The molecular mechanisms of morphine addiction

Addiction to opiates such as morphine is a major public health concern. A more thorough understanding of the molecular mechanisms of opiate addiction can lead to better treatment options in the future. Many of the changes in neuronal activity that occur upon morphine exposure have been known for some time, but until recently, little was known about the changes in gene expression that underlie these effects. Recent advances in molecular biology such as microarray analysis and quantitative (real time) PCR have allowed us to examine the gene expression changes that occur in response to morphine treatments and during morphine withdrawal. This review summarizes many of the known molecular and cellular actions of morphine, and some of the important gene expression changes that occur in response to morphine treatment. Many of these gene expression changes underlie the alterations in neuronal excitability, cell morphology and cell birth or death responsible for producing morphine's rewarding effects, the development of dependence, and withdrawal symptoms after treatment ends.     

Both upstream and intragenic sequences of the human neurofilament light gene direct expression oflacZ in neurons of transgenic mouse embryos

Initial expression of the neurofilament light gene coincides with the appearance of postmitotic neurons. To investigate the molecular mechanisms involved in neuron-specific gene expression during embryogenesis, we generated transgenic mice carrying various regions of the human neurofilament light gene (hNF-L) fused to thelacZ reporter gene. We found that 2.3 or 0.3 kb of the hNF-L promoter region directs expression oflacZ in neurons of transgenic embryos. Addition of 1.8 kb hNF-L intragenic sequences (IS) enlarges the neuronal pattern of transgene expression. The 2.3-kb hNF-L promotelacZ-IS construct contains all regulatory elements essential for both spatial and temporal expression of the hNF-L gene during embryogenesis and in the adult. The use of a heterologous promoter demonstrated that the 1.8-kb hNF-L intragenic sequences are sufficient to direct the expression oflacZ in a NF-L-specific manner both temporally and spatially during development and in the adult. We conclude that these hNF-L intragenic sequences containcis-acting DNA regulatory elements that specify neuronal expression. Taken together, these results show that the neurofilament light gene contains separate upstream and intragenic elements, each of which directslacZ expression in embryonic neurons.     

Lentiviral expression of HIV-1 Vpr induces apoptosis in human neurons

Our recent studies have demonstrated that extracellular, recombinant human immunodeficiency virus type I (HIV-1) Vpr protein is highly neurotoxic in the microenvironment of differentiated mature human neurons and undifferentiated neuronal precursors. Although most of the direct neurotoxic effects of HIV-1 have been attributed previously to the envelope gene product, gp120, and the Tat regulatory protein, it was demonstrated that Vpr protein caused apoptosis comparable to that induced by gp120 protein in a dose-dependent manner in the neuronal system. Having observed the neurocytopathic effects of extracellular Vpr protein previously, the effects of virally expressed Vpr on nondividing, terminally differentiated human neurons were investigated. An HIV-1-based three-plasmid expression vector system was utilized to study the effects of intracellularly expressed Vpr. These virion preparations were then used to transduce neurons generated from the human neuronal precursor NT2 cell-line. Intracellularly expressed Vpr induced apoptosis within terminally differentiated neurons, as demonstrated by TUNEL assays. Additionally, virions lacking Vpr expression did not significantly induce apoptosis within these neurons. These results suggest that HIV-1 Vpr may also be leading directly to selective neurotoxicity through intracellular expression. Furthermore, human apoptosis gene microarray comparisons exhibited an up-regulation of Bcl-2-related mRNA, as well as other apoptosis genes involved in the mitochondrial apoptotic pathway, for the Vpr-transduced neuronal cells, when compared to Vpr-negative controls. Thus, Vpr delivered intracellularly, as well as extracellularly, is involved in the induction of significant neuronal apoptosis and may be one of the molecular mechanisms in HIV-1-induced encephalopathy.     

Cortical Interneurons Upregulate Neurotrophinsin Vivoin Response to Targeted Apoptotic Degeneration of Neighboring Pyramidal Neurons

Intercellular signals provided by growth and neurotrophic factors play a critical role during neurogenesis and as part of cellular repopulation strategies directed toward reconstruction of complex CNS circuitry. Local signals influence the differentiation of transplanted and endogenous neurons and neural precursors, but the cellular sources and control over expression of these molecules remain unclear. We have previously examined microenvironmental control in neocortex over neuron and neural precursor migration and differentiation following transplantation, using an approach of targeted apoptotic neuronal degeneration to specific neuronal populationsin vivo.Prior results suggested the hypothesis that upregulated or reexpressed developmental signal molecules, produced by degenerating pyramidal neurons and/or by neighboring neurons or nonneuronal cells, may be responsible for observed events of directed migration, differentiation, and connectivity by transplanted immature neurons and precursors. To directly investigate this hypothesis, we analyzed the gene expression of candidate and control neurotrophins, growth factors, and receptors within regions of targeted neuronal cell death, first by quantitative Northern blot analysis and then byin situhybridization combined with immunocytochemical analysis. The genes for BDNF, NT-4/5, trkB receptors, and to a lesser extent NT-3 were upregulated specifically within the regions of neocortex undergoing targeted neuronal degeneration and specifically during the period of ongoing pyramidal neuron apoptosis. Upregulation occurred during the same 3-week period as the previously investigated cellular events of directed migration, differentiation, and integration. No upregulation was seen in panels of control neurotrophins, growth factors, and receptors that are not as developmentally regulated in cortex or that are thought to have primary actions in other CNS regions.In situhybridization and immunocytochemistry revealed that BDNF mRNA expression was upregulated specifically by local interneurons adjacent to degenerating pyramidal neurons. These findings suggest specific effects of targeted apoptosis on neurotrophin and other gene expression via mechanisms, including intercellular signaling between degenerating pyramidal neurons and surrounding interneurons. Further understanding of these and other controls over neocortical projection neuron differentiation may provide insight regarding normal neocortical development, intercellular signaling induced by apoptosis, and toward reconstruction and cellular repopulation of complex neocortical and other CNS circuitry.     

Lighting a path: genetic studies pinpoint neurodevelopmental mechanisms in autism and related disorders

In this review, we outline critical molecular processes that have been implicated by discovery of genetic mutations in autism. These mechanisms need to be mapped onto the neurodevelopment step(s) gone awry that may be associated with cause in autism. Molecular mechanisms include: (i) regulation of gene expression; (ii) pre-mRNA splicing; (iii) protein localization, translation, and turnover; (iv) synaptic transmission; (v) cell signaling; (vi) the functions of cytoskeletal and scaffolding proteins; and (vii) the function of neuronal cell adhesion molecules. While the molecular mechanisms appear broad, they may converge on only one of a few steps during neurodevelopment that perturbs the structure, function, and/or plasticity of neuronal circuitry. While there are many genetic mutations involved, novel treatments may need to target only one of few developmental mechanisms.     

Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation

Reactive oxygen species (ROS) are supposed to play an important role in hypoxia- and ischemia/reperfusion-mediated neuronal injury with the characteristics of apoptosis. There are many reports showing that cobalt chloride (CoCl(2)) could mimic the hypoxic responses in some aspects including production of ROS in cultured cells. The cytotoxicity of CoCl(2) and its molecular mechanisms have yet to be elucidated. We report that CoCl(2) triggered neuronal PC12 cells apoptosis in a dose- and time-dependent manner. Apoptosis was demonstrated by morphological changes and DNA fragmentation, and was dependent on macromolecular synthesis. Apoptosis was also confirmed by the decrease of the expression of Bcl-X(L). To our knowledge, this is the first documentation of the apoptotic induction of CoCl(2) on PC12 cells. Furthermore, ROS production in PC12 cells was increased during CoCl(2) treatment. Antioxidants, which could inhibit ROS production, significantly blocked CoCl(2)-induced apoptosis, suggesting that apoptosis is mediated by ROS production. We also observed a significant increase of the DNA-binding activity of AP-1 in response to CoCl(2) and this increase was blocked by antioxidants, showing that CoCl(2)-induced apoptosis is accompanied by ROS-activated AP-1. CoCl(2)-treated PC12 cells may serve as an in vitro model for studies of molecular mechanisms in ROS-linked neuronal disorders.     

急性脑梗死患者外周血单个核细胞114个氧化应激相关基因的差异表达：一项基因芯片筛查实验

目的 了解急性脑梗塞患者溶栓术前后脑缺血/再灌注损伤过程中细胞氧化应激及毒性相关基因的变化。 方法 通过基因芯片筛查急性脑梗塞患者溶栓术前后脑缺血/再灌注损伤过程中相关基因的变化。结果 急性脑梗塞患者组中,溶栓前后显示显著变化的基因有14个,其中表达显著上调的为8个,显著下调的为4个。上调的基因中,细胞生长停滞/衰老相关１个（GADD45A）, 氧化和代谢应激相关1个(PRDX2), 热休克相关3个（HSPD1, DNAJB1, DNAJB2）,DNA损害和修复相关1个(RAD50),及凋亡信号相关2个(TNFSF6, TRADD)。下调的基因中,细胞增殖/癌变相关１个（CCNG1）,氧化和代谢应激相关个（CAT,CYP1A1）,DNA损害和修复相关1个（ATM）。结论 急性脑梗塞患者行溶栓术后脑缺血/再灌注损伤受多种基因的调控,包括氧化代谢应激,热休克,DNA损害和修复及凋亡信号相关基因,整体上了解到脑缺血/再灌注损伤基因调节的复杂性。     

Domoic acid-induced neurotoxicity in the hippocampus of adult rats

Domoic acid (DA), an agonist of non-N-methyl-D-aspartate (non-NMDA) receptor subtype including kainate receptor, was identified as a potent neurotoxin showing involvement in neuropathological processes like neuronal degeneration and atrophy. In the past decade evidence indicating a role for excitatory amino acids in association with neurological disorders has been accumulating. Although the mechanisms underlying the neuronal damage induced by DA are not yet fully understood, many intracellular processes are thought to contribute towards DA-induced excitotoxic injury, acting in combination leading to cell death. In this review article, we report the leading hypotheses in the understanding of DA-induced neurotoxicity, which focus on the role of DA in neuropathological manifestations, the formation of the retrograde messenger molecule nitric oxide (NO) for the production of free radicals in the development of neuronal damage, the activation of glial cells (microglia and astrocytes) in response to DA-induced neuronal damage and the neuroprotective role of melatonin as a free radical scavenger or antioxidant in DA-induced neurotoxicity. The possible implications of molecular mechanism underlying the neurotoxicity in association with necrosis, apoptosis, nitric oxide synthases (nNOS and iNOS) and glutamate receptors (NMDAR1 and GluR2) related genes and their expression in DA-induced neuronal damage in the hippocampus have been discussed.     

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.     

Mechanisms of LRRK2-Mediated Neurodegeneration

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of familial Parkinson’s disease (PD), whereas common variation at the LRRK2 locus is associated with an increased risk of idiopathic PD. Considerable progress has been made toward understanding the biological functions of LRRK2 and the molecular mechanisms underlying the pathogenic effects of disease-associated mutations. The development of neuronal culture models and transgenic or viral-based rodent models have proved useful for identifying a number of emerging pathways implicated in LRRK2-dependent neuronal damage, including the microtubule network, actin cytoskeleton, autophagy, mitochondria, vesicular trafficking, and protein quality control. However, many important questions remain to be posed and answered. Elucidating the molecular mechanisms and pathways underlying LRRK2-mediated neurodegeneration is critical for the identification of new molecular targets for therapeutic intervention in PD. In this review we discuss recent advances and unanswered questions in understanding the pathophysiology of LRRK2.     

Expression Profile of Rat Hippocampal Neurons Treated with the Neuroprotective Compound 2,4-Dinitrophenol: Up-Regulation of cAMP Signaling Genes

2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.     

Use of microarrays in the search of gene expression patterns: application to the study of complex phenotypes

Sequencing the human genome has prompted the development of new technologies, which have emerged as promising methodological and scientific tools for advancing the current knowledge about the causes and mechanisms involved in various complex disorders. Among those, the high-throughput technique known as microarray is particularly powerful in providing a global view of gene expression patterns in biological samples. By the simultaneous determination of the expression levels of thousands of genes, microarrays allow researchers to compare the molecular behaviour of different types of cells lines or specific tissues that have been exposed to pathological or experimental conditions. The method may provide insights into physiological processes and facilitate the identification of novel biological markers for diagnostic, prognostic and pharmacological treatments for a number of diseases. In this article, we present theoretical and methodological concepts underlying the microarray technology, as well as an overview of its advantages, perspectives and future scientific directions. In an attempt to demonstrate the applicability and efficiency of the method in the study of complex phenotypes, initial results on gene expression studies in post mortem brain samples of psychiatric patients and on the molecular and functional consequences of sleep disturbances, which is strongly associated with psychiatric illness, will be described and discussed.     

Live or let die - retinal ganglion cell death and survival during development and in the lesioned adult CNS

Programmed cell death or apoptosis is a common and widespread phenomenon that is important for proper development of the nervous system. In the adult CNS, however, apoptosis contributes to secondary cell loss after various types of lesions. The retino-tectal system has been successfully used as a convenient model system to study the molecular mechanisms of neuronal apoptosis and survival during development and in the lesioned adult CNS. This review describes the current knowledge about the interactions of cell death and survival pathways in general and for retinal ganglion cells specifically.