Proteomics of serum exosomes identified fibulin-1 as a novel biomarker for mild cognitive impairment

Mild cognitive impairment(MCI)is a prodrome of Alzheimer’s disease pathology.Cognitive impairment patients often have a delayed diagnosis because there are no early symptoms or conventional diagnostic methods.Exosomes play a vital role in cell-to-cell communications and can act as promising biomarkers in diagnosing diseases.This study was designed to identify serum exosomal candidate proteins that may play roles in diagnosing MCI.Mass spectrometry coupled with tandem mass tag approach-based non-targeted proteomics was used to show the differentially expressed proteins in exosomes between MCI patients and healthy controls,and these differential proteins were validated using immunoblot and enzyme-linked immunosorbent assays.Correlation of cognitive performance with the serum exosomal protein level was determined.Nanoparticle tracking analysis suggested that there was a higher serum exosome concentration and smaller exosome diameter in individuals with MCI compared with healthy controls.We identified 69 exosomal proteins that were differentially expressed between MCI patients and healthy controls using mass spectrometry analysis.Thirty-nine exosomal proteins were upregulated in MCI patients compared with those in control patients.Exosomal fibulin-1,with an area under the curve value of 0.81,may be a biomarker for an MCI diagnosis.The exosomal protein signature from MCI patients reflected the cell adhesion molecule category.In particular,higher exosomal fibulin-1 levels correlated with lower cognitive performance.Thus,this study revealed that exosomal fibulin-1 is a promising biomarker for diagnosing MCI.

Ultrastructure of focal cerebral cortex tissue from rats with focal cortical dysplasia

BACKGROUND： Developing a model of focal cortical dysplasia in microgyrus and observing the ultrastructure of focal tissue is of important significance for analyzing the pathology of cortical developmental disorder and the factors of structural changes. OBJECTIVE： This study was to observe the pathological characteristics of focal tissue around the microgyrus of rats with cortical developmental disorder using an electron microscope, so as to analyze the causes associated with cerebral cortical developmental disorder. DESIGN： A randomized controlled animal experiment. SETTING： The First Affiliated Hospital of Chongqing Medical University. MATERIALS： This study was carried out in the Chongqing Key Laboratory of Neurology, Room for Electron Microscope of Chongqing Medical University, and Laboratory Animal Center, Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA between January 2004 and August 2006. Eighteen healthy newborn male Wistar rats, weighing 3.0 - 6.0 g, provided by the Laboratory Animal Center, Daping Hospital, Third Military Medical University of Chinese PLA, were involved in this study. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Probes （Chongqing Wire ＆ Cable Factory, China） were made of copper core wire with diameter of 1mm. METHODS： The rats were randomly divided into 3 groups with 6 in each： normal control group, liquid nitrogen injured group and sham-operation group. （1）In the liquid nitrogen injured group, a blunt probe frozen by liquid nitrogen was placed on fronto-parietal crinial bone of rats for 8 s. A 3 - 5 cm of microgyrus was induced in the unilateral cerebral sensory cortical area. In the sham-operation group, probe was placed at the room temperature. In the normal control group, rats were untouched. （2） The conscious state and electrical activity of brain of rats in each group were observed. （3） 2 - 3 mm thickness of hippocampal tissue with coronary section was taken for observing its ultrastructure under a transmission electron microscope. MAIN OUTCOME MEASURES： （1） The ultrastructure of hippocampal tissue. （2）The conscious state and electrical activity of brain of rats. RESULTS： Eighteen rats were enrolled in the final analysis. （1） Observation of hippocampal ultrastructure： Electromicroscopic pathological findings showed that for each rat of the liquid nitrogen injured group, mitochondrium in the pyramidal neuron around the microgyrus was swelled, endoplasmic reticulum was expanded, glial cells were swelled, water gathered around the blood capillary, partial medullary sheath was degenerated, neuropilem was normal and no obviously abnormal synapse was found. （2） Changes in conscious state of rats： Rats in the normal control group and sham-operation group had no convulsive seizure, but those in the liquid nitrogen injured group had occasionally. Most of them showed increased activities, excitation and restlessness, scratching and frequent ＂ watching face-like activities＂. （3）Electrical activity of brain of rats： Electroencephalogram recording of liquid nitrogen injured group showed that small wave amplitude of rhythm took the main part. No typical sharp wave, V wave, sharp and slow wave, V and slow waves were discharged. CONCLUSION： Liquid nitrogen can lead to cerebral cortical developmental disorder. Pathological changes of ultrastructure of focal tissue around the microgyrus can provide pathological basis for epilepsy associated with focal cortical developmental disorder.

Construction and expression of nucleic acid vaccine pVAX1-h-alpha S_(1-140) coding human alpha-synuclein

BACKGROUND： The deposition of α -synuclein （ α -syn） aggregates is a neuropathological feature of Parkinson＇s disease. It remains impossible to involve α-syn aggregation in the treatment of Parkinson＇s disease. A nucleic acid vaccine will provide a new pathway to immunotherapy for Parkinson＇s disease. OBJECTIVE： To construct a recombinant eukaryotic expression vector pVAX1 coding human α -syn and to observe its expression level in COS-7 cells. DESIGN AND SETTING： The present bioengineering and molecular biology experiment was performed at Department of Neurology, First Affiliated Hospital of Chongqing Medical University ＆ Chongqing Key Laboratory of Neurology. MATERIALS： The eukaryotic expression plasmid pVAXI, human embryonic brain tissue, healthy human blood cells, and COS-7 cells were purchased from Promega Company, USA. METHODS： The full-length CDS sequence of the human a -syn gene was amplified by RT-PCR, which contained restriction sites for the enzymes Kpn Ⅰ, Xba Ⅰ and Kozak consensus sequence. Then the PCR products and eukaryotic expression vector pVAX1 were digested with Kpn Ⅰ and Xba Ⅰ simultaneously, and were extracted and ligated by T4 ligase. The recombinant constructs pVAX1-h α -S1-140 were transformed into competent E. coli TOP 1 0 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis, and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells by RT-PCR and Western blotting. MAIN OUTCOME MEASURES： Identification of an eukaryotic expression vector containing the human α -syn gene, pVAX1-h α-S1-140, and detection of the expression in mammalian cell COS-7. RESULTS： The pVAX1 vector was successfully cloned with human α -syn in the correct orientation and in-frame. The DNA vaccine constructs pVAX 1-h α-S1-140 with the human α-syn gene were shown to be expressed in COS-7 cells. Human α-syn was successfully expressed in the mammalian cell line and was detected by RT-PCR and western blotting. CONCLUSION： Nucleic acid vaccine pVAX1-h α S1-140 was successfully constructed and expressed in COS-7 cells.

Rapid construction of phosphatase and tensin homolog-deleted on chromosome ten gene recombinant adenovirus using the AdEasy system

Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely used, greatly simplifies preparation of recombinant adenovirus. Therefore, recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene （Ad-PTEN） was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification. Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro. Results revealed a cytopathic effect in green fluorescent protein expression, which increased with prolonged time. After three cycles of amplification, the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons. The entire process typically requires 4-5 weeks for completion. Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.

Influence of edaravone on Notch1 and nuclear factor-kappaB in rats with cerebral ischemia/reperfusion injury

BACKGROUND： It has been demonstrated that edaravone has a a neuroprotective role, inhibits free radical increase, and reduces celt apoptosis. The Notch pathway is a key factor in neurogenesis and cellular apoptosis The proinflammatory transcription factor nuclear factor-kappaB （NF-κB） plays an important role in inflammation and oxidation. OBJECTIVE： To observe the influence of edaravone on Notchl and NF-κB mRNA and protein expression in rats with focal cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： This randomized controlled neural and molecular biology experiment was performed at the Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, and the Chongqing Key Laboratory of Neurology between July 2007 and May 2008. MATERIALS： Thirty female Wistar rats were used. Edaravone was purchased from Jiangsu Xiansheng Pharmaceutical Limited Company, China. METHODS： Wistar rats were randomly divided into five groups （n = 6）. Thread was inserted into the internal carotid artery of the sham operation group but the middle cerebral artery was not ligated. A focal cerebral ischemia/reperfusion model was established by inserting thread into the right middle cerebral artery. The model rats in the edaravone groups were given tail vein injections of edaravone at 3 mg/kg body weight after ischemia for 2 hours and reperfusion for 12 or 24 hours. Ischemia/reperfusion groups （model group） received intravenous infusion of normal saline at the same volume as the edaravone groups after ischemia for 2 hours and reperfusion for 12 or 24 hours. MAIN OUTCOME MEASURES： The volume of the ischemic region was measured by 2,3,5-triphenyltetrazolium chloride staining. Notchl and NF-κB protein and mRNA expression were measured by immunohistochemistry and RT-PCR. Protein expression was represented by the absorbance value. RESULTS： Edaravone greatly reduced the focal infarct volume. Notchl and NF-κB protein and mRNA expression were rapidly upregulated following cerebral ischemia/reperfusion injury in model and edaravone groups compared with the sham operation group （P 〈 0.01 ）. In addition, edaravone treatment significantly upregulated Notchl expression but down-regulated NF-κB expression compared with model groups （P 〈 0.01）. CONCLUSION： Edaravone possibly protects brain tissue from ischemia/reperfusion injury by upregulating Notchl expression and regulating NF-κB expression.

Effects of fastigial nucleus electrical stimulation on lateral ventricle nestin expression after focal cerebral ischemia/reperfusion in adult rats

BACKGROUND： Previous studies have confirmed that fastigial nucleus electrical stimulation can induce endogenous neuroprotective mechanisms and produce wide and long-lasting neuroprotective effects. Nevertheless, the precise mechanisms remain poorly understood. OBJECTIVE： This study was designed to observe the effects of fastigial nucleus electrical stimulation on nestin-positive cell expression in adult rat lateral ventricle after focal cerebral ischemia/reperfusion, as well as neurological functional changes as a function of time. DESIGN： A randomized controlled animal experiment. SETTING： Department of Neurology, First Affiliated Hospital of Chongqing Medical University; Chongqing Key Laboratory of Neurology. MATERIALS： This study was performed in the Department of Neurology, First Affiliated Hospital of Chongqing Medical University and Chongqing Key Laboratory of Neurology from September 2004 to February 2006. A total of 180 healthy, adult, male Wistar rats, aged 8 weeks old, were provided by the Laboratory Animal Center of Chongqing Medical University. The main reagents and equipments were as follows： rabbit anti-rat nestin monoclonal antibody （Wuhan Boster Company, China）. METHODS： The included male Wistar rats were randomly divided into 5 groups： normal control, sham-operated, model, fastigial nucleus sham-stimulation （sham-stimulation for short）, and fastigial nucleus electrical stimulation （stimulation for short） groups. Six time points （1 hour of ischemia and 1, 3, 7, 14, 21, and 28 days of reperfusion, 6 rats per time point） were allotted to each group. Cerebral ischemia/reperfusion was performed by occlusion to the right middle cerebral artery with suture, followed by suture removal. In the stimulation group, subsequent to reperfusion, the rat left cerebellar fastigial nucleus was immediately subjected to 1 hour of stimulation. After anesthesia, the rat left cerebellar fastigial nucleus was stimulated for 1 hour using a square-wave electronic stimulator with a current intensity of 50 μ A, frequency of 50-100 Hz, and duration of 0.5 ms. In the sham-stimulation group, the procedure was identical to the stimulation group, except the needle was retained for 1 hour and current stimulation was withdrawn. In the model group, rats were subjected to cerebral ischemia/reperfusion, but electrical stimulation was omitted. In the sham-operated group, the internal carotid artery, rather than the middle cerebral artery, was inserted with suture, and simultaneously, electrical stimulation was omitted. In the normal control group, the rats received no treatments. MAIN OUTCOME MEASURES： Nestin-positive cells were detected by immunohistochemical staining in the rat ischemic lateral cerebral ventricle at 1, 3, 7, 14, 21, and 28 days post-reperfusion. RESULTS： Morphological changes of nestin-positive cells in the ischemic lateral ventricle： in the normal control group, very few nestin-positive cells were detected in the choroid plexus, ependyma, and subependymal region of the lateral ventricle. In the model group, the number of nestin-positive cells exhibited a tendency towards a single peak, i.e., cells increased at day 1, reached peak levels by day 7, and then decreased sharply. Fastigial nucleus electrical stimulation was administered following focal cerebral ischemia/reperfusion, and results revealed that nestin-positive cell morphology was similar to horizontal cell morphology on day 7. The number of nestin-positive cells decreased after 14 days; however, the proportion of horizontal cell-like cells increased. In the sham-stimulation group, there was no change in nestin-positive cells, nestin-positive cell expression in the ischemic lateral ventricle： nestin-positive cell expression increased in the ischemic lateral cerebral ventricle, exhibiting a tendency towards unimodality; the number of cells peaked on day 7 （P 〈 0.01） and gradually decreased after 14 days （P 〈 0.01）. Following fastigial nucleus electrical stimulation, the number of nestin-positive cells increased significantly （P 〈 0.05-0.01）, reached peak levels by day 7 （P 〈 0.01）, and remained at very high levels after 14 days （P 〈 0.01）. Neurofunctional changes： neurofunctional deficits were gradually alleviated with prolonged focal cerebral ischemia/reperfusion time. At 1 hour of ischemia, and 6 hours to 7 days of reperfusion, rat neurological scores were significantly lower in the stimulation group than in the model and sham-operation groups （P 〈 0.05-0.01）. CONCLUSION： Fastigial nucleus electrical stimulation increased nestin-positive cells in the rat lateral ventricle after focal cerebral ischemia/reperfusion in a time-dependent manner and simultaneously improved neurological deficits, as well as promoted differentiation of nestin-positive cells towards a cell type with neuronal and neuroglial cellular morphology.

Expression of nerve growth factor mRNA in splenic lymphocytes of bronchial asthma rats and its influencing factors

BACKGROUND： Previous research has proved that nerve growth factor （NGF） participates in the onset of asthma by the induction of neurogenic inflammation. OBJECTIVE： To investigate the effect of interleukin-13 （IL-13） and interferon- γ; （IFN- γ ） on the expression of NGF mRNA in the splenic lymphocytes of bronchial asthma rats. DESIGN, TIME AND SETTING： The experiment, a completely randomized study based on cellular immunology, was performed in the Laboratory of Neurology in Chongqing Medical University and the Department of Clinical Pharmacy in College of Clinical Medicine, Chongqing Medical University （Chongqing, China） from January 2006 to April 2007. MATERIALS： Four adult male Wistar rats were used in this study. Rat IL-13, IFN- γ probe and the total RNA extraction kit were produced by Shanghai Sangon Biological Technology ＆ Services Co., Ltd （China）. The NGF ELISA kit was a product of Wuhan Boster Bioengineering Co., Ltd （China）. A Du-70 automatic UV spectrophotometer was produced by Beckman Company （USA）. METHODS： Rats were subjected to 1-mL intraperitoneal injections each containing 100 mg of ovalbumin, and were sensitized by using antigen solution, which was sensitized with 5×10^9 Bacillus pertussis and 100 mg aluminum hydroxide powder. Four rats were challenged with 1% ovalbumin using an ultrasonic nebulizer for 60 minutes to establish an asthmatic model. After rats were anesthetized, splenic lymphocytes were isolated and cultured in medium, which was supplemented with IL-13 or IFN- γ, for 0, 12, 24 or 48 hours. A parallel study was conducted with cultured splenic lymphocytes, which were divided into a control group, an IL-13 group and an IFN- γ group. Culture medium was added with different concentrations of IL-13 （10, 50, 100 U g/L） and IFN- γ; （1, 10, 50 u g/L）; 24 hours later, all samples were harvested. MAIN OUTCOME MEASURES： The expression levels of NGF mRNA were detected by reverse transcription-polymerase chain reaction. RESULTS： In the control group, the lymphocytes of the asthmatic model expressed NGF mRNA in a time-dependent manner. Alter lymphocytes were cultured with IL-13 at a final concentration of 50 u g/L for 12, 24 or 48 hours, expression of NGF mRNA was upregulated in a time-dependent manner to higher levels than the basal values at the same time points （P 〈 0.01 ）. IL- 13 upregulated the expression of NGF mRNA in a dose-dependent manner, and the NGF mRNA expression levels at middle and high concentrations of IL-3 were significanlly higher than the values at a low concentration of IL-13 （P 〈 0.05）. With increasing IFN- γ concentration, the expression of NGF mRNA was gradually downregulated. The low concentration group showed lower levels of NGF mRNA than the blank control group, while the middle and high concentration IFN- γ, groups showed lower levels than the low concentration group （P 〈 0.05）. CONCLUSION： In asthmatic rats, IL-13, a Th2 cytokine, upregulates the expression ofNGF mRNA, while IFN- γ, a Thl cytokine, downregulates NGF mRNA expression. The effects of both cytokines were time and dose dependent. Th 1/Th2 cytokine immune imbalance may indirectly induce airway neurogenic inflammation by regulating NGF mRNA expression.

Effects of antiseizure medications on alternative psychosis and strategies for their application

Forced normalization(FN)is a unique phenomenon that is often seen in the treatment of epilepsy.FN is characterized by abnormal mental behavior and disordered emotions in epilepsy patients despite a significantly improved electroencephalogram and successful seizure control;the occurrence of FN seriously affects patients’quality of life.The causes of FN include antiseizure medications(ASMs),epilepsy surgery and vagus nerve stimulation,with ASMs being the most common cause.However,with the timely reduction or discontinuation of ASMs and the use of antipsychotic drugs,the overall prognosis is good.Here,we perform an extensive review of the literature pertaining to FN,including its epidemiology,possible mechanisms,clinical features,treatment and prognosis.

The Traf2 and NcK interacting kinase inhibitor NCB-0846 suppresses seizure activity involving the decrease of GRIA1

Epilepsy,one of the most common neurological disorders,is characterized by spon-taneous recurrent seizures.Temporal lobe epilepsy(TLE)is one of the most common medically intractable seizure disorders.Traf2-and NcK-interacting kinase(TNIK)has recently attracted attention as a critical modulation target of many neurological and psychiatric disorders,but its role in epilepsy remains unclear.In this study,we hypothesized the involvement of TNIK in epilepsy and investigated TNIK expression in patients with intractable TLE and in a pilocarpineinduced rat model of epilepsy by western blotting,immunofluorescence,and immunohistochemistry.A pentylenetetrazole(PTZ)-induced epilepsy rat model was used to determine the effect of the TNIK inhibitor NCB-0846 on behavioral manifestations of epilepsy.Coimmunoprecipitation(Co-IP)/mass spectrometry(MS)was used to identify the potential mechanism.Through Co-IP,we detected and confirmed the main potential TNIK interactors.Subcellular fractionation was used to establish the effect of NCB-0846 on the expression of the main interactors in postsynaptic density(PSD)fractions.We found that TNIK was primarily located in neurons and decreased significantly in epilepsy model rats and TLE patients compared with controls.NCB-0846 delayed kindling progression and decreased seizure severity.Co-IP/MS identified 63 candidate TNIK interactors in rat hippocampi,notably CaMKIl.Co-IP showed that TNIK might correlate with endogenous GRIA1,SYN2,PSD-95,CaMKIV,GABRG1,and GABRG2.In addition,the significant decrease in GRIA1 in hippocampal total lysate and PSDs after NCB-0846 treatment might help modify the progression of PTZ kindling.Our results suggest that TNIK contributes to epileptic pathology and is a potential antiepileptic drug target.

Minocycline inhibits neuroinflammation and enhances vascular endothelial growth factor expression in a cerebral ischemia/reperfusion rat model

BACKGROUND： Brain ischemia involves secondary inflammation, which significantly contributes to the outcome of ischemic insults. Vascular endothelial growth factor （VEGF） may play an important role in the vascular response to cerebral ischemia, because ischemia stimulates VEGF expression in the brain, and VEGF promotes formation of new cerebral blood vessels. Minocycline, a tetracycline derivative, protects against cerebral ischemia and reduces inflammation, oxidative stress, and apoptosis. OBJECTIVE： To observe the influence of minocycline on VEGE interleukin-1 beta （IL-119 ）, and tumor necrosis factor alpha （TNF-α） expression in Wistar rats with focal cerebral ischemia/reperfusion injury, and to study the neuroprotection mechanism of minocycline against focal cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： Randomized, controlled experiment, which was performed in the Chongqing Key Laboratory of Neurology between March 2007 and March 2008. MATERIALS： A total of 36 female, Wistar rats underwent surgery to insert a thread into the left middle cerebral artery. Animals were randomly divided into sham-operation, minocycline treatment, and ischemia/reperfusion groups, with 12 rats in each group. Minocycline （Huishi Pharmaceutical Limited Company, China） was dissolved to 0.5 g/L in normal saline. METHODS： A 0.5-1.0 cm thread was inserted into rats from the sham-operation group. Rats in the ischemia/reperfusion group underwent ischemia and reperfusion. The minocycline group received minocycline （50 mg/kg） 12 and 24 hours following ischemia and reperfusion, whereas the other groups received saline at the corresponding time points. MAIN OUTCOME MEASURES： mRNA and protein expression of IL-1β and TNF-α was measured by reverse transcriptase-polymerase chain reaction （RT-PCR） and enzyme linked immnnosorbent assay （ELISA）, respectively. VEGF mRNA and protein expression was examined by RT-PCR, Western blot, and ELISA. RESULTS： Minocycline decreased the focal infarct volume. VEGF, IL-1β, and TNF-α expression was upregulated in the ischemia-perfusion group after injury. Following minocycline treatment, IL-1β and TNF- α expression was significantly downregulated, and VEGF was significantly upregulated, compared with the ischemia/reperfusion group （all P 〈 0.01）. Expression of VEGF, IL-1β, and TNF- α was greater in the ischemia-perfusion and minocycline treatment groups, compared with sham-operated animals （P 〈 0.01 ）. CONCLUSION： Minocycline can reduce expression of IL- 1β and TNF-α, and increase VEGF expression, in the rat brain following cerebral ischemia/reperfusion. From these findings, a hypothesis can be formed that minocycline attenuates neuroinflammation and enhances recovery of vascular integrity during the process of cerebral ischemia/reperfusion.

Angiopoietin-1 mRNA and Bcl-2 expression following estradiol treatment in ovariectomized rats with focal cerebral ischemia/reperfusion injury

BACKGROUND： Estrogen has been clinically demonstrated to attenuate ischemic brain injury. However, the precise mechanisms remain controversial. OBJECTIVE： To investigate the effects of estradiol on angiopoietin-1 mRNA and Bcl-2 expression, as well as apoptosis and cerebral blood flow, in ovadectomized rats with focal cerebral ischemia following reperfusion. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiment. The study was performed at the Central Laboratory, Chongqing Medical University from September to December 2005. MATERIALS： Estradiol benzoate was purchased from Shanghai Ninth Pharmaceutical Factory; corn oil was purchased from Walmart Supercenter; TUNEL kit, rabbit anti-rat Bcl-2 polyclonal antibody, and biotin-labeled goat anti-rabbit antibody were purchased from Wuhan Boster, China. METHODS： Healthy, female, 6-month-old Wistar rats-wild-type and estrogen alpha receptor gene knockout （ERKO）-were randomly divided into estradiol and control groups with 25 animals in each group. The rats were intramuscularly injected with estradiol benzoate （100 μg/kg per day） at 30 days following bilateral ovariectomy or corn oil （1 mL/kg per day） for seven consecutive days. Following administration, cerebral ischemia/reperfusion models were established using the right middle cerebral artery occlusion （MCAO） method. After 30 minutes of MCAO, estradiol and control groups were separately injected with estradiol benzoate and corn oil with the above-mentioned doses. MAIN OUTCOME MEASURES： Cell apoptosis was determined by TUNEL; angiopoietin-1 mRNA and Bcl-2 gene expression was determined, respectively, by immunohistochemical staining and RT-PCR. In addition, changes in cerebral blood flow were measured by laser Doppler flowmetry. RESULTS： Changes in angiopoietin-1 mRNA and cerebral blood flow in estradiol-treated, wild-type, MCAO rats following ischemia/reperfusion were greater than in control rats （P 〈 0.01 or 0.05）. However, no significant difference was observed between estradiol-treated ERKO MCAO rats and control rats. In addition, estradiol-treated wild-type and ERKO MCAO rats exhibited significantly increased Bcl-2 expression （P 〈 0.05） and decreased number of apoptotic cells in brain tissues compared with control groups （P 〈 0.05）. CONCLUSION： Estradiol upregulated angiopoietin-1 mRNA and Bcl-2 expression, suggesting that estradiol might be involved in protective mechanisms of cerebral ischemia/reperfusion injury.

Cerebral furin deficiency causes hydrocephalus in mice

Furin is a pro-protein convertase that moves between the trans-Golgi network and cell surface in the secretory pathway.We have previously reported that cerebral overexpres-sion of furin promotes cognitive functions in mice.Here,by generating the brain-specific furin conditional knockout(ckO)mice,we investigated the role of furin in brain development.We found that furin deficiency caused early death and growth retardation.Magnetic resonance im-aging showed severe hydrocephalus.In the brain of furin cko mice,impaired ciliogenesis and the derangement of microtubule structures appeared along with the down-regulated expres-sion of RAB28,a ciliary vesicle protein.In line with the widespread neuronal loss,ependymal cell layers were damaged.Further proteomics analysis revealed that cell adhesion molecules including astrocyte-enriched ITGB8 and BCAR1 were altered in furin cKO mice;and astrocyte overgrowth was accompanied by the reduced expression of sox9,indicating a disrupted differ-entiation into ependymal cells.Together,whereas alteration of RAB28 expression correlated with the role of vesicle trafficking in ciliogenesis,dysfunctional astrocytes might be involved in ependymal damage contributing to hydrocephalus in furin ckO mice.The structural and mo-lecular alterations provided a clue for further studying the potential mechanisms of furin.

STON2风险变异通过影响Syt1转运和突触功能导致精神分裂症样行为

Synaptic dysfunction is a core component of the pathophysiology of schizophrenia.However,the genetic risk factors and molecular mechanisms related to synaptic dysfunction are still not fully understood.The Stonin 2(STON2)gene encodes a major adaptor for clathrin-mediated endocytosis(CME)of synaptic vesicles.In this study,we showed that the C-C(307Pro-851Ala)haplotype of STON2 increases the susceptibility to schizophrenia and examined whether STON2 variations cause schizophrenia-like behaviors through the regulation of CME.We found that schizophrenia-related STON2 variations led to protein dephosphorylation,which affected its interaction with synaptotagmin 1(Syt1),a calcium sensor protein located in the presynaptic membrane that is critical for CME.STON2307Pro851Ala knockin mice exhibited deficits in synaptic transmission,short-term plasticity,and schizophrenia-like behaviors.Moreover,among seven antipsychotic drugs,patients with the C-C(307Pro-851Ala)haplotype responded better to haloperidol than did the T-A(307Ser-851Ser)carriers.The recovery of deficits in Syt1 sorting and synaptic transmission by acute administration of haloperidol effectively improved schizophrenia-like behaviors in STON2307Pro851Ala knockin mice.Our findings demonstrated the effect of schizophreniarelated STON2 variations on synaptic dysfunction through the regulation of CME,which might be attractive therapeutic targets for treating schizophrenia-like phenotypes.

Practical considerations of dietary therapies for epilepsy in adults

Despite the increasing number of anti-seizure medications becoming available,the proportion of patients with drug-resistant epilepsy remains unchanged.Dietary therapy for epilepsy is well-established practice in paediatric care,but relatively underutilised in adults.Recently,international recommendations have been published to guide the treatment of adults receiving dietary therapy for epilepsy.This review focuses on the specific aspects of care unique to the management of adults receiving dietary therapy for epilepsy,including patient selection,diet composition,initiation,monitoring and cessation of dietary treatment.We emphasise the need for a multidisciplinary team approach with appropriately trained neurologists and dietitians to provide holistic care while the patients are receiving dietary therapy.Future research should focus on the optimal diet composition and meeting the psychosocial needs of adults with epilepsy to maximise efficacy and adherence to dietary treatment.

A Missense Mutation in Epsilon-subunit of Acetylcholine Receptor Causing Autosomal Dominant Slow-channel Congenital Myasthenic Syndrome in a Chinese Family

Background：Congenital myasthenic syndromes are a group orrare disorders that are clinically and genetically heterogeneous and caused by mutations in the genes encoding proteins of the neuromuscular junction.Here,we described a Chinese family that presented with phenotypes of classic slow-channel congenital myasthenic syndrome （SCCMS）.Methods：Clinical characteristics and electrophysiological features of three patients from a Chinese family were examined,and next-generation sequencing followed by direct sequencing was carried out.Results：The patients revealed variability in clinical and electrophysiological features.However,weakness,scoliosis,and repetitive-compound muscle action potential were found in all affected members in the family.A heterozygous C〉T missense mutation at nucleotide 865 in acetylcholine receptor epsilon-subunit （CHRNE） gene that causes a leucine-to-phenylalanine substitution at position 289 （L289F） was found.Conclusions：We reported a SCCMS family of Chinese origin.In the family,classical clinical phenotype with phenotypic variability among different members was found.Genetic testing could help diagnose this rare disease.

KIF17 Modulates Epileptic Seizures and Membrane Expression of the NMDA Receptor Subunit NR2B

Epilepsy is a common and severe brain disease affecting>65 million people worldwide.Recent studies have shown that kinesin superfamily motor protein 17(KIF17)is expressed in neurons and is involved in regulating the dendrite-targeted transport of N-methyl-D-aspartate receptor subtype 2B(NR2B).However,the effect of KIF17 on epileptic seizures remains to be explored.We found that KIF17 was mainly expressed in neurons and that its expression was increased in epileptic brain tissue.In the kainic acid(KA)-induced epilepsy mouse model,KIF17 overexpression increased the severity of epileptic activity,whereas KIF17 knockdown had the opposite effect.In electrophysiological tests,KIF17 regulated excitatory synaptic transmission,potentially due to KIF17-mediated NR2B membrane expression.In addition,this report provides the first demonstration that KIF17 is modified by SUMOylation(SUMO,small ubiquitin-like modifier),which plays a vital role in the stabilization and maintenance of KIF17 in epilepsy.

Sulfuretin exerts diversified functions in the processing of amyloid precursor protein

Sulfuretin is a flavonoid that protects cell from damage induced by reactive oxygen species and inflammation.In this study,we investigated the role of sulfuretin in the processing of amyloid precursor protein(APP),in association with the two catalytic enzymes the a-secretase a disintegrin and metalloproteinase(ADAM10),and the beta-site APP cleaving enzyme 1(BACE1)that play important roles in the generation of β amyloid protein(Aβ)in Alzheimer’s disease(AD).We found that sulfuretin increased the levels of the immature but not the mature form of ADAM10 protein.The enhanced ADAM10 transcription by sulfuretin was mediated by the nucleotides444 to300 in the promoter region,and was attenuated by silencing or mutation of transcription factor retinoid X receptor(RXR)and by GW6471,a specific inhibitor of peroxisome proliferator-activated receptor α(PPAR-α).We further found that sulfuretin preferentially increased protein levels of the immature form of APP(im-APP)but significantly reduced those of BACE1,sAPPβ and β-CTF,whereas Ab1-42 levels were slightly increased.Finally,the effect of sulfuretin on BACE1 and im-APP was selectively attenuated by the translation inhibitor cycloheximide and by lysosomal inhibitor chloroquine,respectively.Taken together,(1)RXR/PPAR-α signaling was involved in sulfuretin-mediated ADAM10 transcription.(2)Alteration of Aβ protein level by sulfuretin was not consistent with that of ADAM10 and BACE1 protein levels,but was consistent with the elevated level of im-APP protein,suggesting that im-APP,an isoform mainly localized to trans-Golgi network,plays an important role in Ab generation.

The effect of NR4A1 on APP metabolism and tau phosphorylation

Alzheimer’s disease(AD)is characterized by senile plaques(SP)composed of b-amyloid protein(Ab)and neurofibrillary tangles(NFTs)composed of intracellular hyperphosphorylated tau.Recently,nuclear receptor subfamily 4 group A member 1(NR4A1)was implicated in synaptic plasticity,long-term memory formation,suggesting that it may play a role in the pathophysiology of AD.Here,we showed that the expression of NR4A1 was significantly increased in the hippocampus of APP/PS1 transgenic mice.In addition,NR4A1 overexpression in HT22 cells up-regulated APP and BACE1 levels,down-regulated ADAM10 expression,and promoted amyloidogenesis as indicated by decreased a-CTF levels and elevated b-CTF levels.Furthermore,a raised level of phospho-tau(p-tau,S396)was accompanied by p-GSK3b(S9)expression reducing,but total tau,p-tau(S262 and T231),CDK5 and ERK remained unchanged in NR4A1-overexpressing cells.Collectively,our results suggest that NR4A1 promotes the amyloidogenic processing of APP by regulating ADAM10 and BACE1 expression in HT22 cells;as well as NR4A1 accelerates tau hyperphosphorylation by GSK3b signal.Therefore,NR4A1 may play an important role in the pathogenesis of AD.

CXCR5 Regulates Neuronal Polarity Development and Migration in the Embryonic Stage via F‑Actin Homeostasis and Results in Epilepsy‑Related Behavior

Epilepsy is a common,chronic neurological disorder that has been associated with impaired neurodevelopment and immunity.The chemokine receptor CXCR5 is involved in seizures via an unknown mechanism.Here,we first determined the expression pattern and distribution of the CXCR5 gene in the mouse brain during different stages of development and the brain tissue of patients with epilepsy.Subsequently,we found that the knockdown of CXCR5 increased the susceptibility of mice to pentylenetetrazol-and kainic acid-induced seizures,whereas CXCR5 overexpression had the opposite effect.CXCR5 knockdown in mouse embryos via viral vector electrotransfer negatively influenced the motility and multipolar-to-bipolar transition of migratory neurons.Using a human-derived induced an in vitro multipotential stem cell neurodevelopmental model,we determined that CXCR5 regulates neuronal migration and polarization by stabilizing the actin cytoskeleton during various stages of neurodevelopment.Electrophysiological experiments demonstrated that the knockdown of CXCR5 induced neuronal hyperexcitability,resulting in an increased number of seizures.Finally,our results suggested that CXCR5 deficiency triggers seizure-related electrical activity through a previously unknown mechanism,namely,the disruption of neuronal polarity.

Mitochondria as a therapeutic target in Alzheimer’s disease

Alzheimer’s disease(AD)remains the most common neurodegenerative disease characterized by b-amyloid protein(Ab)deposition and memory loss.Studies have shown that mitochondrial dysfunction plays a crucial role in AD,which involves oxidative stress-induced respiratory chain dysfunction,loss of mitochondrial biogenesis,defects of mitochondrial dynamics and mtDNA mutations.Thus mitochondria might serve as drug therapy target for AD.In this article,we first briefly discussed mitochondrial theory in the development of AD,and then we summarized recent advances of mitochondrial abnormalities in AD pathology and introduced a series of drugs and techniques targeting mitochondria.We think that maintaining mitochondrial function may provide a new way of thinking in the treatment of AD.