Objective/Rationale:
The death of neurons in the brains of Parkinson’s disease (PD) patients involves a buildup of agents that cause oxidative stress, resulting in damage to proteins, lipids, and DNA. Oxidative stress is alleviated by methionine sulfoxide reductase A (MsrA), an enzyme that repairs oxidatively damaged proteins and neutralizes oxidizing agents. The goal of this project is to determine whether an increase in levels of MsrA alleviates neurodegeneration in pre-clinical models of PD.
Project Description:
The ability of MsrA to protect dopamine neurons will be examined in two animal models of PD: rats over-expressing human alpha-synuclein, and rats exposed to the neurotoxic agent, 6-hydroxydopamine (6-OHDA). DNA encoding MsrA will be introduced into rat substantia nigra using adeno-associated virus (AAV) as a genetic delivery agent. As one approach, the MsrA virus will be administered at the same time as AAV encoding alpha-synuclein. Alternatively, rats infected with MsrA virus will receive a subsequent injection of 6-OHDA in the striatum. Both sets of animals will be analyzed at different times for evidence of motor abnormalities using various behavioral tests. Dopamine neuron survival and alpha-synuclein aggregation will be monitored by staining isolated rat midbrains with labeled antibodies and examining the stained cells by microscopy.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Our project is relevant to the treatment of PD because it will reveal whether an increase in levels of the antioxidant enzyme MsrA can prevent dopamine neuron killing in animal models of the disease. If we prove that an increase in MsrA levels blocks the destruction of dopamine neurons triggered by alpha-synuclein over-expression or 6-OHDA lesion, then this discovery would justify the development of new therapies aimed at upregulating MsrA in the brains of PD patients.
Anticipated Outcome:
Our studies will reveal whether an increase in MsrA levels alleviates dopamine neuron death and the formation of Lewy-like inclusions in rats over-expressing alpha-synuclein in the midbrain. The results will also indicate whether MsrA upregulation inhibits the destruction of dopamine neurons in rats treated with the neurotoxin 6-OHDA. Our findings will help to establish that increasing MsrA expression levels is a reasonable strategy to inhibit neurodegeneration in the brains of PD patients.
Progress Report
Dr. Kirik has generated the tools necessary to proceed with his studies and is currently testing these. He will next begin characterizing the role of MsrA in vivo.
Final Outcome
The project was designed to test the hypothesis that enhancement of the antioxidant response of the dopamine neurons in the substantia nigra by overexpression of the methionine sulfoxide reductase A (MsrA) enzyme would result in protection of dopaminergic neurons in two pre-clinical models of Parkinson’s disease. We found that the overexpression of MsrA using AAV vectors did not result in protection of dopaminergic neurons in the 6-hydroxydopamine lesion model under conditions tested. The second study utilized co-expression of alpha-synuclein and MsrA using the same AAV platform to assess its potential to counteract protein aggregation mediated toxicity. The results from the second experiment have not been conclusive as the toxicity of a-synuclein itself was not sufficient to assess the effect of MsrA. The experiments highlighted the importance of a precise readout of MsrA activity in the brain that can be measured from tissue samples. We are currently investigating methods that can provide robust and specific results regarding this readout. It is anticipated that establishment of techniques providing this information will enable us to revisit the hypothesis tested here and generate conclusive information about their potential role in PD pathogenesis and opportunities for development of novel therapeutics.