an-invasive-method-for-activation-mouse-dentate-gyrus-high-frequency

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Beijing Institute of Basic Medical Sciences

Nantong University

Sema4C is a member of transmembrane semaphorin proteins which regulate axonal guidance in the developing nervous system. The expression of Sema4C was dramatically induced not only during differentiation of C2C12 mouse myoblasts, but also during injury-induced skeletal muscle regeneration. C2C12 cells stably or transiently expressing Sema4C both showed increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. Overexpression of Sema4C elicited p38 phosphorylation directly, and the effects of Sema4C during myogenic differentiation could be abolished by the p38alpha-specific inhibitor SB203580. Knockdown of Sema4C by siRNA transfection during C2C12 myoblasts differentiation could suppress the phosphorylation of p38 followed by dramatically diminished myotube formation. Sema4C could activate the myogenin promoter during myogenic differentiation. This activation could be abolished by p38 inhibitor SB203580. Taken together, these observations reveal novel functional potentialities of Sema4C which suggest that Sema4C promotes terminal myogenic differentiation in a p38 MAPK-dependent manner.

Sema4C participates in myogenic differentiation in vivo and in vitro through the p38 MAPK pathway.

CPNE5 is one of the ubiquitous Ca(2+)-dependent, phospholipid-binding proteins that are highly conserved in animals. It was cloned in the fetal human brain with no exact functions identified yet. We have examined the distribution pattern of CPNE5 mRNA and protein in the developing murine brain by using in situ hybridization, western blotting and immunocytochemistry. Expression of CPNE5 mRNA remains high from embryonic day 9.5 (E9.5) to E15.5 in the developing murine brain. Whole-mount in situ hybridization with the E11.5 and E12.5 embryos showed the strong positive signals in the central nervous system. Western-blot analysis showed that CPNE5 protein is expressed in the developing but not in the adult murine brain. In situ hybridization and immunohistochemistry analysis on the embryonic brain sections indicated that both at RNA and protein levels CPNE5 is mainly expressed in frontal cortex, medial nasal prominence, ganglionic eminence and medulla, particularly in the ventricular zones. Further investigation revealed the co-localization of CPNE5 with Tuj1 and Nestin on embryonic brain sections. In addition to the slight expression in primary cultured neural progenitor cells, CPNE5 is found in soma and neurite projections of primary cultured neurons where Tuj1 is co-localized. Our results demonstrate that CPNE5 is expressed in both neural progenitor cells and the differentiated neurons during the neural development, which suggests that CPNE5 might play an important role in the development of murine central nervous system.

Localization and cellular distribution of CPNE5 in embryonic mouse brain.

Sema4C is a transmembrane protein that belongs to axon guidance molecules of semaphorin family. Previous reports have shown that Sema4C could interact with postsynaptic protein PSD95, etc, but the expression and the role of Sema4C in neurogenesis remains unknown. In this study, whole-mount in situ hybridization result showed that Sema4C was expressed abundantly in the areas of lateral ventricle, the striatum, the wall of midbrain, and the pons/midbrain junction of E11.5 embryos brain. Neural stem/progenitor cells (NSPs) obtained from E13.5 embryonic rat midbrain are also positive for Sema4C immunoreactivity. Sema4C expression was dramatically downregulated during induction of NSP differentiation. In order to confirm the involvement of Sema4C in neurogenesis, we used the rat global cerebral ischemia model to make adult neurogenesis in vivo. The robust proliferative NSPs were monitored by labeling with bromodeoxyuridine (BrdU) within the subventricular zone and dentate gyrus that continues for at least 2 weeks. Immunohistochemistry and Western blot analysis showed that Sema4C expression was dramatically upregulated during neurogenesis after cerebral ischemia-perfusion injury. Double immunostaining and stereologic counting analysis indicated that a high proportion of BrdU-positive proliferative cells were Nestin-positive NSPs, and also, Sema4C was highly expressed in these proliferative populations at specific stages after ischemic injury. These observations provide the evidence to support a putative role of Sema4C during neurogenesis both in vivo and in vitro.

Sema4C expression in neural stem/progenitor cells and in adult neurogenesis induced by cerebral ischemia.

Here we report the negative correlation of phosphorylation of Smad1 and BMPR-IB expression with the development of human glioma. Western blot analysis showed that expression of both phospho-Smad1/5/8 and BMPR-IB were decreased in malignant glioma tissues compared with normal brain tissues. Kaplan-Meier survival curves revealed that lower expression ratio of phospho-Smad1/5/8 to Smad1 expression significantly correlates with poor patient survival. Transient transfection of BMPR-IB activates Smad1 signaling and induces differentiation and apoptosis of U251 and U87 glioblastoma cells. The effects could be blocked by cotransfection of Smad6. These results might provide new molecular marker and target for glioma diagnosis and therapy.

Expression and functional roles of Smad1 and BMPR-IB in glioma development.

Immunofluorescence or IF is a technique allowing the visualization of a specific protein or antigen in cells or tissues by binding a specific antibody chemically conjugated with a fluorescence dye. Immunofluorescent staining is widely used in life science research, particularly for neuroscience. Here, we describe the immunofluorescent staining of whole-mount neonatal mouse diaphragms to study the morphological patterns of the neuromuscular junction (NMJ) by using of presynaptic neuronal marker-neurofilament (NF) and synaptophysin antibodies; postsynaptic acetylcholine receptors (AChRs) were labeled with Alexa Fluor 594-conjugated α-bungarotoxin (α-BTX). Immunofluorescence-stained diaphragms were examined under a confocal microscope.

Morphological analysis of neuromuscular junctions by immunofluorescent staining of whole-mount mouse diaphragms.

It is well known that Notch1 signaling plays a crucial role in embryonic neural development and adult neurogenesis. The latest evidence shows that Notch1 also plays a critical role in synaptic plasticity in mature hippocampal neurons. So far, deeper insights into the function of Notch1 signaling during the different steps of adult neurogenesis are still lacking, and the mechanisms by which Notch1 dysfunction is associated with brain disorders are also poorly understood. In the current study, we found that Notch1 was highly expressed in the adult-born immature neurons in the hippocampal dentate gyrus. Using a genetic approach to selectively ablate Notch1 signaling in late immature precursors in the postnatal hippocampus by cross-breeding doublecortin (DCX) neuron-specific proopiomelanocortin (POMC)-α Cre mice with floxed Notch1 mice, we demonstrated a previously unreported pivotal role of Notch1 signaling in survival and function of adult newborn neurons in the dentate gyrus. Moreover, behavioral and functional studies demonstrated that POMC-Notch1 mutant mice showed anxiety and depressive-like behavior with impaired synaptic transmission properties in the dentate gyrus. Finally, our mechanistic study showed significantly compromised phosphorylation of cAMP response element-binding protein (CREB) in Notch1 mutants, suggesting that the dysfunction of Notch1 mutants is associated with the disrupted pCREB signaling in postnatally generated immature neurons in the dentate gyrus.-Feng, S., Shi, T., Qiu, J., Yang, H., Wu, Y., Zhou, W., Wang, W., Wu, H. Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment.

Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment.

Notch1 signaling regulates B and T lymphocyte development and also in vitro promotes antibody secretion upon B cell activation. However, it is still unclear about the role of Notch1 in antibody production upon in vitro and in vivo mixture lymphocytes activation. We first showed that Notch1 expressed in LPS-activated CD19 B cells and CD19 mediated Notch1 knock-down in LPS-activated B cells. Furthermore, we demonstrated that Notch1 knock-down in B cells reduced antibody production in LPS-stimulated B cells but did not affect antibody production in LPS-stimulated splenocytes and in experimental allergic encephalomyelitis (EAE) mice. Importantly, Notch1 ligands Dll1 and Jag1 expressed in B cells and pre-coated Notch1 protein promotes Notch1-knocked down B cells to produce antibody in LPS-stimulated B cells suggesting that Notch1 in other cells may promote antibody production by binding its ligands Dll1 and Jag1 in B cells. Together, our results suggest that both Notch1 and its ligands in B cells play an important role in antibody production.

Haitao Wu

Department of Neurobiology

Beijing Institute of Basic Medical Sciences

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation