Morphological and Behavioral Impact of AAV2/5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System

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.

Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain

“…We first verified, as demonstrated previously, that our rAAV2/5 expressing GFP is not toxic to nigral dopaminergic neurons of the SNpc (Gombash et al , 2013). Age-related reductions in total nigral dopaminergic neurons are not a factor in our present results as equivalent numbers of nigral THir neurons are present in the SD rat at 20 months as compared to 3 months of age.…”

“…Particles were purified using iodixanol gradients and Q-sepharose chromatography, and dotblot was used to determine vector titer (Zolotukhin et al , 1999). The vector titer used in this study was 5.88 × 10 13 genomes/mL (Gombash et al , 2013). In order to preserve the viral stability and titer, viral preparations were stored at 4°C and never frozen.…”

“…Rats were placed in a stereotaxic frame and two 2μL injections of rAAV2/5 were injected in the left SN at coordinates (from dura) AP −5.3mm, ML +2.0mm, DV −7.2mm, and AP −6.0mm, ML +2.0mm, and DV −7.2mm as previously described (Gombash et al , 2013). A Hamilton syringe fitted with a glass capillary needle (Hamilton Gas Tight syringe 80,000, 26s/2” needle; Hamilton, Reno, NV; coated in SigmaCote) was used for injection.…”

“…The present study sought to determine whether viral vector-mediated transgene expression was reduced in the aged rat nigrostriatal system as compared to the young adult. We utilized the rAAV2/5 serotype to examine this question because of its efficient transduction of nigral neurons (Burger et al , 2004; Gombash et al , 2013). Intranigral injections of rAAV serotype 2/5 expressing green fluorescent protein (rAAV2/5 GFP) resulted in significantly reduced exogenous protein expression in the aged rat brain as compared to the young adult brain.…”

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents

“…Similar to toxin models, viral vectors overexpressing aSyn can induce a robust lesion of the nigrostriatal system leading to behavioral phenotypes in rodents and non-human primates using a system that allows spatiotemporal control and the ability to design studies with an internal control [23][23]. In addition, the level of protein overexpression can easily be altered to reduce the level of overexpression to sub-toxic levels or prolong the time before degeneration if desired [14,24–25]. The main drawback of this model, however, is the supra-physiological level of overexpression required for pathology, leading to the question of generalizability between this model and the human condition.…”