On the Inadequacy of the Current Transgenic Animal Models of Alzheimer's Disease: The Path Forward.

For at least two reasons, the current transgenic animal models of Alzheimer's disease (AD) appear to be patently inadequate. They may be useful in many respects, the AD models; however, they are not. First, they are incapable of developing the full spectrum of the AD pathology. Second, they respond spectacularly well to drugs that are completely ineffective in the treatment of symptomatic AD. These observations indicate that both the transgenic animal models and the drugs faithfully reflect the theory that guided the design and development of both, the amyloid cascade hypothesis (ACH), and that both are inadequate because their underlying theory is. This conclusion necessitated the formulation of a new, all-encompassing theory of conventional AD-the ACH2.0. The two principal attributes of the ACH2.0 are the following. One, in conventional AD, the agent that causes the disease and drives its pathology is the intraneuronal amyloid-ß (iAß) produced in two distinctly different pathways. Two, following the commencement of AD, the bulk of Aß is generated independently of Aß protein precursor (AßPP) and is retained inside the neuron as iAß. Within the framework of the ACH2.0, AßPP-derived iAß accumulates physiologically in a lifelong process. It cannot reach levels required to support the progression of AD; it does, however, cause the disease. Indeed, conventional AD occurs if and when the levels of AßPP-derived iAß cross the critical threshold, elicit the neuronal integrated stress response (ISR), and trigger the activation of the AßPP-independent iAß generation pathway; the disease commences only when this pathway is operational. The iAß produced in this pathway reaches levels sufficient to drive the AD pathology; it also propagates its own production and thus sustains the activity of the pathway and perpetuates its operation. The present study analyzes the reason underlying the evident inadequacy of the current transgenic animal models of AD. It concludes that they model, in fact, not Alzheimer's disease but rather the effects of the neuronal ISR sustained by AßPP-derived iAß, that this is due to the lack of the operational AßPP-independent iAß production pathway, and that this mechanism must be incorporated into any successful AD model faithfully emulating the disease. The study dissects the plausible molecular mechanisms of the AßPP-independent iAß production and the pathways leading to their activation, and introduces the concept of conventional versus unconventional Alzheimer's disease. It also proposes the path forward, posits the principles of design of productive transgenic animal models of the disease, and describes the molecular details of their construction.

Quintessential Synergy: Concurrent Transient Administration of Integrated Stress Response Inhibitors and BACE1 and/or BACE2 Activators as the Optimal Therapeutic Strategy for Alzheimer's Disease.

The present study analyzes two potential therapeutic approaches for Alzheimer's disease (AD). One is the suppression of the neuronal integrated stress response (ISR). Another is the targeted degradation of intraneuronal amyloid-beta (iAß) via the activation of BACE1 (Beta-site Aß-protein-precursor Cleaving Enzyme) and/or BACE2. Both approaches are rational. Both are promising. Both have substantial intrinsic limitations. However, when combined in a carefully orchestrated manner into a composite therapy they display a prototypical synergy and constitute the apparently optimal, potentially most effective therapeutic strategy for AD.

ACH2.0/E, the Consolidated Theory of Conventional and Unconventional Alzheimer's Disease: Origins, Progression, and Therapeutic Strategies.

The centrality of amyloid-beta (Aß) is an indisputable tenet of Alzheimer's disease (AD). It was initially indicated by the detection (1991) of a mutation within Aß protein precursor (AßPP) segregating with the disease, which served as a basis for the long-standing Amyloid Cascade Hypothesis (ACH) theory of AD. In the intervening three decades, this notion was affirmed and substantiated by the discovery of numerous AD-causing and AD-protective mutations with all, without an exception, affecting the structure, production, and intraneuronal degradation of Aß. The ACH postulated that the disease is caused and driven by extracellular Aß. When it became clear that this is not the case, and the ACH was largely discredited, a new theory of AD, dubbed ACH2.0 to re-emphasize the centrality of Aß, was formulated. In the ACH2.0, AD is caused by physiologically accumulated intraneuronal Aß (iAß) derived from AßPP. Upon reaching the critical threshold, it triggers activation of the autonomous AßPP-independent iAß generation pathway; its output is retained intraneuronally and drives the AD pathology. The bridge between iAß derived from AßPP and that generated independently of AßPP is the neuronal integrated stress response (ISR) elicited by the former. The ISR severely suppresses cellular protein synthesis; concurrently, it activates the production of a small subset of proteins, which apparently includes components necessary for operation of the AßPP-independent iAß generation pathway that are absent under regular circumstances. The above sequence of events defines "conventional" AD, which is both caused and driven by differentially derived iAß. Since the ISR can be elicited by a multitude of stressors, the logic of the ACH2.0 mandates that another class of AD, referred to as "unconventional", has to occur. Unconventional AD is defined as a disease where a stressor distinct from AßPP-derived iAß elicits the neuronal ISR. Thus, the essence of both, conventional and unconventional, forms of AD is one and the same, namely autonomous, self-sustainable, AßPP-independent production of iAß. What distinguishes them is the manner of activation of this pathway, i.e., the mode of causation of the disease. In unconventional AD, processes occurring at locations as distant from and seemingly as unrelated to the brain as, say, the knee can potentially trigger the disease. The present study asserts that these processes include traumatic brain injury (TBI), chronic traumatic encephalopathy, viral and bacterial infections, and a wide array of inflammatory conditions. It considers the pathways which are common to all these occurrences and culminate in the elicitation of the neuronal ISR, analyzes the dynamics of conventional versus unconventional AD, shows how the former can morph into the latter, explains how a single TBI can hasten the occurrence of AD and why it takes multiple TBIs to trigger the disease, and proposes the appropriate therapeutic strategies. It posits that yet another class of unconventional AD may occur where the autonomous AßPP-independent iAß production pathway is initiated by an ISR-unrelated activator, and consolidates the above notions in a theory of AD, designated ACH2.0/E (for expanded ACH2.0), which incorporates the ACH2.0 as its special case and retains the centrality of iAß produced independently of AßPP as the driving agent of the disease.

The Amyloid Cascade Hypothesis 2.0 for Alzheimer's Disease and Aging-Associated Cognitive Decline: From Molecular Basis to Effective Therapy.

With the long-standing amyloid cascade hypothesis (ACH) largely discredited, there is an acute need for a new all-encompassing interpretation of Alzheimer's disease (AD). Whereas such a recently proposed theory of AD is designated ACH2.0, its commonality with the ACH is limited to the recognition of the centrality of amyloid-ß (Aß) in the disease, necessitated by the observation that all AD-causing mutations affect, in one way or another, Aß. Yet, even this narrow commonality is superficial since AD-causing Aß of the ACH differs distinctly from that specified in the ACH2.0: Whereas in the former, the disease is caused by secreted extracellular Aß, in the latter, it is triggered by Aß-protein-precursor (AßPP)-derived intraneuronal Aß (iAß) and driven by iAß generated independently of AßPP. The ACH2.0 envisions AD as a two-stage disorder. The first, asymptomatic stage is a decades-long accumulation of AßPP-derived iAß, which occurs via internalization of secreted Aß and through intracellular retention of a fraction of Aß produced by AßPP proteolysis. When AßPP-derived iAß reaches critical levels, it activates a self-perpetuating AßPP-independent production of iAß that drives the second, devastating AD stage, a cascade that includes tau pathology and culminates in neuronal loss. The present study analyzes the dynamics of iAß accumulation in health and disease and concludes that it is the prime factor driving both AD and aging-associated cognitive decline (AACD). It discusses mechanisms potentially involved in AßPP-independent generation of iAß, provides mechanistic interpretations for all principal aspects of AD and AACD including the protective effect of the Icelandic AßPP mutation, the early onset of FAD and the sequential manifestation of AD pathology in defined regions of the affected brain, and explains why current mouse AD models are neither adequate nor suitable. It posits that while drugs affecting the accumulation of AßPP-derived iAß can be effective only protectively for AD, the targeted degradation of iAß is the best therapeutic strategy for both prevention and effective treatment of AD and AACD. It also proposes potential iAß-degrading drugs.

Next Generation Therapeutic Strategy for Treatment and Prevention of Alzheimer's Disease and Aging-Associated Cognitive Decline: Transient, Once-in-a-Lifetime-Only Depletion of Intraneuronal Aß (iAß) by Its Targeted Degradation via Augmentation of Intra-iAß-Cleaving Activities of BACE1 and/or BACE2.

Although the long-standing Amyloid Cascade Hypothesis (ACH) has been largely discredited, its main attribute, the centrality of amyloid-beta (Aß) in Alzheimer's disease (AD), remains the cornerstone of any potential interpretation of the disease: All known AD-causing mutations, without a single exception, affect, in one way or another, Aß. The ACH2.0, a recently introduced theory of AD, preserves this attribute but otherwise differs fundamentally from the ACH. It posits that AD is a two-stage disorder where both stages are driven by intraneuronal (rather than extracellular) Aß (iAß) albeit of two distinctly different origins. The first asymptomatic stage is the decades-long accumulation of Aß protein precursor (AßPP)-derived iAß to the critical threshold. This triggers the activation of the self-sustaining AßPP-independent iAß production pathway and the commencement of the second, symptomatic AD stage. Importantly, Aß produced independently of AßPP is retained intraneuronally. It drives the AD pathology and perpetuates the operation of the pathway; continuous cycles of the iAß-stimulated propagation of its own AßPP-independent production constitute an engine that drives AD, the AD Engine. It appears that the dynamics of AßPP-derived iAß accumulation is the determining factor that either drives Aging-Associated Cognitive Decline (AACD) and triggers AD or confers the resistance to both. Within the ACH2.0 framework, the ACH-based drugs, designed to lower levels of extracellular Aß, could be applicable in the prevention of AD and treatment of AACD because they reduce the rate of accumulation of AßPP-derived iAß. The present study analyzes their utility and concludes that it is severely limited. Indeed, their short-term employment is ineffective, their long-term engagement is highly problematic, their implementation at the symptomatic stages of AD is futile, and their evaluation in conventional clinical trials for the prevention of AD is impractical at best, impossible at worst, and misleading in between. In contrast, the ACH2.0-guided Next Generation Therapeutic Strategy for the treatment and prevention of both AD and AACD, namely the depletion of iAß via its transient, short-duration, targeted degradation by the novel ACH2.0-based drugs, has none of the shortcomings of the ACH-based drugs. It is potentially highly effective, easily evaluable in clinical trials, and opens up the possibility of once-in-a-lifetime-only therapeutic intervention for prevention and treatment of both conditions. It also identifies two plausible ACH2.0-based drugs: activators of physiologically occurring intra-iAß-cleaving capabilities of BACE1 and/or BACE2.

Mycobacterial and Plasmodium ovale-associated destruction of the jaw bones.

OBJECTIVES: The project aims were to identify infectious mechanisms responsible for an extreme form of mandibular osteonecrosis and osteomyelitis in West African populations and test the hypothesis that Mycobacterium tuberculosis plays a pivotal role. MATERIALS AND METHODS: DNA was extracted from mandibular fragments of 9 of 19 patients previously included in a prospective study leading to the mycobacterial hypothesis. Amplified DNAs were used for preparing libraries suitable for next-generation sequencing. For comparison of the whole-genome sequencing data of the 9 patients with DNAs of both microbiota and human tissues, DIAMOND v0.9.26 was used to align sequencing reads to NCBI-nr database and MEGAN 6 for taxonomy binning and identification of Mycobacterium tuberculosis strains. RESULTS: The data show that mandibular bone fragments of all 9 patients not only contain Homo sapiens and Mycobacterium tuberculosis DNAs; they also contain DNAs of Plasmodium ovale wallikeri, Staphylococcus aureus, Staphylococcus hominis, and Prevotella P3-120/intermedia; as well as large numbers of DNAs from other infectious components. CONCLUSIONS: The data obtained provide direct evidence to support the conclusion that combinations of Mycobacterium tuberculosis, Plasmodium ovale wallikeri, and other oral bacteria are involved in this particular type of mandibular destruction in West African individuals of many ages.

Principles of Design of Clinical Trials for Prevention and Treatment of Alzheimer's Disease and Aging-Associated Cognitive Decline in the ACH2.0 Perspective: Potential Outcomes, Challenges, and Solutions.

With the Amyloid Cascade Hypothesis (ACH) largely discredited, the ACH2.0 theory of Alzheimer's disease (AD) has been recently introduced. Within the framework of the ACH2.0, AD is triggered by amyloid-ß protein precursor (AßPP)-derived intraneuronal Aß (iAß) and is driven by iAß produced in the AßPP-independent pathway and retained intraneuronally. In this paradigm, the depletion of extracellular Aß or suppression of Aß production by AßPP proteolysis, the two sources of AßPP-derived iAß, would be futile in symptomatic AD, due to its reliance on iAß generated independently of AßPP, but effective in preventing AD and treating Aging-Associated Cognitive Decline (AACD) driven, in the ACH2.0 framework, by AßPP-derived iAß. The observed effect of lecanemab and donanemab, interpreted in the ACH2.0 perspective, supports this notion and mandates AD-preventive clinical trials. Such trials are currently in progress. They are likely, however, to fail or to yield deceptive results if conducted conventionally. The present study considers concepts of design of clinical trials of lecanemab, donanemab, or any other drug, targeting the influx of AßPP-derived iAß, in prevention of AD and treatment of AACD. It analyzes possible outcomes and explains why selection of high-risk asymptomatic participants seems reasonable but is not. It argues that outcomes of such AD preventive trials could be grossly misleading, discusses inevitable potential problems, and proposes feasible solutions. It advocates the initial evaluation of this type of drugs in clinical trials for treatment of AACD. Whereas AD protective trials of these drugs are potentially of an impractical length, AACD clinical trials are expected to yield unequivocal results within a relatively short duration. Moreover, success of the latter, in addition to its intrinsic value, would constitute a proof of concept for the former. Furthermore, this study introduces concepts of the active versus passive iAß depletion, contends that targeted degradation of iAß is the best therapeutic strategy for both prevention and treatment of AD and AACD, proposes potential iAß-degrading drugs, and describes their feasible and unambiguous evaluation in clinical trials.

The Amyloid Cascade Hypothesis 2.0: Generalization of the Concept.

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-ß (iAß) and occurs in two stages. In the first, relatively benign stage, Aß protein precursor (AßPP)-derived iAß activates, upon reaching a critical threshold, the AßPP-independent iAß-generating pathway, triggering a devastating second stage resulting in neuronal death. While the ACH2.0 remains aligned with the ACH premise that Aß is toxic, the toxicity is exerted because of intra- rather than extracellular Aß. In this framework, a once-in-a-lifetime-only iAß depletion treatment via transient activation of BACE1 and/or BACE2 (exploiting their Aß-cleaving activities) or by any means appears to be the best therapeutic strategy for AD. Whereas the notion of differentially derived iAß 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 AßPP-derived iAß-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 iAß 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.

Effect of Lecanemab in Early Alzheimer's Disease: Mechanistic Interpretation in the Amyloid Cascade Hypothesis 2.0 Perspective.

In clinical trials, lecanemab and donanemab showed statistically significant yet marginal slowdown of Alzheimer's disease (AD)-associated cognitive decline. This could be due to their sub-optimal design and/or deployment; alternatively, their 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 and donanemab within the framework of recently proposed Amyloid Cascade Hypothesis 2.0 and concludes that the second possibility is correct. It suggests that substantial improvement of the efficiency of these drugs in symptomatic AD is unlikely and proposes the alternative therapeutic strategy.

The Amyloid Cascade Hypothesis 2.0: On the Possibility of Once-in-a-Lifetime-Only Treatment for Prevention of Alzheimer's Disease and for Its Potential Cure at Symptomatic Stages.

We posit that Alzheimer's disease (AD) is driven by amyloid-ß (Aß) generated in the amyloid-ß protein precursor (AßPP) independent pathway activated by AßPP-derived Aß accumulated intraneuronally in a life-long process. This interpretation constitutes the Amyloid Cascade Hypothesis 2.0 (ACH2.0). It defines a tandem intraneuronal-Aß (iAß)-anchored cascade occurrence: intraneuronally-accumulated, AßPP-derived iAß triggers relatively benign cascade that activates the AßPP-independent iAß-generating pathway, which, in turn, initiates the second, devastating cascade that includes tau pathology and leads to neuronal loss. The entire output of the AßPP-independent iAß-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 Aß production in the AßPP proteolytic pathway; operation of the former renders the latter irrelevant to the progression of AD by relegating its iAß contribution to insignificant, and brands its manipulation for therapeutic purposes, such as BACE (beta-site AßPP-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 Aß-cleaving activities. Such treatment would collapse the iAß population and 'reset' its levels below those required for the operation of the AD Engine. Any sufficiently selective iAß-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 Aß 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.

News from Mars: Two-Tier Paradox, Intracellular PCR, Chimeric Junction Shift, Dark Matter mRNA and Other Remarkable Features of Mammalian RNA-Dependent mRNA Amplification. Implications for Alzheimer's Disease, RNA-Based Vaccines and mRNA Therapeutics.

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 ßAPP 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.

Alzheimer's Disease is Driven by Intraneuronally Retained Beta-Amyloid Produced in the AD-Specific, ßAPP-Independent Pathway: Current Perspective and Experimental Models for Tomorrow.

A view of the origin and progression of Alzheimer's disease, AD, prevailing until now and formalized as the Amyloid Cascade Hypothesis theory, maintains that the disease is initiated by overproduction of beta-amyloid, Aß, which is generated solely by the Aß precursor protein, ßAPP, proteolytic pathway and secreted from the cell. Consequent extracellular accumulation of Aß triggers a cascade of molecular and cellular events leading to neurodegeneration that starts early in life, progresses as one prolonged process, builds up for decades, and culminates in symptomatic manifestations of the disease late in life. In this paradigm, a time window for commencement of therapeutic intervention is small and accessible only early in life. The outlook introduced in the present study is fundamentally different. It posits that the ßAPP proteolytic/secretory pathway of Aß production causes AD in humans no more than it does in either short- or long-lived non-human mammals that share this pathway with humans, accumulate beta-amyloid as they age, but do not develop the disease. Alzheimer's disease, according to this outlook, is driven by an additional powerful AD-specific pathway of Aß production that operates in affected humans, is completely independent of the ßAPP precursor, and is not available in non-human mammals. The role of the ßAPP proteolytic pathway in the disease in humans is activation of this additional AD-specific Aß production pathway. This occurs through accumulation of intracellular Aß, primarily via ApoE-assisted cellular uptake of secreted beta-amyloid, but also through retention of a fraction of Aß produced in the ßAPP proteolytic pathway. With time, accumulated intracellular Aß triggers mitochondrial dysfunction. In turn, cellular stresses associated with mitochondrial dysfunction, including ER stress, activate a second, AD-specific, Aß production pathway: Asymmetric RNA-dependent ßAPP mRNA amplification; animal ßAPP mRNA is ineligible for this process. In this pathway, every conventionally produced ßAPP mRNA molecule serves potentially as a template for production of severely 5'-truncated mRNA encoding not the ßAPP but its C99 fragment (hence "asymmetric"), the immediate precursor of Aß. Thus produced, N-terminal signal peptide-lacking C99 is processed not in the secretory pathway on the plasma membrane, but at the intracellular membrane sites, apparently in a neuron-specific manner. The resulting Aß is, therefore, not secreted but is retained intraneuronally and accumulates rapidly within the cell. Increased levels of intracellular Aß augment mitochondrial dysfunction, which, in turn, sustains the activity of the ßAPP mRNA amplification pathway. These self-propagating mutual Aß overproduction/mitochondrial dysfunction feedback cycles constitute a formidable two-stroke engine, an engine that drives Alzheimer's disease. The present outlook envisions Alzheimer's disorder as a two-stage disease. The first stage is a slow process of intracellular beta-amyloid accumulation. It results neither in significant neurodegenerative damage, nor in manifestation of the disease. The second stage commences with the activation of the ßAPP mRNA amplification pathway shortly before symptomatic onset of the disease, sharply increases the rate of Aß generation and the extent of its intraneuronal accumulation, produces significant damages, triggers AD symptoms, and is fast. In this paradigm, the time window of therapeutic intervention is wide open, and preventive treatment can be initiated any time, even late in life, prior to commencement of the second stage of the disease. Moreover, there are good reasons to believe that with a drug blocking the ßAPP mRNA amplification pathway, it would be possible not only to preempt the disease but also to stop and to reverse it even when early AD symptoms have already manifested. There are numerous experimental models of AD, all based on a notion of the exceptionality of ßAPP proteolytic/secretory pathway in Aß production in the disease. However, with no drug even remotely effective in Alzheimer's disease, a long list of candidate drugs that succeeded remarkably in animal models, yet failed utterly in human clinical trials of potential AD drugs, attests to the inadequacy of currently employed AD models. The concept of a renewable supply of beta-amyloid, produced in the ßAPP mRNA amplification pathway and retained intraneuronally in Alzheimer's disease, explains spectacular failures of both BACE inhibition and Aß-immunotherapy in human clinical trials. This concept also forms the basis of a new generation of animal and cell-based experimental models of AD, described in the present study. These models incorporate Aß- or C99-encoding mRNA amplification pathways of Aß production, as well as intracellular retention of their product, and can support not only further investigation of molecular mechanisms of AD but also screening for and testing of candidate drugs aimed at therapeutic targets suggested by the present study.

Alzheimer's Disease Prevention and Treatment: Case for Optimism.

A paradigm shift is under way in the Alzheimer's field. A view of Alzheimer's disease, AD, prevailing until now, the old paradigm, maintains that it is initiated and driven by the overproduction and extracellular accumulation of beta-amyloid, Aß; a peptide assumed to be derived, both in health and disease, solely by proteolysis of its large precursor, ßAPP. In AD, according to this view, Aß overproduction-associated neurodegeneration begins early, accumulates throughout the lifespan, and manifests symptomatically late in life. A number of drugs, designed within the framework of exceptionality of the ßAPP proteolytic/secretory pathway in Aß production in Alzheimer's disease, achieved spectacular successes in treatment, even the reversal, of AD symptoms in animal models. Without exception, they all exhibited equally spectacular failures in human clinical trials. This paradigm provides few causes for optimism with regard to prevention and treatment of AD. In its context, the disease is considered untreatable in the symptomatic phase; even prodromal cases are assumed too advanced for treatment because Aß-triggered damages have been accumulating for preceding decades, presumably starting in the early twenties and, to be effective, this is when therapeutic intervention should commence and continue for life. The new paradigm does not dispute the seminal role of Aß in AD but posits that beta-amyloid produced in the ßAPP proteolytic/secretory pathway causes AD in humans no more than it does in non-human mammals that share this pathway with humans, accumulate Aß as they age, but do not develop the disease. Alzheimer's disease, according to this outlook, is driven by the AD-specific pathway of Aß production, independent of ßAPP and absent in animals. Its activation, late in life, occurs through accumulation, via both cellular uptake of secreted Aß and neuronal retention of a fraction of beta-amyloid produced in the ßAPP proteolytic pathway, of intraneuronal Aß, which triggers mitochondrial dysfunction. Cellular stresses associated with mitochondrial dysfunction, or, probably, the integrated stress response, ISR, elicited by it, activate an AD-specific Aß production pathway. In it, every conventionally produced ßAPP mRNA molecule potentially serves repeatedly as a template for production of severely 5'-truncated mRNA encoding C99 fragment of ßAPP, the immediate precursor of Aß that is processed in a non-secretory pathway, apparently in a neuron-specific manner. The resulting intraneuronally retained Aß augments mitochondrial dysfunction, which, in turn, sustains the activity of the ßAPP mRNA amplification pathway. These self-propagating Aß overproduction/mitochondrial dysfunction mutual feedback cycles constitute the engine that drives AD and ultimately triggers neuronal death. In this paradigm, preventive treatment can be initiated any time prior to commencement of ßAPP mRNA amplification. Moreover, there are good reasons to believe that with a drug blocking the amplification pathway, it would be possible not only to preempt the disease but also stop and reverse it even when early AD symptoms are already manifested. Thus, the new paradigm introduces a novel theory of Alzheimer's disease. It explains the observed discordances, determines defined therapeutic targets, provides blueprints for a new generation of conceptually distinct AD models and specifies design of a reporter for the mRNA amplification pathway. Most importantly, it offers detailed guidance and tangible hope for prevention of the disease and its treatment at the early symptomatic stages.

AD "Statin": Alzheimer's Disorder is a "Fast" Disease Preventable by Therapeutic Intervention Initiated Even Late in Life and Reversible at the Early Stages.

The present study posits that Alzheimer's disorder is a "fast" disease. This is in sharp contrast to a view, prevailing until now, that Alzheimer's Disease (AD) is a quintessential "slow" disease that develops throughout the life as one prolonged process. According to this view, beta-amyloid (Aß) is produced and secreted solely by the beta-amyloid precursor protein (ßAPP) proteolytic/secretory pathway. As its extracellular levels increase, it triggers neurodegeneration starting relatively early in life. Damages accumulate and manifest, late in life in sporadic Alzheimer's Disease (SAD) cases, as AD symptoms. In familial AD (FAD) cases, where mutations in ßAPP gene or in presenilins increase production of either common Aß isoform or of its more toxic isoforms, neurodegeneration reaches critical threshold sooner and AD symptoms occur earlier in life, mostly in late 40s and 50s. There are currently no preventive AD therapies but if they were available, according to this viewpoint it would be largely futile to intervene late in life in case of potential SAD or at mid-age in cases of FAD because, although AD symptoms have not yet manifested, the damage has already occurred during the preceding decades. In this paradigm, to be effective, preventive therapeutic intervention should be initiated early in life. The outlook suggested by the present study is radically different. According to it, Alzheimer's disease evolves in two stages. The first stage is a slow process of intracellular beta-amyloid accumulation. It occurs via ßAPP proteolytic/secretory pathway and cellular uptake of secreted Aß common to Homo sapiens, including healthy humans, and to non-human mammals, and results neither in significant damage, nor in manifestation of the disease. The second stage occurs exclusively in humans, commences shortly before symptomatic onset of the disease, sharply accelerates the production and increases intracellular levels of Aß that is not secreted but is retained intracellularly, generates significant damages, triggers AD symptoms, and is fast. It is driven by an Aß generation pathway qualitatively and quantitatively different from ßAPP proteolytic process and entirely independent of beta-amyloid precursor protein, and results in rapid and substantial intracellular accumulation of Aß, consequent significant neurodegeneration, and symptomatic AD. In this paradigm, a preventive therapy for AD, an AD "statin", would be effective when initiated at any time prior to commencement of the second stage. Moreover, there are good reasons to believe that with a drug blocking ßAPP-independent Aß production pathway in the second stage, it would be possible not only to preempt the disease but also to stop and to reverse it even when early AD symptoms have already manifested. The present study posits a notion of AD as a Fast Disease, offers evidence for the occurrence of the AD-specific Aß production pathway, describes cellular and molecular processes constituting an engine that drives Alzheimer's disease, and explains why non-human mammals are not susceptible to AD and why only a subset of humans develop the disease. It establishes that Alzheimer's disease is preventable by therapeutic intervention initiated even late in life, details a powerful mechanism underlying the disease, suggests that Aß produced in the ßAPP-independent pathway is retained intracellularly, elaborates why neither BACE inhibition nor Aß immunotherapy are effective in treatment of AD and why intracellularly retained beta-amyloid could be the primary agent of neuronal death in Alzheimer's disease, necessitates generation of a novel animal AD model capable of producing Aß via ßAPP-independent pathway, proposes therapeutic targets profoundly different from previously pursued components of the ßAPP proteolytic pathway, and provides conceptual rationale for design of drugs that could be used not only preemptively but also for treatment and reversal of the early stages of the disease.

Protein-Encoding RNA-to-RNA Information Transfer in Mammalian Cells: Principles of RNA-Dependent mRNA Amplification.

The transfer of protein-encoding genetic information from DNA to RNA to protein, a process formalized as the "Central Dogma of Molecular Biology", has undergone a significant evolution since its inception. It was amended to account for the information flow from RNA to DNA, the reverse transcription, and for the information transfer from RNA to RNA, the RNA-dependent RNA synthesis. These processes, both potentially leading to protein production, were initially described only in viral systems, and although RNA-dependent RNA polymerase activity was shown to be present, and RNA-dependent RNA synthesis found to occur, in mammalian cells, its function was presumed to be restricted to regulatory. However, recent results, obtained with multiple mRNA species in several mammalian systems, strongly indicate the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. It can result in the rapid production of the extraordinary quantities of specific proteins as was seen in cases of terminal cellular differentiation and during cellular deposition of extracellular matrix molecules. A malfunction of this process may be involved in pathologies associated either with the deficiency of a protein normally produced by this mechanism or with the abnormal abundance of a protein or of its C-terminal fragment. It seems to be responsible for some types of familial thalassemia and may underlie the overproduction of beta amyloid in sporadic Alzheimer's disease. The aim of the present article is to systematize the current knowledge and understanding of this pathway. The outlined framework introduces unexpected features of the mRNA amplification such as its ability to generate polypeptides non-contiguously encoded in the genome, its second Tier, a physiologically occurring intracellular polymerase chain reaction, iPCR, a "Two-Tier Paradox" and RNA "Dark Matter". RNA-dependent mRNA amplification represents a new mode of genomic protein-encoding information transfer in mammalian cells. Its potential physiological impact is substantial, it appears relevant to multiple pathologies and its understanding opens new venues of therapeutic interference, it suggests powerful novel bioengineering approaches and its further rigorous investigations are highly warranted.

Protein-Encoding RNA to RNA Information Transfer in Mammalian Cells: RNA-dependent mRNA Amplification. Identification of Chimeric RNA Intermediates and Putative RNA End Products.

Our initial unidirectional understanding of the flow of protein-encoding genetic information, DNA to RNA to protein, a process defined as the "Central Dogma of Molecular Biology" and usually depicted as a downward arrow, was eventually amended to account for the "vertical" information back-flow from RNA to DNA, reverse transcription, and for its "horizontal" side-flow from RNA to RNA, RNA-dependent RNA synthesis, RdRs. These processes, both potentially leading to protein production, were assumed to be strictly virus-specific. However, whereas this presumption might be true for the former, it became apparent that the cellular enzymatic machinery for the later, a conventional RNA-dependent RNA polymerase activity, RdRp, is ubiquitously present and RdRs regularly occurs in eukaryotes. The strongest evidence for the occurrence and functionality of RdRp activity in mammalian cells comes from viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the host. Eventually, 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, because of 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 RNA 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. Here, we report the in vivo detection in cells undergoing terminal erythroid differentiation of the major model-predicted identifiers of such a process, a chimeric double-stranded/pinhead-structured intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner. We also report the identification of the putative chimeric RNA end product of mRNA amplification. It is heavily modified, uniformly truncated, yet retains the intact coding region, and terminates with the OH group at both ends; its massive cellular amount is unprecedented for a conventional mRNA transcription product and it translates into polypeptides indistinguishable from the translation product of conventional mRNA. Moreover, we describe the occurrence of the second Tier of mammalian RNA-dependent mRNA amplification, a physiologically occurring, RdRp-driven intracellular PCR process, "iPCR", and report the detection of its distinct RNA end products. Whether mammalian mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation in cells programmed for only a short survival span or a general physiological phenomenon was answered in the companion article Volloch et al. Ann Integr Mol Med. 2019;1(1):1004. by the detection of major identifiers of this process for mRNA encoding α1, ß1, and γ1 chains of laminin, a major extracellular matrix protein abundantly produced throughout the tissue and organ development and homeostasis and an exceptionally revealing indicator of the range and scope of this phenomenon. The results obtained introduce the occurrence of RNA-dependent mRNA amplification as a new mode of genomic protein-encoding information transfer in mammalian cells and establish it as a general physiological phenomenon.

RNA-dependent Amplification of Mammalian mRNA Encoding Extracellullar Matrix Proteins: Identification of Chimeric RNA Intermediates for α1, ß1, and γ1 Chains of Laminin.

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, ß1, 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.

Precursor-Independent Overproduction of Beta-Amyloid in AD: Mitochondrial Dysfunction as Possible Initiator of Asymmetric RNA-Dependent ßAPP mRNA Amplification. An Engine that Drives Alzheimer's Disease.

The present study defines RNA-dependent amplification of ßAPP mRNA as a molecular basis of beta-amyloid overproduction in Alzheimer's disease. In this process, ßAPP mRNA serves as a template for RNA-dependent RNA polymerase, RdRp complex. The resulting antisense RNA self-primes its extension utilizing two complementary elements: 3'-terminal and internal, located within an antisense segment corresponding to the coding portion of ßAPP mRNA. The extension produces 3'-terminal fragment of ßAPP mRNA, a part of a hairpin-structured antisense/sense RNA molecule. Cleavage at the 3' end of the hairpin loop produces RNA end product encoding a C-terminal fragment of ßAPP. Since each conventional ßAPP mRNA can be used repeatedly as a template, the process constitutes an asymmetric mRNA amplification. The 5'-most translation initiation codon of the amplified mRNA is the AUG preceding immediately and in-frame the Aß-coding segment. Translation from this codon overproduces Aß independently of ßAPP. Such process can occur in humans but not in mice and other animals where segments of ßAPP antisense RNA required for self-priming have little, if any, complementarity. This explains why Alzheimer's disease occurs exclusively in humans and implies that ßAPP mRNA amplification is requisite in AD. In AD, therefore, there are two pathways of beta-amyloid production: ßAPP proteolytic pathway and ßAPP mRNA amplification pathway independent of ßAPP and insensitive to beta-secretase inhibition. This implies that in healthy humans, where only the proteolytic pathway is in operation, Aß production should be suppressed by the BACE inhibition, and indeed it is. However, since ßAPP-independent pathway operating in AD is by far the predominant one, BACE inhibition has no effect in Alzheimer's disease. It appears that, physiologically, the extent of beta-amyloid overproduction sufficient to trigger amyloid cascade culminating in AD requires asymmetric RNA-dependent amplification of ßAPP mRNA and cannot be reached without it. In turn, the occurrence of mRNA amplification process depends on the activation of inducible components of RdRp complex by certain stresses, for example the ER stress in case of amplification of mRNA encoding extracellular matrix proteins. In case of Alzheimer's disease, such an induction appears to be triggered by stresses associated with mitochondrial dysfunction, a phenomenon closely linked to AD. The cause-and-effect relationships between mitochondrial dysfunction and AD appear to be very different in familial, FAD, and sporadic, SAD cases. In FAD, increased levels or more toxic species of Aß resulting from the abnormal proteolysis of ßAPP trigger mitochondrial dysfunction, activate mRNA amplification and increase the production of Aß, reinforcing the cycle. Thus in FAD, mitochondrial dysfunction is an intrinsic component of the amyloid cascade. The reverse sequence is true in SAD where aging-related mitochondrial dysfunction activates amplification of ßAPP mRNA and enhances the production of Aß. This causes further mitochondrial dysfunction, the cycle repeats and degeneration increases. Thus in SAD, the initial mitochondrial dysfunction arises prior to the disease, independently of and upstream from the increased Aß production, i.e. in SAD, mitochondrial pathology hierarchically supersedes Aß pathology. This is the primary reason for the formulation of the Mitochondrial Cascade Hypothesis. But even in terms of the MCH, the core of the disease is the amyloid cascade as defined in the amyloid cascade hypothesis, ACH. The role of mitochondrial dysfunction in relation to this core is causative in SAD and auxiliary in FAD. In FAD, the initial increase in the production of Aß is mutations-based and occurs relatively early in life, whereas in SAD it is coerced by an aging-contingent component, but both lead to mechanistically identical self-perpetuating mutual Aß/mitochondrial dysfunction feedback cycles, an engine that drives, via RNA-dependent ßAPP mRNA amplification, overproduction of beta-amyloid and, consequently, AD; hence drastic difference in the age of onset, yet profound pathological and symptomatic similarity in the progression, of familial and sporadic forms of Alzheimer's disease. Interestingly, the recent findings that mitochondrial microprotein PIGBOS interacts with the ER in mitigating the unfolded protein response indicate a possible connection between mitochondrial dysfunction and ER stress, implicated in activation of RNA-dependent mRNA amplification pathway. The possible involvement of mitochondrial dysfunction in ßAPP mRNA amplification makes it a promising therapeutic target. Recent successes in mitigating, and even reversing, Aß-induced metabolic defects with anti-diabetes drug metformin are encouraging in this respect.

Results of Beta Secretase-Inhibitor Clinical Trials Support Amyloid Precursor Protein-Independent Generation of Beta Amyloid in Sporadic Alzheimer's Disease.

The present review analyzes the results of recent clinical trials of ß secretase inhibition in sporadic Alzheimer's disease (SAD), considers the striking dichotomy between successes in tests of ß-site Amyloid Precursor Protein-Cleaving Enzyme (BACE) inhibitors in healthy subjects and familial Alzheimer's disease (FAD) models versus persistent failures of clinical trials and interprets it as a confirmation of key predictions for a mechanism of amyloid precursor protein (APP)-independent, ß secretase inhibition-resistant production of ß amyloid in SAD, previously proposed by us. In light of this concept, FAD and SAD should be regarded as distinctly different diseases as far as ß-amyloid generation mechanisms are concerned, and whereas ß secretase inhibition would be neither applicable nor effective in the treatment of SAD, the ß-site APP-Cleaving Enzyme (BACE) inhibitor(s) deemed failed in SAD trials could be perfectly suitable for the treatment of FAD. Moreover, targeting the aspects of Alzheimer's disease (AD) other than cleavages of the APP by ß and α secretases should have analogous impacts in both FAD and SAD.

Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering.

To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.