Where are we with Alzheimer’s?

By: Heather Parker

Alzheimer’s Disease(AD) is being called the “Twenty-first Century Plague” with 35.6 million people living with dementia worldwide in 2010, and a predicted 65.7 million by 2030 (1). The estimated worldwide cost of dementia in 2010 is $604 billion, or 1% of the world’s gross domestic product (1). With no current cure and limited treatment, this disease is at the forefront of medical research, with many different hypotheses, but few hopeful outcomes. It has a complex, multi-faceted pathology that continues to stump researchers, but there are hopeful results in early clinical trials and many avenues that need to be explored.

There are different classifications of AD. Early onset AD typically starts between ages 35 and 60, where late onset AD is typically starts after 55 years of age (2). AD typically begins with a loss of short term memory, and progresses over decades, leading to total loss of cognition and executive function (3). Executive function is defined as a set of cognitive abilities that are necessary for goal-directed behavior, and influence memory, attention, and motor skills (4). AD patients also have symptoms of anxiety, depression, and emotional disturbance (3). Histopathology of the disease involves both extracellular and intracellular deposits of aggregated proteins, which can appear decades before symptoms appear (3).

It is possible that after nearly a century of investigating this disease, we still don’t know what is really causing AD. Alois Alzheimer found two lesions in the brains of patients a hundred years ago, amyloid beta (Aβ) plaques (extracellular) and neurofibrillary tangles (NFTs) (intracellular) made of tau protein, both of which are considered to be types of amyloid (6). The question remains, which is causing the disease? Are the amyloids causing the disease at all? With so many failed clinical trials of AB-targeted therapy and more promising results of tau-targeted therapy, attention is increasingly turning towards tau. However, some scientists have wondered if the plaques are truly the cause of the disease or a side effect. It is not a new discovery that neuroinflammation plays a large role in AD, and studies have shown that anti-inflammatory drugs can protect against AD. An exciting, recent study has shown that an antibody against TNF-alpha, an adipokine involved in systemic inflammation, has proved to be beneficial. Perhaps the key is to treat the inflammation, not the plaques.

This review will cover the hurdles of finding a cure for AD and what the current research and clinical trials are finding. While it is critical to understand and consider the small molecular pieces involved, it is equally critical to step back and keep in mind the big picture. Is it possible that researchers have been overly focused on plaques all along?

Amyloid Β Hypothesis-AD Dogma

Much of the research on Alzheimer’s since the 1960’s has been focused on The Amyloid Βeta Hypothesis which started with the discovery of amyloid plaques in the brains of Alzheimer’s patients (6). “Amyloid”

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means “starch-like” because the deposits stained with iodine, which was normally used to detect starch (5). Amyloid plaques are mainly composed of the amyloid beta peptide, a processed fragment of the amyloid precursor protein, APP (7). APP undergoes proteolytic processing by the aspartyl protease β -secretase (Β-site APP cleaving enzyme 1) BACE1 which cleaves at a unique “B” site (7,8). ϒ secretase then cleaves the left over carboxy-terminus end of APP more than once at position 40 and 42, resulting in peptides Aβ 1-40 and Aβ 1-42 (9). Aβ-42 has two extra hydrophobic amino acids, alanine and isoleucine, which makes it much more likely to aggregate (6). Aβ-40 is ten-fold more abundant in physiological conditions in mammals (6). The creation of Aβ peptides and the formation of plaques activates glia cells (microglia and astroglia), which are found surrounding the plaques (10,11). Activated glia cells are a hallmark of Alzheimer’s disease (11). This may lead to a vicious cycle because activated glia cells overexpress BACE1 in response to stress, and therefore produce Aβ as well (12). The combination of amyloid plaques and neuroinflammation eventually lead to neuronal dysfunction and death.

The “amyloid state” itself is complex and requires further definition. Normally soluble proteins can take on a different state, usually through misfolding, that results in “intractable aggregates” (5). Surprisingly, the amyloid states are not rare occurrences, as one might guess. Many, if not all, proteins have the capability of forming this non-native state (13). Some proteins do not spontaneously fold into globular proteins (based on their amino acid sequence and specific free energy surfaces), but maintain a relatively unfolded structure and are often called “natively unfolded” or “intrinsically disordered” (14). When examining the proteins involved in misfolding diseases, many of the players are these natively unfolded proteins, including the AΒ peptide in Alzheimer’s disease (5). However, there is no similarity in amino acid sequence, native structure or function of these proteins (15).

Of particular interest in amyloid fibrils is the high level of organization, unlike many other aggregated forms of proteins that are more random (5). The structure is called a “cross-β pattern” which consists of β-strands stacked on each other, continuously hydrogen bonded together, perpendicular to the fibril axis, which has a high kinetic and thermodynamic stability (13). These form a protofilament (~4nm wide), many of which wrap around each other to create a mature fibril (~8nm wide) with even more stability and tensile strength (5,6). It is the backbone of the polypeptide chains that naturally hydrogen bond to one another that create this structure, but the lateral association of β-sheets depends on the amino acid sequences, which is why usually these structures are composed of many copies of one peptide (5).

Another angle to consider is it is not just the plaques that cause disease, but also the soluble oligomers of AΒ peptides (16, 17). By introducing only these soluble oligomers into an experimental system, many of the phenotypes seen in Alzheimer’s arise: synaptic loss, impaired hippocampal synaptic plasticity, microgliosis, tau hyperphosphorylation, and neurofibrillary changes (6). These oligomers can act as a “seed” to create more aggregation elsewhere. The plaques themselves may function as reservoirs of oligomers, where the oligomers can diffuse and cause more injury and inflammation elsewhere, developing an equilibrium of plaques sequestering oligomers while they also diffuse away (6).

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In vitro studies (at the cellular level) have looked at the process of amyloid spreading within a cell and between cells (5). It has been shown that the mechanism of spreading is “prion-like” and “self-propagating” for A β, tau, and many other disease-causing amyloids (5, 21). For example, Guo and Lee showed that by adding a small amount of preformed tau to tau producing cells, there was rapid recruitment of soluble tau and formation of new NFTs, confirming a “seeding” mechanism that once started, spreads and propagates in waves (5,22). It is important to note that these have all been studied at the cellular level and we can only hypothesize about what it really happening in the human brain. Another obstacle of creating a laboratory model of AD is that neurodegenerative diseases develops slowly over decades, which is difficult to recreate in a laboratory or at the cellular level (5).

Since the first APP mutation was found that causes AD in 1991, as many as many as 18 different APP mutations have been found that lead to an AD phenotype (18). Nearly all of these mutations are at or near the cleavage sites of α , β, or ϒ secretase, resulting in lower production of AΒ (18). For example, in 2012, Jonsson et al found that coding mutation A673T in APP, which is adjacent to the β -secretase cleavage site, led to a 40% reduction in AΒ formation (19). They searched the genomes of 1,795 Icelanders and found an SNP (single nucleotide polymorphism) in the A allele of alanine to threonine at position 673 that seemed to have a protective effect. They found this APP variant significantly more common in a control group which consisted of people who had lived to at least 85 years old without a diagnosis of AD. They identified three homozygous individuals; one died at 88, and two were still living at 67 and 83, and none of the three had any history of dementia. Interestingly, mutations at the ϒ-secretase site tend to cause a more aggressive form of AD with an onset as early as 34-35 years old, for example Austrian APP T714I (18, 20). These findings also support the amyloid β hypothesis that the cleavage of APP to make AΒ is what causes AD.

However, keep in mind that all of the cases of AD caused by a genetic mutation (in APP, PS1, PS2) or inheritance (familial Alzheimer’s Disease, or FAD) cause early onset AD. This type of AD is rare and accounts for less than 5% of all AD cases. Late onset AD is the more common type of AD, and the cause is still unknown (23).

Presenilins

Lysosomes are an important organelle in the cell that breaks down essentially all of the compounds made and used by the cell, using hydrolytic enzymes. The function of lysosomes is particularly important for neurons because they do not divide and have a longer lifespan, and therefore need to be able to get rid of these waste products (24). Lysosomal storage diseases (LSD’s) are characterized by lysosomal dysfunction. Interestingly, AD has been linked to lysosomal dysfunction in neurons, and is therefore often grouped with LSD’s, but the connection between the two is not understood (24).

People with a mutation of the presenilin protein, PS-1, develop the most common type of Familial Alzheimer’s Disease (FAD). Presenilin is a multi-pass membrane protein with nine transmembrane domains in the endoplasmic reticulum (25). Presenilin is cleaved into two subunits and becomes part of gamma-secretase complex, which is then transported to the cell surface where it cleaves APP to form A

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β (25,26). Gamma-secretase containing mutant PS-1 seem to have a higher affinity for cleaving APP at amino acid 42, therefore increasing the formation of A β and aggregation of plaques (25).

It is unclear how this mutation causes lysosomal dysfunction, and there are conflicting theories. In 2010 Lee et al used PS1 KO mouse blastocysts to show that PS1 acts as a chaperone to V0a1, a subunit of Lysosomal v-ATPase (24, 27,28). Lee hypothesized that PS1 facilitates the proper folding and N-glycosylation of Voa1 in the ER (27). PS1 is thought to interact with oligosaccharyl-transferase (OST) and facilitate the transfer of oligosaccharide precursors to V0a1 during its translational translocation into the rough ER (27,29). This modification of V0a1 is required for transport from the ER to lysosome, and due to the lowered level of V0a1 in the lysosome of PS1 mutants, the ATPase complex cannot form (27,29). Using LysoTracker, Lee found the pH in the lysosome is raised above the optimal level of 4.0-5.0, and proteolytic enzymes, specifically cathepsin D, no longer function efficiently (24,27,28). This leads to impaired autophagy, protein accumulation in lysosomes, and an early onset of AD (27). Because A β is generated and degraded during autophagy, they hypothesize that A β is able to build up in the lysosomes and have a higher likelihood of aggregation and amyloid formation (27).

Interestingly, Coen et al (among others) challenged Lee’s findings (29). First, Coen found no difference in lysosomal pH in PS DKO mice and WT mice (29). Coen argues that LysoTracker is only a “qualitative assessment of pH” and used a different ratiometric measurement to measure pH, LysoSensor Yellow/Blue (29). She also showed that V0a1 is not necessary for lysosomal acidification by looking at knockdowns of V0a1 in WT mice and found no change in acidification of lysosomes. Coen explains that there are three orthologs of V0a1, V0a2,3,and 4, but RNA transcripts were analyzed and there were no reductions in any of these in PS mutants. She goes on to show that N-glycosylation of V0a1 and downstream trafficking is also not affected in PS KO cells and even mutating the N-glycosylstion site on V0a1 does not affect its function. What Coen does find is that lysosomal calcium homeostasis is disturbed in PS KO cells. Fusion of late endosome and lysosome depend on acidification of organelles and lysosomal calcium release, the latter of which may be deficient in PS mutants. When autophagosomes cannot fuse with lysosomes in neurons, autophagy is impaired and the cell is unable to clear long-lived proteins from the cell. This could lead to build up of proteins in autophagosomes causing aggregation and amyloids, and eventually cell death.

After all the controversy and arguments, it was found that Lee’s results may have been accurate but was only found in the cell line he used. It is still unclear exactly what role presenilin mutations play in AD. There are many theories and many ways that presenilin affects the cell. It could be involved in Notch signaling, or CREB-binding (cAMP response element binding), reduction in gamma-secretase activity, or calcium homeostasis as proposed by Coen (25). It is still an active area of research today.

ApoE

While early onset AD has been linked to mutations in APP, and Presenilin I and II, the APOE gene (or ApoE protein) has been identified as another genetic locus that greatly affects an individual’s susceptibility to late onset AD (2). It is important to note that these are not all separate and unrelated. Presenilin likely interacts with ApoE in the endosomal/lysosomal pathway and ApoE binds to

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hydrophobic Aβ, the cleaved fragment of APP. ApoE is also found in senile plaques. While all of these pathways likely intersect, the exact interactions are not known.

ApoE was mapped to chromosome 19 in the mid 80’s, and was then linked to AD in 1990 (2). ApoE has three most common alleles: ε2, ε3, and ε4. Ε2 has a cysteine at position 112 and 158 and is the least common allele(2). More than 10% of the general population inherits at least one ε2 allele (2). Ε3 has a cysteine at position 112 and is the most common allele(2). Ε4 has an arginine at position 112 and 158, and it not rare (2). About 30% of individuals carry at least one ε4 allele (2).

In 1990 Strittmatter discovered that ApoE ε4 had a higher affinity for AB than ε3 (30). In 1993 Corder and Srittmatter joined forces and found that not only is ApoE linked to AD, the risk for AD is dose dependent on AD (31). They studied 46 families with AD, 42 with late onset, and 4 with early onset (2 of these also has the APP mutation), all 60 years of age or older (31). They genotyped the individuals in the study and compared ApoE alleles to AD diagnosis. What they found was shocking. 20% of individuals with a 2/3 or 3/3 genotype were affected by AD. 47% of individuals with 2/4 or ¾ genotypes were affected, and a shocking 91% of individuals with a 4/4 genotype were affected by AD (31). In other words, individuals with two ε4 alleles are more likely than those with one ε4 allele to develop AD, and there was therefore a dose dependence of ApoE ε4! They calculated that the risk for AD went up by a factor of 2.84 for each ε4 allele one carried. An individual with two ε4 alleles is more than 8 times more likely to develop AD than someone with a 2/3 or 3/3 genotype (31).

Corder and Strittmatter went on to examine age of onset. They found that the mean age of onset of AD was 84.3 for individuals with no ε4 allele, 75.5 for those with one, and 68.4 for those with 2 ε4 alleles (31). They even found a correlation between ε4 allele presence and survival age (regardless of disease). They found the mean age of death for those with no ε4 allele was 84.9, one ε4 allele was 79.8, and two ε4 alleles was 78.1 (31). The authors dutifully point out towards the end of the paper that 19 of the 95 affected subjects studied, and 12 of the 42 late onset families that were affected with AD, did not carry an ε4 allele. They propose that there still are other genetic factors involved, and the disease is still more complex than just identifying this one genetic link.

Is ApoE causing the disease or is it just a side-effect? Having the ApoE ε4 allele is also associated with age-related cognitive decline in normal aging as well, so it may just be a sign of neurodegeneration (32). However, ApoE is not only a cholesterol carrier for the brain, but is also involved in injury repair in the brain (32). It plays an important role in clearing the Aβ peptide from the neurons and the different isoforms bind to it with different affinities. ApoE binds to the hydrophobic Aβ which may initiate the pathway that leads to synaptic dysfunction and neurodegeneration. Further research is needed to determine the role of ApoE in disease progression.

Clinical trials targeting AΒ

Researchers hoped that targeting Aβ would cure the disease, but unfortunately, we seem to be missing an important part of the puzzle. Clinical trials of Aβ-targeted therapy have not had promising results.

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There are four major strategies that have been studied to try to reduce the levels of AΒ in the brain: reducing the amount of AΒ by inhibiting α, β, or ϒ secretase, removal of already formed AΒ plagues by immunotherapy, inhibiting the aggregation of AΒ peptides, and the clearance or removal of AΒ (33).

Tarenflurbil, an NSAID and known ϒ-secretase modulator or GSM, was used to inhibit ϒ-secretase to reduce the proteolytic processing of APP and therefore reduce the amount of Aβ42, the more hydrophobic type of Aβ, and therefore the more likely to form amyloids (34). It acts by shifting the cleavage site of APP at the C-terminal end of Aβ. A 21 day study in healthy, older individuals showed a correlation between higher levels of the drug in blood plasma and lower levels of Aβ42 (35). Although the treatment was promising, phase III clinical trials were a disappointment (34). In 2009 Dr. Green et al performed a double-blind trial at 133 different sites between 2005 and 2008 using either Tarenflurbil (800 mg) or placebo twice a day on 1,649 patients with mild AD (35). Dr. Green concluded that Tarenflurbil did not slow cognitive decline or the loss of daily activities. The conclusion was that the drug was not able to cross the blood-brain barrier. This was seen in preclinical trials in transgenic mice (Tg2576 mice which overexpress the mutant form of APP) as well, but still had a therapeutic effect on Aβ levels, so the clinical trials were pursued. There is always the gamble that what we see in mouse models will not translate to humans, and this was the case for Tarenflurbil. Hardy, one of the original researchers to propose the Aβ hypothesis, argues that perhaps if it was administered early on in the disease progression it may be more beneficial (36). Hardy proposes that studies should be done on patients in the earliest stages of the disease, where patients are selected based on ApoE ε4 homozygosity, or by PIB imaging and selected based on the presence of plaques (37). Patients with even mild symptoms of AD may already have extensive cell loss and perhaps the chain reaction is too far advanced to reverse. He also suggests that the dose may not have been high enough to cross the blood-brain barrier, as the EC50 (half the maximal effective concentration)was not reached in the brain (37).

Next Semagacestat , a more potent GSM, was then tested in 2013. Doody et al, funded by Eli Lilly, conducted a double-blind, placebo controlled study where 1,537 patients with probable AD received either 100mg, 140mg, or placebo daily (38). All three groups showed declining cognition, although the patients receiving the drug were scoring lower than placebo patients, and the patients receiving the higher dose scored the lowest. The patients on Seagacestat also developed skin cancer and other infections. Due to these adverse reactions, the study was halted. Importantly, there was a measurable decrease in the amounts of Aβ40 and Aβ42 in the plasma of patients on the drug, a lower amount in patients on the higher dose, and no change in patients on the placebo, confirming that the drug was actually “working” as far as reducing the amount of AΒ in the plasma. However, there was no change in the amounts of Aβ or tau in cerebrospinal fluid (CSF). Selkoe argues that the dose was only given once a day, which was half of what was originally intended due to a low therapeutic index(3-Selkoe first section). He also argues that the adverse effects seen were likely from inhibiting Notch processing, another target of gamma-secretase. He argues that if gamma secretase pathway could be inhibited only when processing APP, not Notch, this therapy might work.

Β-secretase (BASE) inhibitors have also been explored with disappointing results. Remember, BASE1 (Β-site APP cleaving enzyme 1), cleaves the APP at the B site, producing Aβ peptides. Phase I trials of BASE inhibitor LY2886721 lead to excitement when it was reported that AΒ40/42 levels in CSF were reduced

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by 75% (33). In June 2013 Lilly announced it was halting the first phase II trial of a BASE inhibitor (LY2886721)due to abnormal liver biochemical tests (39). Lilly reports that their scientists do not believe the drug was the cause of the liver problems and are continuing to work on developing BASE inhibitors as a treatment for AD (39).

There is hope for BASE inhibitors. Merck is working on a phase III trial on MK-8931, another BASE inhibitor. In Phase II trials, levels in the CSF were reduced by 57%, 79%, and 84% compared to baseline levels, when patients received 12, 40, or 60 mg of medication respectively (40). There were also no serious adverse reactions reported. The phase III trial will be completed in April 2020, and the results may hold the answer everyone has been looking for (41).

Another novel approach to reducing Aβ levels is active or passive immunization, utilizing immune cells to target and clear Aβ peptides. Active immunization is where either an inactive form or part of the pathogen is injected into the body and the immune response is activated for future protection. Passive immunization is when antibodies to a particular pathogen are made, purified and injected into a patient, often due to a lack of endogenous immune system function. Early studies on AD model mice (PDAPP mice that have a human familial APP mutation that causes Aβ plaque formation) using an active immunization of Aβ42 had very hopeful results. Mice that were immunized early did not form plaques as they aged, and even more exciting, older mice with plaques already formed showed reduced numbers of plaques (42).

A 2013 trial by Dodel et al tested the results of injecting IV immunoglobulin, a treatment often used for autoimmune diseases. IVIG treatment causes the neutralization of a wide array of antigens, as the product is prepared from the serum of 1,000-1,500 donors per batch (43). The results showed the treatment was safe, but there was little effect on plasma Aβ levels (44). The study only included 58 patients, and the conclusion was that a larger, longer study was needed, but with such disappointing results, it may not be pursued.

AN1792 was the first active immunization for Aβ (33). The immunization was shown to reduce Aβ plaques and improve cognitive function in AD mouse models (45). In 2007 a phase II clinical trial by Elan Pharmaceuticals treated 372 patients with mild to moderate AD, where 300 patients received the immunization and 72 received the placebo. 59 (19.7%) of the patients who received the immunization developed the predetermined antibody response. The study was halted after 6% of the patients developed meningoencephalitis; however, a small number of patients (eleven), who did respond to the antibody, had significantly lower tau levels, but no change in AΒ in CSF. Memory function was improved in antibody responders, and postmortem reports showed decreased neocortical AΒ. The cause of the adverse reaction was concluded to be from a change in the formulation of the immunization. Polysorbate-80 was added to the drug to prevent precipitation and may be to blame for the encephalitis. There is hope that this treatment may be still show promise if the medium is adjusted.

Boche et al performed post mortem clinicopathological follow-up studies of nine patients that received active immunizations from two Elan Pharmaceutical trials from 2002 and 2007, and their finding were surprising to say the least (46). They found that one patient was misdiagnosed and did not have AD, and

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argue that this may be common and may skew results in clinical trials. They looked at histological patterns of AΒ immunostaining in the cerebral cortex and what they found was a shock. Of the immunized patients, some had plaques that looked exactly like the AD patients that were not immunized (controls), and some had no plaques. They found similar variability in the amount of Aβ42 in the entire frontal lobe of the cerebral cortex. Another important finding was a link between Aβ and tau. They determined that plaques had been removed where they saw “alternating patches of presence and absence of plaques in the cerebral cortex, which coincided with Aβ antibody titers in the blood”. They concede that there is no way to prove the Aβ was removed, as they did not have histology samples from before the treatment. However, they point out that now there is PET imaging available, which was not available then, and it has confirmed that the Aβ immunotherapy reduces the amyloid signal after treatment. They found that where Aβ plaques had been removed, tau NFTs were removed too and where Aβ 42 was low, phosphorylated tau was low as well (in the neocortex and hippocampus).

Most importantly, they point out that synaptic loss and therefore cognitive decline are not necessarily linked to Aβ plaques, and that the correlation is not well understood. They found no correlation between Aβ plaque load and synaptic loss. Even in patients with little to no plaques, they still had synaptic loss. Analyzing 80 patients that received the immunization, they compared cognitive function scores with post mortem neuropathology and found that cognitive function continued to decline despite reduction and even complete removal (or lack of) Aβ plaques. They pose the important question; does the presence or absence of AΒ plaques really correspond to loss cognitive function? They hypothesize that perhaps AΒ plaques are required to start the progression of the disease, but other players are involved in propagating cognitive decline, such as tau, activated microglia, astrocytes, or a number of other players we have discussed.

Bapineuzumab , a monoclonal antibody that binds soluble and fibrillary AB, was tested in PDAPP mice and was not only able to cross the blood-brain barrier, but also clear plaques via microglial cell facilitated phagocytosis (47). The first phase II trial measuring only cognition and function of patients showed little effect, and 9.7% of patients experienced reversible vasogenic edema (48). Selkoe argues in this study that patients who received all six doses showed less decline than those on placebo by some measures of cognitive function (6). Interestingly, a follow up study on the same patients in 2012 measured biomarkers in CSF including AB1-42, ABx-42, ABx-40, total tau and phosphorylated tau, and found promising results. Blennow et al reported a significant decrease in total and phosphorylated tau in patients that received the antibody versus placebo. Surprisingly, they did not see a significant difference in the amount of CSF AB levels (49). These results support Boche’s hypothesis that perhaps a reduction in biomarkers for AD and improved cognition and function may not go hand in hand. Selkoe argues that there were only two patients that had significant clearance of AB plaques who still died with advanced dementia (6).

All arguments aside, Bapineuzumab failed in recent phase III trials (50). There were more patients with edema which increased with dose and there was no significant difference in cognition and function between placebo and treatment groups. Interestingly, in the participants that were carriers of the ApoE ε4 allele, there was a decrease in amyloid plaques and CSF phosphorylated tau biomarkers.

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Another monoclonal antibody that binds soluble AB, Solanezumab, was tested and results just came out this year. Doody et al report that it also had no cognitive benefit and higher rates of edema and hemorrhage than placebo groups (51). Oddly, Eli Lilly reports a 20-42% slowing of cognitive decline but no significant effect on function (52). Four new monoclonal antibodies and six AB vaccines are currently being tested and there is a focus on early intervention to try to get ahead of the disease progression (33).

Many drugs have been developed to try to prevent the aggregation of AB peptides as a different way to combat plaque formation. In mouse models, Tramiprosate showed promise. There was a 30% reduction in plaques , a 20-30% reduction in soluble and insoluble AB40 and AB42 in the brain, and a dose-dependent reduction of up to 60% of AB in plasma (53). Not surprisingly, these results did not translate to humans. It failed in phase III trials with no significant treatment effect and even worse, there was evidence of a reduction in hippocampus volume in patients that received the medication (54). Selkoe argues in his review that there was no evidence that the drug entered the CNS or that it had the opportunity to bind AB (3-Selkoe first section).

PBT2 is a copper/zinc ionophore, meaning it moves metal ions across membranes having far reaching effects on cellular functions (55). PBT2 quickly improves cognition in mice with AD, but in phase II trial in 2006, they found significant reduction in the CSF AB42, but no change plasma markers (56,57). They did find significant improvement in two executive function tests of the NTB (Neuropsychological Test Battery) (57), but no overall correlation between AB42 levels and cognition (56). Faux et al hypothesize that the study was only 12 weeks long and perhaps a longer study is needed (56).

Neprilysin, which is an enzyme that degrades AB in the brain, has been overexpressed in AD model mice and effects plaques but not oligomers, and therefore failed to improve cognition (58). A more recent study used an adeno-associated virus to express the gene more globally in the brain and reduced AB oligomers and improved cognition in mice (59). They inject the virus into the left ventricle of the heart because it is a direct feed to the brain, and is also capable of crossing the blood-brain barrier, although it is not understood how. Of interest, this expression of Neprilysin is concentrated in the endosome, which is where AB accumulation begins. Perhaps clinical trials will follow.

Selkoe also points out that the current AD mouse models may not accurately mirror the human disease (6). He points out that the mouse models used have both mutated APP and tau, but PHF’s do not form in rodents. Also it is still unclear if the Aβ plaques form first, which then trigger tau aggregation, or vice versa, and to just make double transgenic mice may not be an accurate enough model.

In summary, what works in mouse models does not seem to work in humans. The question arises, does preventing amyloid plaques or oligomer aggregation really lead to improvements in AD symptoms? Is the Amyloid Beta hypothesis flawed or incomplete? Perhaps there is another player that needs to be considered: tau.

Tau and NFTs

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30 years ago Tau was discovered by Weingarten in 1975. The involvement of tau and NFTs in AD was downplayed when it was found between 2001-2003 that tau pathology occurred downstream of AΒ formation (6,63). Lewis et al found that tau/APP double mutant mice developed NFTs at a much higher rate than the mice with only a mutant tau transgenic gene, and concluded that the AΒ or the mutant APP caused the higher rate of NFT formation (60). Gotz et al found that by injecting AΒ42 into the brains of mutant tau mice, there was a fivefold increase in NFT formation, concluding that AΒ stimulates the formation of NFTs (61). However, later studies showed that tau is required for AΒ to cause neuronal damage. For example, Shipton et al found in 2011 that tau is required for the AΒ-mediated synaptic dysfunction in the hippocampus (a region that is affected early on in the disease), and that AΒ damage is even mediated by phosphorylated tau (62). In 2007, Roberson et al performed a pivotal study where they reduced endogenous tau levels in transgenic mice expressing hAPP with familial AD mutations which raise AΒ levels, and found that the lowered tau levels had a protective effect (63). Clearly, tau is an important player and perhaps a missing piece to the puzzle.

More and more studies are pointing to the importance of tau in the progression of the disease, and a closer correlation between the tau pathway and clinical symptoms than with AB plaques (Giacobini 1 in clinical trial section). As long ago as 1992, Arriagada et al studied the brains of ten AD patients and quantified NFT’s and senile plaques (AB plaques) and compared their results with the severity of patient dementia (64). They found a close correlation between NFT’s and dementia, but no correlation between senile plaques and dementia! These finding have been reproduced by many, including Bierer et al. in 1995, Goómez-Isla et al. in 1997, Giannakopoulos et al. in 2003, and Ingelsson et al. in 2004 (65). Serrano-Pozo et al reported in 2011 that AB plaques form early in the disease progression, before dementia symptoms arise, but that NFT’s parallel the severity of dementia (65). The plaques seem to “plateau” early on in the disease, where NFT’s continue to form.

Tau has six main isoforms in the human brain, which are all from one gene through alternative splicing (66). MAP Tau is a microtubule-associated protein (MAP), whose main role in a non-diseased state is to stabilize microtubules (MTs), and are found in a high concentration in axons of neurons (67). All of the isoforms of tau have varying numbers of MT-binding domains (67). Human tau is encoded by one gene containing 16 exons on chromosome 17q21 (68). All of the CNS isoforms of tau are made by alternative splicing of 11 of these 16 exons, which result in either three (3R-tau) or four (4R-tau)carboxy-terminal tandem repeats from exons nine through twelve (68).

Tau phosphorylation is developmentally regulated and fetal brains have highly phosphorylated tau, where adult brains have tau that is in a lower phosphorylated state (9,66). Tau plays an important role in MT function and therefore affects the shape of the cell, important to neurons due to their long axon, as well as transport and signaling, also important to neurons due to their shape (67). Tau is normally in equilibrium between its unphosphorylated state bound to MTs, and its phosphorylated state when it is cytosolic. Tau has fast cycles of phosphorylation and de-phosphorylation because tau is needed to stabilize the MTs, but physically blocks transport along the MT, so must fall off temporarily for vesicles to pass (67). When tau gets hyper-phosphorylated, there is a higher than normal level of cytosolic tau which leads to increased possibility of forming aggregation and the formation of fibrils (67). Initially small deposits form called “pretangles” (these do not contain β sheets),which then form paired helical

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filaments (PHFs) containing eight protofilaments, which self-assemble to form NFTs (67). NFTs are aggregates of either straight filament (SF)or PHFs of hyper-phosphorylated tau (8,9). It is this accumulation of abnormal tau that causes all tauopathies, a notion that was not proven until 1998 when tau mutations were linked to these diseases, which will be discussed shortly (68).

There are three main kinases that are involved in phosphorylating tau in vitro: glycogen synthase kinase 3 (GSK3), cyclin-dependent kinase 5 (CDK5), and microtubule-affinity-regulating kinase (MARK), but it is still not known exactly how it occurs in vivo (8,9). GSK3 can be inhibited by lithium and studies have shown it can reduce tau phosphorylation and tauopathies both in vitro (Hong) and in vivo (Noble) (69, 70). There is a second effect of lithium that is also exciting. In addition to lithium’s effect on phosphorylation of tau, it is also interferes with ϒ-secretase processing of APP, and therefore reduces the amount of AΒ produced (71). Shipton’s 2011 study also examined the effect of inhibiting GSK-3 with a specific GSK-3 inhibitor, AR-A014418, and also found a protective affect (62).

In 1998, the first mutations in MAP tau were shown to cause Frontotemporal Dementia (FTD), a degenerative brain disease with features very similar to AD, and since then more than 40 such mutations have been found (18). These result in phenotypes that lack the AΒ plaques, but have the same tangles of tau protein seen in AD, and are therefore called “pure tauopathies” (18). Importantly there is one such mutation, MAPT R406W, that is indistinguishable from AD (18). This points to the central role of tau and its ability to cause neurodegeneration regardless of AΒ involvement.

Another important finding which makes one question the AB Hypothesis, made by “The 90+ Study”, is that 49% of the non-demented patients (all 90 years old or older) have the pathology to be diagnosed with AD and 22% of demented patients did not have the expected pathology in the brain, including tangles and plaques (72). This population-based study looked at over 1,000 patients with and without dementia and autopsied 150 brains. These biomarkers might be a common occurrence in the aging process and might not be the cause of the symptoms at all. However, Selkoe points out quite logically, there are many people who have artherosclerotic plaques in their arteries may not be diagnosed with heart disease and may not have any symptoms of it (6). This does not mean that the plaques have nothing to do with heart disease. In essence, perhaps these patients with AD pathology and no symptoms of dementia, just have not progressed enough to show symptoms yet. Admittedly AD takes decades to progress, so to say plaques and NFTs may be unrelated to AD because they are present in patients without dementia is a bit of a jump.

Tau Clinical Trials

It is important to note that mouse models have been modified to better mimic AD. Originally transgenic mice had the mutated form of APP which gave rise to the AB plaques. However, these mice did not form NFTs. A better model was made that has not only the mutated APP but also a mutated tau gene, and these mice have both AB plaques and NFT’s in their brains (33). These mice are commonly referred to as multigenic mice.

In 2006 Rosenmann et al used human unphosphorylated tau protein to immunize mice (73). Surprisingly, this caused the mice to have histopathologic features of AD, including NFTs, axonal damage

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and gliosis, which was not expected. Mice also had symptoms of neurologic deficits including a limp tail and partial paralysis. Between this trial and the active immunization with AB resulting in encephalitis, active immunization is viewed as extremely dangerous with these types of proteins. However, further studies have been done using truncated forms of tau reported exciting results. In one such study from 2011, three different age groups of mice were inoculated with a 12 amino acid sequence of the human tau protein (aa 395-406)(74). NFT pathology was significantly reduced and phosphorylation of tau was also reduced. Importantly, in the oldest group of mice inoculated, they even found a lower level of astrocyte activation. As we have seen, there is no way to know if this will translate to human trials, but it is hopeful.

Passive immunization using tau has also had promising results. In 2011, Chia et al administered two different antibodies against pathologic forms of tau into two different AD mouse models (75). They showed that this treatment reduced tau pathology in both models, even the mouse model that has more aggressive pathology. Even better, the immunization also “significantly delayed” motor function decline and weight loss. Phase 1 clinical trials are under way for active tau immunizations (76). Tau does not bind to blood vessels, as dos AB, which makes tau a safer choice (Giaconbini). Time will tell if the immunization with tau will have better results than the AB-targeted therapy, but it is hopeful.

Because of tau’s effect on microtubules (MTs) and axon transport, studies have looked at ways to stabilize MTs. Paxceed is one such MT-stabilizing drug, which is thought to take the place of tau sequestered in NFTs. Paxceed, or paclitaxel, is already used as a chemotherapy treatment and is being tested for treatment of rheumatoid arthritis (77). Zhang et al report that injecting this drug into tau transgenic mice(models of human tauopathies) for 12 weeks restored axon transport in spinal axons. MTs and stable tubulin were also increased (78). Motor impairments were greatly improved in mice treated with the drug, versus the control group. This study demonstrates that loss of tau function can be replaced by other MT-stabilizing agents and axonal transport can be restored. Paclitaxel is a natural compound from the Pacific yew tree and acts by promoting the interaction between alpha and beta tubulin heterodimers to form MTs (79). The problem is Paxceed cannot cross the blood-brain barrier (BBB), so a MT-stabilizing drug that can is greatly needed (79). One novel approach has proved successful: ANG1005. ANG1005 is a 19 amino acid peptide names angiopep-2, with three paclitaxel molecules attached to various resides. Amazingly, in 2008 it was shown that this compound can cross the BBB via a lipoprotein receptor (79). This drug is in phase II clinical trials to treat brain tumors, but has not been tested yet on AD (80).

Davunetide, an 8 amino acid peptide, is another MT-stabilizing drug and may even repair MTs (81). Jouroukhin et al used a mouse model for amyotrophic lateral sclerosis (ALS), a neurodegenerative disease accompanied by slower than normal axonal transport, and not surprisingly, hyperphosphorylation of tau. Given chronically, davunetide reduced hyperphosphorylation of tau and normalized axonal transport speed even when given acutely. To further show the protective effect of this drug they gave WT mice colchicine to disrupt axonal transport, then treated these mice with davunetide and it again normalized axon transport. Sadly, in stage III clinical trials, the drug failed to treat Progressive Supranuclear Palsy, a pure tauopathy (82). There was no difference in outcomes in

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placebo versus treated patients. This drug has not been tested on AD, but it is worth investigating with such promising preclinical results.

The hyperphosphorylation of tau is an important and consistent step in both AD and many other tauopathies. Blocking the kinases that carry out the phosphorylation seems like an obvious plan, but these kinases act on many other substrates. Interestingly, one study shows that hypothermia associated with anesthesia induces hyperphosphorylation of tau in vivo, and acts as an easy way to screen for kinase inhibitor efficacy (83). They showed a decrease in tau phosphorylation in glycogen synthase kinase-3 inhibitors LiCl and AR-A014418, and roscovitine, a cyclin-dependent kinase 5 inhibitor. Zhang et al find that small molecules in the diaminothiazole class are effective inhibitors of kinases involved in phosphorylating tau, CDK5 and GSK3β (84). They used two mouse models, 3X-Tg AD which are mice with AB deposits and NFTs, and CK-p25, mice that have upregulated CDK5 and NFTs. They found that treatment resulted in inhibition of tau phosphorylation in both models and in CK-p25 mice, they even found recovered memory loss in fear conditioning assays. This treatment could potentially slow the progression of AD and other tauopathies. One particular agent, LDN-193594, a lead compound, inhibits both kinases equally well, so it is especially effective at treating hyperphosphorylated tau, as these are the main two kinases involved.

There are drugs that prevent the aggregation of tau, such as methylene blue, a dye from the phenothiazine family. Methylene blue was the very first synthetic drug that has been used for various ailments for nearly 120 years (85). In 1996, Wischick et al discovered that methylene blue inhibited the tau-tau interaction (86). In 2008, Wischick reported at the International Conference on Alzheimer’s Disease in Chicago that methylene blue, or RemberTM , had therapeutic effects on AD (85). In a randomized phase II study, 321 patients were given Rember for 50 weeks and showed significant reduction in cognitive decline (79). However, these results were never published, which is puzzling. In Ballatore’s review of this work, he points out that methylene blue has many targets, so it is not clear exactly what pathway was affected, but Wischick’s earlier study showing its inhibitory effect on tau is intriguing (79).

Conclusion to tau/Aβ section:

Hardy makes an unsettling hypothesis by wondering if APP/Aβ are some sort of damage response that is upregulated due to some signal of neuronal damage. This would explain why getting rid of AB does not solve the problem, and why just having Aβ deposits may not correlate with symptoms because they have not started yet. This would mean a lot of research has been done on the wrong target. It is still unclear what the pathway connecting tau and Aβ (37).

Neuroinflammation:

It is not a new notion that neuroinflammation plays a large role in AD. But recent studies are showing that the brain’s innate immune system may contribute to, exacerbate, or even cause the disease (87). As the human brain ages, there is an upregulation of genes involved in the innate immune system (88). Microglia are the macrophages at work in the CNS, and it does not take much to activate them in the brain (88). They are hypersensitive to the environment and are triggered, or “primed”, by any change in

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homeostasis (88). Primed microglia change shape, upregulate antigens on their surface, and multiply in number, but most importantly they secrete cytokines, either pro-inflammatory or anti-inflammatory (88). Microglia are activated by neuronal damage and degeneration, but also by misfolded proteins, such as the amyloids associated with AD (88).

Remember that AB deposits are often surrounded by microglia cells which have the purpose of clearing them by phagocytosis. Pro-inflammatory cytokines, such as IL-1β and TNF-α, are therefore higher in brains of AD patients due to the large number of activated immune cells (89). The question remains, which came first the chicken or the egg; did the plaques cause the inflammation or did the inflammation cause the plaques?

Krstic et al showed that inflammation could cause amyloid aggregation (87). They subjected mice to a viral mimic (poly IC) in utero, which stimulates the innate immune response via the pro-inflammatory response. The mice treated with polyIC developed amyloid deposits and AD-like pathology later in life (87). The WT mice have chronically high levels of inflammatory cytokines, higher levels of hippocampal APP and its cleaved fragments, as well as altered tau phosphorylation and impaired memory (87). When these mice were subjected to another infection in adulthood, all these symptoms were exacerbated. These results imply that systemic infections, and therefore inflammatory responses, both prenatally and in adulthood, could be a risk factor, if not a cause of the disease.

Krabbe et al investigated the microglia that surround the amyloid plaques and wondered if they really were the cause of the inflammation. They used two different models of AD transgenic mice and WT mice of similar age (90). They found that mobility and phagocytic activity of microglia in the AD mice were “strongly” impaired compared to WT mice. This impairment was worse near the plaques and could be rescued when AB load was reduced by administering an AB vaccine. When looking at Krabbe and Krstic’s results, it seems much more likely that inflammation is causing the disease, not the other way around.

Another interesting study that points to inflammation playing a large role in AD, is Jonsson’s analysis of Icelander genomes (91). They analyzed 2,261 genomes and found a rare missense mutation in TREM2 that correlated with a three-fold increased risk factor of AD. TREM2 is expressed on microglial cells and is critical in suppressing the pro-inflammatory cytokine production (88). The mutation, therefore, may be the link to an increased inflammatory response which could lead to a higher risk of AD.

Zhang et al found results implicating microglial cells by looking at gene regulatory networks in 1,647 samples of Late-onset AD (LOAD) patients’ brain tissue and comparing them to non-demented patients (92). They found TYROBP (aka Dap 12), a key regulator in microglia pathogen phagocytosis, is upregulated and activated in LOAD patients (92). Interestingly, TREM2 is known to associate with and signal through TYROBP, so this finding corroborates Jonsson’s findings, which were made at around the same time. Not surprisingly, TYROBP is directly related to AB turnover and neuronal damage (92). Mutations of TYROBP and TREM2 result in Nasu-Hakola Disease, a rare disease that involves chronic inflammatory neurodegeneration(92).

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If it is the inflammation that is causing AD, it would seem easy to cure with anti-inflammatories. As far back as 1996 it was known that chronic use of anti-inflammatory drugs may reduce the risk of AD. McGreer et al combined the results from 17 epidemiological studies from 9 countries to analyze the effects of long-term anti-inflammatory drugs and the incidence of AD (93). They found that arthritis, steroids, and NSAIDS all had a negative correlation to AD. In 1997, 1,686 patients in the Baltimore Longitudinal Study of Aging compared the risk of AD between people taking NSAIDS versus acetaminophen, or other pain medicine with no anti-inflammatory action (94). They found that the risk of AD decreased in relation to the longer duration of NSAID use and the use of acetaminophen did not correlate with a decreased risk of AD.

In 2010, Chou et al wanted to find the connection between Rheumatoid Arthritis (RA), which also involves deposition of amyloid proteins, and AD (95). They studied 42,193 patients with RA, noted treatment with sulfasalazine, prednisone, three anti-tumor necrosis factor (TNF) agents (infliximab, etanercept, adalimumab) and rituximab, and watched for the incidence of AD. They found that treatment of RA with anti-TNF agents correlated to a lowered risk of AD compared to controls and the other treatments.

TNF-α, which is systemically produced as a response to infection or inflammation, is of great importance because it plays an important role of communication between the immune system and the brain (96). TNF-α is the conductor that initiates and regulates the cytokine symphony in the inflammatory response (97). In normal brains, TNF-α mRNA concentration is low or absent; however, TNF-α homozygous KO mice cannot survive a bacterial infection (97). TNF- α maps to chromosome 6p21.3, is 2,676 base pairs long, and contains 3 introns and 4 exons (97). TNF- α is originally secreted as a transmembrane protein, but is later cleaved by TNF- α converting enzyme (TACE) to make a soluble form (97). Many SNP’s (single nucleotide polymorphisms) have been found in the 5’ UTR and promoter regions, and some have been shown to affect transcriptional levels (97).

In 2000, Collins and Perry et al showed a correlation between these SNPs and AD (98). They performed a genome-wide scan of 266 late onset AD families and found a region at 6p21.3 that included the TNF-α gene. They found three polymorphisms in particular that made a haplotype (collection of alleles in a cluster of genes that tend to be inherited together) of -308 TNFpromoter, -238 TNF promoter, and microsatellite TNFa, all of which are associated with higher TNF production. They found these alleles present in a 2-1-2 allele haplotype, respectively. These are significantly associated with AD, which further implicates TNF as a main player in the onset and progression of AD.

TNF-α acts via two main receptors, p55 (TNFR1) and p75 (TNFR2). Interestingly, both receptors can be cleaved from the cell to make a soluble form of the receptor that can compete with the membrane-bound receptors and act to sequester or neutralize TNF-α (97). TNFR2 is actually cleaved by the same TACE that makes the mature form of TNF- α. Perry and Collins et al found another correlation between a polymorphism in exon 6 of TNFR2 in late-onset AD (97).

Holmes et al first looked at 300 mild to severe AD patients, assessing cognition, determining their recent systemic inflammatory events and measuring the levels of TNF- α in their blood over a 6 month period

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(96). Amazingly, they found about half of the patients had acute inflammatory events that were also associated with an increase in serum levels of TNF- α and 2-fold increase in the rate of cognitive decline (96). Patients who had a high baseline level of TNF- α had a 4-fold increase in the rate of cognitive decline (96). Even more convincing, patients with low levels of TNF- α showed no cognitive decline over the 6 month study (96). TNF- α may be a promising therapeutic target.

Holmes et al just presented exciting results at the Alzheimer’s Association International Conference in Copenhagen, Denmark, in July of 2014 (99). They wondered if TNF-α receptor blocker, Etanercept, might block the action of TNF-α and block the progression of AD by eliminating the inflammation. They started with 67 patients with mild to moderate AD, but after eliminations due to TB exposure, 42 patients were included and randomized. 20 patients received subcutaneous injections with 50 mg of Etanercept once a week, and 21 patients received a placebo injection of water. Treatment was administered for six months followed by a one month wash out. They found no adverse reaction or safety issues in the Etanercept group. The placebo group showed a greater rate of decline in measurements of cognition, behavior, and activities of daily living. The Etanercept group remained unchanged compared to baseline measurements. While this is small study, it is hopeful. A larger, longer follow-up study is needed to confirm this finding. This may be the key that unlocks the cure. It would make sense that all of the therapies targeting plaques have been unsuccessful if the cause was the neuroinflammation all along and the plaques are just a downstream effect.

More studies testing the therapeutic effects of blocking neuroinflammation are needed. Finally, researchers may be onto something, after decades of chasing their tails treating the wrong targets. With our nation and world’s elderly population rapidly growing, this disease needs a cure.

In the news just this week, two scientists in Boston have found a way to create human Alzeimer’s disease in a petri dish (100). They grew human neuron cells with the APP and PSN-1 mutations in a gel, where they were able to form synaptic networks, just like in living brain tissue. The neurons developed Aβ plaques and NFT’s, something that has been difficult to do in mice. There is a lot of excitement about this new development, where drugs and treatments can be tested as therapies. However, if neuroinflammation or the immune system are key to AD, this in vitro environment will fall short. It will be interesting to see what lies ahead for AD research.

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