Effects of social isolation on cognitive function and phenotypic transition of hippocampal astrocytes in mice / 中华行为医学与脑科学杂志

Objective:To explore the effect of social isolation (SI) on cognitive function and the phenotypic transition of hippocampal astrocytes in mice.Methods:Twenty male C57BL/6 mice aged 3-4 weeks were randomly divided into normal group house (GH group) and social isolation group (SI Group). The mice in SI group were fed one per cage for 8 weeks to establish a social isolation model, and the mice in GH group were fed five per cage. The cognitive function of mice was detected by the novel object recognition test and novel location recognition test. The expression of astrocyte marker glial fibrillary acidic protein (GFAP) was detected by immunohistochemistry, RT-PCR and Western blot.The astrocyte morphology change was quantitatively analyzed by Sholl Analysis.The expression of the hippocampal A1-A2 astrocytes markers proteasome subunit beta 8(PSMB8) and a member of the S100 family of Ca 2+ -binding proteins (S100A10) were determined by RT-PCR and Western blot. Statistical analysis was performed using GraphPad Prism 6.0 software, and t-test was used for comparison between two groups. Results:The results of cognitive function showed that the exploration index of novel object ((-5.54±3.30)%, (33.42±7.14)%; t=4.680, P=0.001) and the exploration index of novel location((-7.96±4.81)%, (23.55±8.20)%; t=3.670, P=0.008) in SI group were both lower than those in GH group.Immunohistochemical results showed that the number of GFAP positive cells in hippocampus of SI group was significantly lower than that of GH group((369.90±42.97), (544.90±57.64); t=2.480, P=0.023). The results of Sholl analysis showed that the protuberance of hippocampal astrocytes in SI Group retracted.There were significant differences in the number of intersections between the two groups at 6, 8, 10, 12, 14 and 16 μm away from astrocyte cell body(all P<0.05). Western blot showed that the expression of GFAP protein in SI group was lower than that in GH group((0.85±0.05), (1.03±0.06); t=2.527, P=0.028). The results of PCR showed that the expression of GFAP mRNA in SI group was lower than that in GH group ((0.83±0.05), (1.00±0.03); t=2.970, P=0.018). The expression of A1 phenotypic marker PSMB8 mRNA ((1.58±0.17), (1.00±0.06); t=2.931, P=0.011) and A2 phenotypic marker S100A10 mRNA ((1.52±0.14), (1.00±0.07); t=3.121, P=0.007) in the hippocampus of SI group were higher than those in GH group.Compared with the GH group, the expression of the neurotrophic factors IGF-1 mRNA in the SI group was down-regulated ((0.73±0.07), (1.00±0.08); t=2.327, P<0.05), while the expression of LCN2 mRNA((1.12±0.03), (1.00±0.03), t=2.575, P<0.05), IL-1β mRNA(1.76±0.19), (1.00±0.07), t=3.460, P<0.01) and TNF-α mRNA((2.18±0.42), (1.00±0.07), t=2.427, P<0.05) were up-regulated in the SI group. Conclusion:The pathological mechanism of social isolation-induced cognitive impairment in mice may be related with the phenotypic changes of astrocytes.

Roles of m6A modification in neurological diseases / 中南大学学报(医学版)

N6-methyladenosine (m6A) methylation modification is one of the most common epigenetic modifications for eukaryotic mRNA. Under the catalytic regulation of relevant enzymes, m6A participates in the body's pathophysiological processes via mediating RNA transcription, splicing, translation, and decay. In the past, we mainly focused on the regulation of m6A in tumors such as hematological tumors, cervical cancer, breast cancer. In recent years, it has been found that m6A is enriched in mRNAs of neurogenesis, cell cycle, and neuron differentiation. Its regulation in the nervous system is gradually being recognized. When the level of m6A modification and the expression levels of relevant enzyme proteins are changed, it will cause neurological dysfunction and participate in the occurrence and conversion of neurological diseases. Recent studies have found that the m6A modification and its associated enzymes were involved in major depressive disorder, Parkinson's disease, Alzheimer's disease, Fragile X syndrome, amyotrophic lateral sclerosis, and traumatic brain injury, and they also play a key role in the development of neurological diseases and many other neurological diseases. This paper mainly reviewed the recent progress of m6A modification-related enzymes, focusing on the impact of m6A modification and related enzyme-mediated regulation of gene expression on the central nervous system diseases, so as to provide potential targets for the prevention of neurological diseases.

Investigation of the genetic etiology in male infertility with apparently balanced chromosomal structural rearrangements by genome sequencing / 亚洲男科学杂志(英文版)

Apparently balanced chromosomal structural rearrangements are known to cause male infertility and account for approximately 1% of azoospermia or severe oligospermia. However, the underlying mechanisms of pathogenesis and etiologies are still largely unknown. Herein, we investigated apparently balanced interchromosomal structural rearrangements in six cases with azoospermia/severe oligospermia to comprehensively identify and delineate cryptic structural rearrangements and the related copy number variants. In addition, high read-depth genome sequencing (GS) (30-fold) was performed to investigate point mutations causative of male infertility. Mate-pair GS (4-fold) revealed additional structural rearrangements and/or copy number changes in 5 of 6 cases and detected a total of 48 rearrangements. Overall, the breakpoints caused truncations of 30 RefSeq genes, five of which were associated with spermatogenesis. Furthermore, the breakpoints disrupted 43 topological-associated domains. Direct disruptions or potential dysregulations of genes, which play potential roles in male germ cell development, apoptosis, and spermatogenesis, were found in all cases (n = 6). In addition, high read-depth GS detected dual molecular findings in case MI6, involving a complex rearrangement and two point mutations in the gene DNAH1. Overall, our study provided the molecular characteristics of apparently balanced interchromosomal structural rearrangements in patients with male infertility. We demonstrated the complexity of chromosomal structural rearrangements, potential gene disruptions/dysregulation and single-gene mutations could be the contributing mechanisms underlie male infertility.

Preliminary mechanism study on the X-linked parkinsonism caused by RAB39B gene mutation c.536dupA / 中华神经科杂志

Objective:To detect the expression level of RAB39B gene and the effect of RAB39B on autophagy and α-synuclein, and then investigate the role of RAB39B gene mutation c.536dupA in the pathogenesis of Parkinson′s disease.Methods:Based on the novel RAB39B gene c.536 dupamutation identified in the previous work, the recombinant expression plasmid (pcDNA3.1-HA-RAB39B-536) of RAB39B gene with this mutation and wild-type recombinant expression plasmid (pcDNA3.1-HA-RAB39B) of RAB39B gene were constructed, and the recombinant expression plasmid was transfected into N2a cells with liposome as experimental group. The control group was made up with N2a cells transfected with plasmid pcDNA3.1-HA-RAB39B. Real-time polymerase chain reaction, Western blotting, immunofluorescence and immunoprecipitation techniques were used to detect the expression level of mutant RAB39B gene and the effects of RAB39B on autophagy and α-synuclein.Results:In the N2a cell model, the transcription level of mutant RAB39B was about twice that of wild type RAB39B, while the protein level of mutant RAB39B (0.30±0.00) was significantly lower than that of wild type (1.50±0.25, t=8.313, P<0.05). After adding proteasome inhibitor MG132, the protein level of mutant RAB39B increased (0.70±0.10, t=6.925, P<0.05); the level of microtubule-associated protein 1 light chain 3 BⅡ/Ⅰ of mutant RAB39B (3.11±0.30) was significantly lower than that of wild type (7.03±0.20, t=18.831, P<0.05); overexpression of wild type and mutant RAB39B did not affect the level of endogenous α-synuclein; overexpression of wild-type RAB39B resulted in elevated level of exogenous wild-type (p.A53T; from 0.60±0.11 to 1.25±0.08, t=8.254, P<0.05) and mutant (from 0.55±0.08 to 1.15±0.08, t=9.293, P<0.05) α-synuclein. Conclusions:The stability of the RAB39B protein decreased with the appearance of c.536 dupA mutation, the mutant protein may be degraded through the ubiquitin-proteasome pathway, and this mutation may affect the autophagy level of cells. RAB39B protein may interact with α-synuclein in vivo and may be involved in the maintenance of the stable level of α-synuclein.

Protective effect and mechanism of Lactobacillus on cerebral ischemia reperfusion injury in rats

The present study was designed to investigate the protective effects and mechanism of inactivated lactobacillus (ILA) on cerebral ischemia reperfusion injury (CIRI) in rats. In this experiment, 30 male Sprague Dawley rats were randomly divided into control group, IRI groups, and ILA group. A middle cerebral artery occlusion and reperfusion model was prepared. The rats were killed after 24 hours of recovery of blood flow of cerebral ischemia resulting from 60-min occlusion. The cerebral infarction volume and neurological scores were assayed by staining and behavioral observation. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were assayed by biochemical kits. Cell apoptosis was assayed by Tunnel and the Toll-like receptor (TLR)-4, IkB, and A20 were assayed by western blot. The neurobehavioral scores in IRI rats were significantly lower compared to the control group while ILA improved the neurobehavioral scores of the ILA groups. The cerebral infarction volume and neural cell apoptosis of rats in the ILA groups decreased significantly compared with those in the IRI group. In addition, MDA level in the ILA groups decreased whereas SOD activity increased compared to the IRI group. Moreover, ILA also inhibited the expression of TLR-4 and promoted the expression of IkB and A20. ILA inhibited the apoptosis of neural cells, decreased cerebral infarction volume, and reduced oxidative stress through inhibition of TLR-4/NF-kappa B signaling, improving neurobehavioral scores. Thus from the present study it was concluded that ILA has protective effect on CIRI.

The baseline predictive factors of early neurological deterioration among mild ischemic stroke patients / 临床神经病学杂志

Objective To determine the occurrence and baseline predictive factors of early neurological deterioration ( END) among mild ischemic stroke patients.Methods Mild ischemic stroke patients admitted in the hospital were prospectively enrolled.Univariate and multivariate Logistic recession analyses were used to analyze the demographic data, risk factors of ischemic stroke, clinical, brain imaging and laboratory data.Risk factors of END were identified.Results From June 2012 to August 2013, a total of 319 patients with mild ischemic stroke were enrolled, 45 patients (14.1%) of them experienced END.Univariate analysis showed that baseline NIHSS ( U=3522.000,P=0.000), baseline systolic blood pressure (t=2.871,P=0.004), proportion of symptomatic large artery severe stenosis or occlusion (χ2 =52.564,P=0.000) and proportion of large artery atherosclerosis among TOAST subtypes (χ2 =47.287,P=0.000) in END group were significantly higher than those in non-END group. Multivariate logistic regression analysis showed that baseline systolic blood pressure>142 mmHg (1 mmHg=0.133 kPa) (OR=3.954, 95%CI:1.693-9.236, P=0.001), symptomatic large artery severe stenosis or occlusion (OR=3.170, 95%CI:1.170-8.583, P=0.023) and baseline NIHSS (OR=2.038, 95%CI:1.359-3.057, P=0.001) were associated with END.Conclusions About 14.1% of the mild ischemic stroke patients can occur END.Baseline systolic blood pressure>142 mmHg, symptomatic large artery severe stenosis or occlusion and higher baseline NIHSS were the independent risk factors of END.

Predictive factors of outcome and poor outcome in patients with mild ischemic stroke: a prospective cohort study / 国际脑血管病杂志

Objective To investigate the functional outcome in patients with mild ischemic stroke and to identify its risk factors for poor outcome.Methods The patients with mild ischemic stroke treated within 72 hours after onset were enrolled prospectively.According to modified Rankin Scale (mRS) scores at day 90 after onset,the patients were randomly divided into either a poor outcome group (mRS score ＞2) or a good outcome group (mRS scores 0-2).Univariate analysis and multivariate logistic regression analysis were used to compare and analyze the demographic data,vascular risk factors,clinical data,laboratory data,imaging data,and follow-up data.The risk factors for poor outcome in patients with mild ischemic stroke were identified.Results A total of 253 patients with mild ischemic stroke were enrolled,and 71 of them (28.1％) had poor outcome.Univariate regression analysis showed that the patients' proportions of age (t =2.037,P =0.043),baseline National Institutes of Health Stroke Scale (NIHSS) score (U =4 610.000,P =0.000),baseline mRS score (U =5 723.000,P =0.000),as well as previous history of ischemic stroke (x2 =4.950,P =0.026),severe symptomatic artery stenosis or occlusion (x2 =49.037,P =0.000),large artery atherosclerotic stroke (x2 =34.359,P =0.000),early neurologic deterioration (x2 =45.804,P =0.000),complicated by pneumonia (x2 =12.121,P =0.000) and recurrent ischemic stroke (x2 =14.305,P =0.000) of the poor outcome group were significantly higher than those of the good outcome group.Multivariate logistic regression analysis showed that advanced age (odds ratio [OR] 1.049,95％ confidence interval [CI] 1.012-1.086; P =0.008),higher baseline mRS score (OR,2.130,95％ CI 1.212-3.743;P=0.009),higher baseline NIHSS score (OR 1.532,95％ CI 1.064-2.206; P=0.022),severe symptomatic large artery stenosis or occlusion (OR 7.569,95％ CI 3.497-16.380; P=0.000),early neurological deterioration (OR 7.369,95％ CI 2.648-20.510; P =0.000) and recurrent ischemic stroke (OR 10.450,95％ CI 3.071-35.564; P =0.000) were the independent risk factors for poor outcome.Conclusions More than one fourth of the patients with mild ischemic stroke had poor outcome.Advanced age,higher baseline mRS score,higher baseline NIHSS score,severe symptomatic large artery stenosis or occlusion,early neurological deterioration,and recurrent ischemic stroke were the independent risk factors for poor outcome.