Evaluation of the neuroprotective effect of parkin in a a-synuclein-based rat model of Parkinson's disease

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, characterized by progressive loss of nigrostriatal dopaminergic neurons and a reduction in striatal dopamine concentration. Different causal genes linked to rare familial forms of PD have been identified, while late-onset idiopathic PD is thought to result from the interplay between predisposing genes and environmental factors. Two missense mutations (A53T and A30P) in the α-synuclein (α-syn) gene have received great attention with the discovery that abnormal metabolism and accumulation of α-syn in dopaminergic neurons leads to both forms of PD. Parkin functions as an E3 ubiquitin ligase involved in the cellular machinery that tags proteins with ubiquitin, thereby targeting them for degradation by the proteasome: loss of its activity seems to cause an autosomal recessive form of PD. The ubiquitin-proteasome system plays a major role in many vital cellular processes, and its dysfunction has been implicated in the pathogenesis of neurodegenerative disorders. A number of observations suggest that functional interaction between α-syn and parkin may involve the proteasome: α-syn interacts with and may be degraded by the proteasome, over-expression of α-syn inhibits the proteasome and mutant α-syn increases the sensitivity of cells to proteasome inhibition. We described previously a hemi-parkinsonian rat model based on the stereotaxic injection of TAT-α-syn-A30P in the right substantia nigra pars compacta (SNpc). The protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein sequence allows diffusion of the fusion protein across the neuronal plasma membrane and results in a localized dopaminergic cell loss. Here we examined possible neuroprotective effects of TAT-parkin against α-syn-induced neurotoxicity. Rats were stereotaxically injected with TAT-α-syn A30P, TAT-parkin, or both. At different times animals were subjected to different behavioral tests and the brains processed for immunohistochemical analysis. The behavioral tests showed a impairment in motor function after TAT-α-syn A30P, TAT-parkin or their combination, and postural asymmetry in all treated groups compared to control animals. Immuohistochemical analysis showed that the most severe depletion occurs in TAT-α-syn A30P + TAT-parkin treated animals, compared to control animals. Next, animals received an intra-nigral injection of different doses of TAT-parkin to evaluate a possible dose-dependent toxicity of TAT-parkin. Damage was again assessed using two behavioral tests, and showed variable impairments in motor function indicating that only the group receiving the lowest TAT-parkin dose is devoid of a toxic effect. All animals were subjected also to in vivo microdialysis to monitor striatal dopamine release; microdialysis samples were analyzed by HPLC and showed an increase in dopamine release after stimulation with nicotine. Immunohistochemical analysis revealed tissue damage as a significant loss of dopaminergic neurons in the SNpc: only animals receiving the lowest dose of TAT-parkin have a less severe damage compared to control animals. In the last experiment we examined the possible neuroprotective effect of a non-toxic dose of TAT-parkin against a 50% higher dose of TAT-α-syn A30P. Behavioral testing revealed motor function impairment among all treated groups, but the highest TAT-parkin dose seems to ameliorate it better than lower doses. The microdialysis experiment confirms that the two doses of TAT-parkin stimulate the release of DA, and shows an increase in dopamine level even in groups with TAT-α-syn A30P lesion, especially with the lowest dose of TAT-parkin. Immunohistochemical analysis revealed an increase in dopaminergic fiber optical density when TAT-parkin is used with a higher dose of TAT-α-syn A30P. The α-syn-parkin-based model reproduces the pathophysiology of PD and could be of utility to understand the mechanisms leading to dopaminergic neurodegeneration, thereby helping to identify disease-modifying strategies as opposed to therapies which provide only symptomatic relief

Topic: Parkinson's Disease Mechanisms and Treatments
Subfield: Neurology
Field: Medicine
Domain: Health Sciences