Abstract
The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 1013) or varying titers (2.2 x 1012, 1.0 x 1013, 5.9 x 1013, or 1.0 x 1014) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 1014 titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 1013 titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 1013 and 1.0 x 1014 rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 1013 rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.
Highlights
Preclinical animal models that can accurately predict clinical efficacy and recapitulate the pathological aspects of Parkinson’s disease (PD) are a necessity
Our results demonstrate that utilizing two-site nigral injections of rAAV2/5 to overexpress wildtype human α-syn results in the progressive death of dopaminergic nigral neurons and significantly reduces tyrosine hydroxylase (TH) immunoreactive (THir) in the striatum
We confirm that nigral degeneration is directly related to vector titer and that longer durations of α-syn expression appear to increase the magnitude of degeneration. α-syn overexpression resulted in significant impairments in contralateral forelimb use in both the cylinder and adjusting steps task and deficits in Ultrasonic vocalizations (USVs) call intensity and call rate
Summary
Preclinical animal models that can accurately predict clinical efficacy and recapitulate the pathological aspects of Parkinson’s disease (PD) are a necessity. There has been increased use of the viral vectormediated α-syn nigrostriatal overexpression rodent model of PD These α-syn overexpression models recapitulate many of the neuropathological hallmarks of the human form of the disease, including but not limited to, α-syn aggregation, degeneration of nigral dopaminergic neurons and striatal terminals, and neuroinflammation. Both recombinant adenoassociated viral vectors (rAAV) and lentiviral vectors have been used to overexpress the human wildtype and mutated forms of α-syn in the rodent nigrostriatal system [20,21,22,23,24,25,26]. These results establish a titer- and duration-dependent impact of rAAV2/5-mediated expression of human wildtype α-syn and allow for appropriate design and testing of future therapeutic neuroprotective strategies for PD
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