Alzheimer’s Disease is Driven by Beta-Amyloid Generated in the Amyloid Precursor Protein-Independent Pathway and Retained Intraneuronally: Research and Therapeutic Strategies in a New AD Paradigm

Abstract The present article describes a New Paradigm of Alzheimer’s disease (AD). In the Old Paradigm, formalized in the Amyloid Cascade Hypothesis (ACH) theory of AD, beta amyloid (Aβ) is produced, both in health and disease, solely in the amyloid precursor protein (βAPP) proteolytic/secretory pathway. Two βAPP cleavages are involved. First cleavage, by beta-secretase (Beta-site APP Cleaving Enzyme, BACE) between Met671 and Asp672 (numbering according to the βAPP770 isoform), generates the C-terminal fragment of βAPP (C99, reflecting the number of its amino acid residues) and forms the N-terminus of Aβ. Subsequent second cleavage of C99 by gamma-secretase (gamma-site βAPP cleaving enzyme) forms the C-terminus of Aβ, completes its production, and coincides with its secretion. The overproduction of Aβ results in its extracellular accumulation commencing early in life. This triggers a cascade of molecular and cellular events, including formation of neurofibrillary tangles, which results in neurodegeneration. When the extent of neurodegeneration reaches critical levels, symptoms of the disease are manifested. In this Paradigm, Alzheimer’s disorder is a quintessential “slow” disease. The ACH clearly defined therapeutic targets, which included key events of βAPP proteolysis as well as secreted extracellular Aβ. Eventually, a number of candidate AD drugs, highly effective in animal model systems, was developed. Of those, especially successful were inhibitors of beta-sectretase that not only prevented the emergence of AD symptoms, but also reversed them when administered after symptomatic manifestation of the disease in animal models. At this point, there was every reason to hope that a solution to the Alzheimer’s problem is at hand; this, however proved not to be the case. Both the Old and the New Paradigms share the common point of departure, namely that the overproduction of beta-amyloid is the causative basis of AD. The rest of the notions of the New Paradigm are distinctly different from those of the Old one. Formulation of the New Paradigm theory of Alzheimer’s disease was necessitated by the analysis of results of massive human clinical trials of candidate AD drugs that performed outstandingly in animal studies. They all failed in human trials as spectacularly as they succeeded in animal studies. Or did they? Whereas they indeed showed no efficacy whatsoever, they performed perfectly within confines of their design and purpose. For example, a BACE inhibitor verubecestat penetrated the brain of AD patients, greatly inhibited βAPP cleavage, and strongly suppressed extracellular levels of Aβ. It did all this with the same efficiency it exhibited in animal studies, where it indisputably succeeded in mitigating symptoms of the disease. Why did it fail to do so in human clinical trials? This failure, apparently inexplicable within the confines of the ACH, seems as good an occasion as any to apply the central dictum of Sherlock Holmes: “... when you eliminated the impossible, whatever remains, however improbable, must be the truth”. In the case under discussion, after the elimination of the “impossible”, including elements of the ACH, and provided that Alzheimer’s disease is indeed caused by the overproduction of Aβ, a notion strongly supported by experimental data, the “however improbable”, which is consistent with the outcomes of human clinical trials of AD candidate drugs, is the following. 1. In addition to the βAPP proteolytic/secretory process, in Alzheimer’s disease in humans, Aβ is also produced in the βAPP-independent pathway. This pathway is active only in AD patients. It does not operate in animals and healthy humans. 2. The output of the βAPP-independent Aβ generation pathway is retained intraneuronally, and it is this pool of intraneuronal beta-amyloid that causes and sustains Alzheimer’s disease. These notions constitute the core of the New Paradigm theory of AD. Several mechanisms are capable of achieving the above. They include: RNA-dependent βAPP mRNA amplification, a process implicated in overproduction of specific proteins in mammalian cells; the internal initiation of transcription within the human βAPP gene; cleavage within βAPP mRNA; the internal initiation of translation within βAPP mRNA. Conceptually, in the context of the present article, the nature of the mechanism generating Aβ independently of βAPP is not important; in every case, identical therapeutic strategies would be indicated. All potential mechanisms of βAPP-independent Aβ generation share several common features. (a) In each case, the expression of a crucial component(s) required for activation of a specific mechanism is induced by the integrated stress response (ISR) elicited via OMA1-DELE1- HRI signaling pathway activated by mitochondrial dysfunction triggered by over-the-threshold levels of βAPP-derived Aβ accumulated intracellularly through cellular uptake of secreted Aβ as well as by retention of a fraction of beta-amyloid produced in the βAPP proteolytic pathway. (b) In every potential mechanism of βAPP-independent Aβ generation, translation initiates at the AUG normally encoding Met671 of βAPP and results in C100, i.e. N-terminal Met-containing C99, which is subsequently cleaved by gamma-secretase to produce Aβ (or Met-Aβ). (c) N-terminal Met of C100 is removed post- rather than co-translationally. Therefore a steady-state population of C100 and, possibly, of N-terminal Met-containing Aβ should occur within human neuronal cells with the activated βAPP-independent Aβ production pathway; their detection would provide irrefutable proof of operation of the pathway. (d) The Aβ (or Met-Aβ) output of every potential mechanism is retained intraneuronally. (e) Once activated, every potential βAPP-independent mechanism would, through generation of intraneuronally retained Aβ, sustain mitochondrial dysfunction and support the activity of the OMA1-DELE1-HRI signaling pathway, which, in turn, will promote, via elicitation of the ISR, the operation of the βAPP-independent Aβ production pathway. These self-perpetuating {βAPP-independent generation of intracellularly retained Aβ}/{mitochondrial dysfunction} mutual feedback cycles constitute the “Engine” that drives Alzheimer’s disease. The life- long accumulation of intraneuronal βAPP-derived Aβ to critical levels, sufficient to trigger mitochondrial dysfunction, plays the role of a starter motor in getting car engine moving in a self-sustainable manner. Only when the AD “Engine” is activated does the disease commence. In the New Paradigm, therefore, Alzheimer’s disorder is a “fast” disease that can be treated and cured at the symptomatic stages. The present article proposes conceptually novel research and therapeutic strategies and suggests that BACE activation (yes, activation!) could be a valid approach in AD therapy.