Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth.

Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death.

Calpain cleavage of collapsin response mediator proteins in ischemic mouse brain.

Collapsin response mediator proteins (CRMPs) are important brain-specific proteins with distinct functions in modulating growth cone collapse and axonal guidance during brain development. Our previous studies have shown that calpain cleaves CRMP3 in the adult mouse brain during cerebral ischemia [S.T.

A novel adenoviral vector-mediated neuronal selective gene expression in neonatal mouse brain in response to hypoxia.

Selective gene expression targeting neurons is a challenge, which, if successfully overcome, carries an enormous potential for clinical applications in therapeutics against neurodegenerative diseases. We have reported previously the construction of a series of adenoviral vectors capable of selectively expressing a reporter gene luciferase in cultured neurons [D. Huang, A.

Neuropilin-1 is a direct target of the transcription factor E2F1 during cerebral ischemia-induced neuronal death in vivo.

The nuclear transcription factor E2F1 plays an important role in modulating neuronal death in response to excitotoxicity and cerebral ischemia. Here, by comparing gene expression in brain cortices from E2F1(+/+) and E2F1(-/-) mice using a custom high-density DNA microarray, we identified a group of putative E2F1 target genes that might be responsible for ischemia-induced E2F1-dependent neuronal death. Neuropilin 1 (NRP-1), a receptor for semaphorin 3A-mediated axon growth cone collapse and retraction, was confirmed to be a direct target of E2F1 based on (i) the fact that the NRP-1 promoter sequence contains an E2F1 binding site, (ii) reactivation of NRP-1 expression in E2F1(-/-) neurons when the E2F1 gene was replaced, (iii) activation of the NRP-1 promoter by E2F1 in a luciferase reporter assay, (iv) electrophoretic mobility gel shift analysis confirmation of the presence of an E2F binding sequence in the NRP-1 promoter, and (v) the fact that a chromatin immunoprecipitation assay showed that E2F1 binds directly to the endogenous NRP-1 promoter.

Calpain-cleaved collapsin response mediator protein-3 induces neuronal death after glutamate toxicity and cerebral ischemia.

Collapsin response mediator proteins (CRMPs) mediate growth cone collapse during development, but their roles in adult brains are not clear. Here we report the findings that the full-length CRMP-3 (p63) is a direct target of calpain that cleaves CRMP-3 at the N terminus (+76 amino acid). Interestingly, activated calpain in response to excitotoxicity in vitro and cerebral ischemia in vivo also cleaved CRMP-3, and the cleavage product of CRMP-3 (p54) underwent nuclear translocation during neuronal death.

Identification of a functional CRE in the promoter of Fukuyama congenital muscular dystrophy gene fukutin.

We isolated a fragment of the fukutin gene promoter from differentiated human NT2 cells using chromatin immunoprecipitation technique with an anti-CREB antibody. This fragment contained a CRE-like sequence and here we describe its functional validation. The results showed that the element was functional in vitro and in vivo and that CREB in neurons was involved in the transcriptional regulation of the fukutin gene.

P2 receptor antagonist PPADS confers neuroprotection against glutamate/NMDA toxicity.

The present study investigated the role of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium salt (PPADS), a P2 receptor antagonist, in protecting mouse cerebellar granule neurons (CGNs) against glutamate/NMDA-induced neuronal death. Neurotoxicity caused by 50 microM glutamate or 200 microM NMDA was significantly reduced in CGNs treated with PPADS. Such neuroprotection was in a time- and dose-dependent manner.

Characterization of the expression of key adenoviral receptors CAR and integrin beta3/beta5 subunits on the membrane of human NT2 neurons.

Expression of therapeutic gene products in differentiated human NT2 neurons (NT2/Ns) is being explored for ex vivo gene therapy of human neurological diseases. In this study we determined the efficiency of adenovirus (Ad)-mediated gene delivery into NT2/Ns and characterized the expression of several key receptors known to be required for efficient Ad-mediated gene delivery. Undifferentiated NT2 cells and NT2/Ns were infected by Ad expressing green fluorescent protein at an efficiency of 33% and 17%, respectively percentages much lower than the 92% infectivity obtained from a human non-neuronal cell line A549 cells.

Group B vitamins protect murine cerebellar granule cells from glutamate/NMDA toxicity.

The role of B group vitamins in preventing neuronal death against excitotoxicity was investigated. Neuronal death of cultured mouse cerebellar granule neurons (CGNs) caused by glutamate (50 microM) or NMDA (200 microM) was delayed in CGNs that had been treated with riboflavin (B2), folic acid (B9) or cynocobalamin (B12) for 18 h. Such neuroprotection by B2, B9 and B12 was in a dose- and time-dependent manner.

Transcriptional activation of the human brain-derived neurotrophic factor gene promoter III by dopamine signaling in NT2/N neurons.

We have identified a functional cAMP-response element (CRE) in the human brain-derived neurotrophic factor (BDNF) gene promoter III and established that it participated in the modulation of BDNF expression in NT2/N neurons via downstream signaling from the D1 class of dopamine (DA) receptors. The up-regulation of BDNF expression, in turn, produced neuroprotective signals through receptor tyrosine kinase B (TrkB) and promoted cell survival under the conditions of oxygen and glucose deprivation. To our knowledge this is the first evidence showing the presence of a functional CRE in the human BDNF gene and the role of DA signaling in establishing transcriptional competence of CRE in post-mitotic NT2/N neurons.