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Fig. 3 | Acta Neuropathologica Communications

Fig. 3

From: Early-onset impairment of the ubiquitin-proteasome system in dopaminergic neurons caused by α-synuclein

Fig. 3

A53T α-synuclein over-expression in nigral dopaminergic neurons is associated with early-onset of accumulation of the UbG76V-GFP reporter. Adult wild-type rats received unilateral stereotaxic co-injection of AAV-UbG76V-GFP with AAV-A53T, AAV-Empty or AAV-TdTomato into the right SNpc. Animals were culled at 1 wpi (n = 12–14 per group). a Comparison of immunofluorescent staining with anti-tyrosine hydroxylase (TH; cyan), anti-GFP (GFP; green), anti-α-synuclein (SYN; red) antibodies or native TdTomato fluorescence (TdTom; red) in SNpc at 1 wpi. Scale bar 500 μm (inset 50 μm). b Quantification of UbG76V-GFP+/TH+ neurons per mm2 reveals accumulation of the UPS reporter in nigral dopaminergic neurons overexpressing A53T α-synuclein, relative to both empty vector and TdTomato controls at 1 wpi. c Quantification of TH+ neurons per mm2 reveals no difference in the density of nigral dopaminergic neurons 1 week after AAV injection into the ipsilateral SNpc. d Measurement of forelimb use by the cylinder test at 1 wpi shows no significant difference in forelimb asymmetry between groups. Data are percentage mean +/−SEM (one-way ANOVA; n = 7–8 per group). e Representative images from multiplex RNA in situ hybridisation experiment to compare UbG76V-GFP (red) and Actb (green) transcript levels in Th-labelled neurons (cyan) 1 week following stereotaxic administration of AAV-UbG76V-GFP/AAV-Empty, AAV-UbG76V-GFP/AAV-TdTomato or AAV-UbG76V-GFP/AAV-A53T into the right SNpc. Nuclei were counterstained with DAPI (blue). Scale bar 5 μm. f Quantification of UbG76V-GFP mRNA levels normalised to beta-actin transcript levels in Th-labelled neurons 1 week following AAV injection into ipsilateral SNpc reveals no significant difference in expression of the UbG76V-GFP reporter transgene between experimental groups. Data are percentage mean +/− SEM (p = 0.5336; one-way ANOVA; n = 4 per group)

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