The identification of Aβ peptides as the major components of amyloid plaque and the discovery in familial cases of Alzheimer’s diseases (AD) of multiple mutations in the genes implicated in their production led to the formalization of the Amyloid Hypothesis (AHyp) . The AHyp asserts that Aβ peptides misfold into toxic aggregates that initiate and drive the pathological mechanisms leading to neurodegeneration and AD. AHyp has been the dominant working hypothesis in the AD field and the primary paradigm directing the development of drug-based therapies for the last three decades .
However, despite thousands of studies intended to validate the AHyp, and after thousands of other studies presenting alternative AD explanations refuting the AHyp, the etiology of AD remains unsettled. Indeed, the authors of the recent NIA-AA research framework, which surprisingly redefined AD based on biological markers, stated that we are right where Alois Alzheimer, Oskar Fischer, and their colleagues left the field a century ago: “We do not know what causes AD, and we have no effective treatments. Although Aβ plaques and pathologic tau are the hallmark pathologic changes, we have limited understanding of how they came about. In this setting, it is important to examine all possible mechanisms” . How can this be? How many dozens, hundreds, thousands, or even tens of thousands of studies does it take to prove or disprove a hypothesis?
According to their authors and supporters, each AD hypothesis is supported by strong evidence and persuasive arguments, as if there were a bottomless pit of findings and arguments that scientists can cherry pick (often with the help of a versatile reference manager) to support almost any idea. It is not clear, though, whether this disturbing reality has to do with science, in the sense that AD and other enigmatic neurodegenerative diseases, including Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Creutzfeldt-Jakob disease (CJD), just happen to be very difficult to study and to understand, or whether we already have answers (see  for a glimpse of hope), but the problem is with the way the scientific enterprise operates, which precludes a resolution.
Although it would make sense to have hypotheses scientifically proven before they are used to direct very expensive clinical trials involving thousands of innocent patients, in the case of AHyp this rationale was turned upside down, and the clinical trials were projected to represent AHyp falsifiability tests. However, after each unsuccessful clinical trial, even after treatments that reached their objective of completely removing the amyloid, the AHyp falsifiability goalposts were moved (discussed in ).
One approach to advance the AD field would be to embrace a broader and more inclusive multifactorial perspective on AD etiology and on the biological role of Aβ in AD, as suggested two decades ago by Mark Smith, George Perry, and their colleagues [6-8]. Such an approach would advance the field by increasing the explanatory power of all reproducible data and observations, which currently are only regarded as significant within the narrow context of specific hypotheses and cannot be readily explained in the context of AHyp [5-11]. Very importantly, this multifactorial perspective would open AD to a myriad of immediate prevention and therapeutic approaches, which, unlike the amyloid-based therapies promoted by the AHyp and the NIA-AA research framework, would help not only with AD but with many other human health issues and diseases. Interestingly, the U. S. National Plan to address AD and related dementias was just updated  to include actions for promoting healthy aging by reducing risk factors, including physical inactivity, hypertension, smoking or excessive alcohol drinking, unhealthy diet, diabetes, infectious diseases, exposure to toxins, physical brain trauma, depression, and low cognitive/social/educational attainments, among many other damaging factors and conditions associated with aging. These sensible interventions should also relieve some of the anxiety/desperation in persons with dementia and their families (as well as in some of their supporting groups), who have been emphatically primed (or self-primed) to expect (or even demand) pharmaceutical solutions (discussed in [13, 14]).
Another way to advance the AD field would be to replace the current clinical-based falsifiability test of AHyp with a science-based test. Ideally, this test should be applied to other hypotheses in the AD field, as well as to those that address the etiology of PD, ALS, CJD, and other enigmatic neurodegenerative diseases. Furthermore, if this aspiration were not already high enough, the results of this test should also guide the way forward in virtually all basic, biomedical, and public health fields. This objective might appear to be part of a science fiction scenario, but it is based on a single, relatively straightforward genetic test that addresses all human biological traits and conditions by determining and analyzing the extent of human genetic variation. Fortunately, the genome sequencing technologies for generating this data are well established and routinely used in hundreds of laboratories around the world, so the only obstacle that might stand in the way of implementing this extraordinary project has nothing to do with science but rather with a poorly run scientific process.
The AHyp falsifiability test proposed here is straightforward: (i) individuals who do not produce the Aβ peptides (denoted here with the annotation Aβ -/-) due to certain mutations (i.e., genetic variation) in the genes involved in their production are expected to be completely resistant to AD-like dementia (in other words, “No Aβ, No AD”; not to be confused with “No Aβ amyloid/plaques, No AD”—the futile therapeutic approach associated with the therapeutic arm of AHyp), and (ii) individuals with reduced production of Aβ peptides (Aβ +/-) are expected to be at least partially, if not fully, resistant to AD-like dementia. The same rationale applies also to all the other proteins implicated in neurodegenerative diseases, including tau, α-synuclein, prion protein (PrP), and TAR DNA-binding protein 43 (TDP-43).
The major appeal of the AHyp was its rationalization in the context of genetic findings and analysis, which, when compared to biological and pathological findings, are usually more informative. However, the genetic data loses some of its clarity when, in conjunction with environmental factors, it is translated into a phenotype, both in health and disease. For example, although the early genetic data indicated that the Aβ peptides/assemblies play a casual role in familial AD, and possibly in sporadic AD, these genetic findings were neutral with regard to the disease mechanism—gain of (toxic) function (GOF) versus loss of (biological) function (LOF)—both of which were thought to be the result of a protein misfolding process leading to amyloid formation. So, why did the AHyp embrace the GOF paradigm? Was it due to a poor scientific judgment?
Not when judged in the context of two well-established paradigms in protein chemistry and biology, the protein misfolding dogma  and the Nobel Prize winning prion hypothesis , both of which consider the amylogenic process to be incompatible with physiological functions but conducive to pathogenic mechanisms leading to disease. However, the protein misfolding dogma and the prion hypothesis are both questionable and, likely, have confused or misled the thinking on the role of Aβ, tau, α-synuclein, TDP-43, PrP, and other amyloidogenic proteins in the disease process [17-23]. Nevertheless, even if we recognize that the advocates of the AHyp and its surrogate concepts, such as the notion of self-propagating pathogenic protein aggregates , have embraced the protein misfolding dogma and the prion hypothesis in good faith, on account of their (near) universal acceptance by the scientific community, the lack of attention to the biological function of Aβ and the other amyloidogenic proteins implicated in neurodegenerative diseases, which would enable scientists to assess the merit of the GOF and LOF paradigms, is difficult to excuse, especially considering that it was not for lack of warnings. For example, six years after the Nobel Prize for the prion hypothesis, Kurt Wunthrich (himself a Nobel laureate for his work on the three-dimensional structure of biological macromolecules) submitted that: “we must understand the function of the normal prion protein before we can understand prion diseases” (quoted in ). Or, consider the apparent distress of John Hardy, one of the AHyp founders, when critically reappraising his own hypothesis: “A major concern about the amyloid hypothesis is that we have very little idea as to the functions of APP or the possible function of Aβ” .
If the GOF model is indeed problematic, then the alternative paradigm, the LOF (see above), must be correct. Correct? Not so (see below). Nevertheless, unlike the GOF-based AHyp and its supporting paradigms (i.e., the protein misfolding dogma and the prion hypothesis), the LOF hypothesis brings the biological function of all these proteins front and center in explaining the etiology of neurodegenerative diseases, as it prompts the critical question: what kind of biological function, when lost, would lead to neurodegeneration?
Although the LOF paradigm (i.e., loss of biological function due to protein misfolding) in the causation of AD, PD, ALS, and CJD has recently been revived , the current genetic data does not have the power to differentiate between GOF and LOF mechanisms, except perhaps in the case of CJD and other transmissible spongiform encephalopathies (TSEs). In 2012, the first natural genetic variation, a nonsense mutation in the PrP gene, which leads to a PrP -/- phenotype, was discovered among healthy homozygous dairy goats in Norway . These goats did not develop any pathology/disease analogous to TSE, nor did the numerous transgenic PrP knockouts generated in mice, sheep, goats, cows, and other animal models. This also appears to be the case with many Aβ, tau, α-synuclein, and TDP-43 transgenic knockouts, but the findings need to be corroborated by natural knockouts.
As I proposed previously, the aggregation of PrP, Aβ, tau, α-synuclein, and TDP-43 does not induce a LOF, nor a toxic GOF, but rather is an activity associated with their evolutionary selected biological function in innate immunity [22, 23]. The innate immunity function of these proteins confers on them both protective and destructive properties, a characteristic common to many other members of the immune system.
The major strength of the innate immunity hypothesis is that it can explain the results of many studies that have been used to support the other hypotheses in the field, including the majority of the findings believed to support the AHyp. The immune reactivity paradigm is gaining increased support (see [29, 30]), but it needs to be fully evaluated. The human genetic variation study proposed here would also go a long way toward deciphering the biological function of all proteins implicated in AD, as well as the function of all other human proteins. Moreover, by identifying the human genetic variation, including heterozygous and, possibly, homozygous knockouts, which would generate the Aβ +/- and Aβ -/- phenotype, respectively, the results of this extraordinary study would also provide a scientific falsifiability test for the AHyp.
A human genetic variation project of this magnitude might appear daunting. Although the logistics and financing of a study of this magnitude are challenging, they are nevertheless feasible and realistic. For example, a recent exome sequencing and analysis study of 454,787 UK Biobank participants indicates that sequencing five million individuals would facilitate the identification of 500+ heterozygous LOF carriers for the majority of human genes . Considering that the heterozygous or even homozygous knockouts of the genes coding for Aβ, tau, α-synuclein, TDP-43, and PrP are not lethal or highly deleterious, they are likely to be found at expected frequency in outbred human populations and, possibly, at higher frequency in consanguineous populations.
Moreover, a coordinated major project of this nature will increase the productivity and standardization of the sequencing and analysis processes while dramatically reducing the cost, which would be a fraction of the cost of clinical trials. Furthermore, there are already dozens of ongoing or planned human genome sequencing projects throughout the world (e.g., [32, 33]), covering millions of people, a number that at the current rate will soon be in the tens of millions.
In light of the newly proposed Advanced Research Projects Agency for Health (ARPA-H) and its intent “to develop breakthroughs—to prevent, detect, and treat diseases like Alzheimer's, diabetes, and cancer” , the scope and the timing for analyzing the extent of human genetic variation are in full concordance with ARPA-H’s overall mission “to accelerate the pace of breakthroughs to transform medicine and health” .
Claudiu I. Bandea, PhD
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