received more research attention in recent years, treatments that improve cholinergic function remain critical in the management of patients with Alzheimer’s disease. The goal of this review is to characterize the nature of the cholinergic lesion in Alzheimer’s disease, its potential interactions with other components of the pathology, and its relevance to treatment. We do not aim to provide a comprehensive review of Alzheimer’s disease pathogenesis or to rank order the impact of the cholinergic lesion among all other components of this disease. Furthermore, our comments will be limited to late-onset Alzheimer’s disease in patients who do not have disease-causing dominant mutations. We should also point out that the brain contains several cholinergic pathways, each with its unique receptor signature, postsynaptic targets and disease vulnerabilities. Unless noted otherwise, our comments in this review will address the forebrain pathway that originates in the basal forebrain and that innervates the neocortex and limbic system. This review also provides a comprehensive evaluation of the known benefits of cholinergic therapies throughout the various stages of Alzheimer’s disease. We aim to demonstrate the enduring value of cholinergic drugs in the pharmacological therapy of Alzheimer’s disease, especially in the context of future combination therapies that may affect both symptoms and disease progression.

Nature and impact of the cholinergic lesion

Acetylcholine is a major neurotransmitter in the brain, with activity throughout the cortex, basal ganglia, and basal forebrain ( Mesulam, 2013 ). Figure 1 illustrates the key steps in the synthesis, release, and reuptake of the neurotransmitter acetylcholine.

Physiology of the cholinergic synapse. Choline is the critical substrate for the synthesis of acetylcholine. Acetyl coenzyme A (Ac CoA), which is produced by the breakdown of glucose (carbohydrate) through glycolysis (Krebs cycle), along with the enzyme choline acetyltransferase (ChAT) are critical for the synthesis of acetylcholine (Ach). Once the neurotransmitter acetylcholine is released into the synapse, it binds (activates) postsynaptic receptor (M 1 ), thus transmitting a signal from one neuron to the other. The excess neurotransmitter in the synaptic cleft is broken down by the enzyme acetyl cholinesterase (AChE) into choline and acetate, which are returned by an uptake mechanism for recycling into acetyl coenzyme A.

Human studies assessing the neuropathological diagnosis of Alzheimer’s disease have shown that the cholinergic lesion, emerging as early as asymptomatic or prodromal stages of the disease, is mainly presynaptic rather than postsynaptic. In other words, the cholinergic loss is based on the degeneration of NBM cholinergic neurons and of the axons they project to the cerebral cortex. As part of the cholinergic lesion, nicotinic (ionotropic) receptors and muscarinic (metabotropic) receptors of the cerebral cortex also undergo changes. Most studies show a loss of nicotinic receptors in the cerebral cortex. For example, there is a decrease of postsynaptic nicotinic receptors on cortical neurons ( Nordberg and Winblad, 1986 ; Schroder et al. , 1991 ). However, there may also be an equally important presynaptic component based on the loss of nicotinic receptors located on the degenerating cholinergic axons coming from the NBM. With respect to muscarinic receptors of the cerebral cortex, it is interesting that the muscarinic (M)1 receptors (mostly postsynaptic) are not decreased whereas the M2 receptors (mostly presynaptic) are decreased ( Mash et al. , 1985 ). However, there is evidence that the remaining postsynaptic M1 receptors of the cerebral cortex may be dysfunctional ( Jiang et al. , 2014 ). Thus, a progressive loss of basal cholinergic neurons represents a key neurochemical event with a subsequent anterograde cortical cholinergic deafferentation, of the cerebral cortex, hippocampus and amygdala ( Sassin et al. , 2000 ). The alternative possibility of an initial degeneration of cortical cholinergic endings that lead to a retrograde degeneration of NBM neurons cannot be ruled out but is unlikely.

As noted above, in contrast to M1 receptors, which are mostly preserved, there is a loss of cortical nicotinic receptors. Postsynaptic α7 nicotinic receptor enhances the neuronal firing rates contributing to the hippocampal long-term potentiation, a neuronal-level component of learning and memory ( Francis et al. , 2010 ). The application of cholinergic agonists and antagonists to rat hippocampal slices has clarified the role for acetylcholine in long-term potentiation ( Blitzer et al. , 1990 ; Auerbach and Segal, 1996 ). Therefore, altered patterns of nicotinic and muscarinic receptor distribution in Alzheimer’s disease are likely to influence many functions of the cerebral cortex and limbic areas through perturbations of synaptic physiology. An upregulation of cortical choline acetyltransferase neuronal expression has been shown in prodromal Alzheimer’s disease patients, suggesting that such neurochemical events may compensate for the depletion of basal cholinergic neurons ( Ikonomovic et al. , 2007 ). Moreover, it has been shown that Alzheimer’s disease patients have higher levels of α7 nicotinic gene expression compared to healthy controls. The influence of these dynamic changes upon Alzheimer’s disease pathogenesis remains to be elucidated.

There is also evidence implicating acetylcholine in a variety of essential functions that promote experience-induced neuroplasticity, the synchronization of neuronal activity, and network connectivity. For instance, variable stimulation of the rat NBM, an acetylcholine-rich area of the basal forebrain with wide projections to the cortex, has been shown to produce extensive cortical remodelling and to modulate cortical sensory maps ( Kilgard and Merzenich, 1998 ). Through intrinsic (NBM) and extrinsic (perivascular postganglionic sympathetic nerve) innervation, the cholinergic system has also been shown to promote cerebral vasodilation and perfusion ( Claassen and Jansen, 2006 ; Van Beek and Claassen, 2011 ). In mice, electrical and chemical stimulation of cholinergic neurons in the NBM results in a significant increase in cerebral blood flow in several cortical areas ( Lacombe et al. , 1989 ; Sato and Sato, 1990 ; Barbelivien et al. , 1995 ; Lacombe et al. , 1997 ; Vaucher et al. , 1997 ). In addition to disrupting synaptic transmission in cortex and limbic areas, the cholinergic lesion of Alzheimer’s disease may therefore also interfere with multiple aspects of neuroplasticity and with cerebral haemodynamic processes.

Anticholinergic agents and cholinergic therapies

The negative pharmacological effects of anticholinergic drugs on human memory and learning have been reported since at least the 1970s ( Drachman and Leavitt, 1974 ; Petersen, 1977 ; Mewaldt and Ghoneim, 1979 ; Izquierdo, 1989 ), and more recent data support these observations. The use of anticholinergic medications in non-demented older adults has been associated with significantly slower reaction times on a measure of rapid information processing and lower cognitive test scores (Stroop test) ( Uusvaara et al. , 2009 ; Sittironnarit et al. , 2011 ). Moreover, the increased use of anticholinergic medications was correlated with reduced cognitive function in a systematic review of 33 studies performed in older adults ( Fox et al. , 2014 ). The cumulative effect of anticholinergic drugs has also been associated with poorer cognitive abilities, as well as poorer functional outcomes (i.e. activities of daily living) in cohort studies of older populations ( Salahudeen et al. , 2015 ). Furthermore, a recent meta-analysis demonstrated that the exposure of older adults with cardiovascular disease to anticholinergic drugs was associated with an increased risk of cognitive impairment ( Ruxton et al. , 2015 ). In that study, a greater burden of anticholinergic exposure was shown to more than double the odds of all-cause mortality.

Recent data also suggest that the negative cognitive effects of cumulative anticholinergic drugs in older adults may not be transient. Among cognitively healthy individuals in the ADNI (Alzheimer Disease Neuroimaging Initiative) and Indiana Memory and Aging Study, the 52 participants who had been regularly taking one or more medications with medium or high anticholinergic activity prior to study entry demonstrated worse immediate recall and executive function than the 350 participants who were not actively using anticholinergic medications at study entry ( Risacher et al. , 2016 ). Strikingly, cognitively normal adults taking anticholinergic medication were observed to have reduced total cortex volume, increased bilateral lateral ventricle volume, and increased inferior lateral ventricle volume. In addition, across both groups of participants, there was a significant longitudinal association between anticholinergic use and later progression to mild cognitive impairment (MCI) or Alzheimer’s disease dementia ( P = 0.01; hazard ratio, 2.47). Concordantly, in a prospective population-based cohort study of 3434 participants ≥65 years with no dementia at study entry, greater cumulative use of anticholinergic drugs over 10 years (based on computerized pharmacy dispensing data) was linked to a statistically increased risk for incident dementia and for Alzheimer’s disease specifically. Thus, higher estimates of cumulative exposure to anticholinergic therapies were associated with a greater risk for incident dementia or Alzheimer’s disease dementia than were lower levels of cumulative anticholinergic exposure ( Gray et al. , 2015 ). In addition to these findings, doses of anticholinergic medication appear to unmask signs of impending dementia in individuals with preclinical Alzheimer’s disease. In a study of healthy older adults at risk for Alzheimer’s disease, single-dose administration of the anticholinergic drug scopolamine unmasked cognitive deficits and poorer cognitive performance more often in patients with higher brain amyloid-β burden on PET images ( Lim et al. , 2015 ). More recently, impaired performance in response to a low-dose scopolamine challenge test among cognitively unimpaired adults at risk for Alzheimer’s disease predicted both amyloid-β positivity on PET images and a decline in episodic memory at 27 months ( Snyder et al. , 2017 ).

Treatment that promotes cholinergic function in individuals with, or at risk for, Alzheimer’s disease may also have more durable beneficial biological effects on the brain than a temporary augmentation of cognitive function. The French Hippocampus Study Group found, in a placebo-controlled trial in people with suspected prodromal Alzheimer’s disease, that use of the cholinesterase inhibitor donepezil was associated with substantially less regional cortical thinning and basal forebrain atrophy over time ( Cavedo et al. , 2016 , 2017 ). A placebo-controlled study on the same population found a 45% reduction in the rate of hippocampal atrophy after 1 year of treatment with donepezil ( Dubois et al. , 2015 ), a finding previously reported by another research group investigating patients with fully expressed dementia ( Hashimoto et al. , 2005 ). Although these results have not yet been linked to a specific biological mechanism, they raise the possibility of substantial brain structural protective effects of cholinergic treatment during various stages of Alzheimer’s disease. Several studies have also explored the role of cholinesterase inhibitors on cerebrovascular perfusion in Alzheimer’s disease and other dementias ( Geaney et al. , 1990 ; Ebmeier et al. , 1992 ; Arahata et al. , 2001 ; Venneri et al. , 2002 ; Lojkowska et al. , 2003 ; Ceravolo et al. , 2006 ). Patients with Alzheimer’s disease dementia receiving a single dose of cholinesterase inhibitor treatment showed an increase ( Geaney et al. , 1990 ; Ebmeier et al. , 1992 ) or a stabilization of cerebral blood flow ( Venneri et al. , 2002 ; Van Beek and Claassen, 2011 ) in the posterior parieto-temporal and superior frontal regions. A recent study showed decreased regional cerebral blood flow in the parietal cortex, and an increase in the frontal and the limbic cortices after 18 months of treatment with donepezil or galantamine ( Shirayama et al. , 2017 ). Case reports and investigations with small sample sizes have reported increased cerebral blood flow after treatment with cholinesterase inhibitors in patients with vascular dementia, dementia with Lewy bodies, and dementia of Parkinson’s disease ( Arahata et al. , 2001 ; Mori, 2002 ; Lojkowska et al. , 2003 ; Ceravolo et al. , 2006 ). The clinical impact of these haemodynamic events has not been clarified.

The main pathological hallmarks of Alzheimer’s disease include not only amyloid-β plaques and neurofibrillary tangles but also neuroinflammation, altered insulin resistance, oxidative stress and cerebrovascular abnormalities. These pathological hallmarks have complex reciprocal interactions with the cholinergic lesion. Previous post-mortem studies have shown that the loss of cortical cholinergic innervation is associated with and probably caused by the neurofibrillary tangles in the NBM ( Geula and Mesulam, 1994 ; Braak and Del Tredici, 2013 ; Mesulam, 2013 ). The basal forebrain cholinergic neurons are among the cell bodies most susceptible to neurofibrillary degeneration and neurofibrillary tangle formation ( Mesulam, 2013 ). There exists a long-established relationship between cholinergic abnormalities and amyloid-β pathology. Perry et al. (1978) correlated diminishing activity of the acetylcholine-synthesizing enzyme choline acetyltransferase with increasing numbers of neuritic plaques in the post-mortem brains of patients with Alzheimer’s disease ( Perry et al. , 1978 ). This correlation was also shown in cognitively unimpaired persons whose brains at autopsy revealed amyloid-β plaques. More recently, an inverse correlation was found between choline acetyltransferase activity and amyloid-β deposition in the inferior temporal gyrus of persons, at autopsy, who had had normal cognitive function ( Beach et al. , 2000 ). Moreover, presynaptic and postsynaptic markers of cholinergic activity were significantly reduced in non-demented individuals whose brains demonstrated neuritic plaques at autopsy—an effect that was even more pronounced in demented individuals with pathologically confirmed Alzheimer’s disease ( Potter et al. , 2011 ). Studies investigating regional correlations between the loss of cholinergic axons and the density of amyloid-β deposits in Alzheimer’s disease-affected human brains have also shown conflicting results. Although the correlation between cholinergic loss and neurofibrillary tangle (both presynaptically in the NBM and postsynaptically in the cortex) is more robust, this correlation is not uniform throughout the brain—specifically in the cingulate cortex ( Geula et al. , 1998 ; Potter et al. , 2011 ).

Animal experiments have suggested that the cholinergic depletion promotes amyloid-β deposition and tau pathology in ways that contribute to the cognitive impairment ( Ramos-Rodriguez et al. , 2013 ). For example, selective lesions of cholinergic neurons in the basal forebrains of Alzheimer’s disease rodent models have been reported to be associated with increased deposition of amyloid-β and levels of hyperphosphorylated tau in the hippocampus and cortex. These types of effects have been reported in the past but have been difficult to replicate. Cholinergic deficits in rat brains have also been shown to interact with acute proinflammatory mechanisms to produce or exacerbate cognitive impairment ( Field et al. , 2012 ).

Stimulation of α7 nicotinic receptors may have a neuroprotective effect against amyloid-β-induced toxicity trough activation of the PI3K-Ak axis, the anti-apoptotic factor bcl2 and downregulation of glycogen synthase kinase-3 (GSK3) ( Beaulieu, 2012 ). GSK3 over activation is correlated with high levels of toxic amyloid-β oligomers, hyperphosphorylated tau strains and neurofibrillary tangles ( Jaworski et al. , 2011 ; Chu et al. , 2017 ), activation of the α7 nicotinic receptor is associated with anti-inflammatory pathways also through downregulation of NFκB via Jak2 ( Kalkman and Feuerbach, 2016 ).

Nitsch et al. (1992) and Mori et al. (1995) demonstrated that the stimulation of cholinergic receptors either by muscarinic agonists or by cholinesterase inhibitor treatment shifted the processing of amyloid precursor protein (APP) towards non-amiloidogenic pathways.

Additional evidence has shown that muscarinic agonists, mainly M1 and less so M3, can downregulate amyloidogenic and tau-generating pathways. The mechanisms are not fully understood yet. However, it has been shown that M1 agonist may act as functional activators of protein kinase C (PKC) signalling which, in turn, promotes a metabolic shift towards α-secretase activity via upregulating ADAM17 [also known as tumour necrosis factor-α-converting enzyme (TACE)]. In support of this hypothesis, animal studies have demonstrated that orthosteric M1-selective agonists are associated with increased levels of APPs cleaved by alpha secretase ( Cisse et al. , 2011 ; Welt et al. , 2015 ). Conceivably, α7 nicotinic and coupling of M1 to PKC may lead to a downregulation of detrimental cell processes occurring in Alzheimer’s disease such as GSK3-mediated tau hyperphosphorylation ( Espada et al. , 2009 ).

The loss of acetylcholine-mediated vasomotor control of the blood–brain barrier could also potentially lead to an aberrant diffusion and transportation of metabolites between the interstitial fluid and the CSF. One possible consequence for this is the impairment of the clearance of amyloid-β from brain ( Hunter et al. , 2012 ). As shown by Weller and colleagues, cholinergic deafferentation may alter the blood–brain barrier and the dynamics of arterial and perivascular lymphatic drainage of amyloid-β ( Engelhardt et al. , 2016 ).

These observations illustrate the highly complex interactions that are likely to exist between cholinergic denervation and other pathological features of Alzheimer’s disease ( Ramos-Rodriguez et al. , 2013 ; Szutowicz et al. , 2013 ; Hartig et al. , 2014 ; Kolisnyk et al. , 2017 ). In addition, important neurophysiological relationships with other major neurotransmitter (serotonergic, dopaminergic, GABAergic) and neurohormonal (renin-angiotensin) systems that are also likely to take place remain to be elucidated ( Bodiga and Bodiga, 2013 ).

Complex interactions among different neurotransmitter systems are essential for adaptive responses and compensatory mechanisms both in physiological and pathophysiological conditions. For example, the activity of presynaptic α7 nicotinic receptor may facilitate glutamate release, while activation of muscarinic receptors may decrease both the release and the concentration of glutamate in the synaptic cleft ( Higley et al. , 2009 ). Although changes of neurotransmitters other than acetylcholine have been demonstrated in Alzheimer’s disease ( Limon et al. , 2012 ; Chalermpalanupap et al. , 2013 ; McNamara et al. , 2014 ) it should be underlined than no drugs selectively acting on noradrenergic, serotoninergic or GABAergic systems have been approved. Supplementary Table 1 provides an overview of the available evidence regarding the involvement of different neurotransmitter in Alzheimer’s disease, as well as the main molecular mechanisms associated with each receptor activity and their interplay with acetylcholine.

The cholinergic system and APOE genetic risk factor

The APOE ɛ4 allele is the strongest genetic risk factor for sporadic/late onset Alzheimer’s disease. The presence of two APOE ɛ4 alleles has been linked to disruptions of amyloid-β and tau proteostasis ( Liu et al. , 2013 ; Shi et al. , 2017 ), impaired clearance, aberrant post-translational modifications (i.e. hyperphosphorylation), mitochondrial dysfunction, and neuroinflammatory processes in ageing and Alzheimer’s disease. The APOE ɛ4 allele is strongly correlated with faster cognitive and functional decline ( Whitehair et al. , 2010 ). It is still unclear whether the presence of APOE ɛ4 allele affects the NBM neuronal functioning, and if it does whether this happens indirectly through amyloid-β and tau accumulation in the basal forebrain. To date, only two human retrospective post-mortem studies have shown that both healthy older individuals and mild Alzheimer’s disease patients, carrying the ɛ4 allele, had reduced neuronal metabolic activity in the NBM as measured by the size of the Golgi apparatus ( Salehi et al. , 1998 ; Dubelaar et al. , 2004 ). Previous studies showed that APOE genotype does not significantly influence the magnitude of the cholinesterase inhibitor response in mild-to- moderate Alzheimer’s disease ( Miranda et al. , 2015 ; Waring et al. , 2015 ). These studies suffer from methodological limitations that might have remarkably biased their results. In particular, several potentially confounding factors have not been taken into account i.e. stage of pathophysiological processes, pharmacogenomic background, and comorbidities. Interestingly, it has been recently shown that APOE genotype may influence cholinergic compensatory mechanisms. In particular, the APOE ɛ4 allele is associated with deficits in the cholinergic hippocampal compensatory sprouting and remodelling in response to cholinergic deafferentation ( Bott et al. , 2016 ). Based on these considerations, further work needs to be performed to investigate whether the APOE ɛ4 status influences the response to cholinomimetic therapy.

Anatomy, selectivity and specificity of the cholinergic deficit in Alzheimer’s disease

The cholinergic loss is one of the most prominent components of the neuropathology of Alzheimer’s disease. In the mid-1970s in the UK, investigators autopsied the brains of people with Alzheimer’s disease and reported a selective and statistically significant reduction in the activity of choline acetyltransferase in the limbic system and cerebral cortex ( Bowen et al. , 1976 ; Davies and Maloney, 1976 ; Perry et al. , 1977 a , b ). At the time, the origin of this cholinergic innervation was unknown. In 1976, axonal transport studies, combined with cholinergic histochemistry, revealed the NBM as the source of cholinergic innervation in the cerebral cortex of the primate brain ( Mesulam, 1976 ). These studies led to the investigation of the NBM in Alzheimer’s disease and to the post-mortem data from Whitehouse et al. (1981 , 1982 ), which demonstrated a profound loss of cholinergic neurons in the basal forebrain, specifically the NBM, of patients with Alzheimer’s disease. The NBM can be considered a rostral extension of the brainstem reticular formation. It innervates the entire cerebral cortex and limbic system, including the hippocampus, and the entorhinal cortex. It has been well established that cholinergic deficits play a key role in the neuropathology of Alzheimer’s disease, not only in late disease, but in preclinical and early stages as well. Accumulated abnormal phosphorylated tau, in the form of neurofibrillary tangles and pretangles, has been found specifically in the cholinergic neurons of the basal forebrain in cognitively normal elderly subjects and patients with MCI and to correlate significantly with performance in memory tasks ( Mesulam et al. , 2004 ). A progression of abnormalities has been observed in the cholinergic neurons of the basal forebrain of non-demented younger adults, non-demented elderly people, and people with mild or severe Alzheimer’s disease ( Geula et al. , 2008 ). Thickened cholinergic nerve fibres and ballooned terminals, demonstrated in middle-aged adults, have been shown to increase with age, suggesting that cholinergic loss in established Alzheimer’s disease is preceded by this cholinergic pathology ( Geula et al. , 2008 ). Cholinergic function outside of the NBM—namely, in the caudate, putamen, and thalamus—appears relatively spared in this process. There is, therefore, no generalized ‘cholinergic vulnerability’ in Alzheimer’s disease but, instead, a preferential vulnerability of the NBM. The underlying mechanism may be the location of the NBM within the corticoid-limbic belt of the forebrain, which includes other limbic structures such as the hippocampus, amygdala, and entorhinal cortex, areas that are collectively the most vulnerable to neurofibrillary degeneration and neurofibrillary tangle formation in the ageing–MCI–Alzheimer’s disease continuum ( Mesulam, 2013 ). With the use of longitudinal MRIs and amyloid-β biomarkers, it has been shown that volume loss in the NBM precedes and predicts memory impairment and degeneration of the entorhinal cortex ( Schmitz et al. , 2016 ). This observed relationship strengthens the conclusion that the loss of NBM neurons is an early and perhaps also clinically relevant event in Alzheimer’s disease.

Unlike the cholinergic neurons and synaptic terminations of the caudate, putamen, and thalamus, the NBM and medial septum cholinergic neurons are fully dependent on the retrograde transport of nerve growth factor (NGF) for the maintenance of their anatomic and biochemical characteristics and their terminal synapses in the cerebral cortex and hippocampus ( Cuello et al. , 2007 , 2010 ; Cuello, 2013 ). It is well accepted that the interactions of NGF with the forebrain cholinergic system is of significance in Alzheimer’s disease ( Mufson et al. , 2008 ; Schliebs and Arendt, 2011 ; Cattaneo and Calissano, 2012 ; Triaca and Calissano, 2016 ; Turnbull et al. , 2018 ). There is evidence that cholinergic neurons in the NBM may well be deprived of trophic support even before clinical manifestations of Alzheimer’s disease. While the biosynthesis of NGF in the cerebral cortex is not affected in Alzheimer’s disease, experimental animal data and human post-mortem brain material would indicate that trophic support of the NGF-dependent cholinergic neurons in the NBM may be compromised by defective retrograde transport of NGF or the diminished conversion of pre-NGF to mature NGF (neuroguidin) ( Cuello et al. , 2007 , 2010 ; Iulita and Cuello, 2014 ; Iulita et al. , 2017 ). In individuals with Down syndrome, who are at high risk for early-onset Alzheimer’s disease by amyloid-β-mediated mechanisms, rising plasma levels of amyloid-β and inflammatory markers have been associated with biomarker evidence of NGF dysregulation ( Iulita et al. , 2016 a , b ). These data suggest that NGF dysregulation may be precipitated by the accumulation of amyloid-β and amyloid-β-driven inflammation, the end result of which is cholinergic loss in the NBM. The potential downstream effects of amyloid-β on cholinergic neurons in the NBM, by way of dysregulated NGF, deserve further exploration. Therefore, the NGF metabolic pathway remains a potential pharmacological target in the effort to slow the loss of critical cholinergic function in Alzheimer’s disease, especially at preclinical stages ( McDade and Bateman, 2017 ). However, intracerebrally- and exogenously-applied NGF has so far shown to be unsuccessful. It is important to keep in mind that exogenous NGF may reach undesirable ectopic targets producing undesirable effects (pain, anorexia, other). On the other hand, the pharmacological normalization of the NGF metabolic pathway, if attainable at early Alzheimer’s disease pathology stages, could potentially halt the NBM degeneration by selectively boosting the trophic influence of NGF with greater physiological selectivity.

Pathology of the NBM is not unique to Alzheimer’s disease. Synucleinopathies such as Parkinson’s disease and especially Lewy body dementia are also associated with NBM degeneration and the resultant cortical cholinergic denervation. In Lewy body dementia this effect may be even more severe than in Alzheimer’s disease. In contrast to Alzheimer’s disease where the NBM degeneration is based on neurofibrillary tangle formation, in Lewy body dementia the degeneration is associated with intracellular Lewy bodies. It is interesting that cholinesterase inhibitors can improve cognition also in Parkinson’s disease and Lewy body dementia ( Graff-Radford et al. , 2012 ).

The role of cholinergic therapy for Alzheimer’s disease

FDA-approved cholinesterase inhibitors for Alzheimer’s disease a

a Minimum effective dosages are provided.

b Rivastigmine is also available as an oral solution, at a concentration of 2 mg/ml.

FDA = US Food and Drug Administration.

A meta-analysis of 26 studies of donepezil, rivastigmine, and galantamine showed a modest but clinically meaningful overall benefit of these drugs for stabilizing cognition, function, behaviour, and global clinical change ( Hansen et al. , 2008 ). Results from the few existing head-to-head comparisons of cholinesterase inhibitors have been mixed; however, an adjusted analysis of placebo-controlled data suggested that donepezil might have a slight advantage over rivastigmine and galantamine in efficacy and tolerability ( Hansen et al. , 2008 ). These results did not include the rivastigmine transdermal delivery system, which has fewer side effects than the oral formulation of rivastigmine. In a systematic review of seven studies that examined the economics of cholinesterase inhibitors, treatment of Alzheimer’s disease with cholinesterase inhibitors appeared to be a cost-effective, if not a cost-saving, strategy—although a considerable number of variables, such as the length of treatment and medication discounts, contributed to general uncertainty as to their benefits ( Pouryamout et al. , 2012 ). A large Medicare beneficiary study concluded that each additional month of cholinesterase inhibitors treatment is associated with a 1% reduction in total all-cause healthcare costs ( Mucha et al. , 2008 ).

Long-term data indicate that the use of a cholinesterase inhibitor in Alzheimer’s disease reduces the risk for nursing home placement by ∼30% for each year of treatment ( Feldman et al. , 2009 ). In addition, patients with Alzheimer’s disease who are treated with a higher mean dose of cholinesterase inhibitors compared with patients receiving a lower mean dose have been shown to experience delayed nursing home placement ( Wattmo et al. , 2011 ). These data are supported by a post hoc analysis of the DOMINO-AD trial, in which the nursing home placement of community-dwelling patients with moderate-severe Alzheimer’s disease was assessed ( Howard et al. , 2015 ). Patients who were randomized to discontinue donepezil therapy (10 mg/day) were twice as likely to enter a nursing home after 1 year as were individuals who continued treatment with cholinesterase inhibitors; however, this effect lost statistical significance after 3 years. Finally, cholinesterase inhibitors have also been shown to reduce the burden experienced by caregivers of patients with Alzheimer’s disease, by reducing caregiver time devoted to the patient, caregiver stress, and some of the behavioural symptoms ( Feldman et al. , 2003 ; Hashimoto et al. , 2009 ; Schoenmakers et al. , 2009 ; Adler et al. , 2014 ).

A recent meta-analysis carried out on 142 randomized controlled trials (RCTs), quasi-RCTs, and non-randomized studies in individuals with Alzheimer’s disease treated with cholinesterase inhibitors, only patients treated with galantamine showed a decreased odds-ratio of mortality when compared with placebo ( Tricco et al. , 2018 ). It has been reported that cholinesterase inhibitors delay the need for nursing home placement and institutionalization ( Jelic and Winblad, 2016 ). This interesting finding has been linked also to a potential effect of such drugs on behavioural and psychological symptoms of dementia (BPSD) ( Cumbo and Ligori, 2014 ). It is demonstrated that BPSD are positively associated with a faster decline in global functioning and higher caregiver burden ( Lyketsos et al. , 2011 ; Collins et al. , 2016 ). Loss of cerebral dopaminergic tone has been likened to apathetic syndrome, which is one of the most frequent and persistent BPSD in Alzheimer’s disease. The impaired dopamine release in the brain reward system has been hypothesized as a potential trigger of apathy in Alzheimer’s disease. Despite this interesting rationale, RCTs investigating the potential cholinomimetic influence on dopamine release effects have not been performed so far ( Lanctot et al. , 2017 ). It is generally believed that cholinesterase inhibitors are a part of the standard of care for management of Alzheimer’s disease, and the foundation of Alzheimer’s disease pharmacotherapy ( Hort et al. , 2010 ; O’Brien et al. , 2011 ; Segal-Gidan et al. , 2011 ; Moore et al. , 2014 ). In mild–moderate Alzheimer’s disease, the expected treatment benefit of cholinesterase inhibitors is a mean of 3 to 4 points on the cognitive subscale of the ADAS-Cog (Alzheimer’s Disease Assessment Scale), when placebo treatment is the reference standard. This score difference corresponds roughly to the expected cognitive decline in people with mild–moderate Alzheimer’s disease over 6 months if the disease is left untreated at these stages ( Hort et al. , 2010 ).

Additional data from both laboratory-based investigations and clinical trials have suggested that cholinesterase inhibitors may have a broader mechanism of action than enhancing cholinergic activity and that these drugs are associated with a stabilizing effect on the course of Alzheimer’s disease dementia that may be greater than expected by cholinesterase inhibition alone ( Giacobini, 1997 , 2002 ). Prospective long-term observational studies suggest that cholinesterase inhibitors offer a benefit over the long-term course of Alzheimer’s disease ( Atri et al. , 2008 ). Cognitive decline has been observed to occur significantly more slowly with cholinesterase inhibitors compared with no treatment, suggesting a delay relative to typical clinical course ( Giacobini, 2001 ). These observations are supported by other long-term data showing declines in cognitive and global functioning were slower with the persistent use of donepezil over a mean follow-up period of 3 years ( Wallin et al. , 2007 ). At least two other prospective observational Alzheimer’s disease studies offer similar results, demonstrating slower cognitive and functional deterioration with the persistent and continued use of cholinesterase inhibitors ( Rountree et al. , 2009 ; Gillette-Guyonnet et al. , 2011 ).

Suboptimal use of cholinesterase inhibitors is common

Despite clinical data and guideline recommendations supporting the use of cholinesterase inhibitors throughout all stages of Alzheimer’s disease, these drugs are often inappropriately regarded as ineffective in Alzheimer’s disease and therefore are underused. According to a US survey of 25 561 outpatient visits for Alzheimer’s disease specifically or dementia more generally, fewer than half (46%) of patients were prescribed cholinesterase inhibitors, with donepezil being the most frequently prescribed (68%) ( Maneno et al. , 2006 ). Of note, psychiatrists and neurologists were significantly more likely to prescribe cholinesterase inhibitors than were other physicians (odds ratios 5.5 and 2.6, respectively). In a Canadian survey of 803 physicians, doctors reported that they would be more likely to prescribe a cholinesterase inhibitor if it enabled a person with mild Alzheimer’s disease to remain clinically stable for 15 months and a person with moderate Alzheimer’s disease to remain clinically stable for 11 months ( Oremus et al. , 2007 ). Survey responses also suggested that a cholinesterase inhibitor prescription was more likely if a physician had less stringent requirements for clinical efficacy. In another survey, 40 US primary care physicians held mostly ambivalent (51%) or negative (31%) views about cholinesterase inhibitor treatment for dementia ( Franz et al. , 2007 ). Potential barriers to the use of cholinesterase inhibitors were physicians’ lack of knowledge and experience with cholinesterase inhibitor treatment, although these primary care clinicians often yielded to pressure from family members to prescribe it.

Overall treatment persistence with cholinesterase inhibitors is suboptimal. In a large Medicare beneficiary study of more than 3000 patients with Alzheimer’s disease treated between 2001 and 2003, treatment persistence at 1 year among patients with Alzheimer’s disease who initially received cholinesterase inhibitors ranged from 64% to 68% ( Mucha et al. , 2008 ). Persistence of cholinesterase inhibitors therapy was even lower in a large Irish study, drawing on data from 2006 to 2010 ( Brewer et al. , 2013 ). Among elderly patients with Alzheimer’s disease who received cognition-enhancing drugs, rates of non-persistence (a prescription gap exceeding 63 days) were 30% at 6 months and 44% at 12 months; although rates of impersistence were lower in the more recent cohort and in patients taking multiple anti-dementia medications. European and Australian studies suggest that the reasons for not prescribing cholinesterase inhibitors and the impersistence of Alzheimer’s disease therapy are complex and highly variable across clinical settings ( Pariente et al. , 2008 ; Robinson et al. , 2009 ; Hollingworth and Byrne, 2011 ; van den Bussche et al. , 2011 ; Tifratene et al. , 2012 ; Ray and Prettyman, 2013 ; Zilkens et al. , 2014 ).

Despite physician ambivalence about the efficacy of cholinesterase inhibitors in Alzheimer’s disease and their inconsistent and limited use, data support the prescription of cholinesterase inhibitors throughout all stages of the disease. In an analysis of four placebo-controlled studies of people with severe Alzheimer’s disease, statistically significant cognitive improvement, and in some cases improvement in global functioning, was observed at 24 or 26 weeks with donepezil treatment at a dosage of 10 mg daily ( Deardorff and Grossberg, 2016 ). In a pooled analysis of these trials, relative improvement was observed across all levels of cognitive score, including patients with the most severe cognitive impairment ( Cummings et al. , 2010 ). In an expansive compendium of cholinesterase inhibitor trials in patients with more advanced Alzheimer’s disease, including patients in a nursing home setting, less decline in daily and global function was consistently documented with donepezil or rivastigmine treatment, although clinical evidence supporting rivastigmine use was less extensive ( Kerwin and Claus, 2011 ). Although choline acetyltransferase activity in the neocortex, as a marker of cholinergic function, keeps declining, some residual choline acetyltransferase activity can be detected in advanced dementia ( Bierer et al. , 1995 ; Davis et al. , 1999 ). This suggests that residual cholinergic input may be present in severe Alzheimer’s disease and thus provides a biological target for cholinesterase inhibitor therapy in this late stage. Other studies, however, have shown near total destruction of cholinergic axons in the cerebral cortex of patients with advanced Alzheimer’s disease, suggesting that the effect of cholinesterase inhibitors at these stages may be mediated through spared cholinergic pathways of the thalamus and basal ganglia rather than cerebral cortex and limbic regions ( Mesulam, 2013 ).

Dosing cholinesterase inhibitors to achieve maximum benefits

Incremental increases in cholinesterase inhibitor dosages have shown further benefit in Alzheimer’s disease, specifically in more advanced disease. In a phase 3 24-week study of patients with moderate–severe Alzheimer’s disease who were taking a stable dose of 10 mg donepezil per day, a dosage increase to 23 mg per day was associated with statistically significant increases in cognitive scores ( Farlow et al. , 2010 ). A post hoc analysis of individuals with more severe cognitive dysfunction in this study revealed significantly improved cognitive and global function scores for individuals who received the higher dosage ( Sabbagh and Cummings, 2011 ). In both assessments, the magnitude of score change was considered clinically meaningful. Although treatment-emergent adverse events—nausea (6.1% versus 1.9%), vomiting (5.0% versus 0.8%), and diarrhoea (3.2% versus 1.5%)—were higher with the increased cholinesterase inhibitors dosage, these adverse events were reportedly infrequent after 1 month of therapy. Similar clinical data support the use of high-dose rivastigmine in severe Alzheimer’s disease for improvements in cognitive function and activities of daily living at 16 and 24 weeks ( Farlow et al. , 2013 ). When considering the value of pharmacological management of Alzheimer’s disease, it is essential to consider the natural progression of untreated disease ( Geldmacher et al. , 2006 ). The initial stabilization of—or even improvement in—cognition and daily functioning with the use of currently approved anti-dementia drugs cannot be sustained indefinitely. Yet, with consistent treatment, a less precipitous decline can be expected over the long term, relative to the known, progressive manifestations of untreated disease.

It is also interesting to highlight that acetylcholine is one of the core neuromodulators involved in the regulation of the sleep-wake cycle, the preservation of which has been reported to be essential for many cognitive functions related to memory processes ( Aston-Jones et al. , 2001 ; Power, 2004 ). There is a circadian rhythm in central cholinergic transmission with a shift to low levels of acetylcholine release during slow-wave sleep compared with wakefulness. In addition, circadian fluctuations have been reported for cholinergic enzyme activity and cholinergic receptor regulation, raising the possibility that therapeutic strategies may need to consider the diurnal timing of administration and the half-life of the agent. Age-related alterations of this circadian rhythm occur in Alzheimer’s disease in tandem with the progression of clinical features ( Mitsushima et al. , 1996 ). Whether cholinesterase inhibitors influence these altered circadian rhythms in Alzheimer’s disease remains to be determined.

How early to treat with cholinesterase inhibitors?

In patients with early-stage Alzheimer’s disease specifically, an initial lapse in cholinesterase inhibitors therapy may lead to the irretrievable loss of potential treatment benefits. For example, in placebo-controlled studies of rivastigmine, an initial 26-week phase was followed by a 26-week open-label extension in which all patients received rivastigmine ( Farlow et al. , 2000 ; Doraiswamy et al. , 2002 ). For the first 26 weeks, rivastigmine provided statistically significant symptomatic benefits to patients with mild–moderate Alzheimer’s disease or more severe disease compared with patients on placebo. However, when patients initially treated with placebo received rivastigmine for the second 26 weeks, they failed to ‘catch up’ to individuals who received the drug for the full year. In a similar trial of galantamine in people with mild–moderate Alzheimer’s disease, patients originally assigned to placebo for the first phase of the trial did not attain a similar level of cognitive benefit at the end of the open-label phase of the study as did patients originally taking galantamine ( Raskind et al. , 2000 ).

Clinical data to support the use of cholinesterase inhibitors earlier in the trajectory, specifically in patients with MCI who are at risk for Alzheimer’s disease, are mixed ( Russ and Morling, 2012 ; Petersen et al. , 2018 ; Richter et al. , 2018 ). Donepezil, at a dosage of 10 mg daily, showed either marginal or no cognitive benefits, relative to placebo, in two well-controlled studies ( Salloway et al. , 2004 ; Doody et al. , 2009 ). Similar disappointing results were reported with rivastigmine and galantamine ( Feldman et al. , 2007 ; Winblad et al. , 2008 ). Investigators cautioned, however, that cognitive changes during this prodromal phase of Alzheimer’s disease are subtler and therefore harder to measure ( Doody et al. , 2009 ). In a 3-year study, donepezil appeared to reduce the risk for conversion of MCI to Alzheimer’s disease, but only during the first year of treatment ( Petersen et al. , 2005 ). Nevertheless, individuals at higher genetic risk for Alzheimer’s disease (with ≥1 APOE ε4 alleles) experienced greater benefit with donepezil treatment for the entire duration of the study. A recent practice guideline update could find no Level A evidence that cholinesterase inhibitors offer symptomatic improvement at the MCI stage ( Petersen et al. , 2018 ). Some of these negative results may be attributed to the heterogeneity of MCI. In the future, when MCI trials are based exclusively on patients with biomarker evidence of Alzheimer’s disease pathology, results may become more encouraging.

Cholinesterase inhibitors may also provide pathological and anatomical benefits, particularly before the emergence of clinical symptoms of Alzheimer’s disease. As noted earlier, the effects of donepezil (10 mg/day) on brain structure were recently demonstrated in a placebo-controlled longitudinal study of community-based adults with prodromal Alzheimer’s disease ( Dubois et al. , 2015 ; Cavedo et al. , 2016 , 2017 ). Over the course of 4 years, patients who received donepezil demonstrated a statistically significant lessening in the annual rate of hippocampal atrophy on MRI. During the first year of treatment specifically, the rate of hippocampal atrophy was reduced by 45% in donepezil-treated subjects in comparison with untreated patients with Alzheimer’s disease ( Dubois et al. , 2015 ).

In the future, indications for cholinomimetic therapies, including cholinesterase inhibitors, may become limited to patients with biomarker confirmation. This more rational approach may show that cholinergic therapies are even more effective than heretofore suspected when applied to a more homogeneous patient population with cholinergic dysfunction as a known component of dementia pathology. Novel technologies such as quantitative magneto- and electro-encephalogram may also allow the detection of subtle neurophysiological alterations induced by cholinesterase inhibitors and other cholinergic drugs that may not be detected at the clinical level. Thus, besides ‘classical’ clinical outcomes, even electrophysiological outcome measures could be introduced into clinical trials, hopefully helping to identify more effective novel therapies.

Integrating complex disease-related processes: future paradigms and implications

The neuropathological and clinical data summarized above make it very likely that cholinesterase inhibitors or other cholinomimetic interventions will remain essential components of therapy for Alzheimer’s disease. The demonstration of early involvement ( Schmitz et al. , 2016 ; Richter et al. , 2018 ) of the cholinergic system starting at preclinical stages of the disease, suggests that cholinomimetics, along with anti-amyloid and anti-tau interventions, may each have a distinct role in disease prevention. Future research and clinical paradigms related to Alzheimer’s disease may rely more heavily upon the ‘systems biology’ approach to the disease, stressing the interaction of factors such as genetic predisposition, oxidative stress, mitochondrial dysfunction, inflammatory mechanisms, vascular insufficiency, the accumulation of amyloid-β, neurofibrillary degeneration, cholinergic deficits, and other neurotransmitter abnormalities. A systems biology approach explicitly recognizes the multifactorial, dynamic nature of diseases like Alzheimer’s disease and helps clinicians customize therapeutic regimens that are targeted at multiple levels of pathology over the course of the disease.

At its most basic level, the pharmacological management of Alzheimer’s disease will likely incorporate tailored combination therapies—by using, for example, currently available and novel cognition-enhancing treatments [e.g. cholinesterase inhibitors, NMDA ( N -methyl- d -aspartate) receptor antagonists, and other therapies in development] with medications that are potentially disease-modifying (e.g. anti-amyloid-β or anti-tau therapies). As our understanding of Alzheimer’s disease pathophysiology expands and we identify additional clinically useful risk factors and biomarkers, the therapeutic approach to Alzheimer’s disease will likely parallel the way in which physicians currently manage other complex, variable, and highly idiosyncratic diseases.

An extension of tailored therapy for complex diseases lies at the core of precision medicine, which should guide future strategies for preventing or treating Alzheimer’s disease. The ultimate goal of precision medicine is to be able to administer a personalized therapy that specifically targets an individual’s known disease risks and disease process at the molecular level ( Reitz, 2016 ). Given the complexity and heterogeneity of Alzheimer’s disease pathophysiology, precision medicine may involve the determination of genetic risk profiles, the use of brain imaging, and the detection of biomarkers in plasma or CSF to fashion a specific preventive or therapeutic regimen for a particular individual at risk for or with Alzheimer’s disease. To this end, ongoing trials, such as DIAN (Dominantly Inherited Alzheimer Network trial), the Alzheimer’s Prevention Initiative, and the A4 (Anti-Amyloid Treatment in Asymptomatic Alzheimer Disease) trial, are studying people at high risk for Alzheimer’s disease and tracking biomarkers to identify individuals who might be most responsive to specific, targeted, disease-modifying interventions ( Reiman et al. , 2011 ; Mills et al. , 2013 ; Sperling et al. , 2014 ). In the meantime, extensive clinical investigations into cholinesterase inhibitors have already been conducted in broad and largely heterogeneous populations, with success seen across multiple patient ‘types’ defined by severity and other important characteristics. These developments consolidate the role of cholinomimetic agents as essential elements of the combined pharmacologic treatments for Alzheimer’s disease that will be developed in the future.

The cholinergic system is important for neuronal function in memory, learning, and other essential aspects of cognition and plays a wider role in the promotion of neuronal plasticity. Multidisciplinary investigations are revealing how dysfunction in cholinergic networks arising from the basal forebrain, interact with other important pathophysiologic aspects of Alzheimer’s disease—including amyloid-β plaques, neurofibrillary tangles, inflammation, oxidative stress, and vascular insufficiency to undermine cognition. A wealth of clinical literature supports the benefit of promoting cholinergic activity in Alzheimer’s disease through the use of cholinesterase inhibitors. Moreover, new data based on MRI are showing evidence of hippocampal protection and, perhaps, disease course alterations in individuals who receive cholinesterase inhibitors for long periods of time. Interest remains high in understanding the temporal sequence and cascade of these complex interactions and their synergistic feedback mechanisms over the course of Alzheimer’s disease. It is anticipated that optimal Alzheimer’s disease management will integrate a systems biology approach based on precision medicine to help tailor combinatorial therapeutic regimens for different stages of Alzheimer’s disease on the basis of genetic risks, brain imaging, and biomarkers. As we anticipate major developments in the treatment strategies of Alzheimer’s disease, cholinergic interventions are likely to maintain their critical roles in the therapeutic armamentarium.

H.H. is supported by the AXA Research Fund, the ‘Fondation partenariale Sorbonne Université’ and the ‘Fondation pour la Recherche sur Alzheimer’, Paris, France. Ce travail a bénéficié d'une aide de l’Etat ‘Investissements d’avenir’ ANR-10-IAIHU-06. The research leading to these results has received funding from the program ‘Investissements d’avenir’ ANR-10-IAIHU-06 (Agence Nationale de la Recherche-10-IA Agence Institut Hospitalo-Universitaire-6). A.C.C. is supported by the CIHR (Canadian Institutes of Health Research), the NSERC (National Research Council) and the Alzheimer Society of Canada. A.C.C. is a Member of the Canadian Consortium of Neurodegeneration in Aging and has received unrestricted support from Merck Canada, Dr. Alan Frosst and the Frosst Family. A.V. is supported by Rotary Club Livorno ‘Mascagni’/The Rotary Foundation (Global Grant No GG1758249). M.M.M. has received research support from the ADC grant (AG013854).

Axovant supported the travel and lodging expenses of the authors for two meetings of the CWG in New York City. The authors were also paid as consultants at customary rates for the time spent at the two meetings of the CWG; there were no other honoraria provided to the authors. A science writer, paid by Axovant help to compile the contributions of the authors into a coherent document. However, the various sections of the paper were prepared exclusively by the authors who were not paid in any way or the time spent in writing-editing the manuscript. All proceedings of the CWG were independent from Axovant. The support for the meetings was accepted by the CWG with the stipulation that Axovant would have no input to the deliberations of the Workgroup and/or influence in anyway the final conclusions/recommendations of the WG. Thus, no member of the company participated in development, discussion or drafting of this manuscript. Axovant has had under development a compound RVT-101 (aka Intepirdine) an antagonist of the serotonin receptor 6 (5-HT6) (which was found to lack efficacy for the treatment of Alzheimer’s disease), as well as RVT-104 (combination of glycopyrrolate and high-dose rivastigmine) a compound that targets the cholinergic mechanism

H.H. serves as Senior Associate Editor for Alzheimer’s & Dementia ; he received lecture fees from Biogen and Roche, research grants from Pfizer, Avid, and MSD Avenir (paid to the institution), travel funding from Axovant, Eli Lilly and company, Takeda and Zinfandel, GE-Healthcare and Oryzon Genomics, consultancy fees from Jung Diagnostics, Cytox Ltd., Axovant, Anavex, Takeda and Zinfandel, GE Healthcare and Oryzon Genomics, and participated in scientific advisory boards of Axovant, Eli Lilly and company, Cytox Ltd., GE Healthcare, Takeda and Zinfandel, Oryzon Genomics and Roche Diagnostics. H.H. is co-inventor in the following patents as a scientific expert and has received no royalties:

In Vitro Multiparameter Determination Method for The Diagnosis and Early Diagnosis of Neurodegenerative Disorders Patent Number: 8916388. In Vitro Procedure for Diagnosis and Early Diagnosis of Neurodegenerative Diseases Patent Number: 8298784. Neurodegenerative Markers for Psychiatric Conditions Publication Number: 20120196300. In Vitro Multiparameter Determination Method for The Diagnosis and Early Diagnosis of Neurodegenerative Disorders Publication Number: 20100062463. In Vitro Method for The Diagnosis and Early Diagnosis of Neurodegenerative Disorders Publication Number: 20100035286. In Vitro Procedure for Diagnosis and Early Diagnosis of Neurodegenerative Diseases Publication Number: 20090263822. In Vitro Method for The Diagnosis of Neurodegenerative Diseases Patent Number: 7547553. CSF Diagnostic in Vitro Method for Diagnosis of Dementias and Neuroinflammatory Diseases Publication Number: 20080206797. In Vitro Method for The Diagnosis of Neurodegenerative Diseases Publication Number: 20080199966. Neurodegenerative Markers for Psychiatric Conditions Publication Number: 20080131921. G.T.G. is a consultant for Acadia, Allergan, Avanir, Axovant, GE, Lundbeck, Novartis, Otsuka, Roche, Takeda. Research Support for Janssen, NIA. M.F. has received research support from Accera, Biogen, Eisai, Eli Lilly, Genentech, Roche, Lundbeck, Chase Pharmaceuticals, Boehringer Ingelheim, Novartis, and Suven Life Sciences, Ltd.; and has been a consultant and/or advisory or DSMB board member for Accera, AstraZeneca, Avanir, Axovant, AZTherapies, Eli Lilly & Company, FORUM Pharmaceuticals, INC Research, KCRN Research, Longeveron, Medavante, Merck and Co., Inc., Medtronic, Proclara (formerly Neurophage Pharmaceuticals), Neurotrope Biosciences, Novartis, Sanofi-Aventis, Stemedica Cell Technologies, Inc., Takeda, United Neuroscience Inc., and vTv Therapeutics. He also holds a patent for a transgenic mouse model that is licensed to Elan. A.C.C., E.G., P.J.S., E.C. and A.V. have nothing of relevance to declare.

Supplementary material is available at Brain online.

Abbreviations

mild cognitive impairment

nucleus basalis of Meynert

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• acetylcholine
• alzheimer's disease
• cholinergic agents
• cholinesterase inhibitors

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Nucleus basalis of Meynert revisited: anatomy, history and differential involvement in Alzheimer’s and Parkinson’s disease

Alan king lun liu.

1 Neuropathology Unit, Division of Brain Sciences, Department of Medicine, Imperial College London, London, UK

2 Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong S.A.R., China

Raymond Chuen-Chung Chang

3 State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong S.A.R., China

4 Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong S.A.R., China

Ronald K. B. Pearce

Steve m. gentleman.

It has been well established that neuronal loss within the cholinergic nucleus basalis of Meynert (nbM) correlates with cognitive decline in dementing disorders such as Alzheimer’s disease (AD). Friedrich Lewy first observed his eponymous inclusion bodies in the nbM of postmortem brain tissue from patients with Parkinson’s disease (PD) and cell loss in this area can be at least as extensive as that seen in AD. There has been confusion with regard to the terminology and exact localisation of the nbM within the human basal forebrain for decades due to the diffuse and broad structure of this “nucleus”. Also, while topographical projections from the nbM have been mapped out in subhuman primates, no direct clinicopathological correlations between subregional nbM and cortical pathology and specific cognitive profile decline have been performed in human tissue. Here, we review the evolution of the term nbM and the importance of standardised nbM sampling for neuropathological studies. Extensive review of the literature suggests that there is a caudorostral pattern of neuronal loss within the nbM in AD brains. However, the findings in PD are less clear due to the limited number of studies performed. Given the differing neuropsychiatric and cognitive deficits in Lewy body-associated dementias (PD dementia and dementia with Lewy bodies) as compared to AD, we hypothesise that a different pattern of neuronal loss will be found in the nbM of Lewy body disease brains. Understanding the functional significance of the subregions of the nbM could prove important in elucidating the pathogenesis of dementia in PD.

Introduction

Although the identification of Lewy bodies (LB) and neuronal loss in the substantia nigra is considered the gold standard for the neuropathological diagnosis of Parkinson’s disease (PD), these two pathological features were actually first recognised by Friedrich Lewy in the nucleus basalis of Meynert (nbM) in 1913. Within the basal forebrain sublenticular region, there is a broad band of cell clusters commonly known as the nbM. Neuronal loss in the nbM is well established in dementing disorders; however, its pathological significance was first recognised in a series of patients with paralysis agitans (now known as PD) by Lewy where severe neuronal degeneration and intraneuronal globose tangles were noted [ 63 ]. He also observed that concentric hyaline-rich “ Kugeln ” (balls, as originally identified in the globus pallidus) were found in surviving neurons in the nbM and dorsal motor nucleus of the vagus [ 89 ]. These intraneuronal inclusions were later given the name LB and the presence of LB became one of the cardinal neuropathological features of PD. Lewy speculated that the nbM neuronal loss was responsible for some of the motor deficits seen in PD and it was not until the 1930s that Hassler suggested the pathological changes in the nbM were probably related to cognitive function deficits in PD (in [ 94 ]). Subsequently, the nbM has been investigated extensively in many neuropsychiatric disorders including schizophrenia [ 4 , 103 ], Pick’s disease [ 86 ], Alzheimer’s disease (AD) [ 2 , 4 – 6 , 17 , 19 , 25 , 29 , 46 , 47 , 64 , 65 , 73 , 74 , 78 – 81 , 85 , 86 , 94 , 100 – 102 , 105 ], Creutzfeldt–Jakob disease [ 86 ], dementia pugilistica [ 96 ] and Down’s syndrome [ 18 , 86 ]. The functional significance and connections of the nbM were unknown until the 1970s when the cholinergic hypothesis in AD was proposed [ 9 ]. The nbM was then found to be a cholinergic centre, with neurons providing cholinergic afferents to the entire neocortex [ 24 , 55 , 67 , 69 ]. Hence, the decrease in cortical acetylcholine levels seen in dementing disorders was thought to relate to cell death within the nbM.

The nbM is a broad and irregular “nucleus” in the human forebrain and functional subdivision of the nbM has been suggested, based on the topographical projection of cholinergic fibres from the nbM in non-human primates [ 69 ]. However, this topography is not directly translatable to the human brain. It is, therefore, important to revisit this question of anatomical subdivision of the nbM for the investigation of possible clinicopathological correlations in different dementias.

With the advances in imaging of the basal forebrain, and the nbM potentially being the next target for neuromodulation with deep brain stimulation (DBS) [ 37 ], we will review the history of the localisation of this basal forebrain nucleus and look at possible trends in clinicopathological correlation of different nbM subsectors.

Where exactly is the nbM?

The basal forebrain region located above and parallel to the optic nerve, with the medial boundary being the wall of the lateral ventricle was first described by Reil in 1809 as the unnamed medullary substance (Die ungenannte Marksubstanz) [ 84 ] (Fig.  1 ). This region was named substantia innominata (SI) of Reil by Theodore Meynert [ 71 ]. However, the SI that we commonly refer to is actually more poorly defined anatomically and is known as the SI of Reichert. This is an unlabelled area with no boundaries within Reichert’s human brain atlas from the 1850s [ 27 ]. This region has evolved to be known as the anterior perforated substance by Beccari and in a more modern human stereotaxic brain atlas by Schaltenbrand and Bailey simply as basalis to describe the sub-commissural region dorsal to the amygdala [ 88 ]. Despite the change in terminology, many current investigators still refer to the region as the SI. However, instead of labelling a region, some investigators describe the SI as discrete group of magnocellular neurons within the basal forebrain synonymous to the nbM we know today [ 104 ], reflecting a confusion of terminology in this area.

A diagram of the human basal forebrain illustrating the location of the substantia innominata (as outlined). AC anterior commissure, Am amygdala, Cd caudate, GP globus pallidus, IC internal capsule, LV lateral ventricle, Pt putamen, SI substantia innominata

Defining a “nucleus”

As mentioned above, Reil was the first to recognise the distinct group of basal forebrain neurons and labelled it as a “medullary substance”. In fact, Meynert described the group of cells as the ganglion of the ansa peduncularis ( ganglion der Hirnschenkelschlinge ), which is found within the SI of Reil bound by the ansa lenticularis dorsally, the optic tract ventrally and the external capsule laterally [ 71 ]. Koelliker coined the term ‘basal ganglion of Meynert’ ( Meynert’sches Basalganglion ) and extended Meynert’s finding to describe the ganglion in its rostrocaudal extent [ 59 ]. This extends from the mammillary bodies posteriorly to the floor of the inter-hemispheric fissure anteriorly. This ganglion was later called the ganglion of the ansa lenticularis by Edinger (Reviewed in [ 77 ]). However, two problems arose from this terminology. First, the term “ganglion” should be used to describe a collection of cell bodies in the peripheral nervous system instead of the central nervous system. Thus, this collection of cells was more closely known as nucleus of the septal plane ( Nucleo del piano settale ) by Beccari [ 43 ] and nucleus of the ansa lenticularis by Ayala [ 8 ]. Second, the structures ansa peduncularis and ansa lenticularis were difficult to define and the collection of cells was more closely related to the former [ 77 ]. Thus, nomenclature associating these neurons to a particular structure was avoided and the term nucleus basalis or basal nucleus ( Der Basalkern by Brockhaus) was established [ 15 ].

Subdividing the “nucleus”

The nbM is an “open” nucleus with no distinct boundaries and it forms several clusters within the basal forebrain. Attempts have therefore been made to subdivide this ‘nucleus’. Ayala observed two distinct clusters of magnocellular neurons, the first being the previously described nbM and the second located lateral to the anterior commissure and ventral to the putamen for which he coined the term nucleus subputaminalis (NSP) [ 8 ]. The NSP is also known as Ayala’s nucleus and it was proposed to be involved in speech function but there is currently no direct evidence to support this hypothesis [ 92 ]. Later, Brockhaus also tried to subdivide the nbM and he classified the more anterior part as the pars diffusa and a posterior portion as pars compacta [ 15 ].

nbM: the cholinergic nucleus

In the 1970s, retrograde horseradish peroxidase (HRP) tracer experiments on subhuman primates identified that cortical cholinergic innervation originates from the nbM [ 68 ]. Using histochemical and immunohistochemical labelling for acetylcholinesterase (AChE) and choline acetyltransferase (ChAT), Mesulam and colleagues [ 69 , 70 ] were able to identify the various cholinergic loci in the subhuman primates’ basal forebrain and introduced the nomenclature Ch1–Ch4 to describe four cholinergic cell groups rostrocaudally, with the cholinergic component of the nbM designated as Ch4 (Table  1 ).

Table 1

Basal forebrain cholinergic cell groups and their projections in the brain [ 69 ]

Cholinergic topographical projection of the nbM

Mesulam and colleagues [ 69 ] found that over 90 % of the magnocellular neurons in the nbM are cholinergic and that the Ch4 group is the largest out of the four basal forebrain cholinergic groups. In humans, Ch4 is measured 13–14 mm antero-posteriorly and 16–18 mm medio-laterally within the SI [ 67 ]. Furthermore, the Ch4 can be subdivided into five groups in monkeys [ 69 ]—the anterior part (Ch4a) into anteromedial (Ch4am) and anterolateral (Ch4al); the intermediate part (Ch4i) into intermediodorsal (Ch4id) and intermedioventral (Ch4iv); and a posterior group (Ch4p). However, there is an additional sixth subsector of the Ch4 in human as the transition between the anterior and intermediate part is elongated, giving rise to the anterointermediate (Ch4ai) region [ 67 ]. Prior to this classification, most studies involving the nbM stopped at the level of Ch4i, neglecting the caudal extension. In fact, according to Meynert’s original description, the nbM is located at the plane of the intermediate Ch4 region.

Through HRP retrograde tracer and AChE co-localisation studies on macaques, the cortical topographical innervations from the Ch4 subgroups have been mapped out (Fig.  2 ) [ 69 ]. In summary, the anterior Ch4 innervates the limbic regions—Ch4am projects to medial cortical regions including the cingulate cortex and Ch4al projects to fronto-parietal opercular regions and amygdala; Ch4p projects to superior temporal and temporal polar regions; and Ch4i to the remaining cortical regions. It is not known whether these innervation patterns are similar to those in human brain but detailed clinicopathological studies relating to the subdivision of the nbM could provide some clues.

Projected innervation map of the various Ch4 regions (Ch4a, green ; Ch4i, blue ; Ch4p, red ) in the human brain on the lateral surface ( top left ) and at the mid-sagittal plane ( top right ). Cortical projection from the Ch4ai ( turquoise ) is currently unknown in the human brain. Topographical innervation in different subsectors of the nbM according to Mesulam et al. ( bottom ) [ 67 , 69 ]

Problems with the Ch4 subsectors

As pointed out by Mesulam et al. [ 69 ], the basal forebrain cholinergic groups do not have strict anatomical boundaries and overlap considerably, in line with the concept that the nbM is an ‘open’ structure rather than a discrete nucleus. Furthermore, within the Ch4 group, some ChAT-immunopositive cells were scattered in different interstitial locations including the anterior commissure, inter-medullary laminae of the globus pallidus, internal capsule, ansa lenticularis and ansa peduncularis. Also, not all magnocellular neurons within the basal forebrain are cholinergic and the terms nbM and Ch4 are therefore not interchangeable.

Ch4ai is a region unique to the human brain, as it is the “gap” between the Ch4a and Ch4i subsectors when results from subhuman primates were translated to humans. It could be speculated that this region is the caudal extension of Ch4a and the rostral extension of Ch4i due to the larger lateral surface area of the neocortex in human as compared with subhuman primates.

Ch4a and Ch4i were further divided into two clusters according to Mesulam. In Ch4a, a vessel or a rarefaction divides it into the medial and lateral sectors. However, with anatomical variation, a vascular structure might not be present in certain planes [ 47 , 87 ]. Also, the sizes of Ch4am and Ch4al seem not to stay constant throughout [ 92 ]. As one progress rostrocaudally, Ch4am appears and overlaps with Ch2 diagonal band nucleus. Then it gradually decreases in size while Ch4al enlarges and merges with Ch4ai. More caudally, the tract of ansa peduncularis is usually an anatomical landmark that divides the Ch4i into dorsal and ventral subsectors, although it can be difficult to identify on thin nbM sections [ 28 , 47 ]. However, the projection pattern of Ch4id and Ch4iv appears to be similar [ 69 ], so the Ch4i can effectively be considered as a single entity.

Establishment of a simplified nbM subdivisional scheme

Analysis of the entire nbM will not be possible in many studies, and thus a subdivisional scheme could be useful to simplify and standardise future work on the nbM. We have reviewed several human brain atlases and previous publications [ 10 , 36 , 67 , 69 , 83 , 92 , 104 ] and have elaborated a notional definition of anterior, intermediate and posterior subsectors of the nbM (Table  2 ). The subdivisions we propose approximately correspond to the original Ch4 classification according to Mesulam, discarding the Ch4ai label with its lack of any reliable topographical correlate. This is depicted histologically with H&E and immunohistochemical staining with choline acetyltransferase (ChAT) on the human basal forebrain (Fig.  3 ). A protocol for recommended sampling at autopsy is given in Fig.  4 .

Table 2

Proposed macroscopic and microscopic landmark for the definition of anterior, intermediate and posterior subsectors of the nbM

Formalin-fixed, paraffin-embedded basal forebrain sections available from the Parkinson’s UK Tissue Bank at Imperial College, London, stained with H&E ( a – c ; g – i ) and serial sections stained with choline acetyltransferase (ChAT) immunohistochemistry (Millipore AB144P, 1:100 with pressure cooker pretreatment in pH 6.0 citrate buffer) ( d – f ; j – l ). Six subdivisions of the basal forebrain were defined and arranged rostrally ( top left ) to caudally ( bottom right ). a , d Level at nucleus accumbens. This level is defined by the absence of anterior commissure and the presence of a large caudate head with nucleus accumbens. b , e Pre-anterior nbM level. Anterior commissure appears in this section but it is located ventral to the globus pallidus and is rostral to decussation level. A large ventral striatum could be seen clearly with ChAT immunohistochemistry. c , f Most rostral anterior nbM level. This level is defined by the decussation of the anterior commissure. Ch4 neurons are defined by their location being lateral to the supraoptic nucleus and they are orientated at the medial–lateral axis parallel to the basal border of the section. g , j Most caudal anterior nbM level. The anterior commissure is split into two parts with medial end still decussating and lateral end located ventral to the globus pallidus. h , k Intermediate nbM level. At this level, the globus pallidus is split into the external and internal components by an inter-medullary lamina. The anterior commissure is located ventral to the putamen and sometimes the infundibulum could be seen. i , l Posterior nbM level. This is defined by the presence of mammillary body, small or absence of caudate and internal capsule occupying the medial half of the tissue. Asterisk denotes area of maximal density of ChAT-immunopositive cells in the nbM. Zoomed-in figure showing the ChAT-immunopositive neurons in the nbM at ×10 objectives. AC anterior commissure, Cd caudate, fx fornix, GP globus pallidus, GPe globus pallidus externa, GPi globus pallidus interna, ic internal capsule, inf infundibulum, mb mammillary body, nAcc nucleus accumbens, ot optic tract, Pt putamen, son supraoptic nucleus, VS ventral stratum

Photographs showing the anatomical landmarks for the anterior, intermediate and posterior levels of the nucleus basalis of Meynert (nbM, as indicated by asterisk ). At dissection, the first coronal slice is made through the mammillary body (MB), revealing the posterior nbM. Using a 0.5-cm cutting guide two further coronal slices will reveal the intermediate and anterior levels. These are specifically identified by the presence of discernible globus pallidus externa (GPe) and interna (GPi), and midline anterior commissure (AC), respectively. With normal anatomical variation between individuals, this general 0.5 cm interval may need slight modification, depending on brain size

The cholinergic hypothesis and the increased emphasis on nbM research

In the 1970s and 80s, a number of animal and human studies pointed to the importance of acetylcholine in cognition. Subsequently, the “cholinergic hypothesis” was proposed suggesting that a cortical cholinergic deficit leads to cognitive decline in ageing and Alzheimer’s disease (AD) [ 9 ]. As the source of cortical cholinergic innervation, the nbM became one of the ‘hot topics’ in dementia research in the last two decades of the 20th century.

Alzheimer’s disease: a caudorostral pattern of neuronal loss in the nbM

Since Whitehouse and colleagues’ first account of 90 % nbM cell loss in a familial AD case [ 99 ], further studies have reported anything from 8 to 87 % cell loss in AD relative to controls [ 2 , 4 – 6 , 17 , 19 , 25 , 29 , 46 , 47 , 64 , 65 , 73 , 74 , 78 – 81 , 85 , 86 , 94 , 100 – 102 , 105 ]. The reason for such a large variation between studies could relate to varying disease severity, but one of the main causes is that neuronal loss is not homogenous throughout the nbM. Therefore, we reviewed the early reports on the neuropathological correlations of nbM in AD with particular emphasis on the region of nbM sampled. Using the aforementioned guideline to divide the nbM into the anterior, intermediate and posterior subdivisions, we identified the regional susceptibility to neuronal loss in AD (Table  3 ). It appears that in AD, there is a caudorostral gradient of nbM neuronal loss with the posterior sector being the most severely affected. As the posterior nbM contains Ch4p providing cholinergic innervation to the temporal pole and superior temporal cortex [ 69 ], this correlates well with memory loss and language impairment in AD.

Table 3

Studies of the nbM in AD. Studies where a caudorostral pattern of nbM neuronal loss is found are indicated by [●]

n.s. Not significant

a Familial AD

b Moderate AD

c Poor definition of nbM region. According to diagram in the study, area included indicates Ch4a and Ch4i

d NGFR immunohistochemistry as marker for cholinergic neurons in the nbM

This pattern of cell loss was supported by some studies where the entire nbM was examined [ 5 , 73 , 101 ]. However, Doucette and colleagues reported that in moderate AD, the anterior nbM had a 50 % neuronal loss while the decrease in cell number was not significant in intermediate and posterior nbM [ 25 ]. Similar findings were reported by Iraizoz’s group where the greatest decline was found in anterior followed by posterior nbM [ 47 ]. The disagreement concerning the caudorostral pattern of nbM loss in AD could be due to differences in the criteria used to define an nbM neuron for cell counting, as nbM neuronal shrinkage has also been reported in AD [ 97 ]. Also, in some of the studies where sections slightly rostral to the anterior commissure decussation were taken, the distinction between Ch4a and Ch2 could be difficult to define. As Ch2 neurons provide innervation to the hippocampus, which is severely affected in AD, the greater loss in the anterior sector of the nbM could be due to the loss of Ch2 rather than Ch4a neurons, although Ch1/2 cell loss in AD has been reported to be minimal [ 62 , 72 ] or even insignificant [ 32 ] compared with age-matched controls.

Revisiting the nbM in Parkinson’s disease

As mentioned previously, cell loss in the nbM was first identified in PD by Lewy, early in the 20th century. However, quantification of neuronal loss was not attempted until the 1980s where studies reported up to 80 % depletion in the nbM of PD cases (Table  4 ). When directly comparing PD and AD cases, the loss is comparable [ 86 ] or more extensive in PD than in AD [ 17 , 20 , 79 ] and the loss was more apparent among PD with dementia (PDD) cases. Therefore, it is perhaps not be surprising that PDD patients have good neuropsychiatric responses to anticholinesterase medication such as rivastigmine and galantamine (review by [ 1 ]). Furthermore, recent imaging studies using cholinergic makers to label acetylcholinesterase have reported significant cortical cholinergic deficits in PD and PDD patients [ 11 – 13 , 44 , 60 , 90 , 91 ]. This suggests that apart from the dopaminergic deficit, a decrease in cholinergic tone also contributes to cognitive impairment in PD, as supported by the dual syndrome hypothesis where executive dysfunction and visuospatial impairment in PD correspond to dopaminergic and cholinergic deficits, respectively [ 53 ].

Table 4

Studies of the nbM in PD

a Did not distinguish PDD from PD

The cognitive picture of PDD is commonly considered as a “subcortical” type since patients typically present with dysexecutive signs without significant impairment in storage memory as in “cortical” AD-type dementia [ 20 , 23 ]. Hence, with varying cortical regions affected in PD and AD the differing clinical profiles may correlate with neuronal loss in particular nbM subsectors. However, only a small number of studies have investigated PDD separately and the different subregions within the nbM have not been compared in PDD cases. We reviewed the literature, estimated the regions sampled in various studies as mentioned before (Table  5 ) and found a slightly greater deficit in the intermediate nbM region. This supports a recent imaging study [ 58 ] which reported a posterior–anterior gradient of cortical cholinergic deficit and this could be due to the extensive cell loss in the Ch4i affecting the cholinergic innervation to occipital–parietal cortical regions.

Table 5

Studies of the nbM in PDD

A dichotomous pattern of nbM cell loss in PD and AD

Amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFT) are hallmark neuropathological features of AD, the latter being more closely associated with cognitive decline [ 7 ]. It has been well recognised that LB- and AD-type pathologies frequently co-exist in the brains of PDD and dementia with Lewy bodies (DLB) [ 49 ] and that there may be synergistic relationships between the two types of pathologies in the development of dementia [ 21 , 48 ].

Cullen and Halliday [ 22 ] proposed that the cause of neuronal loss in the nbM could differ between PD and AD. They studied cell loss and NFT pathology within the nbM of AD and LB disease with concomitant AD. Severe cell loss in the nbM of AD cases was accompanied by abundant extraneuronal NFT pathology. However, cases of LB disease with AD have equally severe nbM depletion, despite the relatively milder NFT pathology. This suggests that the alpha-synuclein pathology could also be involved in nbM neuronal death in cases with LB pathology. This dichotomous disease process affecting the nbM in PD and AD was also described by Candy and colleagues. They reported that nbM neuronal loss in PD was more extensive than in AD in the absence of co-existing cortical NFT pathology [ 17 ]. Similarly, Gaspar and Gray noted that in 5 of 6 PDD cases, there was severe nbM neuronal depletion despite relatively little or no cortical AD-type pathology [ 34 ]. They concluded that cortical AD pathologies did not seem to affect the reduction of cholinergic cortical afferents in PD. In addition, Nakano and Hirano reported that neuronal loss in the nbM of PD is not associated with NFT in the cortex, hippocampus or in the nbM [ 76 ]. Therefore, in order to study nbM loss in ‘pure’ PD, cases with severe AD pathologies should be excluded. In the studies we reviewed (Tables  4 , ​ ,5), 5 ), most [ 17 , 19 , 20 , 34 , 79 , 80 , 95 ] but not all [ 4 , 86 , 98 , 105 ] have excluded cases with severe co-existing AD pathologies.

Striatal Aβ has been suggested to contribute to the development of dementia in PD and DLB, independently of comorbid AD pathologies [ 51 , 52 ]. Although a couple of studies reported striatal Aβ as specific to DLB not PDD [ 39 , 50 ], and subsequently concluded Aβ load in the striatum affects the temporal relationship between dementia and PD motor symptoms rather than presence of dementia, controversies remain as to whether the severity of striatal Aβ could differentiate PDD from DLB.

Other basal forebrain cholinergic nuclei such as the medial septal nucleus (Ch1) and the vertical limb of the diagonal band nucleus (Ch2) also show differential susceptibility in AD and LB disorders. Fujishiro and colleagues reported the loss of Ch1 and Ch2 ChAT-positive neurons in DLB but not AD cases compared with controls [ 32 ]. However, a recent study reported no significant change in Ch1 and Ch2 ChAT-positive neurons in PD and PDD [ 38 ]. So further work is needed to identify the potentially subtle pathological differences between PDD and DLB. One further consideration, as mentioned previously, is that other neurotransmitter deficits could contribute to cognitive decline in PD. Cell loss in the nbM happens in parallel with dopaminergic neuronal loss in the substantia nigra and ventral tegmental area; and noradrenergic neuronal loss in the locus coeruleus [ 54 ]. Hence, the basal forebrain depletion could also be associated with the decrease in dopaminergic and noradrenergic innervation in PD. Finally, serotoninergic dysfunction, dysregulation of excitatory amino acid and purinergic interactions in PD [ 93 ] should not be neglected as they might also contribute to non-motor symptoms including cognitive impairment in PD.

Possible functional correlate to subdivisional neuronal loss in the nbM in PD

Correlation relating specific cognitive deficits in PD with subsector pathology in the nbM has not been achieved, in part because of a lack of detailed neuropsychological testing in extant postmortem brain studies. An exception is from one study by Chui and co-workers in which three neuropathologically confirmed PD cases with dementia had undergone extensive neuropsychiatric tests before death and detailed cognitive profiles for the patients were available [ 20 ]. Their cognitive impairment included the presence of hallucination, visuospatial impairment and attentional deficits typical in PD cases. When the cell count of nbM (apparently Ch4a) was compared against AD cases, a greater decrease in mean density was observed in PDD (66.1 % loss) than in AD (61.9 % loss). On the other hand, the correlation between cognitive impairment in LB disorders and regional cortical involvement has been well supported by various functional imaging studies. In particular, atrophy and hypometabolism in the occipito-parietal region and, to a lesser extent, the frontal cortex in PDD patients relative to controls have been reported [ 35 ]. One study has compared the specific cognitive impairment in PD with regional fluorodeoxyglucose (FDG) uptake [ 33 ]. The degree of executive dysfunction in PD patients correlated positively to hypometabolism score in the frontal lobe, whereas visuospatial function impairment correlated to occipito-parietal reduction in FDG uptake.

Hence, one could speculate that pathology in Ch4a correlates with executive dysfunction in PDD due to frontal and limbic cortical innervation from the anterior Ch4 area. Moreover, anosmia in PD and PDD has been shown to be associated with limbic cortical cholinergic denervation, which could again correlate with Ch4a pathology [ 14 ]. Similarly, visuospatial impairment in PDD or even in early PD would possibly be due to neuronal loss in the Ch4i subsector. As PD/PDD patients typically have a less amnestic profile than AD, we would expect Ch4p to be relatively spared. However, visual hallucination in PD is a more complex phenomenon which might be due to a combination of occipito-parietal hypometabolism [ 45 ] and the presence of LB in the temporal lobe [ 40 ]. Therefore, pathology in the Ch4i and Ch4p regions may play a role in this characteristic element of cognitive dysfunction in PDD/DLB, along with other brain centres.

In addition, in earlier studies, no distinction was made between PDD and DLB [ 79 , 80 ], as DLB is a more recently established clinical entity [ 66 ], and it would be of interest to investigate whether the pattern of cell loss within the nbM subregions differs in PDD and DLB.

Conclusion and future work

Following our literature review on the studies of the nbM above, we also revisited the original work by Friedrich Lewy [ 30 ] and found that the area he defined as the nbM was from the optic tract to septum pellucidum. This would equate to the anterior/intermediate Ch4 region according to modern classification and thus historical evidence illustrates that LB and severe neuronal depletion were first described in the anterior portion of nbM. Along with the collective evidence and our speculation that there is a relative sparing of posterior nbM involvement in PD patients, it could be hypothesised that pathology in the nbM begins in the anterior portion and progresses caudally in PD. This anatomical progression supports the prion-like propagation hypothesis [ 3 , 16 , 26 , 31 ] and the dual-hit hypothesis of alpha-synuclein [ 41 , 42 ] where pathology starts in the olfactory bulb and spreads towards the basal forebrain region (Fig.  5 ). However, further studies investigating the topographical innervation pattern from different subsectors of the human nbM to target regions and reciprocal connectivity are needed, particularly with the advancement in tractography and other high-resolution imaging techniques.

Projected schema of anatomical progression of pathology within the nbM with possible clinicopathological correlations. Hypothesised progression is indicated by dashed arrows

With the increasing number of imaging studies focusing on basal forebrain changes [ 56 , 57 ] there is a need for this nucleus to be revisited in pathological studies. The potential for neuromodulatory treatment targeting the nbM is now being realised, in particular deep brain stimulation in dementia [ 37 , 61 ] and stereotactic gene delivery of trophic factors [ 82 ]. However, it is important to note that there are many caveats to consider, including distinctly varying pathogenesis of dementia in PD and AD. Better clinicopathological correlations have to be established, especially in relation to the different subregions of the nbM. This will improve our understanding of the pathological basis for different forms of dementing disorders and the role of forebrain cholinergic mechanisms in normal cognition as well as in the setting of cognitive decline.

Acknowledgments

The authors would like to thank Parkinson’s UK, registered charity 258197, for their continual support as well as the donors and family for their invaluable donation of brain tissue to the Parkinson’s UK Tissue Bank. The work in Laboratory of Neurodegenerative Diseases is supported by HKU Alzheimer’s Disease Research Network under Strategic Research Theme of Healthy Aging to RCCC.