Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: Meth exposure causes mitophagy and necroptosis in primary neurons. (A) Volcano plots of differential gene expression analysis. (B) GO pathway enrichment of differential gene expression analysis. (C) KEGG pathway enrichment of differential gene expression analysis. (D) Gene set enrichment analysis (GSEA) of mitophagy‐related and mitochondrial fission‐related RNA‐seq data. (E) PARL, PGAM5, PINK1, Parkin, MLKL, RIP1, and RIP3 mRNA levels in neurons. (F) KEGG pathway enrichment of differential protein expression analysis (Top 10). (G) Volca no plot of differential protein expression analysis. (H) PARL, PGAM5, PINK1, Parkin, MLKL, RIP1, and RIP3 protein levels. (I) Statistical results of the relative protein levels of PARL, PGAM5, PINK1, Parkin, MLKL, RIP1, and RIP3. Data are shown as the mean ± SEM. Student's t‐test was used to measure the significance between two groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus Con group.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Expressing, RNA Sequencing Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PARL overexpression reversed the Meth‐induced neuronal necroptosis. (A) The Calcein‐AM/PI double staining in control and PARL overexpression neurons after Meth treatment. Scale bar = 50 μm. (B) Quantitative analysis of the proportions of dead cells in neurons. (C) LDH levels in different groups. (D) The level of TNF‐α in the cell culture supernatants detected by ELISA. (E) MLKL, RIP3, RIP1, Drp1, and Fis1 protein expression. (F) Statistical results of the relative levels of MLKL, RIP3, RIP1, Drp1, and Fis1 proteins. (G) Tom20 (green) and p‐MLKL (red) co‐staining. Scale bar = 5 μm. (H) Quantitative analysis of colocalization of p‐MLKL and Tom20. Data are shown as the mean ± SEM. One‐way ANOVA was used to determine the statistical significance between multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus Ad group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 for the PARL + Meth group versus Ad + Meth group.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Over Expression, Double Staining, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PARL regulates PINK1 and PGAM5 cleavage after Meth exposure. (A) PINK1 (green) and PARL (red) costaining. Scale bar = 5 μm. (B) PGAM5 (green) and PARL (red) costaining. Scale bar = 5 μm. (C) PINK1 (green) and Tom20 (red) costaining. Scale bar = 5 μm. (D) PGAM5 (green) and Tom20 (red) costaining. Scale bar = 5 μm. (E) Detection of PGAM5 and PINK1 protein expression by Western blot. (F) Statistical results of the relative expression levels of PGAM5 and PINK1 proteins. (G) PGAM5 (green) and p‐MLKL (red) costaining. Scale bar = 5 μm. (H) Quantitative analysis of colocalization of PGAM5 and p‐MLKL. * p < 0.05, ** p < 0.01, and **** p < 0.0001 versus Ad group; ## p < 0.01 for the PARL + Meth group versus Ad + Meth group.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: Knockdown of PGAM5 alleviated neuronal necroptosis in primary neurons. (A) The Calcein‐AM/PI double staining in control and PGAM5 downregulation neurons after Meth treatment. Scale bar = 50 μm. (B) Quantitative analysis of the proportions of dead cells in neurons. (C) The LDH release level was measured in different groups. (D) The expression level of TNF‐α in the cell culture supernatants was analyzed by ELISA. (E) Detection of MLKL, RIP3, RIP1, Drp1, and Fis1 protein expression by Western blot. (F) Statistical results of the relative expression levels of MLKL, RIP3, RIP1, Drp1, and Fis1 proteins. (G) Tom20 (green) and p‐MLKL (red) costaining. Scale bar = 5 μm. (H) Quantitative analysis of colocalization of p‐MLKL and Tom20. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus ShNC group; # p < 0.05, ## p < 0.01, and #### p < 0.0001 for the ShPGAM5 + Meth group versus ShNC + Meth group.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Knockdown, Double Staining, Control, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PARL overexpression ameliorated neuronal loss after Meth exposure. (A) PARL overexpression significantly increased the number of Nissl‐positive cells in the hippocampus after Meth exposure. (B) TEM images showing mitochondrial morphology and autophagosome containing unhealthy mitochondria in the hippocampus. (C) Detection of Drp1, Fis1, MLKL, RIP3, RIP1, PINK1, PGAM5, Parkin, and LC3 protein expression by Western blot. (D) Statistical results of the relative expression levels of Drp1, Fis1, MLKL, RIP3, RIP1, PINK1, PGAM5, Parkin, and LC3 proteins. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus AAV group; # p < 0.05, ## p < 0.01, and ### p < 0.001 for the PARL + Meth group versus AAV + Meth group.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Over Expression, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PGAM5 knockdown ameliorated neuronal loss after Meth exposure. (A) PGAM5 downregulation significantly increased the number of Nissl‐positive cells in the hippocampus after Meth exposure. (B) TEM images showing mitochondrial morphology and autophagosome containing unhealthy mitochondria in the hippocampus. (C) Detection of MLKL, RIP3, RIP1, Drp1, and Fis1 protein expression by Western blot. (D) Statistical results of the relative expression levels of MLKL, RIP3, RIP1, Drp1, and Fis1 proteins. * p < 0.05 and ** p < 0.01, versus AAV group; # p < 0.05 and ## p < 0.01 for the ShPGAM5 + Meth group versus AAV + Meth group.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Knockdown, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: Schematic diagram of the effects of Meth on mitophagy and necroptosis in neurons. Low‐expression PARL induced by Meth regulated the differential cleavage of PINK1 and PGAM5, leading to the accumulation of PGAM5, which in turn recruited p‐MLKL on the mitochondrial membrane, forming “pores” that disrupted mitochondrial homeostasis, aggravating ROS production and TNF‐α release, ultimately triggering mitophagy and neuronal necroptosis. However, this effect could be substantially mitigated by PARL overexpression or PGAM5 downregulation, thereby preventing Meth‐mediated neuronal injury.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Expressing, Membrane, Over Expression