Gene Delivery and Addiction

今年，我们启动了实验，检测甲基苯丙胺对原代小胶质细胞、原代神经元和细胞系中HIV长末端重复序列(LTR)启动子的影响。我们已经优化了从大鼠身上分离小胶质细胞的条件。我们目前正在优化基因转移范例，以提供一种报告基因结构，以测量在甲基苯丙胺存在下HIV LTR启动子的活性。使用细胞系，我们已经成功地交付了报告构建，目前正在准备我们的结果发表。这项工作正在进行中。 我们部门多年来一直在研究的蛋白质之一是骨形态发生蛋白7，特别是它的神经营养/神经再生特性。我们先前证明，外源性应用骨形态发生蛋白7(BMP7)可以减少帕金森病啮齿动物模型中6-羟基多巴胺介导的神经变性。最近，我们正在研究BMP7神经再生效应的细胞和分子机制，重点是影响细胞外基质(ECM)或更广泛的细胞外环境的能力。利用原代培养的大鼠神经元，我们检测了骨形态发生蛋白7的S对细胞外基质修饰酶的影响。总之，我们的数据表明，BMP7和BMP受体信号在不同的神经退行性变模型中具有神经保护和神经再生的作用。 我们构建了一个表达人MU阿片受体(体液)的AAV载体，并正在评估体液在小鼠甲基苯丙胺敏化中的作用。我们的初步发现表明，AAV载体在特定大脑区域的幽默表达改变了甲基苯丙胺的敏化。我们已经提交了这部作品供出版，目前正在审查中。 我们还构建了一个表达谷氨酸转运蛋白(GLT-1)的AAV载体，并证明了它具有功能。我们正在进行使用中风大鼠模型测试GLT-1减少缺血造成的损害的能力的工作。通过微透析法测量，对缺血的保护伴随着缺血诱导的谷氨酸溢出的减少，这项研究发表在今年。AAV-GLT-1是用来调节细胞外谷氨酸水平的。我们已经开始实验检测AAV过量表达GLT-1来减少谷氨酸的兴奋性毒性的能力。我们还开始研究GLT-1在特定脑区的过度表达，以寻找对甲基苯丙胺敏感的变化。这些实验正在进行中。 最后，我们协助完成了与Yavin Shaham博士在NIDA-IRP的合作研究。

This year we initiated experiments examining the effects of methamphetamine on the HIV long terminal repeat (LTR) promoter in primary microglia, primary neurons and cell lines. We have optimized conditions to isolate microglia from rats. We are currently optimizing gene transfer paradigms to deliver a reporter gene construct to measure the activity of the HIV LTR promoter in the presence of methamphetamine. Using cell lines, we have successfully delivered the reporter construct and are currently preparing our results for publication. This work is ongoing. One of the proteins that our section has been studying for many years is bone morphogenetic protein 7, specifically, its neurotrophic/neuroregenerative properties. We previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson's disease. Recently, we are examining the cellular and molecular mechanisms of BMP7 neuroregenerative effects by focusing on the ability to influence the extracellular matrix (ECM) or more generally the extracellular environment. Using a primary rat neurons, we are examining BMP7's ability to influence ECM-modifiying enzymes. Collectively, our data demostrate that BMP7 and BMP receptor signaling have neuroprotective and neuroregenerative effects in various models of neurodegeneration. We generated an AAV vector expressing the human mu opioid receptor (huMOR) and are evaluating the role of huMOR in methamphetamine sensitization in mice. Our preliminary findings show that huMOR expression by an AAV vector in specific brain regions alters methamphetamine sensitization. We have submitted this work for publication and it is currently under review. We have also generated an AAV vector expressing the glutamate transporter (GLT-1) and demonstrated that it functional. We have ongoing work examining the ability of GLT-1 to reduce damage caused by ischemia using a rat model of stroke. The protection against ischemia was accompanied by a decrease in ischemia-induced glutamate overflow as measured by microdialysis and this study was published this year. AAV-GLT-1 was created to modulate the levels of extracellular glutamate. We have begun experiments examining the ability of excess GLT-1 overexpression by AAV to reduce excitoxicity by glutamate. We have also begun examining the GLT-1 overexpression in specfic brain regions for alterations to methamphetamine sensitization. These experiments are ongoing. Lastly, we contributed to the completion of a collaborative study with Dr. Yavin Shaham at the NIDA-IRP.