Distinct Th17 effector cytokines differentially promote microglial and blood-brain barrier inflammatory responses during post-infectious encephalitis.

Group A Streptococcus (GAS) infections can cause neuropsychiatric sequelae in children due to post-infectious encephalitis. Multiple GAS infections induce migration of Th17 lymphocytes from the nose into the brain, which are critical for microglial activation, blood-brain barrier (BBB) and neural circuit impairment in a mouse disease model. How endothelial cells (ECs) and microglia respond to GAS infections, and which Th17-derived cytokines are essential for these responses are unknown. Using single-cell RNA sequencing and spatial transcriptomics, we found that ECs downregulate BBB genes and microglia upregulate interferon-response, chemokine and antigen-presentation genes after GAS infections. Several microglial-derived chemokines were elevated in patient sera. Administration of a neutralizing antibody against interleukin-17A (IL-17A), but not ablation of granulocyte-macrophage colony-stimulating factor (GM-CSF) in T cells, partially rescued BBB dysfunction and microglial expression of chemokine genes. Thus, IL-17A is critical for neuropsychiatric sequelae of GAS infections and may be targeted to treat these disorders.

Innate and Adaptive Immunity of Murine Neural Stem Cell-Derived piRNA Exosomes/Microvesicles against Pseudotyped SARS-CoV-2 and HIV-Based Lentivirus.

By testing pseudotyped SARS-CoV-2 and HIV-based lentivirus, this study reports that exosomes/microvesicles (Ex/Mv) isolated from murine hypothalamic neural stem/progenitor cells (htNSC) or subtype htNSCPGHM as well as hippocampal NSC have innate immunity-like actions against these RNA viruses. These extracellular vesicles also have a cell-free innate antiviral action by attacking and degrading viruses. We further generated the induced versions of Ex/Mv through prior viral exposure to NSCs and found that these induced Ex/Mv were stronger than basal Ex/Mv in reducing the infection of these viruses, suggesting the involvement of an adaptive immunity-like antiviral function. These NSC Ex/Mv were found to be characterized by producing large libraries of P element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) against genomes of various viruses, and some of these piRNAs were enriched during the adaptive immunity-like reaction, possibly contributing to the antiviral effects of these Ex/Mv. In conclusion, NSC Ex/Mv have antiviral immunity and could potentially be developed to combat against various viruses.

A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system.

Microglia are resident immune cells of the central nervous system (CNS). The exact role of microglia in CNS disorders is not clear due to lack of tools to discriminate between microglia and infiltrating myeloid cells. Here, we present a novel reporter mouse model targeting a microglia-specific marker, TMEM119, for studying microglia in health and disease. By placing a reporter cassette (GSG-3xFlag-P2A-tdTomato) between the coding sequence of exon 2 and 3'UTR of the Tmem119 gene using CRISPR/Cas9 technology, we generated a Tmem119-tdTomato knock-in mouse strain. Gene expression assay showed no difference of endogenous Tmem119 in the CNS of Tmem119tdTomato/+ relative to wild-type mice. The cells expressing tdTomato were recognized by immunofluorescence staining using commercially available anti-TMEM119 antibodies. Additionally, immunofluorescence and flow cytometry techniques revealed that tdTomato+ cells are detected throughout the CNS, but not in peripheral tissues of Tmem119tdTomato/+ mice. Aging does not influence TMEM119 expression as tdTomato+ cells were detectable in the CNS of older mice (300 and 540 days old). Further immunofluorescence characterization shows that tdTomato+ cells colocalize with Iba1+ cells in the brain, but not with neurons, astrocytes or oligodendrocytes. Moreover, flow cytometry analysis of brain tissues of adult mice demonstrates that the majority of microglia CD45loCD11b+ cells (96.3%) are tdTomato-positive; and a minority of infiltrating CD45hiCD11b+ myeloid cells (5.3%) are also tdTomato-positive, which we further characterized and found that tdTomato expression is in part of choroid plexus macrophages but not in meningeal and perivascular macrophages. Functionally, using an acute injury model, we measured time-lapse activation of tdTomato-labeled microglia by transcranial two-photon microscopy in live Tmem119tdTomato/+ mice. Taken together, the Tmem119-tdTomato reporter mouse model is a valuable tool to specifically study the role of microglia in health and disease.

ProBDNF/p75NTR/sortilin pathway is activated in peripheral blood of patients with alcohol dependence.

Alcohol dependence is a worldwide problem with a great social and economic burden in many countries. A number of studies have suggested that BDNF (mature BDNF) and its precursor (proBDNF) play important roles in the alcohol dependence. However, what roles of the mBDNF/proBDNF pathways play during the pathological process of alcohol dependence are not clearly understood. In our clinical study, peripheral blood was sampled from 30 male patients with alcohol dependence and 50 healthy males (as control). The protein levels of proBDNF, p75NTR, sortilin, mBDNF, TrkB and mRNA levels of BDNF, p75NTR, sortilin, and TrkB were detected in the peripheral blood in our study. We found that the protein levels of proBDNF and p75NTR were increased, but not the sortilin protein level; while the TrkB protein level was decreased in the alcohol dependence patients compared with healthy controls. Moreover, the mRNA levels of p75NTR and sortilin from the lymphocytes were slightly increased; while BDNF and TrkB were significantly decreased. The ELISA results of mBDNF and TrkB were declined in the alcohol dependence group. The levels of mBDNF and TrkB were negatively correlated with the average amount of daily ethanol consumption, and the levels of proBDNF, p75NTR and sortilin were positively correlated with the average amount of ethanol consumption per day. The ratio of proBDNF to mBDNF was altered in alcohol dependence patients. The balance between the proBDNF/p75NTR and mBDNF/TrkB signalling pathways appeared dysregulated in alcohol dependence. Our results suggested that both pathways may participate in the complex processes of alcohol dependence.

Sortilin inhibits amyloid pathology by regulating non-specific degradation of APP.

Amyloid plaque is one of the hallmarks of Alzheimer's disease (AD). The key component beta-amyloid (Aß) is generated via proteolytic processing of amyloid precursor protein (APP). Sortilin (encoded by the gene Sort1) is a vacuolar protein sorting 10 protein domain-containing receptor, which is up-regulated in the brain of AD, colocalizes with amyloid plaques and interacts with APP. However, its role in amyloidogenesis remains unclear. In this study, we first found that the protein level of sortilin was up-regulated in the neocortex of aged (7 and 9months old) but not young (2 and 5months old) AD mice (APP/PS1). 9months old APP/PS1 transgenic mice with Sort1 gene knockout showed increased amyloid pathology in the brain; and this phenotype was rescued by intrahippocampal injection of AAV-hSORT1. Moreover, the 9months old APP/PS1 mice without Sort1 also displayed a decreased number of neurons and increased astrocyte activation in the hippocampus. In addition, the present study showed that the intracellular domain of sortilin was involved in the regulation of the non-specific degradation of APP. Together, our findings indicate that sortilin is a beneficial protein for the reduction of amyloid pathology in APP/PS1 mice by promoting APP degradation.

Sortilin Fragments Deposit at Senile Plaques in Human Cerebrum.

Genetic variations in the vacuolar protein sorting 10 protein (Vps10p) family have been linked to Alzheimer's disease (AD). Here we demonstrate deposition of fragments from the Vps10p member sortilin at senile plaques (SPs) in aged and AD human cerebrum. Sortilin changes were characterized in postmortem brains with antibodies against the extracellular and intracellular C-terminal domains. The two antibodies exhibited identical labeling in normal human cerebrum, occurring in the somata and dendrites of cortical and hippocampal neurons. The C-terminal antibody also marked extracellular lesions in some aged and all AD cases, appearing as isolated fibrils, mini-plaques, dense-packing or circular mature-looking plaques. Sortilin and ß-amyloid (Aß) deposition were correlated overtly in a region/lamina- and case-dependent manner as analyzed in the temporal lobe structures, with co-localized immunofluorescence seen at individual SPs. However, sortilin deposition rarely occurred around the pia, at vascular wall or in areas with typical diffuse Aß deposition, with the labeling not enhanced by section pretreatment with heating or formic acid. Levels of a major sortilin fragment ~15 kDa, predicted to derive from the C-terminal region, were dramatically elevated in AD relative to control cortical lysates. Thus, sortilin fragments are a prominent constituent of the extracellularly deposited protein products at SPs in human cerebrum.

ProBDNF Signaling Regulates Depression-Like Behaviors in Rodents under Chronic Stress.

Chronic exposure to stressful environment is a key risk factor contributing to the development of depression. However, the mechanisms involved in this process are still unclear. Brain-derived neurotropic factor (BDNF) has long been investigated for its positive role in regulation of mood, although the role of its precursor, proBDNF, in regulation of mood is not known. In this study, using an unpredictable chronic mild stress (UCMS) paradigm we found that the protein levels of proBDNF were increased in the neocortex and hippocampus of stressed mice and this UCMS-induced upregulation of proBDNF was abolished by chronic administration of fluoxetine. We then established a rat model of UCMS and found that the expression of proBDNF/p75NTR/sortilin was upregulated, whereas the expression of mature BDNF and TrkB was downregulated in both neocortex and hippocampus of chronically stressed rats. Finally, we found that the injection of anti-proBDNF antibody via intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) approaches into the UCMS rats significantly reversed the stress-induced depression-like behavior and restored the exploratory activity and spine growth. Although intramuscular injection of AAV-proBDNF did not exacerbate the UCMS-elicited rat mood-related behavioral or pathological abnormalities, i.c.v. injection of AAV-proBDNF increased the depression-like behavior in naive rats. Our findings suggest that proBDNF plays a role in the development of chronic stress-induced mood disturbances in rodents. Central (i.c.v.) or peripheral (i.p.) inhibition of proBDNF by injecting specific anti-proBDNF antibodies may provide a novel therapeutic approach for the treatment of stress-related mood disorders.

Mice with Sort1 deficiency display normal cognition but elevated anxiety-like behavior.

Exposure to stressful life events plays a central role in the development of mood disorders in vulnerable individuals. However, the mechanisms that link mood disorders to stress are poorly understood. Brain-derived neurotrophic factor (BDNF) has long been implicated in positive regulation of depression and anxiety, while its precursor (proBDNF) recently showed an opposing effect on such mental illnesses. P75(NTR) and sortilin are co-receptors of proBDNF, however, the role of these receptors in mood regulation is not established. Here, we aimed to investigate the role of sortilin in regulating mood-related behaviors and its role in the proBDNF-mediated mood abnormality in mice. We found that sortilin was up-regulated in neocortex (by 78.3%) and hippocampus (by 111%) of chronically stressed mice as assessed by western blot analysis. These changes were associated with decreased mobility in the open field test and increased depression-like behavior in the forced swimming test. We also found that sortilin deficiency in mice resulted in hyperlocomotion in the open field test and increased anxiety-like behavior in both the open field and elevated plus maze tests. No depression-like behavior in the forced swimming test and no deficit in spatial cognition in the Morris water maze test were found in the Sort1-deficient mice. Moreover, the intracellular and extracellular levels of mature BDNF and proBDNF were not changed when sortilin was absent in vivo and in vitro. Finally, we found that both WT and Sort1-deficient mice injected with proBDNF in lateral ventricle displayed increased depression-like behavior in the forced swimming test but not anxiety-like behaviors in the open field and elevated plus maze tests. The present study suggests that sortilin functions as a negative regulator of mood performance and can be a therapeutic target for the treatment of mental illness.

Neurotrophin-3 Induces BMP-2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats.

Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT-3, and NT-4 and their Trk receptors. NT-3 and its receptor TrkC showed the highest induction. NT-3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT-3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT-3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP-2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT-3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP-2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT-3 osteogenic effect in vitro because it can be almost completely abrogated by co-addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT-3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co-treatment with anti-VEGF. This study suggests that NT-3 may be an osteogenic and angiogenic factor upstream of BMP-2 and VEGF in bony repair, and further studies are required to investigate whether NT-3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.

Deletion of TRIM32 protects mice from anxiety- and depression-like behaviors under mild stress.

Chronic stress causes a variety of psychiatric disorders such as anxiety and depression, but its mechanism is not well understood. Tripartite motif-containing protein 32 (TRIM32) was strongly associated with autism spectrum disorder, attention deficit hyperactivity disorder, anxiety and obsessive compulsive disorder based on a study of copy number variation, and deletion of TRIM32 increased neural proliferation and reduced apoptosis. Here, we propose that TRIM32 is involved in chronic stress-induced affective behaviors. Using a chronic unpredictable mild stress mouse depression model, we studied expression of TRIM32 in brain tissue samples and observed behavioral changes in Trim32 knockout mice. The results showed that TRIM32 protein but not its mRNA was significantly reduced in hippocampus in a time-dependent manner within 8 weeks of chronic stress. These stress-induced affective behaviors and reduction of TRIM32 protein expression were significantly reversed by antidepressant fluoxetine treatment. In addition, Trim32 knockout mice showed reduced anxiety and depressive behaviors and hyperactivities compared with Trim32 wild-type mice under normal and mild stress conditions. We conclude that TRIM32 plays important roles in regulation of hyperactivities and positively regulates the development of anxiety and depression disorders induced by chronic stress.