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In clinical trials, lecanemab showed statistically significant yet marginal slowdown of Alzheimer’s disease (AD)-associated cognitive decline. This could be due to its sub-optimal design and/or deployment; alternatively, its limited efficiency could be intrinsic. Distinguishing between the two is of great importance considering the acute need of efficient AD therapy and tremendous resources being invested in its pursuit. The present study analyzes the mode of operation of lecanemab within the framework of recently proposed Amyloid Cascade Hypothesis 2.0 and concludes that the second is correct. It suggests that substantial improvement of the efficiency of lecanemab in symptomatic AD is unlikely and proposes the alternative therapeutic strategy.

Recently, we proposed the Amyloid Cascade Hypothesis 2.0 (ACH2.0), a reformulation of the ACH. In the former, in contrast to the latter, Alzheimer’s disease (AD) is driven by intraneuronal amyloid-β (iAβ) and occurs in two stages. In the first, relatively benign stage, Aβ protein precursor (AβPP)-derived iAβ activates, upon reaching a critical threshold, the AβPP-independent iAβ-generating pathway, triggering a devastating second stage resulting in neuronal death. While the ACH2.0 remains aligned with the ACH premise that Aβ is toxic, the toxicity is exerted because of intra- rather than extracellular Aβ. In this framework, a once-in-a-lifetime-only iAβ depletion treatment via transient activation of BACE1 and/or BACE2 (exploiting their Aβ-cleaving activities) or by any means appears to be the best therapeutic strategy for AD. Whereas the notion of differentially derived iAβ being the principal moving force at both AD stages is both plausible and elegant, a possibility remains that the second AD stage is enabled by an AβPP-derived iAβ-activated self-sustaining mechanism producing a yet undefined deleterious “substance X” (sX) which anchors the second AD stage. The present study generalizes the ACH2.0 by incorporating this possibility and shows that, in this scenario, the iAβ depletion therapy may be ineffective at symptomatic AD stages but fully retains its preventive potential for both AD and the aging-associated cognitive decline, which is defined in the ACH2.0 framework as the extended first stage of AD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus-encoded spike protein and fusion between the viral and target cell membranes. We report here that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor-binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity. These receptor-like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc-gamma receptor I, a chimera of their antigen-binding fragment with the transmembrane domain of ACE2 or a membrane-bound B cell receptor, indicating that ACE2 and its specific interactions with the spike protein are dispensable for SARS-CoV-2 entry. These results suggest that antibody responses against SARS-CoV-2 may expand the viral tropism to otherwise nonpermissive cell types; they have important implications for viral transmission and pathogenesis.

We posit that Alzheimer’s disease (AD) is driven by amyloid-β (Aβ) generated in the amyloid-β protein precursor (AβPP) independent pathway activated by AβPP-derived Aβ accumulated intraneuronally in a life-long process. This interpretation constitutes the Amyloid Cascade Hypothesis 2.0 (ACH2.0). It defines a tandem intraneuronal-Aβ (iAβ)-anchored cascade occurrence: intraneuronally-accumulated, AβPP-derived iAβ triggers relatively benign cascade that activates the AβPP-independent iAβ-generating pathway, which, in turn, initiates the second, devastating cascade that includes tau pathology and leads to neuronal loss. The entire output of the AβPP-independent iAβ-generating pathway is retained intraneuronally and perpetuates the pathway’s operation. This process constitutes a self-propagating, autonomous engine that drives AD and ultimately kills its host cells. Once activated, the AD Engine is self-reliant and independent from Aβ production in the AβPP proteolytic pathway; operation of the former renders the latter irrelevant to the progression of AD by relegating its iAβ contribution to insignificant, and brands its manipulation for therapeutic purposes, such as BACE (beta-site AβPP-cleaving enzyme) inhibition, as futile. In the proposed AD paradigm, the only valid direct therapeutic strategy is targeting the engine’s components, and the most effective feasible approach appears to be the activation of BACE1 and/or of its homolog BACE2, with the aim of exploiting their Aβ-cleaving activities. Such treatment would collapse the iAβ population and ‘reset’ its levels below those required for the operation of the AD Engine. Any sufficiently selective iAβ-depleting treatment would be equally effective. Remarkably, this approach opens the possibility of a short-duration, once-in-a-lifetime-only or very infrequent, preventive or curative therapy for AD; this therapy would be also effective for prevention and treatment of the ‘common’ pervasive aging-associated cognitive decline. The ACH2.0 clarifies all ACH-unresolved inconsistencies, explains the widespread ‘resilience to AD’ phenomenon, predicts occurrences of a category of AD-afflicted individuals without excessive Aβ plaque load and of a novel type of familial insusceptibility to AD; it also predicts the lifespan-dependent inevitability of AD in humans if untreated preventively. The article details strategy and methodology to generate an adequate AD model and validate the hypothesis; the proposed AD model may also serve as a research and drug development platform.

Delta’s spike Understanding the molecular mechanisms of the increased transmissibility and immune evasion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is critical to guiding current and future intervention strategies. Zhang et al . determined cryo–electron microscopy structures of the full-length spike protein trimers of the Delta, Kappa, and Gamma variants of SARS-CoV-2 and studied their function and antigenic properties. The Delta spike protein fused membranes more efficiently at low levels of the cellular receptor ACE2, and its pseudotyped viruses infected target cells substantially more rapidly than all other variants tested, possibly at least partly accounting for its heightened transmissibility. Mutations of each variant rearranged the antigenic surface of the N-terminal domain of the spike protein but only caused local changes in the receptor-binding domain, consistent with greater resistance to neutralizing antibodies. These findings elucidate the molecular events that have led these viruses to adapt in human communities and to evade host immunity. —VV

Entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells depends on refolding of the virus-encoded spike protein from a prefusion conformation, metastable after cleavage, to a lower energy, stable postfusion conformation. This transition overcomes kinetic barriers for fusion of viral and target cell membranes. We report here a cryo-EM structure of the intact postfusion spike in a lipid bilayer that represents single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membraneinteracting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.

Molecular Biology, a branch of science established to examine the flow of information from “letters” encrypted into DNA structure to functional proteins, was initially defined by a concept of DNA-to-RNA-to-Protein information movement, a notion termed the Central Dogma of Molecular Biology. RNA-dependent mRNA amplification, a novel mode of eukaryotic protein-encoding RNA-to-RNA-to-Protein genomic information transfer, constitutes the extension of the Central Dogma in the context of mammalian cells. It was shown to occur in cellular circumstances requiring exceptionally high levels of production of specific polypeptides, e.g. globin chains during erythroid differentiation or defined secreted proteins in the context of extracellular matrix deposition. Its potency is reflected in the observed cellular levels of the resulting amplified mRNA product: At the peak of the erythroid differentiation, for example, the amount of globin mRNA produced in the amplification pathway is about 1500-fold higher than the amount of its conventionally generated counterpart in the same cells. The cellular enzymatic machinery at the core of this process, RNA-dependent RNA polymerase activity (RdRp), albeit in a non-conventional form, was shown to be constitutively and ubiquitously present, and RNA-dependent RNA synthesis (RdRs) appeared to regularly occur, in mammalian cells. Under most circumstances, the mammalian RdRp activity produces only short antisense RNA transcripts. Generation of complete antisense RNA transcripts and amplification of mRNA molecules require the activation of inducible components of the mammalian RdRp complex. The mechanism of such activation is not clear. The present article suggests that it is triggered by a variety of cellular stresses and occurs in the context of stress responses in general and within the framework of the integrated stress response (ISR) in particular. In this process, various cellular stresses activate, in a stress type-specific manner, defined members of the mammalian translation initiation factor 2α, eIF2α, kinase family: PKR, GCN2, PERK and HRI. Any of these kinases, in an activated form, phosphorylates eIF2α. This results in suppression of global cellular protein synthesis but also in activation of expression of select group of transcription factors including ATF4, ATF5 and CHOP. These transcription factors either function as inducible components of the RdRp complex or enable their expression. The assembly of the competent RdRp complex activates mammalian RNA-dependent mRNA amplification, which appears to be a two-tier process. Tier One is a “chimeric” pathway, named so because it results in an amplified chimeric mRNA molecule containing a fragment of the antisense RNA strand at its 5’ terminus. Tier Two further amplifies one of the two RNA end products of the chimeric pathway and constitutes the physiologically occurring intracellular polymerase chain reaction, iPCR. Depending on the structure of the initial mRNA amplification progenitor, the chimeric pathway, Tier One, may result in multiple outcomes including chimeric mRNA that produces either a polypeptide identical to the original, conventional mRNA progenitor-encoded protein or only its C-terminal fragment, CTF. The chimeric RNA end product of Tier One may also produce a polypeptide that is non-contiguously encoded in the genome, activate translation from an open reading frame, which is “silent” in a conventionally transcribed mRNA, or initiate an abortive translation. In sharp contrast, regardless of the outcome of Tier One, the mRNA end product of Tier Two of mammalian mRNA amplification, the iPCR pathway, always produces a polypeptide identical to a conventional mRNA progenitor-encoded protein. This discordance is referred to as the Two-Tier Paradox and discussed in detail in the present article. On the other hand, both Tiers are similar in that they result in heavily modified mRNA molecules resistant to reverse transcription, undetectable by reverse transcription-based methods of sequencing and therefore constituting a proverbial “Dark Matter” mRNA, despite being highly ubiquitous. It appears that in addition to their other functions, the modifications of the amplified mRNA render it compatible, unlike the bulk of cellular mRNA, with phosphorylated eIF2α in translation, implying that in addition to being extraordinarily abundant due to the method of its generation, amplified mRNA is also preferentially translated under the ISR conditions, thus augmenting the efficiency of the amplification process. The vital importance of powerful mechanisms of amplification of protein-encoding genomic information in normal physiology is self-evident. Their malfunctions or misuse appear to be associated with two types of abnormalities, the deficiency of a protein normally produced by these mechanisms and the mRNA amplification-mediated overproduction of a protein normally not generated by such a process. Certain classes of beta-thalassemia exemplify the first type, whereas the second type is represented by overproduction of beta-amyloid in Alzheimer’s disease. Moreover, the proposed mechanism of Alzheimer’s disease allows a crucial and verifiable prediction, namely that the disease-causing intraneuronally retained variant of beta-amyloid differs from that produced conventionally by βAPP proteolysis in that it contains the additional methionine or acetylated methionine at its N-terminus. Because of its extraordinary evidential value as a natural reporter of the mRNA amplification pathway, this feature, if proven, would, arguably, constitute the proverbial Holy Grail not only for Alzheimer’s disease but also for the mammalian RNA-dependent mRNA amplification field in general. Both examples are discussed in detail in the present article, which summarizes and systematizes our current understanding of the field and describes two categories of reporter constructs, one for the chimeric Tier of mRNA amplification, another for the iPCR pathway; both reporter types are essential for elucidating underlying molecular mechanisms. It also suggests, in light of the recently demonstrated feasibility of RNA-based vaccines, that the targeted intracellular amplification of exogenously introduced amplification-eligible antigen-encoding mRNAs via the induced or naturally occurring RNA-dependent mRNA amplification pathway could be of substantial benefit in triggering a fast and potent immune response and instrumental in the development of future vaccines. Similar approaches can also be effective in achieving efficient and sustained expression of exogenous mRNA in mRNA therapeutics.

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.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bearing an unusually high number of mutations, has become a dominant strain in many countries within several weeks. We report here structural, functional and antigenic properties of its full-length spike (S) protein with a native sequence in comparison with those of previously prevalent variants. Omicron S requires a substantially higher level of host receptor ACE2 for efficient membrane fusion than other variants, possibly explaining its unexpected cellular tropism. Mutations not only remodel the antigenic structure of the N-terminal domain of the S protein, but also alter the surface of the receptor-binding domain in a way not seen in other variants, consistent with its remarkable resistance to neutralizing antibodies. These results suggest that Omicron S has acquired an extraordinary ability to evade host immunity by excessive mutations, which also compromise its fusogenic capability.

De novo production of RNA on RNA template, a process known as RNA-dependent RNA synthesis, RdRs, and the enzymatic activity conducting it, RNA-dependent RNA polymerase, RdRp, were initially considered to be exclusively virus-specific. Eventually, however, the occurrence of RdRs and the ubiquitous presence of conventional RdRp were demonstrated in numerous eukaryotic organisms. The evidence that the enzymatic machinery capable of RdRs is present in mammalian cells was derived from studies of viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the mammalian host; thus firmly establishing its occurrence and functionality. Moreover, it became clear that RdRp activity, apparently in a non-conventional form, is constitutively present in most, if not in all, mammalian cells. Because such activity was shown to produce short transcripts, given its apparent involvement in RNA interference phenomena, and because double-stranded RNA is known to trigger cellular responses leading to its degradation, it was generally assumed that its role in mammalian cells is restricted to a regulatory function. However, at the same time, an enzymatic activity capable of generating complete antisense RNA complements of mRNAs was discovered in mammalian cells undergoing terminal differentiation. Moreover, observations of widespread synthesis of antisense RNAs initiating at the 3'poly(A) of mRNAs in human cells suggested an extensive cellular utilization of mammalian RdRp. These results led to the development of a model of RdRp-facilitated and antisense RNA-mediated amplification of mammalian mRNA. Recent detection of the major model-predicted identifiers, chimeric RNA intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner, as well as the identification of a putative chimeric RNA end product of this process, validated the proposed model. The results corroborating mammalian RNA-dependent mRNA amplification were obtained in vivo with cells undergoing terminal erythroid differentiation and programmed for only a short survival span. This raises a question of whether mammalian RNA-dependent mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation or a general physiological phenomenon. The present study addresses this question by testing for the occurrence of RNA-dependent amplification of mRNA encoding extracellular matrix proteins abundantly produced throughout the tissue and organ development and homeostasis, an exceptionally revealing indicator of the range and scope of this phenomenon. We report here the detection of major identifiers of RNA-dependent amplification of mRNA encoding α1, β1, and γ1 chains of laminin in mouse tissues producing large quantities of extracellular matrix proteins. The results obtained warrant reinterpretation of the mechanisms involved in ubiquitous and abundant production and deposition of extracellular matrix proteins, confirm the occurrence of mammalian RNA-dependent mRNA amplification as a new mode of genomic protein-encoding information transfer, and establish it as a general physiological phenomenon.