Differential expression of immediate early genes in the hippocampus in the kindling model of epilepsy.

Kindling is a phenomenon in which brief afterdischarges (ADs) evoked by periodic electrical stimulation of the brain eventually result in generalized clonic motor seizures. Once present, the enhanced sensitivity to electrical stimulation is lifelong. The mechanism by which brief ADs produce this long-lasting effect may involve a change in gene expression. To begin to investigate changes in gene expression that occur during kindling, we used in situ hybridization histochemistry to examine the time course of expression of mRNAs of the immediate early genes (IEGs) c-fos, c-jun, NGFI-A, and c-myc within the dorsal hippocampus of rats following a kindling AD. Three principal findings resulted from this study. First, the expression of all mRNAs except c-myc was significantly increased (P less than 0.05) within discrete neuronal populations. Second, the time course of expression of the IEGs differed markedly within the same neuronal population. Third, for a given IEG, the time course and anatomic pattern of expression were strikingly different among different neuronal populations of the hippocampus. The prolonged and distinctly different patterns of IEG expression suggest that target genes are differentially regulated in these neuronal populations for prolonged periods following a kindling AD.     

Programmed cell death in cerebral ischemia.

Programmed cell death (PCD) is an ordered and tightly controlled set of changes in gene expression and protein activity that results in neuronal cell death during brain development. This article reviews the molecular pathways by which PCD is executed in mammalian cells and the potential relation of these pathways to pathologic neuronal cell death. Whereas the classical patterns of apoptotic morphologic change often do not appear in the brain after ischemia, there is emerging biochemical and pharmacologic evidence suggesting a role for PCD in ischemic brain injury. The most convincing evidence for the induction of PCD after ischemia includes the altered expression and activity in the ischemic brain of deduced key death-regulatory genes. Furthermore, studies have shown that alterations in the activity of these gene products by peptide inhibitors, viral vector-mediated gene transfer, antisense oligonucleotides, or transgenic mouse techniques determine, at least in part, whether ischemic neurons live or die after stroke. These studies provide strong support for the hypothesis that PCD contributes to neuronal cell death caused by ischemic injury. However, many questions remain regarding the precise pathways that initiate, sense, and transmit cell death signals in ischemic neurons and the molecular mechanisms by which neuronal cell death is executed at different stages of ischemic injury. Elucidation of these pathways and mechanisms may lead to the development of novel therapeutic strategies for brain injury after stroke and related neurologic disorders.     

A preproenkephalin-neurofilament chimeric promoter in a helper virus-free herpes simplex virus vector enhances long-term expression in the rat striatum

Helper virus-free herpes simplex virus (HSV-1) plasmid vectors are an attractive system for gene transfer into neurons in the brain, but promoters that support long-term, neuronal-specific expression are required. Elucidation of general principles that govern long-term expression would likely assist efforts to develop improved promoters. Although expression from many promoters in HSV-1 vectors is unstable, two neuronal subtype-specific promoters, the preproenkephalin (ENK) promoter and the tyrosine hydroxylase (TH) promoter, support long-term expression. We have previously shown that 5' upstream sequences in the TH promoter are required for long-term expression, and addition of these upstream sequences to a neurofilament heavy gene (NF-H) promoter enhances long-term, neuronal-specific expression. The goal of this study was to determine if the upstream sequences from the TH promoter contain a unique element that enhances expression, or if other neuronal promoters also contain sequences that can enhance expression. To this end, we tested 5' upstream sequences in the ENK promoter. We isolated a vector that fuses upstream sequences from the ENK promoter to the NF-H promoter. This vector supported expression in the striatum for 2 months after gene transfer, the longest time point evaluated. Expression was neuronal specific. As ENK and TH are a peptide neurotransmitter and a classical neurotransmitter biosynthetic enzyme, respectively, these results suggest that a significant number of promoters for neurotransmitter biosynthetic genes may contain elements that can enhance expression from HSV-1 vectors. The strategy of using upstream sequences from neuronal subtype-specific promoters to enhance expression from heterologous promoters is discussed.     

Comparative fine structural distribution of endopeptidase 24.15 (EC3.4.24.15) and 24.16 (EC3.4.24.16) in rat brain

Glial-cell-line--derived neurotrophic factor (GDNF) has been identified as a potent survival and differentiation factor for several neuronal populations in the central nervous system (CNS), but to date, distinct effects of GDNF on motor axon growth and regeneration in the adult have not been demonstrated. In the present study, ex vivo gene delivery was used to directly examine whether GDNF can influence axonal growth, expression of neuronal regeneration-related genes, and sustain the motor neuronal phenotype after adult CNS injury. Adult Fischer 344 rats underwent unilateral transections of the hypoglossal nerve, followed by intramedullary grafts of fibroblasts genetically modified to secrete GDNF. Control animals received lesions and grafts of cells expressing a reporter gene. Two weeks later, GDNF gene delivery (1) robustly promoted the growth of lesioned hypoglossal motor axons, (2) altered the expression and intracellular trafficking of the growth-related protein calcitonin gene-related peptide (CGRP), and (3) significantly sustained the cholinergic phenotype in 84 +/- 6% of hypoglossal neurons compared with 39 +/- 6% in control animals (P < 0.001). This is the first neurotrophic factor identified to increase the in vivo expression of the trophic peptide CGRP and the first report that GDNF promotes motor axonal growth in vivo in the adult CNS. Taken together with previous in vitro studies, these findings serve as the foundation for a model wherein GDNF and CGRP interact in a paracrine manner to regulate neuromuscular development and regeneration.     

Identification of candidate genes for controlling development of the basilar pons by differential display PCR.

The basilar pons, a major hindbrain nucleus involved in sensory-motor integration, has become a model system for studying long-distance neuronal migration, axon-target recognition by collateral branching, and the formation of patterned axonal projections. To identify genes potentially involved in these developmental events, we have performed a differential display PCR screen comparing RNA isolated from the developing basilar pons with RNA obtained from developing cerebellum and olfactory bulb, as well as the mature basilar pons. Using 400 different combinations of primers, we screened more than 11,000 labeled DNA fragments and identified 201 that exhibited higher expression in the basilar pons than in the control tissues. From these, 138 distinct gene fragments were cloned. The differential expression of a large subset of these fragments was confirmed using RNase protection assays. In situ hybridization analysis revealed that the expression of many of these genes is limited to the basilar pons and only a few other brain regions, suggesting that they may play specific roles in pontine development.     

Notch信号通路在培养神经元细胞中的表达

目的 观察在体外培养的神经元细胞内Notch基因的分布和表达.方法 原代神经元培养,免疫组织化学的方法定位.对按不同时间点进行划伤处理的神经元细胞进行Westernblot分析,测定其Notch表达的变化趋势.结果 在培养的神经元细胞内有大量细胞(＞99％)均为Notch基因表达信号阳性;按时间点损伤的培养细胞其Notch 1受体胞内段(N1CD)表达呈动态变化.结论 成熟神经元中存在Notch基因的表达,Notch基因可能参与神经元损伤的病理过程.     

Heme deficiency suppresses the expression of key neuronal genes and causes neuronal cell death

Defective heme synthesis may cause acute porphyrias, which are associated with a wide array of neurological disturbances involving both the central and peripheral nervous systems. Thus, the understanding of the roles of heme in neuronal cell function may provide insights into the molecular events underlying the pathogenesis of neuropathies associated with defective heme synthesis. In this report, we use rat pheochromocytoma (PC12) clonal cells as a model system for studying the role of heme in neuronal cell survival. We examined the effects of inhibition of heme synthesis on signaling pathways and gene expression in nerve growth factor (NGF)-induced PC12 cells. We found that succinyl acetone-induced heme deficiency selectively caused apoptosis in NGF-induced PC12 cells. Further, we found that in succinyl acetone-treated, NGF-induced cells, the pro-survival Ras-ERK1/2 signaling pathway was inactivated and the pro-apoptotic JNK signaling pathway was activated. In these cells, the activation of caspase and the cleavage of nuclear poly (ADP-ribose) polymerase (PARP) were also evident. Importantly, microarray gene expression analysis showed that more than 20 key neuronal genes that were induced by NGF were suppressed by succinyl acetone. These genes include those encoding survival motor neuron protein, synaptic vesicle protein SVOP, and neural cell adhesion molecule NCAM. These results indicate that heme is important for neuronal cell signaling and the proper functioning of neuronal cells.     

Methamphetamine-induced neuronal apoptosis involves the activation of multiple death pathways. Review

The abuse of the illicit drug methamphetamine (METH) is a major concern because it can cause terminal degeneration and neuronal cell death in the brain. METH-induced cell death occurs via processes that resemble apoptosis. In the present review, we discuss the role of various apoptotic events in the causation of METH-induced neuronal apoptosis in vitro and in vivo. Studies using comprehensive approaches to gene expression profiling have allowed for the identification of several genes that are up-regulated or down-regulated after an apoptosis-inducing dose of the drug. Further experiments have also documented the fact that the drug can cause demise of striatal enkephalinergic neurons by cross-talks between mitochondria-, endoplasmic reticulum- and receptor-mediated apoptotic events. These neuropathological observations have also been reported in models of drug-induced neuroplastic alterations used to mimic drug addiction (Nestler, 2001).     

Neuronal Apoptosis Revealed by Genomic Analysis: Integrating Gene Expression Profiles with Functional Information

Apoptosis is a key physiological response that occurs during development of the nervous system, resulting in the death of nearly half of the embryonic neuronal population. Aberrant apoptotic mechanisms are thought to contribute significantly to many neurological disorders including Alzheimer’s disease. Although many experiments in the past have demonstrated the requirement of de novo gene expression during neuronal apoptosis, the complete spectrum of genes involved in distinct temporal domains is mostly unknown. To begin a comprehensive survey of the gene-based molecular mechanisms that underlie neuronal apoptosis, we have used the unprecedented experimental opportunities that genome sequences and the development of DNA microarray technology now provide to perform genome-wide expression analysis in different paradigms of neuronal apoptosis. In order to extract knowledge from gene expression information we have developed new informatics applications that enable clustering methods based on semantic characteristics, such as gene ontologies. This review will highlight the use of a genomic approach to identify the molecular program underlying neuronal apoptosis and illustrate how a semantic clustering method can be useful to extract more knowledge from microarray data.