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. 2016 May 11;7:11445. doi: 10.1038/ncomms11445

Figure 4. SIRT1 overexpression reduces mutant ataxin-3 toxicity and neuronal dysfunction in a striatal lentiviral model of MJD.

Figure 4

(a) Schematic representation of the strategy used to create in vivo striatal lentiviral model of MJD and to overexpress SIRT1/H363Y. Five-week-old C57Bl/6J mice were bilaterally injected with lentiviral vectors encoding for mutant ataxin-3. In the left hemisphere were co-injected lentiviral vectors encoding for H363Y and in the right hemisphere for SIRT1. Four weeks after the surgery, mice were euthanized. (b) SIRT1 immunoreactivity in the striatum of animals of the striatal lentiviral model of MJD co-injected with H363Y or SIRT1 versus a control animal. DAPI, blue, nuclei. Scale bar, 500 μm. (c) Acetylated Forkhead box protein O1 (FOXO1) levels are significantly lower in the striata of lentiviral model of MJD when the active form of SIRT1 was overexpressed, in comparison with control side where the inactive form of SIRT1 was overexpressed. (d) With an anti-ataxin-3 antibody (Ab 1H9), total number of mutant ataxin-3 inclusions were counted and are quantified in e. Scale bar, 500 μm. (f) Western blot analysis demonstrated that SIRT1 overexpression significantly reduced several toxic forms of mutant ataxin-3, namely aggregates, soluble form and ∼34 and ∼26 kDa fragments. (g) Quantitative reverse transcriptase–PCR analysis of Atx3MUT mRNA. (h) DARPP-32 staining revealed a major loss of DARPP-32 immunoreactivity in the striatal hemisphere co-infected with mutant ataxin-3 and H363Y. Scale bar, 500 μm. This was quantified in i as depleted volume of DARPP-32 staining. (j) Cresyl violet staining indicating pyknotic nuclei in both hemispheres. A higher number of pyknotic nuclei were visible around the injection site area in the H363Y-transduced hemisphere. Scale bar, 500 μm (brightfield and left cresyl violet) and 50 μm (right cresyl violet). This was quantified in k. Data represent mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.001 compared with non-treated hemisphere. (c,e–g,i,k) Paired Student's t-test. n=4.