Following the loss of nigrostriatal dopamine (DA), there is an increase in activity of glutamate neurons within the subthalamic nucleus (STN). In animal models and in humans with Parkinson's disease (PD), both a lesion or high frequency stimulation/deep brain stimulation (DBS) of the STN results in improvement in motor function and provides symptomatic relief. However, both DBS and a lesion of the STN could be affecting the fibers of passage. Direct inactivation of the STN neurons without affecting the fibers of passage could answer this concern. Since STN neurons utilize the vesicular glutamate transporter 2 (VGLUT2) for uptake of glutamate into synaptic vesicles, deletion of this gene would selectively inactivate those STN glutamate neurons and block glutamate release. Using the Cre/loxP recombinase gene technology where a specific gene can be silenced, inactivation of the STN glutamate neurons can be achieved through deleting a targeted gene in the specific brain area by injecting AAV-Cre to mice that are floxed for Vglut2 (Vglut2flox/flox). To determine if deletion of the Vglut2 gene in the STN can be neuroprotective against DA terminal and cell loss using the neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), we find that a unilateral infusion of AAV-Cre-GFP (green fluorescent protein) into the STN inactivated approximately 80% of those STN glutamate neurons, as determined by GFP labeling of STN neurons. As measured by tyrosine hydroxylase (TH) immunoreactivity, this resulted in a bilateral protection from the loss of DA terminals in the striatum and DA neurons in the substantia nigra pars compacta (SNpc) following progressive administration of MPTP compared to the wildtype mice treated with the toxin. There was a 35% decrease in activated tyrosine kinase receptor B (TrkB) within the substantia nigra (SN) following MPTP compared to the vehicle treated group. If VGLUT2 deletion occurred following the initiation of MPTP treatment (i.e, neurointervention), we find striatal nerve terminals recovered to nearly 80% of the vehicle treated group. Since MPTP results in a decrease in activated TrkB in the SN, deletion of the Vglut2 gene within the STN, which we found resulted in a decrease in extracellular glutamate in the SN, would allow activated TrkB in the SN to return to control levels and protect the SNpc neurons. To address a possible mechanism of this protection, we find that systemic administration of the TrkB agonist, 7,8-dihydroxyflavone, 2 weeks after the initiation of MPTP treatment (i.e., neurointervention) resulted in a blockade of any further loss of TH within the striatum due to continued MPTP treatment. The overall goal of this project is to determine whether deletion of the Vglut2 gene within the STN initiated following (i.e., restoration) either progressive administration of MPTP or intrastriatal infusion of 6-hydroxydopamine (6-OHDA) can reverse the loss of DA within the nigrostriatal pathway in both young and aged mice. We will also determine the role of TrkB in blocking or reversing the DA depletion due to MPTP.
The specific aims of this proposal are to: 1.) determine if deletion of the Vglut2 gene within the STN initiated 4 weeks after progressive MPTP administration can reverse the loss of DA markers in the striatum/SN and motor function in both young and aged mice, 2.) determine if deletion of the Vglut2 gene within the STN initiated 12 days following infusion of 6-OHDA into the dorsolateral striatum can reverse the loss of DA markers in the striatum/SN and motor function in both young and aged mice, and 3.) determine if the neuroprotection from MPTP following deletion of the Vglut2 gene within the STN is due to maintained levels of activated TrkB in the SN by daily infusion of a TrkB agonist into the SN.
Investigation of movement disorders, especially Parkinson's disease, is a high priority research area within the Department of Veterans Affairs. Because of the high incidence of this disease in the general population over the age of 50 and the fact that the age of the veteran population is slowly increasing, this movement disorder is affecting more and more of our veterans. Specific inactivation of the subthalamic nucleus or pharmacological activation of the TrkB receptor in our animal model of this disease appears to be effective in terms of blocking/reversing the loss of nigrostriatal dopamine needs further investigation. The results from this project have potentially wide implications of great significance to the population cared for by the Department of Veteran Affairs and by the fact that six Parkinson's Disease Research, Education and Care Centers (PADRECCs) currently are funded (one here at the Portland VA Medical Center), to carry out clinical research projects focusing on Parkinson's disease.
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