Sleep-wake body temperature regulates tau secretion in mice and correlates with CSF and plasma tau in humans

Sleep-wake body temperature regulates tau secretion in mice and correlates with CSF and plasma tau in humans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sleep-wake body temperature regulates tau secretion in mice and correlates with CSF and plasma tau in humans Geoffrey Canet*, Felipe Da Gama Monteiro, Emma Rocaboy, Sofia Diego-Diaz, and 27 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4384494/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The sleep-wake cycle regulates interstitial fluid and cerebrospinal fluid (CSF) tau levels in both mouse and human by mechanisms that remain unestablished. Here, we reveal a novel pathway by which wakefulness increases extracellular tau levels in mouse and humans. In mice, higher body temperature (BT) associated with wakefulness and sleep deprivation increased CSF tau. In vitro , wakefulness temperatures upregulated tau secretion via a temperature-dependent increase in activity and expression of unconventional protein secretion pathway-1 components, namely caspase-3-mediated C-terminal cleavage of tau (TauC3), and membrane expression of PIP 2 and syndecan-3. In humans, the increase in both CSF and plasma tau levels observed post-wakefulness correlated with BT increase during wakefulness. Our findings suggest sleep-wake variation in BT may contribute to regulating extracellular tau levels, highlighting the importance of thermoregulation in pathways linking sleep disturbance to neurodegeneration, and the potential for thermal intervention to prevent or delay tau-mediated neurodegeneration. *Geoffrey Canet, Esther M. Blessing, and Emmanuel Planel contributed equally to this work. Biological sciences/Neuroscience/Cellular neuroscience Biological sciences/Neuroscience/Molecular neuroscience Health sciences/Biomarkers/Diagnostic markers tau unconventional protein secretion sleep-wake cycle body temperature Alzheimer’s disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The intraneuronal accumulation of hyperphosphorylated aggregated tau protein is the pathological hallmark of neurodegenerative tauopathies including Alzheimer's disease (AD) 1 . Both the neuron-to-neuron propagation of tau pathology, and higher levels of cerebrospinal fluid (CSF) and plasma tau correlates with cognitive decline 2 . Tau secretion to the extracellular space consequently influences the propagation of tau aggregates in the brain, marking one of the initial steps of pathological tau transmission from diseased to recipient neurons. Further elucidating the key components in pathways underlying tau secretion and key physiological factors regulating their activity may yield insights into therapeutic avenues for slowing the spread of pathological tau. It is now established that tau, as a leaderless protein, is mainly secreted through the unconventional protein secretion pathway-I (UPS-I), consistent with ~ 90% of extracellular tau being free and unbound to vesicular organelles 3–5 . Key components in this pathway include phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which binds to tau at the inner leaflet of the plasma membrane, and heparan sulfate proteoglycans (HSPGs), which facilitate export across cell membrane 4,6 . Furthermore, the main form of extracellular tau is found as its C-terminal-truncated fragment (D421) referred to as TauC3 7–10 . TauC3 cleavage is mediated by caspase-3 and seems to occur intracellularly prior to release 9,11 , and this cleavage at D421 is inhibited by tau phosphorylation at S422 12,13 . Regarding physiological factors regulating tau secretion, previous studies in both mouse and human suggest extracellular tau levels are strongly influenced by the sleep-wake cycle 14 , consistent with the established bidirectional link between sleep disturbance and AD neuropathology 15,16 . During wakefulness, CSF and interstitial fluid (ISF) tau levels substantially increase compared to sleep. Sleep deprivation (SD) is associated with elevated tau levels in ISF and CSF 14 , while decreasing CSF tau phosphorylation level 10 . Moreover, chronic SD reduces tau phosphorylation while promoting its aggregation in AD mice 18 . Previous studies suggested that both increased tau secretion and reduced tau clearance contribute to elevated tau levels during waking 10,19 . However, the precise mechanisms by which wakefulness leads to increased ISF and CSF tau remain unknown. We sought to address this question by testing whether tau secretion levels are modulated by the sleep-wake cycle, and investigating mechanisms underlying this modulation. We previously showed sleep-wake differences in tau phosphorylation were driven by fluctuations in core body temperature (BT) during the sleep-wake cycle 20 . Furthermore, SD prevented sleep-associated tau phosphorylation by disrupting the normal core BT decrease during sleep 20 . Building on these findings, we hypothesized that sleep-wake variations in core BT may drive sleep-wake variation in tau secretion levels by modulating the activity of key components in the UPS-I pathway. Here, we demonstrate in mice and in vitro that variation in CSF and ISF tau levels across the sleep-wake cycle are driven by changes in core BT, owing to temperature-dependent regulatory mechanisms governing tau protein secretion. We found that higher BT, either during wakefulness, SD, or induced by mild-hyperthermia, promotes tau secretion into the CSF via the upregulation of UPS-I-related components in mice. We elucidated a specific intracellular pathway involving (i) caspase-3-mediated TauC3 production, (ii) subsequent binding of TauC3 to PIP 2 at plasma membrane, and (iii) the transmembrane export of TauC3 facilitated by the HSPG family member syndecan-3 (SDC3). In older adults, we found that the rise in BT during wakefulness was positively correlated with the increase in CSF and plasma tau levels. RESULTS Tau secretion is temperature-dependent During wakefulness, CSF and ISF tau levels increase nearly twofold compared to sleep 14 . We first investigated whether temperatures simulating BT variations during the sleep-wake cycle could regulate neuronal tau secretion in SH-Tau3R human cells (Fig. 1 a) and in primary mouse cortical cells (Fig. 1 h). We found that extracellular tau levels plateaued after 72 hours at 37˚C (Fig. 1 b). Using quantitative ELISA and dot blotting, we then showed that higher temperatures (38°C vs. 35°C) led to a ~ 2-fold increase in tau secretion in both human (Fig. 1 c,e,f; Extended Data Fig. 1 a) and mouse neuron-like cells (Fig. 1 i-k), without inducing cytotoxicity, except for 39°C (Extended Data Fig. 1 b,c). In order to more faithfully replicate physiological temperature variations occurring during a full 24-h sleep-wake cycle, we ensured that shorter exposures (6, 24 and 48 hours) corroborated these findings (Extended Data Fig. 1 d). Our prior research indicated that increased core BT induced by sauna-like conditions 21 or wakefulness-like temperature exposure 20 , lead to tau dephosphorylation. We replicated these findings, showing a temperature-dependent reduction in phosphorylation levels of both intracellular (Extended Data Fig. 1 e,f) and extracellular tau (Fig. 1 d,e,g,j,k) in both SH-Tau3R and mouse primary cells. Overall, exposure to higher temperatures similar to those experienced during wakefulness promotes tau secretion, with the secreted tau species being markedly dephosphorylated. To assess whether the effect of temperature is specific to tau secretion, we conducted a comparative analysis of extracellular contents for various proteins, including microtubule-associated protein 2 (MAP2), α-synuclein, fibroblast growth factor 2 (FGF2), caspase-1, and Neurofilament light chain (NfL). MAP2 is also a microtubule-associated protein, and NfL is a cytoskeletal protein often used as negative control for extracellular tau 14,22 . Additionally, α-synuclein, FGF2 and caspase-1 are proteins known to be secreted via the UPS pathways 23,24 . However, temperature did not affect the secretion profiles of these proteins (Extended Data Fig. 1 g,h), pointing to a specific temperature modulation of tau release though unknown underlying mechanisms. Wakefulness temperatures promote tau release through its caspase-3-mediated cleavage We further sought to identify cellular pathways underlying temperature-dependent effects. Given extracellular tau is predominantly present as the TauC3 proteolytic fragment in AD 7,8 , which is thought to facilitate its secretion 9 , we wondered whether temperature affects its caspase-3-mediated cleavage. Our findings revealed that wakefulness temperatures increased caspase-3 activity and protein levels (Fig. 2 a-c) in both human and mouse cells, compared to those exposed at 35 or 37°C. Interestingly, this coincided with the intracellular dephosphorylation of tau at S422 (Fig. 2 b,c), previously shown to facilitate the caspase-3-mediated cleavage at D421 12,13 . As a result, we observed increased levels of both intra- and extracellular TauC3 in cells exposed to 38°C (Fig. 2 b-e). As additional validation, we observed that wakefulness temperatures decreased the levels of intra- and extracellular Tau46 (epitope of 428–441) (Fig. 2 b-e), which does not recognize C-term cleaved tau 8 . To further substantiate the role of caspase-3 in tau secretion, we observed that its inhibition – either with z-DEVD-FMK pharmacological inhibitor or caspase-3 mRNA-targeting siRNA – significantly decreased extracellular tau release (Fig. 2 f-i). Collectively, our data demonstrates that wakefulness temperatures promote caspase-3-mediated cleavage of tau, facilitating its UPS-I mediated secretion. Wakefulness temperatures drives TauC3 secretion through SDC3 upregulation HSPGs have been identified as critical components of the UPS-I pathway due to their ability to bind to intracellular proteins and facilitate their direct export across the plasma membrane 25 . Ubiquitously expressed on cell surfaces, HSPGs consist of a core proteoglycan with heparan sulfate chains, the elongation of which is facilitated by the glycosyltransferase activity of Exostosin-1 (EXT1) in the brain 26 . Among the diverse family of HSPGs, neuronal SDC3 is particularly relevant to AD pathophysiology, promoting the propagation of amyloid pathology and the neuronal uptake of tau 27,28 . To obtain insight into how temperature might drive tau secretion through the UPS-I pathway, we investigated whether temperature influences SDC3 metabolism. We found wakefulness temperatures increased SDC3 and EXT1 protein and mRNA expressions in SH-Tau3R cells and primary neuronal cells (Fig. 3 a-c). To further emphasize that TauC3 is highly releasable, we used confocal microscopy and observed a temperature-dependent increase in the merged staining of SDC3 and TauC3, with numerous puncta (SDC3- and TauC3-positive) mainly localized in the soma and in proximal neurites of primary neurons cultured at 38°C (Fig. 3 d; Extended Data Fig. 2 ). As SDC3 might be also involved in tau internalization inside neurons 27,28 , we observed peri-membranous puncta that were TauC3 and SDC3, although in a lower proportion compared to intracellular ones (Fig. 3 d; Extended Data Fig. 2 ). To confirm the role of SDC3 and EXT1 in tau secretion via the UPS-I pathway, we conducted siRNA-mediated knockdown experiments either for SDC3 or EXT1. In both cases, we observed a significant reduction in the extracellular levels of total and cleaved-tau compared to cells transfected with scrambled siRNA (Fig. 3 d-f). To further examine the interplay between caspase-3 and SDC3 in tau release, we also simultaneously inhibited the expression of caspase-3 and SDC3 resulting in almost complete suppression of intra- and extracellular TauC3 levels (Fig. 3 e,g). Intriguingly, the inhibition of TauC3 expression was associated with a notable increase in tau hyperphosphorylation at S422 (Fig. 3 g). These observations collectively emphasize a complementary role of caspase-3 and SDC3 in mediating the extracellular export of TauC3 during wakefulness. Wakefulness temperatures facilitate tau recruitment and release at plasma membrane Due to their lipidic composition, plasma membranes are highly sensitive to temperature fluctuations. An elevation of temperature leads to increased membrane fluidity and permeability in plant and animal cells 29,30 . Based on these findings, we hypothesized that higher temperature exposure might drive tau secretion by optimizing the properties of the plasma membrane to facilitate tau translocation into the extracellular space. We found that SH-Tau3R cells exhibited increased membrane fluidity at wakefulness temperatures (Fig. 3 i). Moreover, tau requires sequestration at the inner layer of the plasma membrane through PIP 2 binding 6 . We thus wondered if temperature influences PIP 2 , and showed that its expression was temperature-dependent in both human and mouse neuron-like cells (Fig. 3 a,b). Interestingly, the temperature-dependent increase in PIP 2 expression was previously documented in yeast and plant cells 31,32 , suggesting a highly conserved process. To explore whether full-length tau and TauC3 have the same affinity for PIP 2 , we performed co-IP using Tau-DA9, TauC3 or Tau46 antibodies and probed for PIP 2 by Western blot (Fig. 3 j), or co-IP using a PIP 2 antibody to assess the interaction with different tau antibodies (Fig. 3 k). In both cases, we observed that PIP 2 preferentially binds TauC3 rather than full-length tau (Tau46 signal was barely detectable) (Fig. 3 j,k). Finally, we demonstrated that an increase in temperature significantly promotes the binding of TauC3 to PIP2, while the binding of full-length tau to PIP2 tends to slightly decrease (Fig. 3 l-n). Altogether, these results suggest that wakefulness temperatures promote the UPS-I-mediated secretion of TauC3 by facilitating its interaction with PIP2 and SDC3 at the plasma membrane, thereby triggering its vesicle-free release. Wakefulness and sleep deprivation upregulate UPS-I pathway by increasing core BT in mice To assess whether the higher CSF and ISF tau levels during wakefulness and SD 14 , are related to natural elevated BT induced by these conditions 20 , we analyzed UPS-I-related proteins in the cortex of wild-type mice across sleep vs. wakefulness, or following SD. Awake mice exhibited higher core BT (Fig. 4 b), associated with increased cortical expression of caspase-3, TauC3, SDC3 and PIP 2 , along with tau dephosphorylation at S422 (Fig. 4 c,d), compared to sleeping mice. Moreover, we observed that the rectal temperature of mice at the time of euthanasia was significantly correlated with the expression levels of caspase-3, pTau(S422), SDC3 and PIP 2 (Extended Data Fig. 3 a-f). We further showed that 6 hours of SD (Fig. 4 e) prevented the natural decrease in core BT during sleep (Fig. 4 f), and triggered the upregulation of caspase-3, TauC3, and PIP 2 levels, associated with decreased S422 phosphorylation and Tau46 expression (Fig. 4 g,h). Mild-hyperthermia increases CSF tau levels in hTau mice To determine whether induced changes in BT affect CSF tau levels, we subjected hTau mice to hypo- or hyperthermic conditions for 4 hours before CSF collection, and compared to normothermic mice (Fig. 4 i,j). We observed that hyperthermic mice exhibited higher CSF tau concentrations (Fig. 4 k), and these levels significantly correlated with rectal temperatures recorded after thermal interventions (Fig. 4 l). The rise in CSF tau concentrations was associated with increased cortical expression of caspase-3, TauC3, SDC3 and PIP 2 , along with a reduction in tau phosphorylation at S422 and Tau46 expression (Extended Data Fig. 4 a,b), all correlating with rectal temperature (Extended Data Fig. 4 c-h). These findings collectively suggest that core BT variation influences CSF tau levels through the upregulation of the UPS-I pathway. It emphasizes the pivotal role played by sleep-wake temperature variations in regulating the secretion and the propagation of tau via the UPS-I pathway. Body temperature correlates with CSF tau but not CSF NfL in humans To test the relationship between BT and sleep-wake tau dynamics in humans, we utilized two separate data sets from older adults in which BT and tau levels (CSF or plasma) were simultaneously measured at multiple time points across the sleep-wake cycle. We examined the correlation between the magnitude of change in tau levels post-wakefulness (∆Tau) and the concurrent rise in BT during wakefulness (∆BT). Predefined measurement times were selected to optimize the average ∆BT within the constraints of the available datasets (see Methods). Similar to previous findings in CSF, plasma tau levels were significantly higher in the evening compared to the morning (Extended Data Tables 1 and 2). We found a positive correlation between ∆BT and ∆Tau for both CSF tau (r = 0.58, p < 0.05) and plasma tau (r = 0.72, p < 0.005), with no correlation for CSF NfL levels (Fig. 5 a-c), and a consistent relationship across CSF and plasma data sets. Specifically, participants exhibiting a large positive ∆Tau, i.e., substantially higher afternoon-evening levels compared to morning, also showed a large positive ∆BT. By contrast, participants with negligible or negative ∆Tau showed minimal or negative ∆BT (Fig. 5 a,c). The observed ∆BT values for the plasma data set agree with our previous study, and represent the first report of correlations between BT and circulating tau in humans. The increase in CSF tau levels with wakefulness vs sleep aligns with previous studies 10,14 . Although diurnal sampling of plasma tau was previously documented in sedentary young adults 33 , this is the first report of diurnal dynamics in tau under naturalistic conditions representative of physiological BT variation. Our overall finding of ~ 15% higher tau in the evening comprised a broad range of ∆Tau values that were substantially explained by ∆BT, with similar patterns for CSF. These results supported our in vivo and in vitro findings, where higher BT during wakefulness drove higher tau secretion. DISCUSSION The present study investigated the influence of BT variation during the sleep-wake cycle upon tau secretion and its underlying regulatory mechanisms (Fig. 6 ). Our findings indicate that wakefulness temperatures, or conditions affecting core BT such as SD or mild-hyperthermia induction, promotes C-term truncation of tau, leading to its extracellular release through UPS-I pathway. Using in vitro and in vivo approaches, we identified that the physiological increase in core BT during periods of wakefulness triggers some specific intracellular mechanisms such as (i) the caspase-3-mediated cleavage of tau into TauC3, (ii) the sequestration of TauC3 at plasma membrane via its binding to PIP 2 , and (iii) the translocation of TauC3 into the extracellular space facilitated by SDC3, resulting in increased CSF tau levels. This pointed to the involvement of the circadian regulation of BT during the sleep-wake cycle in tau secretion and propagation. The precise mechanisms underlying tau secretion remain unestablished. Our observation that tau release is modulated in a temperature-dependent manner suggests core BT variation may play a significant role in regulating tau secretion. Holth et al previously showed a twofold rise in ISF and CSF tau levels during wakefulness compared to sleep 14 . Here, we replicated these tau level increases by varying temperature alone within physiological range, with tau doubling at 38°C compared to 35°C.. Excitatory neuronal activity, one of the first identified biological processes capable of increasing tau release 34,35 , is potentiated during wakefulness and depressed during sleep 36 . In order to test whether tau release during wakefulness was accounted for by concurrent increases in neuronal activity, Holth et al. used tetrodotoxin (TTX) to inhibit neuronal activity, showing that it prevented tau release during SD 14 . However, TTX also causes rapid hypothermia 37,38 , pointing to a possible role of temperature in these findings. While it is known that a slight 1°C change in brain temperature is sufficient to alter neuronal excitability and activity 39–41 we similarly found that a 1°C change alters tau secretion. Given SH cells and mouse primary neurons lack neuronal activity 42 , our data strongly suggest that temperature directly regulates UPS-I-mediated tau secretion. However, considering that neuronal activity alone can also drive tau release 34 , the interplay between neuronal activity and BT in stimulating tau secretion requires further investigation. Our study emphasizes tau cleavage into TauC3 as pivotal for secretion, with CSF and extracellular tau mainly present as C-terminally truncated 8,9 . While the administration of a TauC3-specific antibody has been shown to impede tau propagation and seeding 43 , the diurnal regulation of these processes remains unknown. We found that wakefulness temperatures induce both tau dephosphorylation at S422, enabling tau cleavage, and upregulation of the caspase-3-mediated TauC3 truncation, leading to its extracellular release. The role of S422 phosphorylation and TauC3 remains debated, with some studies evidencing TauC3 as neuroprotective 44–46 and others linking TauC3 to neurofibrillary tangle assembly and synaptic toxicity 12,47,48 . Our investigation revealed an inverse relationship between TauC3 and S422 phosphorylation, modulated by physiological sleep-wake fluctuations in core BT. These findings also imply a physiological tau release, consistent with prior studies showing tau secretion does not necessarily result in neuronal pathology spreading 34,35 . In favor to this view, treatment with an anti-pS422 antibody has been shown to reduce AD pathology while increasing plasma tau concentrations in AD mice 49 , suggesting that TauC3 might be more prone to brain clearance. However, while wakefulness temperatures induce tau dephosphorylation at multiple epitopes, the relevance of other phosphorylation sites in driving tau secretion remains to be explored. Here, we have made several novel findings regarding the modulation of tau secretion via interactions between multiple temperature-dependent components of the UPS-I pathway. While the UPS-I pathway is known for the release of FGF2 25 , α-synuclein 50 , and tau 4,5 , our observations of a temperature-dependent effect on tau secretion—without similar changes for others proteins—suggest a unique BT-driven tau secretion pathway. Our results suggest that the temperature-dependent cleavage of tau into TauC3 may serve as an initiating factor for finely modulating its secretion. Notably, the loss of microtubule-binding capacity of TauC3 48 might enhance its availability for the secretion pathway, while wakefulness temperatures facilitate the binding of TauC3 to PIP 2 at the inner plasma membrane. Prior research has demonstrated that the tau C-terminal domain contains a low-affinity site that affects its interaction with phosphoinositides 51 , likely explaining the preferential binding of PIP 2 to TauC3, given this fragment lacks a portion of the C-terminal domain. Altogether, these findings suggest that higher core BT during wakefulness, SD or mild-hyperthermia, promotes TauC3 binding to PIP 2 at the plasma membrane, initiating the export process. The increase in BT during wakefulness appears to also promote the extracellular release of TauC3 by enhancing its interaction with SDC3, facilitating the membrane translocation process. While prior studies reported increased levels of SDC3 in the brain of AD mouse models 28 , or following neuronal stimulation 52 , this is the first report of its temperature-dependent expression and metabolism. Notably, one study showed that the glycosyltransferase activity of enzymes such as EXT1—required for the elongation of SDC3 sulfate chains—increases with temperature 53 . Our study extends these findings, showing that wakefulness temperatures enhance EXT1 mRNA expression, potentially improving SDC3 function. We also observed a substantial intracellular co-localization of SDC3 with TauC3 at wakefulness temperatures, contributing to a better understanding of the mechanisms underlying tau secretion during the sleep-wake cycle. Our results suggest a pathway by which sleep-wake BT variation may modulate physiological CSF and plasma tau dynamics in human via temperature-dependent tau secretion and phosphorylation. However, further research is needed to determine additional temperature-dependent processes. Important candidates include neuronal activity 39–41 , as previously discussed, as well as sleep, known to depend upon body and brain temperature fluctuation 54 . Demonstrating a pathway by which temperature influences AD biomarkers via sleep, a previous study showed chronic thermoneutral temperature exposure in AD mice reduced amyloid pathology by enhancing slow-wave sleep 55 . We also note that bidirectional effects may additionally contribute to the observed relationship between BT and tau dynamics in that early tau pathology in thermoregulatory brain areas may influence BT patterns, as recently shown in mice 56 . Finally, in the setting of AD, further research is needed to distinguish between circulating tau derived from unconventional vs vesicular secretion 5 , or other sources such as impaired degradation and clearance pathways 57 , or release after neuronal death 58 . Altogether, our findings suggest that sleep-wake BT variation modulates parallel dynamics in tau secretion and phosphorylation, and provide the first evidence associating BT variation with CSF and plasma tau dynamics in human. By extension, our results suggest that impaired thermoregulation as well as BT alteration caused by sleep disturbance may contribute to the pathogenesis of AD and related tauopathies. It is therefore crucial to understand how naturalistic variation in BT over the sleep-wake interval affects CSF and plasma tau levels used for AD diagnosis, particularly in patients with thermoregulatory or sleep deficits. We note that few previous studies in AD patients measured BT variation over the sleep vs wake interval—rather most reported BT averaged over the sleep wake cycle, and meta-analysis showed little difference (0.1°C) between AD and controls 59 . By contrast, our findings emphasize the importance of assessing BT dynamics over the naturalistic sleep-wake interval in order to understand how BT interacts with tau metabolism. We previously showed that lower waking BT predicted tau pathology, supporting hypotheses that age-associated BT decline may be a risk factor for AD 60–62 . On the other hand, sleep fragmentation or deprivation 16,63 and increased nocturnal activity 64,65 —both risk factors for, and observed in AD— may prevent the nocturnal BT drop 20 , thereby increasing tau secretion and potentially accelerating tau pathogenesis 66,67 . Conclusions Our model (Fig. 6) elucidates how core BT regulates tau secretion by driving UPS-I pathway activity during the sleep-wake cycle in healthy individuals. We posit that wakefulness temperatures facilitate physiological tau release, while sleep temperatures inhibit this pathway and increase tau phosphorylation. Further, wakefulness temperatures might facilitate the secretion of dephosphorylated and cleaved tau species which are less toxic, less prone to aggregation and more manageable for clearance and degradation systems within the brain 45,46,49,68 . By contrast during sleep, tau release slows, potentially aiding its clearance via the glymphatic system 68,69 . This model points toward the importance of maintaining and managing the appropriate core BT at the right phase of the sleep-wake cycle, and the potential for age- or AD-related disorder in this pattern to lead to tau pathology. Interestingly, interventions like sauna bathing, which temporarily increase BT 70 , are beneficial in reducing AD risk, increasing deep sleep in humans 71 , and reducing tau phosphorylation in mice 21 . Future studies may examine whether sauna use can delay tau-mediated neurodegeneration by correcting sleep and core BT misalignment associated with thermoregulatory and sleep-disturbances in aging and early AD. Finally, while the physiological role of extracellular tau remains enigmatic, it may act as a signaling molecule, potentially interacting with muscarinic receptors 72 . Elucidating the physiological role of tau secretion 73 and understanding the normal function of extracellular tau could inform therapeutic strategies to impede tau pathology propagation. METHODS Cell culture In this study, human neuroblastoma cells (SH-SY5Y cells) stably expressing human tau 3 repeat isoform 2+3-10- (designated as SH-Tau3R cells, generously provided by Luc Buée) were used. The SH-Tau3R cells were cultured as previously described 74 . Briefly, the cells were grown in DMEM/High glucose medium (11995-065, ThermoFisher), supplemented with 10% bovine growth serum (BGS, heat inactivated, F1051-500ML, Sigma-Aldrich), 1% glutamine (25030081, ThermoFisher), and 1% penicillin/streptomycin (15140-122, ThermoFisher). The cell cultures were maintained in a humidified incubator with 5% CO 2 at 37°C. The cells were grown either in 10 cm Petri dishes, 6-, 12-, or 96-wells plates. Primary culture of neurons For the primary neuronal culture, cortices of mouse embryos at embryonic day 16 (E15-E17) were used from transgenic mice B6.129S2Emx1tm1(cre)Krj/J, where Emx1-Cre mice were crossed with Red Fluorescent Protein-Lox mice (Jackson Laboratories). Briefly, brains embryos were dissected out, meninges, choroid plexus and hippocampus were removed to avoid contamination and cortices were mechanically and enzymatically disrupted in the presence of trypsin-EDTA 0,25% (Gibco) for 20 min at 37°C. The cell suspension was filtered through a 70 µm cell strainer and plated onto 6-well plates (200,000 cells/well), which were pre-coated with 50 µg/ml poly-D-lysine (A3890401, ThermoFisher), or on coverslips pre-coated with 1 µg/mL polyethylenimine (043896.03, ThermoFisher) and 50 µg/mL poly-D-lysine in 24-well plates (150,000 cells/well). The cells were firstly grown for 2 hours in DMEM/High glucose medium, supplemented with 10% BGS and 1% of streptomycin/penicillin antibiotics in a 5% CO 2 humidified incubator at 37°C. Then, the culture medium was changed per a growth medium (NeurobasalTM medium (21103-049, ThermoFisher), 1% glutamine (25030081, ThermoFisher), 2% B-27 supplement (17504044, ThermoFisher), 1% N-2 (17502-048, ThermoFisher) and 1% penicillin/streptomycin). The cultures were maintained at 37°C in a humid atmosphere with 5% CO2 and a growth period of 4 days was allowed before any experimental treatment was administered. Temperatures exposure and cell treatments Prior to initiating any treatment, the cell culture medium was replaced with fresh DMEM/High glucose medium (without BSA) or Neurobasal medium, for SH-Tau3R or primary neuronal culture, respectively. Then, the cells were placed in dedicated CO 2 incubators set to 35, 37, 38 or 39°C for a duration from 6 to 72 hours (Fig.1a, h). To inhibit caspase-3 activity, cells were treated for a period of 72 hours with the selective caspase-3 inhibitor z-DEVD-FMK (A13503; Adooq Biosciences) at a concentration of 20 µM 9 dissolved in a vehicle solution (phosphate-buffered saline (PBS) containing 0.1% of DMSO). Transfection of small interfering RNA was carried out using Lipofectamine TM RNAiMAX transfection reagent (13778075, ThermoFisher) according to the manufacturer’s instructions. Briefly, for each transfection, cells were cultured for 72 hours in 1 ml of Opti-MEM (ThermoFisher) containing 40 µl of Lipofectamine TM , and 100 nmol of respective siRNAs. The following siRNAs were used: Silencer® Pre-designed EXT1 siRNA (ID116802, ThermoFisher), Stealth RNAi TI SDC3 siRNA (HSS145253, ThermoFisher), and SignalSilence® Caspase-3 siRNA (6466S; Cell Signaling). Silencer TM select negative control siRNA (4390843, ThermoFisher) was used as the scrambled negative control. Animals In this study, three-months-old C57BL6 (males and females) and 18-month-old hTau (males) mice or their littermate control tau knockout (TKO; males) 75 were used. The hTau mice were generated by crossing mice expressing the 6 isoforms of nonmutated human tau (known as 8c mice) 76 with murine TKO mice 77 . The founders of hTau and TKO colonies originated from a C57BL6 background (B6.Cg-Mapttm1(EGFP)Klt-Tg(MAPT)8cPdav/J, Jackson Laboratories). The animals were handled according to procedures endorsed by the “The Animal Care Committee of Université Laval (CPAUL-3, approbation number: CHU-22-1027)” under the guidelines of the Canadian Council on Animal Care. All mice had access to water and food ad libitum. The mice were housed in a 12 h light/12 h dark cycle, with the lights being turned on at 7:15 am. At the end of each experiment, mice were euthanized through decapitation without anesthesia, as anesthesia leads to tau hyperphosphorylation 78,79 . The brains were promptly removed and cortices were dissected on ice, frozen on liquid nitrogen and stored at −80°C for further analysis Sleeping vs. awake mice Mice were subjected to a continuous period of darkness lasting for 3 days. The determination of subjective day was determined as previously described 20 . Briefly, sleeping C57BL6 mice (n=5 males and n=5 females) were euthanized between 10:30 and 11:30 am local time (at Circadian Time 4 (CT4), 16 h after the onset of activity) and active mice (n=5 males and n=5 females) were euthanized between 10:30 and 11:30 pm local time (at CT16, 4 h after the onset of activity) (Fig. 5a). Furthermore, the sleeping criterion corresponded to mice in the nest, in a “resting posture”, as elucidated by Thoman and Carroll: absence of locomotor activity, absence of movement, absence of erect posture 80 . The core BT of mice was assessed just before euthanasia with a rectal probe (RET-3, Brain Tree Scientific Inc) connected to a digital thermometer (Thermalert TH5; Physitemp). Sleep deprivation As previously described by our group 20 , a subset of C57BL6 mice was intentionally kept awake for the first 6 hours of the light period (sleep deprivation (SD) group, n=9, males and females). Naive mice (n=7, males and females) were allowed to sleep without any disturbance. All mice were euthanatized by decapitation at the end of SD period (Fig. 4e). Prior to SD experiment, a subset of five mice of both groups was abdominally implanted with telemetric probes (BodyCap, Anipill) enabling continuous monitoring of their BT. The baseline BT was assessed the day preceding the SD protocol for the same set of animals. Cold and heat exposures On the day preceding the experiment, hTau mice were individually housed to prevent any mutual heating. For the entire duration of the study, the naive group (n=5) and the negative control TKO (n=3) remained at the standard temperature of the animal facility (22°C). As previously described by our group 21,81 , the two other groups of mice underwent a 4-hour exposure period either at temperature of 4°C (n=3) or 38°C (n=5). The core BT of mice was assessed just prior to euthanasia utilizing a rectal probe (RET-3, Brain Tree Scientific Inc) connected to a digital thermometer (Thermalert TH5; Physitemp). CSF collection The mice were anesthetized with isoflurane and positioned on a stereotaxic instrument. To maintain core BT, a water heating pad was used. Under the observation of a dissection microscope, the subcutaneous tissues and muscles (m. biventer cervicis and m. rectus capitis dorsalis major) were gently separated via blunt dissection utilizing forceps. This separation facilitated the exposure of the dura mater of the cisterna magna. A capillary tube was introduced through the dura mater into the cisterna magna in order to induce the CSF flow into the capillary tube. Protein extraction The samples (cell lysates or mice cortices) were homogenized by sonication in Radioimmunoprecipitation assay (RIPA) buffer, then centrifuged for 20 min at 20,000g at 4°C. The resulting supernatant was collected, and the total protein concentration was assayed (Pierce™ BCA Protein Assay Kits, 23225, ThermoFisher). The samples were diluted in sample buffer (NuPAGE LDS; Invitrogen) containing 5% of 2-β-mercapto-ethanol, 1 mM Na 3 VO 4 , 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340; Sigma-Aldrich). The samples were then subjected to denaturation for 10 min at 95 °C. Western blotting Western blot analysis was conducted as previously described 82 . 10-20 μg of the samples were separated on an SDS-10% polyacrylamide gel and transferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were saturated, hybridized with the appropriate antibodies, and revealed as described in 82 . For immunoblots targeting phospho-tau epitopes, the signal was normalized to the total tau protein. Used as a loading control, other proteins were normalized to β-actin. Representative lanes from the immunoblots were exhibited for each specific experimental condition. The dashed lines indicate segments where certain lanes from the same blot were excluded, and the remaining lanes were combined. Brightness levels were adjusted as necessary to enhance visualization and accuracy. Antibodies All antibodies used in this study, in addition to their dilution, are listed in Extended Data Table 3. Dot blotting The cell medium was harvested following appropriate treatments and centrifugated for 10 min at 20,000g at 4°C to remove cell debris. In order to assess extracellular content of proteins by dot blotting, 100 μl of cell medium were deposited onto nitrocellulose membranes (Amersham Biosciences), utilizing a microfiltration blotting apparatus (Bio-Dot Apparatus 1706545, Bio-Rad). The membranes were saturated, hybridized with appropriate antibodies (Extended Data Table 3) and revealed as described in 82 . For dot blots targeting phospho-tau epitopes, the signal was normalized to the total tau protein. In the case of other proteins, the normalization was performed relative to the respective extracellular LDH value (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega). Representative dots signal was exhibited for each specific experimental condition. The dashed lines indicate segments where certain dots from the same blot were excluded, and the remaining dots were combined. Brightness levels were adjusted as necessary to enhance visualization and accuracy. Co-immunoprecipitation Co-immunoprecipitation (co-IP) analyses were performed to determine interactions between PIP 2 total tau and TauC3, following manufacturer’s instructions (Pierce™ Classic Magnetic IP/Co-IP Kit, 88804, ThermoFisher Scientific). Briefly, SH-Tau3R cells were harvested using lysis buffer, incubated at 4 °C for 5 min, and centrifuged at 13,000g to pellet cellular debris. The supernatants were collected, proteins levels were adjusted to 500 µg and primary antibodies (Extended Data Table 3) were added to samples, except for the negative control (NC) sample. The samples were then incubated overnight at 4 °C on a rotating device. Following this step, protein A/G magnetic beads (25 μl) were added to each sample and incubated for 1 hour with agitation at room temperature. The antibody-bound beads were extracted using a magnetic device and washed three times. The beads were dissociated using the elution buffer and separated magnetically. To neutralize the low pH environment, 10 µl of neutralization buffer were added to the supernatant. The resulting sample was diluted with sample buffer (NuPAGE LDS; Invitrogen) containing 5% of 2-β-mercapto-ethanol, 1 mM Na 3 VO 4 , 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail, and finally boiled at 95°C for 5 minutes. The proteins were analyzed using Western blot analysis. Immunocytochemistry Neurons from primary culture were fixed in PBS 1X (311-010-CL, Multicell) /4% paraformaldehyde (19210 Electron Microscopy Sciences, ThermoFisher)/10% sucrose (S53, ThermoFisher) for 20 min at room temperature. After washing 3 times with PBS 1X, cells were permeabilized in 0.2% Triton X-100 (T8787-100ML, Millipore) in PBS 1X for 30 min at room temperature and blocked with 5% Goat Serum Heat Inactivated (G6767, Millipore) in PBS 1X for 1 hour at room temperature. Then, cells were incubated with the primary antibodies against TauC3 and SDC3 (Extended Data Table 3) in 5% goat serum heat inactivated in PBS 1X at 4°C overnight. After washing 3 times with PBS 1X, the secondaries antibodies (anti-mouse Alexa Fluor 488 diluted at 1:1000 (#A-11029, ThermoFisher) and goat anti-rabbit IgG Alexa Fluor 633 diluted at 1:1000 (#A-21070, ThermoFisher)) were added for 2 hours. After 3 washes with PBS 1X, DAPI (4′,6-diamidino-2-phenylindole, (ThermoFisher) 3,5µL of DAPI in 25 mL PBS 1X) was used for nuclei staining and coverslips were mounted with Fluoromont-G (00-4958-02, Invitrogen). Sections were imaged on a Zeiss LSM800 confocal microscope system equipped with 405, 488, 561 and 640 nm lasers. Confocal images were acquired and mosaics created using the Zen Blue Edition software (v. 2.3, Carl Zeiss). Caspase-3 activity assay kit The SH-Tau3R cells were cultured in 96-well plates and treated according to appropriate experimental conditions (Fig. 1a). The colorimetric caspase-3 Assay Kit (ab39401, abcam) was used to determine the activity of caspase-3, and following manufacturer’s instruction. Membrane fluidity The SH-Tau3R cells were cultured within 96-well plates and subjected to treatment as outlined in the experimental groups (Fig. 1a). The membrane fluidity (ab189819, abcam) was assessed according to manufacturer’s instructions. Briefly, the cells were incubated 1 hour at temperatures of 35°C, 37°C or 38°C in a cell medium supplemented with 5 µM of Fluorescent Lipid Reagent and 0.08% Pluronic F127. The fluorescence intensity was then measured (Infinite F200, Tecan) at wavelengths of 400nm and 470nm, using the appropriate filter for excitation at 350nm. The recorded fluorescence values were corrected by subtracting the corresponding blanks from each sample, and the fluorescence ratio of excimer emission (470nm) to monomer emission (400nm) was calculated. ELISA assays of extracellular tau Total and phosphorylated tau concentrations within the cell medium were quantified using ELISA kit: Tau (total) Human KHB0041; Tau [pS199] Human KHB7041; Tau [pT231] Human KHB8051; Tau [pS396] Human KHB7031; Tau (total) Mouse KMB7001 (ThermoFisher). Prior to analysis, the samples were suitably diluted in diluent buffer (1:50 for human tau, 1:2 for mouse tau and phospho-tau). The ELISA assays were performed in accordance with the instructions provided by the manufacturer. Quantitative PCR Total RNA was isolated from SH-Tau3R cells using TrizolÒ reagent (Life Technology) in accordance with the manufacturer’s instructions. The quantification of RNA was conducted, and 1 mg of total RNA was used for cDNA synthesis using the iscript TM cDNA Synthesis Kit (Biorad), containing an optimal blend of oligo-dT and random primers. For subsequent PCR amplification, 1 µl of the resultant cDNA was used as template. The primer sequence used for the PCR amplification are reported in Extended Data Table 3. The qPCR mix was formulated with 18 µL per 2 µL of 20 ng cDNA. The mix consisted of 0.5 µL of both the forward and reverse primers, 10 µL of SYBR Green PCR Master Mix (Applied Biosystems), and 7.5 µL of nuclease free water. The qPCR program began with a hot start at 95°C for 3 minutes, succeeded by 40 cycles at 95°C for 15 seconds, followed by 60°C for 1 minute, using a LightCycler 480 II apparatus (Roche). The melting curves were evaluated to ensure a single PCR product. To quantify cDNA levels, the comparative 2ΔΔCt method was employed. Ct values corresponding to the target gene were normalized to the Ct values of the house-keeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). The results were expressed as n-fold differences relative to the experimental control. Human Studies CSF temperature correlations: Detailed information about participants, CSF collection, and study design can be found in Lucey et al. 83 . Thirteen participants who completed the placebo group of a recently published clinical trial had 6 ml of CSF collected every 2 hours for 36 hours via an indwelling lumbar catheter 83 . All participants were cognitively unimpaired and in good general health except for poor sleep efficiency <85% measured by actigraphy. Body temperature was recorded every four hours with a temporal forehead thermometer (Adc Adtemp 427, American diagnostic Corp, United-States). CSF tau forms (T181, S202, T217) were measured by immunoprecipitation/mass spectrometry as previously described 83 . NfL protein levels were quantified using the NF-light TM ELISA kit (UmanDiagnostics, Umea, Sweden) following the manufacturer’s protocol. The assay’s measurement range is 100 pg/ml to 10,000 pg/ml with a detection threshold of 33 pg/ml. CSF samples were prepared through dilution with an equal volume of Sample Diluent, achieving a 1:100 dilution ratio, to ensure a volume suitable for analysis. The quantitation process entailed the enzymatic conversion of a colorless substrate into a colored product indicative of the NfL concentration in the samples. Absorbance readings were taken at 450 nm with a reference wavelength of 620-650 nm. To ensure consistency, samples with known high levels of NfL (“bloody CSF”) were utilized as positive controls on each plate diluted to 1:1000. CSF tau-181, tau-202, and tau-217 concentrations were averaged at 8AM, 4PM, and 8PM. Differences between temperature, CSF tau levels, and CSF NfL levels were then calculated for use in the analyses, and detailed data for each participant are provided in (Extended Data Table 1). NfL was selected as control protein because its soluble concentration is not affected by sleep-wake activity 14 . We selected time points of 8 AM vs 4 PM for post-sleep vs post-wakefulness tau levels, which also corresponded to the minimum and maximum BT, respectively. These intervals were selected based on the following rationale. First, we assumed little delay between tau secretion and appearance of tau in the CSF, meaning that BT taken at the time of CSF collection would roughly reflect brain temperature at the time of tau secretion. Second, we selected 8 am vs 4 pm as the interval that maximized that difference in sleep vs wake temperatures, given that BT was not recorded during sleep, and for the majority of participants, had already begun to drop between 4 pm and 8 pm (Extended Data Table 1). Plasma temperature correlations : Data were collected from 24 older adults 68.39±5.25 years of age, 17 of whom were female. Subjects were enrolled in cross-sectional study examining the relationship between core BT and plasma and PET AD biomarkers. Subjects were cognitively normal (n=21) or had mild cognitive impairment (n=3) as determined by the clinical dementia rating scale (CDR), and were medically healthy with only mild, or no sleep apnea. Prior to the study, participants were screened with interviews and one week of home actigraphy for sleep-wake disorders including sleep less that 6 hours per night, significant phase advance or phase-delay. Additional exclusion criteria are detailed in 62 and included AD dementia (CDR > 0.5), medical comorbidities and the use of medications that might affect sleep or thermoregulation, major psychiatric disorders and moderate-severe substance use disorders, shift work within the last 6 months, or traveling across 1 or more time zones within 2 weeks of study participation. The study design was a semi-naturalistic protocol fully detailed in 62 . Briefly, participants underwent continuous measurement of core body temperature using an ingestible telemetric device (Cortemp, HQInc) that sampled temperature every 15 seconds with an accuracy of 0.2°C for a minimum of 36 hours spanning 2 nights. During this time, 2 in-lab nocturnal polysomnograms were measured, and participants were free to behave as they chose during the intervening day between the lab nocturnal recordings. The goal of this design was to capture data that most closely represented the typical BT for each participant. Blood draws for plasma tau were collected on four occasions, in the mornings (7:00 am) and evenings (7:00 pm) on both mornings and nights (Extended Data Table 2). Prior to analysis, temperature data were preprocessed to exclude gaps and artifacts as detailed in 62 . Data presented for BT-tau correlations comprised tau levels from night 2 and morning 2, given BT was not always measured prior to blood draw on night 1 (Table S3). Paired tau levels and BT data were obtained for 15 subjects (Extended Data Table 2). For BT–tau correlations, the difference between the average BT between 6–7 pm and 1–2 am was calculated. This interval was chosen because these times represented the sample average minimum and maximum BT, and as such their difference maximized the diurnal BT difference, or ∆BT. Plasma sampling times were selected to maximize efficiency in collecting data. Food intake was not regulated, but the morning sample was typically before the morning meal, whereas the evening sample was typically before the evening meal. Concentrations of plasma tau were measured using the neurology 3-PLEX kit and Simoa HD-X instruments (Quanterix, Billerica, MA, USA) at the NYU Alzheimer’s Disease Center Biomarker Core according to the manufacturer’s instructions. Plasma extraction was performed as described previously. Assays were run in duplicate to obtain inter-assay coefficient of variations (CVs). The inter-assay CV was under 20% for all samples. Statistical analysis A minimum of two distinct experiments were carried out for each experimental condition. Prior to conducting each analysis of variance, an assessment of Gaussian distribution was performed, and its validity was confirmed through a Kolmogorov-Smirnov test (utilizing GraphPad Prism 9.0). Depending on the specific analysis, two-tailed t-tests (or Mann-Whitney tests), as well as one- or two-way ANOVAs (or Kruskal-Wallis tests), were applied. Post hoc analyses, involving either Tukey’s or Dunnett’s tests, were subsequently employed. A significance threshold of P < 0.05 was employed to determine statistical significance. The presentation of data incorporated either box and whisker plots (illustrating the range from minimum to maximum values, encompassing the median) or mean ± standard error of the mean (s.e.m). The scatter plots depicted on each graph provide an indication of the number of data points, and detailed statistical information is provided in Extended Data Table 4. Declarations ACKNOWLEDGEMENTS During the preparation of this work the authors used Chat- GPT-4.0 in order to improve readability and language. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. We are also grateful to the late Dr. Peter Davies (Feinstein Institute for Medical Research, Manhasset, USA) for the generous gift of anti-tau antibodies, to Dr. Luc Buée (Centre de Recherche Jean-Pierre Aubert, Lille, France) for the SH-Tau3R cells, and to France Alzheimer for travel award to present this work. Funding: This work was made possible by grants from the FRSQ, NSERC (RGPIN/05862- 2016), CIHR (IC121689), and Alzheimer Society of Canada (19-04) to EP and VP, and by an NIA/NIH (R01AG070866) award to EMB, by a postdoctoral award from the Alzheimer Society of Canada (to GC), and the National Institutes of Health (K76 AG054863), BrightFocus Foundation (A2016180S), and the Washington University/Centene Personalized Medicine Initiative. The sponsors had no role in the design, execution, interpretation, or writing of the study. Author contributions: GC and EP conceived the preclinical studies and designed experiments. EMB conceived of and designed the human plasma study. BPL conceived and designed the human CSF studies. FDGM performed dissection and seeding of primary cultures. GC, ER, SDD, FL, EB, BK, PFF, and SC performed WB and dot blot experiments. FL performed qPCR experiments. GC performed ELISA assay, co-IP, membrane fluidity assay and caspase-3 activity assay. GC and FDGM performed immunocytochemistry, microscopy imaging and analysis. GC and IG designed and performed sleep-wake and sleep deprivation experiments in wild-type mice, and performed surgical implantation of abdominal probes for temperature recordings. GC and FL performed heat and cold exposures in hTau mice. NF performed mouse CSF collection. FL maintained animal colonies and genotyped animals. BPL, HL, and WL performed human CSF studies. JK, JH and DV conducted the human plasma studies, AY, HTW, and AP processed and analyzed temperature and tau data, EB conducted cognitve screening, DMR, IA, AWV, and RSO supervised sleep disorder and actigraphy screening, LD performed the tau SIMOA assays. GC, EP and EMB wrote the manuscript. All authors reviewed, corrected and approved the final manuscript. Competing interests: All authors declare no competing interests. Data and material availability: The raw data supporting the findings of this study are available on: https://drive.google.com/drive/folders/1iQqmgMdC4GJ04ZQDHb0XIj5Jnd32-iZk?usp=drive_link References Long, J. M. & Holtzman, D. M. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell 179 , 312–339 (2019). Bejanin, A. et al. Tau pathology and neurodegeneration contribute to cognitive impairment in Alzheimer’s disease. Brain 140 , 3286–3300 (2017). Chai, X., Dage, J. L. & Citron, M. Constitutive secretion of tau protein by an unconventional mechanism. Neurobiology of Disease 48 , 356–366 (2012). Merezhko, M. et al. Secretion of Tau via an Unconventional Non-vesicular Mechanism. Cell Reports 25 , 2027-2035.e4 (2018). Merezhko, M., Uronen, R.-L. & Huttunen, H. J. The Cell Biology of Tau Secretion. Front Mol Neurosci 13 , 569818 (2020). Katsinelos, T. et al. Unconventional Secretion Mediates the Trans-cellular Spreading of Tau. Cell Reports 23 , 2039–2055 (2018). Johnson, G. V. W. et al. The τ Protein in Human Cerebrospinal Fluid in Alzheimer’s Disease Consists of Proteolytically Derived Fragments. Journal of Neurochemistry 68 , 430–433 (2002). Borroni, B. et al. Pattern of Tau forms in CSF is altered in progressive supranuclear palsy. Neurobiology of Aging 30 , 34–40 (2009). Plouffe, V. et al. Hyperphosphorylation and Cleavage at D421 Enhance Tau Secretion. PLoS ONE 7 , e36873 (2012). Barthélemy, N. R. et al. Sleep Deprivation Affects Tau Phosphorylation in Human Cerebrospinal Fluid. Annals of Neurology 87 , 700–709 (2020). Gamblin, T. C. et al. Caspase cleavage of tau: Linking amyloid and neurofibrillary tangles in Alzheimer’s disease. Proceedings of the National Academy of Sciences 100 , 10032–10037 (2003). Guillozet-Bongaarts, A. L. et al. Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivo. J Neurochem 97 , 1005–1014 (2006). Sandhu, P. et al. Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease. Bioorganic & Medicinal Chemistry Letters 27 , 642–652 (2017). Holth, J. K. et al. The sleep-wake cycle regulates brain interstitial fluid tau in mice and CSF tau in humans. Science 363 , 880–884 (2019). Stevanovic, K. et al. Disruption of normal circadian clock function in a mouse model of tauopathy. Experimental Neurology 294 , 58–67 (2017). Wang, C. & Holtzman, D. M. Bidirectional relationship between sleep and Alzheimer’s disease: role of amyloid, tau, and other factors. Neuropsychopharmacol. 45 , 104–120 (2020). Rothman, S. M., Herdener, N., Frankola, K. A., Mughal, M. R. & Mattson, M. P. Chronic mild sleep restriction accentuates contextual memory impairments, and accumulations of cortical Aβ and pTau in a mouse model of Alzheimer’s disease. Brain Research 1529 , 200–208 (2013). Di Meco, A., Joshi, Y. B. & Praticò, D. Sleep deprivation impairs memory, tau metabolism, and synaptic integrity of a mouse model of Alzheimer’s disease with plaques and tangles. Neurobiology of Aging 35 , 1813–1820 (2014). Lucey, B. P. et al. Effect of sleep on overnight cerebrospinal fluid amyloid β kinetics. Annals of Neurology 83 , 197–204 (2018). Guisle, I. et al. Circadian and sleep/wake-dependent variations in tau phosphorylation are driven by temperature. Sleep 43 , zsz266 (2020). Guisle, I. et al. Sauna-like conditions or menthol treatment reduce tau phosphorylation through mild hyperthermia. Neurobiology of Aging S019745802200032X (2022) doi:10.1016/j.neurobiolaging.2022.02.011. Hohmann & Dehghani. The Cytoskeleton—A Complex Interacting Meshwork. Cells 8 , 362 (2019). Lee, H.-J., Patel, S. & Lee, S.-J. Intravesicular Localization and Exocytosis of α-Synuclein and its Aggregates. J. Neurosci. 25 , 6016–6024 (2005). Rabouille, C. Pathways of Unconventional Protein Secretion. Trends in Cell Biology 27 , 230–240 (2017). Steringer, J. P. & Nickel, W. The molecular mechanism underlying unconventional secretion of Fibroblast Growth Factor 2 from tumour cells: Unconventional secretion of FGF2. Biol. Cell 109 , 375–380 (2017). Busse, M. et al. Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation. Journal of Biological Chemistry 282 , 32802–32810 (2007). Liu, C.-C. et al. Neuronal heparan sulfates promote amyloid pathology by modulating brain amyloid-β clearance and aggregation in Alzheimer’s disease. Sci. Transl. Med. 8 , 332ra44-332ra44 (2016). Hudák, A., Letoha, A., Vizler, C. & Letoha, T. Syndecan-3 as a Novel Biomarker in Alzheimer’s Disease. IJMS 23 , 3407 (2022). Blicher, A., Wodzinska, K., Fidorra, M., Winterhalter, M. & Heimburg, T. The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics. Biophysical Journal 96 , 4581–4591 (2009). Fan, W. & Evans, R. M. Turning Up the Heat on Membrane Fluidity. Cell 161 , 962–963 (2015). Mishkind, M., Vermeer, J. E. M., Darwish, E. & Munnik, T. Heat stress activates phospholipase D and triggers PIP2 accumulation at the plasma membrane and nucleus. The Plant Journal 60 , 10–21 (2009). Prieto, J. A. et al. Pho85 and PI(4,5)P2 regulate different lipid metabolic pathways in response to cold. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1865 , 158557 (2020). Benedict, C., Blennow, K., Zetterberg, H. & Cedernaes, J. Effects of acute sleep loss on diurnal plasma dynamics of CNS health biomarkers in young men. Neurology 94 , (2020). Pooler, A. M., Phillips, E. C., Lau, D. H. W., Noble, W. & Hanger, D. P. Physiological release of endogenous tau is stimulated by neuronal activity. EMBO Rep 14 , 389–394 (2013). Yamada, K. et al. Neuronal activity regulates extracellular tau in vivo. Journal of Experimental Medicine 211 , 387–393 (2014). Vyazovskiy, V. V., Cirelli, C., Pfister-Genskow, M., Faraguna, U. & Tononi, G. Molecular and electrophysiological evidence for net synaptic potentiation in wake and depression in sleep. Nat Neurosci 11 , 200–208 (2008). Clark, W. G. & Coldwell, B. A. The hypothermic effect of tetrodotoxin in the unanaesthetized cat. The Journal of Physiology 230 , 477–492 (1973). Tambyah, P. A., Hui, K. P., Gopalakrishnakone, P., Chin, N. K. & Chan, T. B. Central-nervous-system effects of tetrodotoxin poisoning. Lancet 343 , 538–539 (1994). Kim, J. A. & Connors, B. W. High temperatures alter physiological properties of pyramidal cells and inhibitory interneurons in hippocampus. Front. Cell. Neurosci. 6 , (2012). Van Hook, M. J. Temperature effects on synaptic transmission and neuronal function in the visual thalamus. PLoS ONE 15 , e0232451 (2020). Kim, T. et al. Thermal effects on neurons during stimulation of the brain. J. Neural Eng. 19 , 056029 (2022). Zhang, J. et al. Recent progresses in novel in vitro models of primary neurons: A biomaterial perspective. Front. Bioeng. Biotechnol. 10 , 953031 (2022). Nicholls, S. B. et al. Characterization of TauC3 antibody and demonstration of its potential to block tau propagation. PLoS ONE 12 , e0177914 (2017). Vana, L. et al. Progression of Tau Pathology in Cholinergic Basal Forebrain Neurons in Mild Cognitive Impairment and Alzheimer’s Disease. The American Journal of Pathology 179 , 2533–2550 (2011). Biundo, F. et al. Abolishing Tau cleavage by caspases at Aspartate421 causes memory/synaptic plasticity deficits and pre-pathological Tau alterations. Transl Psychiatry 7 , e1198–e1198 (2017). Chi, H. et al. Cleavage of human tau at Asp421 inhibits hyperphosphorylated tau induced pathology in a Drosophila model. Sci Rep 10 , 13482 (2020). Zhang, Q., Zhang, X. & Sun, A. Truncated tau at D421 is associated with neurodegeneration and tangle formation in the brain of Alzheimer transgenic models. Acta Neuropathol 117 , 687–697 (2009). Conze, C. et al. Caspase-cleaved tau is senescence-associated and induces a toxic gain of function by putting a brake on axonal transport. Mol Psychiatry 27 , 3010–3023 (2022). Troquier, L. et al. Targeting phospho-Ser422 by active Tau Immunotherapy in the THYTau22 mouse model: a suitable therapeutic approach. Curr Alzheimer Res 9 , 397–405 (2012). Choi, Y. R., Park, S. J. & Park, S. M. Molecular events underlying the cell‐to‐cell transmission of α‐synuclein. The FEBS Journal 288 , 6593–6602 (2021). Surridge, C. D. & Burns, R. G. The Difference in the Binding of Phosphatidylinositol Distinguishes MAP2 from MAP2C and Tau. Biochemistry 33 , 8051–8057 (1994). Lauri, S. E. et al. Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan ( N -syndecan) in Hippocampal Long-Term Potentiation. J. Neurosci. 19 , 1226–1235 (1999). Setlow, V. P., Roth, S. & Edidin, M. Effects of temperature on glycosyltransferase activity in the plasma membrane of L cells. Experimental Cell Research 121 , 55–61 (1979). McGinty, D., Alam, M. N., Szymusiak, R., Nakao, M. & Yamamoto, M. Hypothalamic sleep-promoting mechanisms: coupling to thermoregulation. Arch Ital Biol 139 , 63–75 (2001). Wang, J. et al. Thermoneutral Temperature Exposure Enhances Slow Wave Sleep with a Correlated Improvement in Amyloid Pathology in a Triple-Transgenic Mouse Model of Alzheimer’s Disease. SLEEP zsae078 (2024) doi:10.1093/sleep/zsae078. Warfield, A. E. et al. A brainstem to circadian system circuit links Tau pathology to sundowning-related disturbances in an Alzheimer’s disease mouse model. Nat Commun 14 , 5027 (2023). Rasmussen, M. K., Mestre, H. & Nedergaard, M. The glymphatic pathway in neurological disorders. The Lancet Neurology 17 , 1016–1024 (2018). Medina, M. & Avila, J. The role of extracellular Tau in the spreading of neurofibrillary pathology. Front. Cell. Neurosci. 8 , (2014). Klegeris, A., Schulzer, M., Harper, D. G. & McGeer, P. L. Increase in core body temperature of Alzheimer’s disease patients as a possible indicator of chronic neuroinflammation: a meta-analysis. Gerontology 53 , 7–11 (2007). Holtzman, A. & Simon, E. W. Body temperature as a risk factor for Alzheimer’s disease. Medical Hypotheses 55 , 440–444 (2000). Whittington, R., Papon, M.-A., Chouinard-Decorte, F. & Planel, E. Hypothermia and Alzheimers Disease Neuropathogenic Pathways. CAR 7 , 717–725 (2010). Blessing, E. M. et al. Association between lower body temperature and increased tau pathology in cognitively normal older adults. Neurobiology of Disease 171 , 105748 (2022). Lim, A. S. P., Kowgier, M., Yu, L., Buchman, A. S. & Bennett, D. A. Sleep Fragmentation and the Risk of Incident Alzheimer’s Disease and Cognitive Decline in Older Persons. Sleep 36 , 1027–1032 (2013). Todd, W. D. Potential Pathways for Circadian Dysfunction and Sundowning-Related Behavioral Aggression in Alzheimer’s Disease and Related Dementias. Front Neurosci 14 , 910 (2020). Harper, D. G. et al. Disturbance of endogenous circadian rhythm in aging and Alzheimer disease. Am J Geriatr Psychiatry 13 , 359–368 (2005). Holth, J. K., Patel, T. K. & Holtzman, D. M. Sleep in Alzheimer’s Disease–Beyond Amyloid. Neurobiology of Sleep and Circadian Rhythms 2 , 4–14 (2017). Sadleir, K. R. & Vassar, R. Connections between ApoE, sleep, and Aβ and tau pathologies in Alzheimer’s disease. Journal of Clinical Investigation 133 , e171838 (2023). Ishida, K. et al. Glymphatic system clears extracellular tau and protects from tau aggregation and neurodegeneration. Journal of Experimental Medicine 219 , e20211275 (2022). Xie, L. et al. Sleep Drives Metabolite Clearance from the Adult Brain. Science 342 , 373–377 (2013). Sohar, E., Shoenfeld, Y., Shapiro, Y., Ohry, A. & Cabili, S. Effects of exposure to Finnish sauna. Isr J Med Sci 12 , 1275–1282 (1976). Laukkanen, J. A. & Kunutsor, S. K. The multifaceted benefits of passive heat therapies for extending the healthspan: A comprehensive review with a focus on Finnish sauna. Temperature 11 , 27–51 (2024). Gómez-Ramos, A., Díaz-Hernández, M., Rubio, A., Miras-Portugal, M. T. & Avila, J. Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells. Molecular and Cellular Neuroscience 37 , 673–681 (2008). Pernègre, C., Duquette, A. & Leclerc, N. Tau Secretion: Good and Bad for Neurons. Front. Neurosci. 13 , 649 (2019). Delobel, P. et al. Stable-Tau Overexpression in Human Neuroblastoma Cells: An Open Door for Explaining Neuronal Death in Tauopathies. Annals of the New York Academy of Sciences 1010 , 623–634 (2003). Andorfer, C. et al. Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms. J. Neurochem. 86 , 582–590 (2003). Duff, K. et al. Characterization of Pathology in Transgenic Mice Over-Expressing Human Genomic and cDNA Tau Transgenes. Neurobiology of Disease 7 , 87–98 (2000). Tucker, K. L., Meyer, M. & Barde, Y.-A. Neurotrophins are required for nerve growth during development. Nat Neurosci 4 , 29–37 (2001). Planel, E. et al. Anesthesia Leads to Tau Hyperphosphorylation through Inhibition of Phosphatase Activity by Hypothermia. Journal of Neuroscience 27 , 3090–3097 (2007). Canet, G. et al. Temperature-induced Artifacts in Tau Phosphorylation: Implications for Reliable Alzheimer’s Disease Research. Exp Neurobiol 32 , 423–440 (2023). Carroll, D. A., Denenberg, V. H. & Thoman, E. B. Reliability and validity of computer scoring of behavioral sleep-wake states in rats and rabbits. Physiology & Behavior 54 , 269–273 (1993). Tournissac, M. et al. Repeated cold exposures protect a mouse model of Alzheimer’s disease against cold-induced tau phosphorylation. Molecular Metabolism 22 , 110–120 (2019). Fereydouni-Forouzandeh, P. et al. Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts. in Tau Protein (ed. Smet-Nocca, C.) vol. 2754 309–321 (Springer US, New York, NY, 2024). Lucey, B. P. et al. Suvorexant Acutely Decreases Tau Phosphorylation and Aβ in the Human CNS. Annals of Neurology 94 , 27–40 (2023). Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataFig1.pdf Extended Data Fig. 1: Wakefulness temperatures increase tau secretion while decreasing intracellular tau phosphorylation level. (a, b)72 hours of exposure to temperatures between 35°C and 38°C did not induce cytotoxic LDH release in SH-Tau3R cells or primary mouse cortical ( n = 6-12; Tukey’s; box and whiskers with minimum to maximum and median). (c)The increase in extracellular tau levels is temperature-dependent in SH-Tau3R cells exposed to 35, 37 or 38°C for 72 hours ( n = 6; Tukey’s; box and whiskers with minimum to maximum and median). (d) The temperature-dependent increase in extracellular tau levels is comparable either after 6, 24 or 48 hours of exposure to 35-38°C ( n = 3-5 for 6-hours; Dunn’s; n = 5-6 for 24- and 48-hours; Tukey’s; mean ± s.e.m.). (e, f)The intracellular phosphorylation level of tau at AT270, S199, CP13, T205, AT100, MC6 and PHF1 is decreased at 38°C compared to 35 or 37°C ( n = 5; Tukey’s; mean ± s.e.m.). (g, h) The extracellular levels of MAP2, α-synuclein, FGF2, Caspase-1 and NfL are not affected by temperature ( n = 5-6; Dunnet’s; mean ± s.e.m). (i) The intracellular expression of FGF2 is not affected by temperature ( n = 6; Dunnet’s; box and whiskers with minimum to maximum and median). *p<0.05, **p<0.01 and ***p<0.001. ns: non-significant. ExtendedDataFig2.pdf Extended Data Fig. 2: Representative confocal images of primary mouse cortical neurons stained for red fluorescent protein (RFP), DAPI (blue), SDC3 (purple) and TauC3 (yellow). Cells were exposed at 35, 37 or 38°C for 72 hours. A merged staining is displayed, showing a temperature-dependant increase of colocalization between SDC3 and TauC3, and marked with white arrows. Scale bar represents 50 μm for upper panels, and 20 µm for lower panels (magnification of dotted boxes). Data representative of n = 3 per condition, examined over 2 independent experiments. ExtendedDataFig3.pdf Extended Data Fig. 3: UPS-I protein component expressions correlate with body temperature during the sleep-wake cycle in wild-type mice. (a-f) The cortical expression of caspase-3, pTau(S422), Tau46, SDC3 and PIP 2 are significantly correlated with rectal temperature of mice (Pearson correlation; standard error bars displayed as error envelopes in light grey). *p<0.05 and **p<0.01 ExtendedDataFig4.pdf Extended Data Fig. 4: UPS-I protein component expressions correlate with body temperature in hTau mice. (a, b) The expressions of caspase-3, TauC3, SDC3 and PIP2 are increased in the cortices of hyperthermic mice compared to hypo- or normothermic mice, while tau phosphorylation is decreased at S422 ( n = 5-6; Tukey’s; mean ± s.e.m). (c-h) The cortical expression of caspase-3, pTau(S422), TauC3, Tau46, SDC3 and PIP 2 are significantly correlated with rectal temperature of mice (Pearson correlation; standard error bars displayed as error envelopes in light grey). *p<0.05, **p<0.01 and ***p<0.001. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4384494","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":301825068,"identity":"d3b7f097-2ea4-4211-955b-453aa07d28ef","order_by":0,"name":"Geoffrey Canet*","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-7291-4512","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":true,"prefix":"","firstName":"Geoffrey","middleName":"","lastName":"Canet*","suffix":""},{"id":301825069,"identity":"39bb0139-4a68-449e-b097-a73dafcb7094","order_by":1,"name":"Felipe Da Gama Monteiro","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de médecine moléculaire, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"Da Gama","lastName":"Monteiro","suffix":""},{"id":301825072,"identity":"404cdd0e-5e10-4edd-a94b-47e7e000ddf0","order_by":2,"name":"Emma Rocaboy","email":"","orcid":"","institution":"Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Emma","middleName":"","lastName":"Rocaboy","suffix":""},{"id":301825073,"identity":"8f31a9ea-2387-4fde-aa6f-f0be214ceefd","order_by":3,"name":"Sofia Diego-Diaz","email":"","orcid":"","institution":"Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Sofia","middleName":"","lastName":"Diego-Diaz","suffix":""},{"id":301825074,"identity":"f1212081-c51f-4bd2-8b86-864332762e35","order_by":4,"name":"Boutheyna Khelaifia","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Boutheyna","middleName":"","lastName":"Khelaifia","suffix":""},{"id":301825075,"identity":"e873879a-e8e2-48fa-b125-c1958115ab73","order_by":5,"name":"Jessica Kim","email":"","orcid":"","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Kim","suffix":""},{"id":301825076,"identity":"8945b5f5-fe18-4076-b1dc-a20e4107d602","order_by":6,"name":"Daphne I. Valencia","email":"","orcid":"","institution":"Mount Sinai Integrative Sleep Center, Division of Pulmonary, Critical Care, and Sleep Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA","correspondingAuthor":false,"prefix":"","firstName":"Daphne","middleName":"I.","lastName":"Valencia","suffix":""},{"id":301825077,"identity":"cda1e03c-0f45-40b5-9666-e8bfdcebf114","order_by":7,"name":"Audrey Yin","email":"","orcid":"https://orcid.org/0000-0002-1313-9314","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":false,"prefix":"","firstName":"Audrey","middleName":"","lastName":"Yin","suffix":""},{"id":301825078,"identity":"fad40714-f647-48e6-acac-0fcc5fadaf53","order_by":8,"name":"Hau-Tieng Wu","email":"","orcid":"","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":false,"prefix":"","firstName":"Hau-Tieng","middleName":"","lastName":"Wu","suffix":""},{"id":301825079,"identity":"eac4c0b9-1434-455f-bf58-e33ccdda6fb1","order_by":9,"name":"Jordan Howell","email":"","orcid":"","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":false,"prefix":"","firstName":"Jordan","middleName":"","lastName":"Howell","suffix":""},{"id":301825080,"identity":"68d0b8c5-4c50-411a-9060-827e48408209","order_by":10,"name":"Emily Blank","email":"","orcid":"","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"","lastName":"Blank","suffix":""},{"id":301825081,"identity":"5f499310-0e4d-4740-b888-46628c30621c","order_by":11,"name":"Francis Laliberté","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Francis","middleName":"","lastName":"Laliberté","suffix":""},{"id":301825082,"identity":"5960f774-27b8-4cdc-b620-6e25045ce6b1","order_by":12,"name":"Nadia Fortin","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Fortin","suffix":""},{"id":301825083,"identity":"b4ead2b0-8752-47c5-8acb-d0abab84bafd","order_by":13,"name":"Emmanuelle Boscher","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Emmanuelle","middleName":"","lastName":"Boscher","suffix":""},{"id":301825084,"identity":"fc08891c-d452-49d7-9c54-7151686c4253","order_by":14,"name":"Parissa Fereydouni-Forouzandeh","email":"","orcid":"","institution":"Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Parissa","middleName":"","lastName":"Fereydouni-Forouzandeh","suffix":""},{"id":301825085,"identity":"ace6b78b-38ef-43cc-b562-d9b25dd96a03","order_by":15,"name":"Stéphanie Champagne","email":"","orcid":"","institution":"Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Stéphanie","middleName":"","lastName":"Champagne","suffix":""},{"id":301825086,"identity":"3f2bbeb7-cb8f-4c7a-871f-34a4031a43dd","order_by":16,"name":"Isabelle Guisle","email":"","orcid":"https://orcid.org/0000-0001-8726-4946","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Guisle","suffix":""},{"id":301825087,"identity":"0504b0b8-6acf-4775-bcd6-195a9f12cfbd","order_by":17,"name":"Sébastien S. Hébert","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Sébastien","middleName":"S.","lastName":"Hébert","suffix":""},{"id":301825088,"identity":"04ebc595-ad77-4844-ba00-430ba308c438","order_by":18,"name":"Vincent Pernet","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada; Department of Neurology, Bern University Hospital, Bern 3010, Switzerland","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Pernet","suffix":""},{"id":301825089,"identity":"1b06bb65-ffea-4fb5-9f33-ba0d3cad61fd","order_by":19,"name":"Haiyan Liu","email":"","orcid":"","institution":"Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Liu","suffix":""},{"id":301825090,"identity":"ba926b21-8c1a-486a-b7c7-5036be419d90","order_by":20,"name":"William Lu","email":"","orcid":"","institution":"Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Lu","suffix":""},{"id":301825091,"identity":"1d66e265-4a3f-4f65-9374-eb7db029c499","order_by":21,"name":"Ludovic Debure","email":"","orcid":"","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":false,"prefix":"","firstName":"Ludovic","middleName":"","lastName":"Debure","suffix":""},{"id":301825092,"identity":"e8174a64-6dd8-4408-bdb1-cac25dc5ba98","order_by":22,"name":"David M. Rapoport","email":"","orcid":"","institution":"Mount Sinai Integrative Sleep Center, Division of Pulmonary, Critical Care, and Sleep Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"M.","lastName":"Rapoport","suffix":""},{"id":301825093,"identity":"fd0fae4f-9033-47af-911c-6aaf8b2c9c76","order_by":23,"name":"Indu Ayappa","email":"","orcid":"","institution":"Mount Sinai Integrative Sleep Center, Division of Pulmonary, Critical Care, and Sleep Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA","correspondingAuthor":false,"prefix":"","firstName":"Indu","middleName":"","lastName":"Ayappa","suffix":""},{"id":301825094,"identity":"bdd604b5-ab04-46f9-8916-25b59cfc332e","order_by":24,"name":"Andrew W. Varga","email":"","orcid":"","institution":"Mount Sinai Integrative Sleep Center, Division of Pulmonary, Critical Care, and Sleep Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"W.","lastName":"Varga","suffix":""},{"id":301825095,"identity":"5c9f9cc1-e672-4b2b-928c-588ba55dfd78","order_by":25,"name":"Ankit Parekh","email":"","orcid":"","institution":"Mount Sinai Integrative Sleep Center, Division of Pulmonary, Critical Care, and Sleep Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA","correspondingAuthor":false,"prefix":"","firstName":"Ankit","middleName":"","lastName":"Parekh","suffix":""},{"id":301825096,"identity":"703c432b-2b26-4e79-a1a0-f9e21d1fe83e","order_by":26,"name":"Ricardo S. Osorio","email":"","orcid":"","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":false,"prefix":"","firstName":"Ricardo","middleName":"S.","lastName":"Osorio","suffix":""},{"id":301825097,"identity":"035bf187-c507-4ca1-aa38-046b01040da2","order_by":27,"name":"Steve Lacroix","email":"","orcid":"","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de médecine moléculaire, G1V 0A6, Québec, QC, Canada","correspondingAuthor":false,"prefix":"","firstName":"Steve","middleName":"","lastName":"Lacroix","suffix":""},{"id":301825098,"identity":"a9d8cfe2-b1db-45ed-955c-9e15fad9b81e","order_by":28,"name":"Brendan P. Lucey","email":"","orcid":"","institution":"Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA","correspondingAuthor":false,"prefix":"","firstName":"Brendan","middleName":"P.","lastName":"Lucey","suffix":""},{"id":301825099,"identity":"9f66fc58-9d77-4240-87c5-8bdfbd5df60f","order_by":29,"name":"Esther M. Blessing*","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABQklEQVRIie2QsUrDUBSGTwjcLK11vCGYvsINhYog+Cq5ZOiSloKLQ1tShOtSde1jxMkKDjcEmiXSNZsNgTgJ7dZKBW9idYlV3ATzDf85y8fPOQAlJX8TlIW5lyeALobEvzfkdwVtlUY2f6EIqPOTUqvNpvPlPXSQcpEs4km/dYttwteTvn6oOFJy9lJQ1LGlGOMUTlElbGIaBu27sU28yzBoHI24bDxcFRQSyUircKAM20jktO1GoqXKptSNTKQORwXlZOYjbZMp9adkIZQWEYr3mimP8y8VAhbSIG8BginrmZniV1lPtABSnVVBwZHVVEcci1tscQvjhhumXf+A8QYJ6bkxdIofu/ZSvOLHnZoSJMs1G9RJYN3Ez2ygk8D3Ymez69f4Y/EB9s3tApIDEtulfDIQxXy75OxsKSkpKfk/vAHmDHftx6w04wAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Psychiatry, NYU Grossman School of Medicine, New York, NY 10016, USA","correspondingAuthor":true,"prefix":"","firstName":"Esther","middleName":"M.","lastName":"Blessing*","suffix":""},{"id":301825100,"identity":"2af147c3-f422-4aff-8598-e789ddd421f5","order_by":30,"name":"Emmanuel Planel*","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBADGX4G5gYgbUG8Fh7JBkaQFgkStBgcIFYLv9jpxM+FbTY8xrcbGz8XVEgw8Esfv4BXi+Ts3M3SM9vSeMzuHGyWnnFGgkGyL6cArxaD27kbpHnbDvOY3UhsADIkGAzO8CTg1WJ/O3fzb962/zzGMxKbf/P+I0KLgXTuNqDhB3gMJBLbpHkbQFrYD+DVInE7d5s1z7lkHok7B9useY5J8Ej28ODVwcAP9P5tnjI7Of7ZzYdv89TYyPHzsD/ArwdhH4TiAcURaVqAgGhbRsEoGAWjYIQAAJo0P53IGLB+AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5128-0565","institution":"Centre de Recherche du CHU de Québec – Université Laval, Axe Neurosciences, G1V 4G2, Québec, QC, Canada; Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, G1V 0A6, Québec, QC, Canada","correspondingAuthor":true,"prefix":"","firstName":"Emmanuel","middleName":"","lastName":"Planel*","suffix":""}],"badges":[],"createdAt":"2024-05-07 17:06:06","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4384494/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4384494/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56450891,"identity":"e5d21f88-df99-48db-8408-c53fbb534c8f","added_by":"auto","created_at":"2024-05-14 10:36:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":225234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTau secretion is temperature-dependent in neuronal cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e 24 hours after seeding, SH-Tau3R cells were exposed to 35, 37, 38 or 39°C for 72 hours. (\u003cstrong\u003eb)\u003c/strong\u003e Extracellular accumulation of tau protein (Tau3R; \u003cem\u003en\u003c/em\u003e = 6-12; mean ± s.e.m (error envelopes in light purple)) and LDH (\u003cem\u003en\u003c/em\u003e= 4-9; mean ± s.e.m) over-time in cell medium of neurons cultured at 37°C. (\u003cstrong\u003ec)\u003c/strong\u003eThe increase in extracellular tau levels is temperature-dependent in SH-Tau3R cells exposed to 35, 37 or 39°C (\u003cem\u003en\u003c/em\u003e = 6; Dunnett’s; box and whiskers with minimum to maximum and median). (\u003cstrong\u003ed) \u003c/strong\u003eThe phosphorylation level of extracellular tau at S199, T231 and S396 is decreased at 39°C compared to 35 or 37°C (\u003cem\u003en\u003c/em\u003e = 6; Tukey’s; mean ± s.e.m). (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e) The increase of extracellular tau levels is temperature-dependent (Tau3R, TauC, DA9 and Tau12 antibodies) in SH-Tau3R cells exposed to 35, 37 or 38°C (\u003cem\u003en\u003c/em\u003e = 7-16; Tukey’s; mean ± s.e.m). (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e) The phosphorylation level of extracellular tau at AT270, S199, CP13, T205, AT100, MC6 and PHF1 is decreased at 38°C compared to 35 or 37°C (\u003cem\u003en\u003c/em\u003e = 7-16; Tukey’s; mean ± s.e.m). (\u003cstrong\u003eh)\u003c/strong\u003e4 days after seeding, mouse primary cortical neurons were exposed at 35, 37 or 38°C for 72 hours. (\u003cstrong\u003ei)\u003c/strong\u003e The increase of extracellular tau levels is temperature-dependent in mouse primary neurons exposed to 35, 37 or 38°C (\u003cem\u003en\u003c/em\u003e= 6; Dunnett’s; box and whiskers with minimum to maximum and median). (\u003cstrong\u003ej, k\u003c/strong\u003e) The increase of extracellular tau levels is temperature-dependent (Tau3R antibody), while its phosphorylation level at S199 and T205 is decreased at 38°C compared to 35 or 37°C (\u003cem\u003en\u003c/em\u003e = 11-12; Tukey’s; mean ± s.e.m). *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/b4b54828262ef896f57cb15d.png"},{"id":56451419,"identity":"990c3d01-24ae-418a-9ad5-1e1543c84692","added_by":"auto","created_at":"2024-05-14 10:44:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":237864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWakefulness temperatures promote tau release through its caspase-3-mediated cleavage in neuronal cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e The increase in the proteolytic activity of caspase-3 is temperature-dependent in SH-Tau3R cells (\u003cem\u003en\u003c/em\u003e = 6; Tukey’s; box and whiskers with minimum to maximum and median).\u003cstrong\u003e (b, c) \u003c/strong\u003eThe intracellular expressions of caspase-3, pTau(S422), TauC3 and Tau46 are temperature-dependent in SH-Tau3R (left panels) or in primary neurons (right panels) (\u003cem\u003en\u003c/em\u003e = 5-6; Tukey’s; mean ± s.e.m). \u003cstrong\u003e(d, e)\u003c/strong\u003e The extracellular levels of TauC3 and Tau46 are oppositely temperature-dependent\u003cstrong\u003e \u003c/strong\u003ein SH-Tau3R (left panels) or in primary neurons (right panels) (\u003cem\u003en\u003c/em\u003e = 6-12; Tukey’s; mean ± s.e.m). \u003cstrong\u003e(f)\u003c/strong\u003eThe inhibition of caspase-3 with z-DEVD-FMK (20 µM) decreases total tau (Tau3R, \u003cem\u003en\u003c/em\u003e = 5-12, unpaired t-test) and TauC3 (\u003cem\u003en\u003c/em\u003e = 3-8, Mann Whithney) extracellular levels in SH-Tau3R cells exposed to 35, 37 or 38°C (mean ± s.e.m). The inhibition of caspase-3 with z-DEVD-FMK (20 µM) decreases the extracellular levels of \u003cstrong\u003e(g)\u003c/strong\u003e total tau (ELISA assay, \u003cem\u003en\u003c/em\u003e = 6, unpaired t-test), \u003cstrong\u003e(h)\u003c/strong\u003e Tau3R and TauC3 (Dot blotting, \u003cem\u003en\u003c/em\u003e = 5-6; unpaired t-test) (mean ± s.e.m). \u003cstrong\u003e(i)\u003c/strong\u003e The genetic knockdown of caspase-3 decreases total tau (Tau3R) and TauC3 extracellular levels in SH-Tau3R cells exposed to 37°C (\u003cem\u003en\u003c/em\u003e = 11; unpaired t-test; mean ± s.e.m). *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001. ns: non-significant.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/8446a35a2322859ec3f4913b.png"},{"id":56450894,"identity":"50ff418e-b047-40cc-844f-8f951d32ad30","added_by":"auto","created_at":"2024-05-14 10:36:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":837352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWakefulness temperatures promote TauC3 interaction with PIP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and SDC3 in neuronal cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a, b)\u003c/strong\u003e The intracellular expressions of SDC3 and PIP\u003csub\u003e2\u003c/sub\u003e are temperature-dependent in SH-Tau3R (left panels) or in primary neurons (right panels) (\u003cem\u003en\u003c/em\u003e = 6; Tukey’s; mean ± s.e.m). \u003cstrong\u003e(c)\u003c/strong\u003e The mRNA expression of \u003cem\u003eSDC3\u003c/em\u003e and \u003cem\u003eEXT1\u003c/em\u003e genes are temperature-dependent in SH-Tau3R, and \u003cem\u003eCASP3\u003c/em\u003e mRNA is unchanged (\u003cem\u003en\u003c/em\u003e = 5; Tukey’s; mean ± s.e.m). (\u003cstrong\u003ed, e\u003c/strong\u003e) The genetic knockdown of SDC3 decreases total tau (Tau3R) and TauC3 extracellular levels in SH-Tau3R cells exposed to 37°C, and the co-transfection of caspase-3 siRNA and SDC3 siRNA induces additive effects in the suppression of tau secretion (\u003cem\u003en\u003c/em\u003e = 5-6; Tukey’s; mean ± s.e.m). (\u003cstrong\u003ef) \u003c/strong\u003eThe genetic knockdown of EXT1 decreases total tau (Tau3R) and TauC3 extracellular levels in SH-Tau3R cells exposed to 37°C (\u003cem\u003en\u003c/em\u003e = 11; unpaired t-test; mean ± s.e.m). (\u003cstrong\u003eg) \u003c/strong\u003eThe genetic knockdown of caspase-3 + SDC3 decreases the intracellular expression of TauC while increasing tau phosphorylation at S422 (representative western blot detection; \u003cem\u003en\u003c/em\u003e = 3). (\u003cstrong\u003eh)\u003c/strong\u003e The increase in membrane fluidity is temperature-dependent in SH-Tau3R cells (\u003cem\u003en\u003c/em\u003e = 8; Tukey’s; box and whiskers with minimum to maximum and median). (\u003cstrong\u003ei, j\u003c/strong\u003e) PIP\u003csub\u003e2\u003c/sub\u003e displays a better binding affinity for TauC3 rather than full-length tau (DA9 and Tau46 and TauC antibodies) in SH-Tau3R cells exposed to 37°C. NC: negative control, IP: immunoprecipitation. (\u003cstrong\u003ek-m\u003c/strong\u003e) The increase in binding between PIP2 and TauC3 is temperature-dependent (\u003cem\u003en\u003c/em\u003e = 3; Kruskal-Wallis; mean ± s.e.m). *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001 vs. respective control condition; +p\u0026lt;0.05 and ++p\u0026lt;0.01 vs. indicated condition. (\u003cstrong\u003en\u003c/strong\u003e) A merged staining is displayed, showing a temperature-dependent increase of colocalization between SDC3 (purple) and TauC3 (yellow) in primary mouse cortical neurons, and marked with white arrows. Scale bar: 20 µm (magnification of dotted boxes).\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/78a425c388fa18329cf6e30c.png"},{"id":56450896,"identity":"ca875903-08cc-436b-89fa-15853bf06548","added_by":"auto","created_at":"2024-05-14 10:36:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":306718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWakefulness, sleep deprivation and mild-hyperthermia promote the UPS-I-dependent tau release in CSF in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e C57BL6 mice were euthanatized during their sleeping (Circadian time 4, CT4, 10:00 am) or active period (CT16, 8:45 pm, awake group). (\u003cstrong\u003eb)\u003c/strong\u003e Awake mice display a higher rectal temperature (°C) at sacrifice compared to sleeping mice (\u003cem\u003en\u003c/em\u003e = 7; unpaired t-test; box and whiskers with minimum to maximum and median). (\u003cstrong\u003ec, d) \u003c/strong\u003eThe expressions of caspase-3, TauC3, SDC3 and PIP\u003csub\u003e2\u003c/sub\u003e are increased in the cortices of awake mice compared to sleeping mice, while pTau(S422) is decreased (\u003cem\u003en\u003c/em\u003e = 10; unpaired t-test; mean ± s.e.m). \u003cstrong\u003e(e)\u003c/strong\u003e C57BL6 mice were sleep-deprived (\u003cem\u003en\u003c/em\u003e=9) for the first 6 hours of the light period and compared to naïve mice (n=7) allowed to sleep without disturbance. (\u003cstrong\u003ef)\u003c/strong\u003e Sleep-deprivation inhibits the drop in core body temperature (°C) induced by sleep (\u003cem\u003en\u003c/em\u003e = 5; Tukey’s; mean ± s.e.m as error envelopes). (\u003cstrong\u003eg, h) \u003c/strong\u003eThe expressions of caspase-3, TauC3, and PIP\u003csub\u003e2\u003c/sub\u003e are increased in the cortices of sleep-deprived mice compared to naive mice, while pTau(S422) and Tau46 are decreased (unpaired t-test; mean ± s.e.m). (\u003cstrong\u003ei, j)\u003c/strong\u003e hTau mice were exposed for 4 hours either to 4°C (hypo, \u003cem\u003en\u003c/em\u003e = 6) or 38°C (hyper,\u003cem\u003e n\u003c/em\u003e = 6), and compared to naïve mice (normo,\u003cem\u003e n\u003c/em\u003e = 5) (Šidák's; mean ± s.e.m as error envelopes). ***p\u0026lt;0.001 vs. Normo group at baseline; +++p\u0026lt;0.001 vs. respective group at baseline. (\u003cstrong\u003ek\u003c/strong\u003e) Hyperthermic mice have higher CSF tau levels compared to hypothermic mice (\u003cem\u003en\u003c/em\u003e = 3 mice (Normo); \u003cem\u003en\u003c/em\u003e = 5 mice (Hypo); \u003cem\u003en\u003c/em\u003e = 6 mice (Hyper); Kruskal-Wallis; mean ± s.e.m). (\u003cstrong\u003el\u003c/strong\u003e) CSF tau is significantly correlated with rectal body temperature (°C) (Pearson’s correlation; error envelopes in light grey). *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/093da383ac138aad65b5fdf0.png"},{"id":56450892,"identity":"d414462b-385d-46a5-a814-6e38ba7c5ee9","added_by":"auto","created_at":"2024-05-14 10:36:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBody temperature correlates with CSF and plasma tau levels in humans.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eThe variation in total CSF tau concentrations between 8AM and 4PM is significantly correlated to the variation in oral temperature at the same times (\u003cem\u003en\u003c/em\u003e = 13, Pearson’s correlation), \u003cstrong\u003e(b) \u003c/strong\u003ewhile no correlation is observed for CSF NfL concentrations and oral temperature\u003cstrong\u003e \u003c/strong\u003e(\u003cem\u003en\u003c/em\u003e = 11, Pearson’s correlation). \u003cstrong\u003e(c) \u003c/strong\u003eThe variation in total plasma tau concentrations between 7AM and 7PM is significantly correlated to the variation in core body temperature between 6PM and 1AM (\u003cem\u003en\u003c/em\u003e = 15, Pearson’s correlation). Standard error bars displayed as error envelopes in light purple, *p\u0026lt;0.05 and **p\u0026lt;0.01. standard error bars displayed as error envelopes in light purple.\u003c/p\u003e","description":"","filename":"Binder15.png","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/6c7c3c1ae251f5ebe62fb80e.png"},{"id":56450900,"identity":"c3a75ac6-b06e-4ecf-bccf-3c874fd03820","added_by":"auto","created_at":"2024-05-14 10:36:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":243483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism to elucidate the regulatory effect of BT on tau secretion \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e the UPS-I pathway during the sleep-wake cycle.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring wakefulness, the physiological elevation in BT instigates a series of events triggering tau secretion. \u003cstrong\u003e(1) \u003c/strong\u003eThere is an increase in caspase-3 activity, concomitant with tau dephosphorylation, especially at S422, leading to an augmented cleavage of tau at D421, yielding the TauC3 fragment. \u003cstrong\u003e(2)\u003c/strong\u003e Subsequently, TauC3 is sequestered at the inner leaflet of the plasma membrane due to its strong affinity binding for PIP\u003csub\u003e2\u003c/sub\u003e. \u003cstrong\u003e(3) (4)\u003c/strong\u003e The interplay between TauC3 and SDC3 initiates and facilitates the export process across the plasma membrane, that exhibits heightened fluidity and permeability properties during wakefulness. In contrast, during sleep, the decrease in BT inhibits caspase-3 activity and promotes tau hyperphosphorylation at S422, preventing the generation of TauC3. The sleep phase also leads to reduced expression levels of both PIP\u003csub\u003e2\u003c/sub\u003e and SDC3, as well as to a lower membrane fluidity, resulting in diminished extracellular tau levels.\u003c/p\u003e","description":"","filename":"Binder16.png","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/4c48431d19c0ae730f49fabb.png"},{"id":59605614,"identity":"3e276c4d-1dfa-48e8-800e-2d819f0b8457","added_by":"auto","created_at":"2024-07-03 18:47:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3169789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/f518e265-f1a4-4b36-8d74-83296134c024.pdf"},{"id":56450901,"identity":"e682ab58-d78e-4c2c-9d1f-51f7ee9f4b6e","added_by":"auto","created_at":"2024-05-14 10:36:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1806722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 1: Wakefulness temperatures increase tau secretion while decreasing intracellular tau phosphorylation level.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a\u003c/strong\u003e, \u003cstrong\u003eb)\u003c/strong\u003e72 hours of exposure to temperatures between 35°C and 38°C did not induce cytotoxic LDH release in SH-Tau3R cells or primary mouse cortical (\u003cem\u003en\u003c/em\u003e = 6-12; Tukey’s; box and whiskers with minimum to maximum and median).\u003cstrong\u003e (c)\u003c/strong\u003eThe increase in extracellular tau levels is temperature-dependent in SH-Tau3R cells exposed to 35, 37 or 38°C for 72 hours (\u003cem\u003en\u003c/em\u003e = 6; Tukey’s; box and whiskers with minimum to maximum and median). \u003cstrong\u003e(d)\u003c/strong\u003e The temperature-dependent increase in extracellular tau levels is comparable either after 6, 24 or 48 hours of exposure to 35-38°C (\u003cem\u003en\u003c/em\u003e = 3-5 for 6-hours; Dunn’s; \u003cem\u003en\u003c/em\u003e = 5-6 for 24- and 48-hours; Tukey’s; mean ± s.e.m.). (\u003cstrong\u003ee, f)\u003c/strong\u003eThe intracellular phosphorylation level of tau at AT270, S199, CP13, T205, AT100, MC6 and PHF1 is decreased at 38°C compared to 35 or 37°C (\u003cem\u003en\u003c/em\u003e = 5; Tukey’s; mean ± s.e.m.). (\u003cstrong\u003eg, h) \u003c/strong\u003eThe extracellular levels of MAP2, α-synuclein, FGF2, Caspase-1 and NfL are not affected by temperature (\u003cem\u003en\u003c/em\u003e= 5-6; Dunnet’s; mean ± s.e.m). (\u003cstrong\u003ei\u003c/strong\u003e) The intracellular expression of FGF2 is not affected by temperature (\u003cem\u003en\u003c/em\u003e = 6; Dunnet’s; box and whiskers with minimum to maximum and median). *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001. ns: non-significant.\u003c/p\u003e","description":"","filename":"ExtendedDataFig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/1ddf3a830bcdf206d15b4590.pdf"},{"id":56451420,"identity":"cd3931ba-cd7c-4b22-a321-9f90e943ea8c","added_by":"auto","created_at":"2024-05-14 10:44:25","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":499014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 2: \u003c/strong\u003eRepresentative confocal images of primary mouse cortical neurons stained for red fluorescent protein (RFP), DAPI (blue), SDC3 (purple) and TauC3 (yellow). Cells were exposed at 35, 37 or 38°C for 72 hours. A merged staining is displayed, showing a temperature-dependant increase of colocalization between SDC3 and TauC3, and marked with white arrows. Scale bar represents 50 μm for upper panels, and 20 µm for lower panels (magnification of dotted boxes). Data representative of \u003cem\u003en\u003c/em\u003e = 3 per condition, examined over 2 independent experiments.\u003c/p\u003e","description":"","filename":"ExtendedDataFig2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/eaa4de4ce417660d77882fb1.pdf"},{"id":56451421,"identity":"ccc3804d-a210-4f1e-bdcb-40fb38bec9ef","added_by":"auto","created_at":"2024-05-14 10:44:25","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":210206,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;\u003cstrong\u003eExtended Data Fig. 3: UPS-I protein component expressions correlate with body temperature during the sleep-wake cycle in wild-type mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a-f)\u003c/strong\u003e The cortical expression of caspase-3, pTau(S422), Tau46, SDC3 and PIP\u003csub\u003e2 \u003c/sub\u003eare significantly correlated with rectal temperature of mice (Pearson correlation; standard error bars displayed as error envelopes in light grey).\u003cstrong\u003e \u003c/strong\u003e*p\u0026lt;0.05 and **p\u0026lt;0.01\u003c/p\u003e","description":"","filename":"ExtendedDataFig3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/c05a121b7d3acb24359ab8ec.pdf"},{"id":56450898,"identity":"b3ce7b59-ab53-454e-ad56-04fcdd90c016","added_by":"auto","created_at":"2024-05-14 10:36:25","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1609778,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 4: UPS-I protein component expressions correlate with body temperature in hTau mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a, b)\u003c/strong\u003e The expressions of caspase-3, TauC3, SDC3 and PIP2 are increased in the cortices of hyperthermic mice compared to hypo- or normothermic mice, while tau phosphorylation is decreased at S422 (\u003cem\u003en\u003c/em\u003e = 5-6; Tukey’s; mean ± s.e.m). \u003cstrong\u003e(c-h)\u003c/strong\u003e The cortical expression of caspase-3, pTau(S422), TauC3, Tau46, SDC3 and PIP\u003csub\u003e2 \u003c/sub\u003eare significantly correlated with rectal temperature of mice (Pearson correlation; standard error bars displayed as error envelopes in light grey). *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"ExtendedDataFig4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4384494/v1/bf4b9f8defa85df8178aa013.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Sleep-wake body temperature regulates tau secretion in mice and correlates with CSF and plasma tau in humans","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe intraneuronal accumulation of hyperphosphorylated aggregated tau protein is the pathological hallmark of neurodegenerative tauopathies including Alzheimer's disease (AD) \u003csup\u003e1\u003c/sup\u003e. Both the neuron-to-neuron propagation of tau pathology, and higher levels of cerebrospinal fluid (CSF) and plasma tau correlates with cognitive decline \u003csup\u003e2\u003c/sup\u003e. Tau secretion to the extracellular space consequently influences the propagation of tau aggregates in the brain, marking one of the initial steps of pathological tau transmission from diseased to recipient neurons. Further elucidating the key components in pathways underlying tau secretion and key physiological factors regulating their activity may yield insights into therapeutic avenues for slowing the spread of pathological tau.\u003c/p\u003e \u003cp\u003eIt is now established that tau, as a leaderless protein, is mainly secreted through the unconventional protein secretion pathway-I (UPS-I), consistent with ~\u0026thinsp;90% of extracellular tau being free and unbound to vesicular organelles \u003csup\u003e3\u0026ndash;5\u003c/sup\u003e. Key components in this pathway include phosphatidylinositol 4,5-bisphosphate (PIP\u003csub\u003e2\u003c/sub\u003e), which binds to tau at the inner leaflet of the plasma membrane, and heparan sulfate proteoglycans (HSPGs), which facilitate export across cell membrane \u003csup\u003e4,6\u003c/sup\u003e. Furthermore, the main form of extracellular tau is found as its C-terminal-truncated fragment (D421) referred to as TauC3 \u003csup\u003e7\u0026ndash;10\u003c/sup\u003e. TauC3 cleavage is mediated by caspase-3 and seems to occur intracellularly prior to release \u003csup\u003e9,11\u003c/sup\u003e, and this cleavage at D421 is inhibited by tau phosphorylation at S422 \u003csup\u003e12,13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding physiological factors regulating tau secretion, previous studies in both mouse and human suggest extracellular tau levels are strongly influenced by the sleep-wake cycle \u003csup\u003e14\u003c/sup\u003e, consistent with the established bidirectional link between sleep disturbance and AD neuropathology \u003csup\u003e15,16\u003c/sup\u003e. During wakefulness, CSF and interstitial fluid (ISF) tau levels substantially increase compared to sleep. Sleep deprivation (SD) is associated with elevated tau levels in ISF and CSF \u003csup\u003e14\u003c/sup\u003e, while decreasing CSF tau phosphorylation level \u003csup\u003e10\u003c/sup\u003e. Moreover, chronic SD reduces tau phosphorylation while promoting its aggregation in AD mice \u003csup\u003e18\u003c/sup\u003e. Previous studies suggested that both increased tau secretion and reduced tau clearance contribute to elevated tau levels during waking \u003csup\u003e10,19\u003c/sup\u003e. However, the precise mechanisms by which wakefulness leads to increased ISF and CSF tau remain unknown. We sought to address this question by testing whether tau secretion levels are modulated by the sleep-wake cycle, and investigating mechanisms underlying this modulation. We previously showed sleep-wake differences in tau phosphorylation were driven by fluctuations in core body temperature (BT) during the sleep-wake cycle \u003csup\u003e20\u003c/sup\u003e. Furthermore, SD prevented sleep-associated tau phosphorylation by disrupting the normal core BT decrease during sleep \u003csup\u003e20\u003c/sup\u003e. Building on these findings, we hypothesized that sleep-wake variations in core BT may drive sleep-wake variation in tau secretion levels by modulating the activity of key components in the UPS-I pathway.\u003c/p\u003e \u003cp\u003eHere, we demonstrate in mice and \u003cem\u003ein vitro\u003c/em\u003e that variation in CSF and ISF tau levels across the sleep-wake cycle are driven by changes in core BT, owing to temperature-dependent regulatory mechanisms governing tau protein secretion. We found that higher BT, either during wakefulness, SD, or induced by mild-hyperthermia, promotes tau secretion into the CSF \u003cem\u003evia\u003c/em\u003e the upregulation of UPS-I-related components in mice. We elucidated a specific intracellular pathway involving (i) caspase-3-mediated TauC3 production, (ii) subsequent binding of TauC3 to PIP\u003csub\u003e2\u003c/sub\u003e at plasma membrane, and (iii) the transmembrane export of TauC3 facilitated by the HSPG family member syndecan-3 (SDC3). In older adults, we found that the rise in BT during wakefulness was positively correlated with the increase in CSF and plasma tau levels.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTau secretion is temperature-dependent\u003c/h2\u003e \u003cp\u003eDuring wakefulness, CSF and ISF tau levels increase nearly twofold compared to sleep \u003csup\u003e14\u003c/sup\u003e. We first investigated whether temperatures simulating BT variations during the sleep-wake cycle could regulate neuronal tau secretion in SH-Tau3R human cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and in primary mouse cortical cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). We found that extracellular tau levels plateaued after 72 hours at 37˚C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Using quantitative ELISA and dot blotting, we then showed that higher temperatures (38\u0026deg;C vs. 35\u0026deg;C) led to a\u0026thinsp;~\u0026thinsp;2-fold increase in tau secretion in both human (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,e,f; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and mouse neuron-like cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-k), without inducing cytotoxicity, except for 39\u0026deg;C (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). In order to more faithfully replicate physiological temperature variations occurring during a full 24-h sleep-wake cycle, we ensured that shorter exposures (6, 24 and 48 hours) corroborated these findings (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Our prior research indicated that increased core BT induced by sauna-like conditions \u003csup\u003e21\u003c/sup\u003e or wakefulness-like temperature exposure \u003csup\u003e20\u003c/sup\u003e, lead to tau dephosphorylation. We replicated these findings, showing a temperature-dependent reduction in phosphorylation levels of both intracellular (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f) and extracellular tau (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed,e,g,j,k) in both SH-Tau3R and mouse primary cells. Overall, exposure to higher temperatures similar to those experienced during wakefulness promotes tau secretion, with the secreted tau species being markedly dephosphorylated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess whether the effect of temperature is specific to tau secretion, we conducted a comparative analysis of extracellular contents for various proteins, including microtubule-associated protein 2 (MAP2), α-synuclein, fibroblast growth factor 2 (FGF2), caspase-1, and Neurofilament light chain (NfL). MAP2 is also a microtubule-associated protein, and NfL is a cytoskeletal protein often used as negative control for extracellular tau \u003csup\u003e14,22\u003c/sup\u003e. Additionally, α-synuclein, FGF2 and caspase-1 are proteins known to be secreted \u003cem\u003evia\u003c/em\u003e the UPS pathways \u003csup\u003e23,24\u003c/sup\u003e. However, temperature did not affect the secretion profiles of these proteins (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg,h), pointing to a specific temperature modulation of tau release though unknown underlying mechanisms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWakefulness temperatures promote tau release through its caspase-3-mediated cleavage\u003c/h2\u003e \u003cp\u003eWe further sought to identify cellular pathways underlying temperature-dependent effects. Given extracellular tau is predominantly present as the TauC3 proteolytic fragment in AD \u003csup\u003e7,8\u003c/sup\u003e, which is thought to facilitate its secretion \u003csup\u003e9\u003c/sup\u003e, we wondered whether temperature affects its caspase-3-mediated cleavage. Our findings revealed that wakefulness temperatures increased caspase-3 activity and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c) in both human and mouse cells, compared to those exposed at 35 or 37\u0026deg;C. Interestingly, this coincided with the intracellular dephosphorylation of tau at S422 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c), previously shown to facilitate the caspase-3-mediated cleavage at D421 \u003csup\u003e12,13\u003c/sup\u003e. As a result, we observed increased levels of both intra- and extracellular TauC3 in cells exposed to 38\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e). As additional validation, we observed that wakefulness temperatures decreased the levels of intra- and extracellular Tau46 (epitope of 428\u0026ndash;441) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e), which does not recognize C-term cleaved tau \u003csup\u003e8\u003c/sup\u003e. To further substantiate the role of caspase-3 in tau secretion, we observed that its inhibition \u0026ndash; either with z-DEVD-FMK pharmacological inhibitor or caspase-3 mRNA-targeting siRNA \u0026ndash; significantly decreased extracellular tau release (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-i). Collectively, our data demonstrates that wakefulness temperatures promote caspase-3-mediated cleavage of tau, facilitating its UPS-I mediated secretion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWakefulness temperatures drives TauC3 secretion through SDC3 upregulation\u003c/h2\u003e \u003cp\u003eHSPGs have been identified as critical components of the UPS-I pathway due to their ability to bind to intracellular proteins and facilitate their direct export across the plasma membrane \u003csup\u003e25\u003c/sup\u003e. Ubiquitously expressed on cell surfaces, HSPGs consist of a core proteoglycan with heparan sulfate chains, the elongation of which is facilitated by the glycosyltransferase activity of Exostosin-1 (EXT1) in the brain \u003csup\u003e26\u003c/sup\u003e. Among the diverse family of HSPGs, neuronal SDC3 is particularly relevant to AD pathophysiology, promoting the propagation of amyloid pathology and the neuronal uptake of tau \u003csup\u003e27,28\u003c/sup\u003e. To obtain insight into how temperature might drive tau secretion through the UPS-I pathway, we investigated whether temperature influences SDC3 metabolism. We found wakefulness temperatures increased SDC3 and EXT1 protein and mRNA expressions in SH-Tau3R cells and primary neuronal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). To further emphasize that TauC3 is highly releasable, we used confocal microscopy and observed a temperature-dependent increase in the merged staining of SDC3 and TauC3, with numerous puncta (SDC3- and TauC3-positive) mainly localized in the soma and in proximal neurites of primary neurons cultured at 38\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As SDC3 might be also involved in tau internalization inside neurons \u003csup\u003e27,28\u003c/sup\u003e, we observed peri-membranous puncta that were TauC3 and SDC3, although in a lower proportion compared to intracellular ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the role of SDC3 and EXT1 in tau secretion \u003cem\u003evia\u003c/em\u003e the UPS-I pathway, we conducted siRNA-mediated knockdown experiments either for SDC3 or EXT1. In both cases, we observed a significant reduction in the extracellular levels of total and cleaved-tau compared to cells transfected with scrambled siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). To further examine the interplay between caspase-3 and SDC3 in tau release, we also simultaneously inhibited the expression of caspase-3 and SDC3 resulting in almost complete suppression of intra- and extracellular TauC3 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,g). Intriguingly, the inhibition of TauC3 expression was associated with a notable increase in tau hyperphosphorylation at S422 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). These observations collectively emphasize a complementary role of caspase-3 and SDC3 in mediating the extracellular export of TauC3 during wakefulness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWakefulness temperatures facilitate tau recruitment and release at plasma membrane\u003c/h2\u003e \u003cp\u003eDue to their lipidic composition, plasma membranes are highly sensitive to temperature fluctuations. An elevation of temperature leads to increased membrane fluidity and permeability in plant and animal cells \u003csup\u003e29,30\u003c/sup\u003e. Based on these findings, we hypothesized that higher temperature exposure might drive tau secretion by optimizing the properties of the plasma membrane to facilitate tau translocation into the extracellular space. We found that SH-Tau3R cells exhibited increased membrane fluidity at wakefulness temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). Moreover, tau requires sequestration at the inner layer of the plasma membrane through PIP\u003csub\u003e2\u003c/sub\u003e binding \u003csup\u003e6\u003c/sup\u003e. We thus wondered if temperature influences PIP\u003csub\u003e2\u003c/sub\u003e, and showed that its expression was temperature-dependent in both human and mouse neuron-like cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). Interestingly, the temperature-dependent increase in PIP\u003csub\u003e2\u003c/sub\u003e expression was previously documented in yeast and plant cells \u003csup\u003e31,32\u003c/sup\u003e, suggesting a highly conserved process.\u003c/p\u003e \u003cp\u003eTo explore whether full-length tau and TauC3 have the same affinity for PIP\u003csub\u003e2\u003c/sub\u003e, we performed co-IP using Tau-DA9, TauC3 or Tau46 antibodies and probed for PIP\u003csub\u003e2\u003c/sub\u003e by Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej), or co-IP using a PIP\u003csub\u003e2\u003c/sub\u003e antibody to assess the interaction with different tau antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). In both cases, we observed that PIP\u003csub\u003e2\u003c/sub\u003e preferentially binds TauC3 rather than full-length tau (Tau46 signal was barely detectable) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej,k). Finally, we demonstrated that an increase in temperature significantly promotes the binding of TauC3 to PIP2, while the binding of full-length tau to PIP2 tends to slightly decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el-n). Altogether, these results suggest that wakefulness temperatures promote the UPS-I-mediated secretion of TauC3 by facilitating its interaction with PIP2 and SDC3 at the plasma membrane, thereby triggering its vesicle-free release.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWakefulness and sleep deprivation upregulate UPS-I pathway by increasing core BT in mice\u003c/h2\u003e \u003cp\u003eTo assess whether the higher CSF and ISF tau levels during wakefulness and SD \u003csup\u003e14\u003c/sup\u003e, are related to natural elevated BT induced by these conditions \u003csup\u003e20\u003c/sup\u003e, we analyzed UPS-I-related proteins in the cortex of wild-type mice across sleep vs. wakefulness, or following SD. Awake mice exhibited higher core BT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), associated with increased cortical expression of caspase-3, TauC3, SDC3 and PIP\u003csub\u003e2\u003c/sub\u003e, along with tau dephosphorylation at S422 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d), compared to sleeping mice. Moreover, we observed that the rectal temperature of mice at the time of euthanasia was significantly correlated with the expression levels of caspase-3, pTau(S422), SDC3 and PIP\u003csub\u003e2\u003c/sub\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-f). We further showed that 6 hours of SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) prevented the natural decrease in core BT during sleep (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), and triggered the upregulation of caspase-3, TauC3, and PIP\u003csub\u003e2\u003c/sub\u003e levels, associated with decreased S422 phosphorylation and Tau46 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg,h).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMild-hyperthermia increases CSF tau levels in hTau mice\u003c/h2\u003e \u003cp\u003eTo determine whether induced changes in BT affect CSF tau levels, we subjected hTau mice to hypo- or hyperthermic conditions for 4 hours before CSF collection, and compared to normothermic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei,j). We observed that hyperthermic mice exhibited higher CSF tau concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek), and these levels significantly correlated with rectal temperatures recorded after thermal interventions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el). The rise in CSF tau concentrations was associated with increased cortical expression of caspase-3, TauC3, SDC3 and PIP\u003csub\u003e2\u003c/sub\u003e, along with a reduction in tau phosphorylation at S422 and Tau46 expression (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b), all correlating with rectal temperature (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-h). These findings collectively suggest that core BT variation influences CSF tau levels through the upregulation of the UPS-I pathway. It emphasizes the pivotal role played by sleep-wake temperature variations in regulating the secretion and the propagation of tau \u003cem\u003evia\u003c/em\u003e the UPS-I pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBody temperature correlates with CSF tau but not CSF NfL in humans\u003c/h2\u003e \u003cp\u003eTo test the relationship between BT and sleep-wake tau dynamics in humans, we utilized two separate data sets from older adults in which BT and tau levels (CSF or plasma) were simultaneously measured at multiple time points across the sleep-wake cycle. We examined the correlation between the magnitude of change in tau levels post-wakefulness (∆Tau) and the concurrent rise in BT during wakefulness (∆BT). Predefined measurement times were selected to optimize the average ∆BT within the constraints of the available datasets (see Methods). Similar to previous findings in CSF, plasma tau levels were significantly higher in the evening compared to the morning (Extended Data Tables\u0026nbsp;1 and 2). We found a positive correlation between ∆BT and ∆Tau for both CSF tau (r\u0026thinsp;=\u0026thinsp;0.58, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and plasma tau (r\u0026thinsp;=\u0026thinsp;0.72, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), with no correlation for CSF NfL levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c), and a consistent relationship across CSF and plasma data sets. Specifically, participants exhibiting a large positive ∆Tau, i.e., substantially higher afternoon-evening levels compared to morning, also showed a large positive ∆BT. By contrast, participants with negligible or negative ∆Tau showed minimal or negative ∆BT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,c). The observed ∆BT values for the plasma data set agree with our previous study, and represent the first report of correlations between BT and circulating tau in humans. The increase in CSF tau levels with wakefulness vs sleep aligns with previous studies \u003csup\u003e10,14\u003c/sup\u003e. Although diurnal sampling of plasma tau was previously documented in sedentary young adults \u003csup\u003e33\u003c/sup\u003e, this is the first report of diurnal dynamics in tau under naturalistic conditions representative of physiological BT variation. Our overall finding of ~\u0026thinsp;15% higher tau in the evening comprised a broad range of ∆Tau values that were substantially explained by ∆BT, with similar patterns for CSF. These results supported our \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e findings, where higher BT during wakefulness drove higher tau secretion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study investigated the influence of BT variation during the sleep-wake cycle upon tau secretion and its underlying regulatory mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our findings indicate that wakefulness temperatures, or conditions affecting core BT such as SD or mild-hyperthermia induction, promotes C-term truncation of tau, leading to its extracellular release through UPS-I pathway. Using \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e approaches, we identified that the physiological increase in core BT during periods of wakefulness triggers some specific intracellular mechanisms such as (i) the caspase-3-mediated cleavage of tau into TauC3, (ii) the sequestration of TauC3 at plasma membrane \u003cem\u003evia\u003c/em\u003e its binding to PIP\u003csub\u003e2\u003c/sub\u003e, and (iii) the translocation of TauC3 into the extracellular space facilitated by SDC3, resulting in increased CSF tau levels. This pointed to the involvement of the circadian regulation of BT during the sleep-wake cycle in tau secretion and propagation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe precise mechanisms underlying tau secretion remain unestablished. Our observation that tau release is modulated in a temperature-dependent manner suggests core BT variation may play a significant role in regulating tau secretion. Holth et al previously showed a twofold rise in ISF and CSF tau levels during wakefulness compared to sleep\u003csup\u003e14\u003c/sup\u003e. Here, we replicated these tau level increases by varying temperature alone within physiological range, with tau doubling at 38\u0026deg;C compared to 35\u0026deg;C.. Excitatory neuronal activity, one of the first identified biological processes capable of increasing tau release \u003csup\u003e34,35\u003c/sup\u003e, is potentiated during wakefulness and depressed during sleep \u003csup\u003e36\u003c/sup\u003e. In order to test whether tau release during wakefulness was accounted for by concurrent increases in neuronal activity, Holth \u003cem\u003eet al.\u003c/em\u003e used tetrodotoxin (TTX) to inhibit neuronal activity, showing that it prevented tau release during SD \u003csup\u003e14\u003c/sup\u003e. However, TTX also causes rapid hypothermia \u003csup\u003e37,38\u003c/sup\u003e, pointing to a possible role of temperature in these findings. While it is known that a slight 1\u0026deg;C change in brain temperature is sufficient to alter neuronal excitability and activity \u003csup\u003e39\u0026ndash;41\u003c/sup\u003e we similarly found that a 1\u0026deg;C change alters tau secretion. Given SH cells and mouse primary neurons lack neuronal activity \u003csup\u003e42\u003c/sup\u003e, our data strongly suggest that temperature directly regulates UPS-I-mediated tau secretion. However, considering that neuronal activity alone can also drive tau release \u003csup\u003e34\u003c/sup\u003e, the interplay between neuronal activity and BT in stimulating tau secretion requires further investigation.\u003c/p\u003e \u003cp\u003eOur study emphasizes tau cleavage into TauC3 as pivotal for secretion, with CSF and extracellular tau mainly present as C-terminally truncated \u003csup\u003e8,9\u003c/sup\u003e. While the administration of a TauC3-specific antibody has been shown to impede tau propagation and seeding \u003csup\u003e43\u003c/sup\u003e, the diurnal regulation of these processes remains unknown. We found that wakefulness temperatures induce both tau dephosphorylation at S422, enabling tau cleavage, and upregulation of the caspase-3-mediated TauC3 truncation, leading to its extracellular release. The role of S422 phosphorylation and TauC3 remains debated, with some studies evidencing TauC3 as neuroprotective \u003csup\u003e44\u0026ndash;46\u003c/sup\u003e and others linking TauC3 to neurofibrillary tangle assembly and synaptic toxicity \u003csup\u003e12,47,48\u003c/sup\u003e. Our investigation revealed an inverse relationship between TauC3 and S422 phosphorylation, modulated by physiological sleep-wake fluctuations in core BT. These findings also imply a physiological tau release, consistent with prior studies showing tau secretion does not necessarily result in neuronal pathology spreading \u003csup\u003e34,35\u003c/sup\u003e. In favor to this view, treatment with an anti-pS422 antibody has been shown to reduce AD pathology while increasing plasma tau concentrations in AD mice \u003csup\u003e49\u003c/sup\u003e, suggesting that TauC3 might be more prone to brain clearance. However, while wakefulness temperatures induce tau dephosphorylation at multiple epitopes, the relevance of other phosphorylation sites in driving tau secretion remains to be explored.\u003c/p\u003e \u003cp\u003eHere, we have made several novel findings regarding the modulation of tau secretion \u003cem\u003evia\u003c/em\u003e interactions between multiple temperature-dependent components of the UPS-I pathway. While the UPS-I pathway is known for the release of FGF2 \u003csup\u003e25\u003c/sup\u003e, α-synuclein \u003csup\u003e50\u003c/sup\u003e, and tau \u003csup\u003e4,5\u003c/sup\u003e, our observations of a temperature-dependent effect on tau secretion\u0026mdash;without similar changes for others proteins\u0026mdash;suggest a unique BT-driven tau secretion pathway. Our results suggest that the temperature-dependent cleavage of tau into TauC3 may serve as an initiating factor for finely modulating its secretion. Notably, the loss of microtubule-binding capacity of TauC3 \u003csup\u003e48\u003c/sup\u003e might enhance its availability for the secretion pathway, while wakefulness temperatures facilitate the binding of TauC3 to PIP\u003csub\u003e2\u003c/sub\u003e at the inner plasma membrane. Prior research has demonstrated that the tau C-terminal domain contains a low-affinity site that affects its interaction with phosphoinositides \u003csup\u003e51\u003c/sup\u003e, likely explaining the preferential binding of PIP\u003csub\u003e2\u003c/sub\u003e to TauC3, given this fragment lacks a portion of the C-terminal domain. Altogether, these findings suggest that higher core BT during wakefulness, SD or mild-hyperthermia, promotes TauC3 binding to PIP\u003csub\u003e2\u003c/sub\u003e at the plasma membrane, initiating the export process.\u003c/p\u003e \u003cp\u003eThe increase in BT during wakefulness appears to also promote the extracellular release of TauC3 by enhancing its interaction with SDC3, facilitating the membrane translocation process. While prior studies reported increased levels of SDC3 in the brain of AD mouse models \u003csup\u003e28\u003c/sup\u003e, or following neuronal stimulation \u003csup\u003e52\u003c/sup\u003e, this is the first report of its temperature-dependent expression and metabolism. Notably, one study showed that the glycosyltransferase activity of enzymes such as EXT1\u0026mdash;required for the elongation of SDC3 sulfate chains\u0026mdash;increases with temperature \u003csup\u003e53\u003c/sup\u003e. Our study extends these findings, showing that wakefulness temperatures enhance EXT1 mRNA expression, potentially improving SDC3 function. We also observed a substantial intracellular co-localization of SDC3 with TauC3 at wakefulness temperatures, contributing to a better understanding of the mechanisms underlying tau secretion during the sleep-wake cycle.\u003c/p\u003e \u003cp\u003eOur results suggest a pathway by which sleep-wake BT variation may modulate physiological CSF and plasma tau dynamics in human \u003cem\u003evia\u003c/em\u003e temperature-dependent tau secretion and phosphorylation. However, further research is needed to determine additional temperature-dependent processes. Important candidates include neuronal activity \u003csup\u003e39\u0026ndash;41\u003c/sup\u003e, as previously discussed, as well as sleep, known to depend upon body and brain temperature fluctuation \u003csup\u003e54\u003c/sup\u003e. Demonstrating a pathway by which temperature influences AD biomarkers \u003cem\u003evia\u003c/em\u003e sleep, a previous study showed chronic thermoneutral temperature exposure in AD mice reduced amyloid pathology by enhancing slow-wave sleep \u003csup\u003e55\u003c/sup\u003e. We also note that bidirectional effects may additionally contribute to the observed relationship between BT and tau dynamics in that early tau pathology in thermoregulatory brain areas may influence BT patterns, as recently shown in mice \u003csup\u003e56\u003c/sup\u003e. Finally, in the setting of AD, further research is needed to distinguish between circulating tau derived from unconventional \u003cem\u003evs\u003c/em\u003e vesicular secretion \u003csup\u003e5\u003c/sup\u003e, or other sources such as impaired degradation and clearance pathways \u003csup\u003e57\u003c/sup\u003e, or release after neuronal death \u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAltogether, our findings suggest that sleep-wake BT variation modulates parallel dynamics in tau secretion and phosphorylation, and provide the first evidence associating BT variation with CSF and plasma tau dynamics in human. By extension, our results suggest that impaired thermoregulation as well as BT alteration caused by sleep disturbance may contribute to the pathogenesis of AD and related tauopathies. It is therefore crucial to understand how naturalistic variation in BT over the sleep-wake interval affects CSF and plasma tau levels used for AD diagnosis, particularly in patients with thermoregulatory or sleep deficits. We note that few previous studies in AD patients measured BT variation over the sleep \u003cem\u003evs\u003c/em\u003e wake interval\u0026mdash;rather most reported BT averaged over the sleep wake cycle, and meta-analysis showed little difference (0.1\u0026deg;C) between AD and controls \u003csup\u003e59\u003c/sup\u003e. By contrast, our findings emphasize the importance of assessing BT dynamics over the naturalistic sleep-wake interval in order to understand how BT interacts with tau metabolism. We previously showed that lower waking BT predicted tau pathology, supporting hypotheses that age-associated BT decline may be a risk factor for AD \u003csup\u003e60\u0026ndash;62\u003c/sup\u003e. On the other hand, sleep fragmentation or deprivation \u003csup\u003e16,63\u003c/sup\u003e and increased nocturnal activity \u003csup\u003e64,65\u003c/sup\u003e\u0026mdash;both risk factors for, and observed in AD\u0026mdash; may prevent the nocturnal BT drop \u003csup\u003e20\u003c/sup\u003e, thereby increasing tau secretion and potentially accelerating tau pathogenesis \u003csup\u003e66,67\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur model (Fig. 6) elucidates how core BT regulates tau secretion by driving UPS-I pathway activity during the sleep-wake cycle in healthy individuals. We posit that wakefulness temperatures facilitate physiological tau release, while sleep temperatures inhibit this pathway and increase tau phosphorylation. Further, wakefulness temperatures might facilitate the secretion of dephosphorylated and cleaved tau species which are less toxic, less prone to aggregation and more manageable for clearance and degradation systems within the brain\u0026nbsp;\u003csup\u003e45,46,49,68\u003c/sup\u003e.\u0026nbsp;By contrast during sleep, tau release slows, potentially aiding its clearance via the glymphatic system\u0026nbsp;\u003csup\u003e68,69\u003c/sup\u003e. This model points toward the importance of\u0026nbsp;maintaining and managing the appropriate core BT at the right phase of the sleep-wake cycle, and the potential for age- or AD-related disorder in this pattern to lead to tau pathology. Interestingly, interventions like sauna bathing, which temporarily increase BT\u0026nbsp;\u003csup\u003e70\u003c/sup\u003e, are beneficial in reducing AD risk, increasing deep sleep in humans\u0026nbsp;\u003csup\u003e71\u003c/sup\u003e, and reducing tau phosphorylation in mice\u0026nbsp;\u003csup\u003e21\u003c/sup\u003e.\u0026nbsp;Future studies may examine whether sauna use can delay tau-mediated neurodegeneration by correcting sleep and core BT misalignment associated with thermoregulatory and sleep-disturbances in aging and early AD. Finally, while the physiological role of extracellular tau remains enigmatic, it may act as a signaling molecule, potentially interacting with muscarinic receptors\u0026nbsp;\u003csup\u003e72\u003c/sup\u003e. Elucidating the physiological role of tau secretion\u0026nbsp;\u003csup\u003e73\u003c/sup\u003e and understanding the normal function of extracellular tau could inform therapeutic strategies to impede tau pathology propagation.\u0026nbsp;\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, human neuroblastoma cells (SH-SY5Y cells) stably expressing human tau 3 repeat isoform 2+3-10- (designated as SH-Tau3R cells, generously provided by Luc Buée) were used. The SH-Tau3R cells were cultured as previously described\u0026nbsp;\u003csup\u003e74\u003c/sup\u003e. Briefly, the cells were grown in DMEM/High glucose medium (11995-065, ThermoFisher), supplemented with 10% bovine growth serum (BGS, heat inactivated, F1051-500ML, Sigma-Aldrich), 1% glutamine (25030081, ThermoFisher), and 1% penicillin/streptomycin (15140-122, ThermoFisher). The cell cultures were maintained in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37°C. The cells were grown either in 10 cm Petri dishes, 6-, 12-, or 96-wells plates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary culture of neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the primary neuronal culture, cortices of mouse embryos at embryonic day 16 (E15-E17) were used from transgenic mice B6.129S2Emx1tm1(cre)Krj/J, where Emx1-Cre mice were crossed with Red Fluorescent Protein-Lox mice (Jackson Laboratories). Briefly, brains embryos were dissected out, meninges, choroid plexus and hippocampus were removed to avoid contamination and cortices were mechanically and enzymatically disrupted in the presence of trypsin-EDTA 0,25% (Gibco) for 20 min at 37°C. The cell suspension was filtered through a 70 µm cell strainer and plated onto 6-well plates (200,000 cells/well), which were pre-coated with 50 µg/ml poly-D-lysine (A3890401, ThermoFisher), or on coverslips pre-coated with 1 µg/mL polyethylenimine (043896.03, ThermoFisher) and 50 µg/mL poly-D-lysine in 24-well plates (150,000 cells/well). The cells were firstly grown for 2 hours in DMEM/High glucose medium, supplemented with 10% BGS and 1% of streptomycin/penicillin antibiotics in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37°C. Then, the culture medium was changed per a growth medium (NeurobasalTM medium (21103-049, ThermoFisher), 1% glutamine (25030081, ThermoFisher), 2% B-27 supplement (17504044, ThermoFisher), 1% N-2 (17502-048, ThermoFisher) and 1% penicillin/streptomycin). The cultures were maintained at 37°C in a humid atmosphere with 5% CO2 and a growth period of 4 days was allowed before any experimental treatment was administered.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperatures exposure and cell treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to initiating any treatment, the cell culture medium was replaced with fresh DMEM/High glucose medium (without BSA) or Neurobasal medium, for SH-Tau3R or primary neuronal culture, respectively. Then, the cells were placed in dedicated CO\u003csub\u003e2\u003c/sub\u003e incubators set to 35, 37, 38 or 39°C for a duration from 6 to 72 hours (Fig.1a, h). To inhibit caspase-3 activity, cells were treated for a period of 72 hours with the selective caspase-3 inhibitor z-DEVD-FMK (A13503; Adooq Biosciences) at a concentration of 20 µM\u0026nbsp;\u003csup\u003e9\u003c/sup\u003e dissolved in a vehicle solution (phosphate-buffered saline (PBS) containing 0.1% of DMSO). Transfection of small interfering RNA was carried out using Lipofectamine\u003csup\u003eTM\u003c/sup\u003e RNAiMAX transfection reagent (13778075, ThermoFisher) according to the manufacturer’s instructions. Briefly, for each transfection, cells were cultured for 72 hours in 1 ml of Opti-MEM (ThermoFisher) containing 40 µl of Lipofectamine\u003csup\u003eTM\u003c/sup\u003e, and 100 nmol of respective siRNAs. The following siRNAs were used: Silencer® Pre-designed EXT1 siRNA (ID116802, ThermoFisher), Stealth RNAi\u003csup\u003eTI\u003c/sup\u003e SDC3 siRNA (HSS145253, ThermoFisher), and SignalSilence® Caspase-3 siRNA (6466S; Cell Signaling). Silencer\u003csup\u003eTM\u003c/sup\u003e select negative control siRNA (4390843, ThermoFisher) was used as the scrambled negative control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, three-months-old C57BL6 (males and females) and 18-month-old hTau (males) mice or their littermate control tau knockout (TKO; males)\u0026nbsp;\u003csup\u003e75\u003c/sup\u003e were used. The hTau mice were generated by crossing mice expressing the 6 isoforms of nonmutated human tau (known as 8c mice)\u0026nbsp;\u003csup\u003e76\u003c/sup\u003e with murine TKO mice\u0026nbsp;\u003csup\u003e77\u003c/sup\u003e. The founders of hTau and TKO colonies originated from a C57BL6 background (B6.Cg-Mapttm1(EGFP)Klt-Tg(MAPT)8cPdav/J, Jackson Laboratories). The animals were handled according to procedures endorsed by the “The Animal Care Committee of Université Laval (CPAUL-3, approbation number: CHU-22-1027)” under the guidelines of the Canadian Council on Animal Care.\u0026nbsp;All mice had access to water and food ad libitum. The mice were housed in a 12 h light/12 h dark cycle, with the lights being turned on at 7:15 am. At the end of each experiment, mice were euthanized through decapitation without anesthesia, as anesthesia leads to tau hyperphosphorylation\u0026nbsp;\u003csup\u003e78,79\u003c/sup\u003e.\u0026nbsp;The brains were promptly removed and cortices were dissected on ice, frozen on liquid nitrogen and stored at −80°C for further analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSleeping vs. awake mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were subjected to a continuous period of darkness lasting for 3 days. The determination of subjective day was determined as previously described\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e. Briefly, sleeping C57BL6 mice (n=5 males and n=5 females) were euthanized between 10:30 and 11:30 am local time (at Circadian Time 4 (CT4), 16 h after the onset of activity) and active mice (n=5 males and n=5 females) were euthanized between 10:30 and 11:30 pm local time (at CT16, 4 h after the onset of activity)\u0026nbsp;(Fig. 5a). Furthermore, the sleeping criterion corresponded to mice in the nest, in a “resting posture”, as elucidated by Thoman and Carroll: absence of locomotor activity, absence of movement, absence of erect posture\u0026nbsp;\u003csup\u003e80\u003c/sup\u003e. The\u0026nbsp;core BT of mice was assessed just before euthanasia with a rectal probe (RET-3, Brain Tree Scientific Inc) connected to a digital thermometer (Thermalert TH5; Physitemp).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSleep deprivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs previously described by our group\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e, a subset of C57BL6 mice was intentionally kept awake for the first 6 hours of the light period (sleep deprivation (SD) group, n=9, males and females). Naive mice (n=7, males and females) were allowed to sleep without any disturbance. All mice were euthanatized by decapitation at the end of SD period\u0026nbsp;(Fig. 4e). Prior to SD experiment, a subset of five mice of both groups was abdominally implanted with telemetric probes (BodyCap, Anipill) enabling continuous monitoring of their BT. The baseline BT was assessed the day preceding the SD protocol for the same set of animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCold and heat exposures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn the day preceding the experiment, hTau mice were individually housed to prevent any mutual heating. For the entire duration of the study, the naive group (n=5) and the negative control TKO (n=3) remained at the standard temperature of the animal facility (22°C). As previously described by our group\u0026nbsp;\u003csup\u003e21,81\u003c/sup\u003e, the two other groups of mice underwent a 4-hour exposure period either at temperature of 4°C (n=3) or 38°C (n=5). The\u0026nbsp;core BT of mice was assessed just prior to euthanasia utilizing a rectal probe (RET-3, Brain Tree Scientific Inc) connected to a digital thermometer (Thermalert TH5; Physitemp).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCSF collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice were anesthetized with isoflurane and positioned on a stereotaxic instrument. To maintain core BT, a water heating pad was used. Under the observation of a dissection microscope, the subcutaneous tissues and muscles (m. biventer cervicis and m. rectus capitis dorsalis major) were gently separated via blunt dissection utilizing forceps. This separation facilitated the exposure of the dura mater of the cisterna magna. A capillary tube was introduced through the dura mater into the cisterna magna in order to induce the CSF flow into the capillary tube.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples (cell lysates or mice cortices) were homogenized by sonication\u0026nbsp;in Radioimmunoprecipitation assay (RIPA) buffer, then centrifuged for 20 min at 20,000g at 4°C. The resulting supernatant was collected, and the total protein concentration was assayed (Pierce™ BCA Protein Assay Kits, 23225, ThermoFisher). The samples were diluted in sample buffer (NuPAGE LDS; Invitrogen) containing 5% of 2-β-mercapto-ethanol, 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail (P8340; Sigma-Aldrich). The samples were then subjected to denaturation for 10 min at 95 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was conducted as previously described\u0026nbsp;\u003csup\u003e82\u003c/sup\u003e.\u0026nbsp;10-20 μg of the samples were separated on an SDS-10% polyacrylamide gel and transferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were saturated, hybridized with the appropriate antibodies, and revealed as described in\u0026nbsp;\u003csup\u003e82\u003c/sup\u003e. For immunoblots targeting phospho-tau epitopes, the signal was normalized to the total tau protein. Used as a loading control, other proteins were normalized to β-actin. Representative lanes from the immunoblots were exhibited for each specific experimental condition. The dashed lines indicate segments where certain lanes from the same blot were excluded, and the remaining lanes were combined. Brightness levels were adjusted as necessary to enhance visualization and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll antibodies used in this study, in addition to their dilution, are listed in Extended Data Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDot blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell medium was harvested following appropriate treatments and centrifugated for 10 min at 20,000g at 4°C to remove cell debris. In order to assess extracellular content of proteins by dot blotting, 100 μl of cell medium were deposited onto nitrocellulose membranes (Amersham Biosciences), utilizing a microfiltration blotting apparatus (Bio-Dot Apparatus 1706545, Bio-Rad). The membranes were saturated, hybridized with appropriate antibodies (Extended Data Table 3) and revealed as described in\u0026nbsp;\u003csup\u003e82\u003c/sup\u003e. For dot blots targeting phospho-tau epitopes, the signal was normalized to the total tau protein. In the case of other proteins, the normalization was performed relative to the respective extracellular LDH value (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega). Representative dots signal was exhibited for each specific experimental condition. The dashed lines indicate segments where certain dots from the same blot were excluded, and the remaining dots were combined. Brightness levels were adjusted as necessary to enhance visualization and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCo-immunoprecipitation (co-IP) analyses were performed to determine interactions between PIP\u003csub\u003e2\u003c/sub\u003e total tau and TauC3, following manufacturer’s instructions (Pierce™ Classic Magnetic IP/Co-IP Kit, 88804, ThermoFisher Scientific). Briefly, SH-Tau3R cells were harvested using lysis buffer, incubated at 4 °C for 5 min, and centrifuged at 13,000g to pellet cellular debris. The supernatants were collected, proteins levels were adjusted to 500 µg and primary antibodies (Extended Data Table 3) were added to samples, except for the negative control (NC) sample. The samples were then incubated overnight at 4 °C on a rotating device. Following this step, protein A/G magnetic beads (25 μl) were added to each sample and incubated for 1 hour with agitation at room temperature. The antibody-bound beads were extracted using a magnetic device and washed three times. The beads were dissociated using the elution buffer and separated magnetically. To neutralize the low pH environment, 10 µl of neutralization buffer were added to the supernatant. The resulting sample was\u0026nbsp;diluted with sample buffer (NuPAGE LDS; Invitrogen) containing 5% of 2-β-mercapto-ethanol, 1 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 1 mM NaF, 1 mM PMSF, 10 μl/ml of Proteases Inhibitors Cocktail, and finally boiled at 95°C for 5 minutes. The proteins were analyzed using Western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunocytochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeurons from primary culture were fixed in PBS 1X (311-010-CL, Multicell) /4% paraformaldehyde (19210 Electron Microscopy Sciences, ThermoFisher)/10% sucrose (S53, ThermoFisher) for 20 min at room temperature. After washing 3 times with PBS 1X, cells were permeabilized in 0.2% Triton X-100 (T8787-100ML, Millipore) in PBS 1X for 30 min at room temperature and blocked with 5% Goat Serum Heat Inactivated (G6767, Millipore) in PBS 1X for 1 hour at room temperature. Then, cells were incubated with the primary antibodies against TauC3 and SDC3\u0026nbsp;(Extended Data Table 3)\u0026nbsp;in 5% goat serum heat inactivated in PBS 1X at 4°C overnight. After washing 3 times with PBS 1X, the secondaries antibodies (anti-mouse Alexa Fluor 488 diluted at 1:1000 (#A-11029, ThermoFisher) and goat anti-rabbit IgG Alexa Fluor 633 diluted at 1:1000 (#A-21070, ThermoFisher)) were added for 2 hours. After 3 washes with PBS 1X, DAPI (4′,6-diamidino-2-phenylindole, (ThermoFisher) 3,5µL of DAPI in 25 mL PBS 1X) was used for nuclei staining and coverslips were mounted with Fluoromont-G (00-4958-02, Invitrogen). Sections were imaged on a Zeiss LSM800 confocal microscope system equipped with 405, 488, 561 and 640 nm lasers. Confocal images were acquired and mosaics created using the Zen Blue Edition software (v. 2.3, Carl Zeiss).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCaspase-3 activity assay kit\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SH-Tau3R cells were cultured in 96-well plates and treated according to appropriate experimental conditions (Fig. 1a). The colorimetric caspase-3 Assay Kit (ab39401, abcam) was used to determine the activity of caspase-3, and following manufacturer’s instruction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMembrane fluidity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SH-Tau3R cells were cultured within 96-well plates and subjected to treatment as outlined in the experimental groups (Fig. 1a). The membrane fluidity (ab189819, abcam) was assessed according to manufacturer’s instructions. Briefly, the cells were incubated 1 hour at temperatures of 35°C, 37°C or 38°C in a cell medium supplemented with 5 µM of Fluorescent Lipid Reagent and 0.08% Pluronic F127. The\u0026nbsp;fluorescence intensity was then measured (Infinite F200, Tecan)\u0026nbsp;at wavelengths of 400nm and 470nm, using the appropriate filter for excitation at 350nm. The recorded fluorescence values were corrected by subtracting the corresponding blanks from each sample, and the fluorescence ratio of excimer emission (470nm) to monomer emission (400nm) was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA assays of extracellular tau\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal and phosphorylated tau concentrations within the cell medium were quantified using ELISA kit: Tau (total) Human KHB0041; Tau [pS199] Human KHB7041; Tau [pT231] Human KHB8051; Tau [pS396] Human KHB7031; Tau (total) Mouse KMB7001 (ThermoFisher). Prior to analysis, the samples were suitably diluted in diluent buffer (1:50 for human tau, 1:2 for mouse tau and phospho-tau). The ELISA assays were performed in accordance with the instructions provided by the manufacturer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from SH-Tau3R cells using TrizolÒ\u0026nbsp;reagent (Life Technology) in accordance with the manufacturer’s instructions. The quantification of RNA was conducted, and 1\u0026nbsp;mg of total RNA was used for cDNA synthesis using the iscript\u003csup\u003eTM\u003c/sup\u003e cDNA Synthesis Kit (Biorad), containing an optimal blend of oligo-dT and random primers. For subsequent PCR amplification, 1 µl of the resultant cDNA was used as template. The primer sequence used for the PCR amplification are reported in Extended Data Table 3. The qPCR mix was formulated with 18\u0026nbsp;µL\u0026nbsp;per 2\u0026nbsp;µL\u0026nbsp;of 20 ng cDNA. The mix consisted of 0.5 µL of both the forward and reverse primers, 10\u0026nbsp;µL\u0026nbsp;of SYBR Green PCR Master Mix (Applied Biosystems), and 7.5\u0026nbsp;µL\u0026nbsp;of nuclease free water. The qPCR program began with a hot start at 95°C for 3 minutes, succeeded by\u0026nbsp;40 cycles at 95°C\u0026nbsp;for 15 seconds, followed by 60°C\u0026nbsp;for 1 minute, using a LightCycler 480 II apparatus (Roche). The melting curves were evaluated to ensure a single PCR product.\u0026nbsp;To quantify cDNA levels, the comparative 2ΔΔCt method was employed. Ct values corresponding to the target gene were normalized to the Ct values of the house-keeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). The results were expressed as n-fold differences relative to the experimental control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCSF temperature correlations:\u003c/u\u003e Detailed information about participants, CSF collection, and study design can be found in Lucey et al.\u0026nbsp;\u003csup\u003e83\u003c/sup\u003e. Thirteen participants who completed the placebo group of a recently published clinical trial had 6 ml of CSF collected every 2 hours for 36 hours via an indwelling lumbar catheter\u0026nbsp;\u003csup\u003e83\u003c/sup\u003e. All participants were cognitively unimpaired and in good general health except for poor sleep efficiency \u0026lt;85% measured by actigraphy. Body temperature was recorded every four hours with a temporal forehead thermometer (Adc Adtemp 427, American diagnostic Corp, United-States). \u0026nbsp;CSF tau forms (T181, S202, T217) were measured by immunoprecipitation/mass spectrometry as previously described\u0026nbsp;\u003csup\u003e83\u003c/sup\u003e. NfL protein levels were quantified using the NF-light\u003csup\u003eTM\u003c/sup\u003e ELISA kit (UmanDiagnostics, Umea, Sweden) following the manufacturer’s protocol. The assay’s measurement range is 100 pg/ml to 10,000 pg/ml with a detection threshold of 33 pg/ml. CSF samples were prepared through dilution with an equal volume of Sample Diluent, achieving a 1:100 dilution ratio, to ensure a volume suitable for analysis. The quantitation process entailed the enzymatic conversion of a colorless substrate into a colored product indicative of the NfL concentration in the samples. Absorbance readings were taken at 450 nm with a reference wavelength of 620-650 nm. \u0026nbsp;To ensure consistency, samples with known high levels of NfL (“bloody CSF”) were utilized as positive controls on each plate diluted to 1:1000. \u0026nbsp;CSF tau-181, tau-202, and tau-217 concentrations were averaged at 8AM, 4PM, and 8PM. Differences between temperature, CSF tau levels, and CSF NfL levels were then calculated for use in the analyses, and detailed data for each participant are provided in\u0026nbsp;(Extended Data Table 1). NfL was selected as control protein because its soluble concentration is not affected by sleep-wake activity\u0026nbsp;\u003csup\u003e14\u003c/sup\u003e. We selected time points of 8 AM vs 4 PM for post-sleep vs post-wakefulness tau levels, which also corresponded to the minimum and maximum BT, respectively. These intervals were selected based on the following rationale. First, we assumed little delay between tau secretion and appearance of tau in the CSF, meaning that BT taken at the time of CSF collection would roughly reflect brain temperature at the time of tau secretion. Second, we selected 8 am vs 4 pm as the interval that maximized that difference in sleep vs wake temperatures, given that BT was not recorded during sleep, and for the majority of participants, had already begun to drop between 4 pm and 8 pm (Extended Data Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePlasma temperature correlations\u003c/u\u003e: Data were collected from 24 older adults 68.39±5.25 years of age, 17 of whom were female. Subjects were enrolled in cross-sectional study examining the relationship between core BT and plasma and PET AD biomarkers. Subjects were cognitively normal (n=21) or had mild cognitive impairment (n=3) as determined by the clinical dementia rating scale (CDR), and were medically healthy with only mild, or no sleep apnea. Prior to the study, participants were screened with interviews and one week of home actigraphy for sleep-wake disorders including sleep less that 6 hours per night, significant phase advance or phase-delay. Additional exclusion criteria are detailed in\u0026nbsp;\u003csup\u003e62\u003c/sup\u003e and included AD dementia (CDR \u0026gt; 0.5), medical comorbidities and the use of medications that might affect sleep or thermoregulation, major psychiatric disorders and moderate-severe substance use disorders, shift work within the last 6 months, or traveling across 1 or more time zones within 2 weeks of study participation.\u003c/p\u003e\n\u003cp\u003eThe study design was a semi-naturalistic protocol fully detailed in\u0026nbsp;\u003csup\u003e62\u003c/sup\u003e. Briefly, participants underwent continuous measurement of core body temperature using an ingestible telemetric device (Cortemp, HQInc) that sampled temperature every 15 seconds with an accuracy of 0.2°C for a minimum of 36 hours spanning 2 nights. During this time, 2 in-lab nocturnal polysomnograms were measured, and participants were free to behave as they chose during the intervening day between the lab nocturnal recordings. The goal of this design was to capture data that most closely represented the typical BT for each participant. Blood draws for plasma tau were collected on four occasions, in the mornings (7:00 am) and evenings (7:00 pm) on both mornings and nights\u0026nbsp;(Extended Data Table 2). Prior to analysis, temperature data were preprocessed to exclude gaps and artifacts as detailed in\u0026nbsp;\u003csup\u003e62\u003c/sup\u003e. Data presented for BT-tau correlations comprised tau levels from night 2 and morning 2, given BT was not always measured prior to blood draw on night 1\u0026nbsp;(Table S3). Paired tau levels and BT data were obtained for 15 subjects\u0026nbsp;(Extended Data Table 2). For BT–tau correlations, the difference between the average BT between 6–7\u0026nbsp;pm and 1–2\u0026nbsp;am was calculated. This interval was chosen because these times represented the sample average minimum and maximum BT, and as such their difference maximized the diurnal BT difference, or ∆BT. Plasma sampling times were selected to maximize efficiency in collecting data. Food intake was not regulated, but the morning sample was typically before the morning meal, whereas the evening sample was typically before the evening meal. Concentrations of plasma tau were measured using the neurology 3-PLEX kit and\u0026nbsp;Simoa HD-X instruments (Quanterix, Billerica, MA, USA) at the NYU Alzheimer’s Disease Center Biomarker Core according to the manufacturer’s instructions. Plasma extraction was performed as described previously. Assays were run in duplicate to obtain inter-assay coefficient of variations (CVs). The inter-assay CV was under 20% for all samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA minimum of two distinct experiments were carried out for each experimental condition. Prior to conducting each analysis of variance, an assessment of Gaussian distribution was performed, and its validity was confirmed through a Kolmogorov-Smirnov test (utilizing GraphPad Prism 9.0). Depending on the specific analysis, two-tailed t-tests (or Mann-Whitney tests), as well as one- or two-way ANOVAs (or Kruskal-Wallis tests), were applied. Post hoc analyses, involving either Tukey’s or Dunnett’s tests, were subsequently employed. A significance threshold of P \u0026lt; 0.05 was employed to determine statistical significance. The presentation of data incorporated either box and whisker plots (illustrating the range from minimum to maximum values, encompassing the median) or mean ± standard error of the mean (s.e.m). The scatter plots depicted on each graph provide an indication of the number of data points, and detailed statistical information is provided in Extended Data Table 4.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the authors used Chat- GPT-4.0 in order to improve readability and language. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. We are also grateful to the late Dr. Peter Davies (Feinstein Institute for Medical Research, Manhasset, USA) for the generous gift of anti-tau antibodies, to Dr. Luc Bu\u0026eacute;e (Centre de Recherche Jean-Pierre Aubert, Lille, France) for the SH-Tau3R cells, and to France Alzheimer for travel award to present this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was made possible by grants from the FRSQ, NSERC (RGPIN/05862- 2016), CIHR (IC121689), and Alzheimer Society of Canada (19-04) to EP and VP, and by an NIA/NIH (R01AG070866) award to EMB, by a postdoctoral award from the Alzheimer Society of Canada (to GC), and the National Institutes of Health (K76 AG054863), BrightFocus Foundation (A2016180S), and the Washington University/Centene Personalized Medicine Initiative. The sponsors had no role in the design, execution, interpretation, or writing of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eGC and EP conceived the preclinical studies and designed experiments. EMB conceived of and designed the human plasma study. BPL conceived and designed the human CSF studies. FDGM performed dissection and seeding of primary cultures. GC, ER, SDD, FL, EB, BK, PFF, and SC performed WB and dot blot experiments. FL performed qPCR experiments. GC performed ELISA assay, co-IP, membrane fluidity assay and caspase-3 activity assay. GC and FDGM performed immunocytochemistry, microscopy imaging and analysis. GC and IG designed and performed sleep-wake and sleep deprivation experiments in wild-type mice, and performed surgical implantation of abdominal probes for temperature recordings. GC and FL performed heat and cold exposures in hTau mice. NF performed mouse CSF collection. FL maintained animal colonies and genotyped animals. BPL, HL, and WL performed human CSF studies. JK, JH and DV conducted the human plasma studies, AY, HTW, and AP processed and analyzed temperature and tau data, EB conducted cognitve screening, DMR, IA, AWV, and RSO supervised sleep disorder and actigraphy screening, LD performed the tau SIMOA assays. GC, EP and EMB wrote the manuscript. All authors reviewed, corrected and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eAll authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and material availability:\u003c/strong\u003e The raw data supporting the findings of this study are available on: \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehttps://drive.google.com/drive/folders/1iQqmgMdC4GJ04ZQDHb0XIj5Jnd32-iZk?usp=drive_link\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLong, J. M. \u0026amp; Holtzman, D. M. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e179\u003c/strong\u003e, 312\u0026ndash;339 (2019).\u003c/li\u003e\n \u003cli\u003eBejanin, A. \u003cem\u003eet al.\u003c/em\u003e Tau pathology and neurodegeneration contribute to cognitive impairment in Alzheimer\u0026rsquo;s disease. \u003cem\u003eBrain\u003c/em\u003e \u003cstrong\u003e140\u003c/strong\u003e, 3286\u0026ndash;3300 (2017).\u003c/li\u003e\n \u003cli\u003eChai, X., Dage, J. L. \u0026amp; Citron, M. Constitutive secretion of tau protein by an unconventional mechanism. \u003cem\u003eNeurobiology of Disease\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 356\u0026ndash;366 (2012).\u003c/li\u003e\n \u003cli\u003eMerezhko, M. \u003cem\u003eet al.\u003c/em\u003e Secretion of Tau via an Unconventional Non-vesicular Mechanism. \u003cem\u003eCell Reports\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 2027-2035.e4 (2018).\u003c/li\u003e\n \u003cli\u003eMerezhko, M., Uronen, R.-L. \u0026amp; Huttunen, H. J. The Cell Biology of Tau Secretion. \u003cem\u003eFront Mol Neurosci\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 569818 (2020).\u003c/li\u003e\n \u003cli\u003eKatsinelos, T. \u003cem\u003eet al.\u003c/em\u003e Unconventional Secretion Mediates the Trans-cellular Spreading of Tau. \u003cem\u003eCell Reports\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 2039\u0026ndash;2055 (2018).\u003c/li\u003e\n \u003cli\u003eJohnson, G. V. W. \u003cem\u003eet al.\u003c/em\u003e The \u0026tau; Protein in Human Cerebrospinal Fluid in Alzheimer\u0026rsquo;s Disease Consists of Proteolytically Derived Fragments. \u003cem\u003eJournal of Neurochemistry\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 430\u0026ndash;433 (2002).\u003c/li\u003e\n \u003cli\u003eBorroni, B. \u003cem\u003eet al.\u003c/em\u003e Pattern of Tau forms in CSF is altered in progressive supranuclear palsy. \u003cem\u003eNeurobiology of Aging\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 34\u0026ndash;40 (2009).\u003c/li\u003e\n \u003cli\u003ePlouffe, V. \u003cem\u003eet al.\u003c/em\u003e Hyperphosphorylation and Cleavage at D421 Enhance Tau Secretion. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e36873 (2012).\u003c/li\u003e\n \u003cli\u003eBarth\u0026eacute;lemy, N. R. \u003cem\u003eet al.\u003c/em\u003e Sleep Deprivation Affects Tau Phosphorylation in Human Cerebrospinal Fluid. \u003cem\u003eAnnals of Neurology\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 700\u0026ndash;709 (2020).\u003c/li\u003e\n \u003cli\u003eGamblin, T. C. \u003cem\u003eet al.\u003c/em\u003e Caspase cleavage of tau: Linking amyloid and neurofibrillary tangles in Alzheimer\u0026rsquo;s disease. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 10032\u0026ndash;10037 (2003).\u003c/li\u003e\n \u003cli\u003eGuillozet-Bongaarts, A. L. \u003cem\u003eet al.\u003c/em\u003e Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivo. \u003cem\u003eJ Neurochem\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 1005\u0026ndash;1014 (2006).\u003c/li\u003e\n \u003cli\u003eSandhu, P. \u003cem\u003eet al.\u003c/em\u003e Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer\u0026rsquo;s Disease. \u003cem\u003eBioorganic \u0026amp; Medicinal Chemistry Letters\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 642\u0026ndash;652 (2017).\u003c/li\u003e\n \u003cli\u003eHolth, J. K. \u003cem\u003eet al.\u003c/em\u003e The sleep-wake cycle regulates brain interstitial fluid tau in mice and CSF tau in humans. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e363\u003c/strong\u003e, 880\u0026ndash;884 (2019).\u003c/li\u003e\n \u003cli\u003eStevanovic, K. \u003cem\u003eet al.\u003c/em\u003e Disruption of normal circadian clock function in a mouse model of tauopathy. \u003cem\u003eExperimental Neurology\u003c/em\u003e \u003cstrong\u003e294\u003c/strong\u003e, 58\u0026ndash;67 (2017).\u003c/li\u003e\n \u003cli\u003eWang, C. \u0026amp; Holtzman, D. M. Bidirectional relationship between sleep and Alzheimer\u0026rsquo;s disease: role of amyloid, tau, and other factors. \u003cem\u003eNeuropsychopharmacol.\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 104\u0026ndash;120 (2020).\u003c/li\u003e\n \u003cli\u003eRothman, S. M., Herdener, N., Frankola, K. A., Mughal, M. R. \u0026amp; Mattson, M. P. Chronic mild sleep restriction accentuates contextual memory impairments, and accumulations of cortical A\u0026beta; and pTau in a mouse model of Alzheimer\u0026rsquo;s disease. \u003cem\u003eBrain Research\u003c/em\u003e \u003cstrong\u003e1529\u003c/strong\u003e, 200\u0026ndash;208 (2013).\u003c/li\u003e\n \u003cli\u003eDi Meco, A., Joshi, Y. B. \u0026amp; Pratic\u0026ograve;, D. Sleep deprivation impairs memory, tau metabolism, and synaptic integrity of a mouse model of Alzheimer\u0026rsquo;s disease with plaques and tangles. \u003cem\u003eNeurobiology of Aging\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 1813\u0026ndash;1820 (2014).\u003c/li\u003e\n \u003cli\u003eLucey, B. P. \u003cem\u003eet al.\u003c/em\u003e Effect of sleep on overnight cerebrospinal fluid amyloid \u0026beta; kinetics. \u003cem\u003eAnnals of Neurology\u003c/em\u003e \u003cstrong\u003e83\u003c/strong\u003e, 197\u0026ndash;204 (2018).\u003c/li\u003e\n \u003cli\u003eGuisle, I. \u003cem\u003eet al.\u003c/em\u003e Circadian and sleep/wake-dependent variations in tau phosphorylation are driven by temperature. \u003cem\u003eSleep\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, zsz266 (2020).\u003c/li\u003e\n \u003cli\u003eGuisle, I. \u003cem\u003eet al.\u003c/em\u003e Sauna-like conditions or menthol treatment reduce tau phosphorylation through mild hyperthermia. \u003cem\u003eNeurobiology of Aging\u003c/em\u003e S019745802200032X (2022) doi:10.1016/j.neurobiolaging.2022.02.011.\u003c/li\u003e\n \u003cli\u003eHohmann \u0026amp; Dehghani. The Cytoskeleton\u0026mdash;A Complex Interacting Meshwork. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 362 (2019).\u003c/li\u003e\n \u003cli\u003eLee, H.-J., Patel, S. \u0026amp; Lee, S.-J. Intravesicular Localization and Exocytosis of \u0026alpha;-Synuclein and its Aggregates. \u003cem\u003eJ. Neurosci.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 6016\u0026ndash;6024 (2005).\u003c/li\u003e\n \u003cli\u003eRabouille, C. Pathways of Unconventional Protein Secretion. \u003cem\u003eTrends in Cell Biology\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 230\u0026ndash;240 (2017).\u003c/li\u003e\n \u003cli\u003eSteringer, J. P. \u0026amp; Nickel, W. The molecular mechanism underlying unconventional secretion of Fibroblast Growth Factor 2 from tumour cells: Unconventional secretion of FGF2. \u003cem\u003eBiol. Cell\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 375\u0026ndash;380 (2017).\u003c/li\u003e\n \u003cli\u003eBusse, M. \u003cem\u003eet al.\u003c/em\u003e Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 32802\u0026ndash;32810 (2007).\u003c/li\u003e\n \u003cli\u003eLiu, C.-C. \u003cem\u003eet al.\u003c/em\u003e Neuronal heparan sulfates promote amyloid pathology by modulating brain amyloid-\u0026beta; clearance and aggregation in Alzheimer\u0026rsquo;s disease. \u003cem\u003eSci. Transl. Med.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 332ra44-332ra44 (2016).\u003c/li\u003e\n \u003cli\u003eHud\u0026aacute;k, A., Letoha, A., Vizler, C. \u0026amp; Letoha, T. Syndecan-3 as a Novel Biomarker in Alzheimer\u0026rsquo;s Disease. \u003cem\u003eIJMS\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 3407 (2022).\u003c/li\u003e\n \u003cli\u003eBlicher, A., Wodzinska, K., Fidorra, M., Winterhalter, M. \u0026amp; Heimburg, T. The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics. \u003cem\u003eBiophysical Journal\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 4581\u0026ndash;4591 (2009).\u003c/li\u003e\n \u003cli\u003eFan, W. \u0026amp; Evans, R. M. Turning Up the Heat on Membrane Fluidity. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 962\u0026ndash;963 (2015).\u003c/li\u003e\n \u003cli\u003eMishkind, M., Vermeer, J. E. M., Darwish, E. \u0026amp; Munnik, T. Heat stress activates phospholipase D and triggers PIP2 accumulation at the plasma membrane and nucleus. \u003cem\u003eThe Plant Journal\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 10\u0026ndash;21 (2009).\u003c/li\u003e\n \u003cli\u003ePrieto, J. A. \u003cem\u003eet al.\u003c/em\u003e Pho85 and PI(4,5)P2 regulate different lipid metabolic pathways in response to cold. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids\u003c/em\u003e \u003cstrong\u003e1865\u003c/strong\u003e, 158557 (2020).\u003c/li\u003e\n \u003cli\u003eBenedict, C., Blennow, K., Zetterberg, H. \u0026amp; Cedernaes, J. Effects of acute sleep loss on diurnal plasma dynamics of CNS health biomarkers in young men. \u003cem\u003eNeurology\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, (2020).\u003c/li\u003e\n \u003cli\u003ePooler, A. M., Phillips, E. C., Lau, D. H. W., Noble, W. \u0026amp; Hanger, D. P. Physiological release of endogenous tau is stimulated by neuronal activity. \u003cem\u003eEMBO Rep\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 389\u0026ndash;394 (2013).\u003c/li\u003e\n \u003cli\u003eYamada, K. \u003cem\u003eet al.\u003c/em\u003e Neuronal activity regulates extracellular tau in vivo. \u003cem\u003eJournal of Experimental Medicine\u003c/em\u003e \u003cstrong\u003e211\u003c/strong\u003e, 387\u0026ndash;393 (2014).\u003c/li\u003e\n \u003cli\u003eVyazovskiy, V. V., Cirelli, C., Pfister-Genskow, M., Faraguna, U. \u0026amp; Tononi, G. Molecular and electrophysiological evidence for net synaptic potentiation in wake and depression in sleep. \u003cem\u003eNat Neurosci\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 200\u0026ndash;208 (2008).\u003c/li\u003e\n \u003cli\u003eClark, W. G. \u0026amp; Coldwell, B. A. The hypothermic effect of tetrodotoxin in the unanaesthetized cat. \u003cem\u003eThe Journal of Physiology\u003c/em\u003e \u003cstrong\u003e230\u003c/strong\u003e, 477\u0026ndash;492 (1973).\u003c/li\u003e\n \u003cli\u003eTambyah, P. A., Hui, K. P., Gopalakrishnakone, P., Chin, N. K. \u0026amp; Chan, T. B. Central-nervous-system effects of tetrodotoxin poisoning. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e343\u003c/strong\u003e, 538\u0026ndash;539 (1994).\u003c/li\u003e\n \u003cli\u003eKim, J. A. \u0026amp; Connors, B. W. High temperatures alter physiological properties of pyramidal cells and inhibitory interneurons in hippocampus. \u003cem\u003eFront. Cell. Neurosci.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, (2012).\u003c/li\u003e\n \u003cli\u003eVan Hook, M. J. Temperature effects on synaptic transmission and neuronal function in the visual thalamus. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, e0232451 (2020).\u003c/li\u003e\n \u003cli\u003eKim, T. \u003cem\u003eet al.\u003c/em\u003e Thermal effects on neurons during stimulation of the brain. \u003cem\u003eJ. Neural Eng.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 056029 (2022).\u003c/li\u003e\n \u003cli\u003eZhang, J. \u003cem\u003eet al.\u003c/em\u003e Recent progresses in novel in vitro models of primary neurons: A biomaterial perspective. \u003cem\u003eFront. Bioeng. Biotechnol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 953031 (2022).\u003c/li\u003e\n \u003cli\u003eNicholls, S. B. \u003cem\u003eet al.\u003c/em\u003e Characterization of TauC3 antibody and demonstration of its potential to block tau propagation. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0177914 (2017).\u003c/li\u003e\n \u003cli\u003eVana, L. \u003cem\u003eet al.\u003c/em\u003e Progression of Tau Pathology in Cholinergic Basal Forebrain Neurons in Mild Cognitive Impairment and Alzheimer\u0026rsquo;s Disease. \u003cem\u003eThe American Journal of Pathology\u003c/em\u003e \u003cstrong\u003e179\u003c/strong\u003e, 2533\u0026ndash;2550 (2011).\u003c/li\u003e\n \u003cli\u003eBiundo, F. \u003cem\u003eet al.\u003c/em\u003e Abolishing Tau cleavage by caspases at Aspartate421 causes memory/synaptic plasticity deficits and pre-pathological Tau alterations. \u003cem\u003eTransl Psychiatry\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e1198\u0026ndash;e1198 (2017).\u003c/li\u003e\n \u003cli\u003eChi, H. \u003cem\u003eet al.\u003c/em\u003e Cleavage of human tau at Asp421 inhibits hyperphosphorylated tau induced pathology in a Drosophila model. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 13482 (2020).\u003c/li\u003e\n \u003cli\u003eZhang, Q., Zhang, X. \u0026amp; Sun, A. Truncated tau at D421 is associated with neurodegeneration and tangle formation in the brain of Alzheimer transgenic models. \u003cem\u003eActa Neuropathol\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 687\u0026ndash;697 (2009).\u003c/li\u003e\n \u003cli\u003eConze, C. \u003cem\u003eet al.\u003c/em\u003e Caspase-cleaved tau is senescence-associated and induces a toxic gain of function by putting a brake on axonal transport. \u003cem\u003eMol Psychiatry\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 3010\u0026ndash;3023 (2022).\u003c/li\u003e\n \u003cli\u003eTroquier, L. \u003cem\u003eet al.\u003c/em\u003e Targeting phospho-Ser422 by active Tau Immunotherapy in the THYTau22 mouse model: a suitable therapeutic approach. \u003cem\u003eCurr Alzheimer Res\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 397\u0026ndash;405 (2012).\u003c/li\u003e\n \u003cli\u003eChoi, Y. R., Park, S. J. \u0026amp; Park, S. M. Molecular events underlying the cell‐to‐cell transmission of \u0026alpha;‐synuclein. \u003cem\u003eThe FEBS Journal\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 6593\u0026ndash;6602 (2021).\u003c/li\u003e\n \u003cli\u003eSurridge, C. D. \u0026amp; Burns, R. G. The Difference in the Binding of Phosphatidylinositol Distinguishes MAP2 from MAP2C and Tau. \u003cem\u003eBiochemistry\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 8051\u0026ndash;8057 (1994).\u003c/li\u003e\n \u003cli\u003eLauri, S. E. \u003cem\u003eet al.\u003c/em\u003e Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan ( \u003cem\u003eN\u003c/em\u003e -syndecan) in Hippocampal Long-Term Potentiation. \u003cem\u003eJ. Neurosci.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 1226\u0026ndash;1235 (1999).\u003c/li\u003e\n \u003cli\u003eSetlow, V. P., Roth, S. \u0026amp; Edidin, M. Effects of temperature on glycosyltransferase activity in the plasma membrane of L cells. \u003cem\u003eExperimental Cell Research\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, 55\u0026ndash;61 (1979).\u003c/li\u003e\n \u003cli\u003eMcGinty, D., Alam, M. N., Szymusiak, R., Nakao, M. \u0026amp; Yamamoto, M. Hypothalamic sleep-promoting mechanisms: coupling to thermoregulation. \u003cem\u003eArch Ital Biol\u003c/em\u003e \u003cstrong\u003e139\u003c/strong\u003e, 63\u0026ndash;75 (2001).\u003c/li\u003e\n \u003cli\u003eWang, J. \u003cem\u003eet al.\u003c/em\u003e Thermoneutral Temperature Exposure Enhances Slow Wave Sleep with a Correlated Improvement in Amyloid Pathology in a Triple-Transgenic Mouse Model of Alzheimer\u0026rsquo;s Disease. \u003cem\u003eSLEEP\u003c/em\u003e zsae078 (2024) doi:10.1093/sleep/zsae078.\u003c/li\u003e\n \u003cli\u003eWarfield, A. E. \u003cem\u003eet al.\u003c/em\u003e A brainstem to circadian system circuit links Tau pathology to sundowning-related disturbances in an Alzheimer\u0026rsquo;s disease mouse model. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 5027 (2023).\u003c/li\u003e\n \u003cli\u003eRasmussen, M. K., Mestre, H. \u0026amp; Nedergaard, M. The glymphatic pathway in neurological disorders. \u003cem\u003eThe Lancet Neurology\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1016\u0026ndash;1024 (2018).\u003c/li\u003e\n \u003cli\u003eMedina, M. \u0026amp; Avila, J. The role of extracellular Tau in the spreading of neurofibrillary pathology. \u003cem\u003eFront. Cell. Neurosci.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, (2014).\u003c/li\u003e\n \u003cli\u003eKlegeris, A., Schulzer, M., Harper, D. G. \u0026amp; McGeer, P. L. Increase in core body temperature of Alzheimer\u0026rsquo;s disease patients as a possible indicator of chronic neuroinflammation: a meta-analysis. \u003cem\u003eGerontology\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 7\u0026ndash;11 (2007).\u003c/li\u003e\n \u003cli\u003eHoltzman, A. \u0026amp; Simon, E. W. Body temperature as a risk factor for Alzheimer\u0026rsquo;s disease. \u003cem\u003eMedical Hypotheses\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 440\u0026ndash;444 (2000).\u003c/li\u003e\n \u003cli\u003eWhittington, R., Papon, M.-A., Chouinard-Decorte, F. \u0026amp; Planel, E. Hypothermia and Alzheimers Disease Neuropathogenic Pathways. \u003cem\u003eCAR\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 717\u0026ndash;725 (2010).\u003c/li\u003e\n \u003cli\u003eBlessing, E. M. \u003cem\u003eet al.\u003c/em\u003e Association between lower body temperature and increased tau pathology in cognitively normal older adults. \u003cem\u003eNeurobiology of Disease\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 105748 (2022).\u003c/li\u003e\n \u003cli\u003eLim, A. S. P., Kowgier, M., Yu, L., Buchman, A. S. \u0026amp; Bennett, D. A. Sleep Fragmentation and the Risk of Incident Alzheimer\u0026rsquo;s Disease and Cognitive Decline in Older Persons. \u003cem\u003eSleep\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 1027\u0026ndash;1032 (2013).\u003c/li\u003e\n \u003cli\u003eTodd, W. D. Potential Pathways for Circadian Dysfunction and Sundowning-Related Behavioral Aggression in Alzheimer\u0026rsquo;s Disease and Related Dementias. \u003cem\u003eFront Neurosci\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 910 (2020).\u003c/li\u003e\n \u003cli\u003eHarper, D. G. \u003cem\u003eet al.\u003c/em\u003e Disturbance of endogenous circadian rhythm in aging and Alzheimer disease. \u003cem\u003eAm J Geriatr Psychiatry\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 359\u0026ndash;368 (2005).\u003c/li\u003e\n \u003cli\u003eHolth, J. K., Patel, T. K. \u0026amp; Holtzman, D. M. Sleep in Alzheimer\u0026rsquo;s Disease\u0026ndash;Beyond Amyloid. \u003cem\u003eNeurobiology of Sleep and Circadian Rhythms\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 4\u0026ndash;14 (2017).\u003c/li\u003e\n \u003cli\u003eSadleir, K. R. \u0026amp; Vassar, R. Connections between ApoE, sleep, and A\u0026beta; and tau pathologies in Alzheimer\u0026rsquo;s disease. \u003cem\u003eJournal of Clinical Investigation\u003c/em\u003e \u003cstrong\u003e133\u003c/strong\u003e, e171838 (2023).\u003c/li\u003e\n \u003cli\u003eIshida, K. \u003cem\u003eet al.\u003c/em\u003e Glymphatic system clears extracellular tau and protects from tau aggregation and neurodegeneration. \u003cem\u003eJournal of Experimental Medicine\u003c/em\u003e \u003cstrong\u003e219\u003c/strong\u003e, e20211275 (2022).\u003c/li\u003e\n \u003cli\u003eXie, L. \u003cem\u003eet al.\u003c/em\u003e Sleep Drives Metabolite Clearance from the Adult Brain. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e342\u003c/strong\u003e, 373\u0026ndash;377 (2013).\u003c/li\u003e\n \u003cli\u003eSohar, E., Shoenfeld, Y., Shapiro, Y., Ohry, A. \u0026amp; Cabili, S. Effects of exposure to Finnish sauna. \u003cem\u003eIsr J Med Sci\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1275\u0026ndash;1282 (1976).\u003c/li\u003e\n \u003cli\u003eLaukkanen, J. A. \u0026amp; Kunutsor, S. K. The multifaceted benefits of passive heat therapies for extending the healthspan: A comprehensive review with a focus on Finnish sauna. \u003cem\u003eTemperature\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 27\u0026ndash;51 (2024).\u003c/li\u003e\n \u003cli\u003eG\u0026oacute;mez-Ramos, A., D\u0026iacute;az-Hern\u0026aacute;ndez, M., Rubio, A., Miras-Portugal, M. T. \u0026amp; Avila, J. Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells. \u003cem\u003eMolecular and Cellular Neuroscience\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 673\u0026ndash;681 (2008).\u003c/li\u003e\n \u003cli\u003ePern\u0026egrave;gre, C., Duquette, A. \u0026amp; Leclerc, N. Tau Secretion: Good and Bad for Neurons. \u003cem\u003eFront. Neurosci.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 649 (2019).\u003c/li\u003e\n \u003cli\u003eDelobel, P. \u003cem\u003eet al.\u003c/em\u003e Stable-Tau Overexpression in Human Neuroblastoma Cells: An Open Door for Explaining Neuronal Death in Tauopathies. \u003cem\u003eAnnals of the New York Academy of Sciences\u003c/em\u003e \u003cstrong\u003e1010\u003c/strong\u003e, 623\u0026ndash;634 (2003).\u003c/li\u003e\n \u003cli\u003eAndorfer, C. \u003cem\u003eet al.\u003c/em\u003e Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms. \u003cem\u003eJ. Neurochem.\u003c/em\u003e \u003cstrong\u003e86\u003c/strong\u003e, 582\u0026ndash;590 (2003).\u003c/li\u003e\n \u003cli\u003eDuff, K. \u003cem\u003eet al.\u003c/em\u003e Characterization of Pathology in Transgenic Mice Over-Expressing Human Genomic and cDNA Tau Transgenes. \u003cem\u003eNeurobiology of Disease\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 87\u0026ndash;98 (2000).\u003c/li\u003e\n \u003cli\u003eTucker, K. L., Meyer, M. \u0026amp; Barde, Y.-A. Neurotrophins are required for nerve growth during development. \u003cem\u003eNat Neurosci\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 29\u0026ndash;37 (2001).\u003c/li\u003e\n \u003cli\u003ePlanel, E. \u003cem\u003eet al.\u003c/em\u003e Anesthesia Leads to Tau Hyperphosphorylation through Inhibition of Phosphatase Activity by Hypothermia. \u003cem\u003eJournal of Neuroscience\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 3090\u0026ndash;3097 (2007).\u003c/li\u003e\n \u003cli\u003eCanet, G. \u003cem\u003eet al.\u003c/em\u003e Temperature-induced Artifacts in Tau Phosphorylation: Implications for Reliable Alzheimer\u0026rsquo;s Disease Research. \u003cem\u003eExp Neurobiol\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 423\u0026ndash;440 (2023).\u003c/li\u003e\n \u003cli\u003eCarroll, D. A., Denenberg, V. H. \u0026amp; Thoman, E. B. Reliability and validity of computer scoring of behavioral sleep-wake states in rats and rabbits. \u003cem\u003ePhysiology \u0026amp; Behavior\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 269\u0026ndash;273 (1993).\u003c/li\u003e\n \u003cli\u003eTournissac, M. \u003cem\u003eet al.\u003c/em\u003e Repeated cold exposures protect a mouse model of Alzheimer\u0026rsquo;s disease against cold-induced tau phosphorylation. \u003cem\u003eMolecular Metabolism\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 110\u0026ndash;120 (2019).\u003c/li\u003e\n \u003cli\u003eFereydouni-Forouzandeh, P. \u003cem\u003eet al.\u003c/em\u003e Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts. in \u003cem\u003eTau Protein\u003c/em\u003e (ed. Smet-Nocca, C.) vol. 2754 309\u0026ndash;321 (Springer US, New York, NY, 2024).\u003c/li\u003e\n \u003cli\u003eLucey, B. P. \u003cem\u003eet al.\u003c/em\u003e Suvorexant Acutely Decreases Tau Phosphorylation and A\u0026beta; in the Human CNS. \u003cem\u003eAnnals of Neurology\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 27\u0026ndash;40 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tau, unconventional protein secretion, sleep-wake cycle, body temperature, Alzheimer’s disease","lastPublishedDoi":"10.21203/rs.3.rs-4384494/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4384494/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe sleep-wake cycle regulates interstitial fluid and cerebrospinal fluid (CSF) tau levels in both mouse and human by mechanisms that remain unestablished. Here, we reveal a novel pathway by which wakefulness increases extracellular tau levels in mouse and humans. In mice, higher body temperature (BT) associated with wakefulness and sleep deprivation increased CSF tau. \u003cem\u003eIn vitro\u003c/em\u003e, wakefulness temperatures upregulated tau secretion \u003cem\u003evia\u003c/em\u003e a temperature-dependent increase in activity and expression of unconventional protein secretion pathway-1 components, namely caspase-3-mediated C-terminal cleavage of tau (TauC3), and membrane expression of PIP\u003csub\u003e2\u003c/sub\u003e and syndecan-3. In humans, the increase in both CSF and plasma tau levels observed post-wakefulness correlated with BT increase during wakefulness. Our findings suggest sleep-wake variation in BT may contribute to regulating extracellular tau levels, highlighting the importance of thermoregulation in pathways linking sleep disturbance to neurodegeneration, and the potential for thermal intervention to prevent or delay tau-mediated neurodegeneration.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e*Geoffrey Canet, Esther M. Blessing, and Emmanuel Planel contributed equally to this work.\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Sleep-wake body temperature regulates tau secretion in mice and correlates with CSF and plasma tau in humans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-14 10:36:20","doi":"10.21203/rs.3.rs-4384494/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c83247a1-4651-43ac-8349-b71d2e6e00fe","owner":[],"postedDate":"May 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31842366,"name":"Biological sciences/Neuroscience/Cellular neuroscience"},{"id":31842367,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":31842368,"name":"Health sciences/Biomarkers/Diagnostic markers"}],"tags":[],"updatedAt":"2024-05-16T17:05:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-14 10:36:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4384494","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4384494","identity":"rs-4384494","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}