USherbrooke.ca Alan A Cohen
email: alan.cohen@usherbrooke.ca.

Brian K Kennedy
email: bkennedy@nus.edu.sg.

Ulrich Anglas
email: ulrich.anglas@mail.mcgill.ca.

Anne M Bronikowski

Joris Deelen
email: joris.deelen@age.mpg.de.

Fr Éd Érik Dufour

Gerardo Ferbeyre
email: g.ferbeyre@umontreal.ca.

Luigi Ferrucci
email: ferruccilu@grc.nia.nih.gov.

Claudio Franceschi
email: claudio.franceschi@unibo.it.

Daniela Frasca
email: dfrasca@med.miami.edu.

Bertrand Friguet
email: bertrand.friguet@sorbonne-universite.fr.

Pierrette Gaudreau
email: pierrette.gaudreau@umontreal.ca.

Vadim N Gladyshev
email: vgladyshev@rics.bwh.harvard.edu.

Efstathios S Gonos
email: sgonos@eie.gr.

Vera Gorbunova
email: vera.gorbunova@rochester.edu.

Philipp Gut
email: philipp.gut@rd.nestle.com.

Mikhail Ivanchenko
email: ivanchenko.mv@gmail.com.

Éronique Legault

Jean-Franc ¸ois Lemaître

Thomas Liontis
email: thomas.liontis@mail.mcgill.ca.

Guang-Hui Liu
email: ghliu@ioz.ac.cn.

Mingxin Liu
email: mingxin.liu@usherbrooke.ca.

Ot Andrea B Maier
email: a.b.maier@vu.nl.

T N Óbrega

Marcel G M Olde Rikkert
email: marcel.olderikkert@radboudumc.nl.

Graham Pawelec
email: graham.pawelec@uni-tuebingen.de.

Sylvie Rheault
email: sylvie.rheault@umontreal.ca.

Alistair M Senior
email: alistair.senior@sydney.edu.au.

Andreas Simm
email: andreas.simm@uk-halle.de.

Sonja Soo
email: sonja.soo@mail.mcgill.ca.

Annika Traa
email: annika.traa@mail.mcgill.ca.

Svetlana Ukraintseva
email: svetlana.ukraintseva@duke.edu.

Quentin Vanhaelen

Jeremy M Van Raamsdonk
email: jeremy.vanraamsdonk@mcgill.ca.

Jacek M Witkowski

Anatoliy I Yashin

Robert Ziman
email: robert.ziman@usherbrooke.ca.

Tamàs Fülöp
email: tamas.fulop@usherbrooke.ca.

Brian K Kennedy B-E

Frédérik Dufour
email: frederik.dufour@usherbrooke.ca.

Claudio Franceschi N-P

Véronique Legault
email: veronique.legault@usherbrooke.ca.

Jean-François Lemaître
email: jean-francois.lemaitre@univ-lyon1.fr.

Guang- Hui Liu

Otávio T Nóbrega G,H

Marcel G M Olde

Alistair M Senior N,O

Jeremy M Van Raamsdonk U-W

Fülöp Tamàs

BB Aa

Lack of consensus on an aging biology paradigm? A global survey reveals an agreement to disagree, and the need for an interdisciplinary framework

Keywords: Aged, Aging, Aging paradigm, biology of aging, aging mechanisms, aging interventions, epidemiology of aging, evolution of aging, philosophy of science

 A recent symposium debate highlighted disagreements and confusion in aging biology  Symposium participants followed up by completing an online survey  Survey results show little common ground on most questions in aging biology  However, there is a near-consensus that aging is heterogeneous and multifactorial  Work is needed to achieve a common paradigm in aging biology

Introduction

The authors were all participants at the Biology of Aging Symposium: Understanding Aging to Better Intervene, held November 9-11, 2019 in Montreal, Quebec. The symposium featured 44 speakers with a diversity of expertise related to aging, including basic aging biology, translational geroscience, geriatric medicine, nutrition, immunosenescence, evolutionary ecology, demography, statistics, systems biology, epidemiology, and complex systems theory. During the course of the symposium, a debate was held on the question, "Do we know what aging is?" with Brian Kennedy ostensibly arguing the "Yes" side and Alan Cohen ostensibly arguing the "No" side. There was dynamic audience participation. Most participants agreed that the debate and the subsequent extensive discussion involving many participants were striking in how it highlighted the lack of a clear consensus paradigm [START_REF] Kuhn | The Structure of Scientific Revolutions Second Edition, Enlarged[END_REF] in the field, and collectively we agreed it would be important to describe this for the research community in our field.

Accordingly, we designed a survey that was sent to participants of the symposium, both invited speakers and students/other participants. The survey was meant to capture the opinions on both the key points of disagreement and basic features of aging in general. All participants who responded to the survey are coauthors. We use the term "aging" to refer to "aging biology," though, as will be shown, some but not all participants felt that aging biology cannot be understood in isolation from psychological, social, and cultural factors.

Philosophers of science generally believe that at least some aspects of a shared paradigm or worldview are often critical in helping a field advance, though the precise nature and role of such paradigms is debated [START_REF] Kuhn | The Structure of Scientific Revolutions Second Edition, Enlarged[END_REF][START_REF] Lakatos | Falsification and the methodology of scientific research programmes[END_REF]. Beyond the biology of aging that is our focus here, it has been argued that there is a broad gerontological paradigm spanning from biology to the social sciences [START_REF] Ferraro | The Gerontological Imagination: An Integrative Paradigm of Aging[END_REF], J o u r n a l P r e -p r o o f with six key features: causality, life course analysis, multifaceted change, heterogeneity, accumulation processes, and ageism. Our discussions at the symposium showed no consensus on questions relating to ageism and causality, but generally supported the other four proposed features. Nonetheless, the broad areas of disagreement shown below will pose challenges for the field, and the nature of a paradigm is likely to be quite different for aging biology than in gerontology more broadly. If we cannot agree on what aging is (definitions and mechanisms), how can we identify it, measure it, or know if we are measuring it [START_REF] Belsky | Quantification of biological aging in young adults[END_REF][START_REF] Calimport | To help aging populations, classify organismal senescence[END_REF][START_REF] Horvath | DNA methylation age of human tissues and cell types[END_REF][START_REF] Levine | Modeling the rate of senescence: can estimated biological age predict mortality more accurately than chronological age?[END_REF]? How can we evaluate potential anti-aging interventions [START_REF] Justice | A framework for selection of bloodbased biomarkers for geroscience-guided clinical trials: report from the TAME Biomarkers Workgroup[END_REF]? How relevant are findings from other species in terms of understanding human aging [START_REF] Austad | Methusaleh's Zoo: how nature provides us with clues for extending human health span[END_REF][START_REF] Jones | Diversity of ageing across the tree of life[END_REF]? We do not believe it is possible at this point to propose a paradigm that would be broadly accepted in the field; accordingly, the best we can do is to note the important differences and try to propose a roadmap for what would be needed to achieve consensus on key questions.

Methods

Survey

We used the tool Google Forms to distribute a survey to all participants at the symposium (44 invited speakers, 2 organizers, 14 students, and the 11 others). The survey collected the following information:  There are many species across the tree of life that do not age at a rate that is biologically relevant or appreciable  Broadly speaking, aging mechanisms are similar in most species  Mortality rates or survival curves are generally a reasonable proxy for aging at the organismal level  It is important for the field to have a consensus definition of aging  Aging is an undesirable process that should be treated, cured, or minimized  Aging biology can be understood largely as a cellular and molecular process We obtained a waiver from the ethics committee at the CIUSSS-ECHUS and post-hoc approval to conduct the survey (Project #2021-3728).

Statistics

We provide only descriptive statistics and do not calculate standard errors, confidence intervals, or pvalues, which would only be relevant if we could suppose that our sample was representative of a larger population. We do not claim this to be the case, and prefer to present only raw data. We did assess pairwise Pearson correlations among the 16 Likert-scale variables by transforming them to a 7-point scale, and we also conducted exploratory principal components analysis (PCA) on the covariance matrix of this dataset, though we present only the broad strokes of those results due to fear that a 16-variable PCA with only 37 observations could be highly unstable.

Limitations

Even though the questions were subject to substantial discussion before the actual survey, with rewording performed throughout the rounds of debate if necessary, some questions could be viewed as ambiguous or be understood differently by some participants. In part, this was due to different understanding of phrases such as "we understand", "damage", etc. We acknowledge these limitations, which we think do not affect the core findings of this study.

J o u r n a l P r e -p r o o f

Results

Thirty-seven researchers/students responded to the survey (the authors of this paper). Demographically, 27 of 37 respondents were male, with origins in 18 different countries. No country had more than 3 respondents except Canada, which had 14, though it is worth noting that some respondents marked their current country of residence, some marked their country of origin, some marked both, and some left the field blank. Respondents were at a variety of career stages as early as post-bachelor's, but most (26) were established researchers/professors (Fig. 1A). A variety of expertise was represented, with the great majority having at least some expertise in the basic biology of aging, and many others with expertise in model organisms, metabolism, complex systems theory, evolutionary ecology, etc. (Fig. 1B). Respondents were also invited to volunteer additional expertise; these responses included: "Neuroscience and computer modelling," "Neurodegenerative disease," "Biodemography of aging, health, and longevity," "Bioinformatics," and "Cell signalling, cell death, degenerative diseases, microenvironment." While our respondents do not represent a random sample of aging researchers, they do represent a wide variety of expertise in the field, and most are far enough advanced in their studies to have well-informed opinions on aging biology. Sample size was insufficient to formally compare responses by subgroups, but an informal analysis by sex suggests there are no particularly marked differences in opinions between male and female researchers. Most of the key questions were asked on a 7-level Likert scale, and responses are summarized in Fig. 2.

J o u r n a l P r e -p r o o f

Of 16 questions, 11 had at least one respondent in each of the most extreme categories ("Strongly agree" and "Strongly disagree"), and all 16 had responses ranging from Strongly on one end to Moderately on the other. For three questions, there was nonetheless a clear preponderance of respondents taking one opinion. For whether aging proceeds uniformly across tissues, 86% disagreed (strongly, moderately, or slightly). Regarding the multi-dimensionality of aging, 86% agreed strongly, moderately, or slightly that it is heterogeneous and cannot be measured with a single metric. Most respondents (81%) agreed strongly, moderately, or slightly that it is important for the field to have a consensus definition of aging. For four J o u r n a l P r e -p r o o f other questions, there was a clear tendency in one direction even if there was still substantial disagreement: more respondents than not concurred that aging is largely a cellular/molecular process (75%), that the hallmarks/pillars [START_REF] Kennedy | Geroscience: linking aging to chronic disease[END_REF]López-Otín et al., 2013) do a decent job of summarizing the aging process (72%), that mortality rates are a reasonable proxy for organismal aging (72%), and that aging is undesirable (69%). In other words, for 9 out of 16 questions, there is no clear trend for a consensus of opinion (<65% on one side or the other), and diametrically opposed views were held by comparable numbers of researchers among our relatively small sample. Even for the two questions with the most agreement, it is worth noting that among the three researchers in the opposing camp in each case, two were among the best-known experts in the field (h-index > 100).

J o u r n a l P r e -p r o o f Unsurprisingly, there appeared to be a clear correlation structure among the Likert-scale questions (Fig. 3), though in many cases the limited sample size makes precise estimation of the coefficients doubtful.

Broadly, those who thought aging should be cured/treated also tended to think it was more likely for this to be possible, and that aging was readily quantifiable, programmed, and universal. PCA confirmed this: 34% of the variance was explained by the first axis, and 15% by the second, with the loadings of the first axis qualitatively reflecting the summary of the correlation matrix. While the correlation structure is clear, it is not overwhelmingly strong, indicating that there are not simply two opposing camps with a range of opposing views.

J o u r n a l P r e -p r o o f As for when aging begins, there was also marked disagreement, with a relatively uniform distribution of responses ranging from parental gamete formation to later in life (Fig. 4A). One participant has argued recently that the answer is none of the above: that aging begins very early in life (during embryogenesis), but the exact moment is not defined [START_REF] Kinzina | Patterns of Aging Biomarkers, Mortality, and Damaging Mutations Illuminate the Beginning of Aging and Causes of Early-Life Mortality[END_REF]. For interventions likely to slow aging, there was near-consensus on some potential interventions but not others (Fig. 4B). Only 2 of 35 respondents J o u r n a l P r e -p r o o f thought enhancing cell replication was promising, and 91% (32/35) thought lifestyle interventions were promising, but for the other four interventions, between 29% and 63% thought them promising, showing substantial divergence in opinions of researchers. Furthermore, this was not due to a small set that found all interventions promising and a larger set that found none or only lifestyle promising; there was a relatively uniform distribution of how many interventions were thought promising (Fig. 4C). Some respondents noted additional interventions they thought promising: "Healthy, diversified nutrition, in particular digital tool aided personalized nutrition," "Minimize somatotroph dysfunction," "Combination of pharmacological (including those affecting immune function) and lifestyle," and "Prevention of early changes in the organism (disease-dependent etc)."

J o u r n a l P r e -p r o o f Lastly, there was substantial heterogeneity in how respondents saw the mechanisms of aging (Fig. 4D).

Damage accumulation was perhaps seen as slightly more important than the others on average, J o u r n a l P r e -p r o o f contributing from 5% to 60% with a median of 30%. Next was homeodynamic dysregulation, contributing from 0% to 70% with a median of 25%. Maladaptation and adaptation both had means of 20%, though maladaptation had a larger range (0% to 70%) than adaptation (5% to 50%). Some respondents indicated additional classes of mechanisms, with the following texts and percentages: "This will be different for each individual depending on genetic and environmental exposition. Personalized medicine will help."; "The intrinsic (causal) interactions between the abovementioned mechanisms makes it artificial to rate them separately"; "Pre-programmed cellular death and aging orchestrated by genetics/epigenetics (40%)"; and "reserve depletion (20%); slowdown in metabolism and information processing (20%)."

In addition to this summary of the survey results, we provide the results on a website that will allow other members of the field to fill out the survey, and allows users to visualize and download data by different subsets of respondents (established researchers, domains of expertise, etc.). If many of our colleagues and readers fill out the same survey, we may increase both the sample size and potentially the representativity.

Definitions of aging

Responses to the question "In no more than three sentences or 1000 characters, please describe your understanding of what causes aging and what it is, or is not, at a mechanistic level" were highly diverse.

Some representative examples of this diversity are shown in Table 1, and the full list of definitions can be found in Supplement 1. Note that some definitions refer explicitly to programs, and others are explicitly non-programmed. Some are primarily focused on damage, others on system dynamics. Some refer to cellular mechanisms, other to systemic or organism-level mechanisms. Some definitions suggest that aging rate is modified by the environment, others that it is not. Some situate aging explicitly in an evolutionary context, others focus exclusively on cellular/biochemical mechanisms. While not all these contrasts are mutually exclusive, they represent a remarkable diversity of perspectives. At a mechanistic level, aging (or I should say 'senescence') corresponds to any deterioration of cellular/physiological traits that will ultimately impact fitness." (J.F. Lemaître) "Mechanistically, aging is a disruption of the homeostasis established between cellular processes. Agerelated-diseases are the external symptoms of this disruption. […] Aging is the result of [an] imperfect optimization to maintain a balance between evolutionary constraints, cellular dynamical equilibrium and environmental constraints. […] Aging is not a programmed process but a consequence of this search for an equilibrium." (Q. Vanhaelen) "Time-dependent degradation of interactions or their (quasi)adaptative changes; sluggish and/or ineffective responses to challenges; when more than one such response is biologically relevant, aging would manifest as preference for a response different than one preferred at youth. The latter response may still be effective, but at higher cost for an aging organism (e.g. increased innate, inflammatory responses to pathogens in the old)." (J.M. Witkowski) "Aging is the progressive decline of function with increasing chronological age due to internal factors that are not dependent on environment, which leads to an increased probability of death. Aging is caused by a genetically programmed switch that downregulates cellular pathways involved in J o u r n a l P r e -p r o o f homeostasis, stress response, repair etc. that evolved to limit competition for resources between offspring and parent." (J. Van Raamsdonk) "I believe that some mechanism that clears damage from the germline (which is immortal) is toxic to the rest of the organism. This toxicity interferes with transcription at the DNA or protein level, leading to imbalanced proteostasis. Chromatin condensation therefore becomes abnormal (as measured by epigenetic clocks) which disrupts function of the organism as a whole and likely exponentially through a negative feedback loop." (T. Liontis) "Ageing is caused by a breakdown in repair mechanisms due to a shift of resource allocation after reproduction, modulated according to the environmental niche of the organism." (G. Pawelec) "Aging is not programmed. Aging is the result of complex interactions between the genome and physiological and environmental changes, with feedback loops that lead to physical and mental impairment. Organisms are not adapted to naturally live indefinitely." (F. Dufour) "Repair machineries fail to keep up with internal and external damage; systems become dysregulated and eventually collapse." (V. Gorbunova) "The gradual break-down of cellular components, leading to the eventually death of the organism, associated with time. This is caused by DNA damage, mutations, genetic/epigenetic pre-programmed senescence, protein aggregates and environmental stress." (U. Anglas)

J o u r n a l P r e -p r o o f

Discussion

The results of our survey undeniably confirm the impression at the symposium: there is no clear consensus in the field of aging biology, even on the most fundamental questions. There was a near-consensus (but not complete) that aging is heterogeneous, reflected in a clear preponderance of respondents considering J o u r n a l P r e -p r o o f that aging does not proceed uniformly across tissues and that aging cannot be measured with a single, unidimensional metric. The only other question with such a clear preponderance of support was on the need for the consensus, which we have demonstrated here does not currently exist. Some degree of disagreement in a scientific discipline is expected -it would be surprising to find complete consensus, particularly on questions under active investigation. Nonetheless, we argue that the disagreement is striking for the types of questions we posed, at least for many of them. For example, there was major disagreement as to whether we have a good understanding of the basic biological mechanisms of aging, whether it will soon be possible to reliably measure aging, whether there are many species that do not age appreciably, whether aging mechanisms are similar across species, whether aging is largely a cellular and molecular process, and whether aging is genetically programmed, among others.

Given our relatively detailed knowledge of molecular pathways related to aging [START_REF] Pan | Key proteins and pathways that regulate lifespan[END_REF], this level of disagreement among well renowned, established researchers/professors on such larger questions should perhaps be of major concern to the field.

We note an important caveat on the apparent disagreement we show: it was nearly impossible to formulate questions to be uniformly understood so as to elicit perfectly clear responses, and the wording of the questions was subject to substantial discussion before the final survey was circulated. For example, a participant's definition/conception of aging likely influenced responses to downstream questions.

Perhaps the clearest example of this is when aging begins: if aging is largely damage accumulation, it likely begins before birth [START_REF] Kinzina | Patterns of Aging Biomarkers, Mortality, and Damaging Mutations Illuminate the Beginning of Aging and Causes of Early-Life Mortality[END_REF], but if it is based on mortality/reproduction patterns or maladaptation/antagonistic pleiotropy (mechanisms that are useful early in life become harmful later in life), it begins closer to sexual maturity or later in life [START_REF] Gaillard | The Williams' legacy: A critical reappraisal of his nine predictions about the evolution of senescence[END_REF][START_REF] Williams | Pleiotropy, natural selection and the evolution of senescence[END_REF].

Another example would be a statement like "We have a good understanding of X." Who is "we," the researcher or the field? How good is good? What do we mean by understanding? In this sense, the J o u r n a l P r e -p r o o f apparent disagreements in responses to our questions may thus be somewhat exaggerated. Nonetheless, our questions were formulated in ways that are typical of discussions in the field, and at the very least the disagreement we show indicates that we have major communication issues to overcome. For example, it is common to read that aging is reversible (López-León and Goya, 2017) (and 42% of our respondents felt so to differing degrees), but what do we mean by aging? Mortality rates, or biomarker signatures? Which biomarkers? What exactly is reversible, and to what extent? Likewise, if we ask whether aging proceeds uniformly across tissues (86% of us felt it doesn't), the answer might be different if we are considering indicators of conserved genetic pathways versus epigenetic signals versus tissue functionality.

Despite this caveat, many disagreements are clearly about facts/reality rather than definitions. For example, disagreement as to whether aging is programmed or not might be slightly attributable to communication, but, based on the definitions, also clearly reflects real differences of opinion. Our objective here is not to settle these differences or to take a side, but simply to point them out.

The definitions of aging are particularly enlightening and often explicitly showed the differences in opinions reflected in the guided questions. Answers ranged from mechanistic to evolutionary, with answers sometimes referring explicitly to either a presence or absence of programming [START_REF] Blagosklonny | Aging is not programmed: Genetic pseudo-program is a shadow of developmental growth[END_REF][START_REF] Goldsmith | On the programmed/non-programmed aging controversy[END_REF][START_REF] Kowald | Can aging be programmed? A critical literature review[END_REF]. Some respondents thought the basis is fundamentally cellular and molecular, whereas others saw important roles for higher-order processes, or defined aging around functionality and phenotype rather than underlying processes. While these ideas are not necessarily mutually exclusive, the combination of the survey questions and the definitions indicate substantial disagreement as to whether an understanding of aging biology should include mechanisms beyond cellular/molecular biology. Certain notions were repeatedly referenced, notably damage accumulation (e.g. via imperfect repair mechanisms) [START_REF] Gladyshev | The free radical theory of aging is dead. Long live the damage theory! Antioxidants Redox Signal[END_REF] and loss of homeostasis [START_REF] Cohen | Complex systems dynamics in aging: new evidence, continuing questions[END_REF], reflecting areas of active interest among many researchers. This highlights that while there J o u r n a l P r e -p r o o f is immense disagreement at some levels, certain subsets of researchers are also actively converging on similar ideas, even if these ideas may not be shared by all. The lack of consensus is thus not a complete absence of convergence or coherence.

What should we do as a research community, in the face of this disagreement? We hope that simply becoming aware of the problem may stimulate researchers to consider aging in new ways, and to interact with colleagues that may have a different perspective. Beyond that, we suggest three complementary approaches that may help. First, though basic biologists tend to avoid theory, we think it is important for the field to develop an explicit theoretical paradigm. Of course there will be some disagreement, but this should largely occur within areas of the paradigm that are still unclear. This paradigm should include an attempt to link a variety of sub-disciplines, encompassing mechanistic, evolutionary, and demographic approaches to aging (e.g. 22). It should also link the conserved signaling pathways and the downstream mechanisms, and should integrate comparative, epidemiological, and clinical perspectives. Any understanding of aging cannot be considered complete if it fails to explain or disagrees with a major subdiscipline. For example, our understanding of the mechanistic basis of aging should complement and clarify our understanding of how lifespan evolves across evolutionary time in response to selection pressure, and vice-versa. It should thus be possible to construct an interdisciplinary understanding of aging progressively merging mechanistic and evolutionary aspects [START_REF] Cui | Relaxed Selection Limits Lifespan by Increasing Mutation Load[END_REF][START_REF] Macrae | DNA repair in species with extreme lifespan differences[END_REF]. This may require the application of approaches to clearly define the questions, disagreements, and terminological confusion. Much as the Hallmarks/Pillars of Aging unified what was before a relatively fragmented landscape of mechanistic research of aging [START_REF] Kennedy | Geroscience: linking aging to chronic disease[END_REF]López-Otín et al., 2013), a similar effort is probably required to unify into a single paradigm mechanistic approaches, research on conserved regulatory pathways, epidemiology, evolutionary biology, clinical geriatrics, late-life diseases, and so forth (Fig. 5). Accordingly, the second approach is to bridge some of the gaps between our sub-disciplines, not just with theory, but with concrete collaborations. While there is certainly some movement of information across the sub-disciplines, there are major questions that are not being sufficiently tackled. How might a mechanistic understanding of aging impact clinical geriatrics? What can species differences in aging mechanisms tell us about the evolution of life history strategies? Beyond the construction of a paradigm, J o u r n a l P r e -p r o o f there is a need to conduct research at the interface of sub-disciplines of aging. This would present an opportunity to benefit from the productivity and effectiveness of what has been called the multidisciplinary edge effect [START_REF] Varpio | Philosophy of Science Series: Harnessing the Multidisciplinary Edge Effect by Exploring Paradigms, Ontologies, Epistemologies, Axiologies, and Methodologies[END_REF]: In ecology, the edge effect refers to characteristics observed when the boundaries of two different habitats meet (e.g., when forests meet rocky outcrops). When the edges of ecosystems intersect, a greater biodiversity exists. Likewise, in research on aging biology, we might expect the most productive research questions to exist at the frontiers between our subdisciplines. These two efforts, theoretical and empirical, should nourish each other. Third and complementary, training of young researchers in aging biology should explicitly involve exposure to this multidisciplinary context, with courses and discussions designed to ensure that students are exposed to a wide range of mechanistic, genetic, evolutionary, ecological, clinical, and epidemiological perspectives.

J o u r n a l P r e -p r o o f

What might an emerging paradigm look like? Ideally, it would provide a framework that largely integrates the basic knowledge from these varied domains, identifying key areas of consensus, as well as areas of continued disagreement. It would provide a coherent structure for terminology, and indeed perhaps a more precise vocabulary for words that are used in different ways in different sub-disciplines -including the word "aging" itself [START_REF] Cohen | What if there's no such thing as "aging[END_REF][START_REF] Gladyshev | Aging: progressive decline in fitness due to the rising deleteriome adjusted by genetic, environmental, and stochastic processes[END_REF] -might go a long way toward creating this framework. As needed, it would include a mathematical framework to ensure appropriate rigor.

We are not under any illusion that this will be an easy task. Disagreement on, say, the programmed vs. non-programmed nature of aging is unlikely to be resolved soon, and without consensus on such points it becomes difficult to also have consensus on broader questions such as how to integrate an evolutionary and mechanistic understanding of aging. However, we also argue that cross-talk between sub-disciplines will gradually clarify the points of disagreement and eventually lead to a more effective and valuable paradigm on aging biology. If such a paradigm does not emerge organically over the next several years, it J o u r n a l P r e -p r o o f may be worth considering organizing a more formal consultation process with key experts from various subdisciplines in order to generate a consensus framework.

( 1 )

 1 name; (2) demographic data (sex, country of origin, career stage) (3) domains of expertise (multiple responses permitted); (4) Likert-scale and other limited response questions (see below); and (5) a single open-ended question, "In no more than three sentences or 1000 characters, please describe your understanding of what causes aging and what it is, or is not, at a mechanistic level." J o u r n a l P r e -p r o o f The Likert scale used was, "For each of the following statements, do you (1) Strongly disagree; (2) Moderately disagree; (3) Slightly disagree; (4) Neutral; (5) Slightly agree; (6) Moderately agree; or (7) Strongly agree?" The statements were:  We have a relatively good understanding of the basic biological mechanisms of aging.  Some combination of the nine hallmarks (Lopez-Otin et al. 2013) or the seven pillars (Kennedy et al. 2014) does a relatively comprehensive job of describing the mechanisms of aging.  Aging proceeds uniformly across tissues.  It is or will soon be possible to have relatively reliable metrics of the overall aging process  Aging cannot and should not be measured by a single metric because it is multi-dimensional and heterogeneous  It should be possible to quantify aging well even in the absence of a clear consensus or mechanistic understanding of what aging is.  It should be possible to intervene in aging, and evaluate interventions, even in the absence of a clear consensus or mechanistic understanding of what aging is.

J

  o u r n a l P r e -p r o o f  Aging is genetically programmed and not just a by-product of imperfections or evolutionary constraints. Aging will be reversible with the right technologies.  Anti-aging interventions have the potential to dramatically extend human lifespan (beyond, say, Does aging begin approximately at (1) parental gamete formation; (2) conception; (3) birth; (4) sexual maturity; or (5) later in life?  Which of the following proposed anti-aging interventions are likely to slow aging in humans with tolerable levels of side-effects (check all that apply)? (1) senolytics/targeting senescent cells; (2) pharmacological or gene editing control of known aging pathways (metformin, rapamycin, NAD+, etc.); (3) enhancing cell replication (e.g. telomerase activation such as via TA65); (4) direct attempts to reduce macromolecular damage (e.g. proteasome activators such as 18aplha-GA, quercetin); (5) interventions in the immune system (e.g. growth hormone to reverse thymic involution); (6) lifestyle interventions; (7) other (list all that apply).  What percent of what we observe as aging do you think can be attributed to the following types of mechanisms (understanding that they may coexist and interact with each other): [100 points that should be distributed among the five options below] o Damage accumulation (DNA damage, protein aggregates, structural damage, etc.) o Maladaptation/antagonistic pleiotropy: mechanisms that are useful early in life (e.g. for cancer prevention) become harmful later in life.

J

  o u r n a l P r e -p r o o f o Adaptation: Adjustments the organism makes to either pathological aspects of aging or to changing needs (e.g. immune repertoire) at different ages. o Homeodynamic dysregulation: breakdown in the capacity of complex regulatory networks to maintain homeodynamics o Other [Up to 100 words to specify]

Fig. 1 .

 1 Fig. 1. Profile of the survey respondents in terms of self-declared career stage (A) and expertise (B). For career stage, respondents chose one category. Two respondents (post-bachelor's and family doctor) volunteered responses not in the list. For expertise, each respondent checked as many boxes as they felt applied.

Fig. 2 .

 2 Fig. 2. Responses to Likert-scale questions on aging, ordered from top to bottom by decreasing consensus.

Figure 3 .

 3 Figure 3. Correlation matrix representing Pearson correlation coefficients between the 16 Likert-scale

Fig. 4 .

 4 Fig. 4. Responses to non-Likert questions. (A) Counts (absolute number) of answers to when aging starts. (B) Counts (absolute number) of respondents endorsing intervention classes thought likely to slow aging. (C) Counts (absolute number) of respondents endorsing intervention classes thought likely to slow aging (D) Relative contribution (%) of different classes of mechanisms to the aging process.

Table 1 :

 1 Examples of the diverse mechanistic definitions of aging "We do not know what causes aging. I think the trigger is endogenous but not necessarily programmed." (G. Ferbeyre) "Aging is mainly accumulation of macromolecular damage and failure of adaptation to a continuous changing environment. It is also an epiphenomenon of the outcome of the protective conditions we humans have achieved thanks to civilization over time and to this end somehow it doesn't comply fully with typical Darwinian Biology." (E.S. Gonos)"As an evolutionary biologist, I see 'aging' as the decrease in the age-specific contribution to fitness.[…]

Fig. 5 :

 5 Fig. 5: A conceptual model of how our understanding of aging biology will need to integrate perspectives

J o u r n a l P r e -p r o o f

Acknowledgements A.A.C. is supported by a CIHR New Investigator Salary Award and is a member of the FRQ-S funded Centre de recherche du CHUS and Centre de recherche sur le vieillissement, as well as by a CIHR project grant (153011). A.M.S. is supported by a DECRA fellowship from the Australian Research Council (DE180101520). VG and VNG are supported by grants from US National Institutes of Health. D.F. is supported by NIH AG059719 and AG023717. OTN is supported by the Brazilian Council for Scientific and Technological Development (CNPq) with a research productivity grant (3035402019-2). S.U. is supported by the NIA/NIH grants R01AG062623 and R01AG070487. A.I.Y is supported by the NIA/NIH grants RF1AG046860, R01AG070487, and U19AG063893. CF and MI are supported by a grant of the Ministry of Education and Science of the Russian Federation Agreement No. 074-02-2018-330.


Gabriel Bidaux
email: gabriel.bidaux@univ-lyon1.fr.

Anne-Sophie Borowiec

Natalia Prevarskaya

Dmitri Gordienko

Dr Gabriel

eTRPM8 rules keratinocytes fate Fine-tuning of eTRPM8 expression and activity conditions keratinocyte fate

Keywords: eTRPM8, mild cold, epidermal homeostasis, ATP, superoxide, proliferation, differentiation, cell death

Recently, we reported the cloning and characterization of short isoform of the icilin-activated cold receptor TRPM8 channel in keratinocytes, dubbed eTRPM8. We demonstrated that eTRPM8 via fine tuning of the endoplasmic reticulum (ER)mitochondria Ca 2+ shuttling

regulates mitochondrial ATP and superoxide (O 2 •-) production and, thereby, mediates control of epidermal homeostasis by mild cold. Here, we provide additional information explaining why eTRPM8 suppression and TRPM8 stimulation both inhibit keratinocyte growth. We also demonstrate that stimulation of eTRPM8 with icilin may give rise to sustained oscillatory responses. Furthermore, we show that ATP-induced cytosolic and mitochondrial Ca 2+ responses are attenuated by eTRPM8 suppression. This suggests positive interplay between eTRPM8 and purinergic signaling pathways, what may serve to facilitate the ERmitochondria Ca 2+ shuttling. Finally, we demonstrate that cold (25°C) induces eTRPM8dependent superoxide-mediated necrosis of keratinocytes. Altogether, these results are in line with our model of eTRPM8-mediated cold-dependent balance between keratinocyte proliferation and differentiation.

Introduction

Under influence of ambient factors, the skin epidermis undergoes continuous regeneration through so-called epidermal homeostasis (EH): the fine-tuning of the balance between proliferation, directional migration, differentiation and death of keratinocytes. Deviation of the ambient temperature is one of the most important stimuli which constantly affect mammals' skin. Changes in the ambient temperature are perceived by thermoreceptors, the ion channels which belong to the transient receptor potential (TRP) super family (for a review see ref. [START_REF] Lumpkin | Mechanisms of sensory transduction in the skin[END_REF] ). Keratinocytes may directly transduce thermal stimuli via warmth-activated TRP channels, TRPV3 (with an apparent threshold of about 32-39°C) and TRPV4 (27-34°C). [START_REF] Mandadi | TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP[END_REF][START_REF] Moqrich | Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin[END_REF][START_REF] Dhaka | Trp ion channels and temperature sensation[END_REF] Nevertheless, it is important to note that at ambient temperatures from +10°C to +30°C, the unprotected human skin temperature settles at mean steady-state values within the range of +24°C to +33°C, respectively, [START_REF] Smith | The validity of wireless iButtons and thermistors for human skin temperature measurement[END_REF] which is within the functional range of TRPM8 cold sensitivity. [START_REF] Mckemy | Identification of a cold receptor reveals a general role for TRP channels in thermosensation[END_REF][START_REF] Peier | A TRP channel that senses cold stimuli and menthol[END_REF] Yet, the molecular identification of the cold transducer which translates cooling into downstream signaling events in keratinocytes was lacking.

In a recent investigation, [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] we reported the cloning and characterization of a new 4trasmembrane-domain epidermal isoform of the TRPM8 cold receptor-channel, dubbed eTRPM8. This channel is localized and functions in the keratinocyte endoplasmic reticulum (ER) membrane but not in the plasmalemma. Activation of eTRPM8 within ER-mitochondria contact sites facilitates mitochondrial Ca 2+ uptake, thus, affecting mitochondrial Ca 2+ concentration ([Ca 2+ ] m ). In turn, [Ca 2+ ] m modulates ATP and superoxide (O 2

•-) synthesis. We reported that this fine-tuning of ATP and O ] m (monitored on a time scale of days). Also, due to space limitations, some interesting observations were bypassed in our recent publication. [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] Here we provide these results to reinforce our hypothesis with additional insights. Firstly, we demonstrate that the modalities of eTRPM8 activation define the cell response towards proliferation or differentiation. While sustained stimuli slowed down cell cycle and induced differentiation, repeated transient stimuli increased proliferation rate. Secondly, we provide additional evidence for positive interplay between eTRPM8 and purinergic signaling pathways, which may serve to facilitate the ER-mitochondria Ca 2+ shuttling. Namely, we demonstrate that ATP-induced Ca 2+ release from the ER and concurrent mitochondrial Ca 2+ uptake depend on eTRPM8 activity. Thirdly, we show that icilin, mimicking cold stimulation, induces an eTRPM8 oscillatory activity lasting at least for tens of minutes. Finally, we complement our previously reported observation, that the decrease of the ratio of proliferating to the late differentiated keratinocytes dropped after a 3-days incubation at 25°C is associated with an increase of O 2 •-content in an eTRPM8-dependent manner, [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF][START_REF] Borowiec | Optimal differentiation of in vitro keratinocytes requires multifactorial external control[END_REF] with demonstration that prolonged cold (25°C)-incubation decreases both [Ca 2+ ] m and [Ca 2+ ] ER and facilitates ROSmediated necrosis of keratinocytes.

This addendum along with our recent publication 8 demonstrate that both the eTRPM8 expression level and the modalities of cold-stimulation serve as important determinants of the keratinocyte fate and act via fine tuning of [ATP] and [O 2

•-].

Results & discussion

Either sustained stimulation of eTRPM8 or its knockdown, both decrease cell growth. In two recent studies, [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF][START_REF] Borowiec | Optimal differentiation of in vitro keratinocytes requires multifactorial external control[END_REF] we reported that cold incubation of keratinocytes impairs their growth.

We also cloned and characterized a novel archetype of TRP channels in epidermis, [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] dubbed eTRPM8, and we demonstrated that it was a functional channel with 4 transmembrane domains (Fig. 1A) instead of the 6 original in full-length TRPM8. We also demonstrated that icilin application on the shaved skin of mice deceases the proliferation index of keratinocytes in an eTRPM8-dependent manner. [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] Figure 1B shows that this effect can be reproduced by a 3-day incubation of HaCaT cell line in a medium supplemented with 20µM icilin (renewed daily). Because Icilin is not very stable at 37°C, we have chosen a 20µM concentration to prevent loss of effect due to degradation. eTRPM8 Knockdown prevented pulsed icilinemediated increase in cell growth at 37°C (Fig HaCaT cells incubated at 31°C or in the presence of icilin (Fig. 1D). In contrast, incubation of the cells at 25°C enhanced eTRPM8 expression. This is in a line with the observation that the incubation at 25°C causes stronger reduction of the cell growth than incubation at 31°C or with icilin (Fig. 1B). Yet, the reduction of HaCaT cell growth caused by incubation with icilin or at 31°C was observed only in the cells expressing eTRPM8.

To what extent the modalities (i.e. intensity, duration and frequency) of eTRPM8 stimulation may affect the cell fate? To address this we explored the effect of "pulsed" stimulation

HaCaT cells with icilin. In these experiments, the cells were exposed to icilin for 10 min per hour 8 times a day during 3 days. In contrast to sustained 3-day stimulation, which reduces the cell growth, the pulsed stimulation increases the cell growth about 1.4 times (Fig. 1B).

This demonstrates that the modality of icilin stimulus can evoke the opposite effects on cell growth. This effect is eTRPM8 specific, since pulsed icilin stimulation did not augment the growth of eTRPM8-silenced HaCaT cells (Fig. 1C andE). Notably, sustained icilin stimulation reduces the cell growth to the same extent as eTRPM8 suppression (Fig. 1B andC). It was previously reported that over time, the activity of full-length TRPM8 run down in a PIP 2 -dependent manner. [START_REF] Rohacs | PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain[END_REF] The eTRPM8 activity also strongly depends in bioavailability of PIP 2 [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] and , therefore, should be regulated in the same way. Also, enhanced Ca 2+ leak from the ER caused by sustained stimulation of eTRPM8 may lead to the decrease of the ER calcium load (Fig. 3C andE). Both the ER depletion and eTRPM8 desensitization should, over time, reduce the ER-mitochondria Ca 2+ transfer. It, therefore, seems likely that either suppression of eTRPM8 or its sustained activation by icilin may have similar effect on the cell growth, even though they engage different mechanisms to control keratinocyte cell cycle. This hypothesis was supported by the results of FACS experiments (see in Methods). The proportion of the cells entering cell cycle decreased following sustained incubation with 20µM iciline, while the population of the cells in S-phase increased (Fig. 1F). This suggests that icilin increases the number of proliferating cell while it slows down of cell cycle. Conversely to icilin treatment, the proportion of non-proliferating cells was similar between control cells and eTRPM8 KD cells, while the fraction of the cells in G2/M-phase was larger in eTRPM8 KD keratinocytes than control cellssuggesting a long-lasting arrest at G2/M checkpoint. In summary, this demonstrates that the suppression and sustained stimulation of eTRPM8 engage different signaling pathways, even though the end effect on the cell growth was similar.

A positive interplay between eTRPM8 and purinergic signaling pathways promotes keratinocytes proliferation. In Bidaux et al [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] , we demonstrated correlation between thermodependent dynamics of [ATP] changes and the dynamics of the cell growth, but did not provide any evidence that eTRPM8-mediated proliferation of keratinocytes at 37°C may recruit ATP-linked signaling pathways. Activation of P2Y receptors (P2YRs) via InsP 3 production stimulates the ER-to-mitochondria Ca 2+ shuttling via inositol trisphosphate receptors, InsP 3 Rs. [START_REF] Lee | Purinoceptor-mediated calcium mobilization and proliferation in HaCaT keratinocytes[END_REF][START_REF] Greig | Purinergic receptors are part of a signaling system for keratinocyte proliferation, differentiation, and apoptosis in human fetal epidermis[END_REF] We, therefore, assessed whether eTRPM8 activity affects in any way the InsP 3 R-mediated Ca 2+ shuttling facilitated by P2YR activation. We found that 30 µM suramin, the P2YR inhibitor, decreases the growth of control HaCaT cells by 2.2 fold, while it reduces the growth of eTRPM8 KD HaCaT cells only by 1.4 fold (Fig. 2A). Furthermore, ] m response. This demonstrates that, at 37°C, eTRPM8 is recruited to the ER-to-mitochondria Ca 2+ shuttling process engaged by purinergic signaling pathways, and is in line with observed decrease of the cell proliferation (Fig. 1C). Furthermore, the effect of capsazepine indicates that the ER-to-mitochondria Ca 2+ shuttling, engaged by purinergic stimulation, is regulated by the eTRPM8 channel activity rather than by interaction of the eTRPM8 protein with InsP 3 R.

using
Activation of eTRPM8 induces long-lasting oscillatory Ca 2+ shuttling from the ER to mitochondria and prevents the loss of Ca 2+ in the mitochondrial matrix during hypothermia. We reported previously that cold or icilin-mediated activation of eTRPM8 induced Ca 2+ shuttling from the ER to mitochondria on the time scale of seconds. It is not clear, however, whether a single robust Ca 2+ uptake into mitochondria can maintain [Ca 2+ ] m in the mitochondrial matrix at sufficient level over long term. Here, we demonstrate that a sustained activity of eTRPM8 in HEK and HaCaT cells results in oscillatory Ca 2+ releases from the ER of (Fig. 3A) which were associated with an oscillatory [Ca 2+ ] m changes (Fig. 3B)

reflecting repetitive Ca 2+ re-uptake into the mitochondria over the period of at least 30 minutes. These oscillations were inhibited by capsazepine (Fig. 3B; insets) and were never observed in HaCaT cells with mutated pore region of eTRPM8. [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] Similar cytosolic and mitochondrial Ca 2+ oscillations and their role in the regulation of hepatocyte metabolism have been reported previously. [START_REF] Robb-Gaspers | Integrating cytosolic calcium signals into mitochondrial metabolic responses[END_REF][START_REF] Ld | Coupling between cytosolic and mitochondrial calcium oscillations: role in the regulation of hepatic metabolism[END_REF] Maintenance of this oscillatory Ca 2+ transfer requires (1) refilling of the ER with Ca 2+ and (2) a repetitive Ca 2+ extrusion from mitochondria, which may serve to prevent activation of cyclophilin D and the permeability transition pore in mitochondria (for a review see ref. [START_REF] Rasola | Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis[END_REF] ). By means of FRET-FLIM of D1ER and mtD3CPV biosensors expressed in the ER and mitochondria of HaCaT cells, respectively, we measured steady-state [Ca 2+ ]. We found that incubation of HaCaT at 31°C did not alter [Ca 2+ ] ER (Fig.

3C) but significantly decreased [Ca 2+

] m (Fig. 3D). It should be noted that the overexpression of eTRPM8 partially attenuated the [Ca 2+ ] m decrease at 31°C while but not at 25°C (Fig. 3E).

This is along the line of our previous observation of correlation between eTRPM8-mediated [Ca 2+ ] m elevation and an increased bioavailability of ATP at 31°C 8 . At 25°C, however, both [Ca 2+ ] ER and [Ca 2+ ] m are significantly reduced (Fig. 3C andD). This may reflect a reduction in the efficiency of the Ca 2+ re-uptake into the ER, which, in turn, will lead to a decrease of the Ca 2+ release from the ER and, hence, to attenuation of the mitochondrial Ca 2+ uptake, so that Ca 2+ extrusion from mitochondria will eventually prevail. Even though eTRPM8 mRNA level is increased after a 3-day incubation at 25°C (Fig. 1B), concurrent decrease of the ER Ca 2+ load overrides the effect of the eTRPM8 upregulation and [Ca 2+ ] m continue to decline.

Summing up, at skin average temperature (range 31-33°C), eTRPM8 mediates Ca 2+ oscillations in the mitochondrial matrix, thus preventing drop of ATP production. At colder temperatures, the sustained eTRPM8 activity contributes to the ER Ca 2+ leak that overrides decrease in cell growth) similar to those cased by its suppression.

Acute cold induces an eTRPM8-dependent and ROS-mediated necrosis of keratinocytes.

One striking feature of keratinocytes incubated at 25°C is dramatic decrease of [Ca 2+ ] m , [Ca 2+ ] ER and [ATP], and attenuation of cellular processes: proliferation, late differentiation markers. [START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] General suppresion of cell function with concurrent increase in oxidative stress

(observed elevation of O 2 •-
) is a good predictive marker for necrosis. This prompts us to assess necrosis in the keratinocytes incubated for 3 days at 25°C. As it is depicted by dark blue-stained cells on the images (Fig. 4) and is quantified in bar diagram plot (Fig. 5A), the fraction of necrotic cells was negligibly small in control cells incubated at 37°C and 31°C but was elevated to 12% following 3-day incubation at 25°C. Overexpression of eTRPM8 sensitizes the cells to necrosis at 31°C and dramatically augments necrosis at 25°C (35%).

Interestingly, an augmentation of necrosis by eTRPM8 overexpression can be virtually completely reversed by the concomitant overexpression of the superoxide dismutase 1 (SOD1). This suggested a causal link between [O 2

•-] and necrosis, which was confirmed further by correlation analysis (Fig. 5B).

In conclusion, in our recent study and in this addendum, we characterized a new isoform of the icilin-activated cold receptor TRPM8 channel, eTRPM8, whose four transmembrane domains-based structure and localization in the ER microdomaines confer its unique role as cold transducer regulating mitochondrial bioenergetics in keratinocytes. We demonstrated that the level of expression of eTRPM8 and the modalities of its activation by cold determine the [ATP] / [O 2

•-] ratio via fine tuning of [Ca 2+ ] m and [Ca 2+ ] ER . This consequently determines the keratinocyte fate (Fig. 5C).

Materials and Methods

Cell culture.

HaCaT cell line was grown in Dulbecco's minimal essential media (DMEM) (Gibco) supplemented with 2% fetal calf serum (FCS) and Kanamycin (100 µg/ml), and [Ca 2+ ] was adjusted to 1.8 mM to induce differentiation, as described previously [START_REF] Borowiec | Optimal differentiation of in vitro keratinocytes requires multifactorial external control[END_REF] . Necrosis was assessed by the mean of Trypan Blue.

Transient expression studies.

Plasmid transfection was carried out with X-tremeGENE HP DNA Transfection Reagent CACCAtattccgttcggtcatctaGAGAGtagatgaccgaacggaata (Capital letters refer to the structure of the short hairpin RNA and small letters refer to the siRNA sequence). Control of silencing efficiency was achieved by immunoblotting of total proteins extracted from Hek cells, cotransfected with either shCTL + eTRPM8pcDNA4 or shM8 + eTRPM8pcDNA4.

Control siRNA targeted Luciferase (siCTL) and siRNA anti-eTRPM8 (siTRPM8) targeted exon 20. The sense sequences are CUUACGCUGAGUACUUCGA(dTdT) and GUAUUCUGGACGAGUCAUU(dTdT), respectively.

Analysis of TRPM8 mRNA expression.

Total RNA was isolated from different cell lines using Tri Reagent mix (Sigma-Aldrich).

After a DNase I (Invitrogen) treatment and phenol/chloroform purification, 2 µg of total RNA was reverse transcribed into cDNA at 42°C using random hexamer primers (Applera) and

MuLV reverse transcriptase (Applera).

Qualitative PCR was performed with TaqGold polymerase (Applera) and controlled on agarose gel.

Real-time quantitative PCR was performed on a Cfx C1000 system (Biorad). For each reaction, 12.5 ng of cDNA was placed in 15 μl of final reaction medium containing 7.5 µl of 2x SsoFast™ EvaGreen ® Supermix (Biorad) and 200 nM primer pairs:

CGGTCATCTACGAGCCCTAC and CACACACAGTGGCTTGGACT. The housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to normalize variations in RNA extracts, the degree of RNA degradation and variability in RT efficiency. GAPDH prmiers were: ACCCACTCCTCCACCTTTG and CTCTTGTGCTCTTGCTGGG. To quantify the results, we used the comparative Ct method.

PCR protocol consisted of: 30 s denaturation at 95°C followed by 40 cycles of [4 s at 95°C

and 30 s at 60°C], and final dissociation curve to control specificity of the amplification.

Flow cytometry was conducted with CyAn™ ADP Analyser. Cells were harvested, then suspended in 250 µl PBS / 4% BSA / 0,1% Triton X100 with Ribonuclease A (200 µg/ml) and incubated at room temperature for 15 min. After addition of 250 µl of PBS containing 30 µg/ml of propidium iodide, cells were further incubated at room temperature for 30 min to 1 hour. Data were analyzed with FlowJo software (version 8.7).

Visualization of agonist-induced changes of Ca 2+ concentration in cytosol ([Ca 2+ ] c ) and mitochondria ([Ca 2+ ] m ).

Changes of [Ca 2+ ] c were imaged using the high-affinity fluorescent Ca 2+ indicator fluo-4, as described previously [START_REF] Povstyan | Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries[END_REF][START_REF] Thebault | Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of storeoperated channels in LNCaP human prostate cancer epithelial cells[END_REF] . Fluo

  confocal imaging of fluo-4 and rhod-2 fluorescence, we compared ATP-induced changes in [Ca 2+ ] c and [Ca 2+ ] m in control HaCaT cells (Fig. 2B), in HaCaT cells pre-treated with the TRPM8 inhibitor capsazepine (Fig. 2C) and in eTRPM8 KD cells (Fig. 2D). Either eTRPM8 inhibition or suppression both augmented the ATP-induced [Ca 2+ ] c responses (Fig. 2E, panel a) and significantly attenuated concurrent mitochondrial Ca 2+ uptake (Fig. 2E, panel b), as reported by the reduction of the [Ca 2+

Ca

  2+ uptake into the ER mediated by SERCA, the activity of which is reduced due to the dramatic drop in[ATP]. This leads to a decrease in ER Ca 2+ content what creates an adverse cycle inducing a drop in [Ca 2+ ] m , an increase in [O 2•-] 8 and further decrease in [ATP].[START_REF] Bidaux | Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a colddependent manner[END_REF] Such a decrease in the ER Ca 2+ content mimics the effects of eTRPM8 inhibition/suppression and may explain why sustained activation of eTRPM8 by cold leads to biological outcomes (e.g.

(

  Roche) at the concentration of 4 µg per million cells. SiRNA transfection was performed with 50 nM of HiPerfect transfection reagent (Qiagen).siRNA and shRNA plasmids were built in pENTER vector (Invitrogen) following manufacturer's recommendations. Control shRNA targeted TRPM8 exon 7 and shRNA anti-

  photomultiplier gain and offset were used in all the experiments. Data were processed with ImageJ64 freeware. After setting offset (pixel fluorescence intensity below 20 a.u.) and the mean cell background (pixel fluorescence intensity 100 a.u.) in the control condition (cells grown at 37°C), specific to O 2 •-increase in the CellRox® Deep Red fluorescence was computed according to the formula     ) 255 20 ( ) 255 100 ( / I I Pix Pix

Figure 1 .

 1 Figure 1. Modalities of eTRPM8 channel activation definite opposite response of cell growth. A, Schematic representation of the structure of a monomeric eTRPM8 protein in the ER membrane. B. Bar diagram plot illustrates growth (see Methods) of HaCaT keratinocyte cell line during 3 days. Cells were incubated at different temperature (37°C, 31°C, 25°C) or in presence of, daily-renewed, 10µM icilin (Icilin). Alternatively, Icilin was applied for 10 minutes, and then washed out for 50 minutes; 8 cycles were achieved per day during 3 days (Pulsed Icilin). Data are presented as fold increase of the cell number from day 0 and have

Figure 2 .

 2 Figure 2. Interplay between eTRPM8 and the purinergic pathway controls the proliferation of keratinocytes at 37°C. A. Bar diagram plot illustrates growth of HaCaT keratinocyte cell line during 3 days. Cells were incubated with or without 30 µM Suramin in order to inhibit purinergic receptors. Data are presented as fold increase of the cell number from day 0 and have been normalized by the control condition. n=3. §: p<0.05. B. Effect of eTRPM8 inhibition on cytosolic (fluo-4) and mitochondrial (rhod-2) [Ca 2+ ] transients induced by 100 μM ATP in HaCaT cells. The plots (b) show the time course of ATP-induced changes in the

Figure 3 .

 3 Figure 3. eTRPM8 mediates a sustained and oscillatory Ca 2+ shuttling from the ER to mitochondria. A. Changes of Ca 2+ concentration in cytosol ([Ca 2+ ] c ) in response to external application of 10 µM icilin were monitored using x-y time series imaging of fura-2 in control vs. eTRPM8-overexpressing HEK cell line. B. Concomitant monitoring of relative changes of Ca 2+ concentration (F/F 0 ) in cytosol ([Ca 2+ ] c ) and mitochondria ([Ca 2+ ] m ) in response to external application of 10 µM icilin was performed in eTRPM8-overexpressing HaCaT cells using fluo-4 and rhod-2 fluorescence, respectively. Insets: Inhibition of the incilin-induced

Figure 4 .

 4 Figure 4. A sustained cold-mediated TRPM8 activity induces necrosis of keratinoyctes. Images shows a Trypan blue staining of control HaCaT cells (CTL), eTRPM8-overexpressing HaCaT cells (eTRPM8) or HaCaT cells concomitantly overexpressing eTRPM8 and the superoxide dismutase 1, SOD1 (eTRPM8+SOD1) incubated at 37°C, 31°C or 25°C for 3 days. Necrotic cells appeared as dark blue-stained cells.

Figure 5 .

 5 Figure 5. Cold, below 25°C, induces an adverse cycle leading to ROS-mediated necrosis of keratinocytes. A. Bar diagram plot represents the proportion of necrosis in the HaCaT cell population as quantified by counting of images presented in the figure 4. Values express Mean±SD. n=3. a: p<0.05 between CTL cells and eTRPM8-overexpressing cells (eTRPM8); b: p<0.05 between eTRPM8 cells and cells concomitantly overexpressing eTRPM8 and the superoxide dismutase 1, SOD1 (eTRPM8+SOD1). B. Scatter plot demonstrates the correlation between superoxide concentration ([O 2 .-]) in mitochondria (see Methods) and the

  2

1.C) and thus demonstrate eTRPM8 specific effect of icilin. qPCR experiment revealed no modification in eTRPM8 expression level in

Downloaded by [Hospital Henry Gabrielle], [Gabriel Bidaux] at 07:48 25 March 2016

Acknowledgements:

Authors thank Prof. Julien J-P. at the centre de recherche CHU Laval, QUEBEC, for his generous gift of pcDNA3-FlagN-SOD1 plasmid. proportion of necrosis in the HaCaT cell population. Control cells were compared to cells overexpressing either eTRPM8 or eTRPM8+SOD1 and were incubated at different temperature for 3 days. Values express Mean of 3 independent experiences. C. Scheme illustrating the correlation between eTRPM8 expression level, the modalities (intensity, duration, frequency) of the cold stimulation, [O 2 .-] and [ATP], and cell fate (Proliferation, Apoptosis/differentiation, necrosis).

Funding: This work has been supported by grants from: INSERM, Ministère de l'Education Nationale, Ligue Nationale Contre le Cancer, Region Nord-Pas-de-Calais and ANR Grant: TRPM8/Reprod. The Cosmetics department of Johnson & Johnson laboratories supported Dr. Gabriel Bidaux. Dr. Anne-sophie Borowiec was supported by Fondation pour la recherche médicale. Dr. Dmitri Gordienko was supported by State Found for Fundamental Research (F 46.2/001).