The unfolded protein response mediates reversible tau phosphorylation induced by metabolic stress.

The unfolded protein response (UPR) is activated in neurodegenerative tauopathies such as Alzheimer's disease (AD) in close connection with early stages of tau pathology. Metabolic disturbances are strongly associated with increased risk for AD and are a potent inducer of the UPR. Here, we demonstrate that metabolic stress induces the phosphorylation of endogenous tau via activation of the UPR. Strikingly, upon restoration of the metabolic homeostasis, not only the levels of the UPR markers pPERK, pIRE1α and BiP, but also tau phosphorylation are reversed both in cell models as well as in torpor, a physiological hypometabolic model in vivo. Intervention in the UPR using the global UPR inhibitor TUDCA or a specific small-molecule inhibitor of the PERK signaling pathway, inhibits the metabolic stress-induced phosphorylation of tau. These data support a role for UPR-mediated tau phosphorylation as part of an adaptive response to metabolic stress. Failure to restore the metabolic homeostasis will lead to prolonged UPR activation and tau phosphorylation, and may thus contribute to AD pathogenesis. We demonstrate that the UPR is functionally involved in the early stages of tau pathology. Our data indicate that targeting of the UPR may be employed for early intervention in tau-related neurodegenerative diseases.

Blood cell dynamics during hibernation in the European Ground Squirrel.

Hibernation is a unique natural model to study large and specific modulation in numbers of leukocytes and thrombocytes, with potential relevance for medical application. Hibernating animals cycle through cold (torpor) and warm (arousal) phases. Previous research demonstrated clearance of leukocytes and thrombocytes from the circulation during torpor, but did not provide information regarding the timing during torpor or the subtype of leukocytes affected. To study the influence of torpor-bout duration on clearance of circulating cells, we measured blood cell dynamics in the European Ground Squirrel. Numbers of leukocytes and thrombocytes decreased within 24h of torpor by 90% and remained unchanged during the remainder of the torpor-bout. Differential counts demonstrated that granulocytes, lymphocytes and monocytes are all affected by torpor. Although a decreased production might explain the reduced number of thrombocytes, granulocytes and monocytes, this cannot explain the observed lymphopenia since lymphocytes have a much lower turnover rate than thrombocytes, granulocytes and monocytes. In conclusion, although underlying biochemical signaling pathways need to be unraveled, our data show that the leukocyte count drops dramatically after entrance into torpor and that euthermic cell counts are restored within 1.5h after onset of arousal, even before body temperature is fully normalized.

Dynamics in the ultrastructure of asymmetric axospinous synapses in the frontal cortex of hibernating European ground squirrels (Spermophilus citellus).

Recent theories on the function of arousals from torpor in hibernating mammals focus on the repair of the central nervous system from damage accumulating during prolonged hypothermia. In this framework, we investigated the synaptic ultrastructure in Layer 2 of the frontal cortex from hibernating European ground squirrels (Spermophilus citellus) sacrificed at four different phases in the torpor-arousal cycle. Using electron microscopy, we quantified synapse number and morphometric data on asymmetric axospinous synapses. Length, width, and surface area of postsynaptic densities (PSDs), and the synaptic apposition length of the analyzed synapse were measured. Five groups of animals were compared during entrance into torpor (Torpor Early, TE, n = 6), late torpor (Torpor Late, TL, n = 5), beginning of euthermic arousal episodes (Arousal Early, AE, n = 5), late in the euthermic arousal episode (Arousal Late, AL, n = 5), and during continuous euthermy in spring (EU, n = 6). The results showed that during torpor and at the beginning of arousals the PSD length and synaptic apposition length are significantly increased compared to synapses during late arousal and in spring conditions. In contrast, the width and surface area of the PSDs are decreased in torpor. At the beginning of an arousal the width of the PSD increases and gains maximum value in late arousals (AL), returning to spring (EU) values. No differences were found in total number of synapses during the torpor-arousal cycle. The results indicate reversible changes in ultrastructure of (asymmetric axospinous) synapses in the frontal cortex, which may be critical for the maintenance of cortical neuronal networks and for protection against potential deleterious effects of prolonged hypothermic phases of hibernation.

Phosphorylation of the tau protein sequence 199-205 in the hippocampal CA3 region of Syrian hamsters in adulthood and during aging.

Paired helical filaments formed by the abnormally phosphorylated microtubule-associated tau are a main sign of Alzheimer's disease and other neurodegenerative disorders. The hippocampal CA3 region, a brain region with a high degree of synaptic plasticity, is known to be strongly involved in tau hyperphosphorylation in several neurodegenerative diseases. In addition, reversible tau phosphorylation was observed during hibernation in European ground squirrels. The present study provides data on the tau phosphorylation status in the hippocampus of euthermic Syrian hamsters (Mesocricetus auratus), laboratory animals potentially prone to hibernation. Mossy fibers in the CA3 region of all investigated hamsters were immunostained using an antiserum detecting phospho-serine 199 of tau. A similar staining pattern was obtained with CP-13 detecting phospho-serine 202. In contrast, the monoclonal antibody AT8, recognizing both phosphorylated serine 202 and threonine 205, stained the CA3 region only in old hamsters. These findings implicate an additional link between aging, tau phosphorylation and synaptic plasticity. Furthermore, the presented data allow analyses whether tau phosphorylation is reversible in these facultative hibernators and versatile laboratory animal as it was recently shown for the hibernation cycle of European ground squirrels.

Temporal organisation of hibernation in wild-type and tau mutant Syrian hamsters.

The temporal pattern of hibernation was studied in three genotypes of Syrian hamsters with different circadian periodicity to assess a potential circadian control of alternating torpor and euthermy. We recorded the pattern of hibernation by measuring activity in continuous dim light and constant environmental temperature (6 +/- 1 degrees C). In spite of differences in the endogenous circadian period of three genotypes (tau +/+: approximately equals 24 h, tau +/-: approximately equals 22 h, and tau -/-: approximately equals 20 h) torpor bout duration was statistically indistinguishable (tau +/+: 86.9+/-5.3 h; tau +/-: 94.2+/-3.3 h; tau -/-: 88.8+/-6.2 h). The time between two consecutive arousals from torpor showed unimodal distributions not significantly different between genotypes. The first entry into torpor occurred within the active phase of the circadian cycle in all genotypes whereas the first arousal from torpor appeared to be timed randomly with respect to the prior circadian cycle. The amplitude of the activity rhythm was lower after hibernation compared with the amplitude before hibernation. The results suggest that in the Syrian hamster the circadian system does not control periodicity of torpor and arousal onsets in prolonged hibernation at 6 degrees C.

A social conflict increases EEG slow-wave activity during subsequent sleep.

Electroencephalogram (EEG) slow-wave activity (SWA) during non-rapid eye movement (NREM) sleep is widely viewed as an indicator of sleep debt and sleep intensity. In a previous study, we reported a strong increase in SWA during NREM sleep after a social conflict in rats. To test whether this increase in SWA reflects normal physiological sleep or an unrelated by-product of the stress, we now measured the effect of a conflict in combination with extended sleep deprivation by means of gentle handling. We anticipated that if the social defeat-induced SWA reflects a true sleep debt, the drive for it would persist during the extended wakefulness. Male rats were subjected to a 1-h social conflict followed by 5 h of sleep deprivation by gentle handling or to 6 h gentle handling alone. The manipulations took place during the second half of the dark phase and recovery sleep was recorded during the subsequent light phase. Neither of the two procedures caused a significant change in the total duration of NREM or REM sleep thereafter. Yet, both modes of sleep deprivation induced a strong increase in SWA during NREM sleep. This SWA was significantly higher for 6 h after sleep deprivation consisting of a social conflict followed by gentle handling, as compared to sleep deprivation by handling alone. Thus, the SWA increasing effect of the conflict persisted during the extended wakefulness. The data confirm that social defeat stress accelerates the build up of sleep debt and support the notion that sleep debt and subsequent NREM sleep intensity not only depend on the duration of prior wakefulness but also on what animals experience during that waking.

Forced desynchrony of circadian rhythms of body temperature and activity in rats.

The daily rhythm in body temperature is thought to be the result of the direct effects of activity and the effects of an endogenous circadian clock. Forced desynchrony (FD) is a tool used in human circadian rhythm research to disentangle endogenous and activity-related effects on daily rhythms. In the present study, we applied an FD protocol to rats. We subjected 8 rats for 5 days to a 20 h forced activity cycle consisting of 10 h of forced wakefulness and 10 h for rest and sleep. The procedure aimed to introduce a 10 h sleep/10 h wake cycle, which period was different from the endogenous circadian (about 24 h) rhythm. Of the variation in the raw body temperature data, 68-77% could be explained by a summation of estimated endogenous circadian cycle and forced activity cycle components of body temperature. Free-running circadian periods of body temperature during FD were similar to free-running periods measured in constant conditions. The applied forced activity cycle reduced clock-related circadian modulation of activity. This reduction of circadian modulation of activity did not affect body temperature. Also, the effects of the forced activity on body temperature were remarkably small.

Dissimilarity of slow-wave activity enhancement by torpor and sleep deprivation in a hibernator.

Sleep regulation processes have been hypothesized to be involved in function and timing of arousal episodes in hibernating ground squirrels. We investigated the importance of sleep regulation during arousal episodes by sleep deprivation experiments. After sleep deprivation of 4, 12, and 24 h, starting 4 h after onset of euthermy, a duration-dependent enhancement of slow-wave activity (SWA) of the cortical electroencephalogram during non-rapid eye movement sleep was found, as expected for normal sleep regulation. When sleep deprivation was applied during the initial phase of the arousal episode, in which effects of prior torpor were present in undisturbed recordings, no subsequent recurrence of SWA was found. In addition, prior torpor induced a reduction in the spectral activity of the sigma frequency range (7-14 Hz), which was not observed after sleep deprivation. The effects of torpor and sleep deprivation on subsequent SWA appear qualitatively different. This indicates that effects of deep torpor on sleep are dissimilar to normal sleep regulation.

Sleep during arousal episodes as a function of prior torpor duration in hibernating European ground squirrels.

EEG's were recorded in hibernating European ground squirrels during euthermic arousal episodes at an ambient temperature of 5.5 degrees C. Spontaneous torpor bouts ranged from 6 to 15 days, body temperature during torpor was 7.5 degrees C. The torpor duration prior to EEG measurements was experimentally manipulated: the animals were induced to arouse by gentle handling after torpor of less then 1 day (n = 3), 1-2 days (n = 6), 3-4 days (n = 9) and 5-12 days (n = 9). The animals slept 71.5% of euthermic time, of which 61.4% NREM and 10.2% REM sleep. NREM percentage was slightly positively and REM percentage negatively correlated with prior torpor duration (TD). Spectral analysis showed changes in EEG activity during the euthermic phase in the slow wave frequency range (1-4 Hz) and in higher frequencies. Prior TD specifically affected the slow waves. Slow wave activity decreased exponentially during the euthermic phase. The initial slow wave activity showed a systematic increase with prior TD, which could be described by an exponentially saturating function, albeit with a relatively small time constant compared with spontaneous torpor duration. It is concluded that sleep during arousal episodes following torpor at an ambient temperature of 5.5 degrees C is affected both in structure and intensity by prior TD. The results are consistent with the proposition that torpor inhibits the restorative function of sleep.

Ambient temperature during torpor affects NREM sleep EEG during arousal episodes in hibernating European ground squirrels.

Ambient temperature (Ta) systematically affects the frequency of arousal episodes in mammalian hibernation. This variation might hypothetically be attributed to temperature effects on the rate of sleep debt increase in torpor. We studied this rate by recording sleep electroencephalogram (EEG) in arousal episodes induced after 4 days of torpor at different Ta. Spontaneous torpor bout duration (TBD) varied with Ta: TBD was maximal at 5.5 degrees C (10.7 days), and was reduced at lower (-5 degrees C: 5.0 days, 0 degrees C: 9.3 days) and higher Ta (10 degrees C: 8.7 days, 15 degrees C: 5.0 days). Slow wave activity (SWA) during non-rapid-eye-movement (NREM) sleep, an indicator for sleep debt, showed initial high values after torpor at Ta ranging from -5 degrees C to 10 degrees C. When torpid at 15 degrees C, SWA was not increased in the subsequent arousal episode. The data are thus inconsistent with a rate of sleep debt explanation for the temperature dependence of TBD.

Warming up for sleep? Ground squirrels sleep during arousals from hibernation.

Hypothermia during mammalian hibernation is periodically interrupted by arousals to euthermy, the function of which is unknown. We report that arctic ground squirrels (Spermophilus parryii) consistently sleep during these arousals, and that their EEG shows the decrease in slow wave activity (delta power) that is characteristic of a declining requirement for sleep. These results are consistent with the novel hypothesis that the need for sleep slowly accumulates during torpor, and that returning to euthermy is periodically required to allow sleep. Sleep thus seems to be energetically expensive for a hibernating mammal, and cannot be considered solely a strategy for saving energy.

Effects of scopolamine on performance of rats in a delayed-response radial maze task.

The effects of the cholinergic blocker scopolamine on performance of rats in an 8-arm radial maze were studied. In Experiment 1, rats received injections of scopolamine-HBr (0.2 mg/kg, IP) or saline, 20 min before a trial. The drug impaired performance only when midtrial delays were introduced (10 sec, 5, 20, 60 or 120 min), during which the animals were removed from the apparatus, and to a similar extent at all delays. Injection of scopolamine directly after choice 4 in a 20 min delayed trial affected performance at a dose of 0.4 mg/kg, but not at 0.2 mg/kg. In Experiment 2, the rats were kept in the apparatus during the delay. There was a significant effect of scopolamine at 0.1 and at 0.2 mg/kg, initially irrespective of the length of the delay (10 sec, 2.5 or 5 min). After considerable training, administration of scopolamine (0.2 mg/kg) had no significant effect at the 10 sec delay but did impair performance after longer (5 or 10 min) midtrial intervals. These results suggest that scopolamine has differential effects, depending on dose and degree of training, and that an effect on memory storage may be one of them.

Memory persistence of rats in a radial maze varies with training procedure.

Different estimations of the time-course of spatial working memory have been reported. Some authors found working memory in the radial maze to be relatively long lasting, while others found more rapid exponential decay. In the present experiments it was attempted to account for the conflicting results by investigating the effects of different training procedures. Two types of training were examined under the same circumstances. A group of seven rats was given a series of delayed trials (5, 20, 60, and 120 min). Every delay was repeated at least four times and the delays were presented in an ascending order. The number of errors decreased at every delay except the last one (120 min), where error levels were constant (.50 errors/trial). The good performance was not based on use of intramaze cues or response chaining. In another group of seven rats the same delays were introduced in a quasi-random order and alternated with uninterrupted trials. The number of errors increased exponentially with the length of the delay. However, when this procedure was repeated, the number of errors decreased. These results suggest that training with delayed trials is a major factor to account for the differences in reports of memory persistence in the radial maze.