Many viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo. Mice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo. ZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased. Findings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.
Corticotropin-releasing factor (CRF) receptors have been demonstrated to be widely expressed in the central nervous system and in many peripheral tissues of mammalians. However, it is still unknown whether CRF receptors will function in cerebellar Purkinje neurons. In the present study, we investigated the expression profile of CRF receptors in rat cerebellum and identified a novel functional role of CRFR2 in modulating Purkinje neuron P-type Ca(2+) currents (P-currents). We found that CRFR2alpha mRNA, but not CRFR1 and CRFR2beta, was endogenously expressed in rat cerebellum. Activation of CRFR2 by UCN2 inhibited P-currents in a concentration-dependent manner (IC(50) approximately 0.07 microM). This inhibitory effect was abolished by astressin2B, a CRFR2 antagonist, and was blocked by GDP-beta-S, pertussis toxin, or a selective antibody raised against the G(o)alpha. Inhibition of phospholipase C (PLC) blocked the inhibitory action of UCN2. The application of diacylglycerol (DAG) antagonist, 1-hexadecyl-2-acetyl-sn-glycerol, as well as inhibition of either protein kinase C or its epsilon isoform (PKCepsilon) abolished the UCN2 effect while 1-oleoyl-2-acetyl-sn-glycerol (EI-150), a membrane-permeable DAG analogue, occluded UCN2-mediated inhibition. In addition, UCN2 significantly increases spontaneous firing frequency of Purkinje neurons in cerebellar slices. In summary, activation of CRFR2 inhibits P-currents in Purkinje neurons via G(o)alpha-dependent PLC/PKCepsilon pathway, which might contribute to its physiological functions in the cerebellum.
The mechanisms that regulate mammalian cell size during development and homeostatic maintenance are poorly understood. The tumor suppressor Pten is required for correct maintenance of mammalian neuronal soma size. Selective inactivation of Pten in postnatal granule neurons of the cerebellum and dentate gyrus in mouse causes cell-autonomous hypertrophy as well as more complex phenotypes, including progressive macrocephaly, seizures, and premature death. To determine the contribution of mTor signaling to Pten-mediated growth regulation in the mammalian nervous system, we treated Pten conditional knockout mice with CCI-779, a specific mTor inhibitor. mTor inhibition decreased the seizure frequency and death rate in Pten mutant mice, prevented the increase in Pten-deficient neuronal soma size in young mice, and reversed neuronal soma enlargement in adult mice. mTor inhibition did not decrease the size of wild-type adult neurons. Thus, mTor is required for neuronal hypertrophy downstream of Pten deficiency, but is not required for maintenance of normal neuronal soma size. mTOR inhibitors may be useful therapeutic agents for diseases in brain resulting from PTEN deficiency such as Lhermitte-Duclos disease or glioblastoma multiforme.
Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P-type Ca(2+) channels and action-potential firing in rat Purkinje neurons. Melatonin at 0.1 μm reversibly decreased peak currents (I(Ba)) by 32.9%. This effect was melatonin receptor 1 (MT(R1)) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the Gβ subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors abolished the melatonin-induced decrease in I(Ba). Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1-fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin-induced decrease in I(Ba). Melatonin application increased the membrane abundance of PKCδ, and PKCδ inhibition (either pharmacologically or genetically) abolished the melatonin-induced IBa response. Functionally, melatonin increased spontaneous action-potential firing by 53.0%; knockdown of MT(R1) and blockade of P-type channels abolished this effect. Thus, our results suggest that melatonin inhibits P-type channels through MT(R1) activation, which is coupled sequentially to the βγ subunits of G(i/o)-protein and to downstream PI3K-dependent PKCδ signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.
To study blast-induced traumatic brain injury (bTBI) characteristics in confined space. The goats were placed at the column-like buildings with trinitrotoluene (TNT) as the source of the blast wave. The pressure was recorded at 2-8 m from the explosion center. The systemic physiology, electroencephalogram (EEG), serum level of S-100beta, and neuron specific enolase (NSE) were determined pre and post the exposure. Neuroanatomy and neuropathology were observed 4 hours after the exposure. The blast waveform was composed of two peaks from the incident and reflection wave with a range of pressure-duration from 555/913 kPa-0.663 milliseconds at 2 m to 45/71 kPa-2.7/2.367 milliseconds at 8 m. At 2 m, the goats experienced brain depression while the heart rate and respiratory rate concomitantly increased with bloody foam fluid emission from the nose and the mouth. Of the goats, 88.89% were burned. The distinctive gross neuroanatomical changes were congestive expansion of surface vessels on the hemisphere cerebellum and brainstem along with subarachnoid hemorrhage on the frontal lobe, mesencephalon, and brainstem. Subarachnoid hemorrhage, enlarged perivascular space, vascular dilatation and congestion, and parenchymal hemorrhagic could be easily observed microscopically. High amplitude and low frequency of waveforms appeared in the EEG. The serum concentration of S-100beta and NSE were elevated. Although these pathophysiological changes diminished with increasing distance from the explosive center, these changes existed for the 8 m subjects. Blast-induced traumatic brain injury can be induced by a complex blast wave with a pressure and duration of 45/71 kPa and 2.7/2.367 milliseconds. Its severity is related to the features and waveforms of the blast.
To evaluate the effectiveness of microvascular-decompression (MVD) or MVD+partial-sensory-rhizotomy (PSR) on the treatment of primary trigeminal neuralgia (TN). 210 TN patients were retrospectively studied, among which there're 142 cases underwent MVD and 68 cases underwent MVD+PSR. In MVD group, pain vanished in 128, obviously relieved in 9, and 137 cases were profited from MVD. In 82 cases with a follow-up over 2 years, pain vanished in 74, pain sometime occurred in 5 which could be relieved by oral medicine, 3 cases could not be controlled effectively by medicine. In MVD+PSR group, pain completely vanished in 67 cases, not changed in 1 case. In 47 cases with a follow-up over 2 years, pain sometime occurred in 2 which could be relieved by carbamazepine, the others were completely pain-free. The short-term pain-free rate of MVD+PSR group was obviously higher than MVD group(P < 0.05), after a follow-up over 2 years, the former was still higher than the later, 95.7% and 90.2% respectively, without significant difference in statistics. MVD+PSR was obviously superior to MVD in completely eliminating pain in short-term period after operation, however, longer pain-free rate need even longer time to follow up. Identifying the responsible vascular exactly and handling it reasonably were key to both groups.
To investigate the abnormal diffusion in cerebral white matter and its relationship with the olfactory dysfunction in patients with Parkinson's disease (PD) through diffusion tensor imaging (DTI). Diffusion tensor imaging of the cerebrum was performed in 25 patients with Parkinson's disease and 25 control subjects matched for age and sex. Differences in fractional anisotropy (FA) and mean diffusivity (MD) between these two groups were studied by voxel-based analysis of the DTI data. Correlations between diffusion indices and the olfactory function in PD patients were evaluated using the multiple regression model after controlling for the duration of the disease, Unified Parkinson's Disease Rating Sale (UPDRS), and age. The damaged white and gray matter showed decreased FA or increased MD, localized bilaterally in the cerebellar and orbitofrontal cortex. In addition, in PD patients there was a positive correlation between FA values in the white matter of the left cerebellum and the thresholds of olfactory identification (TOI) and a negative correlation between MD values in the white matter of right cerebellum and the TOI. In patients with PD, there was disruption in the cerebellar white matter which may play an important role in the olfactory dysfunction in patients with Parkinson's disease.
Background-The present study evaluated the efficacy of curcumin as a nutritional supplement in preventing aluminum-induced neurotoxicity in rats. Methods-The rats were segregated into four groups, which included normal controls and aluminum-treated, curcumin- treated, and aluminum- and curcumin-treated animals. Results-Eight weeks of aluminum treatment resulted in a significant increase in the levels of lipid peroxidation (LPO) and reactive oxygen species (ROS) in both cerebellum and cerebrum as compared to normal animals. In contrast, the activities of glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD), and reduced glutathione (GSH) levels were found to be significantly decreased following aluminum treatment. Furthermore, aluminum resulted in anxiety in rats as determined with the elevated plus maze test. In addition, an appreciable decrease was noticed in both muscular and locomotor activity of aluminum-treated animals, as determined by rotarod and actophotometer tests, respectively. However, in aluminum-treated animals that also received curcumin supplements, the already raised levels of LPO and ROS returned to near normal limits in the cerebrum. Moreover, curcumin treatment of the aluminum-treated animals also resulted in a significant improvement in the levels of GSH and enzyme activities of GST in both the cerebrum and cerebellum. Also, improvement was observed in the behavior of aluminum-treated animals upon curcumin supplementation. Conclusion-The present study suggests that curcumin may act as a neuroprotectant against aluminum-induced neurodegenerative and behavioral disorders, but further investigations are needed to understand the exact mechanism of neuroprotection.
Benzo(a)pyrene (BaP) is a widespread environmental contaminant that has been associated with neurotoxicity in mammals. It has strong toxic effects on the developing central nervous system. Cerebellum is associated with locomotor activity and anxiety behavior, but there is very little research about the toxic effects of BaP in cerebellum. The present study aims to investigate the global influence of BaP subacute exposure on the metabolome of rat cerebellum. Male neonatal rats (postnatal day 5) were divided into two groups: control group and BaP-treated group (2 mg/kg/day for 7 weeks). Open field test and transmission electron microscopy were performed to analyze neurobehavior and ultramicrostructure alteration. Gas chromatography-mass spectrometry (GC-MS) was used to analyze metabolites of the cerebellum in both groups. The results revealed that postnatal exposure to BaP promoted pathological changes in the cerebellum and increased locomotor and anxiety activities in early adulthood. Twenty differential significant metabolites were identified by multivariate statistical analysis. Further metabolic pathway impact analysis and network analysis suggested that the primary metabolic pathways affected included pathway involved in energy metabolism, methionine and cysteine metabolism, and glutathione metabolism. These findings suggest that BaP-induced cerebellum injury may be correlated with metabolic changes and provide an area to target to reduce the negative effects of BaP.