Y137F-PARIS-mediated suppression c-Abl-PARIS pathway rescues MDM4 repression, blocks p53 activation and prevents development of motor deficits and dopamine neuron loss in parkin knockout mice. (A) Representative exploratory paths from an open field test of 6-month-old wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) nigrally injected with AAV-GFPCre (3 months) ± AAV-PARIS-Y137F (3 months, phospho-deficient mutant PARIS). (B) Anxiety assessment of each experimental mouse group examining the percentage of exploration time in the border versus the sum of the centre and periphery zones (n = 8 mice per group). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of the vertical pole (n = 8 mice per group). (D) Representative TH immunohistochemical staining of substantia nigra from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) with intranigral injection of AAV-GFPCre ± AAV-PARIS-Y137F. Scale bar = 500 µm. (E) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) of the indicated mouse groups (n = 4 mice per group). (F) Representative TUNEL assay images of ventral midbrain from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) that experienced stereotaxic nigral injection of AAV-GFPCre ± AAV-PARIS-Y137F. The coronal brain sections were counterstained with DAPI. Merged images are shown in the bottom panel. (G) Quantification of the percentage of TUNEL-labelled cells in AAV-GFPCre-injected ventral midbrain regions from wild-type littermate and parkinfl/fl mice ± AAV-PARIS-Y137F (n = 16 sections from four mice per group). (H) Representative immunoblots examining the expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53, and parkin in the AAV-GFPCre ± AAV-PARIS-Y137F-injected ventral midbrain regions from wild-type littermate and parkinfl/fl mice using the indicated antibodies. β-Actin serves as an internal loading control. (I) Quantification of the relative expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53 and parkin proteins normalized to β-actin in the indicated experimental groups (n = 4 mice per group). Data are expressed as mean ± SEM. Statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. ***P < 0.001. WT = wild-type.