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

Abstract

Sleep disturbance is bidirectionally associated with an increased risk of Alzheimer's disease and other tauopathies. While the sleep-wake cycle regulates interstitial and cerebrospinal fluid (CSF) tau levels, the underlying mechanisms remain unknown. Understanding these mechanisms is crucial, given the evidence that tau pathology spreads through neuron-to-neuron transfer, involving the secretion and internalization of pathological tau forms. Here, we combined in vitro, in vivo, and clinical methods to reveal a pathway by which changes in body temperature (BT) over the sleep-wake cycle modulate extracellular tau levels. In mice, a higher BT during wakefulness and sleep deprivation increased CSF and plasma tau levels, while also upregulating unconventional protein secretion pathway I (UPS-I) events including (a) intracellular tau dephosphorylation, (b) caspase 3-mediated cleavage of tau (TauC3), and (c) membrane translocation of tau through binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and syndecan 3. In humans, the increase in CSF and plasma tau levels observed after wakefulness correlated with BT increases during wakefulness. By demonstrating that sleep-wake variation in BT regulates extracellular tau levels, our findings highlight the importance of thermoregulation in linking sleep disturbances to tau-mediated neurodegeneration and the preventative potential of thermal interventions.

Keywords: Alzheimer disease; Cell biology; Neuroscience; Proteoglycans.

Figure 1. Tau secretion is temperature dependent in neuronal cells.

(A) Twenty-four hours after seeding, SH-Tau3R cells were exposed to 35°C, 37°C, 38°C, or 39°C for 72 hours. (B) Extracellular accumulation of tau protein (Tau3R) (n = 6–12; mean ± SEM; error envelopes are shown in light purple) and LDH (n = 4–9; mean ± SEM) over time in cell medium of neurons cultured at 37°C. (C) The increase in extracellular tau levels (pg/mL) was temperature dependent in SH-Tau3R cells exposed to 35°C, 37°C, or 38°C for 72 hours (n = 6; Tukey’s test; box and whiskers show minimum to maximum and median). (D and E) The increase in extracellular tau levels was temperature dependent (Tau3R, TauC, DA9, and Tau12 antibodies) in SH-Tau3R cells exposed to 35°C, 37°C, or 38°C (n = 7–16; Tukey’s test; mean ± SEM). (F and G) The phosphorylation level of extracellular tau at AT270 (T181), S199, CP13 (S202), T205, AT100 (S212/S214), MC6 (S235), and PHF1 (S396/S404) was decreased at 38°C compared with 35°C or 37°C (n = 7–16; Tukey’s test; mean ± SEM. (H) Four days after seeding, mouse primary cortical neurons were exposed at 35°C, 37°C, or 38°C for 72 hours. (I) The increase in extracellular tau levels was temperature dependent in mouse primary neurons exposed to 35°C, 37°C, or 38°C (n = 6; Dunnett’s test; box and whiskers show minimum to maximum and median). (J and K) The increase in extracellular tau levels was temperature dependent (Tau3R antibody), whereas its phosphorylation level at S199 and T205 was decreased at 38°C compared with 35°C or 37°C (n = 11–12; Tukey’s test; mean ± SEM). (K) Data are from a minimum of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2. Wakefulness temperatures promote tau release through its caspase 3–mediated cleavage in neuronal cells.

(A) The increase in the proteolytic activity of caspase 3 was temperature dependent in SH-Tau3R cells (n = 6; Tukey’s; box and whiskers show minimum to maximum and median). (B and C) The intracellular expression levels of caspase 3 (Casp-3), p-Tau (S422), TauC3, and Tau46 were temperature dependent in SH-Tau3R cells (left panels) or primary neurons (right panels) (n = 5–6; Tukey’s test; mean ± SEM). (D and E) Extracellular levels of TauC3 and Tau46 were oppositely temperature dependent in SH-Tau3R cells (left panels) or primary neurons (right panels) (n = 6–12; Tukey’s test; mean ± SEM). (F) The inhibition of caspase 3 with z-DEVD-FMK (20 μM) decreased total tau (Tau3R) (n = 5–12; unpaired, 2-tailed t test) and TauC3 (n = 3–8; Mann-Whitney U test) extracellular levels in SH-Tau3R cells exposed to 35°C, 37°C, or 38°C (mean ± SEM). The inhibition of caspase 3 with z-DEVD-FMK (20 μM) decreased the extracellular levels of (G) total tau (ELISA; n = 6; unpaired, 2-tailed t test) and (H) Tau3R and TauC3 (dot blots, n = 5–6; unpaired, 2-tailed t test; mean ± SEM. (I) The genetic knockdown of caspase 3 decreased total tau (Tau3R) and TauC3 extracellular levels in SH-Tau3R cells exposed to 37°C (n = 11; unpaired, 2-tailed t test; mean ± SEM). Data are from a minimum of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3. Wakefulness temperatures promote TauC3 interaction with SDC3 in neuronal cells.

(A and B) Intracellular expression levels of SDC3 and PIP₂ were temperature dependent in SH-Tau3R cells and in primary neurons (n = 6; Tukey’s test; mean ± SEM). Data are from a minimum of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the respective control condition. (C) Merged staining images show a temperature-dependent increase of colocalization between SDC3 (purple) and TauC3 (yellow) in primary mouse cortical neurons (marked with white arrowheads). Scale bars: 20 μm).

Figure 4. Wakefulness temperatures promote TauC3 secretion though its interaction with PIP₂ and SDC3.

(A and B) SDC3 knockdown decreased total tau (Tau3R) and TauC3 extracellular levels in SH-Tau3R cells exposed to 37°C, and the cotransfection of caspase 3 siRNA and SDC3 siRNA induced additive effects in the suppression of tau secretion (n = 5–6; Tukey’s test; mean ± SEM). ^†P < 0.05 and ^††P < 0.01 versus the indicated condition. (C) EXT1 knockdown decreased total tau (Tau3R) and TauC3 extracellular levels in SH-Tau3R cells exposed to 37°C (n = 11; unpaired, 2-tailed t test; mean ± SEM). (D) Knockdown of caspase 3 and SDC3 decreased the intracellular expression of TauC, while increasing tau phosphorylation at S422 (representative Western blot; n = 3). (E) The increase in membrane fluidity was temperature dependent in SH-Tau3R cells (n = 8; Tukey’s test; box and whiskers show minimum to maximum and median). (F and G) PIP₂ showed a better binding affinity for TauC3 than for full-length tau (anti-DA9, anti-Tau46, and anti-TauC antibodies) in SH-Tau3R cells exposed to 37°C. (H–J) The increase in binding between PIP₂ and TauC3 was temperature dependent (n = 3; Kruskal-Wallis test; mean ± SEM). Data are from a minimum of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the respective control condition.

Figure 5. Wakefulness, SD, and mild hyperthermia promote UPS-I–dependent tau release in CSF in mice.

(A) C57BL6 mice were euthanized during sleep (CT4, 10:00 am) or the active period (CT16, 8:45 pm). (B) Awake mice had higher rectal temperatures (°C) compared with sleeping mice (n = 7; unpaired, 2-tailed t test; box and whiskers with minimum to maximum and median). (C and D) Caspase 3, TauC3, SDC3, and PIP₂ expression levels were increased in the cortices of awake mice compared with levels in sleeping mice, whereas p-Tau (S422) levels were decreased (n = 10; unpaired, 2-tailed t test; mean ± SEM). (E) C57BL6 mice were sleep deprived (n = 9) for 6 hours and then compared with naive mice (n = 7). (F) SD inhibited the drop in BT (°C) induced by sleep (n = 5; Tukey’s; mean ± SEM shown as error envelopes). (G and H) Caspase 3, TauC3, and PIP₂ expression levels were increased in the cortices of sleep-deprived mice compared with naive mice, whereas p-Tau (S422) and Tau46 levels were decreased (unpaired, 2-tailed t test; mean ± SEM). (I and J) hTau mice were exposed for 4 hours to 4°C (hypo, n = 6) or 38°C (hyper, n = 6) and then compared with naive (normo) mice (normo, n = 5; Šidák’s test; mean ± SEM shown as error envelopes). ***P < 0.001 versus the naive (normo) group at baseline; ^†††P < 0.001 versus the respective group at baseline. (K) Hyperthermic (hyper) mice had higher CSF tau levels than did hypothermic (hypo) mice (n = 5 normo; n = 3 hypo; n = 6 hyper; Kruskal-Wallis test; mean ± SEM). CSF tau levels were significantly correlated with rectal BT (°C) (Pearson’s test; error envelopes are shown in gray). (L) Hyperthermic mice had higher plasma tau levels than did hypothermic mice (n = 5 normo; n = 6 hypo; n = 5 hyper; Dunnett’s test; mean ± SEM). Plasma tau levels were significantly correlated with rectal BT (°C) (Pearson’s test; error envelopes are shown in gray). Data are from a minimum of 2 independent experiments. NaN, not a number. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6. BT correlates with CSF and plasma tau levels in humans.

(A) The variation in total CSF tau concentrations between 8 am and 4 pm was significantly correlated to the variation in oral temperature at the same times (n = 13; Pearson’s test), (B) but no correlation was observed for CSF NfL concentrations or oral temperature (n = 11; Pearson’s test). (C) The variation in total plasma tau concentrations between 7 am and 7 pm was significantly correlated to the variation in core BT between 6 pm and 1 am (n = 15; Pearson’s test). Standard error bars are shown as error envelopes in light purple. *P < 0.05 and **P < 0.01.

Figure 7. Proposed mechanism to elucidate the regulatory effect of BT on tau secretion via the UPS-I pathway during the sleep-wake cycle.

During wakefulness, the physiological elevation in BT instigates a series of events triggering tau secretion. (i) There is an increase in caspase 3 activity, concomitant with tau dephosphorylation, especially at S422, leading to augmented cleavage of tau at D421 and yielding the TauC3 fragment. (ii) Subsequently, TauC3 is sequestered at the inner leaflet of the plasma membrane due to its strong affinity binding for PIP₂. (iii and iv) The interplay between TauC3 and SDC3 initiates and facilitates the export process across the plasma membrane, which has 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 PIP₂ and SDC3, along with lower membrane fluidity, resulting in diminished extracellular tau levels. GSK3β, glycogen synthase kinase 3β; JNK, c-Jun N-terminal kinase.