Gene Delivery and Addiction

今年，我们启动了实验，研究甲基苯丙胺对初级小胶质细胞、初级神经元和细胞系中HIV长末端重复序列（LTR）启动子的影响。 我们优化了从大鼠中分离小胶质细胞的条件。 我们目前正在优化基因转移模式，以提供一个报告基因构建体来测量甲基苯丙胺存在下的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体，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。 我们以前证明，外源性应用骨形态发生蛋白7（BMP 7）减少6-羟基多巴胺介导的神经退行性疾病的帕金森氏病的啮齿动物模型。最近，我们正在研究BMP 7神经再生作用的细胞和分子机制，重点是影响细胞外基质（ECM）或更普遍的细胞外环境的能力。 使用原代大鼠神经元，我们正在研究BMP 7影响ECM修饰酶的能力。 总的来说，我们的数据表明，BMP 7和BMP受体信号传导在各种神经退行性变模型中具有神经保护和神经再生作用。 我们产生了表达人μ阿片受体（huMOR）的AAV载体，并且正在评估huMOR在小鼠中甲基苯丙胺致敏中的作用。 我们的初步研究结果表明，通过AAV载体在特定脑区域中表达huMOR改变了甲基苯丙胺的致敏性。 我们已经提交了这项工作的出版，目前正在审查中。 我们还产生了表达谷氨酸转运蛋白（GLT-1）的AAV载体，并证明其功能。 我们正在使用大鼠中风模型研究GLT-1减少缺血引起的损伤的能力。 通过微透析测量，对缺血的保护伴随着缺血诱导的谷氨酸溢出的减少，这项研究于今年发表。 产生AAV-GLT-1以调节细胞外谷氨酸的水平。 我们已经开始实验检查通过AAV过量GLT-1过表达降低谷氨酸兴奋毒性的能力。 我们也开始研究特定脑区GLT-1过表达对甲基苯丙胺致敏的影响。 这些实验正在进行中。 最后，我们与NIDA-IRP的Yavin Shaham博士共同完成了一项合作研究。

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.