Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2.

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson's disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4-TRPML1-ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca2+ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4-TRPML1-ALG2 pathway was also activated by H2 O2 , indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation-induced lysosomal retrograde transport involved both the TMEM55B-JIP4 and TRPML1-ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy-inducing signal.

A metabolic profile of polyamines in parkinson disease: A promising biomarker.

OBJECTIVE: Aging is the highest risk factor for Parkinson disease (PD). Under physiological conditions, spermidine and spermine experimentally enhance longevity via autophagy induction. Accordingly, we evaluated the ability of each polyamine metabolite to act as an age-related, diagnostic, and severity-associated PD biomarker. METHODS: Comprehensive metabolome analysis of plasma was performed in Cohort A (controls, n = 45; PD, n = 145), followed by analysis of 7 polyamine metabolites in Cohort B (controls, n = 49; PD, n = 186; progressive supranuclear palsy, n = 19; Alzheimer disease, n = 23). Furthermore, 20 patients with PD who were successively examined within Cohort B were studied using diffusion tensor imaging (DTI). Association of each polyamine metabolite with disease severity was assessed according to Hoehn and Yahr stage (H&Y) and Unified Parkinson's Disease Rating Scale motor section (UPDRS-III). Additionally, the autophagy induction ability of each polyamine metabolite was examined in vitro in various cell lines. RESULTS: In Cohort A, N8-acetylspermidine and N-acetylputrescine levels were significantly and mildly elevated in PD, respectively. In Cohort B, spermine levels and spermine/spermidine ratio were significantly reduced in PD, concomitant with hyperacetylation. Furthermore, N1,N8-diacetylspermidine levels had the highest diagnostic value, and correlated with H&Y, UPDRS-III, and axonal degeneration quantified by DTI. The spermine/spermidine ratio in controls declined with age, but was consistently suppressed in PD. Among polyamine metabolites, spermine was the strongest autophagy inducer, especially in SH-SY5Y cells. No significant genetic variations in 5 genes encoding enzymes associated with spermine/spermidine metabolism were detected compared with controls. INTERPRETATION: Spermine synthesis and N1,N8-diacetylspermidine may respectively be useful diagnostic and severity-associated biomarkers for PD. ANN NEUROL 2019;86:251-263.

NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.

Parkin-mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N-terminal SKICH domain and C-terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly-ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52-mediated mitophagy, and the NDP52-MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.

Neuroprotective effects of memantine via enhancement of autophagy.

INTRODUCTION: Chemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has been established as a disease-modifying agent against neurodegenerative diseases. METHODS: We screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models. RESULTS: The primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-d-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria. CONCLUSION: These findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.

Ethambutol neutralizes lysosomes and causes lysosomal zinc accumulation.

Ethambutol is a common medicine used for the treatment of tuberculosis, which can have serious side effects, such as retinal and liver dysfunction. Although ethambutol has been reported to impair autophagic flux in rat retinal cells, the precise molecular mechanism remains unclear. Using various mammalian cell lines, we showed that ethambutol accumulated in autophagosomes and vacuolated lysosomes, with marked Zn(2+) accumulation. The enlarged lysosomes were neutralized and were infiltrated with Zn(2+) accumulations in the lysosomes, with simultaneous loss of acidification. These results suggest that EB neutralizes lysosomes leading to insufficient autophagy, implying that some of the adverse effects associated with EB in various organs may be of this mechanism.

A treadmill exercise reactivates the signaling of the mammalian target of rapamycin (mTor) in the skeletal muscles of starved mice.

It has been well established that a starvation-induced decrease in insulin/IGF-I and serum amino acids effectively suppresses the mammalian target of rapamycin (mTor) signaling to induce autophagy, which is a major degradative cellular pathway in skeletal muscles. In this study, we investigated the systematic effects of exercise on the mTor signaling of skeletal muscles. Wild type C57BL/6J mice were starved for 24h under synchronous autophagy induction conditions. Under these conditions, endogenous LC3-II increased, while both S6-kinse and S6 ribosomal protein were dephosphorylated in the skeletal muscles, which indicated mTor inactivation. Using GFP-LC3 transgenic mice, it was also confirmed that fluorescent GFP-LC3 dots in the skeletal muscles increased, including soleus, plantaris, and gastrocnemius, which clearly showed autophagosomal induction. These starved mice were then subjected to a single bout of running on a treadmill (12m/min, 2h, with a lean of 10 degrees). Surprisingly, biochemical analyses revealed that the exercise elicited a decrease in the LC3-II/LC3-I ratio as well as an inversion from the dephosphorylated state to the rephosphorylated state of S6-kinase and ribosomal S6 in these skeletal muscles. Consistently, the GFP-LC3 dots of the skeletal muscles were diminished immediately after the exercise. These results indicated that exercise suppressed starvation-induced autophagy through a reactivation of mTor signaling in the skeletal muscles of these starved mice.

Ferulic acid induces mammalian target of rapamycin inactivation in cultured mammalian cells.

Ferulic acid (FA), a naturally occurring polyphenol abundant in vegetables and rice bran, is known to possess a potent antioxidant activity, thereby protecting cells from oxidative stress. In the present study, we show that in addition to its known anti-oxidant activity, ferulic acid exerts substantial inhibitory activity on cellular mammalian target of rapamycin (mTor)-signaling pathways. In HeLa cells and mouse primary hepatocytes cultured with conventional nutrient-rich media, ferulic acid (1 mM) elicited dephosphorylation of S6 kinase and its substrate ribosomal S6. The dephosphorylating activity of ferulic acid was almost comparable to that of rapamycin, an established mTor inhibitor (TORC1). We next investigated the effect of ferulic acid on autophagy, a major cellular degradative process, which significantly contributes to the maintenance of cell homeostasis. Using a conventional green fluorescent protein-microtubule-associated protein IA/IB light chain 3 (GFP-LC3) dot assay to evaluate autophagy flux, we showed that ferulic acid caused a significant increase in GFP-LC3 dots under serum-rich conditions in HeLa cells. The enhancement of autophagic flux by ferulic acid was almost equivalent to that of rapamycin. Furthermore, ferulic acid significantly enhanced autophagic degradation of (14)C-leucine-labeled long-lived proteins of cultured mouse hepatocytes under nutrient-rich conditions, but not nutrient-deprived conditions. These results indicate that ferulic acid is almost the equivalent of rapamycin in the ability to inhibit mTor (TORC1), which makes it a potent activator of basal autophagy.

The MAP1-LC3 conjugation system is involved in lipid droplet formation.

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.

Iron Supply via NCOA4-Mediated Ferritin Degradation Maintains Mitochondrial Functions.

Iron is an essential nutrient for mitochondrial metabolic processes, including mitochondrial respiration. Ferritin complexes store excess iron and protect cells from iron toxicity. Therefore, iron stored in the ferritin complex might be utilized under iron-depleted conditions. In this study, we show that the inhibition of lysosome-dependent protein degradation by bafilomycin A1 and the knockdown of NCOA4, an autophagic receptor for ferritin, reduced mitochondrial respiration, respiratory chain complex assembly, and membrane potential under iron-sufficient conditions. However, autophagy did not contribute to degradation of the ferritin complex under iron-sufficient conditions. Knockout of the ferritin light chain, a subunit of the ferritin complex, inhibited ferritin degradation by decreasing interactions with NCOA4. However, ferritin light chain knockout did not affect mitochondrial functions under iron-sufficient conditions, and ferritin light chain knockout cells showed a rapid reduction of mitochondrial functions compared with wild-type cells under iron-depleted conditions. These results indicate that the constitutive degradation of the ferritin complex contributes to the maintenance of mitochondrial functions.

p150glued deficiency impairs effective fusion between autophagosomes and lysosomes due to their redistribution to the cell periphery.

Dynein-dynactin has an indispensable role in autophagy and p150glued is the largest component of the dynactin complex. Here, we characterized the effects of knockdown (KD) of endogenous p150glued and of the pathogenic mutation of p150glued found in autosomal dominant p150glued-associated disorders [hereditary motor neuronopathy with vocal paresis (HMN7B) and Perry syndrome] on autophagy. Overexpression of the p150glued pathogenic mutant or siRNA KD of p150glued promoted the localization of lysosomes at the cell periphery and increased the number of autophagosomes, suggesting partial blockage of autophagic flux. Surprisingly, although autophagosomes and lysosomes were redistributed predominantly to the cell periphery in p150glued-KD cells, the autolysosome formation ratio was preserved. However, under autophagy activation conditions induced by starvation, the ratio of autophagosome-lysosome fusion in p150glued-KD cells was decreased in the early phase. Our data demonstrate that functional loss of p150glued may cause autophagic insufficiency, which may be associated with the pathogenesis of p150glued-associated disorders.

Short Overview.

Mitochondrial autophagy (mitophagy) is a mitochondrial quality control mechanism that selectively removes damaged mitochondria via autophagic degradation. Autophagic adaptor/receptor proteins contribute to the selective degradation of damaged mitochondria by autophagy. A part of them containing both ubiquitin binding domains and Atg8 interacting motif (AIM)/LC3 interacting region (LIR) motifs, which bind to the autophagy-related protein 8 (Atg8) family (LC3 and GABARAP family), lead ubiquitylated (damaged) mitochondria to selective removal. On the other hand, some specific outer mitochondrial membrane-anchored proteins containing AIM/LIR motif function as another type of autophagy adaptor/receptor proteins. Here I briefly summarize mechanisms of mitophagy and its related proteins.

Induction of PINK1/Parkin-Mediated Mitophagy.

PINK1/Parkin mitophagy is a key mechanism to contribute mitochondrial quality control, and the defects are thought to be a cause of those Parkinson's disease onsets. Upon loss of mitochondrial membrane potential, PINK1 and Parkin are activated to promote the proteasomal degradation of mitochondrial outer membrane proteins and selective elimination of damaged mitochondria by autophagy. In this chapter, we describe the methods for induction of PINK1/Parkin-mediated mitophagy in tissue culture cell lines.

Perlecan, a heparan sulfate proteoglycan, regulates systemic metabolism with dynamic changes in adipose tissue and skeletal muscle.

Perlecan (HSPG2), a heparan sulfate proteoglycan, is a component of basement membranes and participates in a variety of biological activities. Here, we show physiological roles of perlecan in both obesity and the onset of metabolic syndrome. The perinatal lethality-rescued perlecan knockout (Hspg2-/--Tg) mice showed a smaller mass and cell size of white adipose tissues than control (WT-Tg) mice. Abnormal lipid deposition, such as fatty liver, was not detected in the Hspg2-/--Tg mice, and those mice also consumed more fat as an energy source, likely due to their activated fatty acid oxidation. In addition, the Hspg2-/--Tg mice demonstrated increased insulin sensitivity. Molecular analysis revealed the significantly relatively increased amount of the muscle fiber type IIA (X) isoform and a larger quantity of mitochondria in the skeletal muscle of Hspg2-/--Tg mice. Furthermore, the perlecan-deficient skeletal muscle also had elevated levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) protein. PGC1α expression is activated by exercise, and induces mitochondrial biosynthesis. Thus, perlecan may act as a mechano-regulator of catabolism of both lipids and glucose by shifting the muscle fiber composition to oxidative fibers. Our data suggest that downregulation of perlecan is a promising strategy to control metabolic syndrome.

Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.

Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus-induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.

Decreased long-chain acylcarnitines from insufficient ß-oxidation as potential early diagnostic markers for Parkinson's disease.

Increasing evidence shows that metabolic abnormalities in body fluids are distinguishing features of the pathophysiology of Parkinson's disease. However, a non-invasive approach has not been established in the earliest or pre-symptomatic phases. Here, we report comprehensive double-cohort analyses of the metabolome using capillary electrophoresis/liquid chromatography mass-spectrometry. The plasma analyses identified 18 Parkinson's disease-specific metabolites and revealed decreased levels of seven long-chain acylcarnitines in two Parkinson's disease cohorts (n = 109, 145) compared with controls (n = 32, 45), respectively. Furthermore, statistically significant decreases in five long-chain acylcarnitines were detected in Hoehn and Yahr stage I. Likewise, decreased levels of acylcarnitine(16:0), a decreased ratio of acylcarnitine(16:0) to fatty acid(16:0), and an increased index of carnitine palmitoyltransferase 1 were identified in Hoehn and Yahr stage I of both cohorts, suggesting of initial ß-oxidation suppression. Receiver operating characteristic curves produced using 12-14 long-chain acylcarnitines provided a large area of under the curve, high specificity and moderate sensitivity for diagnosing Parkinson's disease. Our data demonstrate that a primary decrement of mitochondrial ß-oxidation and that 12-14 long-chain acylcarnitines decreases would be promising diagnostic biomarkers for Parkinson's disease.

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle.

The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.

VPS29-VPS35 intermediate of retromer is stable and may be involved in the retromer complex assembly process.

Retromer is a complex of proteins that functions in the endosome-to-Golgi retrieval cargo transport pathway. VPS35 works as the central subunit of retromer to recognize the cargos and binds with VPS29 and VPS26 via distinct domains. We show that deficiency of VPS35 or VPS29 accompanies degradation of other subunits, whereas VPS26 deficiency had no effect on VPS29 and VPS35 levels. Although VPS35 forms VPS26-VPS35 and VPS29-VPS35 sub-complexes with similar efficiency in vitro, VPS26-VPS35 was more easily degradable by the ubiquitin-proteasome-system than VPS29-VPS35. These results indicate that VPS29 and VPS35 form a biologically stable sub-complex in vivo.

CHCHD2 mutations in autosomal dominant late-onset Parkinson's disease: a genome-wide linkage and sequencing study.

BACKGROUND: Identification of causative genes in mendelian forms of Parkinson's disease is valuable for understanding the cause of the disease. We did genetic studies in a Japanese family with autosomal dominant Parkinson's disease to identify novel causative genes. METHODS: We did a genome-wide linkage analysis on eight affected and five unaffected individuals from a family with autosomal dominant Parkinson's disease (family A). Subsequently, we did exome sequencing on three patients and whole-genome sequencing on one patient in family A. Variants were validated by Sanger sequencing in samples from patients with autosomal dominant Parkinson's disease, patients with sporadic Parkinson's disease, and controls. Participants were identified from the DNA bank of the Comprehensive Genetic Study on Parkinson's Disease and Related Disorders (Juntendo University School of Medicine, Tokyo, Japan) and were classified according to clinical information obtained by neurologists. Splicing abnormalities of CHCHD2 mutants were analysed in SH-SY5Y cells. We used the Fisher's exact test to calculate the significance of allele frequencies between patients with sporadic Parkinson's disease and unaffected controls, and we calculated odds ratios and 95% CIs of minor alleles. FINDINGS: We identified a missense mutation (CHCHD2, 182C>T, Thr61Ile) in family A by next-generation sequencing. We obtained samples from a further 340 index patients with autosomal dominant Parkinson's disease, 517 patients with sporadic Parkinson's disease, and 559 controls. Three CHCHD2 mutations in four of 341 index cases from independent families with autosomal dominant Parkinson's disease were detected by CHCHD2 mutation screening: 182C>T (Thr61Ile), 434G>A (Arg145Gln), and 300+5G>A. Two single nucleotide variants (-9T>G and 5C>T) in CHCHD2 were confirmed to have different frequencies between sporadic Parkinson's disease and controls, with odds ratios of 2·51 (95% CI 1·48-4·24; p=0·0004) and 4·69 (1·59-13·83, p=0·0025), respectively. One single nucleotide polymorphism (rs816411) was found in CHCHD2 from a previously reported genome-wide association study; however, there was no significant difference in its frequency between patients with Parkinson's disease and controls in a previously reported genome-wide association study (odds ratio 1·17, 95% CI 0·96-1·19; p=0·22). In SH-SY5Y cells, the 300+5G>A mutation but not the other two mutations caused exon 2 skipping. INTERPRETATION: CHCHD2 mutations are associated with, and might be a cause of, autosomal dominant Parkinson's disease. Further genetic studies in other populations are needed to confirm the pathogenicity of CHCHD2 mutations in autosomal dominant Parkinson's disease and susceptibility for sporadic Parkinson's disease, and further functional studies are needed to understand how mutant CHCHD2 might play a part in the pathophysiology of Parkinson's disease. FUNDING: Japan Society for the Promotion of Science; Japanese Ministry of Education, Culture, Sports, Science and Technology; Japanese Ministry of Health, Labour and Welfare; Takeda Scientific Foundation; Cell Science Research Foundation; and Nakajima Foundation.

P150glued-associated disorders are caused by activation of intrinsic apoptotic pathway.

Mutations in p150glued cause hereditary motor neuropathy with vocal cord paralysis (HMN7B) and Perry syndrome (PS). Here we show that both overexpression of p150glued mutants and knockdown of endogenous p150glued induce apoptosis. Overexpression of a p150glued plasmid containing either a HMN7B or PS mutation resulted in cytoplasmic p150glued-positive aggregates and was associated with cell death. Cells containing mutant p150glued aggregates underwent apoptosis that was characterized by an increase in cleaved caspase-3- or Annexin V-positive cells and was attenuated by both zVAD-fmk (a pan-caspase inhibitor) application and caspase-3 siRNA knockdown. In addition, overexpression of mutant p150glued decreased mitochondrial membrane potentials and increased levels of translocase of the mitochondrial outer membrane (Tom20) protein, indicating accumulation of damaged mitochondria. Importantly, siRNA knockdown of endogenous p150glued independently induced apoptosis via caspase-8 activation and was not associated with mitochondrial morphological changes. Simultaneous knockdown of endogenous p150glued and overexpression of mutant p150glued had additive apoptosis induction effects. These findings suggest that both p150glued gain-of-toxic-function and loss-of-physiological-function can cause apoptosis and may underlie the pathogenesis of p150glued-associated disorders.

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation.

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin-proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.