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CCCP Induces Parkin-Independent p-S65 Ubiquitin of Nuclear Proteins (A) Pathway analysis of the top CCCP-induced, PINK1-dependent, p-S65 ubiquitin-enriched proteins identified by MS, following immunoprecipitation. Proteins that also showed an increase in p-S65 ubiquitin enrichment when PINK1 was knocked down were excluded from this analysis. (B) Identification of p-S65-ubiquitinated proteins from Parkin-negative HeLa cells via MS following immunoprecipitation. x axis, enrichment of p-S65-ubiquitinated proteins induced by CCCP treatment in cells transfected with a non-targeting siRNA (NT + CCCP) compared to DMSO treated (NT + DMSO); y axis, further increases in p-S65 ubiquitin enrichment induced by CCCP when PPEF2 is knocked down (PPEF2 + CCCP) in comparison to NT (NT + CCCP). Red points represent proteins associated with the mitochondria, blue points represent proteins found in the nucleus, and gray points represent proteins localized to non-mitochondrial, non-nuclear compartments (see also Table S1).
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Dysregulation of mitophagy, whereby damaged mitochondria are labeled for degradation by the mitochondrial kinase PINK1 and E3 ubiquitin ligase Parkin with phosphorylated ubiquitin chains (p-S65 ubiquitin), may contribute to neurodegeneration in Parkinson's disease. Here, we identify a phosphatase antagonistic to PINK1, protein phosphatase with EF-h...
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... next sought to investigate the cell types in which PPEF2 may interact with p-S65 ubiquitin to impact mitochondrial function in vivo. We utilized RNA in situ hybridization (RNA-ISH) on a mouse multi-tissue array ( Figure S5A) and, consistent with its known role in the retina ( Sherman et al., 1997;Steele et al., 1992), confirmed that Ppef2 is highly expressed in the rodent eye ( Figure S5B). In addition, we detected expression of Ppef2 in multiple brain regions using this method, including the cortex and hippocampus ( Figures S5C and S5D). ...
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... next sought to investigate the cell types in which PPEF2 may interact with p-S65 ubiquitin to impact mitochondrial function in vivo. We utilized RNA in situ hybridization (RNA-ISH) on a mouse multi-tissue array ( Figure S5A) and, consistent with its known role in the retina ( Sherman et al., 1997;Steele et al., 1992), confirmed that Ppef2 is highly expressed in the rodent eye ( Figure S5B). In addition, we detected expression of Ppef2 in multiple brain regions using this method, including the cortex and hippocampus ( Figures S5C and S5D). ...
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... utilized RNA in situ hybridization (RNA-ISH) on a mouse multi-tissue array ( Figure S5A) and, consistent with its known role in the retina ( Sherman et al., 1997;Steele et al., 1992), confirmed that Ppef2 is highly expressed in the rodent eye ( Figure S5B). In addition, we detected expression of Ppef2 in multiple brain regions using this method, including the cortex and hippocampus ( Figures S5C and S5D). Having already observed that Ppef2 is expressed in primary dissociated cultures from rat cortices and hippocampi ( Figure S2C), we subsequently focused on this culture system to further interrogate the effects of Ppef2 knockdown on mitochondrial quality control. ...
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... already observed that Ppef2 is expressed in primary dissociated cultures from rat cortices and hippocampi ( Figure S2C), we subsequently focused on this culture system to further interrogate the effects of Ppef2 knockdown on mitochondrial quality control. Rat-specific Ppef2 siRNAs encapsulated in nanoparticles reduced transcript levels by nearly 75% in these cultures ( Figure S5E). Next, we examined characteristics of mitochondrial health and function in these cultures upon Ppef2 knockdown. ...
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... indicating an increase in mitochondrial biogenesis ( Figure 4A). Ppef2 knockdown did not impact the morphology of the mitochondrial network ( Figure 4B) and caused a slight reduction in the basal rate and maximum capacity for oxidative phosphorylation ( Figure 4C) without affecting glycolysis (Fig- ure S5F). In addition, Ppef2 knockdown lowered the expression levels of transcription factors (TFs) responsible for encoding genes important for metabolic health and mitochondrial function ( Figure 4D). ...
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... these, 134 of the 449 total proteins exhibited an increase in p-S65 ubiquitin enrichment in the PINK1 siRNA + CCCP conditions and were subsequently omitted from our downstream analyses. Pathway analysis of the remaining 113 proteins exhibiting PINK1-dependent p-S65 ubiquitin enrichment revealed cellular functions in the cytoplasm and nucleus, in addition to the expected Parkinubiquitin proteasomal and mitochondrial processes ( Figure 5A). Importantly, PPEF2 knockdown boosted the signal for PINK1-dependent p-S65 ubiquitin enrichment after CCCP of 182 proteins over NT siRNA ( Figure 5B, log 2 fold change (FC) R 0 on the y axis), including some proteins that did not increase upon CCCP treatment under the NT siRNA condition ( Figure 5B, log 2 FC % 0 on the x axis; see also Table S1). ...
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... analysis of the remaining 113 proteins exhibiting PINK1-dependent p-S65 ubiquitin enrichment revealed cellular functions in the cytoplasm and nucleus, in addition to the expected Parkinubiquitin proteasomal and mitochondrial processes ( Figure 5A). Importantly, PPEF2 knockdown boosted the signal for PINK1-dependent p-S65 ubiquitin enrichment after CCCP of 182 proteins over NT siRNA ( Figure 5B, log 2 fold change (FC) R 0 on the y axis), including some proteins that did not increase upon CCCP treatment under the NT siRNA condition ( Figure 5B, log 2 FC % 0 on the x axis; see also Table S1). Mitochondrial proteins surprisingly constituted only a small fraction of this group (20 out of 182) ( Figure 5B, red points). ...
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... analysis of the remaining 113 proteins exhibiting PINK1-dependent p-S65 ubiquitin enrichment revealed cellular functions in the cytoplasm and nucleus, in addition to the expected Parkinubiquitin proteasomal and mitochondrial processes ( Figure 5A). Importantly, PPEF2 knockdown boosted the signal for PINK1-dependent p-S65 ubiquitin enrichment after CCCP of 182 proteins over NT siRNA ( Figure 5B, log 2 fold change (FC) R 0 on the y axis), including some proteins that did not increase upon CCCP treatment under the NT siRNA condition ( Figure 5B, log 2 FC % 0 on the x axis; see also Table S1). Mitochondrial proteins surprisingly constituted only a small fraction of this group (20 out of 182) ( Figure 5B, red points). ...
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... PPEF2 knockdown boosted the signal for PINK1-dependent p-S65 ubiquitin enrichment after CCCP of 182 proteins over NT siRNA ( Figure 5B, log 2 fold change (FC) R 0 on the y axis), including some proteins that did not increase upon CCCP treatment under the NT siRNA condition ( Figure 5B, log 2 FC % 0 on the x axis; see also Table S1). Mitochondrial proteins surprisingly constituted only a small fraction of this group (20 out of 182) ( Figure 5B, red points). In contrast, twice as many proteins whose p-S65 ubiquitin enrichment signal was boosted by PPEF2 knockdown (54 out of 182) were predominantly associated with the nuclear compartment ( Figure 5B, blue points). ...
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... proteins surprisingly constituted only a small fraction of this group (20 out of 182) ( Figure 5B, red points). In contrast, twice as many proteins whose p-S65 ubiquitin enrichment signal was boosted by PPEF2 knockdown (54 out of 182) were predominantly associated with the nuclear compartment ( Figure 5B, blue points). Notable among these were proteins involved in DNA repair (DHX9, PRKDC, and XRCC6) and with the proteasome or autophagy pathways (CSN1, MYCB2, PRS6/7, and PSA6). ...
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... among these were proteins involved in DNA repair (DHX9, PRKDC, and XRCC6) and with the proteasome or autophagy pathways (CSN1, MYCB2, PRS6/7, and PSA6). We were able to validate some of the hits from each compartment using immunoprecipitation followed by immunoblotting, and also demonstrated their dependency on PINK1 ( Figure 5C). Thus, upon mitochondrial damage, PINK1-dependent p-S65 ubiquitin is observed on both mitochondrial and nuclear proteins, which is counteracted by PPEF2. ...
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... were surprised by our observation of PINK1-dependent p-S65 ubiquitination of proteins that are associated with the nucleus after treatment with CCCP and subsequently sought to validate this finding to explore the functional consequences of nuclear p-S65 ubiquitin. We first visualized patterns of p-S65 ubiquitin localization in the nucleus upon CCCP treatment and found it to be broadly distributed throughout the cell ( Figure 5D). Second, PPEF2 knockdown significantly amplified the nuclear p-S65 ubiquitin signal, again in a PINK1-dependent manner (Fig- ure 5E). ...
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... first visualized patterns of p-S65 ubiquitin localization in the nucleus upon CCCP treatment and found it to be broadly distributed throughout the cell ( Figure 5D). Second, PPEF2 knockdown significantly amplified the nuclear p-S65 ubiquitin signal, again in a PINK1-dependent manner (Fig- ure 5E). We next confirmed this result via western blot of fractionated nuclear and cytoplasmic lysates ( Figure 5F). ...
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... PPEF2 knockdown significantly amplified the nuclear p-S65 ubiquitin signal, again in a PINK1-dependent manner (Fig- ure 5E). We next confirmed this result via western blot of fractionated nuclear and cytoplasmic lysates ( Figure 5F). Interestingly, PINK1 protein was also detectable in the nuclear fraction upon CCCP treatment, in contrast to cytoplasmic or mitochondrial markers ACLY and SDHB ( Figure 5F). ...
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... next confirmed this result via western blot of fractionated nuclear and cytoplasmic lysates ( Figure 5F). Interestingly, PINK1 protein was also detectable in the nuclear fraction upon CCCP treatment, in contrast to cytoplasmic or mitochondrial markers ACLY and SDHB ( Figure 5F). These results indicate that PINK1 and PPEF2 can reciprocally regulate pS65-ubiquitin abundance in the nucleus. ...
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... condition including a phosphatase buffer supplemented with 1mM CaCl 2 was also included, in addition to a calf intestinal phosphatase (CIP) treatment of the p-S65 ubiquitin chains as a positive control. This same assay run without a DUB inhibitor included in the phosphatase buffer is shown in Figure S5C. ...
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... cell line was transfected with a separate species-specific siRNA for PPEF2 (or non-targeting, NT) for 3 days prior to CCCP treatment. (C) Quantitative PCR for absolute PPEF2 mRNA transcript amount after siRNA knockdown in the three cell lines described in (B), n = 3 independent experiments; mean + SEM; * p < 0.05, Figure S5. Expression of Ppef2 in mouse and rat neuronal tissues, related to Figure 4. (A) Negative and positive controls for RNA-in situ hybridization in mouse tissue sections via RNAscope. ...
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Mitophagy is a selective autophagy process that specifically removes damaged mitochondria via general autophagy. The two major recessive Parkinson's disease genes PINK1 and Parkin play essential roles in mitophagy initiation, increasing the interest in mitophagy in both basic and translational research over the past 10 years. Initially, mitophagy w...
Citations
... Also, formation of phospho-ubiquitin, the reaction product of PINK1, on depolarized mitochondria (Fig. 5c, d) was unaffected by α-synuclein upregulation. A small minority of phospho-ubiquitin puncta after valinomycin treatment appeared to be localized in the nucleus (both with and without doxycycline treatment) (Fig. 5c), consistent with a previous report of PINK1-mediated phospho-ubiquitin accumulation in the nucleus after mitochondrial depolarization 30 . ...
The pathogenic effect of SNCA gene multiplications indicates that elevation of wild-type α-synuclein levels is sufficient to cause Parkinson’s disease (PD). Mitochondria have been proposed to be a major target of α-synuclein-induced damage. PINK1/parkin/DJ-1-mediated mitophagy is a defense strategy that allows cells to selectively eliminate severely damaged mitochondria. Here, we quantified mitophagic flux and non-mitochondrial autophagic flux in three models of increased α-synuclein expression: 1/ Drosophila melanogaster that transgenically express human wild-type and mutant α-synuclein in flight muscle; 2/human skin fibroblasts transfected with α-synuclein or β-synuclein; and 3/human induced pluripotent stem cell (iPSC)-derived neurons carrying an extra copy of wild-type SNCA under control of a doxycycline-inducible promoter, allowing titratable α-synuclein upregulation. In each model, elevated α-synuclein levels potently suppressed mitophagic flux, while non-mitochondrial autophagy was preserved. In human neurons, a twofold increase in wild-type α-synuclein was already sufficient to induce this effect. PINK1 and parkin activation and mitochondrial translocation of DJ-1 after mitochondrial depolarization were not affected by α-synuclein upregulation. Overexpression of the actin-severing protein cofilin or treatment with CK666, an inhibitor of the actin-related protein 2/3 (Arp2/3) complex, rescued mitophagy in neurons with increased α-synuclein, suggesting that excessive actin network stabilization mediated the mitophagy defect. In conclusion, elevated α-synuclein levels inhibit mitophagic flux. Disruption of actin dynamics may play a key role in this effect.
... The other isoform, PTEN-L, inhibits mitophagy by antagonizing ubiquitin phosphorylation, preventing Parkin mitochondrial translocation and inhibiting its E3 ubiquitin ligase activity (Wang et al., 2018). In addition, protein phosphatase with EF-hand structural domain 2 (PPEF2) was shown to inhibit PINK1-dependent mitophagy through ubiquitin dephosphorylation, acting as a negative regulator of mitophagy (Wang et al., 2018;Wall et al., 2019). Various PINK1-Parkin-mediated mitophagy process. ...
Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world, and alpha-synuclein (α-syn) abnormal aggregate and mitochondrial dysfunction play a crucial role in its pathological development. Recent studies have revealed that proteins can form condensates through liquid–liquid phase separation (LLPS), and LLPS has been found to be widely present in α-syn aberrant aggregate and mitophagy-related protein physiological processes. This review summarizes the occurrence of α-syn LLPS and its influencing factors, introduces the production and transformation of the related protein LLPS during PINK1-Parkin-mediated mitophagy, hoping to provide new ideas and methods for the study of PD pathology.
... As the cleavage product of the full-length PINK1, sPINK1 is rapidly degraded per the Nend rule, with fully functional proteasomes present in the cell. The pUb level is also in a dynamic flux, as several phosphatases have been identified to remove the phosph 35-37 oryl group from Ub [35][36][37] . As a result, the pUb is usually kept at a very low level 38 . ...
... Upon the over-expression of a long-lived version sPINK1, the pUb level increased by only 45.8% and 61% on the 30 th and 70 th day post-transfection, respectively. Besides the phosphorylation efficiency of the kinase, continuous dephosphorylation of the pUb also accounted for the overall phosphorylation level [35][36][37] . Considering that the phosphatase activity also declines during aging 56,57 , elevating the phosphatase activity holds the therapeutic promise for exerting neuroprotective effects in the aging brain. ...
Ser65-phosphorylated ubiquitin (pUb) was found elevated in neurons of aged and neurodegenerative brains. Yet little is known whether a causative link exists between pUb level and brain aging. Here we show that the knockout of pink1 , a Ub kinase, abolished pUb elevation and decelerated protein aggregation in aged mouse brains and cells with proteasomal inhibition. Conversely, over-expression of PINK1 but not the kinase-dead version increased the pUb level and accelerated protein aggregation by suppressing of proteasomal degradation. Furthermore, PINK1 over-expression in mouse hippocampus neurons increased pUb level and protein aggregation, slowly leading to mitochondrial injury, neurodegeneration, and cognitive impairment. Notably, the neuronal damages induced by PINK1 were rescued by the dominant negative Ub/S65A mutant, while Ub/S65E phosphomimetic mutant caused neuronal death. Together, an incidental increase of Ub phosphorylation can progressively and cumulatively cause the decline of Ub-dependent proteasomal activity, consequenting promotes neurodegeneration in the aging brain.
... We also confirm decreased levels of pS65Ub, which is induced by PINK1 activation, after mitochondrial depolarization in LRRK2 R1441C iPSC-DA neuronal cultures. Importantly, the mechanism of decreased pS65Ub levels in LRRK2 R1441C mutants could involve altered PINK1 activation or increased activity of ubiquitin phosphatases such as PPEF2 or PTEN-L (42,43). Therefore, decreased mitophagy could further exacerbate perturbations on mitochondrial homeostasis because damaged mitochondria are not being degraded and could disturb the mitochondrial network, contributing to molecular pathology in PD. ...
Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilise and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation.
... Inhibition or silencing of PPEF2 (protein phosphatase with EF-hand domain 2) and PTEN-L phosphatase were demonstrated to increase mitophagy. PPEF2 is a PINK1 phosphatase antagonist that dephosphorylates ubiquitin and inhibits PINK1-mediated mitophagy [202,218]. ...
Parkinson's disease (PD) is a common age-related neurodegenerative disorder whose pathogenesis is not completely understood. Mitochondrial dysfunction and increased oxidative stress have been considered as major cause and central event responsible for the progressive degeneration of dopaminergic (DA) neurons in PD. Therefore, investigating mitochondrial disorders plays a role in understanding the pathogenesis of PD and can be an important therapeutic target for this disease. This study discusses the effect of environmental, genetic and biological factors on mitochondrial dysfunction and also focuseson the mitochondrial molecular mechanisms underlying neurodegeneration, and its possible therapeutic targets in PD, including reactive oxygen species generation, calcium overload, inflammasome activation, apoptosis, mitophagy, mitochondrial biogenesis, and mitochondrial dynamics. Other potential therapeutic strategies such as mitochondrial transfer/transplantation, targeting microRNAs, using stem cells, photobiomodulation, diet, and exercise were also discussed in this review, which may provide valuable insights into clinical aspects. A better understanding of the roles of mitochondria in the pathophysiology of PD may provide a rationale design of novel therapeutic interventions in our fight against PD.
... Little is known about how SLRdependent mitophagy is turned off, but several deubiquitylating enzymes (DUBs) have been shown to antagonize parkin-mediated ubiquitylation and mitophagy, including the ubiquitin-specific peptidases USP15, USP30, USP33, USP35 and USP36 (Bingol et al., 2014;Cornelissen et al., 2014;Geisler et al., 2019;Gersch et al., 2017;Niu et al., 2020;Wang et al., 2015). Furthermore, protein phosphatase with EF-hand domain 2 (PPEF2), which dephosphorylates ubiquitin at Ser65, has been shown to inhibit PINK1-dependent mitophagy (Wall et al., 2019). ...
Mitochondria are crucial organelles that play a central role in various cell signaling and metabolic pathways. A healthy mitochondrial population is maintained through a series of quality control pathways and requires a fine-tuned balance between mitochondrial biogenesis and degradation. Defective targeting of dysfunctional mitochondria to lysosomes through mitophagy has been linked to several diseases, but the underlying mechanisms and the relative importance of distinct mitophagy pathways in vivo are largely unknown. In this Cell Science at a Glance and the accompanying poster, we describe our current understanding of how parts of, or whole, mitochondria are recognized by the autophagic machinery and targeted to lysosomes for degradation. We also discuss how this might be regulated under different physiological conditions to maintain mitochondrial and cellular health.
... In the human postmortem brain of PD patients, the novel selective mitophagy marker (directed against phosphorylated S65 ubiquitin moiety; hereafter in short 'p65') increases across age and with PD diagnosis 24 . This mitophagy marker is also associated with Lewy Bodies, showing an increased mitophagy in early PD stages and a decrease in late stages [24][25][26] . On the other hand, there is impaired cytosolic and mitochondrial cargo targeting the autophagosome in Huntington's disease, leading to impaired removal of damaged proteins and organelles 27 . ...
... For instance, ubiquitin phosphorylated on the 65-Serin residue has the potential to cascade routing of damaged mitochondria to degradation in lysosomes and thereby launch the mitophagy flux-machinrey 57 . This process is mediated by activating the Pink/Parkin-mitophagydependent pathway 26 . A comprehensive review of p65 and its role in Pink-Parkin-dependent mitophagy is reviewed in reference 58 . ...
... Moreover, in PD, p65 is also observed on both mitochondrial and nuclear proteins, interacting with damaged DNA and repair machinery 26 . It has recently been shown that p65 exists in low detection levels in the human postmortem brain neurons under homeostatic conditions, with low detection levels. ...
Abstract
Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS), traditionally considered a chronic autoimmune attack against the insulating myelin sheaths around axons. Mitochondria are complex intracellular multifunctional organelles that form the primary source of cellular energy and participate in cellular signaling and homeostasis. Therefore, the adequate physiological functioning of neurons is significantly dependent on the viability of mitochondria. The longevity of neurons relies, among others, on cell capacity to scavenge and 'recycle' impaired mitochondria. This process is called 'mitophagy,' and its dysregulation can lead to neuroinflammation, as demonstrated in several neurodegenerative diseases, such as Parkinson’s disease. Previously, our group has identified mitochondrial physiological abnormalities within neurons in MS postmortem brains. However, little is known about mitophagy in MS and whether dysregulation in mitochondrial dynamics – its elimination or biogenesis- is affected. This internship project aimed to study mitophagy in an MS postmortem brain cohort using immunostaining, confocal laser scanning microscopy, and novel data analysis software. We hypothesized that aberrant aspects of mitophagy could be revealed qualitatively and qualitatively in postmortem tissue. In this view, visualizing mitochondria tagged with unique 'eat-me' markers could indicate maladaptive mitochondrial physiology. For example, mitochondria co-localized with the specific mitophagy-initiation marker – phosphorylated S65 ubiquitin (‘p65’) - could denote damaged mitochondria-targeted to elimination. By quantifying the total volume of mitochondria tagged with p65, we aspired to shed light on aberrant mitophagy physiology in MS. To this end, a multiplex fluorescence immunostaining protocol was developed, and multiple mitophagy-related antibodies were tested under different conditions. Next, three-dimensional confocal images were acquired, then analyzed with a semi-supervised segmentation algorithm. Finally, volumetrics and morphometrics were acquired, and a customized R-language code was compiled for statistics and data visualization. This vital work lays the foundation for further investigating neuronal mitochondria in postmortem brain tissue in an unprecedented level of detail.
... pS65-Ub accumulated upon paraquat exposure then declined over several days post-treatment, which we took to indicate turnover of the conjugated substrates (Fig 2C and D). Importantly, the loss of pS65-Ub was not due to the known pS65-Ub phosphatase PPEF2 (Wall et al, 2019), since the loss of the Drosophila homologue rdgC did not affect pS65-Ub degradation ( Fig EV2). Another pS65-Ub phosphatase, PTEN-L (Wang et al, 2018), is not conserved in flies thus precluding its assessment. ...
Parkinson's disease-related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1-Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1-Parkin pathway operates in vivo, we developed methods to detect Ser65-phosphorylated ubiquitin (pS65-Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1-dependent pS65-Ub production, while pS65-Ub accumulates in unstimulated parkin-null flies, consistent with blocked degradation. Additionally, we show that pS65-Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65-Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat-induced pS65-Ub in an Atg5-null background. Thus, we have established that pS65-Ub immunodetection can be used to analyse Pink1-Parkin function in vivo as an alternative to reporter constructs. Moreover, our findings suggest that the Pink1-Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.
... In comparison, much less is known about the mitophagy braking system. Previously, two enzymes were identified as the phosphatases for pUb 24,25 . In one study, the authors proposed that ubiquitin dephosphorylation indirectly impairs Parkin mitochondrial translocation, but fell short in providing direct evidence for pParkin dephosphorylation 26 . ...
Mitophagy is a selective autophagic process that removes damaged mitochondria. PINK1-Parkin axis is primarily responsible for initiating mitophagy via feedforward mechanism, in which PINK1 phosphorylates ubiquitin and Parkin, and Parkin gains E3 ligase activity at the mitochondrial outer membrane. However, the phosphatase of pParkin is unknown, and the braking mechanism of mitophagy is incomplete. Here we report that protein phosphatase 2A (PP2A) catalyzes the dephosphorylation of Parkin and ubiquitin, with Cα, Aα, and B55α, the catalytic scaffolding and regulatory subunits, respectively. Up- or down-regulation of PP2A protein level in cells by over-expression or transfection of specific siRNA decreases or increases Parkin and ubiquitin phosphorylation levels, respectively. Consequently, PP2A phosphatase activity negatively modulates mitochondrial translocation of Parkin and forestalls mitophagy. Our finding thus places PP2A, an enzyme already known for its involvement in the pathogenesis of neurodegenerative diseases, further intertwined with the PINK1-Parkin signaling pathway.
... [53] Biochemical sub-cellular fractionation experiments have found that both phosphorylated ubiquitin and cPINK1 are also present in the nucleus following mitochondrial depolarisation. [109] Phosphoproteomics suggests that PINK1 phosphorylates nuclear proteins directly and also enables their modification by phospho-ubiquitin. [109,110] F I G U R E 3 PINK1 and Parkin are linked to innate and adaptive immune responses. ...
... [109] Phosphoproteomics suggests that PINK1 phosphorylates nuclear proteins directly and also enables their modification by phospho-ubiquitin. [109,110] F I G U R E 3 PINK1 and Parkin are linked to innate and adaptive immune responses. Some mitochondrial derived vesicles (MDVs) are proposed to traffic mitochondrial cargo to specialized endosomes, for processing and loading onto MHC-II molecules. ...
PTEN-induced kinase 1 (PINK1) is a Parkinson's disease gene that acts as a sensor for mitochondrial damage. Its best understood role involves phosphorylating ubiquitin and the E3 ligase Parkin (PRKN) to trigger a ubiquitylation cascade that results in selective clearance of damaged mitochondria through mitophagy. Here we focus on other physiological roles of PINK1. Some of these also lie upstream of Parkin but others represent autonomous functions, for which alternative substrates have been identified. We argue that PINK1 orchestrates a multi-arm response to mitochondrial damage that impacts on mitochondrial architecture and biogenesis, calcium handling, transcription and translation. We further discuss a role for PINK1 in immune signalling co-ordinated at mitochondria and consider the significance of a freely diffusible cleavage product, that is constitutively generated and degraded under basal conditions.
