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. We published a study this past year showing that transgenic mice expressing a truncated form of the BMP receptor II in dopaminergic neurons of the nigrostriatal pathway were more vulnerable to methamphetamine challenge as determined by TUNEL labeling in the substantia nigra. Our data suggest that a deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA. In a second study, we show that methamphetamine suppressed BMP7 expression in the substantia nigra of mice. Mice that carry only one allele of BMP7 were more vulnerable to methamphetamine toxicity as measured by tyrosine hydroxylase (TH) immunostaining in the nigra reticulate and changes in locomotor activity. Exogenous delivery of BMP7 protein via the lateral ventricle of mouse brain reduced methamphetamine toxicity as measured by locomotor activity and TH immunostaining. 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 presented findings from this study at the 2009 Society for Neuroscience Meeting in Chicago. This work is ongoing. 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. Additionally, we are collaborating with Dr. Roy Wise to examine the ability of GLT-1 to alter cocaine seeking behavior in rats. 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. Recent work with mesencephalic astrocyte-derived neurotrophic factor (MANF) protein suggest that is may play a role in modulating neuronal function. We have initiated experiments examining the ability of MANF overexpression to change behavioral sensitization to methamphetamine. We have also begun looking in vitro to see whether MANF can alter synapse formation, neurotransmitter loading into vesicles and release of vesicles. Lastly, we contributed to the completion of three studies with Drs. Hope, Morales and Su at the NIDA-IRP.
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