The PP2A-B55α phosphatase is a master regulator of mitochondrial degradation and biogenesis

Abstract

Mitochondrial homeostasis relies on a tight balance between mitochondrial biogenesis and degradation. Although mitophagy is one of the main pathways involved in the clearance of damaged or old mitochondria, its coordination with mitochondrial biogenesis is poorly characterized. Here, by unbiased approaches including last-generation liquid chromatography coupled to mass spectrometry and transcriptomics, we identify the protein phosphatase PP2A-B55α/PPP2R2A as a Parkin-dependent regulator of mitochondrial number. Upon mitochondrial damage, PP2A-B55α determines the amplitude of mitophagy induction and execution by regulating both early and late mitophagy events. A few minutes after the insult, ULK1 is released from the inhibitory regulation of PP2A-B55α, whereas 2 to 4 hours later, PP2A-B55α promotes the nuclear translocation of TFEB, the master regulator of autophagy and lysosome genes, to support mitophagy execution. Moreover, PP2A-B55α controls a transcriptional program of mitochondrial biogenesis by stabilizing the Parkin substrate and PGC-1α inhibitor PARIS. PP2A-B55α targeting rescues neurodegenerative phenotypes in a fly model of Parkinson's disease, thus suggesting potential therapeutic application.

Fig. 1.. PP2A-B55α dephosphorylates ULK1 at S556.

(A) Phosphorylation events on ULK1, AMPK, and ATG13. (B and C) Protein extracts of HeLa cells treated with Ok. Ac. for 5 to 15 min (B) or depleted for PP2Ac (C) and immunoblotted for the indicated proteins. MW, molecular weight. (D) Schematic of the in vitro phosphatase (PP) assay. IP, immunoprecipitation. (E) In vitro phosphatase reactions obtained as in (D), negative control (–Purif. PP2Ac), and total protein extract of hemagglutinin (HA)–ULK1 immunoprecipitation were immunoblotted for the indicated proteins. Tot. extr., total extract. (F) Workflow of chromatography techniques coupled with liquid chromatography–mass spectrometry (LC-MS) proteomics to identify the protein phosphatase targeting P-ULK1 S556. Fractions from ULK1/2 double knockout (DKO) murine embryonic fibroblasts (MEFs) cell extracts, separated through chromatography, were tested for their phosphatase activity toward P-ULK1 S556 in an in vitro phosphatase assay. Active fractions were pooled and repurified through chromatography to further separate phosphatases of interest. Fractions from last chromatography column applied [1 to 24 in (G)] were analyzed by LC-MS proteomics. A₂₈₀, absorbance at 280 nm; m/z, mass/charge ratio; WB, Western blot. (G) In vitro phosphatase assays with fractions from the last chromatography column were immunoblotted for P-ULK1 S556. Active fractions (13 to 18) in red. (H) Cell extracts from oligomycin and antimycin A (OA)–treated Parkin-induced HeLa cells depleted for B55α were immunoblotted for the indicated proteins. (I to K) Quantification of the phosphorylated protein immunoblots represented in (H). a.u., arbitrary units. (L) Cell extracts from OA-treated HeLa cells stably expressing mCherry-Parkin, depleted for B55α, and induced for Yellow Fluorescent Protein (YFP)–B55α or not were immunoblotted for the indicated proteins. (M) Quantification of the phosphorylated protein immunoblots represented in (L). (N) PP2A-B55α–mediated regulation of P-ULK1 S556. Red and green arrows indicate inhibition and activation, respectively. ACTIN: Loading control for all immunoblots. Columns represent the mean ± SD relative to the corresponding untreated [−; in (I) to (K)] or control [siCTR; in (M)] sample. Two-way [(I) to (K)] or one-way (M) analysis of variance (ANOVA), and Tukey’s multiple comparison test. Significant P values are reported in the figure.

Fig. 2.. PP2A-B55α inhibits mitochondria priming for recognition by the autophagy machinery.

Cell extracts from Parkin-induced HeLa cells depleted for B55α or transfected with a nontargeting control oligo (siCTR) and treated with OA for the indicated time points were immunoblotted for the indicated proteins. ACTIN: Loading control. (B) Quantification of P-Ubiq. (S65) represented in (A). (C) Cell extracts from OA-treated HeLa cells stably expressing mCherry-Parkin, depleted for B55α or transfected with a nontargeting control oligo (siCTR), and induced for YFP-B55α or not were immunoblotted for the indicated proteins. ACTIN: Loading control. (D) Quantification of P-Ubiq. (S65) represented in (C). (E) Cytosol- and mitochondria-enriched fractions from cells as in (A), treated with OA for 100 min, and immunoblotted for the indicated proteins. ACTIN and cytochrome C oxidase subunit II (COXII): Loading controls. (F) Quantification of P-Ubiq. (S65) represented in (E). Mitoc., mitochondrial. (G) Mitochondria-enriched fractions from cells as in (A), treated with OA for 100 min, and immunoblotted for the indicated proteins. COXII: Loading control. (H) Quantification of OPTN, NDP52, and p62 represented in (G). All columns in the figure represent the mean ± SD of each time point relative to the corresponding control sample (siCTR). P values were calculated by one-way ANOVA, followed by Dunnett’s multiple comparison test. Significant P values are reported in the figure.

Fig. 3.. PP2A-B55α, which limits early mitophagy signaling, is required for the subsequent Parkin-dependent mitochondrial clearance.

(A) Cell extracts from Parkin-induced HeLa cells depleted for B55α and treated with OA were immunoblotted for the indicated proteins. h, hours. (B) Quantification of COXII represented in (A). (C)mt-mKeima flux analyzed by flow cytometry in OA-treated Parkin-induced HeLa cells depleted for B55α. Cell extracts from untreated cells were immunoblotted for the indicated proteins. Columns represent the mean ± SD. ACT., ACTIN. (D) Mitophagy reporter mCherry-GFP-FIS labeling cytosolic and lysosomal mitochondria in yellow and red, respectively. eGFP, enhanced GFP. (E) Cell extracts from untreated cells from (F) immunoblotted for the indicated proteins. (F) mCherry-GFP-FIS flux analyzed by immunoblot in OA-treated Parkin-induced HeLa cells depleted for B55α. Cell extracts were immunoblotted for the indicated proteins. mChe., mCherry; expos., exposure. (G) Quantification of mCherry-FIS1/mCherry-GFP-FIS1 ratio represented in (F). (H) Cells as in (A) were treated with OA for 60 min and immunoblotted for the indicated proteins. (I) Quantification of LC3B lipidation (LC3BII) represented in (H). (J) Cells as in (A) were treated with OA alone or in combination with Bafilomycin A (BafA1) for evaluation of autophagy flux by immunoblotting of LC3B. (K) Quantification of LC3BII represented in (J). (L) Quantification of the number of LC3B dots (in green) per cell, as seen in (M). Cells as in (A) were treated with OA for 60 min and stained for LC3B, Parkin, and Hoechst 33342 (n = 3 independent experiments, with 100 counted cells per experiment). ACTIN: Loading control for all immunoblots. All columns in the figure but in (C) represent the mean ± SD relative to the corresponding control sample. P values were calculated by two-way ANOVA, followed by Tukey’s multiple comparison test in (B); one-way ANOVA, followed by Dunnett’s multiple comparison test in (C), (I), and (K); and unpaired Student’s t test (two-tailed) in (G) and (L). Significant P values are reported in the figure.

Fig. 4.. PP2A-B55α activates TFEB upon mitochondrial damage to support Parkin-dependent mitophagy.

(A) Cell extracts from Parkin-induced HeLa cells depleted for B55α or transfected with a non-targeting control oligo (siCTR) were incubated with the +λ-PP or the vehicle at 30°C for 30 min. Samples were immunoblotted for the indicated antibodies. ACTIN: Loading control. (B) Box plot of single-cell analysis for nuclear-to-cytoplasm ratio of cells as in (A), treated with OA for 4 hours or Torin1 for 1 hour. n = 500 from four biologically independent replicates. Data in box plot are presented as first quartile, median, and third quartile. (C) Protein extracts from nuclear and cytosolic fractions of cells as in (A) treated with OA for 4 hours were immunoblotted for the indicated antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone 3 (H3) were used as cytosolic and nuclear markers, respectively, and as loading controls. (D) Quantification of nuclear TFEB represented in (C). Columns represent the mean ± SD relative to the corresponding control sample. P values were calculated by one-way ANOVA, followed by Tukey’s multiple comparison test. Significant P values are reported in the figure. (E) All significant Cellular Compartments [(CC), left plot] in which the 2276 genes (of 4417) are negatively regulated are mainly enriched. Center: The Venn diagram shows that 1207 of 2276 genes are CLEAR-containing genes. Right plot: CC and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the 1207 CLEAR-containing genes. The enrichment score for each CC cluster is plotted, while for the KEGG pathways, the gene counts are plotted. Cytopl., cytoplasmic; memb.-bound, membrane-bound; Extracell., extracellular; prot., protein; ER, endoplasmic reticulum.

Fig. 5.. PP2A-B55α–mediated regulation of mitophagy depends on TFEB.

(A) Cell extracts from Parkin-induced HeLa cells depleted for B55α or TFEB or transfected with a non-targeting control oligo (siCTR), treated with OA, alone or combined with BafA1 for 60 min, were immunoblotted for the indicated proteins. ACTIN: Loading control. (B and C) Protein extracts from knocked-down untreated cells from (A) where immunoblotted for the indicated antibodies. ACTIN: Loading control. (D) Quantification of LC3BII represented in (A). Columns represent the mean ± SD relative to untreated control sample (siCTR). (E) Cell extracts from Parkin-induced HeLa cells depleted for B55α or transfected with a nontargeting control oligo (siCTR), transiently expressing Flag-TFEB (S142/211A), and treated with BafA1 for 60 min were immunoblotted for the indicated antibodies. ACTIN: Loading control. (F) Cell extracts from untreated cells as in (E) were immunoblotted for the indicated antibodies. ACTIN: Loading control. (G) Quantification of LC3BII represented in (E). Columns represent the mean ± SD relative to untreated control sample (siCTR). (H) Flow cytometry analysis of mt-mKeima flux in Parkin-induced HeLa cells stably expressing mt-mKeima, depleted for B55α or transfected with a nontargeting control oligo (siCTR), transiently transfected with Flag-TFEB (S142/211A), and treated with OA for 4 hours. Columns represent the mean ± SD. All P values in the figure were calculated by one-way ANOVA, followed by Dunnett’s multiple comparison test. Significant P values are reported in the figure.

Fig. 6.. PP2A-B55α inhibits mitochondrial biogenesis.

(A) Parkin-induced HeLa cells depleted for B55α were stained for mtDNA and Hoechst 33342. (B) Quantification of mtDNA/cell area ratio for cells as seen in (A). (C) Reverse transcription qPCR (RT-qPCR) analysis of mtDNA copy number for cells as in (A). (D) RT-qPCR analysis of mtDNA copy number for HeLa cells stably expressing mCherry-Parkin, depleted for B55α, and induced for YFP-B55α or not. (E) All significant CC in which the 2141 genes (of 4417) are positively regulated are mainly enriched. Transf. compl., transfer complex; acetyltransf., acetyltransferase; methyltransf., methyltransfase. (F) RT-qPCR analyses of PGC-1α and TFAM mRNA in cells as in (A). (G) Protein extracts from cells as in (A) were immunoblotted for the indicated antibodies. (H) Quantification of TFAM represented in (G). (I) RT-qPCR analyses of PGC-1α and TFAM mRNA in SH-SY5Y cells depleted for B55α. (J) Protein extracts from cells as in (I) were immunoblotted for the indicated proteins. (K) Quantification of TFAM represented in (J). (L) Cell extracts from cells as in (D) were immunoblotted for the indicated proteins. (M) Quantification of TFAM represented in (L). (N) Protein extracts from cells as in (I) were immunoblotted for the indicated proteins. (O) Quantification of mitochondrial proteins represented in (N). ACTIN: Loading control for all immunoblots. Columns represent the mean ± SD [in (B), (H), (K), (M), and (O)] or the mean ± SEM [in (C), (D), (F), and (I)]. All P values were calculated by one-way ANOVA, followed by Dunnett’s multiple comparison test. Significant P values are reported in the figure.

Fig. 7.. PP2A-B55α regulates the Parkin substrate and PGC-1α inhibitor PARIS.

(A) Protein extracts from SH-SY5Y cells depleted for B55α were immunoblotted for the indicated antibodies. (B) Quantification of PARIS represented in (A). (C) Protein extracts from Parkin-induced HeLa cells depleted for B55α were immunoblotted for the indicated antibodies. (D) Quantification of PARIS represented in (C). (E) Protein extracts from HeLa cells depleted for B55α, and induced or not for Parkin expression (+/−Dox: doxycycline), were immunoblotted for the indicated proteins. (F) Quantification of PARIS represented in (E). (G and H) Protein extracts from cells as in (A), treated with cycloheximide (CHX) for the indicated time points, were immunoblotted for the indicated proteins. In (G), quantification of PARIS represented in (H). (I) Cell extracts from SH-SY5Y cells depleted for B55α, and treated with RA for 72 hours, were immunoblotted for the indicated antibodies. (J) Quantification of PARIS represented in (I). (K) Quantification of TFAM represented in (I). ACTIN: Loading control for all immunoblots. Columns represent the mean ± SD. P values were calculated by one-way ANOVA in (B) and (D) or two-way ANOVA in (G), followed by Tukey’s multiple comparison test, or unpaired Student’s t test (two-tailed) in (F), (J), and (K). Significant P values are reported in the figure.

Fig. 8.. PP2A-B55α rescues neurodegenerative phenotypes in flies in a Parkin-dependent manner.

(A and B) Ultrastructural analysis of the indirect flight muscles and mitochondria from fly thoraces of the indicated genotypes. (C) Graph bar shows mean ± SEM of the percentage of climbing flies of the indicated genotype from at least three independent experiments. P values were calculated by one-way ANOVA, followed by Tukey’s multiple comparison test. Significant P values are reported in the figure; n = 3. (D) Graph bar shows mean ± SEM of the percentage of climbing flies of the indicated genotype from at least three independent experiments. P values were calculated by one-way ANOVA, followed by Tukey’s multiple comparison test. Significant P values are reported in the figure; n = 3 to 4. m, mitochondria; dm, damaged mitochondria. Complete genotypes: Actin-GAL4/+ (WT); Actin-GAL4/+; park25/park25 (Parkin KO); Actin-GAL4/UAS-Tws-RNAi; park25/park25 (Park. KO::Tws KD); Pink1B9/Y; Actin-GAL4/+ (Pink1 KO); Pink1B9/Y; Actin-GAL4/UAS-Tws-RNAi (Pink KO::Tws KD); and Actin-GAL4/UAS-Tws-RNAi (Tws KD).