Fig 1: SIRT2 translocates from the cytoplasm to the nucleus in PD.a, b Subcellular localization of SIRT2 was monitored using dual immunolabeling for TH (red) and SIRT2 (green) in the SNpc in mice treated with normal saline (NS) or MPTP (a) as well as in WT and α-synuclein-A30P*A53T transgenic mice (TG) (b). c Immunofluorescence assays for SIRT2 (green) were conducted in primary culture neurons treated with 50 μM MPP+ for 24 h. d The localization of SIRT2 was detected using nuclear/cytosolic immunoblotting in primary culture neurons treated with 50 μM MPP+ for 0, 6, 12, and 24 h. e Quantification of SIRT2 protein levels in the cytoplasm and nucleus (separate from d). f SH-SY5Y cells were transiently transfected with GFP vector (green) or GFP-α-synuclein-A30P*A53T (green) plasmids for 24 h and subsequently immunostained for SIRT2 (red) and observed using confocal microscopy. g Subcellular localization of SIRT2 examined using dual immunolabeling for TH (red) and SIRT2 (green) in the mouse SNpc 35 days after the striatal stereotactic injection of 2 μl (per side) normal saline (NS), or α-synuclein PFF (Abcam, ab246002, 1 μg/μl). h Localization of SIRT2 detected using nuclear/cytoplasmic immunofluorescence staining in primary culture neurons treated with 4 μg/ml PFF for 7 days. i Localization of SIRT2 detected using nuclear/cytosolic immunoblotting in primary culture neurons treated with 4 μg/ml PFF for 7 days. j Quantification of SIRT2 protein levels in the cytoplasm and nucleus (separate from [i]). The scale bar represents 20 μm. All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in (e). Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in (j). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: UBA52 interacts with α-Syn, and its over-expression in SH-SY5Y cells deter the alteration in PD markers protein abundance, preventing the disease onset. Co-IP (Protein A Sepharose beads attached to the anti-UBA52 antibody/anti-IgG antibody) was performed, and the cell or tissue lysates were incubated with the antibody-beads complex, following which the beads were boiled with 2X-reducing buffer, centrifuged and the supernatant was resolved on a 4–16% gradient SDS-PAGE to detect the interacting proteins. (a,b) Representative images and analysis depicting the in vitro interaction of UBA52 and α-syn, against IgG as a negative control in SH-SY5Y cells with or without rotenone& MG132 treatment; and control& rotenone treated PD-related SN and STR region, respectively; rats used per group in each replicate = 4; nexp = 4. (c) Representative image and analysis depicting the Co-IP experiment to check the interaction of UBA52 and α-Syn, against IgG as a negative control in PBS-administered control& mouse recombinant α-Syn PFF treated PD-related SN and STR region, respectively at 15-, 30- and 45-days post-injection (dpi) to show the underlying mechanism of UBA52 in regulating α-Syn abundance; rats used per group per replicate = 5; nexp = 3. (d) Representative image and analysis depicting the Co-IP experiment to check the interaction of UBA52 and α-Syn, against IgG as a negative control in the PD-related SN and STR region of the control C57BL mice and the C57BL/6J-Tg (Th-SNCA*A30P*A53T) 39Eric/J transgenic (Tg) mice, respectively; mice used per group per replicate = 5; nexp = 2. (e) PcDNA3.1 or Myc-UBA52 transfected SH-SY5Y cells with or without rotenone treatment were immunoblotted for PD-related protein markers; nexp = 3. Quantification is the mean and SEM of at least three independent experiments, and statistical analysis was performed using two-way ANOVA, followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 control vs. Rotenone/αSyn PFFs/Transgenic (Tg); # p < 0.05, ## p < 0.01.
Fig 3: Serpina3n KO ameliorated Mn-induced α-Syn accumulation in the mouse midbrain. A: Histopathological changes indicated by tyrosine hydroxylase staining in mouse midbrain. B-C: Semi-quantitative analysis of TH positive neurons (n = 4). D-F: The protein level of α-Syn and Serpina3n in mice midbrain and their semi-quantification (n = 6). G: The mRNA level of Snca in mice midbrain (n = 6). H: Immunofluorescent co-localization of GFAP with α-Syn and semi-quantification analysis in mice striatum. Scale bar: 50 μm. I: 8 non-overlapping areas of the midbrain of 4 mice (sex ratio 1:1) were assigned for semi-quantitative analysis. The values are presented as means ± SEM. N.A. means not available. * p < 0.05, ** p < 0.01
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