Mice harboring the T316N variant in the GABAAR γ2 subunit exhibit sleep-related hypermotor epilepsy phenotypes and hypersynchronization in the thalamocortical pathway.

OBJECTIVE: Sleep-related hypermotor epilepsy (SHE) is a focal epilepsy syndrome characterized by seizures that predominantly occur during sleep. The pathogenesis of these seizures remains unclear. We previously detected rare variants in GABRG2, which encodes the γ2 subunit of γ-aminobutyric acid type A receptor (GABAAR), in patients with SHE and demonstrated that these variants impaired GABAAR function in vitro. However, the mechanisms by which GABRG2 variants contribute to seizure attacks during sleep remain unclear. METHODS: In this study, we designed a knock-in (KI) mouse expressing the mouse Gabrg2 T316N variant, corresponding to human GABRG2 T317N variant, using CRISPR/Cas9. Continuous video-electroencephalogram monitoring and in vivo multichannel electrophysiological recordings were performed to explore seizure susceptibility to pentylenetetrazol (PTZ), alterations in the sleep-wake cycle, spontaneous seizure patterns, and synchronized activity in the motor thalamic nuclei (MoTN) and secondary motor cortex (M2). Circadian variations in the expression of total, membrane-bound, and synaptic GABAAR subunits were also investigated. RESULTS: No obvious changes in gross morphology were detected in Gabrg2T316N/+ mice compared to their wild-type (Gabrg2+/+) littermates. Gabrg2T316N/+ mice share key phenotypes with patients, including sleep fragmentation and spontaneous seizures during sleep. Gabrg2T316N/+ mice showed increased susceptibility to PTZ-induced seizures and higher mortality after seizures. Synchronization of the local field potentials between the MoTN and M2 was abnormally enhanced in Gabrg2T316N/+ mice during light phase, when sleep dominates, accompanied by increased local activities in the MoTN and M2. Interestingly, in Gabrg2+/+ mice, GABAAR γ2 subunits showed a circadian increase on the neuronal membrane and synaptosomes in the transition from dark phase to light phase, which was absent in Gabrg2T316N/+ mice. CONCLUSION: We generated a new SHE mouse model and provided in vivo evidence that rare variants of GABRG2 contribute to seizure attacks during sleep in SHE.

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/ß-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/ß-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/ß-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

Development and validation of a nomogram to provide individualized predictions of functional outcomes in patients with convulsive status epilepticus at 3 months: The modified END-IT tool.

AIMS: The prediction of outcomes in convulsive status epilepticus (CSE) remains a constant challenge. The Encephalitis-Nonconvulsive Status Epilepticus-Diazepam Resistance-Image Abnormalities-Tracheal Intubation (END-IT) score was a useful tool for predicting the functional outcomes of CSE patients, excluding cerebral hypoxia patients. With further understanding of CSE, and in view of the deficiencies of END-IT itself, we consider it necessary to modify the prediction tool. METHODS: The prediction model was designed from a cohort of CSE patients from Xijing Hospital (China), between 2008 and 2020. The enrolled subjects were randomly divided into training cohort and validation cohort as a ratio of 2:1. The logistic regression analysis was performed to identify the predictors and construct the nomogram. The performance of the nomogram was assessed by calculating the concordance index, and creating calibration plots to check the consistency between the predicted probabilities of poor prognosis and the actual outcomes of CSE. RESULTS: The training cohort included 131 patients and validation cohort included 66 patients. Variables included in the nomogram were age, etiology of CSE, non-convulsive SE, mechanical ventilation, abnormal albumin level at CSE onset. The concordance index of the nomogram in the training and validation cohorts was 0.853 (95% CI, 0.787-0.920) and 0.806 (95% CI, 0.683-0.923), respectively. The calibration plots showed an adequate consistency between the reported and predicted unfavorable outcomes of patients with CSE at 3 months after discharge. CONCLUSIONS: A nomogram for predicting the individualized risks of poor functional outcomes in CSE was constructed and validated, which has been an important modification of END-IT score.

Long-Term Outcomes among Patients with Prolonged Disorders of Consciousness.

PURPOSE: To evaluate the long-term survival and functional outcomes of patients with prolonged disorders of consciousness (pDoC) 1-8 years after brain injuries. METHODS: Retrospective study to assess the long-term survival and functional outcomes of patients with pDoC was conducted. We performed Cox regression and multivariate logistic regression to calculate hazard ratios (HRs) for the outcome of survival and to identify risk factors of the functional outcome. RESULTS: We recruited 154 patients with pDoC. The duration of follow-up from disease onset was 1-8 years. The median age was 46 years (IQR, 32-59), and 65.6% (n = 101) of them were men. During the follow-up period, one hundred and ten patients (71.4%) survived; among them, 52 patients had a good outcome. From the overall survival curve, the 1-, 3-, and 8-year survival rates of patients were about 80.5%, 72.0%, and 69.7%, respectively. Cox regression analysis revealed a significant association between the lower APACHE II score (p = 0.005) (cut-off score ≥ 18) and the presence of sleep spindles (p = 0.001) with survival. Logistic regression analysis demonstrated a higher CRS-R score (cut-off score ≥ 7), and presence of sleep spindles were related to a favorable outcome among patients with pDoC. CONCLUSIONS: Sleep spindles are correlated with both long-term survival and long-term functional outcome in pDoC patients.

Detecting residual brain networks in disorders of consciousness: A resting-state fNIRS study.

Functional near infrared spectroscopy (fNIRS) is an emerging non-invasive technique that allows bedside measurement of blood oxygenation level-dependent hemodynamic signals. We aimed to examine the efficacy of resting-state fNIRS in detecting the residual functional networks in patients with disorders of consciousness (DOC). We performed resting-state fNIRS in 23 DOC patients of whom 12 were in minimally conscious state (MCS) and 11 were in unresponsive wakefulness state (UWS). Ten regions of interest (ROIs) in the prefrontal cortex (PFC) were selected: both sides of Brodmann area (BA) 9, BA10, BA44, BA45, and BA46. Graph-theoretical analysis and seed-based correlation analyses were used to investigate the network topology and the strength of pairwise connections between ROIs and channels. MCS and UWS exhibited varying degrees of the loss of topological architecture, and the regional nodal properties of BA10 were significantly different between them (Nodal degree, PLeft BA10 = 0.01, PRight BA10 < 0.01; nodal efficiency, PLeft BA10 = 0.03, PRight BA10 < 0.01). Compared to healthy controls, UWS had impaired functions in both short- and long-distance connectivity, however, MCS had significantly impaired functions only in long-distance connectivity. The functional connectivity of right BA10 (AUC = 0.88) and the connections between left BA46 and right BA10 (AUC = 0.86) had excellent performance in differentiating MCS and UWS. MCS and UWS have different patterns of topological architecture and short- and long-distance connectivity in PFC. Intraconnections within BA10 and interhemispheric connections between BA10 and 46 are excellent resting-state fNIRS classifiers for distinguishing between MCS and UWS.

Mechanism of opioid addiction and its intervention therapy: Focusing on the reward circuitry and mu-opioid receptor.

Opioid abuse and addiction have become a global pandemic, posing tremendous health and social burdens. The rewarding effects and the occurrence of withdrawal symptoms are the two mainstays of opioid addiction. Mu-opioid receptors (MORs), a member of opioid receptors, play important roles in opioid addiction, mediating both the rewarding effects of opioids and opioid withdrawal syndrome (OWS). The underlying mechanism of MOR-mediated opioid rewarding effects and withdrawal syndrome is of vital importance to understand the nature of opioid addiction and also provides theoretical basis for targeting MORs to treat drug addiction. In this review, we first briefly introduce the basic concepts of MORs, including their structure, distribution in the nervous system, endogenous ligands, and functional characteristics. We focused on the brain circuitry and molecular mechanism of MORs-mediated opioid reward and withdrawal. The neuroanatomical and functional elements of the neural circuitry of the reward system underlying opioid addiction were thoroughly discussed, and the roles of MOR within the reward circuitry were also elaborated. Furthermore, we interrogated the roles of MORs in OWS, along with the structural basis and molecular adaptions of MORs-mediated withdrawal syndrome. Finally, current treatment strategies for opioid addiction targeting MORs were also presented.

Rare variants in GABRG2 associated with sleep-related hypermotor epilepsy.

Sleep-related hypermotor epilepsy (SHE) is a focal epilepsy syndrome. The underlying pathophysiology is presumed to be closely related with disruption of GABAergic neurotransmission, which is mainly medicated by γ-aminobutyric acid type A receptor (GABAAR). Thus, it is reasonable to assume that rare GABAAR variants might contribute to the pathogenesis of SHE. To test this hypothesis, we performed next-generation sequencing in 58 SHE patients and analyzed the functional effects of the identified variants in both neuronal and non-neuronal cells using a combination of electrophysiology recordings, western blot, flow cytometry, and confocal microscopy. In our study, we detected three rare variants (NM_198904.2: c.269C > T, p.T90M; NM_198904.2: c.950C > A, p.T317N and NM_198903.2: c.649C > T, p.Q217X) in GABRG2 (MIM:137,164, encoding GABAAR γ2 subunit) in three unrelated patients. Two of the three rare variants were transmitted unaffected maternally (T90M) or unaffected paternally (Q217X), whereas the T317N variant arose de novo. The mother of proband carrying the T90M variant was unaffected and being mosaicism for this variant. Functional analysis showed that T90M and T317N variants decreased GABA-evoked current amplitudes by diverse mechanisms including impaired surface expression, endoplasmic reticulum retention, and channel gating defects. And Q217X variant reduced synaptic clustering and distribution of GABAAR. While a causal role of these variants cannot be established directly from these results, the functional assessment together with the genetic sequencing suggests that these rare GABRG2 variants may constitute genetic risk factors for SHE. Our study further expands the GABRG2 phenotypic spectrum and supports the view that GABAergic neurotransmission participates in the epileptogenesis of SHE.

γ-secretase inhibitor disturbs the morphological development of differentiating neurons through affecting Notch/miR-342-5p.

During neurodevelopment, differentiation of neural stem/progenitor cells (NSPCs) into neurons are regulated by many factors including Notch signaling pathway. Herein, we report the effect of a Notch signaling blocker, i.e. γ -secretase inhibitor (GSI), on this differentiating process, especially on the morphological development. NSPCs were cultured and induced to differentiate with or without GSI. The neurite outgrowth was impeded by GSI application and the expression of a Notch signaling downstream effector miR-342-5p increased with the downregulated expression of Notch effectors Hes1 and Hes5. Upregulated expression of miR-342-5p in differentiating NSPCs could shorten the neurite length of progeny neurons, which was similar to the effect of GSI. To avoid the possible influence from astrocytes into neurons, we directly applied cultured neurons, on which GSI could shorten the processes and RBP-J knockdown could also reduce the neurite length. Similarly, transfection of miR-342-5p mimics or inhibitors into PC12 cells led to shorter or longer processes of cells compared with control ones. Furthermore, in differentiating NSPCs, GSI-induced shorter neurites could be partially rescued by miR-342-5p inhibitors, and STAT3 was one of the possible targets of miR-342-5p during this differentiating process as indicated by results of Western Blot test, luciferase reporter assay and GFP reporter assay. To further demonstrate the role of STAT3, it was introduced into GSI-treated neurons and the GSI-affected neurites could also be partially rescued. In conclusion, GSI could influence the morphological development of neurons and the possible mechanism involved Notch/miR-342-5p and STAT3. These results would be informative for future therapeutic research.

Inhibition of mu-opioid receptor suppresses proliferation of hepatocellular carcinoma cells via CD147-p53-MAPK cascade signaling pathway.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths. Previous studies have suggested that mu-opioid receptor (MOR), a member of the opioid receptor family, is involved in the pathogenesis of HCC. However, the mechanism by which MOR regulates the biological behavior of HCC is still poorly understood. To address this problem, in this study, we investigated the role of MOR in the proliferation of HCC cell lines and the underlying mechanism. First, RT-PCR, western-blot and immunohistochemistry revealed higher expression of MOR in HCC cells and tissue than in non-tumor cells or adjacent tissue, and elevated expression of MOR was associated with jeopardized survival of HCC patients, as demonstrated by bioinformatic databases. Knockdown of MOR by specific siRNA attenuated the proliferation and migration of HCC cells and this effect could be reversed by rescue experiments, confirming the essential role of MOR in the proliferation of HCC. Moreover, results of colony formation assay, CCK8 test, flow cytometry and western blot suggested that a monoclonal antibody (mAb) specifically against MOR could inhibit proliferation of HepG2 and Huh7 cells via the MOR-CD147-p53-MAPK pathway, and the interaction between MOR and CD147 was verified by immunofluorescence colocalization and co-IP analysis. The mAb against MOR also enhanced the cisplatin-induced apoptosis of HCC cells by downregulating p-ERK, Bcl-2 and upregulating Bax. Taken together, these results suggest that MOR could regulate the proliferation of HCC cells in a CD147-p53-MAPK dependent manner. MOR possesses the potential to be a therapeutic target to treat HCC.

Endocannabinoid system in the neurodevelopment of GABAergic interneurons: implications for neurological and psychiatric disorders.

In mature mammalian brains, the endocannabinoid system (ECS) plays an important role in the regulation of synaptic plasticity and the functioning of neural networks. Besides, the ECS also contributes to the neurodevelopment of the central nervous system. Due to the increase in the medical and recreational use of cannabis, it is inevitable and essential to elaborate the roles of the ECS on neurodevelopment. GABAergic interneurons represent a group of inhibitory neurons that are vital in controlling neural network activity. However, the role of the ECS in the neurodevelopment of GABAergic interneurons remains to be fully elucidated. In this review, we provide a brief introduction of the ECS and interneuron diversity. We focus on the process of interneuron development and the role of ECS in the modulation of interneuron development, from the expansion of the neural stem/progenitor cells to the migration, specification and maturation of interneurons. We further discuss the potential implications of the ECS and interneurons in the pathogenesis of neurological and psychiatric disorders, including epilepsy, schizophrenia, major depressive disorder and autism spectrum disorder.

The efficacy and safety of Hirudin plus Aspirin versus Warfarin in the secondary prevention of Cardioembolic Stroke due to Nonvalvular Atrial Fibrillation: A multicenter prospective cohort study.

Background: To investigate the efficacy and safety of hirudin plus aspirin therapy compared with warfarin in the secondary prevention of cardioembolic stroke due to nonvalvular atrial fibrillation (NVAF). Methods: Patients with cardioembolic stroke due to NVAF were prospectively enrolled from 18 collaborating hospitals from Dec 2011 to June 2015. Fourteen days after stroke onset, eligible patients were assigned to the hirudin plus aspirin group (natural hirudin prescribed as the traditional Chinese medicine Maixuekang capsule, 0.75 g, three times daily, combined with aspirin 100 mg, once daily) or the warfarin group (dose-adjusted warfarin targeting international normalized ratio (INR) 2-3, with an initial daily dose of 1.25 mg). Patients were followed up at 1, 2, 3, 6, 9, and 12 months after stroke onset. Time in therapeutic range (TTR) was calculated according to Rosendaal methodology to evaluate the quality of INR management in the warfarin group. The primary efficacy endpoint was the recurrence of stroke within 12 months after stroke onset. Safety was assessed as the occurrence of the composite event "intracranial hemorrhage and other bleeding events, death, and other serious adverse events". The Cox proportional hazard model and Kaplan-Meier curve were used to analyze the efficacy and safety events. Results: A total of 221 patients entered final analysis with 112 patients in the hirudin plus aspirin group and 109 in the warfarin group. Over the whole duration of our study, TTR for patients taking warfarin was 66.5 % ± 21.5%. A significant difference was not observed in the recurrence of stroke between the two groups (3.57% vs. 2.75%; P = 0.728). The occurrence of safety events was significantly lower in the hirudin plus aspirin group (2.68% vs.10.09%; P = 0.024). The risk for efficacy event was similar between the two groups (hazard ratio (HR), 1.30; 95% confidence interval (CI), 0.29-5.80). The safety risk was significantly lower in the hirudin plus aspirin group (HR, 0.27; 95% CI, 0.07-0.95). Kaplan-Meier analysis revealed significant difference in the temporal distribution in safety events (P = 0.023) but not in stroke recurrence (P = 0.726). Conclusion: Significant difference in efficacy was not detected between warfarin group and hirudin plus aspirin group. Compared with warfarin, hirudin plus aspirin therapy had lower safety risk in the secondary prevention of cardioembolic stroke due to NVAF.

Clinical characteristics and fetal outcomes in women with epilepsy with planned and unplanned pregnancy: A retrospective study.

PURPOSE: To compare the antiepileptic drug (AED) treatment patterns, seizure control, and folic acid supplementation between planned and unplanned pregnancy in women with epilepsy (WWE) and to investigate the effects of planned pregnancy on fetal outcomes. METHODS: A prospectively collected database including WWE with pregnancy from Feb 2010 to Dec 2018 was retrospectively analyzed. Planned pregnancy was defined as WWE being regularly supervised by epileptologists from the time of intended pregnancy until delivery. Clinical characteristics and fetal outcomes were compared between the planned and unplanned pregnancy groups. Logistic regression was used to identify modifiable factors associated with adverse fetal outcomes. RESULTS: A total of 188 planned pregnancies and 289 unplanned pregnancies were enrolled in our study. Among planned pregnancies, 66.0 % took AED monotherapy, and 32.4 % received polytherapy. Among unplanned pregnancies, 58.1 % didn't take AEDs, 28.0 % took monotherapy, and 12.8 % received polytherapy. The planned pregnancies had less generalized tonic-clonic seizures (P = 0.002) and higher proportion of being seizure-free (41.0 % vs. 22.8 %; P <0.001). All planned pregnancies took folic acid while 39.8 % of unplanned pregnancies never took it (P <0.001). The planned pregnancies had less rates of induced abortions (2.7 % vs. 13.5 %; P <0.001), preterm births (3.3 % vs. 20.4 %; P <0.001), and major congenital malformations (1.6 % vs. 7.5 %; P = 0.016). Pregnancy planning was independently associated with adverse fetal outcomes (adjusted OR, 0.14; 95 % CI, 0.08-0.27; P <0.001). CONCLUSION: Planned pregnancy in WWE contributes to more optimized AED pattern, better seizure control, more appropriate folic acid supplementation, and less adverse fetal outcomes.

Prognostic roles of sleep electroencephalography pattern and circadian rhythm biomarkers in the recovery of consciousness in patients with coma: a prospective cohort study.

OBJECTIVE: To investigate the potential prognostic value of sleep electroencephalography (EEG) pattern and serum circadian rhythm biomarkers in the recovery of consciousness in patients at the acute stage of coma. METHODS: A prospective observational study which included 75 patients with coma was conducted. Twenty-four-hour continuous polysomnography (PSG) was performed to determine the sleep EEG pattern according to the modified Valente's Grade (mVG) that we proposed. Serum levels of melatonin and orexin-A at four consecutive time points during the PSG were examined. Patients were then followed for one month to determine their level of consciousness. Multivariate logistic regression analysis was performed to examine associations between demographics, aetiology, baseline clinical features (pupillary and corneal reflex, and neuron-specific enolase [NSE]), clinical scores (Glasgow Coma Scale-Motor Response [GCS-M], Full Outline of Unresponsiveness [FOUR] scale, Acute Physiology and Chronic Health Evaluation II [APACHE II] scale), mVG, serum circadian biomarkers, and recovery of consciousness within one month. RESULTS: Within one month of enrolment, 34 patients regained consciousness and 36 patients remained non-conscious. Spearman rank correlation revealed a significant association between mVG and state of consciousness after one month. Significant variation in serum melatonin or orexin-A was not detected in either the conscious or non-conscious groups. Hypoxic aetiology, APACHE II, and mVG were independently associated with recovery of consciousness within one month. CONCLUSION: Sleep EEG structure, hypoxic aetiology, and APACHE II can independently predict recovery of consciousness in patients with acute coma. Taken together, we encourage neurologists to use sleep elements to assess patients with acute coma.

Stem cells: a promising candidate to treat neurological disorders.

Neurologic impairments are usually irreversible as a result of limited regeneration in the central nervous system. Therefore, based on the regenerative capacity of stem cells, transplantation therapies of various stem cells have been tested in basic research and preclinical trials, and some have shown great prospects. This manuscript overviews the cellular and molecular characteristics of embryonic stem cells, induced pluripotent stem cells, neural stem cells, retinal stem/progenitor cells, mesenchymal stem/stromal cells, and their derivatives in vivo and in vitro as sources for regenerative therapy. These cells have all been considered as candidates to treat several major neurological disorders and diseases, owing to their self-renewal capacity, multi-directional differentiation, neurotrophic properties, and immune modulation effects. We also review representative basic research and recent clinical trials using stem cells for neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and age-related macular degeneration, as well as traumatic brain injury and glioblastoma. In spite of a few unsuccessful cases, risks of tumorigenicity, and ethical concerns, most results of animal experiments and clinical trials demonstrate efficacious therapeutic effects of stem cells in the treatment of nervous system disease. In summary, these emerging findings in regenerative medicine are likely to contribute to breakthroughs in the treatment of neurological disorders. Thus, stem cells are a promising candidate for the treatment of nervous system diseases.

Exosomes derived from human umbilical vein endothelial cells promote neural stem cell expansion while maintain their stemness in culture.

The neural stem cell (NSC) niche in subventricular zone (SVZ) of adult mammalian brain contains dense vascular plexus, where endothelial cells (ECs) regulate NSCs by releasing plenty of angiocrine factors. However, the role of ECs-derived exosomes, a novel type of mediators of intercellular communications, in the regulation of NSCs remains unclear. In the current study, primary NSCs isolated from embryonic mouse brains form more neurospheres when cultured in the presence of human umbilical vein endothelial cells (HUVECs). The supportive role of ECs in the coculture was significantly attenuated when GW4869, a blocker of exosome formation, was included, suggesting that HUVECs-derived exosomes played a significant role in supporting NSCs. In order to investigate the role of ECs-derived exosomes on NSCs, we collected exosomes from HUVECs. We found that HUVECs-derived exosomes could significantly promote the formation of neurospheres by primary murine NSCs. EdU incorporation and TUNEL assays indicated that the proliferation of NSCs increased while apoptosis decreased when cultured in the presence of HUVECs-derived exosomes. NSCs incubated with the HUVECs-derived exosomes maintained their potential of multi-lineage differentiation potentials. The expression of stemness-related genes was up-regulated. These data suggested that ECs-derived exosomes could play an importantly role in NSC niche, and they might be used as a reagent for ex vivo NSC amplification for medical application.

Expression and purification of mouse Ttyh1 fragments as antigens to generate Ttyh1-specific monoclonal antibodies.

Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1. With these Ttyh1 protein products, a set of monoclonal antibodies (mAbs) against the mouse Ttyh1 protein was established by using conventional hybridoma techniques. The specificity of the anti-Ttyh1 mAbs was determined based on their activities in Western blotting and immunofluorescent analysis using embryonic brain tissues and cultured mouse neural stem cells (NSCs). We also show that the mouse Ttyh1 protein was expressed in cultured NSCs, most likely in membrane and cytoplasm. In mouse embryonic brains, it appeared that the Ttyh1 protein was specifically expressed in the apical edge of the ventricular zone as puncta-like structures, as determined by using immunofluorescence. Taken together, our study provided a useful tool for further exploration of the biological functions and pathological significance of Ttyh1 in mice.