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. 2016 Jan 29;139(3):953–970. doi: 10.1093/brain/awv384

Figure 2.

Figure 2

ShCYP46A1 produces spontaneous motor deficits and striatal neuron dysfunctions in a wild-type context. ( A ) Two-month-old C57BL6J mice were stereotactically injected in the striatum with either AAV5-GFP-shCyp46A1 or AAV5-GFP-shScramble (control sequence that does not target Cyp46a1 mRNA). ( B ) All mice ( n = 8 for each group) were blindly submitted to behavioural experiments. The Rotarod test used to evaluate mouse balance and coordination revealed that the latency to fall was decreased in mice injected with shCYP46A1, compared to shScramble-injected mice. ( C ) Two months later, the weight of mice injected with shCYP461 showed a statistically significant reduction at the time of sacrifice (∼16%). ( D–G ) Mice were sacrificed 3 months later and for each mouse, the striatum from one hemisphere was processed for biochemical studies while the other was used for neuropathological studies. ( D ) 24S-OHC measurements were performed by mass spectrometry. A ∼50% reduction in the levels of 24S-OHC was shown in the striatum of shCYP46A1-injected mice, relative to shScramble-injected mice. ( E ) Cresyl violet staining from striata injected with shScramble ( top ) and shCYP46A1 ( bottom ). Note the increased condensed nuclei (degeneration) in the striatum injected with shCyp46A1 when compared to shScramble, where minimal condensed nuclei were observed around the needle tract region. ( F ) DARPP32 immunostaining shows a strong depletion in the striatum of shCYP46A1 injected mice ( lower panel), when compared to shScramble injected mice. ( G ) Quantification of DARPP32 depleted area ( n = 6 for each group). ( H ) Co-labelling of GFP (used as a tag for shRNA sequences) and NeuN immunostaining shows a strong co-localization (merge panel) in shScramble injected neurons. In contrast, no co-localization is observed between GFP and NeuN in animals injected with shCYP46A1, indicating neuronal dysfunction in the transduced area. ( B – G ): Data are represented as mean ± SEM; statistical analyses: Mann whitney’s test. * P < 0.01; ** P < 0.05; *** P < 0.001 shScramble versus shCYP46A1. WT = wild-type.