ABSTRACT
Title
Evaluation of the neuroprotective effect of parkin against α-synuclein-induced neurotoxicity in a rat model of Parkinson’s disease.
Authors
G. Mercanti
Doctoral School of Pharmacological Sciences, Section Molecular and Cellular Pharmacology
Department of Pharmacology and Anesthesiology “E. Meneghetti”, University of Padua, Italy
Abstract
Parkinson’s disease (PD) is a chronic progressive neurodegenerative movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons in the substantia nigra pars compacta (SNpc), leading to a loss of dopamine in the target structure striatum and development of motor symptoms [1]. The etiology of PD is still largely unknown. At least six different casual 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. In particular, two missense mutations (A30P and A53T) 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 sporadic and familial forms of PD [2,3]. Parkin functions as an E3 ubiquitin ligase: loss of its activity seems to cause an autosomal recessive form of PD [4]. We have recently described a hemi-parkinsonian rat model, based on the stereotaxic injection of TAT-α-syn-A30P in the SNpc of the right hemisphere. The TAT sequence allows diffusion of the fusion protein across the neuronal plasma membrane and results in a localized dopaminergiccell loss [3].
The research project is designed to examine possible neuroprotective effects of TAT-parkin against α-syn-induced neurotoxicity in this model. In a first experiment rats were stereotaxically injected with TAT-α-syn-A30P, TAT-parkin, or both. At different times after injection all animals were subjected to Rotarod behavioral test and apomorphine-induced rotation test to evaluate impairment in motor function and the extent of dopaminergic cell damage,respectively. In order to better visualize the extent of the lesion, all animals were sacrificed and the brains processed for immunohistochemical analysis with a specific primary antibody against tyrosine hydroxylase (TH). In agreement with previous experiments, there was an impairment in motor function after TAT-α-syn A30P, TAT-parkin, or a combination of TAT-α-syn A30P and TAT-parkin, at both testing sessions post-lesion. Apomorphine-induced rotation exhibited a postural asymmetry with a significantly different turning behavior in all treated groups compared to control animals. Also the immuohistochemical analysis showed anincreasing depletion of dopaminergic terminals on right SNpc in all treatment groups compared to left SNpc of control animals. The most severe depletion occurs with TAT-α-syn A30P+TAT-parkin treated animals. In a second experiment four groups of animals received an intra-nigral injection of different doses of TAT-parkin to evaluate a possible dose-dependent toxicity of TAT-parkin. The outcome rate of the damage was assessed again with Rotarod test, Footprint test and apomorphine-induced rotation test starting from 2 weeks post-surgery. The animals showed variable impairments in motor function in the two spontaneous motor tests and in the apomorphine-induced rotation test, revealing that only the group that received the lowest TAT-parkin dose is devoid of a toxic effect compared to control animals.
To clearly characterize the extent of damage, all animals were subjected to in vivo microdialysis to monitor striatal dopamine release after d-amphetamine or nicotine administration, while the microdialysis samples were analyzed by HPLC. To evaluate tissue damage brains will be processed for TH immunostaining.
The α-syn-parkin-based model better reproduces the pathophysiology of PD and could be of utility to understand the mechanisms that lead to dopaminergic neurodegeneration. Moreover, it could help identify disease-modifying strategies as opposed to therapies which provide only symptomatic relief.
1) Dauer W et al. (2003) – Neuron vol 39: 889-909.
2) Batelli S et al. (2008) - PLoS ONE 3(4): e1884
3) Recchia A et al. (2008) - Neurobiol Dis 30(1): 8-18.
4) Von Coelln R et al. (2004) - Cell Tissue Res 318(1): 175-84.
The research project is designed to examine possible neuroprotective effects of TAT-parkin against α-syn-induced neurotoxicity in this model. In a first experiment rats were stereotaxically injected with TAT-α-syn-A30P, TAT-parkin, or both. At different times after injection all animals were subjected to Rotarod behavioral test and apomorphine-induced rotation test to evaluate impairment in motor function and the extent of dopaminergic cell damage,respectively. In order to better visualize the extent of the lesion, all animals were sacrificed and the brains processed for immunohistochemical analysis with a specific primary antibody against tyrosine hydroxylase (TH). In agreement with previous experiments, there was an impairment in motor function after TAT-α-syn A30P, TAT-parkin, or a combination of TAT-α-syn A30P and TAT-parkin, at both testing sessions post-lesion. Apomorphine-induced rotation exhibited a postural asymmetry with a significantly different turning behavior in all treated groups compared to control animals. Also the immuohistochemical analysis showed anincreasing depletion of dopaminergic terminals on right SNpc in all treatment groups compared to left SNpc of control animals. The most severe depletion occurs with TAT-α-syn A30P+TAT-parkin treated animals. In a second experiment four groups of animals received an intra-nigral injection of different doses of TAT-parkin to evaluate a possible dose-dependent toxicity of TAT-parkin. The outcome rate of the damage was assessed again with Rotarod test, Footprint test and apomorphine-induced rotation test starting from 2 weeks post-surgery. The animals showed variable impairments in motor function in the two spontaneous motor tests and in the apomorphine-induced rotation test, revealing that only the group that received the lowest TAT-parkin dose is devoid of a toxic effect compared to control animals.
To clearly characterize the extent of damage, all animals were subjected to in vivo microdialysis to monitor striatal dopamine release after d-amphetamine or nicotine administration, while the microdialysis samples were analyzed by HPLC. To evaluate tissue damage brains will be processed for TH immunostaining.
The α-syn-parkin-based model better reproduces the pathophysiology of PD and could be of utility to understand the mechanisms that lead to dopaminergic neurodegeneration. Moreover, it could help identify disease-modifying strategies as opposed to therapies which provide only symptomatic relief.
1) Dauer W et al. (2003) – Neuron vol 39: 889-909.
2) Batelli S et al. (2008) - PLoS ONE 3(4): e1884
3) Recchia A et al. (2008) - Neurobiol Dis 30(1): 8-18.
4) Von Coelln R et al. (2004) - Cell Tissue Res 318(1): 175-84.