The Current State of Alzheimer’s Disease Treatment

Additional Alzheimer’s disease Blog Post here



Regrettably there remains no effective treatment for Alzheimer’s disease (AD). Current therapies target cholinergic (acetylcholine esterase inhibitors) and glutaminergic (NMDA receptor antagonists) neuronal activity in an attempt to improve symptoms largely associated with cognitive decline.1-3 Unfortunately these treatments are limited in their effectiveness because they do not address the cause of the disease, but rather the symptoms. There are other non-pharmacological treatments that address the detriments of cognitive decline like social measures through various support groups and more personal individualized care. However, while these interventions do what they can to help manage AD, without the development of a viable disease-modifying therapy the natural expansion of AD cases, due to an increasing elderly population, will significantly increase global healthcare costs especially in high healthcare cost countries like the United States. In addition to increasing healthcare costs across the board these cases will also significantly reduce the quality of life for millions.

Based on the success of the amyloid beta (Abeta) cascade theory regarding the development of AD one of the principal recent strategies for creating a future treatment has been utilizing an Abeta antibody that will either prevent plaque formation or break plaques apart hopefully producing positive cognitive remediation for those suffering from AD. Unfortunately while this theory appears reasonable, positive empirical evidence supporting this strategy has proven lacking. In fact numerous Phase II and Phase III studies have failed to demonstrate significant positive cognitive outcomes for these types of drugs versus placebo controls.1,4,5 The two most notable recent failures have been Bapineuzumab and Solanezumab; both were able to reduce fibrillar amyloid concentrations, but demonstrated no significant benefit to cognitive processes.5,6

These results should not be surprising because these drugs represent an older way of thinking about Alzheimer’s disease where plaques are the principal deleterious agent and their elimination is essential for recovery. Unfortunately there is ample evidence that soluble Abeta oligomers, and not their fibrillary associates, are the actually deleterious agents responsible for a significantly level of the symptomology of AD. If this different pathway is correct then the elimination of Abeta plaques should serve little benefit, as seen in the multiple Phase II and III trial failures, and could even been considered negative depending on how those plaques are broken apart (possibly increasing the available concentration of Abeta oligomers).

Now it is believed that Solanezumab can bind to soluble Abeta, which could explain why it performed better than Bapineuzumab, which binds to aggregate/fibrillar Abeta.6 However, the binding activity of Solanezumab was still insufficient to produce a meaningful benefit. Solanezumab supporters believe that if applied early, before symptoms, it may be able to produce a meaningful benefit; however, this belief may be misplaced. If this treatment has to begin that early to produce valid benefit then it will not help many individuals overall, assuming that it ever works for at the moment its “potential” is still theoretical.

There is an additional concern that simply changing the strategy from a fibrillary antibody to a soluble one may cause as many problems as it solves. Despite its fame as the chief element responsible for initiating AD, Abeta has innate roles in the brain that could cause problems if natural concentrations are significantly reduced as would occur in a preventative vaccination/treatment strategy. The two major natural roles for Abeta in the brain appear to be that of an indirect neuronal inhibitory agent and an anti-microbial agent.7-10 This anti-microbial activity may be why producing success from a direct antibody therapy is difficult for numerous previous attempts at active immunization against Abeta has resulted in numerous cases of aseptic meningoencephalitis.11

Regarding the issue of Abeta as an anti-microbial agent, there is a wealth of circumstantial evidence that seems to support such a conclusion and small amounts of direct evidence that demonstrate anti-microbial behavior against certain specific targets.7-9 For example one typical piece of evidence is that AD temporal lobe homogenates contain about 25% more activity against C. albicans on average over non-AD samples.7 However, while Abeta is thought to react against C. albicans it is also suggested that microglia are more active in AD patients than in non-AD patients, thus this increased activity maybe derived from the microglia instead of the Abeta. On a side note this anti-microbial behavior has lead some to conclude that AD can be induced by pathogenic response. Overall there could be two different methodologies behind how bacteria and other pathogenic agents could induce AD.

The first method involves observations that several bacteria contain amyloidogenic proteins. For example the periplasmic outer membrane lipoprotein of E. coli demonstrates a similar amino acid sequence to Abeta peptides and have similar visual structures similar to amyloid.12,13 In addition there appears to be pathological similarity between herpes simplex encephalitis (HSE) and AD.13-15 With this information one could come to the conclusion that either certain bacteria have the ability to mimic Abeta and facilitate extracellular receptor interactions similar to that of extracellular Abeta or the bacteria produce their own Abeta. Either scenario would enhance the probability of an individual developing AD in the presence of a specific infection.

The second method involves the overexpressed synthesis and release of Abeta produced in response to an infection. If Abeta does in fact have an anti-microbial effect then it would make logical sense from a biological standpoint that the body would release Abeta in response to an infectious agent, especially in the brain where immune response is limited. Cumulative infections or a single long duration infection could result in an increased probability to develop AD due to the excess production of Abeta to fight off the infection.

Not surprisingly there are some significant concerns about claims that infections play a significant role in the development and progression of AD. Simply from a general understanding it appears that too often researchers want to tie a particular pathogen to the cause of a given disease, “this bacteria/virus causes this particular type of cancer” is one of the most popular. The problem is that most of the time there is no direct evidence to support such a conclusion beyond the fact that the pathogen is present in individuals who have the condition. Another piece of reasoning that individuals who are pro-infection like to report is along the lines of “well, virus/bacteria x has a similar symptomology and/or pathology in the brain” as was previously mentioned above. The problem with that claim is that there are hundreds of diseases that are very similar to each other in many symptomatic respects, yet have small very meaningful differences in how they originate.

Whether or not infection plays a meaningful role in AD is still under debate, but is more unlikely than likely because early developing cases rarely demonstrate any large bacteria/viral concentrations and a significant number of late-developing cases also have a lack of any abnormal bacteria/viral concentrations relative to the age of the patient. The lack of large bacterial concentrations in the early stages of AD progression, and especially for late-developing cases, leads to one of two conclusions regarding any increase in bacteria concentration in AD patients. First, this increase occurs as a later development due to what could almost be viewed as a weakened immune system because microglia are busy trying to clear the excess Abeta. Second, the increase has little to do with AD and can be considered a coincidental occurrence. Neither of these possibilities involves bacteria as a causal agent.

Overall there is sufficient direct and indirect evidence to support the idea that Abeta has anti-microbial properties. An example of indirect evidence is the increased susceptibility to infection possessed by beta-secretase or gamma-secretase knockout mice.16 Also Abeta, in vitro, is clinically active against at least eight common microorganisms, similar activity to pleiotropic LL-37, a common ‘‘antimicrobial peptides.7

However, despite this biological role, there is currently no published evidence that demonstrates an increase in Abeta synthesis and secretion that later leads to the development of AD. Therefore, it is difficult to conclude that infections genuinely facilitate AD through additional Abeta synthesis. This highlights an interesting aspect of Abeta in that eliminating it or dramatically reducing it increases infection susceptibility, yet infections seem to be unable to produce sufficient concentration changes in AD development.

Regarding the role of Abeta as an “inhibitory” agent in neuronal processes one of the most telling pieces of evidence is the fact that benzodiazepines, an inhibitory agent, reduce secretion of Abeta peptides from hippocampal slice neurons.10,17 Also overexpression of amyloid peptide precursor (APP) significantly reduces excitatory activity.10 Other evidence suggests an “inhibitory” similar role through excitatory depression, but under normal physiological conditions this excitatory depression possesses a level of minute control resulting in enough of an impact to quell hyper-excitation, but not enough to cause short-term or long-term damage.

There is some research that suggests a connection between epilepsy and AD. A number of studies claim that increasing Abeta42 concentration in mice increases the probability for progressive epilepsy.18,19 One interesting component is whether the role of Abeta with regards to its influence on neuronal excitability changes as it changes states from monomeric to oligomeric to proto-fibrillar/fibrillar. While Abeta as a monomer/oligomer construct appears inhibitory, the ability of fibrillar Abeta to interfere with membrane fluidity allows it to influence neurons in an excitatory manner.

Another piece of information that supports a role for Abeta in neuronal firing is that calcium imaging shows hyperactive neurons clustering around amyloid plaques in the cortex of APP/PS1 mice.20 There are two common explanations for this result: 1. the neurons begin to hyperexcite, which prompts the release of Abeta peptides (40, 42, 43, etc.) in an attempt to inhibit further excitation. However, during the process of inhibition these peptides begin to coalesce into proto-fibrillars and plaques which then somehow rejuvenate hyper-excitation (most likely by changing membrane fluidity and resting potential) creating a small, but progressively positive feedback loop; 2. the neurons begin to release Abeta peptides in larger than normal concentrations, perhaps due to infection or genetic mutation, these Abeta peptides then form proto-fibrillars and plaques to facilitate a new found hyper-excitation.

One method to determine which of the above explanations is more logical is to distinguish between the influence of the proto-fibrillar Abeta and monomer/oligomer Abeta. Basically ask the question: do proto-fibrillars really induce additional excitatory behavior or are they simply blocking the ability of the smaller Abeta species (monomers, dimers and oligomers) to reduce excitatory activity?

For example it makes sense that an Abeta oligomer binding to an extracellular receptor induces a neuronal depressed response with the receptor eventually discarding the bound Abeta, but that unbound Abeta could still be available to bind another receptor if necessary until it is cleared. If proto-fibrillars act as a form of blockade it would be expected to limit the binding ability of oligomer Abeta reducing the ability to lessen excitatory behavior. However, the reduction of this damping ability does not explain why non-epileptic individuals would develop epilepsy, there must be an additional excitatory agent. Thus it stands to reason that the first answer is more favorable in that proto-fibrillar Abeta induces excitatory activity.

However, there are questions regarding the timing of epilepsy development because hippocampal neurons become hyperactive early in transgenic mice whereas hyperactivity in the cortex is temporally linked to plaque formation.20 One possibility to explain this apparent contradiction is that soluble Abeta has a higher probability of inhibiting inhibitory elements in the hippocampal space versus the cortex due to neuronal architecture. Regardless of this hyperexcitation issue, typically epilepsy in AD patients increases in probability with disease progression, which corresponds to a greater development and concentration of proto-fibrillar Abeta, especially relative to oligomeric Abeta. Therefore, it stands to reason that under normal conditions and early to moderate AD Abeta concentrations are chiefly inhibitory agents (regardless of what type of neurons they are inhibiting), but due to the differing activity Abeta slowly becomes more excitatory as the AD advances.

This method of inhibitory action extends not only to competitive binding between glutamate and Abeta on NMDA and AMPA receptors, but also induce endocytosis of AMPA receptors reducing excitatory binding probability.10,21-23 The endocytosis of AMPA receptors may explain why neurons that are hyperexcited with APP over-expression mutations are not immediately reversible, but over time become reversible with cessation of immediate neuronal firing.10 The lack of Abeta also induces GABAergic neuron sprouting, which may be driven to compensate for the hyperexcitation.24

In the end any future attempts to develop an antibody-based therapy for Abeta will have to determine how the presence of the antibody will influence the two natural Abeta processes. While there has been some initial and isolated success from studies that have demonstrated some protective benefits for auto-antibodies of a AB oligomer subset,25-26 it is difficult to “hand wave” away the negative results associated with previous antibody tests that resulted in cases of aseptic meningoencephalitis.

Recall that the production of Abeta begins with APP, which is a transmembrane protein that has three principal isoforms, 695, 751 and 770, each containing the 4 kDa Abeta peptide and is synthesized in the rough endoplasmic reticulum and glycosylated in the Golgi apparatus.10 Three types of secretase enzymes interact with APP. Endopeptidase alpha-secretase cleaves within the Abeta region, eliminating any opportunity to form an Abeta peptide. If APP is not cleaved by alpha-secretase then APP can be incorporated into endocytic compartments for cleavage by beta-secretase and/or gamma-secretase.

Beta-secretase cleaves APP at the N terminus of the Abeta peptide sequence and gamma-secretase cleaves at the C terminus. When beta-secretase cleaves APP it generates a secreted ectodomain beta-APP and a 10-kD COOH terminal fragment (beta-CTF).27,28 This beta-CTF fragment is the substrate for gamma-secretase, which cleaves the transmembrane domain of APP producing an Abeta fragment.28 Gamma-secretase can cleave at multiples sites creating multiple length Abeta peptides (typically 40, 42 and 43).27 However, if gamma-secretase cleaves APP before beta-secretase, the end product cannot be converted to Abeta. Therefore increasing alpha-secretase concentration/activity will decrease Abeta concentration, increasing beta-secretase concentration/activity will increase Abeta concentration, and increasing gamma-secretase may or may not (depending on other factors and just simple luck) increase in Abeta concentration.

While the functionality of the various secretases is straightforward, the development of a viable inhibitor is challenging for it faces three separate problems. First, secretases, especially beta, have multiple substrates and large substrate binding domains, so competitive inhibitors are typically short-lived and non-competitive inhibitors have difficulty achieving full inhibition. Second, inhibitor candidates must be able to cross the blood-brain barrier. Third, secretases are also involved in other important biological processes, thus inhibition must be conducted carefully otherwise numerous unfavorable side effects will accrue making long-term treatment difficult. For example beta-secretase is thought to be necessary for proper function of muscle spindles due to its interaction with Neuregulin-1, thus long-term beta-secretase inhibition may already be a non-starter.29

Another problem with secretase interaction is the relationship between gamma-secretase and intramembrane cleavage of Notch receptors, most notably Notch1.30 The Notch pathway in general is important for neuronal function, cell communication and cell homeostasis in developed brains. Therefore, due to this relationship while gamma-secretase inhibitors typically reduce Abeta concentrations in plasma and cerebral spinal fluid (CSF) they also produce side effects like haematological and gastrointestinal toxicity, skin rashes and changes in skin color.30 The failure of a Phase III trial of Semagacestat (a highly touted gamma-secretase inhibitor), after a successful Phase II trial, was due to the above mentioned side effects including the development of non-melanoma skin cancer as well as a dose-related worsening of cognitive measures.31

The above problems make it difficult to believe that a secretase inhibitor will be a long-term answer for AD treatment. There was a growing trend towards redirecting attention away from an inhibitor agent and towards a modulator agent. A modulator can shift the APP cleavage site maintaining the relationship with the Notch receptor, thus creating a possibility to reduce Abeta concentration while reducing side effects. Unfortunately one of the first modulators, Tarenflurbil, failed to produce any positive results regarding AD treatment;32 thus modulators may not be a strong choice for future AD therapies.

Also research has been invested in a naturally occurring monosaccharide, NIC5-15, that can function as a gamma-secretase inhibitor that somehow avoids interfering with the Notch relationship as well as increasing tissue sensitivity to insulin reducing insulin concentration.33,34 However, there is the lingering concern about how this inhibitor will influence the natural roles of Abeta in the body and its lack of any meaningful studies beyond a very simple Phase II trial.

One of the more interesting and potentially important elements in the progression of AD is the location of beta-secretase and gamma-secretase relative to each other and APP. A point of interest is what role lipid rafts play in dictating how APP is processed. Lipid rafts are lateral assemblies of cholesterol and sphingolipids which form ordered platforms that move through the matrix of a cellular membrane that can compartmentalize various membrane processes. Due to this compartmentalization they can produce microdomains that provide an efficient environment for molecule assembly and membrane protein trafficking as well as influencing membrane fluidity.

The involvement of lipid rafts in Abeta processing is supported by multiple stages of evidence. First there is reason to suspect that beta-secretase needs to be associated with lipid rafts to even be active let alone interact with its APP substrate.28 Whether or not gamma-secretase is also inactive when outside of a lipid raft is unclear, but it appears that significant activity takes place on lipid rafts.35-37 Bolstering support for the involvement of lipid rafts is the identification of various AD related proteins in lipid rafts from both human and mice brain. Currently Abeta40, Abeta42, presenilin 1, beta-secretase, APP, beta-CTF and alpha-CTF have all been isolated from lipid rafts.28,38,39

In addition beta and gamma-secretase induced cleavage seems to depend on endocytosis of APP. The requirement of endocytosis suggests that beta-secretase interaction does not occur at the cell surface. This requirement may be because surface APP and beta-secretase are either floating freely in the cellular matrix or on separate lipid rafts.28 Therefore, it seems to make more sense that the interaction between beta-secretase and APP occurs after endocytosis during the amalgamation of various lipid rafts within endosomes. Interestingly the appearance of larger endosomes is a typical precursor to AD progression, which could support this idea, i.e. the endosomes grow larger to accommodate the coalescence of the lipid rafts due to greater cholesterol and/or Abeta levels.

There is also evidence that significantly increased concentrations of Abeta begin to appear in lipid rafts before even symptoms begin, but these studies did not compare concentrations of Abeta in the lipid rafts to concentrations of Abeta in the intracellular or extracellular matrix, thus this result cannot be used as conclusive evidence that Abeta synthesis originates or is dependent on lipid rafts.40 Another important distinction is that some estimate slightly over 20% of brain Abeta on lipid rafts,40 with lipid rafts only constituting 0.4 to 0.8% of a given plasma membrane.41 While the estimate relative to lipid raft compartment space is for only one particular cell type, there is little reason to believe that the amount of lipid rafts varies significantly between different cell types.

For the moment assume the following regarding Abeta synthesis and lipid rafts:

- The overall size of a lipid raft is largely dictated by the total amount of elements available to form it;
– beta-secretase is only active on a lipid raft and produces beta-CTF;
- gamma-secretase is in close proximity to lipid rafts; whether or not it only active on a lipid raft is unknown;
- alpha-secretase is not localized on lipid rafts;

Based on the above information it makes sense that removing cholesterol from plasma membranes would significantly increase membrane fluidity (by shrinking the total number and size of lipid rafts). Increasing membrane fluidity would increase lateral movement of APP and alpha-secretase in the plasma membrane possibly increasing alpha-secretase activity. Also reducing the availability of lipid rafts should also limit beta-secretase activity making alpha-secretase interaction more likely, which has been supported experimentally.42

There is an interesting side point here in that recall fibrillar Abeta in the extracellular matrix is thought to change membrane fluidity with a higher probability for an increase in fluidity over a decrease. If this change in fluidity actually occurs then it could act as a negative feedback mechanism. After enough Abeta is secreted into the extracellular matrix that leads to the formation of fibrillar elements, like plaques, an increase in membrane fluidity should occur that would negatively influence lipid rafts reducing the probability for further Abeta synthesis until the fibrils are cleared from the extracellular matrix. If this is the case then plaque busting drugs could worsen AD in multiple ways, not only by breaking down fibrils and plaques making more toxic oligomers, but also eliminating a negative feedback mechanism which could limit Abeta synthesis.

There appears to be two different avenues when APP can associate with a lipid raft: 1) during transit between emergence from the Golgi body and becoming a transmembrane protein, newly synthesized APP could interact with lipid rafts; 2) during the endocytosis process where the APP is re-internalized through clathrin-coated pits.43,44 Without direct evidence it appears more reasonable to assume endocytotic recycling as the dominant lipid raft interaction process simply because it is more frequent of the two.

Further support for the importance of lipid rafts involves the behavior of its building blocks. Cleary numerous studies have demonstrated that increasing cholesterol leads to an increase in AD development probability in addition to lipid rafts, but cholesterol is not the only element that makes up lipid rafts. What happens if sphingomyelin levels are altered? The initial assumption would be that increasing sphingomyelin levels would lead to a corresponding increase in lipid rafts and Abeta synthesis. However, this does not appear to be the case in at least one study. When down-regulating sphingomyelinase (SMase) and up-regulating sphingomyelin-synthase activity, both actions increase available sphingomyelin, intracellular and extracellular Abeta levels decrease.45 The same result was acquired when foregoing enzyme manipulation and directly increasing sphingomyelin levels.

There are two immediate possibilities that could explain this result. First, the ratio between the total levels of cholesterol to sphingomyelin may influence the structure of the lipid raft where higher sphingomyelin levels create a raft formation that reduces beta and/or gamma-secretase activity. Second, maybe cholesterol is not directly responsible for changing Abeta concentrations, but instead an associated molecule that frequently increases and decreases in consort with cholesterol levels is actually influencing Abeta concentrations. While this second possibility is possible it must also address how different variations of ApoE dramatically change the probability of developing AD, which makes it unlikely.

Increasing sphingomyelin also appears to increase concentrations of C99, the byproduct of beta-secretase processing.39,45 Therefore, it can be reasoned that these changes in both C99 and Abeta concentrations are the result of a reduction in gamma-secretase activity more than likely due to a reduced ability to interact with C99 due to lipid raft proximity issues rather than direct reduced gamma-secretase synthesis or increased inhibition.

Unfortunately there could be another positive feedback effect relative to Abeta42 as Abeta42 directly increases SMase activity and reduces sphingomyelin-synthase activity.45 Such a result is interesting because does that mean SMase is also located on lipid rafts? There is no reason to immediately assume that this inhibition/activation will lead to a significant increase in Abeta concentration because if the change between the destruction/creation dynamic is altered too much in favor of destruction it will lead to the breakdown of lipid rafts halting Abeta synthesis. There could be a problem in that any loss of sphingomyelin in the rafts may be accommodated by an increase in cholesterol deposition. If in fact the lipid raft ratio between cholesterol and sphingomeylin does matter with respects to gamma-secretase activity and overall Abeta production then such an outcome could indeed worsen the progression of AD.

Another important element involving lipid rafts is that Abeta appears to inhibit sphingosine kinase-1, an enzyme that is chiefly responsible for balancing ceramide and sphingosine 1-phosphate (S1P).46 Ceramide significantly influences many stress signals, which can result in ceasing cellular growth or even cell death where S1P neutralizes the effects of ceramide.46 Thus Abeta, separate from other AD-related mechanisms, can induce cellular death by increasing ceramide concentrations and decreasing S1P concentrations.

There may also be a relationship between SMase and ceramide, which could also act as a positive feedback mechanism relative to the toxicity of Abeta.46 Finally IGF-1 is able to stimulate sphingosine kinase-1 activity neutralizing ceramide, thus this method may be how IGF-1 provides its neuroprotective effect relative to AD. Overall a strategy that focuses on influencing lipid raft configuration or their associated elements may be an interesting means to help neutralize AD because it avoids direct inhibition of the secretases and could allow for more fine control of Abeta concentration.

As mentioned above although there are still numerous concerns with implementing an Abeta antibody treatment strategy, another potential antibody strategy that has gained some favor in recent years is treatment with intravenous immune (or immuno) globulin (IVIG). In 2002 it was determined that the IVIG element Octagam had the tendency to possess antibodies for Abeta, which fostered the idea that IVIG could be used to treat AD.47 Early evidence from a small Phase II IVIG efficacy study showed improved cognition in mild to moderate AD patients and reduced Abeta in CSF.48,49

Unfortunately this initial promise was marred by the failure of IVIG to demonstrate any improvement in cognitive scores in a larger Phase III study and an additional Phase II study.50,51 The failure to reproduce the positive results from the Phase II study in the Phase III study limits the hope that IVIG could be a useful treatment in the future. Some argue that one bright spot is that IVIG did improve cognitive ability in ApoE4 carriers, otherwise commonly regarded as those who are genetically inclined to develop AD versus more spontaneous development.52 Note that from a safety standpoint both studies did support a positive safety profile for IVIG in AD patients.

Unfortunately even if the Phase III results were positive, one of the major obstacles to producing an effective IVIG based treatment is that lack of uniformity in the concentrations for different samples. According to FDA regulations IVIG samples must be prepared from the plasma contents of at least 1,000 individuals with all IgG subgroups (1-4) present, purified by removing all other blood elements and must be utilized or properly stored within 21 days of its creation. This process produces a non-uniform IVIG product that may have differing concentrations and types of antibodies versus another sample produced in a different laboratory. One sample may contain antibodies to Abeta and tau whereas a second sample may only contain antibodies for tau.53

Another drawback is that it takes approximately 9 months to produce an IVIG sample, thus mass production on a typical pharmaceutical scale is not possible creating a dearth in supply potential. Some have suggested alleviating this problem through new manufacturing processes or use of recombinant strategies, but neither of these suggestions have been incorporated into a large-scale production line, thus limiting their predictive power.54 Also unless more people donate blood in general increasing production with a natural product base will be very difficult.

This supply crunch creates various problems both ethically and economically. Not surprisingly IVIG treatment is expensive, costing about $75 per patent gram or $7,500 - $15,000 for the average person; with a fixed price for reimbursement from Medicare it stands to reason that a number of low-income individuals could be priced out of long-term IVIG therapy55 (new infusions would be expected between every two to five weeks), which is standard protocol and would be applicable for AD. IVIG is also used to treat acute infections and some other conditions most notably immune deficiencies and autoimmune diseases. With the limited supply availability transferring IVIG samples to AD patients would hurt these other patients, that is of course if IVIG was an effective treatment for AD, which has not been sufficiently or appropriately demonstrated.

Finally IVIG treatment is not without its own side effects most notably increased probability for thromboemboli due to increased serum viscosity reducing blood flow, especially in individuals with existing vascular difficulties and/or abnormalities.56,57 Another concern is the increased probability for a decrease in white blood cell, red blood cell and platelet concentrations including increased platelet aggregation, which could also exacerbate thromboemboli potential.58-60 A decrease in hematocrit levels is thought to occur through high-molecular weight IgG complexes binding red blood cells increasing sequestration.61 Despite these drawbacks one positive produced from the IVIG clinical studies is the idea that a multi-antibody therapy should be superior to a monoclonal antibody therapy. However, one of the new problems with such a strategy is identifying the antibodies, outside of Abeta, that should be included in such a therapy.

If IVIG is not a direct treatment option, an indirect treatment option could be gleamed from how IVIG affects the inflammatory response where IVIG inhibits complement activation, modulates chemokine expression and regulatory T cell subsets, and negatively influences inflammatory cytokines.52,62,63 One of the more notable results is a decrease in the ratios of IL-5 and IL-12 relative to IL-10 (i.e. either IL-5/IL-12 concentrations decreased or IL-10 concentrations increased), which is thought to decrease the rate of atopy.64,65

Another important element influencing the inflammation response of IVIG is the role of IgG Fc fragments, which involve a glycan component with a terminal sialic acid.66,67 IgG fragments with sialic acid bind to human receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or its murine orthologue (SIGN-R1).68 These binding targets are thought to stimulate immunosuppressive action reducing inflammation, which could reduce AD severity. However, this anti-inflammatory behavior requires a high dose, if IVIG is the source provider, because only a very small percentage of IVIG contains elements with sialic acid. This high dose may be also be prohibitory for treatment due to side effects.69

Another theory behind the effectiveness of IVIG is how it interacts with a more exotic version of Abeta. For example some evidence has demonstrated a significant decrease in soluble Abeta56 oligomer concentration after IVIG treatment. Similar to Abeta42, Abeta56 is another abnormal Abeta isoform that is thought to increase the probability of AD development and influences cognitive impairment on a concentration dependent level.68 One reason for this influence is that Abeta56 increases the expression rate of tau and its effect is negatively influenced by drebrin and fyn kinase availability available in IVIG treatments.65

A more controversial issue with IVIG treatment is whether or not any positive influence is drawn from its facilitated decrease in CD4/CD8 ratio. Various research has produced results were AD patients have increased,71-72 no significant changes73 or decreased74 CD4/CD8 ratios. Overall it is difficult to conclude, either through IVIG treatment or in general, whether influencing CD4/CD8 ratios is an intelligent therapy strategy for treating AD. In addition to CD4/CD8 ratios, IVIG also appears to decrease the concentration of YKL-40, but outside of being a marker for advanced AD there is little belief that manipulating its concentrations could prove useful as a therapeutic.75

With the failures of Abeta antibiotic therapies and the difficulties associated with IVIG confirmation and production some researchers have turned their attentions to attacking tau as a treatment methodology. One of the major reasons tau looks promising is that its pathology appears to correlate better with dementia severity than Abeta. Based on some research tau supporters argue that tau is actually responsible for Abeta toxicity.4 However, there is a significant problem with this enthusiasm namely that while misfolded and hyperphosphorylated tau does lead to generic dementia, it fails to develop into AD without the influence of Abeta.76,77 Also the argument that Abeta toxicity is dependent on tau only appears applicable to fibrial Abeta not soluble Abeta, which is of little consequence because fibrial Abeta has low direct toxicity overall.78 These issues have created conflict between Abeta and tau proponents regarding which element is worth neutralizing.

Regardless of its lack of AD initiation tau could be an important theoretical therapeutic target. A quick reminder that tau is a microtubule-associated protein (MAP), which is important for the proper stability and functioning of microtubules. The general understanding behind tau toxicity follows a similar pattern to that of AD. Hyperphosphorylation of tau negatively influences its affinity for microtubules increasing microtubule structural degradation and increases the probability that monomer tau form oligomers, paired helical filaments (PHF) and neuron fibrial tangles (NFTs). The breakdown of microtubules reduces in axonal transport leading to synaptic starvation and retrograde degeneration.

However, originally most believed that the neurotoxicity of tau was born from NFTs whereas more and more recent evidence supports the tau oligomers being responsible for a majority of damage.79-81 Some may suggest that this more severe oligomer toxicity does not correlate with increased NFT load and distribution markers for AD progression. This result is not contradictory because more tau oligomers equals more damage, but also increases the probability for more NFTs.

One of the interesting elements of tau is the idea of a positive feedback mechanism that makes it self-propagating, similar to a prion.82,83 If such a methodology is correct, then AD treatment would require one of two strategies: 1) treat AD before this self-propagating positive feedback mechanism is activated by Abeta; 2) the tau mechanism must be neutralized to a point that disallows the occurrence of this self-propagating mechanism. Otherwise tau is not addressed, the treatment may neutralize AD, but the continued expression of tau could lead to another form of dementia.

There is two major schools of thought with regards to neutralizing the effects of tau: 1) influence tau phosphorylation; 2) influence tau aggregation. The first option typically involves using elements that will inhibit phosphorylation of tau whereas the second option typically involves using elements that will either prevent tau aggregation or enhance aggregate disassembly.

Not surprisingly the first option has been explored on a greater level than the second owing to the idea that hyperphosphorylation stems from an abnormal ratio of activation between glycogen-synthase-kinase-3 (GSK3), which is responsible for phosphorylation, and phosphatase PP2A, which is responsible for removing phosphates from tau.84 There was some early promise seen for influencing tau through the inhibition of GSK3 by way of either lithium or valproate, two treatments that are commonly used in psychiatric disorders with relatively stable safety histories and protocols. In addition both are thought to enhance neuroprotective effects by upregulating anti-apoptotic factor BCL2.85

Unfortunately despite these positive effects, small studies involving lithium treatment in patients with mild Alzheimer’s disease demonstrated no change in CSF biomarkers or any cognitive benefit.86 Granted some explanations for this result could be the study’s short time frame (6 weeks) and the mild condition of Alzheimer’s could limit the overall effectiveness of a tau-based therapy because the detrimental nature of hyperphosphorylation has yet to fully occur. Studies with valproate have generated similar results with no positive effects on cognitive or functional status.85,87

Another natural compound that has been targeted as a potential tau therapy is nicotinamide, the biologically active form of niacin (Vitamin B3) and precursor of coenzyme NAD+. Studies in mice have demonstrated that orally administered nicotinamide limits cognitive deficits and reduces concentrations of phosphorylated tau.30,88 There is also some evidence that nicotinamide upregulates acetyl-alpha-tubulin, protein p25 and MAP2c, which are all thought to increase microtubule stabilization, thus increasing the probability of neuron survival.88

There is limited understanding regarding the biological effects of nicotinamide in AD methodology; however, nicotinamide appears to exert two effects relative to tau and microtubule stabilization. First, it upregulates p25 and dowregulates p35, which is thought to increase microtubule stabilization.88 Second, it inhibits SIRT2, which functions as an alpha-tubulin deacetylase.88 While the exact method is still unclear, increasing acetylated alpha-tubulin levels, along with alpha-synuclein activity, increases microtubule stabilization and reduces cognitive degradation, possibly through aggregation stimulation among microtubules.88

The principal method in which Abeta influences the progression of tau phosphorylation leading into hyperphosphorylation and possible altered tau conformations is increasing activation of GSK3, which is activated downstream of NMDA-receptor signaling.89 Other more minor signaling pathways that are also involved are CAMKK2-AMPK kinase and C-Jun N-terminal kinase.90,91 Obviously multiple pathway activation eliminates the ability to fully neutralize Abeta activation of tau with a single molecule, a result that explains in part why a treatment like Mematine is not as effective as it should be theoretically.

In general the progression of tau to a hyperphosphorylated deleterious agent occurs in consistent manner where concentrations of tau dramatically increase in the transentorhinal cortex eventually producing sufficient quantities of NFTs, then concentrations increase in the hippocampal CA1 (II-IV) later advancing into the temporal (V) and isocortical areas (VI).92-94 While this progression can occur through normal aging it is dramatically accelerated in the presence of Abeta despite a lack of direct proximity/compartment relationship, i.e. brain regions low AB concentrations with no plaques can still see accelerated tau phosphorylation due to only a neuronal connection with a concentration heavy region.95,96

The exact method in which Abeta induces greater tau phosphorylation through the above enzymes is unclear, but the three major options are: 1) direct interaction from specific binding of monomeric and oligomeric Abeta to a variety of neuronal receptors; 2) indirect action involving induced inflammation via glial and microglial cells; 3) cross-seeding between Abeta and tau dramatically increasing misfolding and hyperphosphorylation probabilities for future tau proteins.84

Overall the chief problem with attempting to utilize a treatment for tau as a principal therapy is that it is a downstream actor. While tau may produce a meaningful amount of neuronal damage in advanced versions of AD, it is not the only damage producing agent and any treatment would be a chronic one for it would not influence Abeta concentration, the chief upstream effector of tau toxicity. This reality should not eliminate the idea for a tau based aspect to an AD treatment, but should end the idea that only neutralizing tau would be enough.

The importance of ApoE is clear in the development and progression of AD. Therefore, there has been significant study regarding its transcription including the important elements of activation and heterodimerization of the nuclear receptor retinoid X receptor (RXR) along with peroxisomes activated receptors (PPAR) or liver X receptors (LXR).97 In addition to facilitating ApoE transcription these elements also activate lipidators which are thought necessary for the proper functionality of ApoE.98,99 Thus, it seems reasonable that increasing RXR agonists should increase ApoE transcription and possibly even ApoE efficiency. This was the thought process that lead to the utilization of Bexarotene, an older cancer drug, as a possible new therapy.

Early in its testing a result produced by Bexarotene appeared very promising as it upregulated ApoE and other lipidators like ABCA1 in transgenic mouse models of Abeta amyloidosis.100 Furthermore after this upregulation there was rapid reduction in Abeta plaques and increased cognitive abilities.100 However, this success was short-lived for a number of other groups have failed to replicate this plaque reduction and improved cognition result despite also replicating the increased upregulation of ApoE and ABCA1.97,101,102

This lack of replication is troubling because various testing has demonstrated that Bexatrotene is able to active its RXR and lipidator targets effectively, but despite this action it appears that this increased upregulation is not able to consistently and/or effectively remove Abeta. Part of the problem is that there is no single formulation for Bexatrotene, thus the one used in the original research demonstrating a positive Abeta removal result may be significantly different from the formulations used by later work attempting replication.97 Unfortunately it appears that the researchers in the original work have yet to release their Bexatrotene formulation eliminating the ability to address this potential discrepancy.

Another possibility to explain the differences may be the interaction between Bexarotene and the blood brain barrier. Studies identified enhanced Abeta peptide clearance at the blood brain barrier moving peptides from the brain into the blood through an ApoE and LRP1-mediated process.103,104 What this result means is still unclear because of the overwhelming failure to replicate the original results. An additional concern created by this discrepancy is that Bexarotene has some negative side effects like weight loss, hyslipidemia, hypersensitivity, hypothyroidism and leukopenia.105 Therefore, as it stands Bexatrotene or other agents influencing RXR do not appear to be viable treatment agents.

As mentioned numerous times the interaction with cholesterol and AD is important, especially with regards to ApoE, thus some believed that statins could provide an effective means to manage or even treat AD. Despite evidence in animal models supporting the neuroprotection and improved pathology for statins, these benefits have not consistently or effectively transferred to human trials.106 Thus, the question of whether or not statins are an effective therapy option for AD patients is a controversial issue.

If one ignores the results from animal models and humans the initial premise seems plausible in that statins reduce available cellular cholesterol concentrations, which based on the relationship between cholesterol and ApoE, or even cholesterol and lipid rafts, should reduce Ab levels. Lower Ab levels should reduce, if not outright cease, the progression of AD, if provided at an early enough stage. While the initial premise seems to flow logically there are questions to whether or not a high enough concentration of statins enters the brain. In addition cholesterol accumulation and behavior function differently between the brain and the rest of the body due to the blood brain barrier.

In the brain cholesterol is produced almost exclusively from de novo synthesis instead of relying on a combination of de novo synthesis and lipoprotein uptake through LDL, HDL, etc. Without this particular lipoprotein cholesterol relationship the efflux of cholesterol from the brain utilizes 24-S-hydroxycholesterol.106 Not surprisingly patients with early-onset AD have elevated concentrations of 24-S-hydroxycholesterol, which suggests a higher intracellular cholesterol level.106

Suppose that statins are unable to pass through the blood brain barrier at high enough concentrations to significantly influence cholesterol levels in the brain, how can one explain the results that statins do provide some effect, especially in non-AD individuals? If one is to believe that there is an effect, then one possible explanation is that by reducing the cholesterol level in the body, the natural synthesis of Ab outside of the brain is reduced. Thus in individuals with ApoE4, which can bind Ab and transport it across the blood brain barrier, less Ab will be available for transport potentially reducing the amount of Ab inside the brain.107

However, the same probably cannot be said for those with ApoE2 or E3 as there does not appear to be significant Ab transport into the brain from these versions of ApoE. Therefore, if this assessment is accurate then statins could provide a small therapeutic effect for individuals with AD and the ApoE4 isoform, but would be relatively useless for individuals with AD and the ApoE2 or E3 isoform. This theory could also explain why animal models typically demonstrate positive results because a number of models stimulate AD development with ApoE4 mutations.

In the brain ApoE also uses its lipid transport function to aid in the repair of neuronal cells. This attribute was hypothesized when experimenters identified a rapid and dramatic increase (200 fold) in APOE concentration after neuron injury followed by a return to normal levels after sufficient time for repair had pasted.108 The reason behind this dramatic increase is that under normal conditions almost all of the ApoE in the brain is produced by astrocytes, but under states of stress neurons start rapid synthesis of ApoE and more ApoE can be produced by active microglia and even neurons.109

The ability to aid in neuronal repair from most helpful to least helpful among the various ApoE isoforms is E2 > E3 > E4.106 Interestingly it appears that ApoE4 has a negative effect on neuronal repair where ApoD has to fill the repair facilitation role.110 ApoE4 is also thought to play a role in the general mental decline that occurs with normal aging. The principle element of misrepair seems to stem from an inability to support neurite outgrowth, which leads to loss of synapto-dendritic communication in certain parts of the brain.109

With the general failures of existing therapy strategies, some unconventional therapies have been explored like Latrepirdine. Latrepirdine was first introduced in Russia as a non-selective anti-histamine.111 Its mechanisms of action involve the weak inhibition of acetylcholinesterase and butyrylcholinesterase along with inhibition of NMDA receptors and voltage-gated calcium channels.112 Interestingly it also has a secondary mitochondrial protective effect preserving structure and function, especially under stressful conditions. This protection is thought to occur through inhibition of the mitochondrial permeability transition pore, which can be activated by Abeta.113 Based on the reasonable success of Memantine along with the ability of other anti-histamine drugs to demonstrate some positive benefits in treating neurodegenerative disorders, some believed that Latrepirdine could be a boon in AD treatment.

An initial Phase II study demonstrated safe tolerance and a statistically significant improvement in cognitive function and psychiatric symptoms, including an anti-depressive effect, for patients with mild or moderate AD.114 Unfortunately like so many other treatments before it Latrepirdine failed to carry these improvements over in Phase III studies.115,116 Some claim that the failure was in the design of the Phase III protocols not in Latrepirdine. However, looking at the result the benefits seen in the Phase II study were amplified by the placebo group worsening versus having no significant loss of cognitive function in the Phase III study. Therefore, the significance of the Phase II success may have been derived from mischaracterization of how severe the AD cases were in the placebo group versus the actual efficacy of Latrepirdine. Despite this drawback there is still hope that Latrepirdine could be a useful therapeutic agent in the future.

Another somewhat less conventional therapy strategy was the utilization of thiazolidinediones. The two most notable thiazolidinediones available for treatment are rosiglitazone and pioglitazone, which were originally developed to treat type 2 diabetes. Both function by stimulating nuclear peroxisomes proliferator-actived receptor gamma (PPARg) which reduces the expression probability of beta-secretase and APP as well as increasing the probability of APP degradation through ubiquitination.117

In addition to the direct action against APP, the loose connection between insulin action and AD lead some to believe that both of these agents could be used to increase insulin sensitivity reducing insulin concentration. Interestingly enough there is some similarities in the degradation of insulin and Abeta, thus leading to the belief that reducing the concentration of insulin would eliminate an “indirect” inhibition effect on Abeta degradation enzymes.117 Unfortunately neither of these agents have demonstrated positive clinical trial results with rosiglitazone reporting no improvement in cognition or global function and has been further derailed by new cardiac risks from the FDA.118

While developing quality therapies for AD is the principal goal, the extent of damage that is produced during the progression of AD makes the timing of therapy application critical. Therefore, it is important to develop diagnostic methodologies that can detect AD development at early enough levels so a therapy can be utilized to ensure no significant change in quality of life rather than simply hoping for some quality of life. The most reliable non-genetic means to determine if an individual is at an increased risk for developing AD is to observe Abeta42 or tau (both total and phosphorylated concentrations) in CSF. CSF is utilized because despite having a lower protein content versus serum, CSF directly interacts with the extracellular space in the brain, thus it produces an accurate assessment regarding the biological contents of the brain.

As previously mentioned the generally accepted neuropathology of AD occurs decades before the expression of symptoms leading to three main phases of AD development: 1) pre-symptomatic (where most of the damage is conducted); 2) prodromal (mild symptoms mostly focused around episodic memory failures); 3) large-scale memory issues and similar symptomatic features common with dementia;119 Interestingly enough among these three phases CSF derived Abeta42 and tau concentrations only appear to significantly change during the pre-symptomatic stage, i.e. there are only minimal changes during the prodromal and dementia stages.120,121 In addition outside of very specific genetic conditions, Abeta42 concentrations change (increasing then decreasing) before significant changes are seen in tau concentrations.122

The decrease in Abeta42 concentration is thought to occur due to oligomeric concentrations being removed from circulation when they become incorporated into plaques. However, if this behavior is accurate then Abeta42 production must decrease at a greater level than plaque formation during the advancement of the condition; this result would speak to a negative feedback associated between plaque formation and Abeta synthesis similar to the one discussed earlier.

Unfortunately the characteristics of these changes make meaningful detection difficult. The general accuracy of current diagnostic methods is actually rather low with sensitivities ranging from 71% to 88% and specificities ranging from 44% to 71%.123 In general diagnostic biomarkers should produce a sensitivity and specificity of at least 85% to be medically useful.124 Another problem is that inter-assay and inter-laboratory variability produces additional inaccuracies ranging from 20% to 35%.125,126 Note that a sensitivity of 100% indicates a 100% identification of subjects with AD where a specificity of 100% indicates a 100% accuracy in distinguishing between AD patients and non-AD patients.

Overall these inaccuracies creates problems in clinical drug testing as 10% to 35% of individuals clinically diagnosed with AD have negative amyloid PET scans, which calls into question whether or not these individuals actually have AD.127 Some have assumed that if these diagnostic tests are accurate then there should be serious consideration to divide AD diagnosis into two sub-categories: “amyloid-first” and “neurodegeneration-first”.5 Another problem with using Abeta and tau as biomarkers is differentiating between simple old age and AD as old age appears to follow a similar pattern.128

A newer direct method to produce information regarding early progression of AD is amyloid imaging. Researchers at the University of Pittsburgh were the first to produce a reliable imaging strategy by modifying the structure of thioflavine T to include 11C has a positron emitter.5 This altered thioflavine T could cross the blood brain barrier and selectively bind to Abeta. Various other imaging methodologies have been commercially developed, but these strategies forego the use of 11C in favor of 18F because the short half-life of 11C demands PET imaging with immediate access to a cyclotron for accurate measurements versus PET imaging alone.5 The first commercial compound to receive FDA approval was Florbetapir, but the Center for Medicaid and Medicare Services have yet to approve its coverage.5 However, that may have changed recently due to the passage of the Affordable Care Act.

One of the chief concerns about using direct Abeta imaging for diagnosis or even detection purposes is that it tends to prefer fibrils instead of oligomers. However, fibrils tend to form after oligomers and there is ample evidence to suggest that oligomers play an important role in the development and progression of AD. Therefore, not only could imaging fail to properly capture the full extent of Abeta expression, but also will lag behind identifying the actual progression of AD.

Another concern for this lag is that neutralizing Abeta strategies will have to proceed before any other negative methodology, like tau progression, accelerates otherwise treatment becomes much more difficult. For example model mouse studies demonstrated that vaccination prior to plaque initiation prevented all amyloidosis versus vaccination after initiation only eliminating about 50%.129 Another important element in diagnosis that has emerged in recent years is that approximately 1/3 of patients with clinical AD do not produce Abeta plaques in the brain, which would make differentiating between AD and non-AD (normal aging) more difficult for this method.

Another strategy to rectify the time delay for an individual between AD development and displaying symptoms of AD is to identify biological and genetic biomarkers that demonstrate significantly increased probability of AD development or currently active AD development.
Unfortunately most attempts to identify genetic biomarkers involve genome-wise association studies, which can produce erroneous results or produce more broad results with little known probabilities. For example one of the more important identified genes is CLU, which encodes clusterin (a.k.a. apolipoprotein J (ApoJ)).130,131 Clusterin is important because it is involved in Abeta clearance, inhibition and neuronal apoptosis and while it is expressed in numerous tissues throughout the body, expression is higher than average in the brain.132-134

This higher than average brain expression, especially in patients with AD, has raised hopes that clusterin could be used as an early identification biomarker for AD.135,136 Unfortunately this hope has not faired well against empirical evidence where higher levels of brain clusterin have not consistently preceded AD development.137,138 In fact as previously mentioned in the blog post linked to above, biomarker analyses in general, including meta-analyses, are plagued by larger, typically bias induced, effect estimates.

However, research has shown an increase in clusterin concentration in association with depression.138 This could explain some of the contradicting results between clusterin concentration and AD for some individuals with AD get depressed, for obvious reasons, and some do not. One of the disappointments with the failure to confirm plasma based clusterin as a biomarker for AD is the plasma aspect for the ease and efficiency of plasma testing is superior to collecting CSF.

Research on disease modifying drugs for AD has covered a lot of ground in recent years, but unfortunately unlike the existing symptomatic treatments there was yet to be a significant success. Even more troubling is that available results from the multitude of Phase III studies on disease modifying drugs suggest that a quality drug is not forthcoming. One strategy to improving the probability of developing a critical treatment is to ensure proper coordination between Phase II and Phase III studies as it is sometimes difficult to reconcile a glowing success in a Phase II study with a significant failure in the corresponding Phase III study.

Another issue that was previously discussed on this blog is studying the importance of multi-drug therapies in clinical studies. While individuals like to think of AD as an Abeta disease that later involves tau, there are numerous pathways involved in the development and progression of AD that can inflict significant cellular damage and produce neurological degeneration. Attacking and neutralizing Abeta is clearly the optimal solution, but current research implies that unless this neutralization is achieved very early in the disease progression, long before the development of symptoms, then it may not be an effective target. Therefore, it stands to reason that AD will commonly involve attacking multiple neurodegenerative pathways. However, there are almost no clinical trials involving treating AD patients with multiple drugs at the same time, clinical trials continue to be conducted with only one drug versus a placebo.

Some may conclude that this multi-drug therapy could be better consolidated into a single drug that attacks multiple targets (multi-target directed ligand design), which would make treatment less complicated from the patient’s perspective. However, such designs are more complicated from a regulatory standpoint and a biological one as the combined effects of a single drug may prove less potent in triggering each pathway versus two separate drugs, one for each pathway.

Despite significant levels of effort and research the immediate future for developing an effective treatment for AD does not seem promising. Some claim that AD research is woefully under-funded given the potential havoc that AD could bring against the healthcare system in the near future. However, the cry for more funding does not appear to be a valid response to the setbacks currently experienced in the AD research community. It may be that the focus of research must change from attempting to find a single compound that will address AD to creating a multi-drug treatment regimen and this multi-drug treatment may require investigating more indirect compounds.

For example flotillin 1 knockout mice express less Abeta and less amyloid plaques, but in levels that are not sufficient for treatment.139 However, if flotillin 1 inhibitors were paired with another Abeta therapy a therapeutic level result could be produced. Also the important of lipid rafts are somewhat acknowledged in the research community, but their important component and interactive elements are typically not investigated for future therapeutic effect. There are high hopes that adherence to a Mediterranean diet will reduce the probability of developing AD, but there have been mixed results regarding whether or not the diet provides a significant protective effect.140-143 Overall while more funding would be nice, a change in perspective regarding how to treat AD may be the most important step to producing an effective treatment.

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Deciphering Alzheimer’s disease – Searching for Successful Therapies

Alzheimer’s disease (AD) is primarily characterized by neuronal damage and loss over specific areas in the brain. The neocortex and entorhinal area lose the large glutaminergic pyramidal neurons; in the hippocampus pyramidal cells are damaged in the CA1 and CA2 regions; the nucleus basalis, medial septal nucleus and diagonal band of the Broca area all lose cholinergic neurons.1 Studies have demonstrated that this neuronal loss creates degeneration in signaling in the temporal and parietal lobes as well as the cingulated gyrus and possibly the frontal cortex.1 This damage to the brain results in indiscriminate memory loss (both long and short-term) as well as dementia eventually leading to death.2 Unfortunately there is no viable treatment, although some drug combinations can diminish magnitude of symptoms, nor is there a diagnostic regiment that can definitively confirm Alzheimer’s sans a brain biopsy.1,3

Three major theories have been postulated to explain the cause of AD. The oldest theory, the specific and significant reduction in acetylcholine concentration, a generally excitatory neurotransmitter, loses more and more popularity as time progresses due to known treatments that should enhance acetylcholine production and retention not curing cognitive degradation. Realistically a better idea regarding acetylcholine would be to focus on inhibition of acetylcholine receptors rather than a direct lack of acetylcholine.4 Currently the most popular theory for AD is the amyloid hypothesis where amyloid beta (Aβ) deposits in ‘senile’ plaques and soluble oligomers cause AD. This theory is commonly referred to as the ‘amyloid cascade hypothesis’.5 The third theory, the tau theory, suggests that disease progression depends on the rapid phosphorylation of a mutated tau protein which then combines with other tau proteins creating large neurofibrillary tangles (NFTs) in neuronal cell bodies. These tangles then somehow destabilize microtubules leading to the systematic breakdown of neuronal processes.6

A significant factor explaining the popularity of the amyloid hypothesis is the location of the beta amyloid precursor protein (APP) gene on chromosome 21. A major reason that this location is deemed important is that those suffering from trisomy 21 (Down Syndrome) almost always suffer from AD (frequently early onset AD).7,8 Also excess amyloid plaques (dense insoluble deposits of beta amyloid peptide and cellular material) buildup in the brain shortly before or just after the occurrence of AD symptoms.1 In addition transgenic mice carrying a mutant APP gene develop fibrillar amyloid plaques and similar AD symptomology.9 Other research target non-plaque derived Aβ oligomers because they are believed to bind to prion protein receptor to induce AD type physiology.10,11

Based on the two above valid prevailing theories, AD can be classified as either proteopathy or tauopathy. Proteopathy describes a disease or condition that results from abnormal protein structures due to protein misfolding. In the case of AD the misfolded proteins are beta amyloid and tau. Beta amyloid is a fragment from the larger trasmembrane protein APP. Tauopathy describes diseases that are a result of pathological aggregation of the tau protein [a microtubule associated protein (MAP)] and typically lead to the formation of NTFs.

There are a number of genetic indicators/risk factors that are believed to play a role in the development and/or progression of AD: 1. mutations in the APP gene on chromosome 21;1 2. mutations in the presenilin 1 gene on chromosome 14;1 3. mutations in the presenilin 2 gene on chromosome 1;1 4. alleles for apolipoprotein E (ApoE) positioned on the proximal long arm of chromosome 19;7,8,1 5. the potential mutation in the alpha-2 macroglobulin gene on chromosome 12;1 6. CLU (ApoJ) gene on chromosome 8;12,13 7. complement receptor 1 (CR1) gene on chromosome 1; 12,13 8. PICALM gene. 12,13 The first 3 genes are associated with early onset AD (40-59), the next 2 genes are associated with late onset AD (60+) and the last 3 have only been recently characterized as playing a role in AD and have yet to be confirmed with only one or both onset cases of AD.

APP is a transmembrane protein, which has three principal isoforms, 695, 751 and 770, each which contains the 4 kDa Aβ peptide and is synthesized in the rough endoplasmic reticulum and glycosylated in the Golgi apparatus.1 APP is typically found in dendrites, cell bodies and axons, which allows for effective Aβ concentration dispersement. Endopeptidase α-secretase cleaves within the Aβ region, eliminating any opportunity to form an Aβ peptide. If APP is not cleaved by α-secretase then APP can be incorporated into endocytic compartments for cleavage by β-secretase and/or γ-secretase. β-secretase cleaves APP at the N terminus of the Aβ peptide sequence and γ-secretase cleaves at the C terminus.1 γ-secretase can cleave at multiples sites creating multiple length Aβ peptides (typically 40, 42 and 43).1

Genetic mutations in APP seem to increase the probability for cleavage of higher number Aβ peptides. Amyloid comprises large fibrils and a b-sheet secondary structure – characterized by Congo red or thioflavin S staining.11 Under normal conditions a vast majority of the formed Aβ is Aβ1-40; however, in AD larger quantities of Aβ1-42 and Aβ1-43 are synthesized which nucleate more rapidly into amyloid plaques.1 This action may explain why most people of an advanced age have some amyloid plaques (the natural synthesis of Aβ1-42 and Aβ1-43 just in very small quantities), but in AD there are so many more amyloid plaques because of the significant increase in Aβ1-42 and Aβ1-43 synthesis.

One of the earliest theories pertaining to AD pathology involved the loss of cholinergic neurons (neurons that release acetylcholine). The reason disruption in the acetylcholine pathway was suggested as a rational for AD pathology was the symptomology of AD focusing on the loss of cognitive functions, especially memory and learning, functions in which acetylcholine plays a critical role. In effort to combat these losses early treatments focused on administering acetylcholine precursors and muscarinergic agonists, but neither strategy worked very well. One reason to explain the unsatisfactory results is that early in AD before any significant neuronal loss Aβ1-42 has the potential to bind to acetylcholine receptors and act as a reversible direct inhibitor against post-synaptic acetylcholine binding.4

Therefore, a portion of the disruption of cognitive function in the early stages of AD may be the direct result of this inhibitory effect instead of the loss of cholinergic neurons. This change in acetylcholine functionality may be why the influence of acetylcholine precursors is less prominent than that of cholinesterase inhibitors. The rate of synaptic release of acetylcholine is still under a specific level of neuronal control as well as the resultant released concentration of acetylcholine from synaptic vesicles which may not necessarily result in an increase in acetylcholine residing in the synaptic cleft whereas a cholinesterase inhibitor has more influence on acetylcholine concentrations in the synaptic cleft. This differing influence is probably why reducing the activity of the cholinesterase with a cholinesterase inhibitor reduces symptoms more effectively.

This pathology is further supported by the various deficits in acetylcholine neurotransmission both due to the loss of cholinergic neurons in later stages of AD and a reduced rate of neurotransmitter release.14 Of the two different types of acetylcholine receptors, muscarinic and nicotinic, Aβ1-42 is known to bind with a high affinity to both α-7 nicotinic receptors and non-α-7 nicotinic receptors,4,15,16 and it does not appear that there is any significant inhibition of muscarinic receptors.16

Binding to the nicotinic receptors reduces current amplitude by 39% +- 3% when using caged carbachol as a binding agent, topping out at a Aβ1-42 concentration of 500 nM, although inhibition was demonstrated at concentrations as low as 100 nM and 59% +- 7% under pressure application.16 It is believed that most of the disperity between these inhibition values is due to a more rapid densensitization. With these concentrations early strategies for dealing with the symptoms of AD focused on outcompeting Aβ1-42 by treating patients with cholinesterase inhibitors to reduce the degradation rate of acetylcholine in the synaptic cleft. Unfortunately these methods do not work over the long-term nor do they seem to effectively treat any of the underlying causes of neuronal death brought on by AD.

One reason for their lack of effectiveness could be that typical concentrations of Aβ1-42 in AD are believed to range from 10-50 nM, although such an estimate may be on the low side due to a suspected non-uniform distribution; these concentrations may not be significantly large enough where increasing the concentration of acetylcholine would drive a significant change in neuronal firing.16,17 Once cholinergic neurons begin to die, no amount of cholinesterase inhibitor will help because the source of acetylcholine is no longer able to produce acetylcholine. Therefore, it is reasonable to suggest that anti-cholinesterase drugs will only be useful in treatment of early to mid stages of AD progression.

A concern that does not appear to be entertained in the pathology of AD is that the application of cholinesterase inhibitors may actually be detrimental in the long-term. While cholinesterase inhibitors demonstrate the potential to increase cognitive abilities in the short-term their use could increase the overall speed of AD progression. The reason for such a counterintuitive statement is that β-secretase activity/influence on APP appears to have an association with neuronal activity in that the more depolarized the cell for the longer period of time the higher probability of β-secretase interaction with APP.18 This increased action seems to be brought on by a greater frequency of endocytosis of surface APP which closes the proximity between the APP and the β-secretase in endosomal recycling increasing the probability of interaction.18

Recall that Aβ peptides, especially Aβ1-42, bind to nicotinic acetylcholine receptors and reduce neuronal activity.15,16 Therefore, β/γ-secretase based Aβ peptides have a principle negative feedback effect as Aβ peptides seem to serve a role as, somewhat ironically, an excitotoxicity inhibitor.19 However, the application of cholinesterase inhibitors has a positive/excitatory effect on neuronal activity which leads to the increase in γ-secretase activity which in turn increases the synthesis of γ-secretase based peptides.18 Thus, it appears reasonable to suggest that cholinesterase inhibitors may decrease the lifespan of those suffering from AD due to their influences on neuronal activity and the overall resultant concentration of Aβ1-42 and other γ-secretase based Aβ peptides in the brain if indeed Aβ peptides are responsible in some part for neuronal death, which is difficult to dispute.

Recently an association between the prion protein and Aβ1-42 oligomers was identified with the prion protein acting as the receptor for the Aβ1-42 oligomer.20 The prion protein tested was of normal conformation (PrPc) not the pathogenic conformation (PrPsc), thus there is no distinction regarding whether or not Aβ1-42 can bind to PrPsc. The prion protein-Aβ1-42 complex seems to have an inhibitory effect on long-term potentiation (LTP) in the hippocampus while influencing the CA1 and CA3 regions.20 However, the cellular pathway that induces this LTP inhibition function was not fully identified. The specific region of Aβ1-42 binding appears to be the charged region of the prion protein between residues 95 and 110.20

There are a couple of questions with this finding in that the prion protein-Aβ1-42 complex did not induce any conformational changes in or interact with GluR1–4 receptors and NR-2B and -2D containing receptors for heterologous X. laevis oocyte system. Also the study concluded that the binding affinity between Aβ1-42 and α-7 nicotinic acetylcholine receptor was almost non-existent which is in direct contrast to other studies.4,15,16 Despite these questions some believe that this result is the turning point in AD treatment and if one can successfully block the Aβ1-42-prion interaction significant progress will be made in finding a cure for AD. Unfortunately such a philosophy heavily simplifies the relationship between Aβ1-42 and PrPc to the point where blocking the interaction may be disastrous.

Between PrPc and PrPsc, PrPsc has received a majority of the attention due to its believed role in neurodegenerative diseases. The divergence in study may also be a reaction to the difficulty of evaluating PrPc pathways because PrP-null mice, unlike most other null gene mice, have not been very clean-cut in highlighting a sensory pathway of action.21,22 On its own PrPc is a glycoprotein with two N-linked oligosaccaride chains and most are localized on the cell surface attached to the lipid bilayer via a C-terminal, glycosyl-phosphatidylinositol (GPI) anchor.23,24 Lipid rafts also seem to play a role in hosting cell-surface PrPc.25

Although there are not many clear roles for PrPc in normal neuronal function, there seems to be reason to believe that PrPc can acts in an apoptosis resistant pathway due to its ability to interact with apoptosis inducer Bax.26,27,28 The ability to interact with Bax reduces the probability of cellular death when PrPc is activated. The protective influence of PrPc was further demonstrated when deleting the residue sequences 32-121 or 32-134 resulted in progressive neurodegenerative illness in mice when lacking both gene copies of endogenous PrP (Prn-p), but not when lacking only a single allele.29 The most likely candidate for the interaction behavior between PrPc and Bax appears to be the direct interaction between the cytoplasmic portion of PrPc and Bax either through direct contact or a secondary messenger type system, with the secondary messenger system being more probable.27,28

PrPc action may also play a role in the prevention of damage due to oxidative stress as mice that lack both Prn-p suffer a higher probability of neuronal damage and/or death from oxidative stress.30,31,32 The protective effect of PrPc relative to damage induced by oxidative stress is somewhat controversial, but is thought to occur primarily through the function of superoxide dismutase (SOD) either directly (the PrPc in specific situations undertakes behavior/action similar to SOD)33 or indirectly by up-regulating other SOD proteins like Cu-Zn SOD.34 Currently the latter option of indirect action seems to be more probable due to inconsistencies in PrPc copper binding affinities.35

If one ties the protective effects of PrPc together with the repolarization influence of Aβ peptide, the formation of the Aβ-PrPc complex may not actually be a negative, but a positive biological action. Initially such a statement may seem foolish as Aβ1-42 binding to PrPc appears to demonstrate inhibition of LTP and induction of long-term depression (LTD).20 However, taking a step back, under normal biological (non-AD) conditions the influence of the Aβ1-42-PrPc complex would not be long-term because of the very low natural concentration of Aβ1-42 peptide. Instead production of that peptide would be increased when a cell was overexcited and possibly facing excitotoxicity and not only interact with PrPc to not only reduce the depolarization duration and rate through some secondary pathway influence on NMDA and/or AMPA receptors, but also activate defenses against apoptosis due to any excitotoxicity because of the over-activation. After a specific period of time, the Aβ1-42 dissociates somehow from the PrPc and the inhibitory activity stops.

Unfortunately in AD, the extracellular concentration of Aβ1-42 is dramatically increased which significantly increases the probability that PrPc remains active in the Aβ1-42-PrPc complex, which continues the inhibitory effects. These inhibitory effects may still slow down the progression of AD because instead of neuronal death being induced by excitotoxicity it is induced by a slower LTD derived axonal and dendritic retraction. On a side note although copper binding is prevalent in PrPc, it does not seem to influence Aβ1-42 binding.20

With all that has been said, the most interesting potential action very well may be the fact that antibody-induced cross-linking of PrPc on a neuroectodermal cell line stimulated non-receptor tyrosine kinase fyn.10 This stimulation of fyn required an interaction between PrPc and caveolin and later resulted in the stimulation of NADPH oxidase and extracellular-regulated kinases (ERKs).36 The reason activation of fyn is interesting will be explained later. Overall at the moment until an actual pathway for pathogenesis can be uncovered with the normal conformational prion protein, it unclear how useful targeting the prion protein would be at treating AD, if even useful at all for if it does act in a more protectionist manner over detrimental then blocking its action may actually increase the rate of progression in AD patients.

Despite a lot of support in the scientific community, which may be in the process of eroding depending on who one talks to, there are significant questions regarding the influence of the amyloid plaques in AD and the role these plaques play in the progression of the disease. Multiple amyloid plaque degradation treatments have been experimented with and none have generated enough positive statistically relevant results to be included in mainstream treatments. The failure in a Phase III trial of Flurizan (tarenflurbil) after a successful Phase II trial was somewhat shocking and disappointing to the medical community.

In culture Aβ1-42 protofibrils that eventually become plaques have a tendency to kill cells through application of oxidative stress and can induce a greater frequency of excitatory post-synaptic potentials,37,38 but there is no definitive evidence that plaques themselves actually have a neurotoxic influence. Also there appears to be no proportional ratio between the number of plaques and the level of neurological disfunction, in addition to lingering questions regarding proximity of plaques to neuronal damage.39,40,41 In fact some studies have shown that neuronal damage occurs outside of or in absence of plaque formation.11,42,43 Note that there are three different classifications of plaque: diffuse, fibrillar and dense-cored where diffuse plaques lack an identifiable or distinguishable morphology, fibrillar plaques have a central mass of β-amyloid with compact spoke-like extensions and dense-cored plaques have a compacted central mass surrounded by an outer sphere of β-amyloid.43 Early in the progression of AD the majority of the plaques are diffuse whereas the ratio shifts to favor fibrillar and dense-cored plaques as the disease reaches later stages.44 This change is probably most influenced by the increasing concentration of Aβ peptides creating an increased opportunity for peptide aggregation.

A serious concern is that researchers may have simply assumed a negative role for the plaques because of their association with AD and neuronal death. For example the plaques are the bystander covered in blood in a room with a recently murdered individual. The police arrive and naturally assume that this bystander is the murderer instead of considering that he happened upon the scene and maybe even tried to help the dying individual. Could the plaques be markers of a counter-response to the overproduction of Aβ1-42 instead of a detrimental element generated by Aβ1-42 or something else entirely? Such a conclusion would explain the failure of plaque degradation treatments because if the plaques were positive or neutral then destroying them does nothing to help the patient, thus there would be no statistical difference between these drugs and placebos.

Initially the statement that the amyloid plaques are not negative seems foolhardy largely because it is believed that Aβ1-42 fragments makeup a significant portion of a plaque, which are neurotoxic and there are various pieces of empirical evidence that seem to support a negative role for plaques. However, there are two different rationalities that can be applied to explain the relationship between Aβ1-42 fragments and plaques. First, when Aβ1-42 fragments aggregate into a plaque they loses their toxicity because they are no longer able to bind to a specific receptor (nicotinic acetylcholine, etc.) initiating a toxic influence. This explanation implies that Aβ1-42 has one of three destinies when secreted from a neuron: aggregate to an oligomer and bind to a receptor, self-aggregate with other Aβ1-42 to form fibrils or plaques or be destroyed or removed via something like a microglia or other clearance method.

Second, what if the amyloid plaques are not solely comprised of Aβ1-42, but are comprised of both Aβ1-42 and Aβ1-43. Aβ1-42 and Aβ1-43 are remarkably similar compounds both are insoluble and create fibrils rather easily. There is no evidence to demonstrate that Aβ1-42 and Aβ1-43 cannot bind together creating plaques probably because it was not viewed as an important point of study (understandably). Both Aβ1-42 and Aβ1-43 can form plaques independently. The action of Aβ1-43 may in fact reduce the neurotoxicity of Aβ1-42 by hastening its ammelgamation into a plaque. Overall the first option seems more viable than the second option as it is difficult to believe that a neuron would self-trigger Aβ1-43 cleavage.

The explanation for why plaques appear toxic in certain research when they really aren’t could be explained by addressing plaque stability. There is no reason to assume that plaques are 100% stable, once formed they do not go through any further change, thus small portions of the plaques could break off into Aβ1-42 dimers, trimers and oligomers which are toxic and proceed to induce the cascade that influences neuronal death. The real question regarding any negative influence of plaques is does their size induce any accelerated microglia or other inflammatory responses or is any resultant inflammatory response a reaction to nearby oligomers?

If Aβ1-42 is a critical component in the onset and advancement of AD then it stands to reason that the administration of Aβ1-42 antibodies would limit the neurotoxic influence of Aβ1-42. Various studies have confirmed this position where peripheral antibodies for Aβ1-40 and Aβ1-42 were applied in transgenic mice models and non-human primates resulting in a reduction in neuritic dystrophy, synaptic degeneration and early tau tanglement.45,46,47,48,49 The chief method through which antibodies work is by binding the Aβ peptide which eliminates its ability to bind to nicotinic acetylcholine receptors or other receptor targets and later trigger phagocytosis or another form of clearance destroying the Aβ peptide.42 However, there is an interesting concern in that the increased phagocytosis/cytokine release may also increase microglia activation which may neutralize the positive effects of the Aβ antibody due to collateral damage generated from the microglia.50,51

This concern of excess immune response may very well be true because unfortunately the use of Aβ antibodies in an actual therapeutic environment has not been very successful largely due to an increased T-cell autoimmune response against the Aβ sometimes resulting in aseptic meningoencephalitis,52,53 inconsistent results where certain populations of patients improve and others do not and little reduction in tau-based late NTFs.52,54 New strategies are being investigated to eliminate this T-cell response, which if successful could make the administration of Aβ antibodies a possible therapeutic treatment once again.

Another theory relating to neuronal death in AD focuses not on Aβ1-42 as the principle actor in the damage leading to death, but more as an instigator that leads to overreaction by microglia, which actually release the toxins that drive neuronal death. Microglia are the principle macrophage in the brain due to the fact that most antibodies and other immune system components once fully differentiated cannot penetrate the blood-brain barrier (BBB). There are typically four types of activation states for microglia: ameboid (principle scavenger role), ramified (inactive/resting/central body motionless probing with branch processes), activated non-phagocytic (partially active, secretes cytotoxic factors and recruitment molecules, uptake of MHC class proteins and proliferation) and activated phagocytic (fully active, secretes cytotoxic and pro-inflammatory factors, antigen presenting and available to phagocytose).

There is evidence that demonstrates microglia have the capacity to bind to the N-terminus of either soluble Aβ1-40 or Aβ1-42, which activates them to at least an activated non-phagocytic state resulting in the active secretion of neurotoxins in effort to clear away the binding target.55 Unfortunately based on the generic close proximity of the Aβ1-40 or Aβ1-42 to the neurons these deleterious agents produced by the microglia not only act against the Aβ1-40/42, but also the neurons. One of the chief agents from microglia thought to induce neuronal death is a neurotoxic version of phenolic amine that binds to NMDA receptors and could begin an excitotoxicity cascade similar to that of glutamate.55

In addition microglia have been shown to infiltrate amyloid plaques in AD.56 However, this infiltration is interesting in the context that one of the chief histological features of AD is amyloid plaques surrounded by microglia and astrocytes. In these AD environments the amyloid plaques are still intact which leads to the question, are microglia able to effectively clear away amyloid plaques? This question also ties into the issue regarding the fact that there are studies that identify significant plaque formation in areas away from significant neuronal death without close proximity to plaques. Note that only dense/core plaques, not diffuse plaques seem to attract and have the potential to activate microglia.55 If microglia did destroy plaques with a high degree of specificity then it would stand to reason that these plaques would eventually be destroyed because once a neuron died the excess secretion of Aβ peptides would significantly decrease.

There appears to be three possible explanations for these lingering plaques. First, microglia are not activated or have a very low activation potential due to fibril/plaque based Aβ peptides; instead only soluble Aβ1-40 or Aβ1-42 have the requisite N-terminus to facilitate binding and high probable activation. Therefore, the microglia that are incorporated into the plaques are those that are binding to the unstable portions of the plaque that break-off to become soluble peptides. Second, the microglia have become inactive due to reaching their phagocytosis limit (basically these incorporated microglia cells have taken their gitter form) or lose the necessary receptors to induce phagocytosis. However, the loss of thes types of receptors, like Toll-like receptors, does not make very much sense. Third, the microglia are supplementing the elimination of the Aβ peptides and their associated plaques by secreting their own Aβ peptides,57 creating a dynamic equilibrium between the clearance of Aβ peptide and its secretion.58

If the third option is correct, then the relationship between microglia and Aβ peptides becomes complicated. The enhanced release of additional Aβ peptides from microglia could either increase the rate of AD progression due to increasing the overall concentration of neurotoxic agents (especially Aβ1-42) in the brain or could decrease the rate of AD progression by increasing the rate of Aβ1-42 plaque formation which significantly limits/eliminates their neurotoxic influence. Or a third option exists in that this release creates an dynamic equilibrium between Aβ peptide synthesis and clearance doing little to help those suffering from AD and in fact possibly being a net detriment due to the occurrence of any ‘collateral damage’ neuronal death because of the release of cytotoxic factors when binding Aβ peptides. In addition to microglia, localized astrocyte populations seem to increase in the presence of Aβ1-42, but neuronal death does not seem to be increased or decreased via the action of astrocytes.55 Instead of clearing away Aβ peptides and plaques astrocytes seem to play the role of barrier formation by forming a wall between the plaques and neuropils.59

It is also reasonable to suggest that microglia would not be able to bind to an Aβ1-42 oligomer that is bound to a receptor due to the fact that it is highly probable that the N-terminus binding site is concealed. Therefore, for microglia to be responsible for significant neuronal death it appears that they would have to bind to a soluble Aβ1-40 or Aβ1-42 to activate and release of their degradation agents in reasonably close proximity, not necessary in direct contact, to the neuron. There is no reason to suspect that phagocytosis would induce neuronal death. Due to the liklihood of such a situation, it is difficult to theoretically view microglia as a chief element in neuronal death in AD, that is not to say that there is no microglia activation, but probably not enough to warrant its role in neuronal death as significant.

However, reality seems to differ from the above hypothesis in that when transgenic mice and AD patients are treated with anti-inflammatory medication there appears to be a reduction in neuronal death.60,61,62 If microglia influence is apparently so difficult to induce via interaction with Aβ1-40 or Aβ1-42, why do anti-inflammatory treatments reduce neuronal death in the short-term? Perhaps the answer lies in the fibril plaques that are created as AD advances. Previously it was suggested that these plaques have no inherent toxicity despite various studies that seem to indicate the contrary. To explain this alleged contradiction it was reasoned here that plaques are in a quasi-dynamic equilibrium state where small oligomers are continually being added and subtracted from the plaque. If this were the case then it would go a long way to explaining why neuronal cell death is significantly reduced because the anti-inflammatory agent is preventing microglia from activating due to interaction with the oligomers that are breaking off from the plaque. It may be reasonable to suggest that plaque-based (fibril) Aβ1-42 is about only 1/5th as toxic as Aβ1-42 oligomers.63 Such a result makes sense if one considers small oligomers breaking off from the plaque to either interact with microglia or other receptor targets vs. the same amount of Aβ1-42 available to interact when in a non-fibrillar state when concentrations are initially equal.

Most of the studies touting the benefits of anti-inflammatory agents are short-term. The reason neuronal death is reduced in the short-term, but not in the long-term is because microglia could very well act as one of the faster pathways when inducing neuronal death. This belief seems to make sense when considering the methods of neuronal death involved in AD. Instead of having to wait on the destruction or excitatory collapse of nearby neurons to induce significant excess glutamate release or anticipate a calcium secondary messenger system over-activation, which triggers hyperphosphorylation of a MAP protein (tau) finally resulting in axonal collapse/retraction, the microglia toxicity acts immediately on NMDA receptors to generate a cascade failure relatively quickly.

Also it is highly probable that the microglia can act over a wider range of immediate influence than glutamate or tau based death, thus not only are neurons killed faster, but more could die in shorter period of time. Unfortunately preventing microglia activation through anti-inflammatory agents or other means is not a cure for AD because they are only addressing one potential neuronal death pathway, other pathways are not neutralized and the neurons that are salvaged due to the anti-inflammatory agent will probably be eventually killed later. Basically microglia action can be regarded as a fast secondary means of neuronal death.

A bright spot in significant microglia induced death may be its action against NMDA receptors. If the neurotoxic activity demonstrated by microglia does indeed influence NMDA receptors and resultant calcium influx then anti-inflammatory agents may not be a necessary element to reducing the influence of microglia on neuronal death. A drug that will be discussed later, memantine, may very well serve a double beneficial purpose in the treatment of AD due to its antagonistic action against NMDA receptors and channel opening. Thus, instead of having to get rid of initial microglia action, its overall neurotoxicity against neurons can be neutralized.

Unfortunately phenolic amine and its action against NMDA receptors may not be the only cytokine derived from microglia that plays a significant role in AD. Microglia can also release interleukin-1(IL-1) α and β, primarily β, when interacting with APP and Aβ1-42.64 IL-1β can bind to surface receptors and activate p38 mitogen-activated protein kinase (p38-MAPK) which seems to have the ability to both reduce the concentration of synaptophysin and phosphorylate tau.64 This phosphorylation could be a step in the hyperphosphorylation of tau and the formation of paired helical filaments (PHFs) and NTFs that are a trademark of AD, because P38-MAPK phosphorylates tau at five sites that are phosphorylated in PHFs.65 Inhibition of IL-1β using an anti-IL-1β antibody or blocking the IL-1 receptor with IL-1ra reduced neuronal tau phosphorylation and increased the concentration of synaptophysin when exposed to APP-activated primary microglia vs. control samples.64 This pathway could demonstrate a secondary means of microglia derived neuronal death. In addition it may muddy the waters with regard to inhibiting the hyperphosphorylation of tau by introducing valid evidence to support the action of another kinase.64,66,67

In the microglia based p38 MAPK pathway, hyperphosphorylation of tau is only one issue; the loss of synaptophysin may also play a role in cognitive degradation. Synaptophysin is an integral membrane protein that is typically phosporylated by tyrosine kinases and is thought to regulate synaptic vesicle release.1 A reduction in synaptic vesicle release would reduce the total concentration of neurotransmitter released which in turn will more than likely reduce depolarization of neighboring neurons reducing signaling.

The story on anti-inflammatory drugs does not end with reducing the activity of microglia. Some anti-inflammatory drugs, most notably non-steroidal anti-inflammatory drugs (NSAIDs), have the ability to inhibit γ-secretase, which reduces the synthesis of Aβ1-42 and Aβ1-43.68 Unfortunately it is difficult to evaluate how influential γ-secretase and β-secretase inhibitors would be as therapeutic strategies because it seems reasonable to suggest that as AD progresses further to mid and late stages the less useful these inhibitors would be in alleviating symptoms due to the large concentrations of Aβ peptide already synthesized. Improvement in AD early diagnostic procedures would go a long way to improving the prospects of secretase inhibitors as treatment agents. Also there is a concern that γ-secretase inhibitors would interfere with other cleavage targets performed by γ-secretase like Notch 1.68

Another method that has been explored to reduce Aβ1-42 concentration is stimulation of the M1 muscarinic acetylcholine-receptor which has been shown to increase the activation of α-secretase, which eliminates the ability of γ-secretase and β-secretase to create Aβ peptides.69,70 Unfortunately M1 activation enhancement has not been extensively tested, so their actual therapeutic value in the long-term is still unknown.

MAP tau is thought to play an important role in neuronal differentiation and axonal development as well as axonal maintenance.71,72 The primary attention on its role in these functions focuses on its ability to influence microtubule assembly and stability. In fact because the phosphorylation of tau alters its ability to bind to microtubules, there are many that believe changes in the phosphorylation rate of tau plays a significant role in the neuronal death witnessed in AD due to a reduced microtubule stability.73,74 The general theory seems to be that under normal conditions tau is a normal elongated protein that aids in promoting microtubule assembly, stability to the microtubular ‘roadway’ and bundles microtubules in the marginal band allowing synaptic vesicles and organelles to move freely and efficiently from the neuronal cell body to the synaptic bouton. However, in the case of AD a hyperphosphorylated tau becomes destabilized and no longer binds to microtubules causing the axon to become destabilized and the axon retracting leading to abnormalities and shortfalls in the delivery of vesicles and organelles impairing communication between neurons.

Support for this theory is largely derived from the two primary characteristics of tau in AD, the abnormally high presence of several phosphorylated serines and threonines 75,76 and the presence of NFTs which are comprised of PHFs and straight filaments of which hyperphosphorylated taus are a principle component.1,77 There is also the probability that the loss of tau solubility is brought on by hyperphosphorylation and increases the probability of NFT/PHF formation. Due to this behavior of tau in AD, one of the more common treatments is to break up NFTs/PHFs with the hopes that it will reduce the probability of neuronal death. The immediate problem with this strategy is if the treatment strategy does not involve dephosphorylating the tau or preventing the phosphorylation in the first place then breaking up the NFT/PHFs will probably do little to stem neuronal death because the phosphorylation itself is what drives the neuronal death, the fact that tau eventually forms NFT/PHFs is just a secondary symptom.

Due to its relationship with microtubules, the axon and possibly other cytoskeletal proteins, neutralizing the mechanism behind the hyperphosphorylation of tau looks to be a promising strategy in the treatment of AD and the reduction of neuronal death. Currently there are two major strategies that are being utilized to address this issue, down-regulation or inhibition of tau phosphorylating kinases or up-regulation of dephosphorylating protein phosphatases. Between these strategies most of the focus have been on kinases GSK-3β and cdk5/p25 and phosphatase (PP)-2A.

There is evidence to suggest that cdk5/p25 plays a role in the development of AD.78,79 Cdk5 is a Cyclin-dependent kinase with an associated regulatory subunit at p35. Proteolytic cleavage of p35 generates p25, which frequently results in abnormal Cdk5 activation, especially in AD.78 Overexpression of p25 results in the hyperphosphorylation of endogenous tau and the eventual formation of NFTs.78 Inhibition of Cdk5 or p25 reduces the amount of tau hyperphosphorylation and neuronal death, but does not eliminate tau aggregates or NFTs. Therefore, it appears that cdk5/p25 has some form of catalytic effect on the hyperphosphorylation of tau. Unfortunately there may not be anything that can be done regarding cdk5 in a long-term treatment regiment because inhibition of cdk5 activity tends to also inhibit fast anterograde axonal transport and the redistribution of cellular proteins.80 Fortunately this lack of inhibition is not a game-breaker because the principle kinases that are thought to be responsible for hyperhosphorylation can still be inhibited.

The aforementioned glycogen synthase kinase-3 beta (GSK-3β) is thought to be a principle actor in hyperphosphorylation of tau. Mammalian GSK-3 has two isoforms, α and β, and on average is constitutively more active in neurons than other kinases.81 Inhibition of GSK-3β via multiple inhibitors demonstrates a reduction in tau aggregation levels as well as a reduced level of neuronal death while overexpression of GSK-3β results in hyperphosphorylation of tau and NFTs.82,83 The inhibition of GSK-3β occurs through two pathways, the inhibitor either competes with magnesium to limit activation or phosphorylates the serine9 residue, which aids inhibition.82

However, there are some concerns with GSK-3β being the driving kinase behind tau hyperphosphorylation. First, in single tau transgenic mice, an increase in GSK-3β activity appeared to reduce neuropathology and motor impairments, basically doing the exact opposite of what would be rationally expected with an increase in tau phosphorylation.84 Second, reduction in tau phosphorylation using a GSK-3β inhibitor does not neutralize all of the phosphorylation of serines on tau. For example treatment with lithium, a known inhibitor of GSK-3β, reduces the level of phosphorylation at Ser202 and Ser 396/404, but not at Ser 262 or Ser422.82 The lack of phosphorylation preventation at Ser422 is of note because Ser422 is commonly regarded as a phosphorylation site that is specific for disease, including AD.85,86 Although it can be argued that Ser202 andSer396/404 are more important in the facilitation of tau-based NFTs.87 Third, there is the question of the natural constitutively activation of GSK-3β and why this activity does not induce more spontaneous NFTs? Maybe it does, but the rate of generation is not large enough to induce any significant changes to microtubule organization? Maybe tau needs to be pre-treated in some fashion to place it in closer proximity to GSK-3β before excessive phosphorylation? Maybe there is associated phosphatase activity that neutralizes natural GSK-3β influence? Fourth, while tau aggregation levels are reduced when GSK-3β is inhibited, the total number of NFTs that form in the transgenic mice are not necessarily reduced.82

At one point in time MAP kinase ERK2 was also viewed as a potential agent in the hyperphosphorylation of tau due to increased co-distribution with the neurofibrillary changes in Alzheimer’s disease and the fact that it appeared to phosphorylate all tau relevant serine-theronine residues at the maximal stoichiometry 88,89 However, it seems more probable that this co-distribution is reflective of a secondary pathway not attributable to hyperphosphorylation tau progression because in studies where MAP kinase ERK2 activity was stimulated or inhibited no significant change in tau progression occurred in kind.90,91,92

Phosphatase PP-2A seems to act as an inhibitor of hyperphosphorylation as when PP-2A is inhibited or down-regulated there is significant tau hyperphosphorylation at Ser202/Thr205 and Ser422.93,94 Clearly it makes sense that in the case of hyperphosphorylation there would be a decrease in respective phosphatase activity. However, there do not appear to be any identifying studies with regard to the interaction between tyrosine kinase fyn and phosphatase PP-2A. It may make more sense that the conformational change that occurs when tau is phosphorylated at tyrosine18 by fyn prevents phosphatase PP-2A from dephosphorylating tau at Ser202/Thr 205 and Ser422 rather than a decreased rate of dephosphorylation due to down-regulation of phosphatase PP-2A.

It is also believed that one of the elements responsible for phosphorylation of tau could be a src family tyrosine kinase, called fyn.75,95,96 Bolstering this claim is that when cells are co-transfected with only fyn or tau AΒ1-42 toxicity is statistically eliminated when accounting for neuronal death.75,97 However, this conclusion leads to an interesting question. Tyrosine kinase fyn phosphorylates tau at the tyrosine18 residue, not at any serine or threonine.72 If this phosphorylation site is accurate, then tau may not induce neuronal death exactly through the aforementioned method of axonal regression due to microtubule destabilization. When the tyrosine18 residue is phosphorylated there is no apparent reduction in probability of tau binding microtubules.75 The ability of tau to bind microtubules seems more dependent on whether or not the serines and threonines are phosphorylated. Due to this action the pathology of neuronal death due to hyperphosphorylation of tau may be more reminiscent of how tau behaves during neuronal development instead of neuronal maintenance.

During neuronal development a subpopulation of tau persists in the distal portion of the axon and the growth cone.98,99 and aids in its outgrowth. In addition various src-family non-receptor tyrosine kinases also exist in the growth cone 100 meaning that it is likely that fyn is among those tyrosine kinases. Tau localization is disrupted when exposed to tyrosine phosphatase inhibitors101; therefore, a fyn and tau interaction could drive tau action in neuronal development. Specific action of tau is related to altering the actin based growth cone to facilitate dynamic microtubule incursion. Then tau binds to the new microtubules in order to organize them to drive the forward advance of the growth cone.102 This action could identify a new mode of action to induce neuronal death in that the breakdown of microtubules is not due to lack of tau binding, but instead is the result of tau programmed regression due to new growth behavior.

For example assume that in early development most of the phosphorylation of tau is induced by fyn on the tyrosine18 residue which drives neuronal differentiation and axonal development whereas after development phosphorylation of tau is induced by other tyrosine kinases that focus on serine and threonine residues which aids in microtubule stabilization; it could be possible that no further phosphorylation by fyn takes place for critical functions after development. Whether or not this is true is dependent on the direct function of fyn in the PrPc neuroprotective function.

In the case of AD, the action of Aβ1-42 triggers a renewal in phosphorylation of tau by fyn creating a sensory trigger that causes tau to breakdown the current axon and attempt to rebuild a new axon, similar to the first axon created during development. However, before this new axon can be developed, tau forms NFTs with other taus ceasing the process. Unfortunately there is only speculation regarding how Aβ1-42 eventually activates fyn. Another question is why doesn’t tyrosine kinase fyn in pyramidal cells in the hippocampus hyperphosphoryalze tau during LTP? A possible answer to the latter question is that the rate of activation under normal LTP conditions is not long enough to induce sufficient phosphorylazation or fyn is not the first step in the fyn activation pathway.

Of the four AD drugs that are currently used for therapeutic purposes the most successful appears to be memantine. Memantine’s success is believed to be derived from its slow inhibition of Ca2+ influx after long-term activation of NMDA receptors and its associated ion channel.103 In the brain there are three classes of ionotropic glutamate dependent ion channels where glutamate must bind to a receptor to trigger opening: NMDA, AMPA and kainite. The most important of these three receptors is NMDA due to its elevated permeability for Ca2+. If a NMDA-based channel is open for too long, then the concentration of Ca2+ that enters the neuron will be too large and will disrupt Ca2+ homeostasis of the neurons leading to a secondary messenger system cascade that will more than likely lead to cell death. This death can come from many different avenues, oxidative damage, proteolytic processes, mitochondria driven apoptosis, etc. Due to the threat of this detrimental possibility NMDA receptors have a magnesium ion that normally blocks the ion channel, which requires the neuron in question to have some level of depolarization before the magnesium ion is repelled enough to clear the channel. This required depolarization typically occurs through the AMPA and kainite channels that open in response to glutamate and allow sodium to flow into the neuron.

Under normal circumstances activation of NMDA receptors and Ca2+ influx is strictly controlled mostly through LTP feedback functions (open for only a few milliseconds at most). However, AD upstream neuronal death due to Aβ1-42 toxicity can lead to the release of large quantities of previously isolated intracellular glutamate that can hyper-activate NMDA and AMPA receptors beginning the neuronal death cascade largely in the CA1 and CA2 regions of the hippocampus. Previous NMDA antagonist treatments were designed to completely block NMDA function in effort to prevent neuronal death, but because NMDA is required for normal learning and memory as well as certain brainstem functions like wakefulness, these 100% block antagonists had severe side effects.103 Memantine works because it acts as uncompetitive antagonist, where its blocking effectiveness increases with channel activation time.103 Basically if the channel is open for a short period of time, very little inhibition occurs vs. if it is open for a long period of time, a significant amount of inhibition occurs. Overall memantine does not stop AD and progressive neuronal death, but does reduce neuronal death on some level with few side effects.

Previously it was hypothesized that Aβ1-42 acted as an inhibitor against the NMDA family of receptors leading to loss of LTP in AD patients and the advancement of LTD in the affected neurons reasoned from a loss of dendrite density.104 However, this result seems to be somewhat confusing in that NMDA activation inhibition would be detrimental because of the success of memantine. Such confusion is understandable and the nature of this confusion will be addressed later.

Another avenue for neuronal death in AD that has gained traction in recent years is mitochondrial induced apoptosis. Recall from freshman biology that the mitochondria is the ‘powerhouse’ of the cell where a majority of the energy production reactions occur (TCA Cycle, electron transport, etc.) that create a majority of the ATP and other energy storage molecules. However, in addition to its duties in providing energy, the mitochondria also possesses a wide variety of signaling molecules that induce cellular apoptosis like Apoptosis Inducing Factor (AIF), Smac/DIABLO and cytochrome C.1 The chief family of proteins that are responsible for driving the mitochondria apoptotic pathway is the bcl-2 proteins. Some of the bcl-2 proteins induce apoptosis (Bax 1, Bak, Bik and Bad) and others resist apoptosis (bcl-2, bcl-W and bcl-XL).1

Under normal circumstances apoptosis inducing factors are normally scattered through the cytosol in effort to detect any cellular stress/damage. If the protein detects a form of stress (triggered via phosphorylation or some other pathway), then that apoptosis inducing factor migrates to the surface of the mitochondria to interact with an apoptosis resisting factor. If enough inducing factors bind to resisting factors transport pores form in the outer mitochondrial membrane. These pores allow cytochrome C and other more isolated apoptosis inducing factors from the intermembrane space (the area between the outer and inner membranes of the mitochondria) to interact with Apaf-1 forming the apoptosome (pro-caspase 9 + cytochrome C + Apaf-1) and finally activating caspase 9 induced apoptosis.1

In AD it appears possible that most of the initial steps in apoptosis are circumvented by intracellular Aβ1-42 binding to Cyclophilin D (CypD) which forms the necessary mitochondrial permeability transport pores to release cytochrome C and start the apoptosis cascade.105,106 However, there is a question in that it is believed that CypD interacts with AΒ1-42 within the intermembrane space.105 So how does Aβ1-42 pass through the outer mitochondrial membrane to reach the intermembrane space and interact with CypD? Is there an intermediate transmembrane protein that facilitates the interaction without requiring a pore? Also how does the Aβ1-42 get into the cytoplasm in the first place to even have the possibility of passing through the outer mitochondrial membrane?

Addressing the second question first, when the various secretases cleave APP the resultant Aβ peptide fragments are typically secreted out of the neuron or to lysosomes. However, there is the possibility that certain concentrations of Aβ1-42 escape this mechanism leaving them in the cytoplasm until cleaned out by other means possibly driven by APOE4 mutations. While lingering in the cytoplasm the Aβ1-42 could migrate towards the mitochondria. Although the probability exists that Aβ1-42 could be in the appropriate proximity to the mitochondria it is still unclear how it could penetrate the outer membrane. Another means of cytoplasmic concentration increase could involve Aβ1-42 from the extracellular space (largely derived from another neuron or any microglia) passing into a neuron through the endocytic pathway. In fact in late-onset AD early endosomes increase in volume up to approximately 2 times.107 Such an increase could very well increase the input of Aβ1-42 into the neuron and increase the probability that newly internalized Aβ1-42 interacts with CypD leading to apoptosis.

The type of APOE gene that the individual possesses influences endosome size. Apolipoprotein E4 (APOE4) genes eventually produce endosomes that are 1.5 times the size of endosomes derived from APOE2 or APOE3 genes.107 However, as late-onset AD progresses the difference in volume between APOEx and APOE4 endosomes shrinks because for some unknown reason APOE4 endosomes shrink.107 There is reason to believe that this increase in endosome volume occurs years before the legitimate onset of AD107, which may provide a new diagnostic mechanism to identify the probability that an individual will suffer from AD in the future. If an effective method for categorizing endosomes, especially those derived from cortical pyramidal neurons, in vivo can be developed it could dramatically improve the effectiveness of therapeutic treatments that use β-secretase and/or γ-secretase.

There are a couple of avenues of exploration for such a diagnostic methodology. The optimal way, based on certainty and non-invasiveness, would be to design a radioligand that could bind exclusively to early endosomes in vivo and observe any significant size or pattern changes through some form of brain scan like PET. A second idea would be to attempt free-flow zone electrophoresis. Unfortunately such a technique would probably require either a spinal tap or a form of brain bioposy. For a diagnostic procedure that has little single-shot prediction power (if endosomes are not enlarged there is no reason to assume that they will not become enlarged in the future) such a process may not be beneficial overall.

Another aspect of the endocytic pathway is its management and interaction with cholesterol. After cholesterol is synthesized in the endoplasmic reticulum it is processed in the Golgi and shipped to the extracellular matrix via a secretory vesicle. Also LDL is shipped from the extracellular matrix into an endosome where the apoprotein B portion of the LDL is dissolved in a lysosome and the remaining cholesterol is freed and released into the cytoplasm.1 APP cleavage rates appear to be influenced by intracellular free cholesterol levels where higher levels lead to higher rates of cleavage and lower levels generate lower rates of cleavage.108,109 However, there is conflicting data regarding whether or not the administration of statins improves cognitive function. Some retrospective epidemiological studies indicated that statins reduced the probability of developing dementia, but other studies failed to demonstrate any statistically significant protective effects associated with the loss of cognitive functions.110,111 Clearly there are elements that need to be better identified before prescribing statins as a form of treatment for AD.

One of those elements that deserve further investigation may be the LDL receptor-related protein (LRP). LRP is a member of the LDL receptor family and is known to bind and mediate endocytosis of soluble APP (APP cleaved by α-secreatase) and cell surface APP (APP yet to be cleaved by any secreatase) if the APP isoform contains a Kunitz proteinase inhibitor (KPI) domain.112,113,114 The importance of LRP is that it appears to have an influence on whether α-secreatase or β-secreatase/γ-secreatase is the dominant form of action on APP. Blocking LRP with RAP increased both the amount of cell surface APP levels (largely due to an increase in APP synthesis) and increased α-secreatase processing due to the existence of more soluble APP.114 Also after treatment with RAP secretion of Aβ peptides dropped significantly. Technically it cannot be fully concluded that RAP reduces Aβ peptide synthesis due to β-secreatase/γ-secreatase as blocking LRP could simply reduce the amount of Aβ peptide secreted, which would result in much higher intracellular Aβ peptide concentrations, but such a result is unlikely.

There are three possible methodologies with which blocking LRP interferes with the endocytic pathway that favors Aβ peptide synthesis. First, LRP association blocks α-secreatase action before formation of an endosome guaranteeing that β-secreatase/γ-secreatase will have an opportunity to cleave. Second, LRP association induces a conformation change in the APP better exposing the β-secreatase/γ-secreatase cleavage site increasing the probability of a successful cut. Third, LRP association increases the total time within an endosome vs. other endocytic pathway associated proteins, which could then increase the probability that the APP interacts with β-secreatase/γ-secreatase.

The relationship between LRP, APP and AD progression is also strengthened by the fact that silent polymorphism in the LRP gene is associated with increased risk for AD.115 This polymorphism probably influences Aβ peptide concentrations in two different ways. First, it shifts the interaction of APP in favor of β-secreatase/γ-secreatase over α-secreatase. Second, LRP is one of the major receptors used in the transport of Aβ peptides across the BBB. Based on this information it seems reasonable to suggest that increasing the number of LRP for a given cell will increase the APP interaction and the number of synthesized Aβ peptides. This proportional relationship between Aβ peptide synthesis and LRP expression/availability could explain the link between the previous studies that associates a higher cholesterol level with a greater probability of developing AD. The higher the cholesterol level leads to greater LDL and probably LRP expression, which would lead to greater synthesis rate and concentration of AD peptides. If correct this mechanism not only explains a portion of diet association with the development of AD, it also explains why statins have a sketchy history alleviating AD sympotoms.

The principal function of statins is to reduce the overall level of cholesterol synthesis in the liver by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Reducing cholesterol synthesis in a given cell will cause the cell to increase its expression of LDL receptors in effort to balance the difference in cholesterol levels between the cytoplasm and extracellular matrix. This up-regulation could also result in the up-regulation of LRP, which as discussed increases the probability of Aβ peptide synthesis. However, if that were the case it would be more probable that statins would hasten the progression of AD instead of possibly slow its progression.

The situation with statins differs from that with high levels of cholesterol because for high cholesterol patients a dynamic equilibrium of LDL receptor expression is attained and typically only increases as extracellular cholesterol levels increase. With statins, the inhibition of HMG-CoA reductase creates a greater LDL receptor expression rate than seen in high cholesterol patients, remember that a vast majority of statin users initially have high cholesterol, in the initial administration of the statins. As time passes this expression rate decreases as a new dynamic equilibrium of LDL receptor expression is reached due to the action of the statin; this new dynamic equilibrium of expression is lower than that of the high cholesterol no-statin patient. Therefore, statins probably do have a small therapeutic role in the treatment of AD symptoms, but this positive effect probably only occurs in high cholesterol patients over a significant time frame. If the above behavior is correct, short-term studies would probably conclude that statins did little to nothing to reduce AD progression or symptoms. Also the effect of statins will be muted because LRP expression will not be eliminated, just reduced.

An increase in β-secretase and/or γ-secretase activity is not the only means responsible for increasing the concentration of Aβ peptides in the brain. There is also a question of the breakdown of the typical clearance mechanisms largely situated near the periphery along the BBB. If clearance mechanisms begin to function improperly even normal synthesis levels of AB peptides can quickly become excessive and increase the probability of detrimental influence in the brain.

Although there are multiple proteins that are responsible for Aβ peptide clearance, the three principle proteins thought to exert the most influence are receptor for advanced end glycation products (RAGE), apoE and LRP.115,116 As previously discussed LRP expression and its resultant interaction with APP is thought to alter various secretase production rates, but LRP also plays a role in the transport of Aβ peptides across the BBB expelling Aβ peptides from the brain into the circulating blood.116 RAGE is a multi-ligand receptor in the immunoglobulin (IgG) superfamily and is the counter agent to LRP as it ferries Aβ peptides from the blood to the brain.116 Over-expression of RAGE or under-expression of LRP can increase the concentration of Aβ peptides in the brain. Fortunately there are self-correcting mechanisms where it appears that if RAGE or LRP have unbalanced interactions with Aβ peptides it facilitates the up-regulation of the under-binding receptor.116 Although disruptions in the RAGE-LRP relationship could increase Aβ peptide concentrations, it seems more probable that the clearance mechanism malfunction responsible for increasing Aβ peptide concentrations in the brain is APOE, which was previously discussed.

In addition to possibly inducing apoptosis, Aβ1-42 is also thought to have a positive effect on the production of superoxides, which generate cell damage and inhibits aconitase, which is an enzyme used in the TCA cycle, significantly reducing the ability of the cell to produce energy.63 Although these studies do identify that increased levels of SOD and deferoxamine (an iron chelator) reduce the total level of toxicity of Aβ1-42,63 the role of other neuronal death elements like tau phosphorylation, microglia recruitment and NMDA hyperactivity are not identified, so it is unclear whether mitochondrial based toxicity is a primary neuronal death factor or a secondary one induced much later in the process after neuronal death is a foregone conclusion, kind of like the linebacker that jumps on the pile long after the running back has been tackled.

For example the latter rationality would offer an explanation to how Aβ1-42 could pass through the outer membrane in that a hyperphosphorylated tau could be a trigger for an apoptotic inducer to bind to an apoptotic resistor opening a pore in the membrane and allowing the Aβ1-42 to pass through. However, if that were the case then blocking CypD would not terminate neuronal death, but simply somewhat reduce the speed of neuronal death.

An element that may play a significant role in AD pathology that has been on the backburner until recently is apolipoprotein J (APOJ) or clusterin. The verification of the APOJ gene as a potential link to the progression of AD has refocused attention on its role in AD.12,13 Clusterin is a disulfide-linked heterodimeric glycoprotein117 that plays a role in many biological and pathologic processes such as phagocyte recruitment, tissue reconstruction, cytolysis inhibition and apoptosis.118 Most of the research surrounding clusterin involves its role in cancer and classification as a small quasi-heat shock protein. In cancer clusterin is acts as a stress-associated cytoprotective chaperone118, which is up-regulated during activation of the apoptotic pathway and is able to bind and inhibit activated Bax to reduce the probability of mitochondria pore formation, cytochrome C release and resultant apoptosis.119

Clusterin also plays a role in the AD, although that role is not exactly clear. Similar to cancer, clusterin synthesis is increased during the progression of AD. However, it is unclear whether or not this increases is beneficial or detrimental to the survival of neurons. Originally it was observed that clusterin bound Aβ peptides to prevent further association with other Aβ peptides to neutralize fibrillization and plaque formation.120 Also a Aβ peptide bound to clusterin has a much higher potential of interacting with megalin receptors on glial cells increasing extracellular clearance through endocytosis. Despite these protection methods, naturally produced clusterin does not appear to be able to prevent AD progression.

One reason for the inability of clusterin to derail AD is that there is contradictory information in that in some scenarios clusterin seems to enhance the level of oxidative stress and neuronal death not prevent neuronal death.121,122 Also clusterin increases the probability for the formation of Aβ-derived diffusible ligands (ADDLs),63 which have a higher probability of surface receptor binding over fibril Aβ peptides.

So does clusterin have both a protective and a destructive role in AD? One way to explain this apparent contradiction is characterize clusterin as an element with a type of catalytic effect on Aβ1-4x. At lower concentrations clusterin enhances the aggregation of Aβ peptides hastening their transition from monomers to oligomers, which have the ability to bind to cell surface receptors. At higher concentrations clusterin is able to bind for a longer range of time facilitating greater aggregation into plaques or removal via glial cells lowering the probability of Aβ peptide interaction with surface receptors. However, if this is the case then the binding affinity of clusterin to the Aβ peptides cannot be very significant. Despite this concern such a methodology seems to make sense in that fact that clusterin prevents rapid self-aggregation of Aβ peptides, especially 42 and 43, with preference for a slower aggregation process with clusterin as a centerpiece.

If the above conclusion is correct then clusterin is a valid therapeutic target for treatment of AD through two different pathways. First, an inhibitor of clusterin can be applied in effort to drastically reduce the rate of aggregation of Aβ peptides reducing the probability that they interact with nicotinic and other receptors. Second, stimulation of clusterin can be attempted to supersaturate the extracellular matrix with clusterin which would eliminate the ability of Aβ peptides to bind to receptors due to conformational change brought on by clusterin binding and eventually result in the clearance of both the clusterin and the Aβ peptide commonly via glial cells.

As previously discussed some have concluded that Aβ somehow directly induces LTD via interaction with some portion of a NMDA receptor, which presents a confusing and contradictive result because NMDA antagonists have been noted to improve AD symptoms. In rudimentary terms LTD can be viewed as basically a biological driven NMDA antagonist as dephosphorylation of AMPA receptors prevents them from opening in response to glutamate which in turn prevents them from depolarizing the neuron which removes the blocking magnesium ion in the NMDA receptor channels. If clinically applied NMDA antagonists like memantine improve AD symptoms, it stands to reason that the ‘natural’ method of applying a NMDA antagonist would do the same instead of increase the severity of AD.

The confusion from this issue can be resolved when considering two separate factors. First, a vast majority of AD experimentation, understandably, occurs in a vacuum where condition or pathway is not measured against another pathway. This form of isolation largely results in the single question of: does eliminating or enhancing factor x lead to neuronal damage/death? The failure to consider other pathways results in a failure to consider the second factor, speed of neuronal death.

There can be no logical argument that LTD is a detrimental outcome, but when all other options that result in neuronal death are considered, LTD is a much better outcome. The following example better illustrates the above reasoning. Suppose that an individual had to select one of the following five options: be shot in the head, have the throat slit, be given radiation poisoning, be injected with HIV or receive a subdural hematoma. Although none of the presented options are desirable, the best option would be to be injected with HIV because although there is a high probability of death, the quality of life after making the choice is significantly better than any of the remaining choices.

This circumstance may describe Aβ driven LTD, which can be classified as a condition that will probably lead to death, but will take a longer time to reach that outcome over other detrimental pathways influenced by Aβ peptides in AD. Basically it is slowest means of neuronal death.

Overall the biggest problem with AD may be all the results from all of the knockout experiments or isolation experiments seem to generate contradictory views on what elements in the pathology are important. Various studies knockout component x and seem to demonstrate that without that component neuronal death due to AD either no longer occurs or is significantly reduced. Unfortunately these types of results drive the ‘silver bullet’ mindset or drug development in that if a drug can be developed to neutralize component x then instant AD cure. However, these experiments rarely touch on the other elements that could induce neuronal death. Basically all of these experiments seem to take place in a vacuum where only component x is important. Such is not the case in reality for AD appears remarkably complex.

The best way to illustrate this apparent disconnect between AD drug testing and biological reality is with the following example. Suppose that a criminal is being put to death, but the individuals responsible for deciding upon the method of death cannot agree to any particular method, so instead of selecting a single method they elect to apply all five methods they have been debating. Therefore, the criminal is lead out to the killing ground and set on fire, shot in the heart, electrocuted, injected with poison and has his throat slit. Now reset the situation and suppose that just before the man is executed an anti-death penalty advocate comes running forward and puts a metal collar around the criminal’s neck believing that it will save his life. Unfortunately for the advocate, no such luck as even though the collar does protect the criminal from getting his throat slit, it does not protect him from getting set on fire, shot in the heart, electrocuted or injected with poison.

Clearly any bystander would view such a situation with confusion, for how could the advocate believe that preventing only one method of death would spare the criminal? The same logic can be applied to AD drug testing. No wonder most AD drug investigations fail when reaching Phase III testing; investigating to see if drug x can extend lifespan and mental conditions by neutralizing death condition y does not seem to be productive if death condition z is not also lessened/neutralized by drug x. It can be argued that electrocuting the criminal does make it easier to set him aflame, but setting him on fire is not dependent on electrocuting him. Therefore, it makes more sense that when testing for possible AD drugs that can limit neuronal death and progression of the condition that a drug cocktail be used instead of a single drug. Attacking AD on multiple fronts is the only guaranteed way to develop an effective and successful treatment.

So the big question is ‘where to attack’? To know where to attack, one must have a general idea of the pathology of AD. Early onset AD is driven primarily by mutations in either the APP, presenilin 1, presenilin 2 which accelerate the rate at which Aβ1-42 is cleaved from APP. Due to the existence of APP in dendrites, cell bodies and axons the additional Aβ1-42 concentrations can become diffuse throughout the local region of the mutation. Under normal conditions the vast majority of Aβ1-40, Aβ1-42 and any other Aβ peptides are neutralized. In this situation concentrations of Aβ1-42 are small enough that there is no significant neuronal damage. Recall that Aβ1-42 is able to interact with clusterin or other monomers of Aβ1-42 outside of the cell and begin to form dimers, trimers and larger oligomers. There is reason to believe that self-Aβ1-42 aggregation is more rapid than clusterin induced Aβ1-42 aggregation. This rapid form of self-Aβ1-42 aggregation may be more beneficial then detrimental because plaques probably limit the neurotoxicity of Aβ1-42. If clusterin concentrations are saturating then it is likely that most Aβ1-42 will be neutralized. If not, then there is reason to believe that clusterin could aid in the formation of deleterious Aβ1-42 oligomers.

One result from the formation of these Aβ1-42 oligomers is that they are able to bind to nicotinic acetylcholine receptors (preferably the α-7 family) providing a sufficient level of direct inhibition. This inhibition forces the channel to stay open longer to generate a depolarized state in the neuron. The additional time open hastens the already inherent rapid desensitization processes in nicotinic receptors.123

The desensitization process for nicotinic ACh receptors involves the activation of protein kinase A (PKA), protein kinase C (PKC) or tyrosine kinase. PKA phosphorylates the gamma and the delta subuints of the receptor. PKC phosphorylates the alpha and delta subunits of the receptor. Tyrosine kinase phosphorylates the beta, gamma and delta subunits of the receptor.1 These three elements inactivate nicotinic acetylcholine receptors. Although it initially takes time, the desensitization process initiated in part by a family of tyrosine kinases could be responsible for the hyperphosphorylation of tau. Eventually more and more tau are hyperphosphorylated causing a retraction in the axon due to the tau either losing the ability to stabilize the microtubules or tau reverting back to its development role of axonal development. If the latter option is correct then it is probable that the tau proteins that are driving this new axonal development tangle with each other forming NFTs before the new axon can be rebuilt.

There is little reason to question that interrupted axonal regrowth or significant axonal destabilization would trigger an apoptotic reaction in the cell. It is also possible that the formation of NFTs could also induce an apoptotic response through normal mitochondria apoptotic influences (inducer binds resistor). Apoptosis destroys the neuron causing large concentrations of intracellular glutamate to leak out of the cell into the extracellular matrix and various nearby synaptic clefts. This non-signalled glutamate is of significant concentration that it is highly probable that it interacts with AMPA and NMDA receptors on surrounding neurons, most notably in pyramidal cells in the CA1 and CA2 regions of the hippocampus.

Binding to the AMPA receptors initially depolarizes the cell removing the magnesiums blocking the NMDA receptor channels allowing for the dramatic increase in the influx of calcium. Due to the excessive concentrations of glutmate remaining in the synaptic cleft, due to the sizable intracellular concentrations of glutamate in normal neurons, the depolarization of the given neuron continues for an extended period far beyond normal excitation. The calcium driven secondary messenger system activates various kinases in the neurons in the hippocampus affected by the released glutamate including tyrosine kinase fyn and p38 MAPK, which phosphorylates tau and initiates the first series of steps in the apoptotic pathway.

This activation of fyn and p38 MAPK implies that glutaminergic neurons in the hippocampus that die in AD may have their apoptotic pathway activated via multiple different means. Also such a situation would explain the existence of NFTs in the hippocampus where cholinergic neurons are not as plentiful as other regions. Significant neuronal death in the hippocampus would also interfere with LTP and thus interfere with memory, learning and other cognitive abilities, hallmark symptoms of AD, and similar to inhibition of cholinergic neurons. This dual detrimental effect would compound any learning problems that arise from the partial inhibition of the acetylcholine receptors and their channels.

There is also the possibility of long term inhibition of NMDA receptors through long term depression of AMPA receptors if the neuron is not killed via apoptosis or Aβ1-42 somehow binds to the NMDA receptor. These processes (the excess calcium influx or Aβ1-42 binding) lead to the activation of calcineurin. Calcineurin dephosphorylates inhibitor-1, increasing the activity of phosphatase-1 resulting in dephosphorylation of the AMPA receptors.1 This dephosphorylation closes the AMPA channels heavily reducing the influx concentration of Na+, leading to the repolarization of neuron and the return of the magnesium channel blocker which would stop in the influx of calcium through the NMDA gated channels. This process would prevent LTP inducing cognitive damage, but it is unclear if it would eventually kill the neuron (although it is likely). Also phosphatase-1 may play a role in dephosphorylating tau which would reduce the probability of neuronal death via microtubule destabilization.

It is also highly probable that internal Aβ1-42 driven apoptosis plays a role in neuronal death, although the magnitude of that role is unknown, albeit at the moment it seems probable that its role is secondary and limited. The reason that this pathway of neuronal death is thought to be limited is that first in either early or late onset a genetic mutation is responsible for the necessary increase in Aβ1-42 concentration to generate the required concentrations for a high enough probability that Aβ1-42 will remain in the neuron and achieve the proximity required to act with CypD to induce apoptosis. Therefore, in this scenario there would need to be a large number of genetic mutations throughout specific regions of the brain, for internal Aβ1-42 driven apoptosis to be a primary means of cell death, which is unlikely. Second, it is highly unlikely that even if certain oligomer structures of Aβ1-42 could enter the neuron through an open ion channel or an endosome, that most of these oligomers would be available to do so as most of these oligomers would either be bound into a plaque or bound to a receptor site. There is the possibility that AD patients will high cholesteral levels could have a higher probability of suffering from internal Aβ1-42 driven apoptosis due to increased levels of Aβ1-42 production due to greater LRP expression.

Microglia derived neuronal death is the trickiest of all pathways to classify. The significance of microglia interaction and neuronal death is that it seems probable that it follows a somewhat inversely proportional rate of influence vs. the progression of the AD. In early and mid-stages of AD microglia derived death could be a primary means of neuronal death due to its fast action both in breadth and toxicity damage. However, as AD progresses the role of microglia derived death more than likely becomes less significant due to higher concentrations of Aβ1-42 in the extracellular matrix, glutamate release due to neuronal death and possible inactivation of microglia cells due to loss of receptors. So in the early stages of AD the primary means of neuronal death can be attribute to microglia and Aβ1-42 binding. As AD progresses NMDA excitotoxicity due to excess glutamate more than likely begins to outpace microglia influenced NMDA excitotoxicity.

The role of Aβ1-42 binding to prions may be the most interesting. There is reason to believe that formation of the Aβ1-42-prion complex somehow influences inhibition of NMDA channel activation through the calcineurin pathway. Such a result is interesting because the Aβ1-42-prion complex may actually extend the lifespan of the AD patient by reducing the severity of any excitotoxicity influence on hippocampal neurons in favor of the more muted LTD response that will typically kill the neuron, but do so at a slower rate and with less collateral damage. Therefore, the Aβ1-42-prion complex may actually be ‘biologically’ therapeutic relative to other Aβ1-42 pathways vs. its popularly detrimental reputation.

The figure below illustrates the various pathways towards neuronal death in AD. Note that there is little difference between early onset and late onset AD. The chief difference is that late onset involves a mutation in the APOE4 gene which influences the endocytic pathway in a cell whereas early onset typically involves a mutation in the APP, pre1 or pre2 gene.

So if the general pathogensis of the AD is properly described and illustrated above, what are possible treatments? Plaque busters like flurizan do not appear to have any role in AD treatment because destroying plaques that do not appear to have toxic affects can be nothing but detrimental (potentially releasing new non-fibril Aβ1-42 oligomers). Inhibiting β and/or γ-secretase could serve a useful purpose limiting the total amount neuronal damage, but it seems reasonable to suggest that the usefulness of β and/or γ-secretase inhibitors is inversely proportional to the progression of AD. Realistically β and/or γ-secretase inhibitors only appear to be really useful in the interim stages of AD as later excitotoxicity reduce its influence.

Currently inhibiting the CypD/Aβ1-42 interaction would probably only slightly reduce neuronal death because it seems to play a secondary pathway for neuronal death despite its speed. Anti-inflammatory drugs would work well early in AD preventing microglia collateral damage, but their effectiveness should fade as AD progresses; however, there is reason to hypothesize that the influence of anti-inflammatory drugs would fade at a slower rate than β or γ-secretase inhibitors.

It would be difficult to recommend the continuation of cholinesterase inhibitors if in fact Aβ has a repolarizing/inhibitory effect as reducing the inhibitory effect associated to Aβ binding nicotinic receptors seems to increase the probability of Aβ peptide synthesis which would exasperate the progression of AD for the sake of limited short-term gains.

Until the T-cell issues are effectively neutralized the application of Aβ antibodies appear to be too dangerous and unpredictable to be used as an effective therapeutic option. Inhibition of clusterin, which plays a role in the formation of Aβ1-4x oligomers from monomers, may be an interesting inhibitory target as Aβ1-4x monomers do not appear to be able to bind to intra and extracellular receptor sites.

Inhibition of tau hyperphosphorylation seems to be a potential important treatment strategy, but also the most complicated. There are at least 3 different kinases (fyn, GSK-3β and p38 MAPK) that have empirical backing for playing some role in tau phosphorylation. Unfortunately these studies typically only targeted one of the three kinases and did not account for either of the other two. Therefore, it is difficult to identify if there is any hierarchy within these kinases where only one needs to be blocked to prevent hyperphosphorylation of tau or if more than one needs to be blocked to fully prevent tau. The best option may be looking at fyn first because fyn appears to play a more limited role in normal homeostasis than GSK-3β or p38 MAPK. If tau phosphorylation on trysone18 can be blocked it may prevent the N terminus of tau from interacting with the proline-rich area creating a conformation change that could reduce the probability of phosphorylation at other sites.

Overall there appear to be a variety of different neuronal death elements that occur during AD, thus there are multiple avenues of therapeutic attack, but such attacks can be short-circuited if considerations are not made for the other mechanisms of neuronal death. Therefore, drug trials cannot longer be limited to single drug at a time, but instead must use multiple drug cocktails in addition to single trials to increase the probability of successfully treating AD. In short when it comes to AD drug companies need to stop thinking about unilateral control of AD treatments and develop Phase II and Phase III testing partnerships for 25-50% of an AD treatment is much better than 100% of failure.


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