Fig 1: Loss of cardiac FAO inhibits mitophagy.a Representative images of heart tissue and (b) quantification of cardiac mitophagy in wild-type or CPT2H-KO mice expressing mt-Keima. The emission signal obtained after excitation with the 458-nm laser is shown in green (neutral), and that obtained after excitation with the 561 nm laser is shown in red (acidic). The fold change of mitophagy was quantified by comparing the red/green signal. Values are normalized to wild-type levels of mitophagy (n = 4). Scale bars, 20 μm. c Representative western blot and (d) quantification of mitophagy receptors, OPTN and p62, in heart tissue derived from wild-type or CPT2H-KO mice (n = 3). e Representative western blot and (f) quantification of PINK1 and PDKs (PDK1, PDK2, PDK3, PDK4) expression in heart tissue derived from wild-type or CPT2H-KO mice (n = 3). g Representative western blot and (h) quantification of PARL expression in heart tissue derived from wild-type or CPT2H-KO mice (n = 3). PARL knockout HeLa cell lysates are employed for antibody validation. i Representative western blot and (j) quantification of Cleaved (CL) PGAM5 expression in heart tissue derived from wild-type or CPT2H-KO mice (n = 3). PGAM5 knockout HeLa cell lysates are employed for antibody validation. The loss of Mitochondrial Membrane Potential following FCCP treatment induces the cleavage of full-length PGAM5 (PGAM5-FL) into its cleaved form (PGAM5-CL) in HeLa cells. Data represent mean ± s.d. The P-values were calculated using an unpaired two-tailed Student’s t test (b, d, f, h, j). Source data are provided as a Source Data file.
Fig 2: PARL was regulated by Nur77 to alleviate apoptosis in MPP+-lesioned SH-SY5Y cells.A Western blot analysis of extracts Nur77, PARL, BCL-2, and BAX. Overexpression of Nur77 increased PARL and reduced apoptosis of SH-SY5Y cells. Data are representative of three independent experiments, which gave similar results. B SH-SY5Y cells were co-transfected with Nur77 plasmid for 24 h and treated with (or without) MPP+ for an additional 24 h. Cell lysates were extracted for immunoblot analysis of Nur77, PARL, BCL-2, and BAX. Representative WB is shown. C Nur77 was overexpressed in SH-SY5Y cells with low PARL expression, and the expressions of BCL-2, BAX, Caspase3 and Cleaved-Caspase3 were detected by western blotting. D, E Fluorescence MitoTracker Green staining in Control and MPP+-treated SH-SY5Y cells; mitochondria (GREEN) and Nur77 (RED). Bar = 5 μm. F, G Fluorescence MitoTracker Green staining in Nur77-Flag and MPP+-treated Nur77-Flag cells; mitochondria (GREEN) and Nur77-Flag (RED). Bar = 5 μm. (ns not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the control group; n = 3, mean ± SEM).
Fig 3: PARL deficiency exacerbates MPTP-induced motor and cognitive deficits in PD mice models.A, D, E Open-field behavior test analysis: Total Track route, Distance and Total Central square time ratio. B, F Elevated Plus Maze: Test the Total Track route and Mean latency to enter the open arms of the elevated plus maze in a 5-min test period. C, G Morris water maze test: representative swimming tracks of mice (time to find the platform) in Morris water maze test. The number of entries in the platform was significantly increased in shPARL mice compared to Sham mice on the last day. H At the same time, the escape latency was significantly decreased in shPARL mice compared to Sham mice. I Latency to fall of various groups assessed by the rotarod test. (ns not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA followed by Turkey’s test for multiple comparisons, n = 6 per group. Bars represent mean ± SD).
Fig 4: Nur77 promotes PARL-BCL-2 interaction to attenuate apoptosis in MPP+-treated SH-SY5Y cells.A Predicted Nur77-PARL binding interface by AlphaFold2. Nur77 (purple) interacts with PARL (blue) via key residues: salt bridge (Nur77-GLU525/PARL-ARG41), hydrogen bonds (Nur77-GLN519/PARL-ARG42; Nur77-ARG318/PARL-ARG9). B Co-immunoprecipitation (co-IP) validation of Nur77-PARL interaction in MPP+ (1 mM, 24 h)-treated SH-SY5Y cells. Lysates immunoprecipitated with anti-Nur77 antibody. IgG: isotype control. C Schematic of PARL truncations (GST-tagged fragments) used for binding assays. Numbers denote amino acid positions. D GST pull-down assay showing Nur77 binds preferentially to PARL-GST1 (1-167 aa). Input: 100% lysate; GST control: empty vector. E, F Western blot analysis of BCL-2 levels after PARL peptide inhibition. β-actin: loading control. G AlphaFold2-predicted PARL-BCL-2 complex. PARL (yellow) binds BCL-2 (blue) via hydrogen bonds (PARL-TRP158/BCL-2-GLU13; PARL-ARG76/BCL-2-SER50) and a π-π interaction (PARL-TRP11/BCL-2-HIS20). H Co-IP confirms PARL-BCL-2 interaction in MPP+-treated cells. Lysates immunoprecipitated with anti-PARL antibody. I, J GST pull-down assays demonstrate Nur77 knockdown disrupts PARL-BCL-2 binding. PARL-GST2 (1-167 aa) shows strongest BCL-2 affinity.
Fig 5: Effect of PARL knockdown and overexpress on apoptosis in PD cellular.A, B PARL knockdown (via shRNA) significantly increased TUNEL+ cells (red fluorescence), demonstrating enhanced DNA fragmentation and apoptosis in SH-SY5Y cells. Quantitative analysis B confirms a significantly increase in apoptotic cells compared to controls (p < 0.001). C, D Overexpression of PARL-Myc reduced TUNEL+ cell counts versus knockdown groups (p < 0.01), supporting PARL’s role in suppressing caspase-dependent apoptotic pathways. (Bar = 20 μM). E, F PARL knockdown exacerbated mitochondrial vacuolar swelling (red arrows) and nuclear chromatin aggregation (orange arrows). PARL overexpression partially restored cristae structure (black arrows) and reduced chromatin condensation. Quantification F shows 54% reduction in damaged mitochondria (p < 0.05). Analyzed by two-tailed Student’s t test or one-way ANOVA with Tukey’s post hoc test (ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the control group; n = 3, mean ± SEM Bar = 20 μM).
Supplier Page from Abcam for Anti-PARL antibody