François Marcouiller

Propofol Directly Increases Tau Phosphorylation

In Alzheimers disease (AD) and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A) activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of neurofibrillary pathology.

Deregulation of Protein Phosphatase 2A and Hyperphosphorylation of τ Protein Following Onset of Diabetes in NOD Mice

The histopathological hallmarks of Alzheimer disease (AD) include intraneuronal neurofibrillary tangles composed of abnormally hyperphosphorylated τ protein. Insulin dysfunction might influence AD pathology, as population-based and cohort studies have detected higher AD incidence rates in diabetic patients. But how diabetes affects τ pathology is not fully understood. In this study, we investigated the impact of insulin dysfunction on τ phosphorylation in a genetic model of spontaneous type 1 diabetes: the nonobese diabetic (NOD) mouse. Brains of young and adult female NOD mice were examined, but young NOD mice did not display τ hyperphosphorylation. τ phosphorylation at τ-1 and pS422 epitopes was slightly increased in nondiabetic adult NOD mice. At the onset of diabetes, τ was hyperphosphorylated at the τ-1, AT8, CP13, pS262, and pS422. A subpopulation of diabetic NOD mice became hypothermic, and τ hyperphosphorylation further extended to paired helical filament-1 and TG3 epitopes. Furthermore, elevated τ phosphorylation correlated with an inhibition of protein phosphatase 2A (PP2A) activity. Our data indicate that insulin dysfunction in NOD mice leads to AD-like τ hyperphosphorylation in the brain, with molecular mechanisms likely involving a deregulation of PP2A. This model may be a useful tool to address further mechanistic association between insulin dysfunction and AD pathology.

Hypothermia-induced hyperphosphorylation: a new model to study tau kinase inhibitors

Tau hyperphosphorylation is one hallmark of Alzheimers disease (AD) pathology. Pharmaceutical companies have thus developed kinase inhibitors aiming to reduce tau hyperphosphorylation. One obstacle in screening for tau kinase inhibitors is the low phosphorylation levels of AD-related phospho-epitopes in normal adult mice and cultured cells. We have shown that hypothermia induces tau hyperphosphorylation in vitro and in vivo. Here, we hypothesized that hypothermia could be used to assess tau kinase inhibitors efficacy. Hypothermia applied to models of biological gradual complexity such as neuronal-like cells, ex vivo brain slices and adult non-transgenic mice leads to tau hyperphosphorylation at multiple AD-related phospho-epitopes. We show that Glycogen Synthase Kinase-3 inhibitors LiCl and AR-A014418, as well as roscovitine, a cyclin-dependent kinase 5 inhibitor, decrease hypothermia-induced tau hyperphosphorylation, leading to different tau phosphorylation profiles. Therefore, we propose hypothermia-induced hyperphosphorylation as a reliable, fast, convenient and inexpensive tool to screen for tau kinase inhibitors.

Dimethyl Sulfoxide Induces Both Direct and Indirect Tau Hyperphosphorylation

Dimethyl sulfoxide (DMSO) is widely used as a solvent or vehicle for biological studies, and for treatment of specific disorders, including traumatic brain injury and several forms of amyloidosis. As Alzheimer’s disease (AD) brains are characterized by deposits of β-amyloid peptides, it has been suggested that DMSO could be used as a treatment for this devastating disease. AD brains are also characterized by aggregates of hyperphosphorylated tau protein, but the effect of DMSO on tau phosphorylation is unknown. We thus investigated the impact of DMSO on tau phosphorylation in vitro and in vivo. One hour following intraperitoneal administration of 1 or 2 ml/kg DMSO in mice, no change was observed in tau phosphorylation. However, at 4 ml/kg, tau was hyperphosphorylated at AT8 (Ser202/Thr205), PHF-1 (Ser396/Ser404) and AT180 (Thr231) epitopes. At this dose, we also noticed that the animals were hypothermic. When the mice were maintained normothermic, the effect of 4 ml/kg DMSO on tau hyperphosphorylation was prevented. On the other hand, in SH-SY5Y cells, 0.1% DMSO induced tau hyperphosphorylation at AT8 and AT180 phosphoepitopes in normothermic conditions. Globally, these findings demonstrate that DMSO can induce tau hyperphosphorylation indirectly via hypothermia in vivo, and directly in vitro. These data should caution researchers working with DMSO as it can induce artifactual results both in vivo and in vitro.

The nuclear progesterone receptor reduces post-sigh apneas during sleep and increases the ventilatory response to hypercapnia in adult female mice.

We tested the hypothesis that the nuclear progesterone receptor (nPR) is involved in respiratory control and mediates the respiratory stimulant effect of progesterone. Adult female mice carrying a mutation in the nPR gene (PRKO mice) and wild-type controls (WT) were implanted with an osmotic pump delivering vehicle or progesterone (4 mg/kg/day). The mice were instrumented with EEG and neck EMG electrodes connected to a telemetry transmitter. The animals were placed in a whole body plethysmograph 7 days after surgery to record ventilation, metabolic rate, EEG and neck EMGs for 4 consecutive hours. The animals were exposed to hypercapnia (5% CO2), hypoxia (12% O2) and hypoxic-hypercapnia (5% CO2+12% O2–5 min each) to assess chemoreflex responses. EEG and EMG signals were used to characterize vigilance states (e.g., wake, non-REM, and REM sleep). PRKO mice exhibited similar levels of minute ventilation during non-REM and REM sleep, and higher frequencies of sighs and post-sigh apneas during non-REM sleep compared to WT. Progesterone treatment increased minute ventilation and metabolic rate in WT and PRKO mice during non-REM sleep. In WT mice, but not in PRKO mice, the ventilation under hypercapnia and hypoxic hypercapnia was enhanced after progesterone treatment. We conclude that the nPR reduces apnea frequency during non-REM sleep and enhances chemoreflex responses to hypercapnia after progesterone treatment. These results also suggest that mechanisms other than nPR activation increase metabolic rate in response to progesterone treatment in adult female mice.

Anesthesia-induced hypothermia mediates decreased ARC gene and protein expression through ERK/MAPK inactivation

Several anesthetics have been reported to suppress the transcription of a number of genes, including Arc, also known as Arg3.1, an immediate early gene that plays a significant role in memory consolidation. The purpose of this study was to explore the mechanism of anesthesia-mediated depression in Arc gene and protein expression. Here, we demonstrate that isoflurane or propofol anesthesia decreases hippocampal Arc protein expression in rats and mice. Surprisingly, this change was secondary to anesthesia-induced hypothermia. Furthermore, we confirm in vivo and in vitro that hypothermia per se is directly responsible for decreased Arc protein levels. This effect was the result of the decline of Arc mRNA basal levels following inhibition of ERK/MAPK by hypothermia. Overall, our results suggest that anesthesia-induced hypothermia leads to ERK inhibition, which in turns decreases Arc levels. These data give new mechanistic insights on the regulation of immediate early genes by anesthesia and hypothermia.

Estradiol Protects Against Cardiorespiratory Dysfunctions and Oxidative Stress in Intermittent Hypoxia

Study Objectives We tested the hypothesis that estradiol (E2) protects against cardiorespiratory disorders and oxidative stress induced by chronic intermittent hypoxia (CIH) in adult female rats. Methods Sprague‐Dawley female rats (230‐250 g) were ovariectomized and implanted with osmotic pumps delivering vehicle or E2 (0.5 mg/kg/d). After 14 days of recovery, the rats were exposed to CIH (21%‐10% O2: 8 h/d, 10 cycles per hour) or room air (RA). After 7 days of CIH or RA exposure, we measured arterial pressures (tail cuff), metabolic rate (indirect calorimetry), minute ventilation, the frequency of sighs and apneas at rest, and ventilatory responses to hypoxia and hypercapnia (whole body plethysmography). We collected the cerebral cortex, brainstem, and adrenal glands to measure the activity of NADPH and xanthine oxidase (pro‐oxidant enzymes), glutathione peroxidase, and the mitochondrial and cytosolic superoxide dismutase (antioxidant enzymes) and measured lipid peroxidation and advanced oxidation protein products (markers of oxidative stress). Results CIH increased arterial pressure, the frequency of apnea at rest, and the hypoxic and hypercapnic ventilatory responses and reduced metabolic rate. CIH also increased oxidant enzyme activities and decreased antioxidant activity in the cortex. E2 treatment reduced body weight and prevented the effects of CIH. Conclusions E2 prevents cardiorespiratory disorders and oxidative stress induced by CIH. These observations may help to better understand the underlying mechanisms linking menopause and occurrence of sleep apnea in women and highlight a potential advantage of hormone therapy.

Hypothermia mediates age-dependent increase of tau phosphorylation in db/db mice.

Accumulating evidence from epidemiological studies suggest that type 2 diabetes is linked to an increased risk of Alzheimers disease (AD). However, the consequences of type 2 diabetes on AD pathologies, such as tau hyperphosphorylation, are not well understood. Here, we evaluated the impact of type 2 diabetes on tau phosphorylation in db/db diabetic mice aged 4 and 26weeks. We found increased tau phosphorylation at the CP13 epitope correlating with a deregulation of c-Jun. N-terminal kinase (JNK) and Protein Phosphatase 2A (PP2A) in 4-week-old db/db mice. 26-week-old db/db mice displayed tau hyperphosphorylation at multiple epitopes (CP13, AT8, PHF-1), but no obvious change in kinases or phosphatases, no cleavage of tau, and no deregulation of central insulin signaling pathways. In contrast to younger animals, 26-week-old db/db mice were hypothermic and restoration of normothermia rescued phosphorylation at most epitopes. Our results suggest that, at early stages of type 2 diabetes, changes in tau phosphorylation may be due to deregulation of JNK and PP2A, while at later stages hyperphosphorylation is mostly a consequence of hypothermia. These results provide a novel link between diabetes and tau pathology, and underlie the importance of recording body temperature to better understand the relationship between diabetes and AD.

Tau hyperphosphorylation in the brain of ob/ob mice is due to hypothermia: Importance of thermoregulation in linking diabetes and Alzheimer's disease

Over the last few decades, there has been a significant increase in epidemiological studies suggesting that type 2 diabetes (T2DM) is linked to a higher risk of Alzheimers disease (AD). However, how T2DM affects AD pathology, such as tau hyperphosphorylation, is not well understood. In this study, we investigated the impact of T2DM on tau phosphorylation in ob/ob mice, a spontaneous genetic model of T2DM. Tau phosphorylation at the AT8 epitope was slightly elevated in 4-week-old ob/ob mice while 26-week-old ob/ob mice exhibited tau hyperphosphorylation at multiple tau phospho-epitopes (Tau1, CP13, AT8, AT180, PHF1). We then examined the mechanism of tau hyperphosphorylation and demonstrated that it is mostly due to hypothermia, as ob/ob mice were hypothermic and normothermia restored tau phosphorylation to control levels. As caffeine has been shown to be beneficial for diabetes, obesity and tau phosphorylation, we, therefore, used it as therapeutic treatment. Unexpectedly, chronic caffeine intake exacerbated tau hyperphosphorylation by promoting deeper hypothermia. Our data indicate that tau hyperphosphorylation is predominately due to hypothermia consequent to impaired thermoregulation in ob/ob mice. This study establishes a novel link between diabetes and AD, and reinforces the importance of recording body temperature to better assess the relationship between diabetes and AD.

Membrane progesterone receptor-β, but not -α, in dorsal brain stem establishes sex-specific chemoreflex responses and reduces apnea frequency in adult mice

We tested the hypothesis that membrane progesterone receptors (mPR) contribute to respiratory control in adult male and female mice. Mice were implanted with osmotic minipumps for continuous infusion of small interfering RNA (siRNA) directed against mPRα, mPRβ, or a control solution in the fourth ventricle (to target brain stem respiratory areas) for 14 days. We then performed respiratory and metabolic recordings by whole body plethysmography at rest and in response to hypoxia (12% O2) or hypercapnia (5% CO2, 5 min each). For each treatment, we have verified with immunohistochemistry that the staining intensity of mPRα or mPRβ in the brain stem is decreased. At rest, the siRNA against mPRα and mPRβ increased respiratory frequency in males only. The siRNA against mPRβ almost tripled the frequency of apneas in male and in female mice, while the siRNA against mPRα had no effect. Regarding respiratory chemoreflex, the siRNA against mPRβ suppressed the response to hypoxia in male and female mice and reduced by ∼50% the response to hypercapnia, while the siRNA against mPRα had more limited effects. Interestingly, control females had higher ventilatory response to hypoxia and hypercapnia than males, and these sex-specific effects were suppressed by the siRNA against mPRβ, whereas they were still present after treatment with the siRNA against mPRα. We conclude that mPRβ reduces apnea frequency in male and female mice and establishes sex-specific ventilatory chemoreflex.