MD Andrea Vergallo

Keywords: aging, senescence, adaptation, resilience, stress response, Alzheimer's disease

Systems biology and systems neurophysiology generate comprehensive mechanistic models of the spatial-temporal evolution of body system networks and their interplay during the dimensional spectrum from physiological to pathophysiological conditions. Alzheimer's disease (AD)-related pathophysiological alterations converge with overexpressed agerelated functional decline, i.e. aging, which is induced by genetic-and stochastic time-dependent events.

Accumulation of cellular senescence has a casual role in aging-related disease and senolytics drugs have already shown encouraging results for counteracting senescence detrimental effect.

However, the non-linear complex nature of AD pathophysiology calls for a systems-level integration of aging dynamics, from molecules until large-scale networks.

We need a holistic systems-wide comprehensive model of aging which is constituted by a nonlinear spatial-temporal weakening of adaptive responses resulting in the activation of compensatory mechanisms that ensure biological robustness, resilience, and finally homeodynamics.

After exceeding the threshold of compensated (resilient) aging, a cascade of decompensatory events occurs, ultimately triggering irreversible systems failure that at some advanced stage reflect widespread "pathophysiological hallmarks of AD".

INTRODUCTION

Complex chronic diseases including cancer, immune diseases, diabetes, and neurodegenerative diseases -such as Alzheimer's disease (AD) -are characterized by: 1) a multifactorial nature, due to the coexistence of (epi)genetic, biological, and environmental susceptibility 2) concurrent pathomechanistic alterations due to the perturbation of multiplex pathways and networks resulting in several disease modules and interactomes 3) the involvement of highly conserved cross-talks among different body systems [START_REF] Arnsten | Stress weakens prefrontal networks: molecular insults to higher cognition[END_REF][START_REF] Bassett | Network neuroscience[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF].

The continuous failure of late stage clinical AD drug trials, raises fundamental theoretical and conceptual questions [START_REF] Cummings | Clinical Trials for Disease-Modifying Therapies in Alzheimer's Disease: A Primer, Lessons Learned, and a Blueprint for the Future[END_REF][START_REF] Dourlen | The new genetic landscape of Alzheimer's disease: from amyloid cascade to genetically driven synaptic failure hypothesis?[END_REF]Hampel et al., 2018b;[START_REF] Panza | A critical appraisal of amyloidbeta-targeting therapies for Alzheimer disease[END_REF] A critical step for developing effective drugs with disease-modifying properties is to explore and predict the comprehensive effect of a compound on three basic levels (i.e. proof of pharmacology), including 1) hitting the intended target, 2) altering the relevant pathophysiological mechanism, and 3) impacting clinical outcomes with a long-lasting effect It seems crucial to enrich drug Research and Development (R&D) pipelines with the perspective of an integrative, exploratory, and holistic multi-paradigm or systems level concept, at both experimental and computational level and with a strong biomarker-drug co-development strategy throughout all development stages [START_REF] Bokde | Assessing neuronal networks: understanding Alzheimer's disease[END_REF][START_REF] Cummings | Clinical Trials for Disease-Modifying Therapies in Alzheimer's Disease: A Primer, Lessons Learned, and a Blueprint for the Future[END_REF][START_REF] Hampel | Biomarker-Drug and Liquid Biopsy Co-development for Disease Staging and Targeted Therapy: Cornerstones for Alzheimer's Precision Medicine and Pharmacology[END_REF] Systems biology and systems neurophysiology paradigms enable to accomplish unbiased experimental predictions of pathophysiological dynamics through knowledge-driven or entirely explorative designs [START_REF] Cummings | Clinical Trials for Disease-Modifying Therapies in Alzheimer's Disease: A Primer, Lessons Learned, and a Blueprint for the Future[END_REF][START_REF] Dourlen | The new genetic landscape of Alzheimer's disease: from amyloid cascade to genetically driven synaptic failure hypothesis?[END_REF]Hampel et al., 2018b;[START_REF] Panza | A critical appraisal of amyloidbeta-targeting therapies for Alzheimer disease[END_REF]. In this sense, integrative analyses allow to track meaningful biological signatures reflecting key pathomechanistic alterations as well as their spatial and temporal evolution over the course of a certain disease.

In the context of neurodegenerative diseases, the contribution of systems biology and systems neurophysiology is to generate comprehensive models of multilevel networks interactions, from molecular pathway(s), to cellular machineries, synaptic circuits, until large-scale network patterns, in either physiological or pathophysiological conditions and according different temporal and spatial scales [START_REF] Arnsten | Stress weakens prefrontal networks: molecular insults to higher cognition[END_REF][START_REF] Bartsch | Network Physiology: How Organ Systems Dynamically Interact[END_REF][START_REF] Bassett | Network neuroscience[END_REF][START_REF] Medland | Whole-genome analyses of whole-brain data: working within an expanded search space[END_REF][START_REF] Richiardi | Correlated gene expression supports synchronous activity in brain networks[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF]. The objective of this article is first to provide a comprehensive overview of the major networks ensuring systems hemodynamics, at the different biological levels, and propose a theoretical framework to explore their changes along a system-wide aging, including cellular senescence, with regards to the risk of developing AD. We will list and describe the most relevant stress response and long-term resilience mechanisms as well as their relationship with lifespan, healthspan (the time during which the organism maintains its functional capacity), and age-related diseases such as AD. In addition, we will depict the main alterations that slow down adaptation and resilience in aging and AD, throughout a continuum from adaptation to compensation until systems failure, and from the molecular level to the body systems level. We will eventually deliver argumentation about the fact that a potential workflow, for the in-vivo systemslevel integration of all aging variables, from molecules (encompassing senescence signatures) to large-scale networks, including adaptive and compensatory patterns will be feasible within a short time. The accomplishment of a holistic systems-scaled comprehensive model of aging is a key step for finally testing the Geroscience Hypothesis"… any intervention that retards the aging process will simultaneously delay the onset of multiple diseases.." [START_REF] Kennedy | Geroscience: linking aging to chronic disease[END_REF]. In line with this postulate, an innovative pathway (mechanism)-based pharmacological paradigm aimed at developing effective biomarker-guided targeted and tailored therapies for preventive "resilience enhancing" and aging-freezing strategies. We will further clarify and define useful terminology and taxonomy relating to the discussed fundamental scientific concepts.

The continuum from homeostasis to adaptation, compensation, decompensation and system failure Homeostasis or more correctly the homeodynamic space is essential for protecting all core physiological processes necessary for optimal functioning and survival and its preservation is highly related to the interplay between intrinsic (e.g., genetic and mechanistic factors) and environmental factors [START_REF] Berg | Gene-environment interplay[END_REF][START_REF] Rattan | Aging is not a disease: implications for intervention[END_REF]. Adaptive responses and compensatory mechanisms account for the overall biological robustness and resilience of an organism and are hierarchically organized across molecular /cellular pathways, cell to cell and tissue dynamics, until large-scale networks, and body systems [START_REF] Arnsten | Stress weakens prefrontal networks: molecular insults to higher cognition[END_REF][START_REF] Bartsch | Network Physiology: How Organ Systems Dynamically Interact[END_REF][START_REF] Bassett | Network neuroscience[END_REF][START_REF] Medland | Whole-genome analyses of whole-brain data: working within an expanded search space[END_REF][START_REF] Richiardi | Correlated gene expression supports synchronous activity in brain networks[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF].

The preservation of homeodynamics under stressful conditions implies a functional shift to face a higher energetic demand, adaptation is essential to reallocate bioenergetic resources through a reprogramming which confers the proper robustness to copy with stressor(s). Biological adaptation (adaptive response) consists in a wide range of functional switches regarding a set of network(s) through different spatiotemporal scales according levels and systems in which they occur [START_REF] Arnsten | Stress weakens prefrontal networks: molecular insults to higher cognition[END_REF][START_REF] Bashan | Network physiology reveals relations between network topology and physiological function[END_REF][START_REF] Harrison | Rapamycin fed late in life extends lifespan in genetically heterogeneous mice[END_REF]Kaushik and Cuervo, 2015;[START_REF] Marder | Variability, compensation and homeostasis in neuron and network function[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF] Indeed, the bioenergetic shift which enables to maintain a proper effective functional output and ultimately homeodynamics, consists of distinct biological networks (spatial scales) as well as their sequential or simultaneous recruitment (temporal scales) [START_REF] Arnsten | Stress weakens prefrontal networks: molecular insults to higher cognition[END_REF][START_REF] Hermans | Stress-related noradrenergic activity prompts large-scale neural network reconfiguration[END_REF][START_REF] Menche | Disease networks. Uncovering disease-disease relationships through the incomplete interactome[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF] The magnitude and duration of an adaptive response match the downstream effect(s) induced by the stressor (see Figure 1). Moreover, all organisms from nematode to humans have an intrinsic property to develop a gradual self-regulation of the stress responses consistent with typology and time exposure of the stressor [START_REF] Berg | Gene-environment interplay[END_REF][START_REF] Lu | REST and stress resistance in ageing and Alzheimer's disease[END_REF] For instance, at the cellular level, evolutionarily conserved transduction signals enable to sense the stress levels, thus triggering antistress responses which promote either cell survival or apoptosis, or senescence itself as stress escalates and becomes unsustainable. In addition, low exposure to a given stressor may trigger biopositive effects representing appropriate cues which strengthen homeodynamics (hormesis) [START_REF] Brose | Hydroxyurea attenuates oxidative, metabolic, and excitotoxic stress in rat hippocampal neurons and improves spatial memory in a mouse model of Alzheimer's disease[END_REF][START_REF] Rattan | Hormesis in aging[END_REF][START_REF] Ruhlmann | Long-term caloric restriction in ApoE-deficient mice results in neuroprotection via Fgf21-induced AMPK/mTOR pathway[END_REF].

This dynamic modulation of adaptive responses is important even at the extra-individual level, where adaptation is a pivotal point for natural selection to promote population-scaled evolutionary transitions (evolutionary adaptation) optimizing large-scale survival and reproduction across ecosystem [START_REF] Behrens | Impact of genomic damage and ageing on stem cell function[END_REF].

Multidisciplinary approaches and in-silico modeling studies have proven that, besides molecules and cells, adaptive responses are elicited at different brain levels and that an interaction between neurobiological and synaptic network changes exists within short-long-term homeostatic mechanisms [START_REF] Arnsten | Stress weakens prefrontal networks: molecular insults to higher cognition[END_REF][START_REF] Bartsch | Network Physiology: How Organ Systems Dynamically Interact[END_REF][START_REF] Bassett | Network neuroscience[END_REF][START_REF] Medland | Whole-genome analyses of whole-brain data: working within an expanded search space[END_REF][START_REF] Richiardi | Correlated gene expression supports synchronous activity in brain networks[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF].

When maladaptive dynamics occur, compensatory mechanisms are essential to ensure resilience and maintain homeodynamics. Compensation is a protective process in which a morpho-functional alteration is counteracted by another morpho-functional alteration without any significant change in the biological output, thus ensuring resilience. Compensation provides a viable balance between the biological robustness and the degree of disruptive effect exerted by the stressor over time [START_REF] Apostolova | Hypermetabolism in the hippocampal formation of cognitively impaired patients indicates detrimental maladaptation[END_REF][START_REF] Arenaza-Urquijo | Resistance vs resilience to Alzheimer disease: Clarifying terminology for preclinical studies[END_REF][START_REF] Bokde | Assessing neuronal networks: understanding Alzheimer's disease[END_REF]Hampel et al., 2018b;[START_REF] Marder | Variability, compensation and homeostasis in neuron and network function[END_REF][START_REF] Scheller | Brain Aging and APOE epsilon4 Interact to Reveal Potential Neuronal Compensation in Healthy Older Adults[END_REF][START_REF] Stern | Different brain networks mediate task performance in normal aging and AD: defining compensation[END_REF].

Like adaptation, the compensatory dynamics are mainly described at the molecular and cellular level. However, in the last ten years, it has been shown that brain compensation range from neuronal / glial metabolic reconfiguration, to large-scale network dynamics, until complex intersystems cross-talks (for instance the brain-innate system interface) [START_REF] Apostolova | Hypermetabolism in the hippocampal formation of cognitively impaired patients indicates detrimental maladaptation[END_REF][START_REF] Elman | Neural compensation in older people with brain amyloid-beta deposition[END_REF]Kaushik and Cuervo, 2015;[START_REF] Marder | Variability, compensation and homeostasis in neuron and network function[END_REF][START_REF] Perez-Nievas | Dissecting phenotypic traits linked to human resilience to Alzheimer's pathology[END_REF].

Animal and human studies have untangled that compensation may be associated with a re-activation of developmental program, such as synaptogenesis, occurring within self-organized multilevel systems [START_REF] Arendt | Alzheimer's disease as a disorder of dynamic brain self-organization[END_REF]. On the flipside, overcompensation may trigger a vicious cycle of feedback loops culminating in loss of function, i.e. decompensation (breakdown of resilient dynamics) [START_REF] Arendt | Alzheimer's disease as a disorder of dynamic brain self-organization[END_REF].

We argue that pervious failing drug R&D programs may have underestimated that an individual genetic-biological vulnerability to stressors exists as well as an individual genetic / epigenetic / sex driven threshold of failure of adaptive responses and compensatory capacities For the neuroscientist the question arises about the biological nature of aging and how does aging biologically relate or contribute to age-related brain diseases, such as AD.

It has previously been postulated that aging is characterized by genetic-and time-dependent decline in adaptive and compensatory properties (Lopez-Otin et al., 2013;[START_REF] Mchugh | Senescence and aging: Causes, consequences, and therapeutic avenues[END_REF].

A remarkable research effort has been generated in the last ten years to identify the hallmarks of aging which are currently related to the (sub)cellular level only. We propose a systems biologybased conceptual framework of brain aging i.e., non-linear functional-structural alterations due to a decline of biological adaptation, compensation, and ultimately robustness and resilience which ensure homeodynamics at the molecular, cellular, synaptic, and large-scale brain network level until the complex brain-body systems cross-talks (see Figure 2). Relying on several translational study results, we hypothesize that systems-scaled aging processes including but not restricted to cellular senescence, may be expressed either in AD cognitive decline or normal cognitive condition depending on the individual threshold of genetically and biologically determined resilience [START_REF] Arenaza-Urquijo | Resistance vs resilience to Alzheimer disease: Clarifying terminology for preclinical studies[END_REF][START_REF] Arnold | Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer's disease[END_REF]Hampel et al., 2018b;[START_REF] Perez-Nievas | Dissecting phenotypic traits linked to human resilience to Alzheimer's pathology[END_REF]. Thus, aging and AD may belong to a continuum of network breakdowns, from decompensation to systems failure, inside brain and body systems.

We assume that the decompensatory cascade that draws aging towards AD has a nonlinear fashion since multiple pathomechanistic alterations evolve differently according to distinct spatial and temporal components, driven by complex genetic-epigenetic interplays.

Conversely, it is conceivable that as long as adaptation and compensation ensure homeodynamics systems networks survive to disrupting signals and AD pathophysiology does not evolve (i.e., "normal" or compensated aging). Hence, after exceeding the limit of compensated aging, a cascade of breakdown events occurs, and, at the final step, detrimental pathways trigger irreversible systems failure reflecting widespread pathophysiological hallmarks of AD: i.e. brain proteinopathies, synaptic failure, loss of brain plasticity, neuroinflammation, axonal damage, and neurodegeneration [START_REF] Arnold | Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer's disease[END_REF][START_REF] Bero | Neuronal activity regulates the regional vulnerability to amyloid-beta deposition[END_REF][START_REF] Bokde | Assessing neuronal networks: understanding Alzheimer's disease[END_REF][START_REF] Cirrito | Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo[END_REF][START_REF] Dourlen | The new genetic landscape of Alzheimer's disease: from amyloid cascade to genetically driven synaptic failure hypothesis?[END_REF].

The devastating rate of failure of AD clinical trials within the last 25 years suggests that when decompensation commences across core biological networks and then systems failure takes place, every therapeutic intervention is likely to be less effective with no possibility to recover physiological functions and the homeodynamic space.

We hypothesize that the key points to prevent AD will be represented first by identifying those clusters of genetic, biological, and environmental factors that increase the vulnerability to stressors leading to an escalation of accumulation of senescence and other aging alterations, maladaptive responses, ineffective compensatory mechanisms that are likely to enter a vicious cycle of decompensation and generate irreversible systems failure. Second mapping in-vivo the adaptive capacity and the degree of resilience under initial pathophysiological dynamics (intermediate endophenotypes). We argue that the presence of active compensation ensuring resilience has the potential to reshape or even replace the current pathophysiological hallmarks of AD.

Achieving these two objectives would represent a crucial step for time-sensitive and effective rejuvenation therapies capable of strengthening resilience, i.e. "compensated aging", counteracting its transition to systems failure and at the same time prolonging health span.

AGING, CELLULAR SENESCENCE, AND AD: a comprehensive overview

Even though aging is primarily a time-dependent stochastic process recent genetic manipulation studies have demonstrated that the "switch off" of specific genes can promote longevity (longevity genes) and prolong lifespan [START_REF] Ayyadevara | Remarkable longevity and stress resistance of nematode PI3K-null mutants[END_REF][START_REF] Kennedy | Geroscience: linking aging to chronic disease[END_REF]Lopez-Otin et al., 2013;[START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF] Such finding has raised the concept of a genetically driven (programmed) aging.

The discovery of an intrinsic (genetic) regulation of aging-related processes has raised the not yet unresolved intellectual conflict between evolutionary theorist which debate on whether aging may represent either an evolutionary adaptive response (adaptive aging ultimately called aging by design) or not [START_REF] Goldsmith | Aging, evolvability, and the individual benefit requirement; medical implications of aging theory controversies[END_REF][START_REF] Kyryakov | Empirical verification of evolutionary theories of aging[END_REF]. An adaptive aging counteracts the traditional evolutionary approach which rules out the possibility of an intrinsically determined selfdefeating biological program (in terms of survival and reproduction) that does not bring about any short-or long-term benefit (non-programmed aging theory). According to the non-programmed theory only time-related stochastic insults account for the aging process. Answering such open question may represent a breakthrough in the development of aging targeted therapeutic avenues [START_REF] Kowald | Can aging be programmed? A critical literature review[END_REF]. However, we think that a deeper argumentation on this intriguing research topic goes beyond the aim of the present article.

It is noteworthy to highlight that a comprehensive understanding of the overall biological background of longevity and aging, especially with regard to the brain, has not been achieved yet.

Molecular and cellular hallmarks of aging: the antagonistic role of cellular senescence and the causal role of its accumulation

Cellular senescence is one the most important hallmark of aging alongside stem cell exhaustion, genomic instability, epigenetic aberrant dynamics, telomere shortening, proteostatic dysfunction, deregulation of intracellular signaling and nutrient-sensing pathways, and mitochondrial dysfunction (Lopez-Otin et al., 2013).

According the role each hallmark may play, a hypothetical categorization has been proposed: causal mechanisms (genomic instability, epigenetic aberrant dynamics, telomere shortening, loss of proteostasis), antagonistic mechanisms (nutrient-sensing pathways, mitochondrial dysfunction, cellular senescence), downstream detrimental mechanisms (deregulation of intracellular signaling and stem cell exhaustion).

Cellular senescence is a cell fate characterized by an irreversible cell cycle arrest coupled with improper activation of mitogenic and growth-promoting pathways (Munoz-Espin and Serrano, 2014).

Senescence represents an anti-tumor defense mechanism halting the propagation of damaged cells and setting off the immune system surveillance (i.e. antagonistic hallmark of aging) (Lopez-Otin et al., 2013;Munoz-Espin and Serrano, 2014). Therefore, senescence constitutes a resilient tissuespecific mechanistic alteration counteracting the functional biological aging-related decline.

Furthermore, senescence plays a crucial role in embryogenic development, adult vascular remodeling, wound healing and lymphocytes reprogramming (Lopez-Otin et al., 2013;Munoz-Espin and Serrano, 2014).

Several experimental data suggested that an accurate stimulation of the senescence-inducing tumor suppressor pathways may extend longevity (Lopez-Otin et al., 2013;Munoz-Espin and Serrano, 2014). Despite senescence's physiological aging-antagonistic roles, the accumulation of senescent cells has causal role for aging aging-related functional decline and is associated with chronic inflammatory state, increased risk of age-related disease, tumorigenesis, and shorter lifespan (Baker et al., 2016;[START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF].

Experimentally induced accumulation of senescent cell through transplantation or stress-mediated induction can directly draw aging-related physical dysfunction and diseases while post-mortem human studies have confirmed such a strong association (Baker et al., 2016;Bussian et al., 2018).

By contrast, removal of senescence cells or the shutdown of pro-senescence pathways delay agerelated pathologies and extend lifespan (Baker et al., 2016;Bussian et al., 2018) (see also "Cellular senescence and senotherapeutics: a pharmacological opportunity for extending heathspan").

Cellular senescent phenotype can be tracked through well-accepted biomarkers (see Table 1).

There are three alternative mechanisms that may trigger senescent transformation and may have distinct phenotypic features (Lopez-Otin et al., 2013;Munoz-Espin and Serrano, 2014).

The replicative senescence is the first type of senescence described and is primarily induced by the age-associated and progressive telomere shortening and attrition taking place over multiple cell cycles. The stress-induced (premature) senescence is caused by either environmental stressors such as ionizing radiation, UV, chemotherapeutic drugs or endogenous factors mainly: oxidative stress (OS), oncogenes, epigenetic dysregulation, mitochondrial dysfunction.

Besides replicative and stress-induced mechanisms of senescence, a third mechanism was demonstrated with a genetically programmed senescence serving as key regulation factor for development pathways such as embryonic morphogenesis, vascular remodeling, and wound healing (Munoz-Espin et al., 2013).

The key process of senescence is represented by the cell cycle arrest which is mainly due to a tissue-and organ-specific hyperexpression of p16 Ink4a , p15 Ink4b , and p21 Waf1 which are cyclindependent kinase inhibitors (CDKI) and key tumor suppressor factors.

Many of the pro-senescent stimuli lead to DDR which in turns induces the upregulation and hyperphosphorylation of p53 which promotes several downstream pathways including transactivation of p21 Waf1 which has a CDK inhibitory effect that culminates in the hyperphosphorylation of the retinoblastoma tumor suppressor (Lopez-Otin et al., 2013;Munoz-Espin and Serrano, 2014).

The epigenetic deregulation of the Ink4a locus during replicative senescence is associated to hyperexpression of both p16 Ink4a and AFR. The latter reinforces the p53-mediated pathways through slowdown of p53 degradation machineries (Munoz-Espin and Serrano, 2014).

Upregulation of p16 Ink4a , p15 Ink4b , and p21 Waf1 leads to permanent cell cycle arrest at G1/S transition (Lopez-Otin et al., 2013;Munoz-Espin and Serrano, 2014). The transcriptional activation of p16 Ink4a is currently one of the core biomarkers for the in-vivo assessment of replicative cellular senescence (see Table 1).

Another core feature of senescence is the apoptosis resistance, a phenomenon that has physiological implications for tissue homedynamics but detrimental consequences, in case of overaccumulation of senescent cells. Indeed, apoptosis resistance and pathway underlying its presence have been associated to chemotherapy resistance in cancer (Baar et al., 2017).

Several molecular factors can induce either senescence with resistance to apoptosis or apoptosis with a "dose"-dependent fashion and according to the cell line they originate from [START_REF] Childs | Cellular senescence in aging and age-related disease: from mechanisms to therapy[END_REF][START_REF] Mchugh | Senescence and aging: Causes, consequences, and therapeutic avenues[END_REF]. A reversible shift from the apoptotic resistant state to the nonresistant one has recently been demonstrated, thus disclosing a promising therapeutic target for diseases with accumulation of senescent cells (Baar et al., 2017).

Most senescent cells produce and release the senescence-associated secretory phenotype (SASP), in autocrine and paracrine manner, that is essential for tumour suppression, tissue regeneration and modelling. The SASP induces the local amplification of the senescence program and boosts the intercellular communication through the secretion of chemokines, cytokines, growth factors, metalloproteinase, and other proinflammatory response mediators (Acosta et al., 2013).

Overexpression of the SASP leads to a downstream pro-inflammatory cascade which associates with detrimental chronic inflammation and over accumulation of senescent cells in a selfreinforcing vicious cycle.

Cellular senescence and AD

In the context of neurodegenerative diseases, the attention towards cellular senescence and the related therapeutic potential, has been drawn by the fact that both dividing CNS resident immune cells and post-mitotic neurons develop senescence or "senescent-like" alterations respectively [START_REF] Cohen | Astrocyte senescence: Evidence and significance[END_REF]Jurk et al., 2012;Stojiljkovic et al., 2019).

Most of the prominent feature of cellular senescence reported for non-CNS cellular lines (see Table 1) match the senescent transformation of neurons and glia cells [START_REF] Cohen | Astrocyte senescence: Evidence and significance[END_REF]Jurk et al., 2012;Stojiljkovic et al., 2019). Indeed, in the last ten years, several studies characterized neurons, astrocytes, and microglia senescent phenotype the well-accepted markers, including telomere length, telomerase activity, expression of p16, p21, p53, senescence-associated βgalactosidase, and a SASP However, given the poor anatomical accessibility of the brain, data on the link between cellular senescence and neurodegenerative disease are lacking compared with other medical field such as oncology. To date, whether cellular senescence plays a causal role in neurodegenerative disease is still largely unknown, however, a rising evidence has shed light on the link between cellular senescence and early pathomechanistic alterations of AD.

First the stem cell exhaustion, an integrative hallmark of aging and a consequence of senescence, constitutes the biological background of the age-related decline in adult hippocampal neurogenesis which in turn is associated with impairment of synaptic plasticity essential for memory learning (see also" Senescence and synaptic aging: state of art") [START_REF] Lazarov | Hippocampal neurogenesis: Learning to remember[END_REF].

Second, rodent model of AD cellular senescence and post-mortem human studies have shown that senescent microglia (also called dystrophic) is characterized by functional decline and associates (or colocalizes) with neuroinflammatory lesions and accumulation of tau-mediated threads and tangles which are likely to occur prior to neurodegeneration [START_REF] Streit | Dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in Alzheimer's disease[END_REF].

Several studies have demonstrated that p16 INK4a -positive astrocytes and microglia surround Aβ plaques and neurofibrillary tangles supporting the hypothesis of: i) a physiological contribution of astrocytes and glia in ensuring Aβ and tau proteostasis ii) a central role of brain cellular senescence for AD-related proteinopathies (Bussian et al., 2018). Such findings have also been confirmed in preliminary and encouraging trials with senolytic drugs (see also "Rejuvenation strategies: a focus on senotherapeutics as a pharmacological opportunity for extending heathspan").

Senescence triggered by stressors such as OS, toxic cerebral byproducts (including Aβ and tau) associates with higher levels of SASP compared to replicative senescence. Moreover, animal and human investigations of AD showed that senescent immune cells undergo an upregulation of some cytokines, including IL-6, IL-1β, TGF-β, and TNF-α that are core components of the SASP, inducing microglia overactivation and astroglyosis and eventually sustaining neuroinflammation, a core pathophysiological process of early preclinical stages of AD [START_REF] Cohen | Astrocyte senescence: Evidence and significance[END_REF].

Astrocytes and microglia contribute to synaptic homeaodynamics therefore overaccumulation of senescent immune cells may account for the aging-related synaptic functional decline (see also" Senescence and synaptic aging: state of art").

Senescence can also occur in another line of non-dividing cells such as oligodendrocytes and their progenitors (OPC) supporting the evidence of an age-related increasing vulnerability to white matter damage as well as of a defective remyelination [START_REF] Erten-Lyons | Neuropathologic basis of white matter hyperintensity accumulation with advanced age[END_REF]. Moreover, a fine regulation of the myelination process is likely involved in hippocampal synaptic plasticity suggesting a distinct path whereby senescence may impair memory functions in aging and AD.

A significant spatial relationship between accumulation of senescent OPC and brain Aβ plaques has been reported in human AD patients as well as in a mouse model of AD, indicating that Aβ toxic monomers induce senescent transformation in OPC whose role in AD still remains unclear [START_REF] Zhang | Senolytic therapy alleviates Abeta-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model[END_REF].

Third, endothelial cells of the blood-brain barrier (BBB) undergo senescence which in turn is associated to peculiar anatomical features of BBB breakdown such as, for instance, loss of tight junction with alterations in permeability. Also, the astrocytes senescence plays a role in the BBB dysfunction [START_REF] Yamazaki | Vascular Cell Senescence Contributes to Blood-Brain Barrier Breakdown[END_REF]. Indeed, astrocyte endfeet-terminated processes interact with endothelial cells of the brain capillaries and contribute to BBB integrity, perivascular homeostasis, crosstalk with the peripheral immune system, endothelial transport. Astroglia also mediates the neurovascular signaling to capillaries (to modulate the vasodilatation) and facilitate, through cell surface aquaporin-4, paravascular pathways for clearance of brain metabolites.

Brain immunosenescence and immunosurveillance: a brief overview on emerging concepts in

AD

Regardless the presence of senescent phenotypic transformation, immunosenescence -age-related phenotypical and functional shift / decline of the innate and adaptive immune system -takes place during aging [START_REF] Nikolich-Zugich | The twilight of immunity: emerging concepts in aging of the immune system[END_REF]. Immunosenescence itself is characterized by loss of brain homeostasis at multiple levels, including synapses, proteostasis machineries, BBB dynamics, and inflammatory responses [START_REF] Malavolta | Peripheral mononuclear cell rejuvenation for senescence surveillance in Alzheimer disease[END_REF][START_REF] Rawji | Immunosenescence of microglia and macrophages: impact on the ageing central nervous system[END_REF].

Experimental models of aging as well as in-human findings have recently suggested candidate hallmarks of immunosenescence: i) overall decrease of naïve T cells and circulating B cells (mainly CD19+), ii) accumulations of late-stage differentiated CD8 + memory cells and, decreased CD4+ Tcell, iii) macrophage and neutrophil defects in receptor expression or signaling linked to loss of homeodynamics and functions (including phagocytosis, chemotaxis, and ROS production), iv) a systemic low-grade inflammatory state (ultimately called inflammaging), v) phenotypical alterations of NK cells (including protein receptors expression), vi) lymphocytes and myeloid cells senescent transformation vii) lymphocytes impaired cytokine-responsiveness, viii) lymphocytes (T, NK, and NKT) lack of functional cytotoxicity (Baar et al., 2017;[START_REF] Nikolich-Zugich | The twilight of immunity: emerging concepts in aging of the immune system[END_REF][START_REF] Ovadya | Impaired immune surveillance accelerates accumulation of senescent cells and aging[END_REF][START_REF] Rawji | Immunosenescence of microglia and macrophages: impact on the ageing central nervous system[END_REF]Stervbo et al., 2015aStervbo et al., , 2015b).

An age-related deficit in immunosurveillance, -a complex mechanism whereby the immune system recognizes (pre-)malignant or senescent cell motifs and promote their suppression -has been reported [START_REF] Ovadya | Impaired immune surveillance accelerates accumulation of senescent cells and aging[END_REF].

Immunosenescnce and loss of immunosurveillance are associated with decline of brain homeodynamics since, for instance, lymphocytes and myeloid cells derived interferon-γ, IL-4, BDNF, among others are crucial effectors for social behavior and memory learning [START_REF] Baruch | Aging. Aginginduced type I interferon response at the choroid plexus negatively affects brain function[END_REF][START_REF] Meyer-Luehmann | Myeloid cells in Alzheimer's disease: culprits, victims or innocent bystanders?[END_REF].

Immunosenescence is a key feature of AD experimental models and recent explorative studies have also shown it occurring in AD human patients [START_REF] Larbi | Dramatic shifts in circulating CD4 but not CD8 T cell subsets in mild Alzheimer's disease[END_REF][START_REF] Meyer-Luehmann | Myeloid cells in Alzheimer's disease: culprits, victims or innocent bystanders?[END_REF][START_REF] Pellicano | Immune profiling of Alzheimer patients[END_REF].

The causal nexuses of brain immunosenescence are still poorly understood in humans. However, it has been shown that aging is associated with altered microglia functions and with an imbalance of brain-homing peripheral immune cells that populate meninges and choroid plexus (lymphocytes and dendritic cells) and form a direct interface with the brain parenchyma [START_REF] Baruch | Aging. Aginginduced type I interferon response at the choroid plexus negatively affects brain function[END_REF][START_REF] Li | Microglial priming in Alzheimer's disease[END_REF][START_REF] Meyer-Luehmann | Myeloid cells in Alzheimer's disease: culprits, victims or innocent bystanders?[END_REF]. Alterations of brain T cells subpopulation ratio and cytokines secretory patterns as well as priming (hyperactivation with sustained pro-inflammatory activity) of dysfunctional microglia have increasingly been recognized as key features of immunosenescence during early stages of neurodegenerative diseases [START_REF] Baruch | Aging. Aginginduced type I interferon response at the choroid plexus negatively affects brain function[END_REF][START_REF] Li | Microglial priming in Alzheimer's disease[END_REF][START_REF] Meyer-Luehmann | Myeloid cells in Alzheimer's disease: culprits, victims or innocent bystanders?[END_REF].

Notably, recent Omic-based studies have indicated that microglia age differently from peripherallyderived macrophages in either healthy condition or disease [START_REF] Malavolta | Peripheral mononuclear cell rejuvenation for senescence surveillance in Alzheimer disease[END_REF][START_REF] Rawji | Immunosenescence of microglia and macrophages: impact on the ageing central nervous system[END_REF].

Moreover, since infiltrating peripheral immune cells contribute to brain homeostasis and immunosurveillance and that such cells go towards an age-related functional decline, a causative role of the peripheral immune for brain immunosenescence has also been hypothesized [START_REF] Malavolta | Peripheral mononuclear cell rejuvenation for senescence surveillance in Alzheimer disease[END_REF][START_REF] Rawji | Immunosenescence of microglia and macrophages: impact on the ageing central nervous system[END_REF].

AGING, CELLULAR SENESCENCE, AND AD: THE ANTI-STRESS RESPONSE

Molecular and cellular mechanisms of adaptation and their decline during aging

To date, molecular, cellular and brain tissue homeostatic mechanisms have been explored more than higher complexity levels. Sub cellular / cellular adaption refer to properly coordinated molecular machineries needed to face and prevent potential stress-induced damage, thus providing functional and bioenergetic stability for the whole organism and finally ensuring homeostasis. Cellular homeostasis is ensured by reprogramming bioenergetic / metabolic performance through: 1) proteostasis networks (mechanisms quality control, from protein synthesis to protein degradation) (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015), and 2) highly conserved cellular transduction pathways, i.e. multiple intracellular regulatory signals acting at the (epi)genetic, pre and post translational level and ensuring preservation of core process including proteostasis, oxidative balance, cellular trafficking (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015).

Proteostasis networks and age-related decline: general concerns

There is a complex interplay consisting of highly conserved feedback loops and cross-talks between an array of quality control mechanisms and protein clearance pathways across cells, tissues, and body systems. It has been demonstrated that proteostasis machineries and the related hierarchical organization across tissues and body systems decline with an age-related and tissue-specific fashion. These mechanisms can alter the entire network through an self-reinforcing detrimental downstream cascade which in turn accelerates aging (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015).

Studies conducted in model organisms including nematodes (Caenorhabditis elegans), Drosophila melanogaster, and mice indicate that the capability to adapt through the maintenance of proteostasis is tightly associated with both lifespan and healthspan (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015).

Experimental evidence suggest that the protection and preservation of the core properties of each proteostasis network as well as the reciprocal modulation among several machineries can significantly extend longevity (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015). The understanding of how gradual mechanistic perturbation of proteostasis occur during aging is of widespread interest and now feasible through comparative gene expression-proteomic network analysis and emerging high-throughput approaches. In Table 2 andFigure 3, we illustrate the prominent alterations that are likely to occur in aging We expect that systems biology approach will help confirm and quantify such observations in neurons and neuronal circuits in reliable experimental models and even human individuals.

The anti-stress transduction signals along the dimensions of aging and neurodegeneration

Cellular stress-responsiveness consists of multiple evolutionarily conserved and highly-organized biological pathways (cellular stress response network). The whole organ systems rely on the efficiency of these regulatory signals which elegantly modulate gene expression patterns, epigenetic processes, DRR activity level, proteostasis, transmembrane transport and vesicles trafficking dynamics, microtubule stability and cytoskeletal remodeling, lipid membrane organization, mitochondrial biogenesis and turn over, bioenergetic / metabolic cycles and resources storage [START_REF] Cole | GSK3 as a Sensor Determining Cell Fate in the Brain[END_REF][START_REF] Harrison | Rapamycin fed late in life extends lifespan in genetically heterogeneous mice[END_REF][START_REF] Mair | Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB[END_REF]. There is an elegant and complex interplay among transduction pathways, from ligand-receptor biding to downstream effector(s) activation, in which several pleiotropic master orchestrators modulate the biological output through up-down regulation of cross-talks and feedback / feedforwards loops.

The dysregulation (either hypoactivation or hyperactivation) of several distinct pathways has been associated with brain aging and to the increased risk of developing neurodegenerative diseases. In the case of AD, two pathways seem to be tightly linked to the disease etiology and pathophysiology: the mammalian target of rapamycin (mTOR) and the glycogen synthase kinase-3β (GSK3β). mTOR is a nutrient-sensing cellular pathway activated by several cues as nutrients, hormones, growth factors and external stressors. mTOR is a central regulatory node upstream of the transcriptional regulation of autophagy shifting cellular metabolism to anabolic processes, including mitochondrial biogenesis (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015) see Figure 4. mTOR complex 1 constitutively slows down autophagic processes and is activated by environmental nutrients (for example, amino acids) (Herranz et al., 2015). By contrast, mTOR is inhibited by sensors of cellular caloric restrictions such as by AMP-activated protein kinase (AMPK), a key orchestrator of the metabolic changes of cellular senescence. Overall, mTOR is upregulated in cellular senescence and plays an essential role for the production of the SASP (Herranz et al., 2015). GSK3β is abundantly expressed in the CNS particularly in the hippocampus, where several physiologic processes such as neurogenesis (the process of generating functional neurons from precursors), neurotransmission and synaptic transmission and plasticity are tightly linked to its physiological activity. GSK3β has been linked to core pathophysiological processes occurring in AD such as tau hyperphosphorylation, Aβ overproduction, neuroinflammation and inhibition of cytoprotective signals leading to neuronal and synaptic loss [START_REF] Beurel | Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases[END_REF][START_REF] Cole | GSK3 as a Sensor Determining Cell Fate in the Brain[END_REF].

It has become clear that all the pathomechanistic alterations occurring in AD are generated by the aberrant regulation of physiological multiple transduction pathways leading to a loss of homeostasis. For instance, hyperphosphorylation of truncated proteins such as tau and Aβ is likely to result from an imbalance between phosphatases and kinases (such as GSK3β) [START_REF] Beurel | Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases[END_REF][START_REF] Cole | GSK3 as a Sensor Determining Cell Fate in the Brain[END_REF]. However, whether hyperphosphorylation occurs after or before misfolding and accumulation and which is the exact temporal sequence of this and other post-translational modifications has to be fully mapped yet.

Answering to this question would have a critical role in developing (multi-)targeted, upstream pathway-based therapies for preventive strategies.

The role of Calcium homeodynamics in cellular senescence, aging, and AD

Calcium and its downstream mediators -i.e. all Calcium-dependent enzymes including calpain, protein kinase C, and Calcium/calmodulin-dependent protein kinase -are essential, in neurons, microglia and astrocytes, for neurotransmission, the fine regulation of neural and synaptic plasticity (synapses/dendrites/terminals growth versus regression), synaptic adaptation, and overall synaptic homeostasis [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF][START_REF] Laferla | Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease[END_REF][START_REF] Yu | Calcium dysregulation in Alzheimer's disease: from mechanisms to therapeutic opportunities[END_REF].

The upstream role of neuroexcitability and Calcium dysregulation, in aging and AD, have been underestimated for long time even though a Calcium hypothesis of AD was proposed in 1994.

The upstream triggers and drivers of Calcium dysregulation have not completely untangled yet, especially in humans.

Also the role of Calcium in cellular senescence is not fully described, however, a recent in-vitro trials showed that treatment of senescent cells with Calcium chelators is associated with upregulation of autophagic pathways (whose dysfunction is associated to cellular senescence) and inhibition of senescent transformation under OS [START_REF] Borodkina | Calcium alterations signal either to senescence or to autophagy induction in stem cells upon oxidative stress[END_REF]. Such study has indicated that Calcium intracellular levels indirectly contribute to the cellular transition towards a senescent state under exposure to OS. Previous studies showed that intracellular Calcium fluctuations modulate second messengers' activity in pathways associated with either replicative or other types of stress-induced senescent transformation.

At a more biologically complex level, Calcium dysregulation along with astroglia senescence are associated with BBB breakdown dysfunctions since Calcium transients in astrocyte endfeet are essential for the fine regulation of the vessels tone, and consequently the cerebrovascular blood flow and BBB permeability, which is essential for homeodynamics of circulating byproducts [START_REF] Mulligan | Calcium transients in astrocyte endfeet cause cerebrovascular constrictions[END_REF]. Moreover, accumulating evidence, have demonstrated that an initial perturbation of cellular Calcium homeodynamics can be both caused and downstream affect intracellular Calciumharboring organelles such as the ER, mitochondria, lysosomes, and autophagosomes, which are involved in proteostatic pathways, membrane transport dynamics (including mitochondria), vesicles trafficking. As reported in previous sections, the above mentioned subcellular compartments have a critical role in anti-stress responses and cellular adaptation [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF][START_REF] Laferla | Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease[END_REF][START_REF] Yu | Calcium dysregulation in Alzheimer's disease: from mechanisms to therapeutic opportunities[END_REF].

Hence, Calcium dyshomeostasis is likely to play a crucial and upstream role in early AD-related pathomechanistic alterations.

Animal models of AD have indicated that Calcium dysregulation is characterized by elevated intracellular Calcium levels which might be due to: i) increased Calcium influx through L-type voltage-gated Calcium channels (VGCCs), ii) enhanced Calcium release from intracellular stores through Inositol 1,4,5-Trisphosphate receptor (InsP3R) and the ryanodine receptor (RYR), iii) reduced Glutamatergic N-methyl-D-aspartate (NMDA) receptor-mediated effect, iv) impaired ability of mitochondria to buffer or cycle Calcium, v) reduced cytosolic Calcium buffering capacity and activation of calcineurin and calpains [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF][START_REF] Laferla | Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease[END_REF][START_REF] Yu | Calcium dysregulation in Alzheimer's disease: from mechanisms to therapeutic opportunities[END_REF].

Recent microarray-based trials, Genome-Wide Association Studies (GWAS), and gene expression profiling investigations indicated Calcium-linked susceptibility genes such as CLUSTERIN, ITPKB, RGS4, RAB3A, STMN1, SYNGR3, PICALM, RYR3, MRVI1, RGN, APOC2, AMY2B, and APOE (among others) may account for Calcium dysregulation as the causal nexus of AD and other neurodegenerative diseases [START_REF] Saetre | Age-related changes in gene expression are accelerated in Alzheimer's disease[END_REF].

A long-lasting perturbation in intracellular Calcium-regulating systems is a candidate signature of aging. Biomarkers to track in vivo Calcium dynamics are currently under development in rodent model of aging and AD. In humans, brain-derived exosomes and metabolomics-based signatures seem promising in-vivo approaches [START_REF] Mustapic | Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes[END_REF][START_REF] Varma | Brain and blood metabolite signatures of pathology and progression in Alzheimer disease: A targeted metabolomics study[END_REF].

In line with our hypothetical model of systems-wide aging processes, we argue that a Calcium imbalance hampers intracellular signals which in turn may affect the overall neural and synaptic plasticity until large-scale networks activity, even potentially before any cellular bioenergetic failure, loss of homeodyanmics, and neuronal damage occurs [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF][START_REF] Mustaly-Kalimi | Calcium Signaling Deficits in Glia and Autophagic Pathways Contributing to Neurodegenerative Disease[END_REF][START_REF] Yu | Calcium dysregulation in Alzheimer's disease: from mechanisms to therapeutic opportunities[END_REF].

Calcium dysregulation has been demonstrated to potentially occur upstream to brain proteinopathies with Aβ monomers production and tau hyperphosphorylation that can be triggered by sustained increase of cytosolic Calcium concentrations over basal levels [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF].

In particular, abnormal intracellular levels of Calcium may induce cytosolic pH fluctuations with downstream modification of protein thermodynamic core properties such as free surface energy, which are key determinants of proper folding process [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF]. Moreover, Calcium plays a crucial role in Aβ-mediated synaptic dysfunction and neuronal loss since Aβ oligomers exert neurotoxicity through over activation of intracellular Calcium-release channels, massive Calcium influx which elicits OS, and mitochondrial dysfunction in a self-perpetuating cycle which culminates in pro-apoptotic signals [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF].

Calcium-dependent signaling pathways are crucial for hippocampal synaptic function and plasticity.

Age-related Calcium-systems dependent alterations are likely to be responsible for aberrant neuronal and glial excitability causing long-term potentiation (LTP) and long-term depression (LTD) threshold shifts (see Figure 3 for more details). These alterations reflect those neural desynchronization and abnormal spatial and temporal brain networks oscillatory activity found in experimental aging models and which may account for the age-related risk of epilepsy, neuropsychiatric disorders and neurodegenerative disease including AD [START_REF] Demuro | Calcium signaling and amyloid toxicity in Alzheimer disease[END_REF][START_REF] Yu | Calcium dysregulation in Alzheimer's disease: from mechanisms to therapeutic opportunities[END_REF].

Given the crucial role of Calcium in AD etiology and pathophysiological evolution, a sustained research into the therapeutic perspectives of restoring Calcium homeostasis is demanded.

From a pharmacological standpoint three upstream mechanisms accounting for cellular loss of Calcium homeostasis, have gained increased interest in recent years: the ER stress-response (see Table 1 for more details), ryanodine receptor [START_REF] Lacampagne | Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits[END_REF] and the Sigma 1 receptors (S1R) [START_REF] Hayashi | Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival[END_REF][START_REF] Mori | Sigma-1 receptor chaperone at the ERmitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival[END_REF] Alongside mitochondria, ER is the most important Calcium buffering organelle playing a constitutive role in maintaining resting Calcium levels into cytosolic space. The ER membrane Calcium channel receptors, InsP3R and the RYR are activated by low Calcium contractions and promote its release into the cytoplasm. It has been demonstrated that aberrant post-translational remodeling of the RYR channels is associated with abnormal leak of Calcium and downstream to AD-related pathophysiological processes [START_REF] Lacampagne | Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits[END_REF].

The S1R is a ligand-regulated transmembrane protein localized to mitochondria-associated ER membranes/interface and is finely regulated by ER-mediated calcium dynamics [START_REF] Hayashi | Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival[END_REF][START_REF] Maurice | The pharmacology of sigma-1 receptors[END_REF][START_REF] Mori | Sigma-1 receptor chaperone at the ERmitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival[END_REF]. Therefore, S1R are crucial molecules in the ER stress response, in the subcellular cross-talks between ER and mitochondria and play a key role in the calcium mobilization and homeostasis [START_REF] Hayashi | Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival[END_REF][START_REF] Maurice | The pharmacology of sigma-1 receptors[END_REF][START_REF] Mori | Sigma-1 receptor chaperone at the ERmitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival[END_REF].

The full understanding of the physiological mechanism of S1R is not achieved yet, however, it has been shown in animal models of AD, that under sustained ER stress activation, S1R 1) promote an increase of the mitochondrial calcium influx and 2) after translocation, may serve as enhancer of calcium-dependent intracellular transduction pathways, including phospholipase C and protein kinase C which in turn finally stimulate calcium homeostasis [START_REF] Hayashi | Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival[END_REF][START_REF] Maurice | The pharmacology of sigma-1 receptors[END_REF][START_REF] Mori | Sigma-1 receptor chaperone at the ERmitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival[END_REF]. Stimulation of S1R is associated to significant inhibition of the NMDA receptor activity finally ameliorating dendritic spines and axonal sprouting, i.e. neural plasticity [START_REF] Hayashi | Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival[END_REF][START_REF] Maurice | The pharmacology of sigma-1 receptors[END_REF][START_REF] Mori | Sigma-1 receptor chaperone at the ERmitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival[END_REF].

For its large spectrum neuroprotective effect due to an extensive capacity to modulate intracellular calcium shutting, S1R have been increasingly recognized as novel main candidate targets to prevent or delay AD-related pathophysiological processes.

SYNAPTIC AGING: the primary pathophysiological node of AD is the failure of synaptic networks.

Brain aging may associate with a decline in synaptic functions and overall robustness [START_REF] Morrison | The ageing cortical synapse: hallmarks and implications for cognitive decline[END_REF][START_REF] Zhou | Shrinkage of dendritic spines associated with long-term References Table[END_REF] (for which we propose the term of synaptic aging). An agedependent slowdown of the synaptic homeodynamics, including Calcium dysregulation, OS, and cellular senescence-related decline of adaptation is more likely to occurs in some brain regions such as the neocortex which display increased synaptic vulnerability over time. Indeed, it is well established that key brain networks are composed by neuronal subpopulations with a constitutive bioenergetic and electrophysiologic properties serving at specific physiological functions. For instance, the DMN neurons show a high rate of basal metabolic performance is coupled with low energy reserves and higher intrinsic stress levels which confers to them an intrinsic high degree of vulnerability to stressors compared with resistant neurons [START_REF] Arendt | Alzheimer's disease as a disorder of dynamic brain self-organization[END_REF][START_REF] Bero | Neuronal activity regulates the regional vulnerability to amyloid-beta deposition[END_REF][START_REF] Cirrito | Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo[END_REF][START_REF] Machulda | Effect of APOE epsilon4 status on intrinsic network connectivity in cognitively normal elderly subjects[END_REF]. Hence, a long-lasting synaptic overactivity is associated with a bioenergetic demand that can increase the risk of OS, Calcium dysregulation, and genotoxicity.

The DMN (among other networks) displays highly active connectivity dynamics that ensure flexible pattern re-organization and plastic remodeling (synaptic network compensation). On the flipside, over boosted compensation may drive the exhaustion of cellular resources which ultimately escalates to failure of molecular, cellular and system itself [START_REF] Hatanpaa | Loss of proteins regulating synaptic plasticity in normal aging of the human brain and in Alzheimer disease[END_REF][START_REF] Masliah | Quantitative synaptic alterations in the human neocortex during normal aging[END_REF][START_REF] Morrison | The ageing cortical synapse: hallmarks and implications for cognitive decline[END_REF][START_REF] Palop | Network abnormalities and interneuron dysfunction in Alzheimer disease[END_REF].

Translational findings have shown that non-neurodegenerative age-related cognitive decline is tightly linked to subtle synaptic alterations which may evolve into synaptic failure despite the absence of protein-mediated neurotoxicity, and neurodegeneration [START_REF] Arnold | Cellular, synaptic, and biochemical features of resilient cognition in Alzheimer's disease[END_REF][START_REF] Hatanpaa | Loss of proteins regulating synaptic plasticity in normal aging of the human brain and in Alzheimer disease[END_REF][START_REF] Masliah | Quantitative synaptic alterations in the human neocortex during normal aging[END_REF][START_REF] Morrison | The ageing cortical synapse: hallmarks and implications for cognitive decline[END_REF][START_REF] Thiagarajan | Adaptation to synaptic inactivity in hippocampal neurons[END_REF]. To date, there is little insight regarding causal mechanisms between dysfunction of established vulnerable brain regions, such as those involved in the default mode network (DMN), and proteinopathies or neurodegeneration [START_REF] Bero | Neuronal activity regulates the regional vulnerability to amyloid-beta deposition[END_REF][START_REF] Koch | Effects of aging on default mode network activity in resting state fMRI: does the method of analysis matter?[END_REF].

In spite of the presence of toxic proteins accumulation with evidence of initial neural dysfunction, individuals may display normal cognitive function suggesting that synaptic dynamics from the subcellular compartments to the synaptic cleft until large-scale connectivity networks (i.e. the dynamic integration and coordination of different functional areas of the brain), may underlie resilience compensating preclinical pathophysiological alterations [START_REF] Cirrito | Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo[END_REF][START_REF] Franzmeier | Left frontal hub connectivity delays cognitive impairment in autosomal-dominant and sporadic Alzheimer's disease[END_REF][START_REF] Hatanpaa | Loss of proteins regulating synaptic plasticity in normal aging of the human brain and in Alzheimer disease[END_REF][START_REF] Husi | Proteomic analysis of NMDA receptor-adhesion protein signaling complexes[END_REF][START_REF] Palop | Network abnormalities and interneuron dysfunction in Alzheimer disease[END_REF][START_REF] Perez-Nievas | Dissecting phenotypic traits linked to human resilience to Alzheimer's pathology[END_REF][START_REF] Westlye | Increased hippocampal default mode synchronization during rest in middle-aged and elderly APOE epsilon4 carriers: relationships with memory performance[END_REF].

From a functional standpoint, it has been proposed that "synaptogenic" mechanisms of AD-related cognitive decline, i.e. network activation and deactivation deficits, abnormal oscillatory rhythmic activity and network hypersynchrony have all recognized as key synaptic functional alteration that may account for AD-related synaptic failure [START_REF] Palop | Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease[END_REF][START_REF] Palop | Network abnormalities and interneuron dysfunction in Alzheimer disease[END_REF][START_REF] Westlye | Increased hippocampal default mode synchronization during rest in middle-aged and elderly APOE epsilon4 carriers: relationships with memory performance[END_REF] For next-generation human studies on aging, resting-state and task related functional magnetic resonance studies (fMRI) is the gold standard technique to untangle signatures of synaptic aging at the large-scale network level (for instance the age-related decline of functional connectivity inside the DMN) as well as synaptic compensation (or synaptic resilience). Moreover, emerging hybrid techniques such as electroencephalogram (EEG) -or magnetoencephalogram (MEG) -fMRI, may provide the proper spatial and temporal frames to explore in vivo stress responses and short-longterm biological-neural compensation [START_REF] Niessing | Hemodynamic signals correlate tightly with synchronized gamma oscillations[END_REF][START_REF] Scheeringa | Trial-by-trial coupling between EEG and BOLD identifies networks related to alpha and theta EEG power increases during working memory maintenance[END_REF].

In this regard, some relevant insights have already been accomplished. fMRI studies have demonstrated that hippocampal and prefrontal hyperactivity may represent compensation of genetic or pathomechanistic predispositions that increase the risk to develop AD [START_REF] Franzmeier | Left frontal hub connectivity delays cognitive impairment in autosomal-dominant and sporadic Alzheimer's disease[END_REF][START_REF] Putcha | Hippocampal hyperactivation associated with cortical thinning in Alzheimer's disease signature regions in non-demented elderly adults[END_REF]. Moreover, compensatory mechanisms seem to manifest over a connectivity anterograde shift as well, which is characterized by a functional slowdown in the posterior midline cortex along with increased activity in medial frontal cortex [START_REF] Davis | Que PASA? The posterior-anterior shift in aging[END_REF][START_REF] Koch | Effects of aging on default mode network activity in resting state fMRI: does the method of analysis matter?[END_REF][START_REF] Park | The adaptive brain: aging and neurocognitive scaffolding[END_REF].

Very recently, for the first time, it has been shown that genetic risk factors with a pleiotropic biological effect such as the APOE ε4 allele impact the trajectories of the DMN changes over time in a cohort of cognitively healthy individuals at risk for AD [START_REF] Chiesa | Differential default mode network trajectories in asymptomatic individuals at risk for Alzheimer's disease[END_REF].

In summary, we believe that it is of primary importance to identify the multilevel organization of resilient mechanisms, including synaptic compensation since, it has been shown that tuning some critical factors accounting for brain network dynamics, either in homeostasis or early decompensation, can improve network functional output and physiological properties, i.e. cognition and or behavior.

Moreover, overexpressed synaptic aging features and the related functional failure have been largely reported in experimental models of AD even in absence of brain proteinopathies, pathomechanistic expression of neurodegenerative processes, and clinical manifestations, suggesting a highly upstream role of synaptic homeostasis in AD pathophysiology.

State of art on established and candidate molecular-cellular-tissue-based biomarkers including cellular senescence

The in-vivo exploration of biomarkers of synaptic aging and synaptic failure is challenging because of the anatomical inaccessibility of the brain and the lack of a systematized approach.

Shrinking of dendritic spines is an established typical feature of synaptic aging [START_REF] Morrison | The ageing cortical synapse: hallmarks and implications for cognitive decline[END_REF][START_REF] Zhou | Shrinkage of dendritic spines associated with long-term References Table[END_REF].

However, the primary features of synaptic aging and even more AD-related synaptic failure may encompass: selective (mainly hippocampal and prefrontal) imbalance in axonal remodeling (sprouting versus pruning), depletion of axonal boutons / dendritic spines / synaptic terminals with overall loss of synaptic density. Shrinking of dendritic spines is established as a typical feature of synaptic aging [START_REF] Morrison | The ageing cortical synapse: hallmarks and implications for cognitive decline[END_REF][START_REF] Zhou | Shrinkage of dendritic spines associated with long-term References Table[END_REF].

At the ultrastructural level, some highly frequent features of synaptic aging have been reported: (i) impairment of both post-synaptic and astrocytic-mediated and re-uptake; (ii) deficits in transmembrane transporter expression; (iii) abnormal binding to ligands; (iv) compromised cellular vesicle trafficking of neurotransmitters; (iv) decrease of synaptic proteins involved in neural adaptation and structural plasticity (remodeling) of axons and dendrites, (v) receptor profile shifts such as decrease of hippocampal NMDA [START_REF] Hatanpaa | Loss of proteins regulating synaptic plasticity in normal aging of the human brain and in Alzheimer disease[END_REF][START_REF] Husi | Proteomic analysis of NMDA receptor-adhesion protein signaling complexes[END_REF][START_REF] Morrison | The ageing cortical synapse: hallmarks and implications for cognitive decline[END_REF][START_REF] Piacentini | Reduced gliotransmitter release from astrocytes mediates tau-induced synaptic dysfunction in cultured hippocampal neurons[END_REF].

All these alterations are associated with hippocampal neuronal hypoexcitability with stimulation thresholds for LTP induction shifted to higher frequencies.

The molecular drivers of such synaptic alterations are not completely elucidated yet, especially in humans. However, recent GWAS-based findings have indicated that the loss of synaptic plasticity is the core of AD pathophysiology and that a (epi)genetically driven intracellular signaling, may draw the synaptic failure [START_REF] Dourlen | The new genetic landscape of Alzheimer's disease: from amyloid cascade to genetically driven synaptic failure hypothesis?[END_REF].

Regarding the relationship between cellular senescence and synaptic decline in aging and AD, very recently, studies employing innovative cellular profiling approaches have delivered crucial insights.

Hippocampal neurogenesis and neural plasticity are essential for memory learning and resilience to stress-induced neuroendocrine and behavioral impairment [START_REF] Lazarov | Hippocampal neurogenesis: Learning to remember[END_REF]. It has been demonstrated in humans that a decrease of adult hippocampal neurogenesis distinguishes the ventral dentate gyrus of AD patients compared to age matched cognitively normal individuals.

Robust evidence indicated that the stem cells exhaustion, an integrative hallmark of aging and consequence of cellular senescence, is mechanistically linked to the impairment of adult hippocampal neurogenesis in brain aging and even more in AD [START_REF] Lazarov | Hippocampal neurogenesis: Learning to remember[END_REF][START_REF] Moreno-Jimenez | Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer's disease[END_REF].

Moreover, senescence may impact the quad-partite model of synapsis consisting of an axon terminal, a dendritic spine which is simultaneously contacted by an astrocytic and microglial process. Astrocytes and microglia are essential for CNS development and synaptic homeaodynamics during adulthood. The former promotes synaptogenesis (axonal and dendritic spines sprouting), regulates the synaptic strength, take part in the spatial-temporal integration of multiple synaptic processes and modulate the neurotransmission. Microglia plays a crucial role for immune surveillance of the presynaptic microenvironment as well as for the synaptic remodeling towards dendritic and axon terminals pruning through remodeling of proteolytic and phagocytic mechanisms. The microglia can either recruit astroglia or being recruited by the latter [START_REF] Cohen | Astrocyte senescence: Evidence and significance[END_REF]Jurk et al., 2012;Stojiljkovic et al., 2019).

Senescent glial cells may draw the regional synaptic vulnerability versus regional synaptic resistance (for which we would suggest using the term "synaptic robustness"). Indeed, a recent study carried out via comparative proteomic profiling coupled to in-silico analyses have shown that p16 INK4a -positive astrocytes microglia are highly expressed in prefrontal cortex of AD subjects compared with non-AD individuals. Moreover, peculiar patterns of SASP composition, i.e. TGF-β in the case of senescent microglia, seems to guide a regionally selective age-related synaptic vulnerability [START_REF] Bhat | Astrocyte senescence as a component of Alzheimer's disease[END_REF][START_REF] Graham | Regional Molecular Mapping of Primate Synapses during Normal Healthy Aging[END_REF]. Further studies are urgently need to fill the gap in evidence regarding the link between CNS-resident cells senescence and synaptic dysfunction throughout aging and AD.

Further investigations are needed to unravel the dynamics between the senescence of CNS immune system resident cells and the synaptic shutdown occurring in aging and AD.

Synaptic aging: neurochemical compensation

Several neuropathological studies have provided crucial information on the synaptic compensation that may take place over the course of AD along its continuum. A few neurochemical remodeling occur during the progressive depletion of basal forebrain (BF) cholinergic neurons associated with a subsequent anterograde cortical cholinergic deafferentation particularly involving hippocampal and prefrontal regions [START_REF] Dekosky | Upregulation of choline acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment[END_REF][START_REF] Ikonomovic | Superior frontal cortex cholinergic axon density in mild cognitive impairment and early Alzheimer disease[END_REF][START_REF] Poirel | Moderate decline in select synaptic markers in the prefrontal cortex (BA9) of patients with Alzheimer's disease at various cognitive stages[END_REF]. However, the progressive rate of functional impairment affecting large parts of the cholinergic system shows a nonlinear temporal and spatial evolution. Indeed, both post-mortem and in-vivo human studies have demonstrated that either cognitively normal or mild cognitive impairment individuals displaying AD-related pathophysiology, may have developed compensation for synaptic resilience. In these subpopulations, a regional hyperregulated choline acetyltransferase (ChAT) activity has been reported (particularly in the frontal cortex and in hippocampal) as well as a remodeling of BS cholinergic cortical projections (cholinergic axonal sprouting in hippocampi) [START_REF] Dekosky | Upregulation of choline acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment[END_REF][START_REF] Ikonomovic | Superior frontal cortex cholinergic axon density in mild cognitive impairment and early Alzheimer disease[END_REF][START_REF] Poirel | Moderate decline in select synaptic markers in the prefrontal cortex (BA9) of patients with Alzheimer's disease at various cognitive stages[END_REF]. The degree of these pathomechanistic changes matches the rate of BF cholinergic neurons depletion. These mechanisms may compensate for impaired cholinergic firing, contributing in the maintenance of physiological synchronization of neural oscillations which is a key event for cortical and subcortical networks, particularly those regarding memory and learning [START_REF] Dekosky | Upregulation of choline acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment[END_REF][START_REF] Ikonomovic | Superior frontal cortex cholinergic axon density in mild cognitive impairment and early Alzheimer disease[END_REF][START_REF] Poirel | Moderate decline in select synaptic markers in the prefrontal cortex (BA9) of patients with Alzheimer's disease at various cognitive stages[END_REF].

Given the primary role of the cholinergic system in AD, it is essential to highlight that ACh projections may trigger several evolutionary highly conserved functional synergistic effects among different neurochemical systems involved in cognition and behavior [START_REF] Higley | Cholinergic modulation of multivesicular release regulates striatal synaptic potency and integration[END_REF][START_REF] Poirel | Moderate decline in select synaptic markers in the prefrontal cortex (BA9) of patients with Alzheimer's disease at various cognitive stages[END_REF][START_REF] Salamone | Inhibitory effects of beta-amyloid on the nicotinic receptors which stimulate glutamate release in rat hippocampus: the glial contribution[END_REF]. Such complex interactions seem to be fundamental for for ensuring overall synaptic robustness and resilience. For instance, surface N receptors of astrocytes activate Ca2+ -mediated intracellular signaling essential for the spatial-temporal synchronization of astrocytes with the hippocampal LTP. Moreover, astrocytic and microglial N receptors activate autophagic processes, glutamate and serotonin synthesis and re-uptake as well as modulating neuroinflammatory response [START_REF] Guerra-Gomes | Functional Roles of Astrocyte Calcium Elevations: From Synapses to Behavior[END_REF][START_REF] Higley | Cholinergic modulation of multivesicular release regulates striatal synaptic potency and integration[END_REF][START_REF] Takata | Astrocyte calcium signaling transforms cholinergic modulation to cortical plasticity in vivo[END_REF]. It has been shown that a remodeling of N astrocytic and microglial cell surface may occur during agerelated gliopathy which can compensate for all the important physiological processes that involve glial cells [START_REF] Guerra-Gomes | Functional Roles of Astrocyte Calcium Elevations: From Synapses to Behavior[END_REF][START_REF] Higley | Cholinergic modulation of multivesicular release regulates striatal synaptic potency and integration[END_REF][START_REF] Takata | Astrocyte calcium signaling transforms cholinergic modulation to cortical plasticity in vivo[END_REF]. Even the noradrenergic system undergoes compensatory remodeling during aging and given the widespread noradrenergic cortical projections the catecholaminergic system plays a prominent role in AD. It is well known that an age-related and pathological pre-cortical tau-deposition in the LC occurs (pre-tangle stage of AD) [START_REF] Andres-Benito | Locus coeruleus at asymptomatic early and middle Braak stages of neurofibrillary tangle pathology[END_REF][START_REF] Chalermpalanupap | Targeting norepinephrine in mild cognitive impairment and Alzheimer's disease[END_REF]. A progressive noradrenergic denervation is associated with higher expression levels of hippocampal and amygdala α2A post-synaptic receptors which may represent a compensatory mechanism since stimulation of α2A promotes dendrite sprouting [START_REF] Andres-Benito | Locus coeruleus at asymptomatic early and middle Braak stages of neurofibrillary tangle pathology[END_REF][START_REF] Chalermpalanupap | Targeting norepinephrine in mild cognitive impairment and Alzheimer's disease[END_REF]. Moreover, a progressive slowdown of cortical levels of noradrenaline is likely to be initially counterbalanced by upregulation of both tyrosine hydroxylases and NA transporter binding sites gene expression [START_REF] Andres-Benito | Locus coeruleus at asymptomatic early and middle Braak stages of neurofibrillary tangle pathology[END_REF][START_REF] Chalermpalanupap | Targeting norepinephrine in mild cognitive impairment and Alzheimer's disease[END_REF]. A cholinergic modulation of dopamine circuits has also been reported during aging and even more pronounced in AD where it may associate with typical pattern of dopaminergic-related synaptic plasticity [START_REF] Higley | Cholinergic modulation of multivesicular release regulates striatal synaptic potency and integration[END_REF].

OPEN ISSUES: overcoming a priori hypotheses and exploring the whole aging narrative

The road towards the possibility to dynamically track in-vivo aging within a systems-level integration paradigm poses considerable methodological challenges.

However, some conceptual crucial issues need to be addressed before setting up a blueprint for exploring aging applying a system-wide integrative approach.

1) The perspective of a 'remote control' of brain proteostasis and immunosurveillance through extra-CNS compensatory mechanisms (peripheral compensation) calls for a paradigm shift: from a CNS-centric to a body-centric disease framework.

Network analysis studies have shown that highly conserved cross-talks underlie the interactions between tissue-specific and ubiquitous housekeeping proteins. Experimental models of aging demonstrated that the modulation of this output can be mastered from either side the tissue specific interactome or the whole-body proteome components. These observations corroborate the existence of a complex systems-scaled proteostasis network which may synchronize and coordinate the proteome with a tissue specific manner (Labbadia and Morimoto, 2015;[START_REF] Wang | A systemic view of Alzheimer diseaseinsights from amyloid-beta metabolism beyond the brain[END_REF].

In the context of neurodegenerative brain proteinopathies, recent systems biology studies have demonstrated that peripheral proteostasis machineries can compensate for incipient cerebral Aβ misfolding and toxic accumulation (Labbadia and Morimoto, 2015;[START_REF] Wang | A systemic view of Alzheimer diseaseinsights from amyloid-beta metabolism beyond the brain[END_REF]. On the flipside, the decline of peripheral protein clearance pathways is associated with higher risk of developing brain amyloidosis (de [START_REF] De Leon | Therapeutic potential for peripheral clearance of misfolded brain proteins[END_REF]Labbadia and Morimoto, 2015).

Discovering a reliable open-access to the brain is an essential step to overcome the high degree of inaccessibility of the brain to pharmacological therapies [START_REF] Wang | A systemic view of Alzheimer diseaseinsights from amyloid-beta metabolism beyond the brain[END_REF]Sala et al., 2017).

Approaching proteostasis from a systems biology perspective would allow to targeting the whole body systems-wide effect that a single aberrant proteostasis machinery may originally induce in a certain tissue [START_REF] Bossi | Tissue specificity and the human protein interaction network[END_REF][START_REF] Miller-Jensen | Common effector processing mediates cell-specific responses to stimuli[END_REF].

In Oncology, a peripheral approach to proteostasis is currently under experimental development [START_REF] Bossi | Tissue specificity and the human protein interaction network[END_REF][START_REF] Miller-Jensen | Common effector processing mediates cell-specific responses to stimuli[END_REF]Labbadia and Morimoto, 2015) Moreover, recent human studies reported that medical procedures such as peritoneal dialysis may promote peripheral clearance of neurotoxic protein facilitating brain protein homeodynamics (de [START_REF] De Leon | Therapeutic potential for peripheral clearance of misfolded brain proteins[END_REF]Labbadia and Morimoto, 2015).

As further step, the system-scaled approach to aging can definitively corroborate (or even disprove) results from animal models of AD indicating Aβ-mediated key physiological signaling which is essential for modulating cellular trafficking, apoptosis, cholesterol homeostasis, neurogenesis, and angiogenesis [START_REF] Abramov | Amyloid-beta as a positive endogenous regulator of release probability at hippocampal synapses[END_REF][START_REF] Morris | Questions concerning the role of amyloid-beta in the definition, aetiology and diagnosis of Alzheimer's disease[END_REF]Zimbone et al., 2018;[START_REF] Dourlen | The new genetic landscape of Alzheimer's disease: from amyloid cascade to genetically driven synaptic failure hypothesis?[END_REF][START_REF] Panza | A critical appraisal of amyloidbeta-targeting therapies for Alzheimer disease[END_REF].

A systems-scaled approach is also required to untangle the multiple molecular cross-talks at the innate immune system-brain interface which may be therefore used for a peripheral rejuvenation of brain microglia eventually enhancing and sustaining immunosurveillance. In this context, studies conducted on mice models of AD have shown that the migration of bone marrow-derived cells into the CNS is associated with partial recover of brain damage in presence of brain proteinopathies and early neurodegeneration [START_REF] Malavolta | Peripheral mononuclear cell rejuvenation for senescence surveillance in Alzheimer disease[END_REF][START_REF] Rawji | Immunosenescence of microglia and macrophages: impact on the ageing central nervous system[END_REF]. In humans, the proof-ofmechanism for rejuvenation strategies to enhance brain immunosurveillance has not been established yet [START_REF] Malavolta | Peripheral mononuclear cell rejuvenation for senescence surveillance in Alzheimer disease[END_REF][START_REF] Rawji | Immunosenescence of microglia and macrophages: impact on the ageing central nervous system[END_REF].

Moreover, studies based on bone marrow and parabiosis chimeras, as well as novel transgenic mice, have supported emerging rejuvenation strategies employing systemic (blood) factors, metabolic manipulations (such us manipulation of the host microbiota), and cellular reprogramming. All of these novel approaches can represent a pursuable path for preventing neurodegenerative diseases [START_REF] Erny | Host microbiota constantly control maturation and function of microglia in the CNS[END_REF][START_REF] Katsimpardi | Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors[END_REF][START_REF] Villeda | The ageing systemic milieu negatively regulates neurogenesis and cognitive function[END_REF].

2) The aging process as well as the individual thresholds of resilience / venerability are (epi)genetically driven.

Genome-wide association studies (GWAS) are contributing to map the main genetic factors associated with increased risk of AD-related pathomechanistic alterations, thus decompensated aging. APOE ε4 allele is still the prominent genetic risk factor of late onset AD accounting for several pathophysiological processes not fully understood yet. It is well known that APOE plays a key role in cellular lipid turn-over, influencing membrane lipid raft composition. Lipid homeodynamics is fundamental for neurotransmission and synaptic plasticity [START_REF] Bott | APOE-Sensitive Cholinergic Sprouting Compensates for Hippocampal Dysfunctions Due to Reduced Entorhinal Input[END_REF][START_REF] Seeley | Divergent network connectivity changes in healthy APOE epsilon4 carriers: disinhibition or compensation?[END_REF]. APOE ε4 allele is associated with defective compensatory mechanisms, including the cholinergic hippocampal compensatory sprouting while cortical cholinergic deafferentation progresses [START_REF] Bott | APOE-Sensitive Cholinergic Sprouting Compensates for Hippocampal Dysfunctions Due to Reduced Entorhinal Input[END_REF]. Moreover, distinctive large-scale brain network patterns are related to the APOE ε4 allele [START_REF] Bott | APOE-Sensitive Cholinergic Sprouting Compensates for Hippocampal Dysfunctions Due to Reduced Entorhinal Input[END_REF][START_REF] Seeley | Divergent network connectivity changes in healthy APOE epsilon4 carriers: disinhibition or compensation?[END_REF] in cognitively healthy individuals. Particularly, APOE ε4 carrier individuals show specific and consistent features in the DMN connectivity, including decreased connectivity in the middle and posterior regions and increased connectivity in the frontal and lateral structures [START_REF] Chen | Disrupted functional and structural networks in cognitively normal elderly subjects with the APOE varepsilon4 allele[END_REF][START_REF] Machulda | Effect of APOE epsilon4 status on intrinsic network connectivity in cognitively normal elderly subjects[END_REF][START_REF] Westlye | Increased hippocampal default mode synchronization during rest in middle-aged and elderly APOE epsilon4 carriers: relationships with memory performance[END_REF]. Whether these features reflect compensatory patterns recruited to counterbalance the loss / gain of function of ApoE ε4 has to be elucidated yet.

Several single nucleotide polymorphisms (SNPs) in different genes, related to distinct molecular pathways and intracellular signals, have been identified as upstream drivers of vulnerability to AD: PCALM, BIN1, CR1, CLU, SORL1, RAGE, AQP4, among others [START_REF] Medland | Whole-genome analyses of whole-brain data: working within an expanded search space[END_REF][START_REF] Nho | Whole-exome sequencing and imaging genetics identify functional variants for rate of change in hippocampal volume in mild cognitive impairment[END_REF][START_REF] Parikh | An intronic PICALM polymorphism, rs588076, is associated with allelic expression of a PICALM isoform[END_REF][START_REF] Wang | Meta-analysis of the Association between Alzheimer Disease and Variants in GAB2, PICALM, and SORL1[END_REF].

Gene co-expression network analysis bridged to high-throughput RNA sequencing have provided evidence that different polymorphic variants of the same genes may be associated with either gain or loss of function of downstream interactomes with consequent detrimental or protective effect [START_REF] Bashan | Network physiology reveals relations between network topology and physiological function[END_REF][START_REF] Padmanabhan | Toward Personalized Network Biomarkers in Alzheimer's Disease: Computing Individualized Genomic and Protein Crosstalk Maps[END_REF][START_REF] Starnawska | Epigenome-Wide Association Study of Cognitive Functioning in Middle-Aged Monozygotic Twins[END_REF]. This observation has raised the intriguing and unresolved question if specific gene-gene interactions, gene encoding editing, transcriptional and splicing dysregulation may account for downstream molecular crosstalks which affect the whole network function [START_REF] Johnson | Deep proteomic network analysis of Alzheimer's disease brain reveals alterations in RNA binding proteins and RNA splicing associated with disease[END_REF][START_REF] Starnawska | Epigenome-Wide Association Study of Cognitive Functioning in Middle-Aged Monozygotic Twins[END_REF][START_REF] Hidalgo | A dynamic network approach for the study of human phenotypes[END_REF][START_REF] Mills | RNA-Seq analysis of the parietal cortex in Alzheimer's disease reveals alternatively spliced isoforms related to lipid metabolism[END_REF][START_REF] Rolland | A proteomescale map of the human interactome network[END_REF]. Also, cellular senescence itself is a complex, non-linear pre-post-transcriptional dysregulation of key genes.

We expect that multiplex omics-based single cell approach bridged with network analysis methodologies may soon provide a reliable and accessible blueprint for tracking the genetic, epigenetic, and downstream molecular underpinnings of aging and disease vulnerability versus resistance, at the individual [START_REF] Blokland | Heritability of working memory brain activation[END_REF][START_REF] Glahn | Genetic control over the resting brain[END_REF][START_REF] Miller | A systems level analysis of transcriptional changes in Alzheimer's disease and normal aging[END_REF].

3) Adaptation and resilience are influenced by sex-biased biological factors. Therefore, it is conceivable that a systems-wide sexual dimorphism exists throughout aging [START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF]Hampel et al., 2018c). In the context of AD, a higher vulnerability of women to AD is well-known [START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF][START_REF] Nyarko | Profiles of beta-Amyloid Peptides and Key Secretases in Brain Autopsy Samples Differ with Sex and APOE epsilon4 Status: Impact for Risk and Progression of Alzheimer Disease[END_REF].

It is well established that highly conserved sex-biased genes and gene expression patterns underlie the sexual dimorphism through lifespan, including aging (both under physiological and pathophysiological condition) [START_REF] Parsch | The evolutionary causes and consequences of sex-biased gene expression[END_REF][START_REF] Penaloza | Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells[END_REF][START_REF] Reinius | An evolutionarily conserved sexual signature in the primate brain[END_REF]. The sexual dimorphism is associated with: a) different genetic architecture, b) sex-biased gene expression, c) sex-related regulatory mechanisms of gene-gene and gene-environment interactions [START_REF] Parsch | The evolutionary causes and consequences of sex-biased gene expression[END_REF][START_REF] Penaloza | Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells[END_REF][START_REF] Reinius | An evolutionarily conserved sexual signature in the primate brain[END_REF][START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF]. It is not understood yet whether genetic factors closely associated with sex are modifiers or primary determinants of the higher intrinsic vulnerability to AD that women express [START_REF] Parsch | The evolutionary causes and consequences of sex-biased gene expression[END_REF][START_REF] Penaloza | Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells[END_REF].

Recently, it has been shown that sexual dimorphism impacts upstream regulator of disease hallmarks, including brain Aβ accumulation. For instance, an estrogen effect on the expression and activity levels of β-secretase 1 (BACE1). A sexual dimorphism exists also at the synaptic and network connectivity level [START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF]Hampel et al., 2018c).

Consistent with our theoretical model of systems-wide aging, it is expected to discover that the sexbiased genetic and biological background is coupled with sex-related differences across multi-level systems until large scale network organization, i.e. sex-biased endophenotypes of aging and AD [START_REF] Parsch | The evolutionary causes and consequences of sex-biased gene expression[END_REF][START_REF] Penaloza | Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells[END_REF][START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF].

We assume that underestimation of the impact of sexual dimorphism on AD pathophysiology may partially explain the failure of previous clinical trials which did not assign sex-biased dosage of active treatment nor analyzed outcomes in a sex-wide manner.

Untangling distinctive weakness to stressors and identifying sex-related intrinsic deficiencies in adaptive and compensatory dynamics is expected to be a key step to achieve individualized therapies aimed at strengthening brain and body robustness over-time [START_REF] Ferretti | Sex differences in Alzheimer disease -the gateway to precision medicine[END_REF]Hampel et al., 2018c). 4) Time and space coordinates are differentially scaled within body systems as well as brain networks. The non-liner integration of multiple processes with peculiar spatial-temporal scales is necessary for any physiological output such a cognitive task [START_REF] Buzsaki | Space and time in the brain[END_REF]. Aging and AD related dynamics, including compensatory mechanisms and network(s) failure, are highly dependent on the system levels (space) and the temporal context they are operating in.

The missing step for the comprehensive understanding of aging, resilience, and AD pathophysiological evolution is the accomplishment of a quantitative and flexible integrative multilevel modeling to detect multiple spatial-temporal patterns and for eventually dissecting causal mechanisms and downstream cascades [START_REF] Hermans | Stress-related noradrenergic activity prompts large-scale neural network reconfiguration[END_REF].

Implementing the computational analysis with brain / body system space and time coordinates will also help identify those primary disease-modules that significantly contribute to decompensatory cascades throughout the aging-AD continuum [START_REF] Bartsch | Network Physiology: How Organ Systems Dynamically Interact[END_REF].

For instance, whether the neuronal bioenergetic failure associated with loss of cellular homeostatic dynamics is upstream or downstream to large-scale network desynchronization and the uncoupling of neural oscillator is likely to be predict by incoming next generation of in-silico modeling [START_REF] Bartsch | Network Physiology: How Organ Systems Dynamically Interact[END_REF][START_REF] Bashan | Network physiology reveals relations between network topology and physiological function[END_REF][START_REF] Bassett | Network neuroscience[END_REF][START_REF] Miller | A systems level analysis of transcriptional changes in Alzheimer's disease and normal aging[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF].

Thus, predictive and disease-modifying strategies aimed at tuning and enhancing resilience-prone factors shall be able to match the different space scales and time windows that are disease-relevant.

PERSPECTIVES

Next steps along the roadmap for deciphering aging-related dynamics underlying the transition to AD, is the standardization and harmonization of a protocol for an accurate detection and profiling of CNS resident cells senescence (a suitable target for senotherapeutic) and then mapping the key drivers of brain adaptive responses and compensatory mechanisms, at the systems-level integration.

A general consensus on the panel of markers of cellular senescence has not been achieved yet because of methodological limitation to discriminate between the several mechanisms and stimuli that may trigger the cellular program responsible for the phenotypic and functional transitional shift (Sharpless and Sherr, 2015).

Omics-based techniques such as proteomic profiling enables a cell-type approach are promising tools for detecting in-vivo cellular senescence and provide reliable outcomes for next generation of senolytic-based clinical trials. The evolution of functional neuroimaging-based hybrid techniques, including fMRI-EEG(-MEG) co-registration trials is significantly supporting the mapping of higher complexity levels of aging-related changes such as synaptic aging signatures.

In addition, unlikely adipose tissue, muscles, blood, or skin, the anatomical complexity of brain does not allow biopsy and tissue assessment for evaluating primary outcome measures such as changes in senescent cells accumulation rates.

Several lines of evidence suggest that peripheral lymphocytes and monocytes may reflect CNS alterations due the intense crosstalk between the peripheral immune and the CNS resident immune cells. For instance, brain-derived toxic byproducts may enter the blood stream through a dysfunctional BBB, and trigger aberrant intracellular signaling in peripheral cells [START_REF] Bartolome | Impaired apoptosis in lymphoblasts from Alzheimer's disease patients: cross-talk of Ca2+/calmodulin and ERK1/2 signaling pathways[END_REF]. It has been reported that immortalized lymphocytes from AD patients display apoptosisresistance and upregulation of p21 CDKi [START_REF] Bartolome | Simvastatin overcomes the resistance to serum withdrawal-induced apoptosis of lymphocytes from Alzheimer's disease patients[END_REF].

Neural-Derived Exosome Protein Abnormalities represent an emerging possibility for prediction of preclinical AD, other neurodegenerative diseases and cerebrovascular disease is quantification of pathogenic proteins in plasma neural cell-derived exosomes [START_REF] Goetzl | High complement levels in astrocyte-derived exosomes of Alzheimer disease[END_REF][START_REF] Mustapic | Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes[END_REF].

Exosomes derived from any type of CNS cell may be harvested efficiently from human plasma by initial physical precipitation followed by immunoabsorption with antibodies that selectively recognize a surface antigen on that CNS cell-type. This two-step process permits separate enrichment of exosomes secreted by neurons, astrocytes, microglia and chondroitin sulfate proteoglycan type 4 (CSPG4) cells. We argue that reliable senescent signatures, previously assessed in-vivo in CSF, may be recovered from circulating plasma Neural-Derived Exosomes [START_REF] Goetzl | High complement levels in astrocyte-derived exosomes of Alzheimer disease[END_REF][START_REF] Mustapic | Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes[END_REF].

Next-generation sequencing and high-throughput immune profiling technologies offer the platform for characterizing innate immune responses during aging and AD [START_REF] Larbi | Dramatic shifts in circulating CD4 but not CD8 T cell subsets in mild Alzheimer's disease[END_REF][START_REF] Meyer-Luehmann | Myeloid cells in Alzheimer's disease: culprits, victims or innocent bystanders?[END_REF][START_REF] Pellicano | Immune profiling of Alzheimer patients[END_REF].

As a next challenge, the combination of aging-related variables (including cellular senescence biomarkers) into a systems-levels integration -integrative disease modelling (IDM) paradigm -will enable to identify aging modules and compensatory mechanisms [START_REF] Bartsch | Network Physiology: How Organ Systems Dynamically Interact[END_REF][START_REF] Bashan | Network physiology reveals relations between network topology and physiological function[END_REF][START_REF] Bassett | Network neuroscience[END_REF][START_REF] Miller | A systems level analysis of transcriptional changes in Alzheimer's disease and normal aging[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF].

To date, IDM studies have suggested that while brain (sub)cellular adaptation occurs through a bioenergetic / metabolic molecular reprogramming, synaptic-scaled adaptation implies timedependent neural oscillatory changes to synergistically modulate and recruit synaptic network for stress response dynamics and resilience [START_REF] Bartsch | Network Physiology: How Organ Systems Dynamically Interact[END_REF][START_REF] Bashan | Network physiology reveals relations between network topology and physiological function[END_REF][START_REF] Bassett | Network neuroscience[END_REF][START_REF] Miller | A systems level analysis of transcriptional changes in Alzheimer's disease and normal aging[END_REF][START_REF] Shine | Human cognition involves the dynamic integration of neural activity and neuromodulatory systems[END_REF].

It is expected that cross-disciplinary and multi-omics approaches such as neuroimaging-omics will allow to depict which gene expression patterns and protein-protein interactions, in both cycling and senescent cells, are linked to distinct large-scale synaptic networks patterns [START_REF] Mcintosh | Two-back makes step forward in brain imaging genomics[END_REF][START_REF] Medland | Whole-genome analyses of whole-brain data: working within an expanded search space[END_REF][START_REF] Richiardi | Correlated gene expression supports synchronous activity in brain networks[END_REF] Neuroimaging-omics and computational approaches to identify and quantify networks of physiologic interactions, will also help mapping how (sub)cellular bioenergetic shifts can impact large scale neural oscillations and vice versa forecasting either physiological or pathophysiological trajectories [START_REF] Mcintosh | Two-back makes step forward in brain imaging genomics[END_REF][START_REF] Medland | Whole-genome analyses of whole-brain data: working within an expanded search space[END_REF][START_REF] Van Keuren-Jensen | Bringing RNA-seq closer to the clinic[END_REF]. This step is essential to establish whether the trajectories of two / or more systems-scaled events are linked one another and also to cognitive outcomes. Network analysis of physiological and pathophysiological interactions across body systems and time will enable to accomplishing the "human map of brain resilience"

Rejuvenation strategies: a focus on senotherapeutics as a pharmacological opportunity for extending heathspan

Very recent proof-of-concept trials conducted in rodent models of aging and AD delivered evidence that senescent cells represent a druggable target for rejuvenation strategies (treatments to delay and potentially even reverse the ageing process), and thus for counteracting age-related disease in humans [START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF]. Two different promising senotherapeutic approaches (compounds able to target senescent cells) have been investigated so far: senolytic drugs (small compounds capable to selectively target senescent cells and promote their clearance) and senomorphic drugs (small compounds capable to slowdown senescent cells' phenotype).

One of the most investigating senotherapeutic approach is the combination of dasatinib, a tyrosine kinase inhibitor approved for leukemia and quercetin which is an experimental compound with pleotropic effects including modulation of the NF-κB, PI3K/Akt, and mTOR axis (Bussian et al., 2018;[START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF].

A proof-of-concept study has recently demonstrated that treating mice transplanted with senescent cells with a senolytic drug (the combination of dasatinib plus quercetin) can recover from illness and prolong health-and lifespan [START_REF] Xu | Senolytics improve physical function and increase lifespan in old age[END_REF].

The same senotherapeutic combination has recently shown a strong effect in inducing necrosis of senescent OPC surrounding cerebral Aβ plaque in brains of both human AD patients and mouse model of the disease. In the latter group, the senolytic effect was also associated with improvement of cognitive outcomes and neuroinflammation markers [START_REF] Zhang | Senolytic therapy alleviates Abeta-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model[END_REF].

The old compound AP20187, an antibiotic ineffective in normal mice or humans, has been proven with a senolytic activity in transgenic mouse named INK-ATTAC where AP20187 promotes the dimerization of a pro-apoptotic factor expressed under p16 INK4A upregulation. AP20187 administration was associated with removal of p16 INK4A -positive senescent astrocytes and microglia coupled with reduction of tau aggregation, neuroinflammation, and amelioration of the cognitive performance [START_REF] Baker | Clearance of p16Ink4a-positive senescent cells delays ageingassociated disorders[END_REF]Bussian et al., 2018). This initial proof-of-concepts have indicated a therapeutic path to pursue. However, the translation of senolysis into a large-scale human clinical trial needs to first identify the most suitable target population(s) that may benefit of the treatment as well as a standardized biomarker panel to estimate target druggability, evaluate treatment response, and anticipate potential resistance mechanisms.

As above mentioned, another potential senotherapeutic approach may be represented by the pharmacological induction of a senomorphic effect, i.e. the suppression of the cell senescence phenotypes, such as the compound capable of apparently "rejuvenate" senescent cells.

Ax example of senomorphic effect has been demonstrated with trials investigating compounds capable to modulate senescent cells' pro-inflammatory and self-reinforcing activity through the downregulation or even the suppression of the key factors of the SASP.

In this context, emerging SASP inhibitors are represented by drugs capable of slowing down the molecular factors upregulating the SASP production and secretion.

The proinflammatory transcription factor NF-κB, the upregulation of the mTOR axis, the ROS induced upregulation of p38MAPK cascade, the upregulation of the JAK/STAT pathway, among others are the main trigger of the SASP (Acosta et al., 2013;Herranz et al., 2015).

Small compounds with a JAK/STAT inhibitory effect (ruxolitinib), or mTOR suppressing effect (rapamycin) or combined, mTOR plus NF-κB, inhibitory activity (metformin) among others, have shown a good efficacy in reducing tissue damage due to SASP [START_REF] Moiseeva | Metformin inhibits the senescence-associated secretory phenotype by interfering with IKK/NF-kappaB activation[END_REF][START_REF] Xu | JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age[END_REF] .

Trials with transient administration of rapamycin have shown good outcomes in mice transplanted with senescent cells [START_REF] Bitto | Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice[END_REF][START_REF] Harrison | Rapamycin fed late in life extends lifespan in genetically heterogeneous mice[END_REF].

Navitoclax (ABT-263) is an experimental compound with a BCL-2 inhibitory effect that has recently shown a remarkable senolytic effect [START_REF] Chang | Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice[END_REF].

The translation of senotherapeutics approaches into human clinical trials has recently started to explore safety and efficacy of dasatinib plus quercetin in subjects displaying organ illness and tissue-specific accumulation of senescent cellular population (for instance ClinicalTrials.gov identifier: NCT02848131).

The anatomical challenges posed by the CNS must be overcome before setting the first in human anti-senescence R&D pipeline.

Systems pharmacology for enhancing adaptation and compensation: towards a pharmacological modulation of aging

Identifying compensatory mechanisms which ensure normal biological and functional outputs during aging as well as understanding the key points to modulate compensation upstream to pathophysiological failures, will open up novel therapeutic avenues to target aging and draw it towards a prolonged compensated phase.

A blueprint for the systems-scaled in-vivo exploration of drug-induced effects and trades off is urgently needed.

Systems pharmacology, an integrative interdisciplinary modelling approach, is an emerging conceptual paradigm that aims at exploring and predicting the entire effect of a drug modeling several multidimensional variables encompassing body systems and their biological interactions across inter-individual variability.

Systems pharmacology provides a blueprint for generating comprehensive experimental designs, especially where the systems encompass complex cross-talks. Systems pharmacology enables to model snapshots of human drug-induced interactomes and intracellular signals, drug-related (epi)genetic regulation, transcription regulatory networks, drug-induced immune responses, drugdrug interactions and drugs-multi targets interaction [START_REF] Harrold | Network-based approaches in drug discovery and early development[END_REF][START_REF] Zhao | Systems pharmacology: network analysis to identify multiscale mechanisms of drug action[END_REF][START_REF] Geerts | Quantitative systems pharmacology as an extension of PK/PD modeling in CNS research and development[END_REF]. Thus, systems pharmacology explorative strategy generates a unique and innovative opportunity to extend traditional pharmacological test and analysis to construct a holistic drug effect modeling grounded on bioinformatic tools and computational algorithms capable to provide the overall output. This integrative approach is in line with the perspective of multi-targets treatment for complex diseases, as AD, and is essential to overcome the limitation inherent the traditional "onedrug-one-target" model [START_REF] Harrold | Network-based approaches in drug discovery and early development[END_REF][START_REF] Zhao | Systems pharmacology: network analysis to identify multiscale mechanisms of drug action[END_REF][START_REF] Geerts | Quantitative systems pharmacology as an extension of PK/PD modeling in CNS research and development[END_REF]. This paradigm can lead to the characterization of pathway-based molecular signatures that will allow a mechanistic stratification of individuals by clustering a given phenotype for underlying hidden pathomechanisms. Thus, disease affected individuals will be able to access stratified pathway-based therapies [START_REF] Harrold | Network-based approaches in drug discovery and early development[END_REF][START_REF] Zhao | Systems pharmacology: network analysis to identify multiscale mechanisms of drug action[END_REF][START_REF] Geerts | Quantitative systems pharmacology as an extension of PK/PD modeling in CNS research and development[END_REF].

Precision pharmacology is a novel conceptual paradigm that aims at accelerating drug R&D programs informing all decision-making processes, from the proof of pharmacology to the clinical trials development, and until the real-word approval. This, in turn, will dramatically lower the rate of attrition, generating a high quality and cost-effective support to researchers for accomplishing individualized treatments. Indeed, applying precision pharmacology is assumed to serve at the following long-term goals: I) developing multi-target therapeutic approaches for multifactorial polygenic diseases, such as AD, II) providing predictive models / quantitative frameworks of therapeutic efficacy and risk of adverse events for individuals, and III) developing "molecularly" biomarker-guided targeted therapies (in an unbiased fashion) -i.e., treatments specifically adapted ("tailored") to the individual and the disease stage, in line with the precision medicine paradigm [START_REF] Hampel | Biomarker-Drug and Liquid Biopsy Co-development for Disease Staging and Targeted Therapy: Cornerstones for Alzheimer's Precision Medicine and Pharmacology[END_REF](Hampel et al., , 2018a) ) The broad goal of PM is to optimize the effectiveness of disease prevention and therapy starting from customized interventions tailored on the individual's biological make-up -e.g. genetic, biochemical, phenotypic, lifestyle, and psychosocial characteristics -through the "P4 Medicine" (P4M) model that is predictive, preventive, personalized, and participatory (Hampel et al., 2018a[START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF]. To ensure the development of the PM paradigm in AD, the international Alzheimer PM Initiative (APMI) and its connected Cohort Program (APMI-CP) were launched and conceptually associated with the large-scale U.S. Precision Medicine Initiative (PMI) (available at https://obamawhitehouse.archives.gov/precision-medicine) and the U.S. "All of Us Research

Program" -originated from the previous U.S. PMI Cohort Program (available at https://allofus.nih.gov/). Therefore, we think that it is now essential to bring aging at the systems-level integration untangling how molecular and cellular pathways dynamically interact with large-scale network to substantially transform the R&D pipelines and paving the way for developing "molecularly" biomarker-guided aging targeted therapies -i.e., treatments specifically adapted ("tailored") to the individual aging phenotype.

Pathway-based therapies are reality in oncology were several phenotypes of cancer diverging for histology, cytology topography and clinical manifestations are already treated with the same drug after targeting the molecular pathway they share [START_REF] Drilon | Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children[END_REF].

We believe that systems pharmacology and in-silico drug development may support future senotherapeutic-oriented R&D pipelines.

For instance, in-silico modeling can deliver key insights to cut down the risk of adverse off-target effects on non-senescent cells. Furthermore, systems pharmacology may generate reliable model of most suitable target population determining optimal benefit/risk trade-off according genetic, biological, and functional variables. Indeed, the precision pharmacology paradigm shift is expected also to facilitate the drug repositioning process -also known as drug re-profiling or drug repurposing process -indicating that a drug registered for a specific disease spectrum may prove potential off-target biological effects that may be beneficial for diseases for which it has not been registered [START_REF] Harrold | Network-based approaches in drug discovery and early development[END_REF][START_REF] Zhao | Systems pharmacology: network analysis to identify multiscale mechanisms of drug action[END_REF]. Drug repositioning programs may provide molecules capable of inducing metabolic / bioenergetic-rewiring as well as hermetic responses (for instance, caloric restrictions) and or promoting compensatory mechanisms to alterations in the proteostasis machineries, calcium homeostasis, oxidative balance, among others.

In this sense, GSK3β inhibitors as lithium chloride (a therapeutic approved for bipolar disorders), have been found to attenuate aberrant overactivation of GSK3β thus increasing resilience and slowing down those downstream pathways leading to decompensated aging and AD [START_REF] Hampel | Lithium trial in Alzheimer's disease: a randomized, single-blind, placebo-controlled, multicenter 10-week study[END_REF][START_REF] Machado-Vieira | Lithium, Stress, and Resilience in Bipolar Disorder: Deciphering this key homeostatic synaptic plasticity regulator[END_REF][START_REF] Phiel | GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides[END_REF].

In conclusion, we assume that the system-level integration of aging processes will allow to attain the "human map of brain resilience "whereas systems pharmacology can probe, in a near future, multi-target drugs capable of targeting senescence and simultaneously other aging-related detrimental mechanisms ("aging targetome"), thus prolonging or even freezing aging at the compensated phase. Age-related decline in mitochondrial trafficking due to decreased size, number, density mitochondrial dynamics: fusion, fission, movement, and mitophagy (Munoz-Espin and Serrano, 2014;Wang et al., 2009) Apoptosis resistance ▲ BCL-2 ▼ Bax Upregulation of the antiapoptotic protein BCL-2 due to chronic activation of the transcription factor cAMP response element-binding protein (CREB), a stress response key mediator Epigenetic downregulation of the proapoptotic gene Bax *Other factors: modulation of the PI3K-AKT-mTOR-FOXO axis, the Mek-Erk axis, the p53-p21 axis (p53 levels and p53 post-translational modifications), and the rate of oxidative stress. (Baar et al., 2017;Lopez-Otin et al., 2013) ER-UPR ▲ ATF6α Upon stress conditions, as accumulation of unfold proteins, the ER stress-response, restore ER homeostasis through: a) promote the translocation of chaperones from the ER lumen to the cytosol, b) to stimulate three ER transmembrane sensors triggering downstream complex signals i.e. ER-UPR: PERK, IRE1α and ATF6α. Upregulation of ATF6α is the strongest candidate biomarker for hyperactivation of the ER-UPR in senescent cells. (Pluquet et al., 2015) Notes: Most of the prominent feature of cellular senescence reported for non-CNS cellular lines (in the table) match the senescent transformation of neurons and glia cells. Indeed, in the last ten years, several studies characterized neurons, astrocytes, and microglia senescent phenotype the well-accepted markers, including telomere length, telomerase activity, expression of p16, p21, p53, senescence-associated β-galactosidase, and a SASP (Bussian et al., 2018;Jurk et al., 2012;Stojiljkovic et al., 2019)

HSPs (chaperones)

▼chaperones gene expression ▼ chaperones transcription factor ▲ degradation (cellular imbalance) Chaperones: 1) ensure correct folding to native states; 2) bind to molecular clients thus inducing allosteric (or orthosteric) changes, 3) are highly selective mediators of cytosolic small proteins degradation in lysosomes (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015) ER-UPR ▲ ATF6α

Upon stress conditions, as accumulation of unfold proteins, the ER stress-response, restore ER homeostasis through: a) promote the translocation of chaperones from the ER lumen to the cytosol, b) to stimulate three ER transmembrane sensors triggering downstream complex signals i.e. ER-UPR: PERK, IRE1α and ATF6α. Upregulation of ATF6α is the strongest candidate biomarker for hyperactivation of the ER-UPR in senescent cells. (Pluquet et al., 2015) Autophagy ▼macroautophagy (even mitophagy) ▼microautophagy ▼chaperones-mediated autophagy (?) accumulation autophagosomes Autophagy strongly contributes to life span while aging negatively impacts on autophagy machineries (vicious cycle).

Autophagy ensure proper protein and mitochondrial (mitophagy) turn-over essential for copying with continuous energetic-metabolic demands and is highly controlled for recycling of non-critical proteins to a pool of amino acids. (Komatsu et al., 2006) Proteasome ▼proteasomal subunits (turn-over or assembly) ▲inactivation of the proteasome (?) Alterations ubiquitin enzymatic cascade.

Proteasome (large multisubunit complexes) activity can be either ubiquitin mediated or not.

Proteasome is responsible for clearance of abnormal, denatured, short-lived or damaged proteins

The ability to maintain a functional proteome declines during the aging process Inactivation of the proteasome occurs through direct interaction with age-induced protein-aggregates. (Kaushik and Cuervo, 2015;Komatsu et al., 2006) Post-translational modifications ▲ covalent modifications byproducts ▲ cleavage end-products PTMs of a protein alters its native structure with: 1) loss of function and a change in thermodynamic properties (i.e. insolubility, misfolding and aggregation) 2) prevents ubiquitin binding to protein and thus degradation Covalent additions to protein side chains: phosphorylation, acylation, alkylation, glycosylation, and oxidation The homeodynamic space i.e. genome / epigenome and ecosystem factors. The homeodynamic space is ensured by adaptive responses and compensatory mechanisms scaled in time and space across multi-level system networks: from molecular pathways, to cellular stress responses, to brain cell-to-cell and synaptic dynamics, to large-scale brain network activity, to brain-periphery crosstalks. If a decompensatory cascade occurs, homeostasis progressively breaks down until final systems failure.

Unrevealing the spatial-temporal coordinates of multilevel adaptive and compensatory dynamics (from molecules to large-scale networks) is essential to enhance resilience and prevent diseases The orange circle represents the entire spectrum of pathophysiological mechanisms across all systems levels, during the disease evolution. Such nonlinear interactions support the hypothesis that the network failure might originate at any level initiating a detrimental cascade.

The subcellular level may show, for instance, compartmental impairment of proteostasis or mitochondrial functions dysfunction of membrane dynamics and cellular trafficking.

The cellular level dysfunction originates from the sum of distinct aberrant molecular pathways with the impairment of anti-stress responses and activation of signaling driving the cell fate.

The tissue level may display loss of structural and functional organization in terms of cell-to cell communication.

At brain system level, functional shifts in large scale network organization allow a spatial and temporal resources redistribution to cope with stress (i.e. systems-wide adaptation and comepsation).

The organ systems level. The existence of many cross-links-talks between CNS and the periphery might account for the hypothesis that brain diseases can originate or be substantially related to peripheral systems failure.

The colored pyramid represents the potential drug response at each level (from green to red and from the base to the peak there is a decreasing amplitude of effect) from adaptation to compensation towards decompensation and finally systems failure, a hypothetical "point of no return". Proteostasis is ensured by evolutionarily conserved networks which act at different levels. The translation control level is regulated by ribosome and ribosomal factors. The protein folding quality control (transition from unfolded to intermediates until native states) is orchestrated by the endoplasmic reticulum "unfolded protein response" (ER-UPR) and the chaperones. The latter also serve as regulator of protein trafficking across different cellular-subcellular compartments. The final step is represented by protein degradation or autophagy. Two pathways allow protein degradation control: the endosomal/lysosomal degradation and proteasome degradation that can undergo either a ubiquitin dependent (UPS) or independent fashion. There is a complex, interplay consisting of highly conserved feedback and cross-talks between protein quality control machineries and clearance pathways and inside each component. The proper functioning of each proteostasis network accounts for longevity and potentially all the components ensuring the integrity of proteome are affected by aging. See also Table 2 for detailed and integrative information [Adapted from Kaushik S and Cuervo AM. Proteostasis and aging. Nat Med. 2015 Dec;21(12):1406-15] Figure 4. mTOR, its downstream factors, and physiological functions mTOR is a nutrient-sensing cellular pathway activated by several clues as nutrient, hormones, growh factors and external stressors. mTOR is a central regulatory node upstream of the transcriptional regulation of autophagy shifting cellular metabolism to anabolic processes. mTOR constitutively slow down autophagic processes and is stimulated by cellular sensors of caloric restrictions. Increased cytoplasmatic concentrations of amino acids and grow factors, activate the so-called lysosomal nutrient-sensing complex with subsequent lysosomal membrane-tethering of transducer of regulated CREB (mTORC1) which, in turn activates the mTOR kinase complex (binding to mTORC2). Active mTORC1 exerts a tonic autophagy-inhibitory function downregulating, via phosphorylation, two crucial autophagy-inducing transcription factors, i.e. the helix-loop-helix transcription factor (TFEB) and the forkhead family of transcription factors (FOXO). In addition, mTOR enhances Nrf-1 activity associated with expression of proteasome subunit and promotes cellular shift to anabolic metabolism, including mitochondrial biogenesis. Aging is tightly associated with the risk of a broad range of activating gene mutations, either upstream or downstream mTOR pathways or specifically concerning the kinase DNA sequence, leading to a sustained over enhancement of mTOR pathways through the cross-talking and feedback interacting networks.

Edited from

[Adapted from Cho CH. Frontier of epilepsy research -mTOR signaling pathway. Exp Mol Med. 2011 May 31;43(5):231-74]

Figure 1 .

 1 Figure 1. Trajectory of pathophysiological mechanisms across the continuum of systems multiscale hierarchical self-organization, from systems homeostasis to systems failure: conceptual basis for molecular pathway-based therapies.The homeodynamic space i.e. genome / epigenome and ecosystem factors. The homeodynamic space is ensured by adaptive responses and compensatory mechanisms scaled in time and space across multi-level system networks: from molecular pathways, to cellular stress responses, to brain cell-to-cell and synaptic dynamics, to large-scale brain network activity, to brain-periphery crosstalks. If a decompensatory cascade occurs, homeostasis progressively breaks down until final systems failure. Unrevealing the spatial-temporal coordinates of multilevel adaptive and compensatory dynamics (from molecules to large-scale networks) is essential to enhance resilience and prevent diseases The orange circle represents the entire spectrum of pathophysiological mechanisms across all systems levels, during the disease evolution. Such nonlinear interactions support the hypothesis that the network failure might originate at any level initiating a detrimental cascade. The subcellular level may show, for instance, compartmental impairment of proteostasis or mitochondrial functions dysfunction of membrane dynamics and cellular trafficking. The cellular level dysfunction originates from the sum of distinct aberrant molecular pathways with the impairment of anti-stress responses and activation of signaling driving the cell fate. The tissue level may display loss of structural and functional organization in terms of cell-to cell communication.At brain system level, functional shifts in large scale network organization allow a spatial and temporal resources redistribution to cope with stress (i.e. systems-wide adaptation and comepsation). The organ systems level. The existence of many cross-links-talks between CNS and the periphery might account for the hypothesis that brain diseases can originate or be substantially related to peripheral systems failure.The colored pyramid represents the potential drug response at each level (from green to red and from the base to the peak there is a decreasing amplitude of effect) from adaptation to compensation towards decompensation and finally systems failure, a hypothetical "point of no return".

Figure 2 .

 2 Figure 2. Hypothetical trajectories of aging according individual threshold of resilience and vulnerability.Each individual has a genetically, biologically, and environmentally determined thresholds of resiliency capacity. Also, an individual vulnerability to stressors and disease exists.We illustrate hypothetical trajectories of aging according individual threshold of resilience and vulnerability across spatial-temporal system-wide shifts and along 4 stages: from adaptation to irreversible failure. STAGE 1 -ADAPTATION STAGE -STRESS RESPONSES: Metabolic and energetic reconfiguration associated with functional switch in Molecular / Cellular / Tissue / Brain systems / body system network activity STAGE 2 -COMPENSATION STAGE -RESILIENCY MECHANISMS: Structural and functional counterbalancing of one or more initial pathomechanistic alterations. STAGE 3 -BREAKDOWN OF RESILIENCY MECHANISMS:

Figure 3 .

 3 Figure 3. Proteostais networks ensuring cellular adaptation and their declining over aging.Proteostasis is ensured by evolutionarily conserved networks which act at different levels. The translation control level is regulated by ribosome and ribosomal factors. The protein folding quality control (transition from unfolded to intermediates until native states) is orchestrated by the endoplasmic reticulum "unfolded protein response" (ER-UPR) and the chaperones. The latter also serve as regulator of protein trafficking across different cellular-subcellular compartments. The final step is represented by protein degradation or autophagy. Two pathways allow protein degradation control: the endosomal/lysosomal degradation and proteasome degradation that can undergo either a ubiquitin dependent (UPS) or independent fashion. There is a complex, interplay consisting of highly conserved feedback and cross-talks between protein quality control machineries and clearance pathways and inside each component. The proper functioning of each proteostasis network accounts for longevity and potentially all the components ensuring the integrity of proteome are affected by aging. See also Table2for detailed and integrative information [Adapted fromKaushik S and Cuervo AM. Proteostasis and aging. Nat Med. 2015 Dec;21(12):1406-15]

Probing the continuum from aging to AD Aging

  has been a vibrant topic of discourse among many generations of Philosophers and William Shakespeare defined the perceived polarity of young age and old age. "Crabbed age and youth cannot live together; Youth is full of pleasure, age is full of care; Youth like summer morn, age like winter weather; Youth like summer brave, age like winter bare. Youth is full sport, age's breath is short; Youth is nimble, age is lame; Youth is hot and bold, age is weak and cold; Youth is wild, age is tame. Age, I do abhor thee; youth, I do adore thee."

  depression of hippocampal synapses. Neuron 44, 749-757. https://doi.org/10.1016/j.neuron.2004.11.011 Zimbone, S., Monaco, I., Giani, F., Pandini, G., Copani, A.G., Giuffrida, M.L., Rizzarelli, E., 2018. Amyloid Beta monomers regulate cyclic adenosine monophosphate response element binding protein functions by activating type-1 insulin-like growth factor receptors in neuronal cells.

		wideness
	Mitochondria	▼volume
	ultrastructure	▼fusion and fission
	and morphology	abnormally elongated
	Aging Cell 17. https://doi.org/10.1111/acel.12684

Table 2 .

 2 The main alterations of proteostasis machineries in aging

	Proteostasis	Aging-related features
	networks

  Hampel H et al. Precision pharmacology for Alzheimer's disease. Pharmacol Res. 2018. doi 10.1016/j.phrs.2018.02.014. Reproduced with permission.

Morimoto, 2015; Sala et al., 2017) HSPs: heat shock proteins; SAHFs: Senescence-associated heterochromatin foci; ER-UPR: endoplasmic reticulum unfolded protein response; PTMs: post-translational modifications

ACKNOWLEDGEMENTS

This research benefited from the support of the Program "PHOENIX" led by the Sorbonne University Foundation and sponsored by la Fondation pour la Recherche sur Alzheimer.

HH is supported by the AXA Research Fund, the "Fondation partenariale Sorbonne Université" and the "Fondation pour la Recherche sur Alzheimer", Paris, France. Ce travail a bénéficié d'une aide de l'Etat "Investissements d'avenir" ANR-10-IAIHU-06. The research leading to these results has received funding from the program "Investissements d'avenir" ANR-10-IAIHU-06 (Agence Nationale de la Recherche-10-IA Agence Institut Hospitalo-Universitaire-6).

CONTRIBUTORS TO THE ALZHEIMER PRECISION MEDICINE INITIATIVE -WORKING GROUP (APMI-WG):

Mohammad AFSHAR (Paris), Lisi Flores AGUILAR (Montréal), Leyla AKMAN-ANDERSON (Sacramento), Joaquín ARENAS (Madrid), Jesus AVILA (Madrid), Claudio BABILONI (Rome), Filippo BALDACCI (Pisa), Richard BATRLA (Rotkreuz), Norbert BENDA (Bonn), Keith L. BLACK (Los Angeles), Arun L.W. BOKDE (Dublin), Ubaldo BONUCCELLI (Pisa), Karl BROICH (Bonn), Francesco CACCIOLA (Siena), Filippo CARACI (Catania), Juan CASTRILLO † (Derio), Enrica CAVEDO (Paris), Roberto CERAVOLO (Pisa), Patrizia A. CHIESA (Paris), Jean-Christophe CORVOL (Paris), Augusto Claudio CUELLO (Montréal), Jeffrey L. CUMMINGS (Las Vegas), Herman DEPYPERE (Gent), Bruno DUBOIS (Paris), Andrea DUGGENTO (Rome), Enzo EMANUELE (Robbio), Valentina ESCOTT-PRICE (Cardiff), Howard FEDEROFF (Irvine), Maria Teresa FERRETTI (Zürich), Massimo FIANDACA (Irvine), Richard A. FRANK (Malvern), Francesco GARACI (Rome), Hugo GEERTS (Berwyn), Filippo S. GIORGI (Pisa), Edward J. GOETZL (San Francisco), Manuela GRAZIANI (Roma), Marion HABERKAMP (Bonn), Marie-Odile HABERT (Paris), Harald HAMPEL (Paris), Karl HERHOLZ (Manchester), Felix HERNANDEZ (Madrid), Dimitrios KAPOGIANNIS (Baltimore), Eric KARRAN (Cambridge), Steven J. KIDDLE (Cambridge), Seung H. KIM (Seoul), Yosef KORONYO (Los Angeles), Maya KORONYO-HAMAOUI (Los Angeles), Todd LANGEVIN (Minneapolis-Saint Paul), Stéphane LEHÉRICY (Paris), Alejandro LUCÍA (Madrid), Simone LISTA (Paris), Jean LORENCEAU (Paris), Dalila MANGO (Rome), Mark MAPSTONE (Irvine), Christian NERI (Paris), Robert NISTICÒ (Rome), Sid E. O'BRYANT (Fort Worth), Giovanni PALERMO (Pisa), George PERRY (San Antonio), Craig RITCHIE (Edinburgh), Simone ROSSI (Siena), Amira SAIDI (Rome), Emiliano SANTARNECCHI (Siena), Lon S. SCHNEIDER (Los Angeles), Olaf SPORNS (Bloomington), Nicola TOSCHI (Rome), Steven R. VERDOONER (Sacramento), Andrea VERGALLO (Paris), Nicolas VILLAIN (Paris), Lindsay A. WELIKOVITCH (Montréal), Janet WOODCOCK (Silver Spring), Erfan YOUNESI (Esch-sur-Alzette).

Time for the systems-level integration of aging: resilience enhancing strategies to prevent Alzheimer's disease

Harald Hampel 1 , Simone Lista 1,2,3 , Christian Neri 4 , and Andrea Vergallo 1,2,3 for the Alzheimer Precision Medicine Initiative (APMI) CONFLICTS OF INTEREST HH serves as Senior Associate Editor for the Journal Alzheimer's & Dementia; he received lecture fees from Biogen and Roche, research grants from Pfizer, Avid, and MSD Avenir (paid to the institution), travel funding from Functional Neuromodulation, Axovant, Eli Lilly and company, Takeda and Zinfandel, GE-Healthcare and Oryzon Genomics, consultancy fees from Qynapse, Jung Diagnostics, Cytox Ltd., Axovant, Anavex, Takeda and Zinfandel, GE Healthcare, Oryzon Genomics, and Functional Neuromodulation, and participated in scientific advisory boards of Functional Neuromodulation, Axovant, Eisai, Eli Lilly and company, Cytox Ltd., GE Healthcare, Takeda and Zinfandel, Oryzon Genomics and Roche Diagnostics.

HH is co-inventor in the following patents as a scientific expert and has received no royalties: The upregulation of p16 Ink4a , a cyclin-dependent kinase inhibitor (CDKI), associates with epigenetic mechanism (through inhibition of a DNA metiltrasferase) which causes the demethylation of the p16Ink4a promoter. (Baker et al., 2016;Lopez-Otin et al., 2013;Munoz-Espin et al., 2013;Munoz-Espin and Serrano, 2014) SASP ▲ IL-6, IL-8 ▲ CCL2 ▲ MMP-1, MMP-3

Cytokines, chemokines, and metalloproteinases are key components of the senescence-associated secretory phenotype (SASP). (Acosta et al., 2013;Herranz et al., 2015) DNA damage repair network ▲ γ-H2AX foci ▲ phosphorylated p53

The presence of γH2AX (H2A histone family member X) foci is a marker of sustained DNA damage.

The phosphorylation of p53 triggers a crucial pathway culminating in the activation of the DNA damage response (DDR) . *A set of unrecoverable DNA damage including double strand breaks may lead to either senescence or apoptosis in a "dose"-dependent fashion. In the senescent cell, the resulting genomic instability triggers a sustained activation of DNA damage response (DDR), also via p53, until it turns into a defective process. Nuclear changes ▼LaminB1 ▲CCF ▲SAHF ▲H3K9me3 ▲HP1γ

Reduced levels of LaminB1, a structural protein of the nuclear lamina, are due to its genetic downregulation exerted by p53 and p16 Hypo-condensed constitutive heterochromatin (chromatin enriched in repressive marks)

Presence of cytoplasmic chromatin fragments (CCF)

Senescence-associated heterochromatin foci (SAHF) composed by repressive histone marks such as H3K9me3 (epigenetic downregulation of the proapoptotic factor Bax) and HP1γ. SAHF are peculiar of oxidative stress induced senescence * The most relevant epigenetic alterations leading to cellular senescence are represented by the overactivity of DNA methylases, histones methylases and deacetylases, including H3K9me3, H3K27me3, and H4K20me3 (age-related changes in epigenetically silenced loci) (Baar et al., 2017;Munoz-Espin et al., 2013) Cell morphology ▲ cytoplasm nucleus ratio ▲ size ▲ flatness and Morphological changes are prominently due to improper activation of mitogenic stimulation with a decrease in the rate of protein degradation, cytoskeletal remodeling, and electrolytes dyshomeostasis mTOR pathways and the endoplasmic reticulum unfolded protein response (ER-URP) ATF6α play a central role in driving senescent transformation (Munoz-Espin et al., 2013;Munoz-Espin and Serrano, 2014)

Mechanism

Biomarker Key notes References

Cell cycle arrest ▲CDKI p16 ▲CDKI p21

The upregulation of p16 Ink4a , a cyclin-dependent kinase inhibitor (CDKI), associates with epigenetic mechanism (through inhibition of a DNA metiltrasferase) which causes the demethylation of the p16Ink4a promoter.

Cytokines, chemokines, and metalloproteinases are key components of the senescence-associated secretory phenotype (SASP). (Acosta et al., 2013;Herranz et al., 2015) DNA damage repair network ▲ γ-H2AX foci ▲ phosphorylated p53

The presence of γH2AX (H2A histone family member X) foci is a marker of sustained DNA damage.

The phosphorylation of p53 triggers a crucial pathway culminating in the activation of the DNA damage response (DDR) . *A set of unrecoverable DNA damage including double strand breaks may lead to either senescence or apoptosis in a "dose"-dependent fashion. In the senescent cell, the resulting genomic instability triggers a sustained activation of DNA damage response (DDR), also via p53, until it turns into a defective process.

( Nuclear changes ▼LaminB1 ▲CCF ▲SAHF ▲H3K9me3 ▲HP1γ

Reduced levels of LaminB1, a structural protein of the nuclear lamina, are due to its genetic downregulation exerted by p53 and p16 Hypo-condensed constitutive heterochromatin (chromatin enriched in repressive marks)

Presence of cytoplasmic chromatin fragments (CCF)

Senescence-associated heterochromatin foci (SAHF) composed by repressive histone marks such as H3K9me3 (epigenetic downregulation of the proapoptotic factor Bax) and HP1γ. SAHF are peculiar of oxidative stress induced senescence * The most relevant epigenetic alterations leading to cellular senescence are represented by the overactivity of DNA methylases, histones methylases and deacetylases, including H3K9me3, H3K27me3, and H4K20me3 (age-related changes in epigenetically silenced loci) (Baar et al., 2017;Munoz-Espin et al., 2013) Cell morphology ▲ cytoplasm nucleus ratio ▲ size ▲ flatness and wideness Morphological changes are prominently due to improper activation of mitogenic stimulation with a decrease in the rate of protein degradation, cytoskeletal remodeling, and electrolytes dyshomeostasis mTOR pathways and the endoplasmic reticulum unfolded protein response (ER-URP) ATF6α play a central role in driving senescent transformation (Munoz-Espin et al., 2013;Munoz-Espin and Serrano, 2014) Mitochondria ultrastructure and morphology ▼volume ▼fusion and fission abnormally elongated Age-related decline in mitochondrial trafficking due to decreased size, number, density mitochondrial dynamics: fusion, fission, movement, and mitophagy (Munoz-Espin and Serrano, 2014;Wang et al., 2009) Apoptosis resistance ▲ BCL-2 ▼ Bax Upregulation of the antiapoptotic protein BCL-2 due to chronic activation of the transcription factor cAMP response element-binding protein (CREB), a stress response key mediator Epigenetic downregulation of the proapoptotic gene Bax *Other factors: modulation of the PI3K-AKT-mTOR-FOXO axis, the Mek-Erk axis, the p53-p21 axis (p53 levels and p53 post-translational modifications), and the rate of oxidative stress. (Baar et al., 2017;Lopez-Otin et al., 2013) ER-UPR ▲ ATF6α Upon stress conditions, as accumulation of unfold proteins, the ER stress-response, restore ER homeostasis through: a) promote the translocation of chaperones from the ER lumen to the cytosol, b) to stimulate three ER transmembrane sensors triggering downstream complex signals i.e. ER-UPR: PERK, IRE1α and ATF6α. Upregulation of ATF6α is the strongest candidate biomarker for hyperactivation of the ER-UPR in senescent cells. (Pluquet et al., 2015) Notes: Most of the prominent feature of cellular senescence reported for non-CNS cellular lines (in the table) match the senescent transformation of neurons and glia cells. Indeed, in the last ten years, several studies characterized neurons, astrocytes, and microglia senescent phenotype the well-accepted markers, including telomere length, telomerase activity, expression of p16, p21, p53, senescence-associated β-galactosidase, and a SASP (Bussian et al., 2018;Jurk et al., 2012;Stojiljkovic et al., 2019) Table 2. The main alterations of proteostasis machineries in aging

Proteostasis networks

Aging-related features Key points References

Protein folding ▲ protein insolubility (aggregation) ▲ folding in non-native states Age-related decline in protein solubility (risk of aggregation), partially due to over-production of proteins with different thermodynamic features. Other contributing factors: electrolytes dyshomeostasis, pH fluctuations Misfolded proteins over-expose hydrophobic stretches that alter solubility coefficients Age-related intrinsic tendency to aggregate is related to defective DRR with abnormal aminoacidic sequences (Daggett and Fersht, 2003;Knowles et al., 2014) HSPs (chaperones) ▼chaperones gene expression ▼ chaperones transcription factor ▲ degradation (cellular imbalance) Chaperones: 1) ensure correct folding to native states; 2) bind to molecular clients thus inducing allosteric (or orthosteric) changes, 3) are highly selective mediators of cytosolic small proteins degradation in lysosomes (Kaushik and Cuervo, 2015;Labbadia and Morimoto, 2015) ER-UPR ▲ ATF6α

Upon stress conditions, as accumulation of unfold proteins, the ER stress-response, restore ER homeostasis through: a) promote the translocation of chaperones from the ER lumen to the cytosol, b) to stimulate three ER transmembrane sensors triggering downstream complex signals i.e. ER-UPR: PERK, IRE1α and ATF6α. Upregulation of ATF6α is the strongest candidate biomarker for hyperactivation of the ER-UPR in senescent cells. (Pluquet et al., 2015) Autophagy ▼macroautophagy (even mitophagy) ▼microautophagy ▼chaperones-mediated autophagy (?) accumulation autophagosomes Autophagy strongly contributes to life span while aging negatively impacts on autophagy machineries (vicious cycle).

Autophagy ensure proper protein and mitochondrial (mitophagy) turn-over essential for copying with continuous energetic-metabolic demands and is highly controlled for recycling of non-critical proteins to a pool of amino acids. (Komatsu et al., 2006) Proteasome ▼proteasomal subunits (turn-over or assembly) ▲inactivation of the proteasome (?) Alterations ubiquitin enzymatic cascade.

Proteasome (large multisubunit complexes) activity can be either ubiquitin mediated or not.

Proteasome is responsible for clearance of abnormal, denatured, short-lived or damaged proteins

The ability to maintain a functional proteome declines during the aging process Inactivation of the proteasome occurs through direct interaction with age-induced protein-aggregates. (Kaushik and Cuervo, 2015;Komatsu et al., 2006) Post-translational modifications ▲ covalent modifications byproducts ▲ cleavage end-products PTMs of a protein alters its native structure with: 1) loss of function and a change in thermodynamic properties (i.e. insolubility, misfolding and aggregation) 2) prevents ubiquitin binding to protein and thus degradation Covalent additions to protein side chains: phosphorylation, acylation, alkylation, glycosylation, and oxidation (Labbadia and Morimoto, 2015;Sala et al., 2017) HSPs: heat shock proteins; SAHFs: Senescence-associated heterochromatin foci; ER-UPR: endoplasmic reticulum unfolded protein response; PTMs: post-translational modifications