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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A Discussion about Autism and Possible Treatment Pathway

Diseases come in many different shapes and sizes inflicting multiple tiers of damage on the sufferer and his/her family and friends. Obviously those diseases that significantly and rapidly shorten an individual’s life span are the worst; however, some would argue that non-fatal neurological diseases like Alzheimer’s and autism are insidiously devastating, taking away the enjoyment from life in a way that cannot be alleviated by simple pain killers or anti-psychotics. Among detrimental neurological conditions it can be argued that autism is one of the worst because most neurological conditions manifest at advanced ages at least giving the individual a “normal” life for a considerable period of time whereas autism typically arises during the first few years of existence negatively impacting life from the beginning.

The symptoms of autism are passively detrimental and heterogeneous with various levels of mental delay, increased probabilities for epilepsy, various levels of attention deficit, language and learning impairments, obsessive compulsive disorders and difficulty interacting with others.1-3 Also like Alzheimer’s, the incident of autism and other similar disorders (autistic
spectrum disorders (ASDs)) among the general population continues to expand increasing from 4 per 10,000 in the 1960s to 30-60 (depending on the strict definition applied) per 10,000 in 2000s.4 While life expectancies of autistic individuals are only slightly reduced, the level of supplemental care that these individuals require demands significant resource expenditure.

A number of individuals believe that this expansion in autism is driven by new genuine cases. To support this position individuals cite that there have been no corresponding significant decreases in other similar diseases to compensate for the increase in autism.5 Basically individuals are not being diagnosed with condition x and then later having the diagnosis reevaluated as autism. However, while there are few substitutions, there is little evidence to contest the position of past under-diagnosis, which would consist of individuals with autism not being properly diagnosed due to the specific diagnostic condition(s) used at the time or due to lack of experience/knowledge of the individual performing the diagnosis. Now with better therapeutic and clinical diagnostic experience and ability, this undiagnosed segment of the population is now being properly diagnosed, thus there is actually no genuine increase. While this reasoning is logical, the rate of the increase seems too great to simply be accounted for by under-diagnosis alone. The rate of increase also seems too large to be explained by genetic mutations alone, thus it stands to reason that if this increase is legitimate then environmental causes are also influencing the change.

The cause(s) of autism, either genetically or environmentally, is currently unknown notwithstanding an incredible display of stupidity from certain individuals attempting to foolishly link vaccination and an increased probability of developing autism despite zero valid scientific evidence supporting such a position. Incidentally these people must also believe that little green men dance the samba on Mars everyday at 4:45 EST because there is an equal amount of empirical evidence supporting either position. Currently there are no specific biomarkers that have been identified to reliably diagnose autism,6-8 although a number of biomarkers have been identified to have differing concentrations between autistic and non-autistic individuals. However, these differences are marginalized because of their non-specificity towards autism versus other metabolic and neurological conditions.

Most modern study of autism has focused on searching for a genetic cause and some progress has been made for the belief is that a majority of autism cases result from probabilistic interactions between multiple common gene variants where each variant contributes some small influence on the overall condition.9,10 Another 10-20% of autism cases are believed to occur through known genetic effects including Synapsin 1, SynGAP1, SHANK3, NLGN4, CNTNAP2, retinoic acid-related orphan receptor-alpha (RORA), MET and cytochrome P450 genes, some which may have significant heritability rates.11-20

However, with all of the hundreds of genes involved in autism, focusing on genetic research and any potential genetically engineered treatment appears to be an inefficient strategy. Instead it seems reasonable that more attention should be paid, by both researchers and the media (especially the media), to the mechanisms associated with the development and maintenance of ASD for addressing the mechanism provides a higher probability of general treatment versus genetic strategies. Two common biological symptoms of autism are an increase in brain size and weight and an increased probability of suffering epileptic seizures. One mechanism to explain these symptoms is a deficiency in the synaptic elimination process during development (a.ka. synaptic pruning).

Soon after birth an overabundance of synapses are formed as neurons produce multiple synapses between a singe post-synaptic and pre-synaptic neuron. During development important synapses, those that fire more frequently and/or have greater firing duration, are strengthened and neighboring synapses are weakened and non-selectively eliminated until there is a one-to-one relationship between axons and cell body.21,22 The reason this process occurs is because unlike mature neuron-axon relationships where electrical impulses either facilitate an action potential or nothing, immature neuron-axon relationships, especially in Purkinje cells (PCs), operate on an intensity mechanism.23,24 This intensity mechanism exists because there is no initial molecular method for the brain to identify which synapses are important and which are not, thus importance is determined by firing duration/rate. Most notably this firing is driven from visual stimulus, especially before a recognized conscious understanding develops in the individual.

In abnormal pruning conditions the process does not create the typical one-one ratio instead it creates a neurological environment where numerous synapses from a single pre-synaptic neuron feed into single post-synaptic neurons. These additional synapses create additional brain volume offering an explanation for the autistic symptomology, where very young children with autism (ranging from 18 months to 4 years) have 5-10% more total brain volume.25-27 The additional weight can be explained by additional myelination because the myelination process is based on proximity, thus all axons in a given proximity will express the necessary oligodendritic targets to drive myelination. MRI studies have demonstrated growth abnormalities concerning gray and white matter supporting the belief that myelination accounts for weight variances.28,29

A lack of effective pruning could also explain the typical autistic symptom of early overgrowth followed by abnormal slowed growth.26,30 The excess synapses create the overgrowth, but due to conflicting excitatory and inhibitory signaling due to unnecessary action potentials, incomplete synchronization, incomplete action potentials and/or neuronal strengthening patterns, like long-term potentiation, autistic individuals have disadvantages hindering learning and other neuronal expansion. Disruption of these processes result in a developmental regression for autistic children of 20-40% between 15 to 24 months of age31 with higher probabilities corresponding to higher autism severity.32

An interesting and ironic side effect to a lack of pruning in the development of autism could be that over time if signal interaction is continuously disrupted by confliction between the almost competing synapses then all of the synapses can be negatively affected to the point where the neuron itself dies due to inactivity. This result may explain some of the outcomes in post-mortem autistic brains that are ripe with stunted neurons, especially PCs.33,34

Overall there are four major chronological phases in the process of creating a normal functional synapse: 1) functional differentiation among multiple creeping fiber inputs; 2) evolution of a superior synapse resulting in dendritic translocation; 3) early phase synapse elimination; 4) late phase synapse elimination.24 For the purpose of autism it appears that the third and fourth phases are the significant ones. Between the two elimination phases late phases appears to be more important due to its parallel fiber (PF)-PC synapse requirements and its longer period of operation in mice and assumed longer period of operation in humans (P9-P17 versus P7-P8 in mice).21

In general early elimination removes synapses that do not form properly and late elimination removes synapses that do not “win” the synapse augmentation competition. Therefore, there is a higher probability that a faulty late elimination process will create a more detrimental outcome than a faulty early elimination process because the late elimination process has a higher probability of eliminating functional synapses or not eliminating competing synapses. However, while in general short-term occurs prior to long-term elimination the exact timing of this elimination is not homogeneous throughout the brain (different areas go through the elimination process at different times during development).35 It is thought that pruning occurs first in primary sensory and motor areas followed by temporal and parietal areas and ends with the frontal cortex.35,36 Interestingly if this pruning order is accurate then sensory and motor abnormalities should be the first autistic indicator over emotional or social interaction.

A key molecule in late stage synaptic elimination is glutamate. The principle reason for its importance is that glutamate drives neuronal excitation probabilities and magnitudes in both non-mature neuronal networks and mature ones. Due to the importance of glutamate the late stage pruning process could find its roots late in stage one during functional differentiation when creeping fibers synthesize type-2 vesicular glutamate transporters during the formation of their initial synaptic butons.24 These transporters are important because they play a key role in determining the amount of glutamate that is released into the synaptic cleft after electrical stimulation of the given neuron. Synapses that express more vesicular glutamate transporters have an advantage in the synapse competition that determines which synapses are eliminated in stage four.

The strength of the particular synapse is largely dictated by the transient rise of glutamate within the cleft at any point in time. Clearly the availability of more synaptic vesicles will increase glutamate concentration and indicate a stronger connection, but that is only one element. Another important element is expression of metabotropic glutamate receptor 1 (mGluR1). As a metabotropic receptor mGluR1, when bound, activates an intracellular G-protein in order to initiate a signal cascade. mGluR1 is a C type G-protein receptor, over the more common A type, and their activation typically results in the activation of calcium channels and protein kinase C (PKC) resulting in a single calcium concentration spike.37 Additional less common downstream effectors include phospholipase D, casein kinase 1, cyclin dependent protein kinase 5, Jun kinase, mitogen-activated protein kinase/extracellular receptor kinase (MAPK/ERK) pathway and mammalian target of rapamycin (MTOR)/p70 S6 kinase pathway, which influence among other things synaptic plasticity.38-41

mGluRs have a ligand binding site on their N-terminal domain, which is formed by two hinged globular domains, commonly known as the Venus Flytrap Domain, due to its structure and action.42 These Venus domains also have allosteric binding sites for agonist and antagonist molecules. When glutamate binds these hinged domains close initiating the activation of the associated G-protein. There is some question to whether or not glutamate binding to a single site will lead to activation (no activation versus negative cooperativity),43 but agonist binding to a protomer seems to induce cis and trans-activation.44 This cis and trans-activation may provide an interesting association with autism development.

mGluR1 can be found both on post-synaptic and pre-synaptic membranes in the cerebellum, thalamus and the hippocampus.42 mGluR1 that is pre-synaptically located could drive feedback on the pre-synaptic cell resulting in a longer duration of glutamate release; thus in such a situation greater expression of pre-synaptic mGluR1 will increase the probability of strengthening the particular synapse. Other feedback activity can also be mediated by depolarization of the post-synaptic cell and the release of endocannabinoids.45-47 Post-synaptic receptor location is highly specific regionalized around the post-synaptic density, but not within it and induces post-synaptic cellular depolarization.42 This localization is thought to be achieved by interaction with Homer family regulatory proteins.48

Deficiencies in mGluR1 operation result in cerebellar gait problems, deficits in long-term depression and long-term potentiation and abnormal levels of regression of climbing fibers from cerebellar Purkinje cells49-50 basically reducing the probability that any fiber creates a strong one-one connection to an individual PC. In addition certain GluR1 mutations generate conditions that facilitate spontaneous ataxia through changes in the ligand-binding domain.51 Other important elements in pruning are GluRdelta2 and Cbln1, which bind to each other and are essential for the formation, organization and maintenance of PF-PC synapses52,53 and the proper execution of late phase synapse elimination as well as PLCbeta4 and PKCgamma which are part of the mGluR1 signaling cascade.24,54

Once the “winning” synapse has been identified, the creeping fibers that make up the synapse finish translocation to dendrites after the early stage elimination while the “losing” synapses remain around the soma. This proximity association creates two questions: 1) does the “winning” synapse produce some molecule that drives translocation towards the dendrites or do the “losing” synapses produce some molecule that inhibits translocation; 2) are somas “cluster pruned” or do each “losing” synapse produce a signal of sorts that targets it for elimination?

For the first question studies of the neuromuscular junction developed the “punishment model” of synapse competition where the strong synapse, created through the process discussed above, “punishes” other synapses by inducing two post-synaptic signals, one that protects itself and one that increases the probability of pruning for other synapses in the area.55,56 However, while this model works at the neuromuscular junction there is no direct evidence suggesting its accuracy for the central nervous system, although it has been the suggestion that complement proteins act as the protectors and punishers and induce microglia-based pruning.57,58

Recently microglia have become a leading candidate in the pruning process, including the process during development. The chief evidence to support this candidacy is electron microscopy imaging demonstrating microglia in the general proximity of RGC pre-synaptic inputs during the period of time when pruning is most likely to occur in developing dLGN.59,60 Further support comes from experiments where disruption of microglia during early development leads to incomplete and abnormal synaptic pruning.58 Based on this information one theory regarding synaptic pruning involves microglia CR3 receptors binding to C3 complement proteins produced in the pre-synaptic compartments due to a lack of glutamate release and appropriate feedback, which leads to synaptic engulfment.

However, there is an interesting immediate question regarding this potential role microglia play in developmental pruning? For example assume the pruning system fails and as a result autism develops. If microglia were involved in this process then how could microglia be available to eliminate/prune damaged neuronal connections later on in life? The general life span of individuals with autism, without another detrimental condition like epilepsy, implies that their brains are able to neutralize detrimental effects stemming from damaged neurons otherwise one would expect their life spans to be significantly shorter. How is this neutralization accomplished if the pruning process were damaged? The most plausible explanation is that there is a different mechanism to govern pruning during development versus during normal non-developmental operation. If so then specific experiments would need to be designed to determine the development pruning and developed pruning mechanisms.

Epilepsy frequency in autistic individuals is much higher than in non-autistic individuals with an occurrence rate ranging from 5% to 39-44% versus around 0.63%.61,62 The driving feature of epilepsy is excessive and abnormally consistent excitation of various neurons leading to abnormal synchronization resulting in spontaneous seizures and movements and potential brain damage. This additional excitation is thought to occur through one (or both) of two principle pathways: 1) excess excitatory neurotransmitters, usually glutamate, in synaptic clefts, which increase synaptic residence times increasing the probability of post-synaptic depolarization; 2) a disrupted inhibitory pathway usually due to a lack of GABA neurotransmitter release, which increases the probability of post-synaptic depolarization. Autistic individuals are thought to have an increased probability for developing epilepsy because of the loss of GABAergic interneurons in the cortical mini-columns and other areas.63,64 The general though is that because these mini-columns are less compact, but of greater number there is more extensive innervation for increased activation, but diminished lateral inhibition.64

It is thought that the relationship between autism and epilepsy is one in which the emergence of one increases the probability of developing the other. There is evidence to support the idea that autism develops prior to the clinical seizures that emphasize epilepsy and there is evidence to support the development of autism in individuals with more severe cases of epilepsy due to excessive neuronal damage.61,65 The development of epilepsy in autistic individuals could explain the level of neuronal death seen in some post-mortem autistic brains. Finally there appear to be two peak times of development of epilepsy in autistic individuals: early childhood and early adolescences.3 Not surprisingly younger preadolescent autistic children only have an epilepsy probability of slightly under 10%,66-68 due to limited compounded neuron damage, where adolescences and adults have an epilepsy probability of slight over 39%.69,70 This probability result makes sense as discussed above the nature of neuronal death and under/over excitement neuronal networks takes time to develop detrimental influence outside of severe cases of autism.

One of the interesting elements of autism is that the disorder shows a bias towards males by a ratio of around four to one.71 Numerous studies have supported this ratio through association of increased concentrations of testosterone in individuals with autism, but there is no clear understanding of any molecular mechanism that governs testosterone-based autistic augmentation.71-74 A recent study identified retinoic acid-related orphan receptor-alpha (RORA) as a possible candidate gene that influences autism development.16 RORA is responsible for regulating aromatase, an enzyme that converts testosterone to estrogen, thus a lack of aromatase will result in an increased concentration of testosterone.

There may be at least two different means of testosterone action within the pruning mechanism that could explain its role in increasing the probability of autism. First, testosterone may act directly to support synaptic pruning creating a situation where instead of multiple synapses being created through a “masking” process no strong synapses are created between certain pre-synaptic and post-synaptic cells.75 This action could be driven by testosterone augmenting the expression of complementary protein C, which may be the signal marker that induces microglia-based synapse elimination. It would seem that this type of process would be competitive with mGluR1 processes, thus if a given synapses was producing enough glutamate release and mGluR1 interaction it could ward off the effect of testosterone.

Successful application of such a response could create gaps in neuronal signaling disrupting synchronization and creating the under-connectivity environment associated with autism. An explanation to why such an outcome is not fatal could be explained by the existence of two different pruning systems, as mentioned earlier one that operates during development and one that operates after development. Therefore, over time due to brain plasticity the pre-synaptic cell may be able to partially extend an axon creating a smaller synaptic cleft, thus increasing synchronization probability. Whether or not more potent hormones like 5-adihydrotestosterone (DHT) have similar influence is unknown.

Second, the change in estradiol concentration, which is produced from cholesterol and as an active metabolic product of testosterone, may act as a neuroprotectant through two different mechanisms. The first mechanism involves enhancing RORA expression, which in turn reduces testosterone concentration limiting possible testosterone synapse elimination augmentation or other mechanism in which testosterone seems to enhance autism.16 The second mechanism involves estradiol down-regulation of mGluR1 and mGluR5.76 Down-regulation of mGluR1 could reduce the probability of abnormal mGluR1 enhancement through environmental or mutation-derived agonists, which would increase the probability of multiple synapses through masking, while also reducing the probability of excessive competition from testosterone. However, it should be acknowledged that too much estradiol may increase mGluR1 down-regulation to the point where it becomes more detrimental than beneficial.

One might ask how these testosterone influences could occur without genetic mutations. Recall that even without mutations, autism, and most neurological detrimental conditions for that instance, does not develop the same way in all individuals. There are issues of random quantum proximity where testosterone concentrations may change minutely to the point that exerts an effect that otherwise may not occur at a slight smaller concentration. For example even without mutations, testosterone has a negative feedback effect on RORA, which can create a small cascade under certain conditions greatly enhancing testosterone concentrations. Basically there is an element of randomness as well as other direct environmental elements.

While a defective pruning mechanism may not explain all autism cases, obvious there some cases that are dictated by certain genetic mutations that exist outside the pruning mechanism, it appears to be a plausible explanation for most cases. One of the major lingering questions regarding the failure of the pruning mechanism is whether it prunes too many synapses or not enough. While one of the hallmarks of autism appears to be functional underconnectivity that leads to neuronal hypo-synchronization and a general lack of neuronal firing/excitation there is also a level of overconnectivity.77-80 For example autistic individuals suffer underconnectivity when having to process dynamic events like facial movements and auditory elements, but exhibit overconnectivity when having to process static images.81

This general neuronal trend in autism has been expanded through fMRI to demonstrate reduced functional long-range connectivity, which would affect synchronization, versus short-range connectivity.82-84 This reduced long-range connectivity could center around an imbalance between short-range excitatory and long-range inhibitory connections, involve the death of PCs.85 and negatively influence the interaction between cortical and limbic systems producing extreme negative emotional responses to change.86 So how does this over-connectivity/under-connectivity contrast develop from the pruning process?

The overall mechanism of pruning could be viewed in the following manner. Numerous synapses form, but none are dominant. The multiple synapses can be thought to be in competition with each other, so when receiving neuronal input from the cell body and dendrites the total potential of that input is divided between the synapses with the total weight of that division unknown. Suppose for the purpose of this example there are five synapses receiving inputs of 15%, 15%, 20%, 20% and 30%. Over time due to higher weighted percentages, more glutamate vesicles, greater feedback from post-synaptic response, etc. one synapse starts to exert dominance receiving more of the initial excitation where the weaker synapses receive less. Returning to the above breakdown after a short period the inputs change to something like 7%, 8%, 14%, 17% and 54%. Eventually the weaker synapses are eliminated and the single dominant synapse receive 100% of the dendritic input.

In scenarios where pruning is not functioning properly one of two situations can arise: 1) pruning fails to eliminate all but one of the existing synapses leaving more than one synapse feeding into post-synaptic dendrites; 2) pruning removes all synapses leaving no synapse feeding into post-synaptic dendrites. It is understandable to assume that the first situation is more frequent than the second because in the second the post-synaptic neuron would have a higher probability of future apoptosis and potential brain damage. The first means of failure (multiple synapses surviving) can be further broken down into two failures: 1) a breakdown in the pruning system itself during development where a dominant synapse is created, but the weaker synapses are not pruned because of a deficiency in the pruning pathway; 2) weakness masking where weaker synapses are masked and interpreted by the pruning pathway as stronger and unworthy of pruning.

The first means of failure is rather self-explanatory where one or more of the agents responsible for pruning are not functioning properly. The second is a little more complicated because of multiple potential targets. The most obvious means is some external environmental agent acting as an agonist for mGluR1. Most likely the system that evaluates the winning synapse does not function on a relative scale (synapse 1 is releasing 70% of the glutamate versus synapse 2 releasing 30% so synapse 2 should be pruned), but instead on an absolute scale with a competition threshold (synapse 1 is releasing 70 mmols of glutamate versus synapse 2 releasing 30 mmols of glutamate so synapse 2 should be pruned because it is not releasing enough “don’t prune me” compounds stemming from that glutamate release). Thus mGluR1 agonist binding could increase overall glutamate release from all synapses “masking” synapse weakness resulting in less overall pruning. This additional agonist from an environmental agent could also help explain the increased rate of autism if the agent is a toxin of some sort.

Again it is difficult to reconcile the bigger brain seen in most autistic patients and less connectivity from excessive pruning, so one must assume for the moment that the above dynamic is correct that there are regions of the brain with multiple synapses instead of just one. How does an environment of multiple synapses create a reduced level of connectivity and activation for dynamic visual and/or audio processes? One possible explanation is that these synapses function differently than normal synapses.

In a multiple synapse situation none of the synapse may extend as far as the single dominant synapse situation, thus there is a larger than normal synaptic cleft. This scenario could make sense because translocation involves signals from both the pre and post-synaptic regions and one could expect a limited amount of signal from the post-synaptic region. If this signal is spread between multiple targets versus a single target shorter translocation is possible. This larger synaptic cleft reduces the total amount of neurotransmitter that is able to bind to receptors on the appropriate post-synaptic cleft. In such a scenario even if the multiple synapses were able to provide more total neurotransmitters due to firing similarity and rate, the additional space that the neurotransmitter has to cover should limit the total binding potential. For example if one were to standardize the one-one situation at a binding potential of 1 a larger synaptic cleft in a multiple synapse scenario would have a binding potential less than 1. This lower binding potential would reduce the activation probability of the post-synaptic cell resulting in slower and/or less neuronal synchronization, which is required for optimal processing of dynamic stimulation where multiple isolated firings of single neurons is limited.

Two possible synaptic explanations for the over-excitation when an autistic individual experiences a static stimulus could involve a lack of axonal evolution and a lack of feedback. First, as previously mentioned developing synapses function on a graduated release system over an all or nothing system, which later develops in more mature neuronal systems. Perhaps this evolution does not occur in the presence of multiple synapses and the system maintains the graduated release response. With this response it would be easier for the system to induce multiple firings during periods of intent focus (static stimulus), thus releasing more neurotransmitters than normal synaptic areas creating a state of over-excitation or over-synchronization. Second, non-pruned synaptic areas could have abnormal feedback systems, which could result in multiple firings and excess neurotransmitters in the cleft. Taking the binding potential example from above instead of being less than 1 in the situation of static stimuli autistic individuals would have a binding potential at these multiple synapses of greater than 1.

As mentioned above there is a question to how the rate of autism prevalence is increasing at the current rate. It stands to reason that natural genetic mutations are not the cause of this increase due to the required increased variance rate. Therefore, it makes sense to assume that there is some environmental catalyst that is increasing autism prevalence rates. Assume for a moment that these environmental factors exist how do they influence autism rates? There appear to be two valid rationalities: 1) These environmental factors influence the rate of genetic mutation in one or more key genetic neuronal development elements; 2) These factors influence the rate of activity, either positively or negatively, for neuronal development. For example one popular rational could be different factors acting as agonists or antagonists on mGluR1, which could influence pruning rates resulting in the increase in autism rates.

From a standpoint of treatment if abnormal pruning is the chief problem then addressing the pruning would be a little tricky because of the multiple elemnets involved in the pruning mechanism. The principle target would be mGluR1 and based on initial assumptions one would have to neutralize multiple synapse masking, thus one would pharmaceutically introduce an mGluR1 antagonist. However, the dosage and effect this antagonist would have on other parts of the brain is unclear. Autistic diagnosis perhaps could be improved by early observation of visual and motor function over emotional and communication abnormalities based on assumed abnormal pruning alterations. Overall while the pruning theory for autism has been proposed before at points in the past87 it seems to have been neglected in the mist of more gene research, which is unfortunate and is more than likely detrimental to the future treatment of autism.


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