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The PP2A-B55-alpha 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-B55a/PPP2R2A as a Parkin-dependent regulator of mitochondrial number. Upon mitochondrial damage, PP2A-B55a determines the amplitude of mitophagy induction and execution, by regulating both early and late mitophagy events. Few minutes after the insult, ULK1 is released from the inhibitory regulation of PP2A-B55a, whereas 2- 4 hours after mitochondrial damage PP2A-B55a promotes the nuclear translocation of TFEB, the master regulator of autophagy and lysosome genes, to support mitophagy execution. Moreover, PP2A-B55a controls a transcriptional program of mitochondrial biogenesis by regulating the stability of the Parkin substrate and PGC1-a inhibitor PARIS. Importantly, PP2A-B55a targeting rescues neurodegenerative phenotypes in a fly model of Parkinson’s disease, thus representing a potential therapeutic target in this disease.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Kidney Epithelial Cell

DISEASE(S): Disease Free

SUBMITTER: Sergio Lilla

LAB HEAD: Sergio Lilla

PROVIDER: PXD065566 | Pride | 2025-09-24

REPOSITORIES: Pride

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			Action	DRS
	20160512_Elite_DaSu_VAC_EXT_100316_A01.raw	Raw
	20160512_Elite_DaSu_VAC_EXT_100316_A02.raw	Raw
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	20160512_Elite_DaSu_VAC_EXT_100316_A04.raw	Raw
	20160512_Elite_DaSu_VAC_EXT_100316_A05.raw	Raw

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Project description:Mutations in the E3 ubiquitin ligase Parkin cause autosomal recessive Parkinson’s disease. In concert with PINK1, Parkin regulates the clearance of dysfunctional mitochondria via lysosomes. In response, new mitochondria are generated through an interplay of nuclear- and mitochondrial-encoded proteins. Mouse and overexpression models suggests that Parkin also influences these processes, both in the nuclear cascade and at the level of the mitochondrial genome. Additionally, Parkin has been shown to prevent mitochondrial membrane permeation, impeding the escape of mitochondrial DNA (mtDNA). In line with this, serum from Parkin mutation carriers showed higher levels of circulating cell-free mtDNA (ccf-mtDNA) and inflammatory cytokines – a result of the innate immune response - which can be triggered by cytosolic mtDNA. However, Parkin’s relationship with the mitochondrial genome and the mitogenesis pathway has not been explored in patient-derived neurons. To investigate this aspect of Parkin’s cellular function endogenously, we generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from Parkin mutation carriers and healthy controls. In Parkin-deficient cells, several factors in the mitochondrial biogenesis pathway were significantly reduced, resulting in impaired mtDNA homeostasis - a phenomenon that was exacerbated in dopaminergic neurons. Moreover, in response to a lack of freely accessible NAD+, the energy sensor Sirtuin 1, which simultaneously controls mtDNA maintenance processes and mitophagy, was downregulated in Parkin-deficient neurons. However, while impaired lysosomal degradation of mitochondria was only detectable in oxidative conditions, biogenesis defects were already apparent in untreated patient neurons. This may suggest that mitophagy disruption occurs in response to acute stress. By contrast, the biogenesis pathway may be continually impaired in Parkin-associated Parkinson’s disease. Next, using a mutagenic stress model in combination with Parkin knockdown, we detected an increase in ccf-mtDNA and the cytosolic DNA sensor cGAS. To explore if ccf-mtDNA can act as damage-associated molecular pattern in the brain, we used postmortem tissue from a Parkin mutation carrier and performed single-cell RNA sequencing. In the midbrain lacking Parkin, we found a higher percentage of microglia along with an upregulation of proinflammatory cytokines in these cells. Together, our findings suggest a role for Parkin in the control of mitochondrial biogenesis and mtDNA maintenance, which protects midbrain neurons from neuroinflammation-induced degeneration. Future research in iPSC-derived neuron-microglia co-culture systems could aim at developing PD treatment approaches that target the neuronal release or microglial uptake of ccf-mtDNA.

Project description:This study aims to investigate the role and mechanism of DEK in asthmatic airway inflammation and in regulating PTEN-induced putative kinase 1 (PINK1)-Parkin mediated mitophagy, NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasome activation, and apoptosis. We found that recombinant DEK protein (rmDEK) promoted eosinophils recruitment, mitochondrial fragmentation, and outer membrane 20 (TOM20) and LC3 co-localization representing mitophagosomes in bronchoalveolar lavage fluid (BALF) in house dust mite (HDM) induced-asthma. rmDEK also reduced co-localization of mitochondrial fusion protein mitofusin1 (MFN1) and mitochondria, and the protein level of manganese superoxide dismutase (MnSOD), enhanced microtubule-associated protein1 light chain 3 (LC3) and voltage-dependent anion channels (VDAC) co-localization which also represent the mitophagosomes in airway epithelial cells, furthermore, increased dynamin-related protein 1 (DRP1) expression, PINK1-Parkin-mediated mitophagy, NLRP3 inflammasome activation, and apoptosis. In the DEK knockout mice, HDM induced asthmatic airway inflammation, MnSOD, PINK1-Parkin protein level, Parkin mediated mitophagy characterized by LC3 and Parkin co-localization in the airways, ROS generation, NLRP3 inflammation and apoptosis were fully reversed. Similar effects of rmDEK were also observed in the BEAS-2B cells, which were rescued by the autophagy inhibitor 3-MA. Moreover, DEK silencing diminished the Parkin, LC3, DRP1 translocation to mitochondria; as well as mitochondrial ROS; TOM20 and mitochondrial DNA mediated mitochondrial oxidative damage. ChIP-sequence analysis showed that DEK was enriched on the AAA domain-containing protein 3A (ATAD3A) promoter and could positively regulate ATAD3A expression. Additionally, ATAD3A was highly expressed in HDM-induced asthma models. Furthermore, ATAD3A interacted with DRP1, and knockdown of ATAD3A could down-regulate DRP1 and mitochondrial oxidative damage. Conclusively, DEK deficiency alleviates airway inflammation in asthma by down-regulating PINK1-Parkin mitophagy, NLRP3 inflammasome activation, and apoptosis. The mechanism may be through the DEK/ATAD3A/DRP1 signaling axis. Our findings may provide new potential therapeutic targets for asthma treatment.

Project description:Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

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The PP2A-B55-alpha phosphatase is a master regulator of mitochondrial degradation and biogenesis

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