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 (ϩ76 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. The expression of p54 was colocalized with the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive nuclei in glutamatetreated cerebellar granule neurons (CGNs) and in ischemic neurons located in the infarct core after focal cerebral ischemia, suggesting that p54 might be involved in neuronal death. Overexpression studies showed that p54, but not p63, caused death of human embryonic kidney cells and CGNs, whereas knock-down CRMP-3 expression by selective small interfering RNA protected neurons against glutamate toxicity. Collectively, these results reveal a novel role of CRMP-3 in that calpain cleavage of CRMP-3 and the subsequent nuclear translocation of the truncated CRMP-3 evokes neuronal death in response to excitotoxicity and cerebral ischemia. Our findings also establish a novel route of how calpain signals neuron death.

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. Hou et al. (2006) J. Neurosci., 26, 2241-2249]. Here, the expression of all CRMP family members (1-5) was examined in mouse brains that were subjected to middle cerebral artery occlusion. Among the five CRMPs, the expressions of CRMP1, CRMP3 and CRMP5 were the most abundant in the cerebral cortex and all CRMPs were targeted for cleavage by ischemia-activated calpain. Sub-cellular fractionation analysis showed that cleavage of CRMPs by calpain occurred not only in the cytoplasm but also in the synaptosomes isolated from ischemic brains. Moreover, synaptosomal CRMPs appeared to be at least one-fold more sensitive to cleavage compared with those isolated from the cytosolic fraction in an in-vitro experiment, suggesting that synaptosomal CRMPs are critical targets during cerebral ischemia-induced neuronal injury. Finally, the expression of all CRMPs was colocalized with TUNEL-positive neurons in the ischemic mouse brain, which further supports the notion that CRMPs may play an important role in neuronal death following cerebral ischemia. Collectively, these studies demonstrated that CRMPs are targets of calpains during cerebral ischemia and they also highlighted an important potential role that CRMPs may play in modulating ischemic neuronal death.

Brain-derived neurotrophic factor (BDNF) 1 is a member of neurotrophin family, structurally related to nerve growth factor, neurotrophin-3, and neurotrophin-4/5. Its biological activity is mediated by tyrosine kinase receptor B (TrkB) and its downstream signaling (1). The gene is highly expressed and widely distributed in the central nervous system, and it plays a significant role in the maintenance of function and survival of neurons (for review, see Ref.2). Evidence accumulated in recent years suggests that BDNF is also involved in the modulation of synaptic activity in the adult brain, producing long lasting changes in synaptic structure and function (for reviews, see Refs. 3-5).There is a growing interest in BDNF as a potential therapeutic agent for neurodegenerative diseases, because its deficiency was found in brains of both Alzheimer's and Parkinson's patients (6 -10). Indeed, BDNF treatment has been shown not only to potentiate synaptic transmission in vivo (11, 12) but also to increase neuronal survival and augment some behavioral changes in animal models (13,14). However, more recent data indicate that BDNF can also induce behavioral sensitization by causing an overexpression of dopamine D3 receptors and could, actually, contribute to the amplification of pathophysiologies associated with conditions such as epilepsy, drug addiction, schizophrenia, and Parkinson's disease (15, 16). Clearly, further work is required to resolve some of these potential side-effects. In contrast to a large body of work on the temporal and spatial patterns of BDNF expression in neurodevelopment and neurodegeneration, relatively little is known about the transcriptional regulation of the human BDNF gene. This is partially due to the fact that the genomic structure of the human gene has not yet been fully elucidated. The gene was first localized to chromosome11p13 and predicted to consist of multiple exons (17), but the existence of multiple transcripts, derived from different exons, was demonstrated only recently by Aoyama et al. (18) in human neuroblastoma cells. A more detailed transcript mapping of an 810-kb region of chromosome 11p13-14 further defined its genomic localization, although no additional information on the actual structure of the gene itself was presented (19).The existence of multiple human BDNF transcripts (18) is consistent with a genomic structure similar to that of the rat gene, which consists of four short 5Ј exons, each controlled by a distinct promoter, and one 3Ј exon encoding the mature BDNF protein (20,21). In the rat, the four promoters direct expression of the BDNF gene in a tissue-specific manner, i.e. promoters I and II are active preferentially in neurons, whereas promoters III and IV are active both in neurons and in a limited number of non-neuronal tissues such as lung and heart (20, 21). Thus far, only a limited characterization of a 3.2-kb genomic fragment of the human gene containing some structural elements of a promoter was reported (22).Recently, two transcription factors, CREB and ...

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. Interestingly, the temporal induction in cerebral ischemia-induced E2F1 binding to the NRP-1 promoter correlated with the temporal-induction profile of NRP-1 mRNA, confirming that E2F1 positively regulates NRP-1 during cerebral ischemia. Functional analysis also showed that NRP-1 receptor expression was extremely low in E2F1 ؊/؊ neurons, which led to the diminished response to semaphorin 3A-induced axonal shortening and neuronal death. An NRP-1 selective peptide inhibitor provided neuroprotection against oxygenglucose deprivation. Taken together, these findings support a model in which E2F1 targets NRP-1 to modulate axonal damage and neuronal death in response to cerebral ischemia.