Fig. 3: Overexpression of PIAS2 alone in neurons is sufficient to cause neurodegeneration, motor and cognitive impairments, and PD-like dementia in mice.

a Schematic of the experimental design. WT, Ifnb^–/–, and Ifnar1^–/– mice injected with AAV6 PIAS2-mCherry or AAV6 CTR (mCherry) into the substantia nigra and cortex. Tests were performed at 15 and 30 days post injection. b Barnes maze test. Data in seconds ± SEM. ^€€€P < 0.001 by two-way ANOVA; *P < 0.05, **P < 0.01 by Student’s t-test. c Rotarod motor coordination test. Data in seconds ± SEM; ^ΨΨΨP < 0.001 by two-way ANOVA between groups and *P < 0.05, **P < 0.01, ***P < 0.001 by post hoc Dunnett’s multiple comparisons test. n = 6 /WT-mCherry CTR, 7/WT-PIAS2-mCherry, and n = 4/each mCherry CTR- and PIAS2-mCherry-injected in Ifnb^–/– and Ifnar1^–/– mice; total n = 29. d NeuN IHC of the nigrostriatal region of brain of WT mice injected with AAV6 PIAS2-mCherry or AAV6 CTR mCherry and respective quantification. Data presented as number of positive cells ± SEM. ***P < 0.001 by Student’s t-test. n = 9/CTR mCherry and 12/PIAS2-mCherry. e IF for tyrosine hydroxylase (green) in SN in WT mouse brain injected with AAV6 PIAS2 or AAV6 CTR (mCherry) and respective quantification. Neurons stained with Nissl. Data on graph represent the number of TH⁺ neurons ± SEM, *P < 0.05 by Student’s t-test. n = 7/CTR and 6/PIAS2. f Schematic of the experimental design in a familial model of PD. WT mice unilaterally co-injected into the nigrostriatal region with AAV6 empty control and mCherry control on one side and human α-syn (hSNCA) together with either PIAS2-mCherry or AAV6 mutPIAS2-mCherry on the other side. g–i Motor coordination by climbing test at 16 days post injection: g latency to climb, h total rearing time, and i average rearing time. Data are in seconds ± SEM, *P < 0.05, **P < 0.01 by unpaired Student’s t-test. j Motor coordination by cylinder test at 30 days post AAV6 hSNCA/AAV6 PIAS2-mCherry or AAV6 hSNCA-mutPIAS2-mCherry injections showing left forepaw usage. Data in seconds ± SEM; n = 6/group. *P < 0.05, **P < 0.01 by unpaired Student’s t-test. k Motor performance by gait test at 30 days post AAV6 hSNCA/AAV6 PIAS2-mCherry or AAV6 hSNCA/AAV6 mutPIAS2 injections; left paw locomotion analysis by footprint. Data in cm ± SEM, *P < 0.05, **P < 0.01 by unpaired Student’s t-test. n = 6/group. l Quantification of m, n IF staining of TH⁺ (green) in the substantia nigra. Nuclei were stained with DAPI (blue); data in graph represent the number of TH-positive neurons ± SEM. ANOVA *P < 0.05, and **P < 0^.01 by post hoc correction for multiple tests. n = 3. o, p IHC of phosphorylated (p)α-syn in brain of mice injected with AAV6 hSNCA/AAV6 PIAS2-mCherry or AAV6 hSNCA/mutPIAS2-mCherry, o STR, and p SN and q quantification. Scale bar is 1.25 mm. Data are mean ± SEM; n = 3. ^€€P < 0.01 by ANOVA and **P < 0.01 by post hoc correction with Dunnett’s multiple correction test. r, s IF of pα-syn (red) and β−III-tubulin (green) in SN (r) and (s) with TH staining in SN. Nuclei were stained with DAPI; t quantification. Data in graph represent the number of pα-syn-positive neurons ± SEM of n = 3; ^€€€P < 0.001 by ANOVA and **P < 0.01 and ***P < 0.001 by post hoc correction for multiple tests, applied to log-normal distribution. u IHC of hSNCA in SN and STR of mice injected with AAV6 hSNCA/AAV6 PIAS2-mCherry or AAV6 hSNCA/mutPIAS2-mCherry. Scale bars, 1 mm. v Quantification of IHC of hSNCA in SN (left) and STR (right). Bars show normalized mean percentage of control ± SEM for n = 4; *P < 0.05, **P < 0.01 by unpaired Student’s t-test.