Clearance Of Mitochondria Research Articles

HDAC6 inhibition induces mitochondrial fusion, autophagic flux and reduces diffuse mutant huntingtin in striatal neurons

Striatal neurons are vulnerable to Huntington's disease (HD). Decreased levels of acetylated alpha-tubulin and impaired mitochondrial dynamics, such as reduced motility and excessive fission, are associated with HD; however, it remains unclear whether and how these factors might contribute to the preferential degeneration of striatal neurons. Inhibition of the alpha-tubulin deacetylase HDAC6 has been proposed as a therapeutic strategy for HD, but remains controversial — studies in neurons show improved intracellular transport, whereas studies in cell-lines suggest it may impair autophagosome–lysosome fusion, and reduce clearance of mutant huntingtin (mHtt) and damaged mitochondria (mitophagy). Using primary cultures of rat striatal and cortical neurons, we show that mitochondria are intrinsically less motile and more balanced towards fission in striatal than in cortical neurons. Pharmacological inhibition of the HDAC6 deacetylase activity with tubastatin A (TBA) increased acetylated alpha-tubulin levels, and induced mitochondrial motility and fusion in striatal neurons to levels observed in cortical neurons. Importantly, TBA did not block neuronal autophagosome–lysosome fusion, and did not change mitochondrial DNA levels, suggesting no impairment in autophagy or mitochondrial clearance. Instead, TBA increased autophagic flux and reduced diffuse mHtt in striatal neurons, possibly by promoting transport of initiation factors to sites of autophagosomal biogenesis. This study identifies the pharmacological inhibition of HDAC6 deacetylase activity as a potential strategy to reduce the vulnerability of striatal neurons to HD.

MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

Abstract 174: The BH3-Only Protein BNIP3 Induces Mitochondrial Clearance via Multiple Pathways

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein which is known to cause mitochondrial dysfunction and cell death in the myocardium. Interestingly, BNIP3 can also protect against cell death by promoting removal of dysfunctional mitochondria via autophagy (mitophagy). We have previously reported that BNIP3 is a potent inducer of mitophagy in cardiac myocytes and that BNIP3 contains an LC3 Interacting Region (LIR) that binds to LC3 on the autophagosome, tethering the mitochondrion to the autophagosome for engulfment. However, the molecular mechanism(s) underlying BNIP3-mediated mitophagy are still unclear. In this study, we discovered that BNIP3 can mediate mitochondrial clearance in cells even in the absence of a functional autophagy pathway. We found that overexpression of BNIP3 led to significant clearance of mitochondria in both wild type (WT) and autophagy deficient Atg5-/- MEFs. BNIP3 caused an increase in LC3II levels in WT MEFs, indicating increased formation of autophagosomes. In contrast, LC3II was undetectable in Atg5-/- MEFs. Furthermore, we found that BNIP3-mediated clearance in WT and Atg5-/- MEFs did not require the presence of Parkin, an E3 ubiquitin ligase which plays a critical role in clearing dysfunctional mitochondria in cells. Also, overexpression of Parkin did not enhance BNIP3-mediated mitochondrial clearance. When investigating activation of alternative cellular degradation pathways, we found that BNIP3 induced activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. Mutating the LC3 binding site in BNIP3 did not interfere with the activation of the endosomal pathway and clearance of mitochondria in Atg5-/- MEFs. Thus, these findings suggest that BNIP3 can promote clearance of mitochondria via multiple pathways in cells. The role of autophagy in removing mitochondria is already well established and we are currently exploring the roles of the endosomal and alternative autophagy pathways in BNIP3-mediated mitochondrial clearance in myocytes.

Abstract 243: The E3 Ubiquitin Ligase Parkin Mediates Clearance of Damaged Mitochondria via Two Distinct Pathways

Damaged mitochondria release reactive oxygen species and pro-death factors which can lead to loss of cardiac myocytes. To protect against such damage, myocytes have developed several mechanisms of quality control that act both on the protein and organelle levels. We have previously identified the E3 ubiquitin ligase Parkin as an important regulator of mitochondrial clearance via autophagy in the myocardium. Here, we report that Parkin can also mediate clearance of mitochondria via the endosomal-lysosomal pathway. We found that Parkin promotes clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Mitochondrial damage leads to rapid activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. We further observed increased activation of Rab5, a protein involved in early endosome formation, in both WT and Atg5-/- MEFs after treatment with FCCP. In addition, we observed sequestration of damaged mitochondria in Rab5+ and Rab7+ early and late endosomes, respectively. Mitochondria also colocalized with Lamp2+ vesicles in Atg5-/- MEFs indicating that the mitochondria are ultimately being delivered to the lysosomes for degradation. Overexpression of Rab5S34N, a dominant negative of Rab5, reduces FCCP-mediated clearance and increases cell death in Atg5-/- MEFs. Pharmacological inhibition of the endosomal-lysosomal pathway also results in increased FCCP-mediated cell death. Furthermore, we confirmed that FCCP treatment or simulated ischemia reperfusion exposure induces Rab5 activation with subsequent mitochondrial sequestration in early endosomes in neonatal myocytes. Interestingly, the activation of Rab5 is abrogated in the presence of the mitochondrial targeted antioxidant Mito-Tempo, suggesting that mitochondrial ROS is involved in the activation the endosomal pathway. Mitochondrial clearance via this pathway is also dependent on Parkin, as FCCP treatment fails to activate Rab5 and induce mitochondrial clearance in both WT and Atg5-/- MEFS in the absence of Parkin. Thus, our data suggest that Parkin can mediate clearance of damaged mitochondria via two distinct pathways in cells.

Abstract 393: MCL-1 Promotes Survival and Influences Mitochondrial Dynamics in Cardiac Myocytes

The BCL-2 family proteins are important regulators of mitochondrial structure and integrity. MCL-1 is an anti-apoptotic BCL-2 protein that is highly expressed in the myocardium compared to the other anti-apoptotic proteins BCL-2 and BCL-X L. Recently, we reported that MCL-1 is essential for myocardial homeostasis. Cardiac-specific deletion of MCL-1 in mice led to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Surprisingly, MCL-1 deficient myocytes did not undergo apoptotic cell death. Instead, the cells displayed signs of mitochondrial deterioration and necrotic cell death, suggesting that MCL-1 has an additional role in maintaining mitochondrial function in cardiac myocytes. Similarly, deletion of MCL-1 in fibroblasts caused rapid mitochondrial fragmentation followed by cell death at 72 hours. Interestingly, the MCL-1 deficient fibroblasts retained cytochrome c in the mitochondria , confirming that the cells were not undergoing apoptotic cell death. We have also identified that MCL-1 localizes to the mitochondrial outer membrane (OM) and the matrix in the myocardium and that the two forms respond differently to stress. MCL-1 OM was rapidly degraded after myocardial infarction or fasting, whereas MCL-1 Matrix levels were maintained. Similarly, starvation of MEFs resulted in rapid degradation of MCL-1 OM , whereas MCL-1 Matrix showed delayed degradation. Treatment with the mitochondrial uncoupler FCCP led to rapid degradation of both forms. This suggests that the susceptibility to degradation is dependent on its localization and the nature of the stress. Our data also suggests that these two forms perform distinct functions in regulating mitochondrial morphology and survival. Overexpression of MCL-1 Matrix promoted mitochondrial fusion in fibroblasts under baseline conditions and protected cells against FCCP-mediated mitochondrial fission and clearance by autophagosomes. Thus, our data suggest that MCL-1 exists in two separate locations where it performs different functions. MCL-1 Matrix promotes mitochondrial fusion, which protects cells against excessive mitochondrial clearance during unfavorable conditions.

Abstract 242: Mitophagy is Required for Mitochondrial Biogenesis and Myogenic Differentiation of Myoblasts

Myogenesis is a crucial process governing muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNML1/DRP1-mediated fragmentation and subsequent removal of mitochondria via p62/SQSTM-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α-mediated biogenesis. Mitochondrial fusion protein OPA1 is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that blocking autophagy with various inhibitors during differentiation results in a blockade in myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

Abstract 208: Parkin Contributes to the Development of Cardiac Hypertrophy in Response to Cardiac Pressure Overload

Parkin is an E3 ubiquitin ligase known to mediate mitochondrial clearance by marking damaged mitochondria for autophagy. Our lab has previously shown that Parkin is important for stress adaptation following myocardial infarction, and that loss of Parkin leads to accumulation of dysfunctional mitochondria. However, whether Parkin plays a role in cardiac adaptation to pressure overload is currently unknown. Here we investigated the functional importance of Parkin in cardiac hypertrophy and development of heart failure in response to hemodynamic stress. Wild type (WT), Parkin knock out (Parkin -/- ), and cardiac-specific Parkin transgenic (Parkin-TG) mice were subjected to trans-aortic constriction (TAC). Cardiac anatomy and function was evaluated by histology and echocardiography. Inflammation and hypertrophy gene expression profiles were assessed using qPCR and immunohistochemistry. We discovered that after 2 weeks of TAC, cardiac hypertrophy markers were not increased in hearts from Parkin -/- mice, and there was no increase in the heart weight to body weight ratio (HW/BW). However, after 8 weeks of TAC, Parkin -/- mice showed similar cardiac hypertrophy and loss of function as WT hearts. Parkin deficient hearts also displayed increased interstitial and perivascular fibrosis compared to WT hearts after 8 weeks of TAC. This suggests that there is a delay in activating the hypertrophy program in the absence of Parkin, and that lack of Parkin leads to excessive fibrosis. In contrast, Parkin-TG mice showed a rapid development of hypertrophy and progression to heart failure compared to WT mice. Interestingly, we observed no differences in either mitochondrial content or LC3 levels after two weeks of TAC in Parkin-TG hearts, suggesting that the rapid development of hypertrophy and early progression to heart failure was not due to excessive mitophagy. These data suggest that Parkin plays an important role in the activation of the cardiac hypertrophy program and that this function may be independent of its role in regulating mitophagy. Thus, this study provides novel insight into the functional importance of Parkin in the heart. Additional studies are needed to determine the mechanism of how Parkin regulates cardiac hypertrophy.

Defective autophagy through epg5 mutation results in failure to reduce germ plasm and mitochondria.

Autophagy is an evolutionarily conserved catabolic process that transports cytoplasmic components to lysosomes for degradation. In addition to the canonical view of strict stress-response-induced autophagy, selectively programmed autophagy was recently reported in the context of gonad development of flies and worms, where autophagy seems to be necessary for clearance of germ plasm components. Similar functions have not been described in vertebrates. We used the medaka fish to study the role of autophagy in gonad formation and gametogenesis for the first time in a vertebrate organism for which the germ line is specified by germ plasm. Using a transgenic line deficient in the Ol-epg5 gene—a new critical component of the autophagy pathway—we show that such deficiency leads to an impaired autophagic flux, possibly attributed to compromised maturation or processing of the autophagosomes. Ol-epg5 deficiency correlates with selectively impaired spermatogenesis and low allele transmission rates of the mutant allele caused by failure of germ plasm and mitochondria clearance during the process of germ cell specification and in the adult gonads. The mouse epg-5 homolog is similarly expressed in the maturating and adult testes, suggesting an at least partially conserved function of this process during spermatogenesis in vertebrates.

Mitochondrial dysfunction and Parkinson disease: a Parkin-AMPK alliance in neuroprotection.

Although a subject of intense research, the etiology of Parkinson disease (PD) remains poorly understood. However, a wide range of studies conducted over the past few decades have collectively implicated aberrant mitochondrial homeostasis as a key contributor to the development of PD. Particularly strong support for this came from the recent demonstration that parkin, a familial PD-linked gene, is a critical regulator of mitochondrial quality control. Indeed, Parkin appears to be involved in all stages of the mitochondrial life cycle (i.e., from biogenesis to its exit from the cell (via mitophagy). Interestingly, the role of Parkin in the biogenesis and clearance of mitochondria is akin to that performed by the energy sensor AMP-activated protein kinase (AMPK), suggesting that the two proteins might act in a functionally converging manner to maintain the quality of cellular mitochondria. In this review, we discuss the contribution of mitochondrial dysfunction to PD pathogenesis and the role of Parkin and AMPK in preserving neuronal mitochondrial homeostasis. Alongside this, we will also articulate our thoughts on the potential alliance between Parkin and AMPK in offering neuroprotection through their ability to maintain energy balance in the brain.

Daily heat stress treatment rescues denervation-activated mitochondrial clearance and atrophy in skeletal muscle.

Traumatic nerve injury or nerve disease leads to denervation and severe muscle atrophy. Recent evidence shows that mitochondrial loss could be a key mediator of skeletal muscle atrophy. Here, we show that daily heat stress treatment rescues denervation-induced loss of mitochondria and concomitant muscle atrophy. We also found that denervation-activated autophagy-dependent mitochondrial clearance (mitophagy) was suppressed by daily heat stress treatment. The molecular basis of this observation is explained by our results showing that heat stress treatment attenuates the increase of key proteins that regulate the tagging step for mitochondrial clearance and the intermediate step of autophagosome formation in denervated muscle. These findings contribute to the better understanding of mitochondrial quality control in denervated muscle from a translational perspective and provide a mechanism behind the attenuation of muscle wasting by heat stress. Traumatic nerve injury or motor neuron disease leads to denervation and severe muscle atrophy. Recent evidence indicates that loss of mitochondria and the related reduction in oxidative capacity could be key mediators of skeletal muscle atrophy. As our previous study showed that heat stress increased the numbers of mitochondria in skeletal muscle, we evaluated whether heat stress treatment could have a beneficial impact on denervation-induced loss of mitochondria and subsequent muscle atrophy. Here, we report that daily heat stress treatment (mice placed in a chamber with a hot environment; 40°C, 30minday(-1) , for 7days) rescues the following parameters: (i)muscle atrophy (decreased gastrocnemius muscle mass); (ii)loss of mitochondrial content (decreased levels of ubiquinol-cytochrome c reductase core proteinII, cytochrome c oxidase subunitsI and IV and voltage-dependent anion channel protein); and (iii)reduction in oxidative capacity (reduced maximal activities of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase) in denervated muscle (produced by unilateral sciatic nerve transection). In order to gain a better understanding of the above mitochondrial adaptations, we also examined the effects of heat stress on autophagy-dependent mitochondrial clearance (mitophagy). Daily heat stress normalized denervation-activated induction of mitophagy (increased mitochondrial microtubule-associated protein 1A/1B-light chain3-II (LC3-II) with and without blocker of autophagosome clearance). The molecular basis of this observation was explained by the results that heat stress attenuated the denervation-induced increase in key proteins that regulate the following steps: (i)the tagging step of mitochondrial clearance (increased mitochondrial Parkin, ubiquitin-conjugated, P62/sequestosome 1 (P62/SQSTM1)); and (ii)the elongation step of autophagosome formation (increased Atg5-Atg12 conjugate and Atg16L). Overall, our results contribute to the better understanding of mitochondrial quality control and the mechanisms behind the attenuation of muscle wasting by heat stress in denervated skeletal muscle.

Tu1848 Prohibitin Deficiency Leads to Defective Mitochondrial Clearance by Mitophagy in Intestinal Epithelial Cells

Tu1848 Prohibitin Deficiency Leads to Defective Mitochondrial Clearance by Mitophagy in Intestinal Epithelial Cells

MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction.

Haematopoietic progenitor cells show special sensitivity to mitochondrial DNA (mtDNA) mutagenesis, which suggests that increased mtDNA mutagenesis could underlie anemias. Here we show that elevated mtDNA mutagenesis in mice with a proof-reading deficient mtDNA polymerase (PolG) leads to incomplete mitochondrial clearance, with asynchronized iron loading in erythroid precursors, and increased total and free cellular iron content. The resulting Fenton chemistry leads to oxidative damage and premature destruction of erythrocytes by splenic macrophages. Our data indicate that mitochondria actively contribute to their own elimination in reticulocytes and modulate iron loading. Asynchrony of this sequence of events causes severe mitochondrial anaemia by depleting the organism of red blood cells and the bone marrow of iron. Our findings account for the anaemia development in a progeroid mouse model and may have direct relevance to the anemias associated with human mitochondrial disease and ageing.

Mitochondria: key players in the neurotoxic effects of amphetamines.

Amphetamines are a class of psychotropic drugs with high abuse potential, as a result of their stimulant, euphoric, emphathogenic, entactogenic, and hallucinogenic properties. Although most amphetamines are synthetic drugs, of which methamphetamine, amphetamine, and 3,4-methylenedioxymethamphetamine ("ecstasy") represent well-recognized examples, the use of natural related compounds, namely cathinone and ephedrine, has been part of the history of humankind for thousands of years. Resulting from their amphiphilic nature, these drugs can easily cross the blood-brain barrier and elicit their well-known psychotropic effects. In the field of amphetamines' research, there is a general consensus that mitochondrial-dependent pathways can provide a major understanding concerning pathological processes underlying the neurotoxicity of these drugs. These events include alterations on tricarboxylic acid cycle's enzymes functioning, inhibition of mitochondrial electron transport chain's complexes, perturbations of mitochondrial clearance mechanisms, interference with mitochondrial dynamics, as well as oxidative modifications in mitochondrial macromolecules. Additionally, other studies indicate that amphetamines-induced neuronal toxicity is closely regulated by B cell lymphoma 2 superfamily of proteins with consequent activation of caspase-mediated downstream cell death pathway. Understanding the molecular mechanisms at mitochondrial level involved in amphetamines' neurotoxicity can help in defining target pathways or molecules mediating these effects, as well as in developing putative therapeutic approaches to prevent or treat the acute- or long-lasting neuropsychiatric complications seen in human abusers.

Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants.

PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. Here, we show that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. We found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. We also provide evidence that loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, our data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions.

AMBRA1-induced mitophagy: A new mechanism to cope with cancer?

Dysfunctions in mitophagy, the process by which mitochondria are eliminated, are associated with cancer. We found that the proautophagic protein AMBRA1 (activating molecule in beclin 1 regulated autophagy) binds the autophagosome adapter LC3, and that this interaction is crucial for mitochondrial clearance with or without involvement of the E3-ligase PARKIN. The discovery of a novel mitophagy pathway has the potential to promote new anticancer strategies.

Inhibition of Mitochondrial Clearance and Cu/Zn-SOD Activity Enhance 6-Hydroxydopamine-Induced Neuronal Apoptosis.

Parkinson's disease (PD) is a common movement disorder among neurodegenerative diseases, involving neuronal cell death in the substantia nigra of the midbrain. Although mechanisms of cell death in PD have been studied, the exact molecular pathogenesis is still unclear. Here, we explore the relationship between two types of cell death, autophagy and apoptosis, which have been studied separately in parkinsonian mimetic model of 6-hydroxydopamine (6-OHDA). 6-OHDA induced autophagy firstly and then later inhibition of autophagy flux occurred with apoptosis. The apoptosis was prevented by treatment of pan-caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-VAD-fluoromethylketone (zVAD)), or early phase inhibitor of autophagy, 3-methyladenine (3-MA), indicating that autophagic induction was followed by the apoptosis. Interestingly, late step inhibitor of autophagy, bafilomycin A1 (BafA), aggravated 6-OHDA-induced apoptosis. This was associated with mitochondrial abnormality such as the inhibition of damaged mitochondrial clearance and aberrant increase of extracellular oxygen consumption. Furthermore, treatment of BafA did not inhibit 6-OHDA-mediated superoxide formation but strongly reduced the hydrogen peroxide production to below basal levels, indicating failure from superoxide to hydrogen peroxide. These results were accompanied by a lowered expression and activity of copper/zinc superoxide dismutase (Cu/Zn-SOD) but not of manganese SOD (MnSOD) and catalase. Thus, the present study suggests that crosstalk among apoptosis, autophagy, and oxidative stress is a causative factor of 6-OHDA-induced neuronal death and provides a mechanistic understanding of PD pathogenesis.

Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-β hydroxysteroid dehydrogenase type 10.

Mutations of the PARK2 and PINK1 genes, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, respectively, cause autosomal recessive early-onset Parkinson's disease (PD). Parkin and PINK1 cooperate in a biochemical mitochondrial quality control pathway regulating mitochondrial morphology, dynamics and clearance. This study identifies the multifunctional PD-related mitochondrial matrix enzyme 17-β hydroxysteroid dehydrogenase type 10 (HSD17B10) as a new Parkin substrate. Parkin overproduction in cells increased mitochondrial HSD17B10 abundance by a mechanism involving ubiquitin chain extension, whereas PARK2 downregulation or deficiency caused mitochondrial HSD17B10 depletion in cells and mice. HSD17B10 levels were also found to be low in the brains of PD patients with PARK2 mutations. Confocal and Förster resonance energy transfer (FRET) microscopy revealed that HSD17B10 recruited Parkin to the translocase of the outer membrane (TOM), close to PINK1, both in functional mitochondria and after the collapse of mitochondrial membrane potential (ΔΨm). PD-causing PARK2 mutations impaired interaction with HSD17B10 and the HSD17B10-dependent mitochondrial translocation of Parkin. HSD17B10 overproduction promoted mitochondrial elongation and mitigated CCCP-induced mitochondrial degradation independently of enzymatic activity. These effects were abolished by overproduction of the fission-promiting dynamin-related protein 1 (Drp1). By contrast, siRNA-mediated HSD17B10 silencing enhanced mitochondrial fission and mitophagy. These findings suggest that the maintenance of appropriate mitochondrial HSD17B10 levels is one of the mechanisms by which Parkin preserves mitochondrial quality. The loss of this protective mechanism may contribute to mitochondrial dysfunction and neuronal degeneration in autosomal recessive PD.

Autophagy-independent functions of UVRAG are essential for peripheral naive T-cell homeostasis

UV radiation resistance-associated gene (UVRAG) encodes a tumor suppressor with putative roles in autophagy, endocytic trafficking, and DNA damage repair but its in vivo role in T cells is unknown. Because conditional homozygous deletion of Uvrag in mice results in early embryonic lethality, we generated T-cell-specific UVRAG-deficient mice that lacked UVRAG expression specifically in T cells. This loss of UVRAG led to defects in peripheral homeostasis that could not be explained by the increased sensitivity to cell death and impaired proliferation observed for other autophagy-related gene knockout mice. Instead, UVRAG-deficient T-cells exhibited normal mitochondrial clearance and activation-induced autophagy, suggesting that UVRAG has an autophagy-independent role that is critical for peripheral naive T-cell homeostatic proliferation. In vivo, T-cell-specific loss of UVRAG dampened CD8(+) T-cell responses to LCMV infection in mice, delayed viral clearance, and impaired memory T-cell generation. Our data provide novel insights into the control of autophagy in T cells and identify UVRAG as a new regulator of naïve peripheral T-cell homeostasis.

PINK1 Kinase Catalytic Activity Is Regulated by Phosphorylation on Serines 228 and 402

Mutations in the PINK1 gene cause early-onset recessive Parkinson disease. PINK1 is a mitochondrially targeted kinase that regulates multiple aspects of mitochondrial biology, from oxidative phosphorylation to mitochondrial clearance. PINK1 itself is also phosphorylated, and this might be linked to the regulation of its multiple activities. Here we systematically analyze four previously identified phosphorylation sites in PINK1 for their role in autophosphorylation, substrate phosphorylation, and mitophagy. Our data indicate that two of these sites, Ser-228 and Ser-402, are autophosphorylated on truncated PINK1 but not on full-length PINK1, suggesting that the N terminus has an inhibitory effect on phosphorylation. We furthermore establish that phosphorylation of these PINK1 residues regulates the phosphorylation of the substrates Parkin and Ubiquitin. Especially Ser-402 phosphorylation appears to be important for PINK1 function because it is involved in Parkin recruitment and the induction of mitophagy. Finally, we identify Thr-313 as a residue that is critical for PINK1 catalytic activity, but, in contrast to previous reports, we find no evidence that this activity is regulated by phosphorylation. These data clarify the regulation of PINK1 through multisite phosphorylation.

Development of a Novel Assay for Pink1-Parkin-Miro1 Pathway

Mutations in the parkin and PINK1 genes are a major cause of autosomal recessive early-onset Parkinson disease (PD). Several studies have shown that PINK1 phosphorylates Parkin, Parkin substrates, and ubiquitin, thereby stimulating ubiquitination of proteins on the mitochondrial surface (i.e. Miro1) by the Parkin E3 ligase. The ubiquitinated proteins then are degraded, which signals for mitochondrial clearance. Although it is imperative to understand these events at the molecular level, current biochemical methods are cumbersome and lack the resolution to answer these questions thoroughly. Most importantly, physiological E2 partners for Parkin ligase are contested in the field. Here, we report a novel assay to study the PINK1-Parkin-Miro1 pathway that fundamentally addresses the aforementioned problems. In our assay, the ubiquitin c-terminus is chemically activated as a thioester that can undergo transthiolation with the catalytic cysteine of the Parkin E3 ligase, thereby directly charging ubiquitin to Parkin E3s without the need for E1, E2 and ATP. We demonstrate that this simplified two-component system recapitulates two native functions of Parkin, Miro1 ubiquitination and autoubiquitination. This simplified E2-bypassing assay will be generally useful to study the Parkin mechanism and to screen small molecular Parkin activators to treat PD.