Abstract
α-Synuclein is a protein implicated in the etiopathogenesis of Parkinson’s disease (PD). AAV1/2-driven overexpression of human mutated A53T-α-synuclein in rat and monkey substantia nigra (SN) induces degeneration of nigral dopaminergic neurons and decreases striatal dopamine and tyrosine hydroxylase (TH). Given certain advantages of the mouse, especially it being amendable to genetic manipulation, translating the AAV1/2-A53T α-synuclein model to mice would be of significant value. AAV1/2-A53T α-synuclein or AAV1/2 empty vector (EV) at a concentration of 5.16 x 1012 gp/ml were unilaterally injected into the right SN of male adult C57BL/6 mice. Post-mortem examinations included immunohistochemistry to analyze nigral α-synuclein, Ser129 phosphorylated α-synuclein and TH expression, striatal dopamine transporter (DAT) levels by autoradiography and dopamine levels by high performance liquid chromatography. At 10 weeks, in AAV1/2-A53T α-synuclein mice there was a 33% reduction in TH+ dopaminergic nigral neurons (P < 0.001), 29% deficit in striatal DAT binding (P < 0.05), 38% and 33% reductions in dopamine (P < 0.001) and DOPAC (P < 0.01) levels and a 60% increase in dopamine turnover (homovanilic acid/dopamine ratio; P < 0.001). Immunofluorescence showed that the AAV1/2-A53T α-synuclein injected mice had widespread nigral and striatal expression of vector-delivered A53T-α-synuclein. Concurrent staining with human PD SN samples using gold standard histological methodology for Lewy pathology detection by proteinase K digestion and application of specific antibody raised against human Lewy body α-synuclein (LB509) and Ser129 phosphorylated α-synuclein (81A) revealed insoluble α-synuclein aggregates in AAV1/2-A53T α-synuclein mice resembling Lewy-like neurites and bodies. In the cylinder test, we observed significant paw use asymmetry in the AAV1/2-A53T α-synuclein group when compared to EV controls at 5 and 9 weeks post injection (P < 0.001; P < 0.05). These data show that unilateral injection of AAV1/2-A53T α-synuclein into the mouse SN leads to persistent motor deficits, neurodegeneration of the nigrostriatal dopaminergic system and development of Lewy-like pathology, thereby reflecting clinical and pathological hallmarks of human PD.
Highlights
Parkinson’s disease (PD) is the second most common neurodegenerative disorder with pathological hallmarks consisting of degeneration of dopaminergic neurons within the substantia nigra (SN), loss of dopaminergic terminals projecting into the striatum and accumulation of insoluble protein aggregates, termed Lewy bodies and Lewy neurites
Unilateral AAV1/2-A53T-aSyn delivery into the substantia nigra leads to behavioral deficits in mice To examine if unilateral AAV1/2-A53T-aSyn injection results in a measurable behavioural deficit in mice, we assessed motor behavior by the cylinder test at two time points, 5 and 9 weeks, during the 10 week in-life phase of the experiment
At the same time most of the remaining tyrosine hydroxylase (TH)+ SN neurons co-localized with human A53T-aSyn as depicted by double immunofluorescence staining showing a production of the A53TaSyn transgene by dopaminergic neurons (Fig. 1d-f)
Summary
Parkinson’s disease (PD) is the second most common neurodegenerative disorder with pathological hallmarks consisting of degeneration of dopaminergic neurons within the substantia nigra (SN), loss of dopaminergic terminals projecting into the striatum and accumulation of insoluble protein aggregates, termed Lewy bodies and Lewy neurites. The pathogenic role of insoluble aSyn in PD is even less clear [3], the fact that hereditary forms of PD can be caused by duplication, triplication and missense mutations in the aSyn encoding gene (e.g. A53T [22]), A30P [16]) has strongly linked this protein to the pathogenesis of PD [10, 16, 22] Based on these observations, numerous mouse models for PD with pathogenic aSyn expression have been generated. In view of the advantages of knock-out or transgenic mouse models for studying molecular mechanisms of neurodegeneration and resilience our aim was to translate the AAV1/2A53T-aSyn mediated rat PD model to the mouse system
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