Rapid eye movement sleep deprivation enhances vulnerability of striatal dopaminergic neurons to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine neurotoxicity in mice

Abstract

Neurodegeneration of the nigrostriatal dopaminergic pathway in Parkinson’s disease (PD) is believed to start long before the defining motor dysfunction is apparent. Non-motor symptoms seem to long antedate the onset of motor symptoms in PD patients. Individuals with rapid eye movement (REM) sleep behaviour disorder (RBD), in particular, have a high risk for developing PD. Interestingly, abnormalities in brain regions known to be related to sleep and circadian rhythms in PD have been reported. Until now, it has been unclear whether these abnormalities appear before or after the onset of the disease and whether these sleep disturbances participate in the pathogenesis of PD. The aim of the present study was to investigate whether REM sleep deprivation (REMSD) enhances susceptibility to 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a Parkinson-inducing toxin, in mice. Eight-week-old male C57BL/6N mice were purchased from Charles River Japan (Atsugi, Japan) and housed in standard conditions. Use of the animals and protocol procedures were approved by the Animal Care Committee at Hokuriku University (no. 18–02). REMSD was applied to mice by the platform method as reported by Niijima et al. In brief, each mouse was placed on a platform (5 cm high, 3 cm diameter) that was fixed to the centre of a plastic cage (23 × 16 × 13 cm). In the plastic cage, we placed a volume of water 4 cm deep, obligating the mice to stay on the platforms. Each mouse stayed on its platform for 20 h/day. Food and water were given ad libitum. After 20 h, each mouse was placed in a plastic cage alone for rest and 4 h of REMSD. These steps were repeated for 3 days. The mice in the control group were exposed to the same conditions, except there was no water in their cages. We confirmed that REMSD increased the level of locomotor activity of mice relative to the control mice, as described in a previous report. Mice received MPTP hydrochloride (20 mg/kg of free base; Sigma-Aldrich, St Louis, MO, USA) in saline intraperitoneally just after the last REMSD. The dose of MPTP used in this study was selected based on our previous study, which showed this dose causes only weak toxicity. In the present study, the striatal tissues were collected from the mice 4 days after MPTP administration. This was based on a previous observation that acute restraint stress led to enhanced susceptibility that was detectable 72 h after administration of low-dose MPTP. Western blot analysis was performed as described previously. The proteins intended for evaluation of striatal dopaminergic neurodegeneration were detected by using primary antibodies, including rat monoclonal antibody against the dopamine transporter (DAT) (MAB369; Millipore, Temecula, CA, USA) and mouse monoclonal antibody against the glial fibrillary acidic protein (GFAP) (Sigma-Aldrich). The actin protein was detected for a loading control by using mouse monoclonal antibody against the actin (MAB1501; Millipore). Data analysis was performed with the statistical analysis system StatMate III (ATMS Co., Ltd., Tokyo, Japan). The statistical significances of the differences with raw data were assessed by one-way ANOVA and then by Tukey’s post-test. Differences of less than 5% were considered statistically significant. We investigated the effect of REMSD on striatal dopaminergic neurons in MPTP-treated mice by evaluating striatal protein levels of DAT and GFAP (Fig. 1a– c). DAT is used as a marker of dopaminergic nerve terminals, and GFAP is a well-known marker of reactive astrocytes in response to neuronal damage. After MPTP injection, striatal protein levels of DAT and GFAP changed to 97% and 215% of control, respectively, but these changes were not statistically significant. Administration of MPTP immediately after the last REMSD significantly altered DAT and GFAP protein levels to 63% and 277% of control, respectively. We confirmed that REMSD alone without MPTP treatment did not affect the striatal protein levels of DAT or GFAP (data not shown).