ACT on Alzheimer’s

Alzheimer’s Disease Curriculum

Module: Science of Alzheimer’s

GUIDELINES AND RESTRICTIONS ON USE OF DEMENTIA CURRICULUM MODULES

This curriculum was created for faculty across multiple disciplines to use in existing coursework and/or to develop as a stand-alone course in dementia. Because not all module topics will be used within all disciplines, each of the ten modules can be used alone or in combination with other modules. Users may reproduce, combine, and/or customize any module text and accompanying slides to meet their course needs.

Use restriction: The ACT on Alzheimer's®-developed dementia curriculum cannot be sold in its original form or in a modified/adapted form.

NOTE: Recognizing that not all modules will be used with all potential audiences, there is some duplication across the modules to ensure that key information is fully represented (e.g., the screening module appears in total within the diagnosis module because the diagnosis module will not be used for all audiences).

© 2016

Acknowledgement

We gratefully acknowledge the funding organizations that made this curriculum development possible: the Alzheimer’s Association MN/ND and the Minnesota Area Geriatric Education Center (MAGEC), which is housed in the University of MN School of Public Health and is funded by the Health Resources and Services Administration (HRSA).

W We also specially acknowledge the principal drafters of this curriculum module, including Kathleen Zahs, Ph.D., Maureen Handoko, Ph.D., Hoang Hoa Nguyen, B.S., Samantha Shapiro, B.A., Katelynn Splett, B.S. and John Reichl

This project is/was supported by funds from the Bureau of Health Professions (BHPr), Health Resources and Services Administration (HRSA), Department of Health and Human

Services (DHHS) under Grant Number UB4HP19196 to the Minnesota Area Geriatric Education Center (MAGEC) for $2,192,192 (7/1/2010—6/30/2015). This information or

content and conclusions are those of the author and should not be construed as the official position or policy of, nor should any endorsements be inferred by the BHPr, HRSA, DHHS

or the U.S. Government.Minnesota Area Geriatric Education Center (MAGEC)

Grant #UB4HP19196Director: Robert L. Kane, MD

Associate Director: Patricia A. Schommer, MA

Overview of Alzheimer’s Disease Curriculum

This is a module within the Dementia Curriculum developed by ACT on Alzheimer’s. ACT on Alzheimer’s is a statewide, volunteer-driven collaboration seeking large-scale social change and community capacity-building to transform Minnesota’s response to Alzheimer’s disease. An overarching focus is health care practice change to ensure quality dementia care for all.

All of the dementia curriculum modules can be found online at www.ACTonALZ.org.

Module I: Disease Description

Module II: Demographics

Module III: Societal Impact

Module IV: Effective Interactions

Module V: Cognitive Assessment and the Value of Early Detection

Module VI: Screening

Module VII: Disease Diagnosis

Module VIII: Dementia as an Organizing Principle of Care

Module IX: Quality Interventions

Module X: Caregiver Support

Module XI: Alzheimer’s Disease Research

Module XII: Science of Alzheimer’s

Glossary

ACT on Alzheimer's has developed a number of practice tools and resources to assist providers in their work with patients and clients who have memory concerns and to support their care partners. Among these tools are a protocol practice tool for cognitive impairment, a decision support tool for dementia care, a protocol practice tool for mid- to late-stage dementia, care coordination practice tools, and tips and action steps to share with a person diagnosed with Alzheimer's. These best practice tools incorporate the expertise of multiple community stakeholders, including clinical and community-based service providers:

• Clinical Provider Practice Tool• Electronic Medical Record (EMR) Decision Support Tool• Managing Dementia Across the Continuum• Care Coordination Practice Tool• Community Based Service Provider Practice Tool• After A Diagnosis

While the recommended practices in these tools are not location-specific, many of the resources referenced are specific to Minnesota.  The resource sections can be adapted to reflect resources specific to your geographic area. To access ACT practice tools and resources, as well as video tutorials on screening, assessment, diagnosis, and care coordination, visit: http://actonalz.org/provider-practice-tools

Alzheimer’s disease (AD) is a progressive neurodegenerative disease – symptoms become more severe over time until eventually the brain can no longer control autonomic functions like swallowing, and death results. AD is thought to begin as a disorder of synaptic function; as disease progresses, synapses are lost and finally neurons die. During the early phases of the disease, before there is a widespread loss of neurons, symptoms are subtle or might even be absent. Understanding the pathobiological mechanisms that lead to neuron death is essential for the development of therapies to halt or delay the disease process. Currently, the field of AD research resembles a large, unfinished jigsaw puzzle with many missing pieces – the big picture is known (the clinical manifestations of disease) and some pieces are in place, but many pieces have yet to be found and/or fit together. This module will review what we have learned about the biology of AD from studies of model systems (e.g., cultured brain cells and mice genetically modified to mimic particular aspects of AD). Studies with human subjects will be discussed in another module.

In addition to pronounced synapse and neuron loss, AD is characterized by the presence of two neuropathological lesions: extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs). Amyloid plaques are formed from aggregated amyloid beta (Aβ) protein, while NFTs are comprised of abnormally processed tau, a protein normally associated with the microtubules that form the cytoskeleton. The identification of Aβ and tau as major components of the lesions seen in AD patients led researchers to focus their attention on these proteins. Subsequent genetic studies supported the idea that corrupt forms of Aβ and tau are harmful. Although most cases of AD arise sporadically, there are rare genetic mutation that destine a person to develop the disease; all of these AD-causing mutations increase the production of Aβ or its propensity to aggregate. Geneticists also discovered mutations in the tau gene that cause frontotemporal dementia, another neurological disorder characterized by NFTs and neuron loss. The discoveries of these mutations not only confirmed the disease relevance of Aβ and tau, but allowed scientists to create transgenic animals in which to study disease processes.

Reflecting the state of AD research over the past two decades, much of this module is devoted to the roles that Aβ and tau might play in the disease process. However, these are certainly not the only active areas of research within the AD field. Genome-wide association studies (GWAS) continue to provide clues about the molecular pathways involved in the pathogenesis of AD. In particular, variants in genes involved in neuroinflammation have been found to associate with disease risk, and interest in neuroinflammation is growing rapidly. There are several excellent on-line resources for keeping up with hot topics in AD research, including the Alzheimer Research Forum and the National Institute on Aging’s Alzheimer’s Disease Education and Referral Center.

Proposed Causes of Alzheimer’s Disease

Before discussing mechanistic studies, we will briefly review the history of the etiology of AD. The causes of AD are not known. In less than one percent of all cases, AD is strongly associated with a genetic mutation; individuals possessing this mutation are certain to get the disease. However, most cases of AD arise sporadically, with age being the greatest risk factor for the disease. Whatever the underlying causes(s) may be, many scientists believe that both the genetically-determined and the sporadic forms of AD share common biochemical and pathogenic mechanisms.

The Aluminum HypothesisOne of the earlier hypotheses to be put forward linked aluminum and Alzheimer’s

disease. In the 1960s, researchers found that injecting rabbit brains with aluminum salts caused progressive neurodegeneration that was accompanied by neurofibrillary pathology similar to the NFTs seen in AD (1, 2). This discovery was reinforced by later findings of elevated aluminum levels in the brains of AD patients (3) and a correlation between incidence of AD and levels of aluminum in drinking water (4). In recent years, support for the aluminum hypothesis has waned as methodological flaws in the original studies have surfaced and subsequent, larger studies have failed to establish any link between aluminum and AD (5, 6).

The Pathogen HypothesisThe pathogen hypothesis posits that AD may be caused by microbial infections.

A variety of microbes, including viruses, bacteria, and fungi have been reported to be more common in the brains of AD patients than in control subjects or to be specifically associated with amyloid plaques (7-11). Interestingly, intranasal inoculation with Chlamydia pneumoniae induces the formation of amyloid plaques in the brains of normal mice (12, 13).

Because findings of increased pathogen burden in the brains of AD patients were not universally replicated, the pathogen hypothesis has not received much attention from the broader AD research community. However, recent reports that Aβ has potent anti-microbial activity in vitro1 (14) and in vivo2 (15) have renewed interest in the pathogen hypothesis (16). The authors of these studies suggest that Aβ may have a protective role in fighting infection. Whether Aβ generated in response to infection then triggers a pathological cascade culminating in AD is food for speculation.

The Cholinergic Hypothesis

1 In vitro. Test tube experiments (literally, “in glass”). In the cited study, Aβ peptides made in the lab killed bacteria and yeast grown in cultures. Most interesting, Aβ from the brains of AD patients also killed yeast grown in vitro.2 In vivo. In a live organism. In the cited study, transgenic animals (mice, worms) expressing human Aβ were protected from bacterial and yeast infections, compared to control animals that did not express Aβ.

One of the oldest hypotheses as to the cause of AD is the cholinergic hypothesis. In AD, there is a loss of basal forebrain cholinergic neurons3 (17, 18). Proponents of the cholinergic hypothesis point to studies indicating that administration of anticholinergic drugs can produce cognitive deficits similar to those seen in AD (19, 20). Cholinesterase inhibitors – drugs that inhibit the breakdown of acetylcholine – are one of only two classes of drugs currently approved by the FDA for the treatment of AD symptoms. Donepezil (Aricept), galantamine (Razadyne), and tacrine (Cognex) belong to this class of drugs. Challenges to the cholinergic hypothesis predominantly come from studies that failed to find a decrease in choline acetyltransferase4 (ChAT) activity in the brains of subjects with mild cognitive impairment (MCI) or mild AD (21-23). In fact, one of these studies actually found elevated ChAT activity in the hippocampi and frontal cortices of MCI subjects (22). Collectively, the evidence tends to suggest that cholinergic deficits and pathology are not the primary causes of AD, but are rather features of late-stage AD that might contribute to the cognitive symptoms observed at this stage.

The Oxidative Stress HypothesisThe oxidative stress hypothesis focuses on the late onset and slow progression of

AD, in that the accumulation of oxidative damage over time is a central feature of AD pathogenesis. Oxidative stress is associated with the presence of reactive oxygen species and is caused by the biological system’s inability to detoxify the reactive species or repair resulting damage (24). Due to its high oxygen consumption, high concentration of polyunsaturated fatty acids, and relatively low antioxidant enzymes, the brain is susceptible to oxidative damage (25). Support for the oxidative stress hypothesis has come from observations that – compared to control brains – AD brains show increased oxidative damage to nucleic acids, proteins, and lipid membranes (26, 27). Multiple studies have found elevated markers of oxidation in the brains and cerebrospinal fluid (CSF) of AD patients (28). Furthermore, studies of oxidative stress in animal models of AD have found evidence of oxidative damage (29). Unfortunately, clinical trials of antioxidants have failed to mitigate AD progression (30, 31).

The Type III Diabetes HypothesisYet another hypothesis points to a link between diabetes and Alzheimer’s disease,

with some even calling for AD to be renamed “Type III diabetes.” This hypothesis is based on the observation that when AD brains are imaged with fluorodeoxyglucose positron emission tomography (FDG-PET), there is reduced glucose utilization in characteristic brain regions (32). This finding led some scientists to suggest that defective insulin signaling was the cause of these FDG-PET abnormalities (33).

3 These neurons project to many regions of the cortex.4 Choline acetyltransferase (ChAT) is the enzyme responsible for the synthesis of the neurotransmitter acetylcholine. ChAT levels or activity are often used to monitor the functional state of cholinergic neurons.

However, it must be stressed that factors such as decreased energy demands by malfunctioning neurons, impaired blood flow to the diseased brain, and/or deranged coupling between neuronal demand and blood vessel response might also underlie the observed decreases in glucose uptake.

Nonetheless, both decreased insulin production and increased insulin resistance have been associated with AD (34). It is the combination of these aspects (elements of both Type I and Type II diabetes) that has led to the somewhat controversial “Type III diabetes” label. Analysis of postmortem AD brains has revealed that components of the insulin- and insulin-like growth factor- signaling pathways are decreased (35), and that these decreases become more pronounced with increasing disease severity (36). Evidence from animal models has provided further support for the Type III diabetes hypothesis. Rats exposed to streptozotocin (STZ) – a compound that causes diabetes when metabolized in insulin-producing cells – exhibit cognitive deficits and significant neurodegeneration similar to what is seen in AD (37). Vervet monkeys with STZ-induced diabetes also exhibit AD-related changes in their brains, including elevated levels of Aβ and increased tau phosphorylation (see below) (38).

Additionally, it has been observed that when individuals with AD are given insulin, they show improved performance on cognitive tasks (39, 40); however, it should be noted that insulin also improves performance in “normal” subjects, suggesting that insulin acts as a general cognitive enhancer (41). In another study, the diabetes drug liraglutide stabilized brain glucose metabolism in AD patients, but the small number of subjects precluded researchers from drawing conclusions about whether the drug improved cognition (42).

The Amyloid Cascade HypothesisThe Amyloid Cascade Hypothesis posits that aberrant aggregation of Aβ triggers

a multi-step cascade in which synaptic dysfunction, pathologic processing of tau, and neuroinflammation eventually lead to neuron death and dementia (43, 44). The strongest evidence in favor of the Amyloid Cascade Hypothesis comes from genetic studies. As mentioned above, there are rare genetic mutations that destine a person to develop AD; all of these AD-causing mutations increase the production of total Aβ or its more aggregation-prone forms5 (reviewed in (45)). Amyloid beta is formed from the sequential cleavage of amyloid precursor protein (APP) by the enzymes beta (β)- and gamma (γ)-secretase. AD-linked mutations have been found in the gene encoding APP and in the genes encoding presenilins (PS) 1 and 2, sub-units of γ–secretase (an up-to-date compendium of these mutations is maintained by the Alzheimer Research Forum). Conversely, a mutation in APP that results in decreased production of Aβ protects against sporadic AD (46). Taken together, these studies are consistent with the part of the

5 The Aβ peptide can have different lengths, depending upon exactly where the -secretase enzyme cuts its precursor. The most abundant form of Aβ is 40 amino acids long, but the rarer 42-amino acid form has a stronger tendency to aggregate.

Amyloid Cascade Hypothesis that postulates that aggregated Aβ initiates the disease process.

There is also experimental support for the hypothesis that tau acts downstream of Aβ. When injected into the brains of mice expressing transgenic human tau, Aβ exacerbates tau pathology (47, 48). Genetic depletion of tau has been shown to prevent Aβ-induced toxicity in vitro (49) and cognitive deficits in mice that express transgenic human APP (50).

Critics of the Amyloid Cascade Hypothesis raise two major objections. First, transgenic mice overexpressing human APP with AD-linked mutations exhibit increased Aβ aggregation but do not develop neurofibrillary tangles or neuronal loss, features of human AD (51). Proponents of the Amyloid Cascade Hypothesis point to the many differences between mice and humans as possible explanations; these differences include forms of tau, neuroinflammatory responses, susceptibility to synaptic-activity-related neuron damage, and lifespan. The second critique of the Amyloid Cascade Hypothesis concerns the fact that drugs targeting Aβ have failed to mitigate AD progression in clinical trials (reviewed in (52)). However, the likely reason for the failures of these drugs is that they were administered too late. The Amyloid Cascade Hypothesis predicts that anti-amyloid therapies should be effective when administered very early in the disease course, before processes downstream of Aβ become self-sustaining.

Despite the criticisms raised against it, the Amyloid Cascade Hypothesis is supported by a preponderance of the evidence and is the predominant model driving most AD research today.

Amyloid-β

Why Does Aβ Accumulate?If, as the Amyloid Cascade Hypothesis proposes, the accumulation of Aβ

aggregates is indeed the event that triggers AD, the question naturally arises as to what causes this accumulation. The rate of accumulation of Aβ in the brain is a result of the relative rates of production and clearance of Aβ. In the rare familial forms of AD (discussed above), genetic mutations lead to the overproduction of Aβ aggregates. Very recently, it has been reported that Aβ production increases in some people early in the course of sporadic AD (53). However, in sporadic AD, accumulation is most likely due to a decline in the ability to eliminate Aβ from the brain (54). This being the case, scientists are very interested in how Aβ is cleared from the brain and in whether these clearance mechanisms become less efficient as we age.

There are at least three pathways for Aβ clearance: 1) via bulk fluid flow from the interstitial space6 into the cerebrospinal fluid, 2) degradation by enzymes within the brain, and 3) receptor-mediated transport from the brain into the blood.

6 The space surrounding the cells in the brain

Bulk-flow clearanceUntil recently, it was believed that this process was too slow and inefficient to

account for more 10-15% of total Aβ clearance (55). However, experiments conducted in the past ~five years, using modern imaging techniques, have caused scientists to re-evaluate the contribution of bulk flow to the clearance of proteins from the interstitial fluid (56). Maiken Nedergaard and colleagues at the University of Rochester have introduced the concept of a “glymphatic” system7, where cerebrospinal fluid moves into the brain along the outer surface of arteries, then moves through the brain tissue, and finally drains from the brain along the outer surface of the veins (57, 58). As the fluid moves it flushes extracellular proteins before it. Nedergaard’s group showed that Aβ clearance via this pathway was dramatically impaired as mice aged (59). Additionally, they observed a decrease in glymphatic flow in the brains of transgenic mice that make Aβ, prior to the formation of amyloid plaques (60).

Enzymatic degradationTwo enzymes are able to degrade Aβ: insulin degrading enzyme (IDE) (61) and

neprilysin (NEP) (62). In transgenic mice with reduced levels of IDE, there is an increased level of Aβ in the brain (63); conversely, transgenic overexpression of IDE results in substantial reductions in soluble Aβ, amyloid plaques and premature death in mice expressing transgenic APP (64). Overexpression of neprilysin in cultured neurons (65) and in mice (62) has been found to increase the rate of degradation of Aβ and decrease the number of amyloid plaques in APP transgenic mouse brains (66).

Receptor-mediated transportReceptor-mediated transport is also responsible for a large portion of Aβ

clearance. The lipoprotein receptor-related protein (LRP) binds to Aβ and facilitates its binding to brain capillaries. Once bound to the capillaries, Aβ is transferred across the blood-brain barrier into the bloodstream (67). Relatively low LRP activity has been found in AD patients (68), and mutation or dysfunction of LRP have been shown to correlate with increased Aβ deposition in APP transgenic mouse brains (69).

Apolipoprotein EIn sporadic AD, there are also genetic factors that influence an individual’s risk of

developing the disease; these genetic factors are distinct from the rare mutations (discussed above) that predestine a person to develop the disease. People with specific

7 “Glymphatic” = “glial” + “lymphatic.” This process acts like a lymphatic system for the brain. Astrocytes, a type of glial cell, play an important role in this process, as these cells control the amount of extracellular fluid around blood vessels, using water channels (aquaporins) in their membranes.

alleles of certain genes have a greater or lesser chance than average of developing AD. The gene with the strongest effect on the risk of sporadic AD encodes apolipoprotein E (ApoE). Compared to the general population, individuals with two copies of the “ε4” allele of this gene are 10-15 times more likely to develop AD (70). While nobody knows the exact role that ApoE plays in AD, animal studies have shown that the rate of Aβ clearance from the brain is differentially regulated by the various isoforms of ApoE (71).

What is the toxic form of Aβ?In the early years of Alzheimer’s disease research, scientists focused their

attention on the neuropathology visible under the light microscope: amyloid plaques and neurofibrillary tangles. However, studies in both humans and animal models of AD generally have shown that plaques do not correlate with cognitive dysfunction (72-75). These observations suggest that other forms of Aβ, rather than plaques, lead to memory decline in AD.

Amyloid plaques are composed of fibrillar Aβ and are insoluble in the detergent-containing solutions commonly used to extract proteins from tissues. By contrast, “oligomers” are detergent-soluble Aβ assemblies. Amyloid beta oligomers can be formed in vitro from synthetic Aβ, and such oligomers impair synaptic function when applied to cultured neurons or injected into the brains of rodents (reviewed in (76)). However, the Aβ oligomers created in a test tube are of uncertain relevance – they may not mimic species that actually form in diseased brains. A few laboratories have attempted to identify and study the effects of oligomers that are naturally generated in the brains of AD patients or mice genetically engineered to express human amyloid precursor protein (reviewed in (77)). This work has invited controversy, due to the technical challenges of isolating oligomers (discussed in more detail below) and, more recently, to reports that newly-discovered APP fragments had been mis-identified as Aβ oligomers (78, 79). Nonetheless, we will briefly review the literature describing putative Aβ oligomers found in brain, as well as newer studies describing hitherto unrecognized cleavage products of APP, while recognizing that research in this area is currently in a state of flux.

Aβ*56 In Tg2576 mice (mice carrying a human APP transgene with an AD-linked

mutation), cognitive decline begins at around 6 months of age – months before plaques appear in the brain (75). This observation led researchers to look for an Aβ species whose appearance coincided with the onset of cognitive decline. In 2006, scientists at the University of Minnesota described “Aβ*56,” a 56-kDa Aβ assembly8 that might be responsible for memory dysfunction (80). In Tg2576, Aβ*56 first appears at 6 months of

8 Aβ*56 is most likely an aggregate of 12 Aβ molecules.

age, and its levels correlate with cognitive dysfunction in the Morris water maze – a test of spatial memory function. To test whether Aβ*56 is sufficient to induce memory problems, researchers isolated Aβ*56 from the brains of transgenic mice and injected it into the brains of young, healthy rats; these rats rapidly displayed memory problems, as evidenced by poor performance in both the Morris water maze and a lever-pressing task.

Tg2576 mice, like other APP transgenic mice, do not fully recapitulate AD – they have subtle cognitive deficits but no NFTs or widespread neuron loss – and are thought to model the earliest stages of the disease. For this reason, it was suggested that Aβ*56 has a role very early in AD, and that it might even be the Aβ species that triggers the Amyloid Cascade (80). Support for this idea came from a study published in 2013. To understand the results of these studies, one must recall that AD has a long presymptomatic phase, possibly lasting two decades or more. During this time, people appear to be cognitively healthy, but sensitive memory tests, brain imaging studies, or analysis of cerebrospinal fluid can reveal evidence of an unhealthy brain (81-85). If Aβ*56 really does have a role during the earliest phase of AD, it should be found in the brains of people who appear cognitively normal but who show signs of a compromised brain. This is indeed what was found. In the brains of subjects who were cognitively normal at the time of death, levels of Aβ*56 correlated negatively with levels of proteins found in synapses (i.e., higher levels of Aβ*56 were associated with lower levels of synaptic proteins) and positively with pathological forms of tau that precede the appearance of NFTs (86).

DimersAfter the discovery of Aβ*56, researchers sought to identify other synaptotoxic9

Aβ oligomers that might be present in the brains of individuals with AD. In 2008, it was first shown that extracts of AD brain can modulate two forms of synaptic plasticity believed to be the cellular bases for learning and memory: long-term potentiation (LTP)10 and long-term depression (LTD)11. When applied to slices of rodent brain, AD brain extracts inhibited LTP and enhanced LTD. After looking into which component of the brain extract was responsible for these effects, researchers concluded that Aβ dimers (pairs of Aβ proteins assembled together) were acting as the toxic species (87). Further studies showed that injecting rodent brains with dimer-containing cerebrospinal fluid (88) or dimer-containing brain extracts (87) resulted in impaired memory function or synaptic plasticity. However, recent research has shown that Aβ dimers quickly form stable protofibrils12, which can mediate synaptotoxicity. That being said, it has been suggested

9 Harmful to synapses.10 Long-term Potentiation (LTP) is a long-lasting increase in the strength of transmission at particular synapses.11 Long-term Depression (LTD) is a long-lasting decrease in the strength of transmission at particular synapses.12 Protofibrils are considered a fibrillar intermediate which acts as a precursor to plaques.

that dimers are not neurotoxic in themselves; but that said toxicity requires their further assemblage into protofibrils (89). It should be noted that dimers are elevated in people with Alzheimer’s dementia (86, 90).

AmylospheroidsAlthough they have not been as thoroughly studied as other oligomers,

amylospheroids are suggested to play a role in AD. Amylospheroids are spherical aggregates of Aβ protein and, importantly, are found in the brains of people with AD but not in cognitively healthy, elderly individuals. Additionally, amylospheroids isolated from AD brains kill cultured neurons (91) with the most toxic form of amylospheroid appearing to be an aggregate of ~32 Aβ molecules (92). Although these initial findings are intriguing, an effect of brain-derived amylospheroids on synaptic plasticity or cognition has yet to be demonstrated.

OtherUsing a novel protocol designed to minimize disruptions of naturally-occurring

Aβ oligomers, David Brody and colleagues at Washington University found large non-fibrillar assemblies (some > 500 kDa molecular weight, or > 100 Aβ peptides per oligomer) unlike any previously reported. These results are preliminary and have thus far been reported only at a conference, but if confirmed, might change the approach that scientists use to study oligomers.

It has recently been suggested that Aβ oligomers can be classified according to their spatial, temporal, and structural relationships to amyloid fibrils. By comparing the complements of oligomers generated by different transgenic mouse lines, Karen Ashe and colleagues at the University of Minnesota defined two categories of oligomers (93). Type 2 oligomers are present only in plaque-bearing brains, are found only in the immediate vicinity of plaques, and share a particular structural feature with amyloid fibrils – a structure described as in-register parallel beta sheet. Type 1 oligomers appear independently of amyloid plaques; while their structure is as yet undefined, these oligomers are not composed of in-register parallel beta sheets. Mice that generate only type 1 oligomers were found to have memory problems, while mice that generated only type 2 oligomers appeared to be cognitively intact even at very old ages. If these findings extend to humans, they could have important implications for the development of therapies directed against Aβ. One would predict that therapies (for example, antibodies) that target type 1 oligomers would be beneficial, while those that target type 2 oligomers might provide little functional benefit.

Challenges of Studying Aβ OligomersWhile the evidence discussed above supports the existence of toxic Aβ oligomers,

there is some debate as to whether these specific oligomers are present in vivo (94). It is

difficult to determine which oligomers are actually present in living brains and to demonstrate their effects in situ (i.e., located within the brain where they are produced, in contrast to their effects when dispersed and applied to cultured cells or host brains). Currently, there are no known reagents that bind to specific Aβ oligomers and can thus be used to demonstrate the presence of a particular oligomer in situ. Biochemical studies, combining some method of separating proteins by size with immunological detection of Aβ, have provided evidence for the presence of specific assemblies in brain homogenates or cerebrospinal fluid. These biochemical studies, while theoretically straightforward, are actually quite challenging. When present in the brain, Aβ oligomers are generally found at low levels; isolation and/or characterization usually require detergent-containing buffers, which can both artificially create and break apart oligomers. Additionally, some oligomeric species may be too unstable to reliably detect and measure in brain homogenates. As a result of these experimental challenges, leaders in the field came together to discuss more stringent criteria for supporting claims that a specific oligomer has disease relevance.

An additional complication has arisen with the discovery of new fragments of APP that can masquerade as Aβ oligomers. In many studies, oligomers were identified using Western blots. In this technique, proteins in a tissue homogenate are first separated by size using gel electrophoresis13. Then particular proteins are visualized using antibodies that specifically recognize that protein. When scientists observed species that reacted with anti-Aβ antibodies but were too big to be a single Aβ peptide, they concluded that these species were Aβ oligomers. However, many of the antibodies that have been used to probe for Aβ also recognize other fragments of APP (these APP fragments contain part of the Aβ sequence and thus are “seen” by antibodies made against Aβ).

Are Plaques Neuroprotective?A majority of current research implies that plaques are not responsible for

cognitive impairment, but does this mean that plaques might actually protect brain function by sequestering toxic Aβ species? Given the finding that amyloid plaques are relatively inert14 unless they are solubilized to release Aβ dimers, it has been proposed that plaques actually help to detoxify harmful, synaptotoxic molecules by quarantining them into insoluble, harmless assemblies (87). Based on autopsy and neuroimaging studies, it has been estimated that ~40% of cognitively healthy individuals have substantial numbers of amyloid plaques in their brains (45). Many scientists have interpreted this observation as evidence that plaques form very early in the disease course, long before cognitive symptoms are apparent. However, Charles Glabe and colleagues have offered the intriguing suggestion that preferential aggregation along a

13 Gel electrophoresis is a technique in which an electric current is used to move proteins through a gel-like sheet. Generally, smaller proteins move faster and larger proteins move more slowly through the gel.14 (i.e., not inhibiting long-term potentiation)

fibrillar pathway might explain why these individuals remain cognitively intact: they do not produce synaptotoxic oligomers (95). This theory is supported by research identifying small molecules that reduce Aβ toxicity by accelerating fibril formation (96, 97).

Do fragments of APP other than Aβ play a role in Alzheimer’s disease?The amyloid precursor protein can be cut by enzymes into smaller fragments.

Until recently, the types of cleavage events were thought to be rather limited. Cleavage along the “amyloidogenic” pathway is a two-step process: 1) cleavage of APP by β-secretase first generates sAPPβ and the β C-terminal fragment (CTF-β); 2) subsequent cleavage of CTF-β by -secretase generates the amyloid intracellular domain (AICD) and Aβ. Cleavage along the “non-amyloidogenic pathway” is also a two-step process: 1) cleavage of APP by α-secretase first generates sAPPα and the α C-terminal fragment (CTF-α); 2) subsequent cleavage of CTF-α by -secretase generates AICD and the p3 fragment.

Canonical processing of APP. The blue bars represent the amyloid precursor protein (APP) inserted into the cell membrane (pink shading). Arrows show the cleavage sites for α-, β- and -secretase. NH2- represents the amino (“N”) terminal; -COOH represents the carboxy (“C”) terminal of the protein.

In 2009, a Swedish group reported finding an array of other APP fragments in the cererbrospinal fluid of AD patients; these fragments start N-terminally to Aβ (i.e., before the β-secretase cleavage site) and end near the middle of the Aβ sequence (98). Since then even more APP fragments have been found – some start N-terminal to Aβ and extend all the way to the end of Aβ (i.e., the -secretase cleavage site) (79, 99); others

look like Aβ that is missing part of its N-terminal (100). Both N-terminally extended and N-terminally truncated fragments were reported to be toxic to neurons. Most recently, fragments have been found that are generated when APP is first cleaved at a site far upstream of the β site (this site has been named the eta (η) cleavage site) and the resulting product is then cleaved by either α- or β-secretase (101). The product formed by α-secretase cleavage, named Aη-α, impairs synaptic function.

It is not known whether any of these APP fragments contribute to neurological impairment in AD. How these fragments fit with the genetic data on familial autosomal dominant AD (AD caused by mutations in APP or the presenilins) is also unknown. It is clear that more study is needed to understand the regulation and consequences of different forms of APP processing.

Potential Targets of AβAmyloid beta may mediate its neurotoxic effects by binding to a variety of

targets. Many groups have attempted to identify these molecular targets of Aβ and to determine which interactions induce pathological changes in neurons. On the one hand, it has been reported that Aβ oligomers insert themselves into cell membranes, creating pores that allow ions to flow into and out of cells. An abnormal influx of calcium ions may then activate calcium-sensitive enzymes which promote synaptic dysfunction and/or cell death (102). On the other hand, there is a plethora of reports showing interactions between Aβ species and specific membrane receptors or intracellular proteins. A few of these targets are described below; this selection is meant to convey the diversity of Aβ targets and is by no means complete. Other posited targets can be found in reviews by Ashe and Zahs (45) and Larson and Lesné (77).

Cellular Prion Protein (PrPc)Cellular prion protein (PrPc) was identified as a potential target of Aβ in an

unbiased screen searching for proteins that bind to Aβ oligomers (103). This observation generated a great deal of excitement, as PrPc is the parent form of the aberrantly folded proteins that causes spongiform encephalopathies (e.g., Creutzfeldt–Jakob disease in humans, mad cow disease in cattle, and scrapie in sheep); many researchers quickly embraced the idea that AD was part of a larger family of neurodegenerative diseases. It was reported that antibodies that block the binding of Aβ to PrPc prevented deleterious effects of Aβ on synaptic plasticity in vitro (104) and that APP transgenic mice lacking PrPc had normal memory function (105). However, other laboratories have challenged these findings, reporting that Aβ-induced deficits in memory function (106) and LTP (107, 108) cannot be rescued by ablation of PrPc. These discrepant results are probably due to differences in the precise species of Aβ that were responsible for inducing neuronal dysfunction in each study. These studies employed a variety of synthetic oligomers and different lines of transgenic mice, each of which generates its own, age-

dependent complement of specific oligomers. Among brain-derived oligomers, only dimers have been shown to interact with PrPc (109). While dimer-mediated deficits might be mediated by PrPc, it is likely that Aβ*56-mediated deficits are PrPc-independent.

Aβ bound to PrPc forms a complex with the metabotropic glutamate receptor mGluR5, and this complex in turn activates Fyn kinase15 (110). Fyn kinase regulates the trafficking of the N-methyl-D-aspartate type of glutamate receptor (NMDAR) at the synapse (111). NMDARs play a critical role in learning and memory, but excessive activation of these receptors is thought to lead to neuron death (112). Binding of Aβ to PrPc has a biphasic effect on NMDARs in cultured neurons, leading to an initial increase in the number of receptors at the cell surface, followed by a decrease in cell-surface receptors (113).

Glutamate transportAs mentioned above, NMDARs are believed to play a role in neuron survival as

well as in synaptic plasticity. “Synaptic NMDARs” are present in dendritic spines, directly apposed to pre-synaptic terminals; activation of these receptors stimulates intracellular pathways that promote neuron survival (114). NMDARs can also be located farther away from the pre-synaptic terminals; activation of these “extrasynaptic” NMDARs suppresses the pro-survival pathways and promotes cell death (115). Normally, glutamate is confined to the immediate area of the synapse; some glutamate is recycled back into the presynaptic terminal by transporters located on the terminal, but most is taken up by transporters located on astrocytes (glial cells) whose processes surround the synapse. However, under some conditions, glutamate uptake cannot keep up with glutamate release, and glutamate “spills over” from the synapse. When this happens, extrasynaptic NMDARs are activated. Amyloid beta oligomers have been shown to increase activation of extrasynaptic NMDARs (116), likely by inhibiting glutamate uptake (117).

Alpha7-nicotinic Acetylcholine ReceptorsA significant decrease in the number of cholinergic neurons is characteristic of

AD brains (see above). Very low (10-8 M) concentrations of Aβ have been shown to reduce cholinergic synaptic transmission (118). In particular, the alpha7-nicotinic acetylcholine receptor (a7nAchR), a pre-synaptic receptor, has been shown to have a high affinity for Aβ (119). Aβ1-42, acting via a7nAchRs, disrupts the release of multiple neurotransmitters (120).

Aβ-ABADIn AD, widespread mitochondrial dysfunction leads to decreased cellular

15 Fyn kinase is a protein that phosphorylates a specific amino acid of target proteins. Generally, the proteins phosphorylated by Fyn kinase are involved in signaling pathways.

metabolism (reviewed in (121)). Amyloid beta peptide-binding alcohol dehydrogenase (ABAD) is localized in the mitochondria and is a candidate for the receptor through which Aβ exerts its effects on the mitochondria. APP transgenic mice that also overexpress ABAD have increased cell death and an increase in reactive oxygenated species (ROS); experiments have indicated that these ROS originate from the mitochondria (122). The interaction between Aβ and ABAD is a possible mechanism through which Aβ exerts toxic effects on neuronal metabolism.

It should be stressed that in this case, Aβ would act intracellularly (for the targets discussed above, extracellular Aβ would act on receptors or transporters in the cell’s plasma membrane). Despite decades of research, scientists still do not completely understand where Aβ is produced and how it is trafficked at a cellular level. A study published in 2016 suggests that -secretases containing presenilin-216 are responsible for the production of intracellular Aβ (123).

Its multiple forms and tendency to stick to itself and to other proteins makes the identification of Aβ’s actual targets quite challenging. One approach to this problem is to genetically manipulate levels of potential targets in Aβ-generating transgenic mice and observe how these manipulations affect disease-related phenotypes. However, this approach has its drawbacks: compensatory changes in other proteins or developmental abnormalities might obscure the effects of the Aβ-target interaction. Despite the challenges of identifying the actual targets of Aβ, this work is essential for the development of new and effective therapies for AD.

Tau

Genetic evidence, cited above, provides strong support for the hypothesis that the accumulation of Aβ triggers AD. What, then, is the role of tau? Abnormally processed tau is the major component of neurofibrillary tangles, one of the pathological hallmarks of AD. However, until the end of the 20th century there was some debate as to whether abnormal tau contributed to the pathogenesis of AD or whether it was merely a by-product of the disease (124). In 1998, it was reported that thirteen separate families with an inherited form of frontotemporal dementia (FTD)17 all had mutations in the gene coding for tau (125). These genetic studies thus provided evidence that abnormal forms of tau can cause neurodegeneration. Subsequently, it was shown that transgenic mice expressing human tau with an FTD-linked mutation exhibit pronounced neurodegeneration (126, 127). Notably, many of the post-translational modifications in 16 The -secretase enzyme contains multiple pieces or sub-units. Either presenilin-1 or presenilin-2 can form the catalytic sub-unit of -secretase, the part of the enzyme that actually cleaves the substrate molecule.17 “Frontotemporal dementia and parkinsonism linked to chromosome 17,” formerly known as Picks’ disease, is a progressive neurodegenerative disorder characterized by disturbances of behavior, cognition and movement

tau that are promoted by FTD-linked mutations are also seen in AD brains (126, 128). These observations are consistent with the hypothesis that AD-related forms of Aβ promote pathological processing of tau, but that it is misprocessed tau that mediates neuron loss. Exactly which form of tau is toxic is one of the outstanding questions in AD research today.

Hyperphosphorylated Tau in ADDespite being most widely known for its role in AD, the tau protein is actually

found in all brains – even those brains that are perfectly healthy. In a normal brain, tau’s primary role is in assembling tubulin18 into microtubules19 and then stabilizing and maintaining these microtubules in the cytoskeleton of axons. Tau is a phosphoprotein, which means that there are sites on the protein that can bind to – and be modified by – phosphate (PO4) groups. Although there are dozens of these sites on the tau protein, normal, non-pathogenic tau generally contains 2-3 moles of phosphate per mole of tau (129). During the 1980’s, researchers analyzing autopsied AD brains discovered that the paired helical fragments (PHFs) which form into neurofibrillary tangles (NFTs) are made up of abnormally phosphorylated tau (130, 131). Additional research revealed that this abnormal tau contains 5-9 moles of phosphate per mole of protein – a phosphorylation level that is 3-4 times higher than what is seen in normal, healthy tau (132-134). Interestingly, the manner in which tau is phosphorylated (e.g., the amount of phosphorylation, where on the tau protein the phosphorylation occurs, etc.) regulates how tau interacts with microtubules. When tau becomes hyperphosphorylated (too many sites on the tau protein are phosphorylated), it loses its ability to bind to microtubules (135, 136).

Possible Causes of HyperphosphorylationAlthough the specific mechanisms underlying tau hyperphosphorylation in AD

are unknown, there are many theories as to what factors drive the change from healthy tau to hyperphosphorylated tau. Some of the more well-studied theories are outlined below.

The Effect of Aβ on TauA number of studies have shown that Aβ can facilitate tau hyperphosphorylation

and NFT formation. When Aβ is added to rat or human neuronal cultures20, it induces tau phosphorylation and loss of microtubule binding (137). It has also been demonstrated

18 The constituent protein of microtubules of cells, which provide a skeleton for maintaining cell shape and is thought to be involved in cell motility (http://medical-dictionary.thefreedictionary.com).19 Composed chiefly of tubulin, microtubules are involved in maintaining cell shape and moving organelles around within the cell. Microtubules are also involved in cell division.20Neuronal culturing is a process in which cells are grown under controlled conditions, outside of their natural environment (http://en.wikipedia.org/wiki/Cell_culture).

that injecting Aβ into the brains of transgenic mice21 markedly increases the number of NFTs in the amygdala (47). Additionally, experiments have shown that crossing a transgenic line overexpressing mutant APP with another, independent line overexpressing mutant tau leads to enhanced tau pathology (48). Conversely, administration of antibodies directed against Aβ reversed tau pathology in a transgenic mouse line that expresses human genes for both mutant APP and tau (138). Taken together, these findings suggest that Aβ promotes the formation of pathological forms of tau.

GSK-3Glycogen synthase kinase 3-beta (GSK-3β) is a microtubule-associated protein

(MAP) that facilitates the phosphorylation of proteins at serine and threonine residues. It has long been known that GSK-3β can phosphorylate tau (139-141), and it turns out that Aβ is able to activate GSK-3β by interfering with the pathway that normally leads to GSK-3β inhibition (142). Therefore, it is possible that GSK-3β activation is one of the paths through which Aβ facilitates tau hyperphosphorylation.

Caspase ActivityThe caspases are a family of twelve proteolytic22 enzymes that are involved in

inflammation and cell death. Activated caspases-8 and -9 have been found in AD brains, where their presence appears to precede tau hyperphosphorylation and NFT formation (143). In vitro studies have shown that caspases-3, -7, and -8 (and, to a lesser degree, caspases-1 and -6) are able to cleave tau into a truncated form that more rapidly assembles into filaments (144, 145). While the exact mechanisms by which the caspases affect tau are unknown, caspase-driven cleavage of tau appears to modify the protein in such a way as to facilitate the formation of NFTs.

Other Possible SuspectsIn addition to looking at Aβ, GSK-3β, and the caspase family, researchers have

studied many other factors that could potentially promote tau hyperphosphorylation. These factors include (but are not limited to):

The interaction of diet, genetics, and cell oxidation (146, 147) Certain insulin deficiencies (148, 149) Vitamin deficiencies (147, 150) Cholesterol level (146, 151)

21 The transgenic mouse line used for this study overexpresses a form of tau that is linked to frontotemporal dementia22 Proteolytic enzymes act on proteins by cleaving them into smaller fragments.

The exact mechanism by which normal tau is modified into hyperphosphorylated tau remains unknown, but it is well known that hyperphosphorylated tau aggregates into NFTs. Unfortunately, the exact role that NFTs play in AD is not entirely clear.

The Role of NFTsPrior to ~2005, NFTs were believed to be highly involved in mediating

neurodegeneration and dementia in AD. In fact, a number of studies showed that the presence of NFTs is associated with cognitive decline and synaptic malfunction (152-155). However, recent studies have shown that while NFTs are one of the primary markers of AD, they do not appear to be the cytotoxic23 form of tau.

In 2001, it was shown that overexpression of tau in Drosophila (fruit flies) led to neurodegeneration without the formation of NFTs (156). This finding was reinforced in 2005 with the discovery that NFT formation is separate from neuron loss and cognitive dysfunction in transgenic mice. When transgenic tau was suppressed in mice expressing a regulatable tau transgene24, NFTs continued to accumulate but neuron loss was arrested and memory function recovered (127). In the same line of transgenic mice, it was seen that neuron loss preceded neurofibrillary pathology in some regions of the brain and that this pathology occurred without neuron loss in other areas (157). In 2006, another study dissociated NFTs from functional deficits in transgenic mice, showing that inhibition of tau hyperphosphorylation delayed the development of motor dysfunction without affecting the number of NFTs (158). Finally, in 2014, it was shown that NFT-bearing neurons in the visual cortex of tau transgenic mice respond normally to visual stimuli (159, 160). Taken together, these results provide strong support for the theory that NFTs do not cause neuronal dysfunction.

The Search for the Toxic Form(s) of TauIf NFTs are not the toxic form of tau, what is? Several candidates have been

proposed. None of these have been proven to be the primary factor underlying tau-induced neuronal dysfunction or neurodegeneration, but initial results indicate that they may be involved in the disease process.

Tau acetylationJust as phosphate groups can attach to proteins to modify their function, so can

acetyl groups (CH3CO-). Li Gan and her colleagues at the University of California, San Francisco found that AD brains, but not the brains of healthy control subjects, contained tau in which an acetyl group was attached to a specific amino acid, the lysine occurring at position 17425. They then overexpressed either normal tau or tau mutated to mimic this acetylation in the brains of normal mice, and found that the acetylation mimic caused 23 Cell-death causing24 A transgene that allows tau production to be turned on or off

twice as much neuron death as did the normal tau. Finally, the researchers took transgenic mice that express human tau with a mutation linked to frontotemporal dementia and gave these mice either a placebo or a drug that inhibits the enzyme that attaches the acetyl group to tau. In the mice given the placebo, the hippocampus shrank and the animals developed memory loss. In the animals given the drug, hippocampal volume and memory function were preserved (161).

Gan’s group also found increased acetylation at lysines 274 and 281 in the brains of AD patients compared to control subjects (162). To study the effects of this tau modification, the researchers created transgenic mice that express tau mutated to mimic acetylation at these sites, and demonstrated that the mice were impaired in a variety of memory tests. They then tracked the source of this impairment at the molecular level, finding that the tau acetylation mimic caused the loss of KIBRA, a post-synaptic protein involved in synaptic plasticity.

Tau oligomersLike Aβ, tau has the ability to form oligomers. Although research into this topic

is ongoing, it is possible that tau oligomers, not tangles, are the primary pathological aggregates in AD. Preparations of tau oligomers are more toxic to cultured neuroblastoma cells than are preparations of tau filaments – the form of tau that makes up NFTs (163). A 170-kDa tau oligomer, possibly a dimer of hyperphosphorylated tau, was found to correlate significantly with cognitive dysfunction in transgenic mice expressing human tau with a mutation linked to frontotemporal dementia (i.e., higher levels of the tau multimer are associated with reduced cognitive function) (164). A 170-kDa tau oligomer has also been seen in another tau transgenic mouse line, and interventions that reduced the levels of this 170-kDa species improved memory function (165). Immunization of tau transgenic mice with an antibody directed against tau oligomers improved motor and memory function (166).

The idea that tau oligomers may be important in the disease process has gained traction in the field. It remains to be seen whether the same issues that have complicated the study of Aβ oligomers arise for tau oligomers.

Toxic Tau FragmentsTau can be cleaved by certain enzymes, and it is possible that the smaller tau

fragments that arise from this cleavage may contribute to neurotoxicity. When tau is cut by caspase-3, a truncated 50-kDa cleavage product is formed. In

vitro studies have correlated high levels of this cleavage product with an increased incidence of cell death (167) and faster, more extensive tau filament assembly (145). In addition to caspase, tau can also be cleaved by calpain, another protease (168). It has been reported that, in vitro, aggregated Aβ stimulates calpain cleavage of tau to generate 25 The amino acids composing a protein are numbered beginning at the N-terminal and proceeding to the C-terminal of the protein.

a 17-kDa neurotoxic fragment (169). While it is unknown whether these tau fragments are actually toxic in vivo, they may turn out to play a key role in the disease process.

In 2014, it was reported that tau can be cleaved by the enzyme asparagine endopeptidase (170). Two lines of evidence support the idea that the tau fragments formed from this cleavage are toxic in vivo: 1) Genetic ablation of this enzyme rescues memory function in transgenic mice that express tau with a mutation linked to frontotemporal dementia, and 2) when the disease-linked tau gene is mutated in the lab so that it can no longer be cleaved by asparagine endopeptidase, mice expressing this form of the gene do not develop cognitive problems.

Tau MislocalizationWhen the tau protein becomes hyperphosphorylated, it loses its ability to bind to

(and maintain) microtubules. In addition to aggregation and NFT formation, the overexpression of hyperphosphorylated tau has been found to result in tau mislocalization – a process in which tau, which is normally found in the axon and soma, diffuses into the dendritic spines (171). This mislocalized tau is distinct from paired helical filaments/NFTs. In cultured neurons, tau mislocalization to spines is accompanied by deficits in synaptic transmission (171) (it should be noted that in this study, neurons expressed tau with mutations linked to frontotemporal dementia or tau with mutations that mimic hyperphosphorylation). Adding a wrinkle to this story, there is some evidence that neuronal activity can drive “normal” tau into dendritic spines, where tau plays a role in synaptic plasticity (172). It remains to be seen whether either of these phenomena, observed in cultured neurons made to overexpress transgenic tau, occur s in vivo. If it turns out that tau mislocalization causes synaptic and cognitive deficits in vivo, preventing tau from mislocalizing may help to rescue some of the cognitive symptoms seen in AD.

Spread of AD Pathology Throughout the Brain

Neuropathological studies have shown that plaques and NFTs spread systematically through the brain, between synaptically connected regions (173). NFTs first appear in the entorhinal cortex26 before spreading to the limbic27 and association28 cortices, while amyloid plaques are first seen in association cortices and subsequently

26 The entorhinal cortex is located in the medial temporal lobe, and is involved in memory and spatial navigation.27 The limbic cortex is involved in a range of factors including emotion, behavior, long-term memory, and motivation.28 The association cortex contain the expanses of the cerebral cortex that are associated with advanced stages of sensory information processing, multisensory integration, and sensorimotor integration (http://medical-dictionary.thefreedictionary.com/association+cortex).

appear in primary cortical areas and the hippocampus. These observations led to the question of whether tau and amyloid pathology can spread between neurons.

Evidence that tau can spread between neurons has been provided by both in vitro and in vivo studies. Tau aggregates can be transferred between cultured mammalian cells (174), and injection of brain extracts from NFT-bearing mice into wild-type, non-transgenic mice induces the appearance of tau fibrils in previously healthy neurons, a phenomenon known as “tau seeding” (175). Using transgenic mice in which human tau is expressed primarily in the entorhinal cortex, two independent groups demonstrated the spread of neurofibrillary pathology first from the entorhinal cortex to the dentate gyrus, then from the dentate gyrus to other subfields of the hippocampus (176, 177). These observations are consistent with the trans-synaptic spread of tau “seeds.” The identity of the tau seeds is not known with certainty, but tau oligomers may be the culprits (178). Scientists are also unsure of the mechanisms by which tau is released, but it has been shown that more active neurons release more tau (179, 180). Microglial cells (discussed below) may also mediate the transfer of tau between neurons (181).

The proliferation of amyloid pathology has also been studied. Several laboratories have shown that injection of Aβ into the mouse brain initiates plaque deposition at the injection site and that this deposition then spreads between axonally-interconnected regions (182-184). Additionally, when transgenic human APP is expressed in the entorhinal cortex, amyloid plaques spread from the entorhinal cortex to the dentate gyrus (185). Under very restricted circumstances, amyloid pathology may transfer between humans: in 2015, researchers from University College London reported finding abundant amyloid plaques in the brains of a small group of relatively young people (36 – 51 years of age) who had received injections of human growth hormone extracted from the brains of cadavers (186). Sadly, these people had died from a transmissible neurodegenerative disorder, Creutzfeldt-Jakob Disease, that they developed as a result of the injections. There was no evidence of AD-like tau pathology in these brains.

Taken together, the results of these in vivo and in vitro studies suggest that amyloid and tau pathology may undergo neuron-to-neuron transmission. However, pathophysiological consequences of the spread of these entities have yet to be convincingly demonstrated. It is important to mention that media reports of these studies have been misinterpreted to mean that AD is a contagious disease – this is not the implication of these studies.

Neuroinflammation

The study of neuroinflammation is one of the most rapidly expanding areas of AD research. In the context of AD, the term “neuroinflammation” has come to refer to chronic “activation” of microglial cells and astrocytes. Unfortunately, the term activation

is not always well defined, but generally means changes in the structure and function of the cells that are observed during injury or disease. Microglia are brain macrophages that respond to infection or injury by ingesting pathogens or cellular debris. These cells accumulate around amyloid plaques, but exactly what they are doing there is uncertain and controversial. Astrocytes were traditionally considered “housekeeping” cells that maintained the ionic balance of the brain and took up neurotransmitters from the synapse and recycled them back to neurons. Neuroscientists have come to appreciate that astrocytes can actually regulate synaptic transmission through a set of complex interactions with neurons, and that these cells also interact with microglia (e.g., astrocytes release signals that cause microglia to migrate to sites of injury (187)). Reactive astrocytes proliferate and their processes hypertrophy. When activated, both microglia and astrocytes release a large array of signaling molecules, some of which might damage neurons and others that might protect them (see (188) for review).

AD researchers initially questioned wither neuroinflammation contributed to the disease process or was merely a response to the accumulation of abnormal proteins (e.g., Aβ fibrils in plaques) and ongoing neurodegeneration. Genetic studies and increased understanding of the functions of microglia and astrocytes strongly suggest that neuroinflammation contributes to the disease process.

Rare genetic mutations that destine a person to develop AD were discussed above under the Amyloid Cascade Hypothesis. Scientists have been diligently searching for other genetic variants that influence an individual’s risk of developing the disease29. One such risk factor is a variant of the gene encoding Apolipoprotein E (discussed above); possession of one copy of the “ε4” allele (variant) triples a person’s risk of AD compared to the risk for someone who does not have a copy of that allele. In 2013, two groups published their findings that variants of the TREM230 gene could also triple a person’s risk of developing AD (189, 190). TREM2 is a membrane receptor found on microglia. Other genetic studies identified variants in additional genes involved in neuroinflammation that either increased or decreased a person’s risk of developing AD (188, 191, 192).

The strong influence of TREM2 on AD risk prompted several laboratories to study how TREM2 affects microglial function. Initially, investigators ablated one or both copies of the TREM2 gene in APP transgenic mice and asked whether manipulating TREM2 had any effect on amyloid plaques – the reasoning being that, because of their close association with plaques, microglia might phagocytize plaques and limit their size or spread. Interestingly, one study found that TREM2 ablation did not affect the number of plaques, but that it did alter their appearance and the responses of surrounding neurons. Plaques in animals lacking TREM2 were not fully surrounded by microglia, the plaques

29 The rationale behind such studies is that identification of genes that have a role in AD will provide clues as to the molecular pathways underlying the disease. These molecular pathways might then be targets for therapeutic interventions.30 triggering receptor expressed on myeloid cells 2

appeared more diffuse when stained to reveal Aβ, and the surrounding neuronal processes appeared more damaged compared to those in mice expressing TREM2 (193). Another pressing question was the identity of the ligand(s) that bind TREM2. In July of 2016, this question was answered by a group of scientists from Genentech (194). They reported that TREM2 binds to Apolipoprotein E and to clusterin31, and that this binding facilitates the uptake of apolipoprotein/Aβ complexes into microglia. These findings are very exciting, as they link three genetic risk factors for AD and suggest a mechanism –altered Aβ clearance – through which risk is altered.

Microglia might also contribute to disease progression by actively eliminating synapses. During normal brain development, synapses are produced in excess and extra synapses are then eliminated as the brain matures. During the past decade, Beth Stevens and colleagues have described an unexpected mechanism underlying this synaptic pruning – synapses marked for destruction are tagged with complement and then phagocytized by microglia (195). This pathway may be re-activated in AD. Stevens’s lab has shown that complement and microglia mediate synapse loss in mature APP transgenic mice, an event likely triggered by Aβ oligomers (196). It has also been reported that astrocytes make complement in response to Aβ (197), so it is conceivable that they, too, contribute to synapse elimination in AD.

Polyproteinopathy of Alzheimer's Disease

Recently, researchers have started to move beyond the notion that pathogenic Aβ and tau are the sole culprits in Alzheimer's disease and have examined the possible involvement of additional proteins normally associated with other neurodegenerative diseases (198).

Alpha-synuclein is a major component of Lewy bodies, the pathognomonic lesions of Parkinson’s disease. In addition to plaques and NFTs, Lewy bodies have been observed in a substantial number of individuals who meet the neuropathological criteria for AD (199-201), and synuclein co-pathology increases the risk of clinical dementia in those with AD pathology (plaques and NFTs) (202-204). Levels of soluble alpha(α)-synuclein were reported to be two-fold higher in AD brains than in control brains, and cognitive function prior to death more strongly correlated with levels of soluble α-synuclein than with levels of soluble tau or A in these brains (205). In vitro, Aβ promotes the formation of abnormal α-synuclein species (206), and Aβ and α-synuclein can induce recombinant human tau to form cytotoxic aggregates (207). Introduction of a transgene encoding α-synuclein (with a Parkinson disease-linked mutation) exacerbates cognitive dysfunction and amyloid plaque and NFT pathology in mice that also express transgenes for human APP (with an AD-linked mutation) and tau (with a mutation linked 31 Variants in the gene encoding clusterin (also known as Apolipoprotein J) have also been associated with AD risk.

to frontotemporal dementia) (208). These studies of the interactions between A, tau, and α-synuclein in vitro and in vivo are consistent with the hypothesis that AD is a “polyproteinopathy” in which A, tau, and α-synuclein act synergistically or additively to impair cognition and cause neurodegeneration.

Alpha-synuclein, Aβ, and tau are not the only proteins that might play a role in AD. TAR DNA-binding protein 43 (TDP-43) inclusions, which form the characteristic neuropathological lesions of amyotrophic lateral sclerosis and the FTLD-U variant of frontotemporal degeneration, have been reported in 25% to 50% of AD cases (209-212). It is not known whether the co-existence of additional lesions with amyloid plaques and NFTs represents a pathological state that promotes protein misaggregation, the coincidental co-occurrence of independent pathological processes, or an essential contribution of α-synuclein and/or TDP-43 to the pathogenesis of AD.

Conclusion/Future Directions

Elucidating the mechanisms of Alzheimer’s disease is a daunting task. Alzheimer’s is a chronic disease; it is now believed that, including its presymptomatic phase, AD lasts for 30 years or more. Yet the most commonly used model systems for studying the disease have lifespans of only a few weeks (cultured neurons) to 2-3 years (transgenic mice). Researchers do not know how to reconcile the results of the relatively short term experiments in these systems to the decades-long disease course. Furthermore, there is no “mouse model of Alzheimer’s disease” that reproduces all of the cardinal features of the human disease: amyloid plaques, neurofibrillary tangles, and most importantly, widespread neuron loss. Developing such a model is one of the most urgent priorities in the field. As some scientists attempt to create improved animal models of the disease, others have turned to cells derived from patients. Patients’ skin cells have been “re-programmed” to become neurons (213, 214) that scientists are using to study pathophysiological processes and to test responses to drugs. One study has already reported that such human-derived cells respond quite differently than mouse neurons to a -secretase inhibitor (215).

Despite large challenges, scientists have made significant advances toward understanding the etiology of AD. One of the most important insights is that plaques and tangles are themselves unlikely to be toxic to neurons; rather, these lesions are indications of abnormal processing of Aβ and tau. Identification of the pathogenic forms of these proteins and their molecular targets should allow us to define the pathways through which they exert their toxicity. The identification of genetic risk factors for sporadic disease is also providing clues about the molecular pathways that go awry in AD. And rather than studying potential molecular players one by one, researchers are employing

systems biology techniques to search for disease- and aging- related changes in entire molecular networks.

We seek to understand this disease in order to prevent or cure it. The ultimate validation of any proposed mechanism must come from studies of the actual disease in human subjects – testing whether interventions that block putative harmful processes, or promote protective processes, prevent dementia in people at risk for AD.

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