Disfunction of dorsal raphe nucleus-hippocampus serotonergic-HTR3 transmission results in anxiety phenotype of Neuroplastin 65-deficient mice.

Anxiety disorders are the most common psychiatric condition, but the etiology of anxiety disorders remains largely unclear. Our previous studies have shown that neuroplastin 65 deficiency (NP65-/-) mice exhibit abnormal social and mental behaviors and decreased expression of tryptophan hydroxylase 2 (TPH2) protein. However, whether a causal relationship between TPH2 reduction and anxiety disorders exists needs to be determined. In present study, we found that replenishment of TPH2 in dorsal raphe nucleus (DRN) enhanced 5-HT level in the hippocampus and alleviated anxiety-like behaviors. In addition, injection of AAV-NP65 in DRN significantly increased TPH2 expression in DRN and hippocampus, and reduced anxiety-like behaviors. Acute administration of exogenous 5-HT or HTR3 agonist SR57227A in hippocampus mitigated anxiety-like behaviors in NP65-/- mice. Moreover, replenishment of TPH2 in DRN partly repaired the impairment of long-term potentiation (LTP) maintenance in hippocampus of NP65-/- mice. Finally, we found that loss of NP65 lowered transcription factors Lmx1b expression in postnatal stage and replenishment of NP65 in DRN reversed the decrease in Lmx1b expression of NP65-/- mice. Together, our findings reveal that NP65 deficiency induces anxiety phenotype by downregulating DRN-hippocampus serotonergic-HTR3 transmission. These studies provide a novel and insightful view about NP65 function, suggesting an attractive potential target for treatment of anxiety disorders.

Neuroplastin 65 mediates cognitive functions via excitatory/inhibitory synapse imbalance and ERK signal pathway.

Neuroplastin 65 (NP65) is a brain-specific glycoprotein component of synaptic membrane, which is predominantly located in the forebrain such as the cortex, amygdala and striatum and hippocampus. Previous studies have shown that NP65 is implicated in synaptic plasticity, so it was hypothesized to play roles in cognitive functions. To test this hypothesis, we generated NP65 knock-out (KO) mice and found that the null mice exhibited enhanced hippocampus-dependent learning and memory as manifested by Morris water maze test and step-through passive avoidance test, but showed anxiety-like behaviors as manifested by open field test and light/dark exploration test. In addition, molecular and cellular studies revealed several alterations including: (1) the enhanced ratio of excitatory to inhibitory synapses; (2) increased expression of NMDA receptors NR2A; (3) enhanced activation of ERK signaling; (4) lowered number of the mushroom- and bifurcate-shaped dendritic spines in NP65 KO mice. Together, our findings suggest that NP65 may mediate cognitive functions.

Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model.

Gene targeting is a critical tool for construction of disease models. However, the application of traditional homologous recombination-mediated gene knockout technology is limited by the absence of rapid frequency-guaranteed targeting methods. Although conventional small hairpin RNA (shRNA)-mediated gene silencing offers an alternative for gene targeting, its application is frequently compromised by lower expression efficiency via RNA interference compared to gene knockout. Here we provide an efficient gene targeting strategy involving drug-inducible synergistic silencing with multiple shRNA molecules. On induction, the levels of the target proteins decreased to undetectable levels in all the tested stable transgenic mammalian cell lines, including HEK293 and embryonic stem cell-derived progenies carrying shRNA silencing cassettes. In a transgenic mouse model carrying a silencing cassette targeting the rhodopsin gene, short-time inducer treatment was sufficient to ablate the rhodopsin protein in the retina, resulting in similar retinal phenotypic changes as those observed in rhodopsin mutant mice. Therefore, on a broad basis, this inducible shRNA gene targeting strategy offers a true gene knockout alternative comparable to conventional RNA interference approaches.

Emodin-mediated cross-linking enhancement for extracellular matrix homeostasis.

The extracellular matrix (ECM) is an essential element of mammalian organisms, and its cross-linking formation plays a vital role in ECM development and postnatal homeostasis. Defects in cross-link formation caused by aging, genetic, or environmental factors are known to cause numerous diseases in mammals. To augment the cross-linking formation of ECM, the present study established a ZsGreen reporter system controlled by the promoter of lysyl oxidase-like 1 gene (LOXL1), which serves as both a scaffold element and a cross-linking enzyme in the ECM. By using this system in a drug screen, we identified emodin as a strong enhancer of LOXL1 expression that promoted cross-linking formation of ECM in all the tested systems, including human fibroblast cells, cultured human skin tissues, and animals that received long-term emodin treatment. Collectively, the results suggest that emodin may serve as an effective drug or supplement for ECM homeostasis.

Inhibiting 5-hydroxytryptamine receptor 3 alleviates pathological changes of a mouse model of Alzheimer's disease.

Extracellular amyloid beta (Aß) plaques are main pathological feature of Alzheimer's disease. However, the specific type of neurons that produce Aß peptides in the initial stage of Alzheimer's disease are unknown. In this study, we found that 5-hydroxytryptamin receptor 3A subunit (HTR3A) was highly expressed in the brain tissue of transgenic amyloid precursor protein and presenilin-1 mice (an Alzheimer's disease model) and patients with Alzheimer's disease. To investigate whether HTR3A-positive interneurons are associated with the production of Aß plaques, we performed double immunostaining and found that HTR3A-positive interneurons were clustered around Aß plaques in the mouse model. Some amyloid precursor protein-positive or ß-site amyloid precursor protein cleaving enzyme-1-positive neurites near Aß plaques were co-localized with HTR3A interneurons. These results suggest that HTR3A -positive interneurons may partially contribute to the generation of Aß peptides. We treated 5.0-5.5-month-old model mice with tropisetron, a HTR3 antagonist, for 8 consecutive weeks. We found that the cognitive deficit of mice was partially reversed, Aß plaques and neuroinflammation were remarkably reduced, the expression of HTR3 was remarkably decreased and the calcineurin/nuclear factor of activated T-cell 4 signaling pathway was inhibited in treated model mice. These findings suggest that HTR3A interneurons partly contribute to generation of Aß peptide at the initial stage of Alzheimer's disease and inhibiting HTR3 partly reverses the pathological changes of Alzheimer's disease.

Neuroplastin 65 deficiency reduces amyloid plaque formation and cognitive deficits in an Alzheimer's disease mouse model.

Introduction: Alzheimer's disease (AD) is characterized by increasing cognitive dysfunction, progressive cerebral amyloid beta (Aß) deposition, and neurofibrillary tangle aggregation. However, the molecular mechanisms of AD pathologies have not been completely understood. As synaptic glycoprotein neuroplastin 65 (NP65) is related with synaptic plasticity and complex molecular events underlying learning and memory, we hypothesized that NP65 would be involved in cognitive dysfunction and Aß plaque formation of AD. For this purpose, we examined the role of NP65 in the transgenic amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD. Methods: Neuroplastin 65-knockout (NP65-/-) mice crossed with APP/PS1 mice to get the NP65-deficient APP/PS1 mice. In the present study, a separate cohort of NP65-deficient APP/PS1 mice were used. First, the cognitive behaviors of NP65-deficient APP/PS1 mice were assessed. Then, Aß plaque burden and Aß levels in NP65-deficient APP/PS1 mice were measured by immunostaining and western blot as well as ELISA. Thirdly, immunostaining and western blot were used to evaluate the glial response and neuroinflammation. Finally, protein levels of 5-hydroxytryptamin (serotonin) receptor 3A and synaptic proteins and neurons were measured. Results: We found that loss of NP65 alleviated the cognitive deficits of APP/PS1 mice. In addition, Aß plaque burden and Aß levels were significantly reduced in NP65-deficient APP/PS1 mice compared with control animals. NP65-loss in APP/PS1 mice resulted in a decrease in glial activation and the levels of pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, and IL-4) as well as protective matrix YM-1 and Arg-1, but had no effect on microglial phenotype. Moreover, NP65 deficiency significantly reversed the increase in 5-hydroxytryptamine (serotonin) receptor 3A (Htr3A) expression levels in the hippocampus of APP/PS1 mice. Discussion: These findings identify a previously unrecognized role of NP65 in cognitive deficits and Aß formation of APP/PS1 mice, and suggest that NP65 may serve as a potential therapeutic target for AD.

Neuroplastin-65 and a mimetic peptide derived from its homophilic binding site modulate neuritogenesis and neuronal plasticity.

Neuroplastin-65 (Np65) is a brain-specific cell adhesion molecule belonging to the immunoglobulin superfamily. Homophilic trans-interaction of Np65 mediates adhesion between cells and modulates synaptic plasticity. This interaction solely occurs through the first immunoglobulin (Ig) module of Np65, but the exact binding mechanism has not yet been elucidated. In this study, we identify the homophilic binding motif of Np65 and show that a synthetic peptide modeled after this motif, termed enplastin, binds to Np65. We demonstrate that both Np65- and enplastin-induced intracellular signaling depends on fibroblast growth factor receptor, p38 mitogen-activated protein kinase, Ca(2+) /calmodulin-dependent protein kinase, and cytoplasmic Ca(2+) concentration. In addition, we show that interference with Np65 homophilic binding by enplastin has an inhibitory effect on Np65-mediated neurite outgrowth in vitro and on the initial phase of spatial learning in rats.

Effects of primary microglia and astrocytes on neural stem cells in in vitro and in vivo models of ischemic stroke.

Transplantation of neural stem cells (NSCs) can protect neurons in animal stroke models; however, their low rates of survival and neuronal differentiation limit their clinical application. Glial niches, an important location of neural stem cells, regulate survival, proliferation and differentiation of neural stem cells. However, the effects of activated glial cells on neural stem cells remain unclear. In the present study, we explored the effects of activated astrocytes and microglia on neural stem cells in vitro stroke models. We also investigated the effects of combined transplantation of neural stem cells and glial cells after stroke in rats. In a Transwell co-culture system, primary cultured astrocytes, microglia or mixed glial cells were exposed to glutamate or H2O2 and then seeded in the upper inserts, while primary neural stem cells were seeded in the lower uncoated wells and cultured for 7 days. Our results showed that microglia were conducive to neurosphere formation and had no effects on apoptosis within neurospheres, while astrocytes and mixed glial cells were conducive to neurosphere differentiation and reduced apoptosis within neurospheres, regardless of their pretreatment. In contrast, microglia and astrocytes induced neuronal differentiation of neural stem cells in differentiation medium, regardless of their pretreatment, with an exception of astrocytes pretreated with H2O2. Rat models of ischemic stroke were established by occlusion of the middle cerebral artery. Three days later, 5 × 105 neural stem cells with microglia or astrocytes were injected into the right lateral ventricle. Neural stem cell/astrocyte-treated rats displayed better improvement of neurological deficits than neural stem cell only-treated rats at 4 days after cell transplantation. Moreover, neural stem cell/microglia-, and neural stem cell/astrocyte-treated rats showed a significant decrease in ischemic volume compared with neural stem cell-treated rats. These findings indicate that microglia and astrocytes exert different effects on neural stem cells, and that co-transplantation of neural stem cells and astrocytes is more conducive to the recovery of neurological impairment in rats with ischemic stroke. The study was approved by the Animal Ethics Committee of Tongji University School of Medicine, China (approval No. 2010-TJAA08220401) in 2010.

Artemisinin protects DPSC from hypoxia and TNF-α mediated osteogenesis impairments through CA9 and Wnt signaling pathway.

Dental pulp stem cells (DPSCs) possess the ability of multi-lineage differentiation, and are excellent sources of tissue engineering and regenerative medicine. Oxygen concentration and inflammation are two critical environmental factors that affect the osteogenic differentiation of DPSCs. We aimed to study the role of the antimalarial drug artemisinin on the osteogenic differentiation of human DPSCs under the hypoxia and inflammation conditions. We demonstrated that hypoxia (5% O2) and inflammation (20 ng/mL TNF-α), alone or in combination, significantly diminished in vitro cell survival and increased apoptotic rates. Notably, hypoxia and TNF-α exerted accumulative effect in suppressing the osteogenic differentiation of DPSCs, as evidenced by reduced expression levels of osteogenesis-associated genes including ALP, RUNX2 and OCN in osteogenic condition, as well as reduced mineral nodules formation as indicated by alizarin red staining. Artemisinin at the dose of 40 µM markedly reversed the suppression in cell survival caused by hypoxia or inflammation, and reduced apoptotic rates and the expressions of pro-apoptotic proteins. Additionally, artemisinin restored osteogenic differentiation of DPSCs under the hypoxia or/and inflammation conditions. Moreover, the beneficial effect of artemisinin was dependent on upregulated expression of CA9 and CA9-mediated antioxidant responses, as CA9 knockdown abolished the protective role of artemisinin on DPSC osteogenesis. Furthermore, while hypoxia or/and inflammation significantly inactivated the Wnt/ß-catenin signaling in DPSCs, additional exposure to artemisinin re-activated this pathway to promote osteogenic differentiation of DPSCs. Our results provide novel insight on the link between artemisinin and DPSC osteogenesis, and suggest promising artemisinin-based strategies for better dentin/pulp tissue engineering.

Multiple inflammatory profiles of microglia and altered neuroimages in APP/PS1 transgenic AD mice.

Aß plaques of Alzheimer's disease (AD) are believed to precede cognitive deficits or clinical manifestation by decades. However, validated biomarkers for early diagnosis of the AD disease are still not available. In this present study, we combined MRI-based neuroimages and histological assessment of the glial response and altered cytokines, neurogenesis during the early course of Aß deposits in TgAPP/PS1 mice to find potential early biomarkers for AD. We found that microglia and astrocytes were initially activated and clustered around Aß plaques at the age of 6 months and significantly increased with age from 6-12 months of age. Confocal microscope analysis revealed that microglia not astrocytes began to phagocytose Aß in 6-month-old TgAPP/PS1 mice, evidenced by the intracellular Aß in Iba1 positive microglia not in GFAP positive astrocytes. In parallel with these observations, we found that mainly clustered microglia significantly upregulated the production of proinflammatory factors including TNF-α, iNOS and IL-1ß, and anti-inflammatory cytokines including IL-4, TGF-ß and extracellular protecting matrix YM-1 and enzyme arginase 1 (Arg1) at 6-12 months of age. Interestingly, reactive astrocyte did not express these cytokines and YM-1 and Arg1. These results may suggest that microglia rather than astrocytes play crucial roles in clearing Aß and neuroinflammation in early stage of AD. In addition, the number of neural stem cells labeled by BrdU and immature neurons labeled by doublecortin was significantly decreased in 3-month-old TgAPP/PS1 mice ahead of Aß deposits. Finally, DTI conforms that reduced fractional anisotropy (FA) in dentate gyrus of hippocampus and rs-MRI shows an increased connectivity in the networks of somatosensory cortex-caudoputamen and insula in TgAPP/PS1 mice at 6 months. These findings provide a clue to early biomarkers for diagnosis of the AD disease.

Electroacupuncture promotes peripheral nerve regeneration after facial nerve crush injury and upregulates the expression of glial cell-derived neurotrophic factor.

The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor (GDNF) has been shown to protect neurons by binding to N-cadherin. Our previous results have shown that electroacupuncture could increase the expression of N-cadherin mRNA in facial neurons and promote facial nerve regeneration. In this study, the potential mechanisms by which electroacupuncture promotes nerve regeneration were elucidated through assessing the effects of electroacupuncture on GDNF and N-cadherin expression in facial motoneurons of rabbits with peripheral facial nerve crush injury. New Zealand rabbits were randomly divided into a normal group (normal control, n = 21), injury group (n = 45) and electroacupuncture group (n = 45). Model rabbits underwent facial nerve crush injury only. Rabbits in the electroacupuncture group received facial nerve injury, and then underwent electroacupuncture at Yifeng (TE17), Jiache (ST6), Sibai (ST2), Dicang (ST4), Yangbai (GB14), Quanliao (SI18), and Hegu (LI4; only acupuncture, no electrical stimulation). The results showed that in behavioral assessments, the total scores of blink reflex, vibrissae movement, and position of apex nasi, were markedly lower in the EA group than those in the injury group. Hematoxylin-eosin staining of the right buccinator muscle of each group showed that the cross-sectional area of buccinator was larger in the electroacupuncture group than in the injury group on days 1, 14 and 21 post-surgery. Toluidine blue staining of the right facial nerve tissue of each group revealed that on day 14 post-surgery, there was less axonal demyelination and fewer inflammatory cells in the electroacupuncture group compared with the injury group. Quantitative real time-polymerase chain reaction showed that compared with the injury group, N-cadherin mRNA levels on days 4, 7, 14 and 21 and GDNF mRNA levels on days 4, 7 and 14 were significantly higher in the electroacupuncture group. Western blot assay displayed that compared with the injury group, the expression of GDNF protein levels on days 7, 14 and 21 were significantly upregulated in the electroacupuncture group. The histology with hematoxylin-eosin staining and Nissl staining of brainstem tissues containing facial neurons in the middle and lower part of the pons exhibited that on day 7 post-surgery, there were significantly fewer apoptotic neurons in the electroacupuncture group than in the injury group. By day 21, there was no significantly difference in the number of neurons between the electroacupuncture and normal groups. Taken together, these results have confirmed that electroacupuncture promotes regeneration of peripheral facial nerve injury in rabbits, inhibits neuronal apoptosis, and reduces peripheral inflammatory response, resulting in the recovery of facial muscle function. This is achieved by up-regulating the expression of GDNF and N-cadherin in central facial neurons.

Neuroplastin 65 modulates anxiety- and depression-like behavior likely through adult hippocampal neurogenesis and central 5-HT activity.

Neuroplastin 65 (Np65) is a brain-specific cell adhesion molecule that is highly expressed in the hippocampus, amygdala, and cortex, regions of the brain that are associated with memory and emotions. However, the role of Np65 in regulation of emotional behavior is still unclear. In the present study, we show that Np65 knock-out (Np65 KO) mice display enhanced anxiety-like behavior, a reduction in some aspects of depressive-like behaviors, and increased sociability and memory. Biochemical investigations revealed that Np65 KO mice show increased adult-born neurons and proliferation in the hippocampus. In addition, the level of 5-hydroxytryptamine (5-HT) in the hippocampus was reduced. The expression of tryptophan hydroxylase 2 in the brainstem and the expression of the 5-HT3A receptor were also decreased. Electrophysiological recordings confirmed an impaired maintenance of long-term potentiation in the hippocampus of Np65 KO mice. Together, our findings uncover a role for Np65 in regulating anxiety- and depressive-like behaviors and suggest that Np65 may be essential for the maintenance of emotional stability, indicating that it might be an attractive potential target for treatment of psychiatric disorders.

Protective effects of mesenchymal stem cells overexpressing extracellular regulating kinase 1/2 against stroke in rats.

OBJECTIVE: Although transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has shown beneficial effects on stroke, lower survival of MSCs limits effects. Extracellular regulating kinase 1/2 signaling (ERK1/2) is crucial for cell survival, differentiation, and proliferation. This study was designed to explore whether MSCs modified by over-expressing ERK1/2 may reinforce beneficial effects on stroke in rats. METHODS: rat MSCs transfected with ERK1/2 and empty lentivirus to generate MSCs overexpressing ERK1/2 (ERK/MSCs) and MSCs (as a control), respectively. In vitro, ERK/MSCs were plated and exposed to glutamate-induced condition, and viability of ERK/MSCs was measured. Furthermore, neural induction of ERK/MSCs was investigated in vitro. Cerebral ischemic rats were induced by occluding middle cerebral artery, and then were stereotaxically injected into ipsilateral right lateral ventricle with ERK/MSCs or MSCs 3 days after stroke and survived for 7 or 14 days after injection. RESULTS: ERK/MSCs showed better viability in physiological and glutamate-induced neurotoxic conditions compared to MSCs. After neural induction, more neurons were be differentiated from ERK/MSCs than from MSCs. After transplantation, more numbers of grafted cells and improved functional recovery were observed in ERK/MSCs-treated rats compared with MSCs-treated rats. Compared with MSCs treatment, ERK/MSCs treatment significantly increased proliferation of neural stem cells in the subventricle zone (SVZ) and the MAP2/nestin double-labeled cells adjacent to the SVZ, enhanced the numbers of reactive astrocytes while suppressed microglial activation. Besides, TNF-α level was elevated in ERK/MSCs-treated rats. CONCLUSION: ERK/MSCs transplantation showed better functional recovery after stroke in rats, likely in part through enhancing survival of MSCs and possibly by modulating the proliferation, neuronal de-differentiation and neuroinflammation.

[Effects of Ginsenoside RB1 on neural cell apoptosis and expressions of Bcl-2 and Bax in rats following subjected to cerebral ischemia-reperfusion].

OBJECTIVE: To observe the effects of Ginsenoside Rb1 (GRb1) on neuronal cell apoptosis and the expressions of Bcl-2 and Bax in rats after cerebral ischemia-reperfusion so as to investigate the neuroprotective mechanism of GRb1. METHDOS: The model of cerebral ischemia-reperfusion was established by occluding rat middle cerebral artery for 2 h. The rats were randomly divided into two groups: ischemia-reperfusion group (I/R group) and GRb1 treat group (GRb1 group). GRb1 (40 mg/kg, i.p.) was administered immediately to rats after the onset of reperfusion. Two groups were further subdivided 7 subgroups according to various reperfusion time (3 h, 12 h, 1 d, 2 d, 3 d, 5 d and 10 d, n=4 per time point). HE staining was used to observe histological features. TUNEL and immunohistochemical method were used to analyze the cell apoptosis and expressions of Bcl-2 and Bax, respectively. RESULTS: Compared with I/R group, GRb1 reduced pathological changes, and decreased the number of apoptotic neural cells (P<0.05 on 12 h, 1 d, 2 d and 3 d) and up-regulated the number of Bcl-2 positive cells (P<0.05 on 12 h, 1 d, 3 d, 5 d and 10 d), and meanwhile down-regulated the number of Bax positive cells (P<0.05 on 3 h, 12 h, 1 d, 2 d, 3 d, 5 d and 10 d) in the ipsilateral hemisphere. CONCLUSION: The neuroprotective effect of GRb1 on cerebral ischemia-reperfusion injury is related to inhibit neuronal apoptosis and to up-regulate the expression of Bcl-2 with down-regulating the expression of Bax.

Microarray Analysis of Gene Expression Changes in Neuroplastin 65-Knockout Mice: Implications for Abnormal Cognition and Emotional Disorders.

Neuroplastin 65 (Np65) is an immunoglobulin superfamily cell adhesion molecule involved in synaptic formation and plasticity. Our recent study showed that Np65-knockout (KO) mice exhibit abnormal cognition and emotional disorders. However, the underlying mechanisms remain unclear. In this study, we found 588 differentially-expressed genes in Np65-KO mice by microarray analysis. RT-PCR analysis also revealed the altered expression of genes associated with development and synaptic structure, such as Cdh1, Htr3a, and Kcnj9. In addition, the expression of Wnt-3, a Wnt protein involved in development, was decreased in Np65-KO mice as evidenced by western blotting. Surprisingly, MRI and DAPI staining showed a significant reduction in the lateral ventricular volume of Np65-KO mice. Together, these findings suggest that ablation of Np65 influences gene expression, which may contribute to abnormal brain development. These results provide clues to the mechanisms underlying the altered brain functions of Np65-deficient mice.

Immunofluorescent Localization of Non-myelinating Schwann Cells and Their Interactions With Immune Cells in Mouse Thymus.

The thymus is innervated by sympathetic/parasympathetic nerve fibers from the peripheral nervous system (PNS), suggesting a neural regulation of thymic function including T-cell development. Despite some published studies, data on the innervation and nerve-immune interaction inside the thymus remain limited. In the present study, we used immunofluorescent staining of glial fibrillary acidic protein (GFAP) coupled with confocal microscopy/three-dimensional (3D) reconstruction to reveal the distribution of non-myelinating Schwann cells (NMSC) and their interactions with immune cells inside mouse thymus. Our results demonstrate (1) the presence of an extensive network of NMSC processes in all compartments of the thymus including the capsule, subcapsular region, cortex, cortico-medullary junction, and medulla; (2) close associations/interactions of NMSC processes with blood vessels, indicating the neural control of blood flow inside the thymus; (3) the close "synapse-like" association of NMSC processes with various subsets of dendritic cells (DC; e.g., B220+ DCs, CD4+ DCs, and CD8+ DCs), and lymphocytes (B cells, CD4+/CD8+ thymocytes). Our novel findings concerning the distribution of NMSCs and the associations of NMSCs and immune cells inside mouse thymus should help us understand the anatomical basis and the mechanisms through which the PNS affects T-cell development and thymic endocrine function in health and disease.

Increased Susceptibility to Ischemic Brain Injury in Neuroplastin 65-Deficient Mice Likely via Glutamate Excitotoxicity.

Cell adhesion molecules (CAMs) are involved in synaptic plasticity and neuronal survival in the adult brain. Neuroplastin 65 (Np65), one member of the immunoglobulin superfamily of CAMs, is brain-specific and highly expressed in rodent forebrain. The roles of Np65 in synaptic plasticity have been confirmed, however, whether Np65 affects neuronal survival remains unknown. To address this gap, we generated, to our knowledge, the first Np65 knockout (KO) mice. By occluding middle cerebral artery to perform ischemic stroke model, we showed that Np65 KO mice exhibited more severe neurological deficits and larger infarction volume measured by TTC staining and more apoptotic cells confirmed by TUNEL staining compared to wild type (WT) mice. Besides, western blot analysis showed that the vesicular glutamate transporter-1(VGluT1), and N-Methyl D-Aspartate receptors, including NR1, NR2A, and NR2B were significantly increased in Np65 KO mice compared with WT mice. In contrast, vesicular gamma amino butyric acid transporter (VGAT) levels were unchanged in two genotypes after stroke. Additionally, phosphorylated-extracellular signal-regulated kinase 1/2 levels were significantly increased in Np65 KO mice compared with WT mice after stroke. Together, these results suggest that Np65 KO mice may be more susceptible to ischemic events in the brain.

Immunofluorescent characterization of non-myelinating Schwann cells and their interactions with immune cells in mouse mesenteric lymph node.

The central nervous system (CNS) influences the immune system in a general fashion by regulating the systemic concentration of humoral substances, whereas the autonomic nervous system communicates specifically with the immune system according to local interactions. Data concerning the mechanisms of this bidirectional crosstalk of the peripheral nervous system (PNS) and immune system remain limited. To gain a better understanding of local interactions of the PNS and immune system, we have used immunofluorescent staining of glial fibrillary acidic protein (GFAP), coupled with confocal microscopy, to investigate the non-myelinating Schwann cell (NMSC)-immune cell interactions in mouse mesenteric lymph nodes. Our results demonstrate i) the presence of extensive NMSC processes and even of cell bodies in each compartment of the mouse mesenteric lymph node; ii) close associations/interactions of NMSC processes with blood vessels (including high endothelial venules) and the lymphatic vessel/sinus; iii) close contacts/associations of NMSC processes with various subsets of dendritic cells (such as CD4+CD11c+, CD8+CD11c+ dendritic cells), macrophages (F4/80+ and CD11b+ macrophages), and lymphocytes. Our novel findings concerning the distribution of NMSCs and NMSC-immune cell interactions inside the mouse lymph node should help to elucidate the mechanisms through which the PNS affects cellular- and humoral-mediated immune responses or vice versa in health and disease.

GDNF Enhances Therapeutic Efficiency of Neural Stem Cells-Based Therapy in Chronic Experimental Allergic Encephalomyelitis in Rat.

Multiple sclerosis (MS) is an autoimmune disease in the CNS. The current immunomodulating drugs for MS do not effectively prevent the progressive neurological decline. Neural stem cells (NSCs) transplantation has been proven to promote repair and functional recovery of experimental allergic encephalomyelitis (EAE) animal model for MS, and glial cell line-derived neurotrophic factor (GDNF) has also been found to have capability of promoting axonal regeneration and remyelination of regenerating axons. In the present study, to assess whether GDNF would enhance therapeutic effect of NSCs for EAE, GDNF gene-modified NSCs (GDNF/NSCs) and native NSCs were transplanted into each lateral ventricle of rats at 10 days and rats were sacrificed at 60 days after EAE immunization. We found that NSCs significantly reduced the clinical signs, and GDNF gene-modification further promoted functional recovery. GDNF/NSCs more profoundly suppressed brain inflammation and improved density of myelin compared with NSCs. The survival of GDNF/NSCs was significantly higher than that of transplanted NSCs. Transplanted GDNF/NSCs, in contrast to NSCs, differentiated into more neurons and oligodendrocytes. Moreover, the mRNA expression of oligodendrocyte lineage cells in rats with GDNF/NSCs was significantly increased compared to rats with NSCs. These results suggest that GDNF enhances therapeutic efficiency of NSCs-based therapy for EAE.

Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons.

Ginsenoside Rg1 (Rg1) has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25-35 (Aß25-35), and to explore whether the extracellular signal-regulated kinase (ERK) and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aß25-35 for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2 (Akt inhibitor) and PD98059 (MAPK/ERK kinase inhibitor), but not by SP600125 or SB203580 (inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively). Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aß25-35-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aß25-35-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aß25-35-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling.