Antiproliferative activities through accelerating autophagic flux by basidalin and its analogs in human cancer cells.

Basidalin, isolated from the basidiomycete Leucoagaricus naucina, has previously demonstrated antibacterial and antitumor properties against murine cancer cells in vivo, but its effects on human cancer cells remain unknown. In this study, we found that basidalin possesses antiproliferative activity against human cancer cell lines. To elucidate the antiproliferative mechanism of basidalin, we focused on autophagy. Treatment with basidalin led to an increase in LC3-II expression level, and accelerated autophagic flux through an mTOR-independent pathway. Moreover, according to the structure-activity relationship analysis-including newly synthesized basidalin analogs-the formyl group, not the amino group, contributes to the antiproliferative activities of basidalin against human cancer cells. Additionally, the antiproliferative activity of basidalin analogs was strongly correlated with autophagy-inducing activity, indicating that basidalin exhibits antiproliferative activity through autophagy induction. These data suggest that basidalin, characterized by its ability to upregulate autophagic flux, emerges as a novel anticancer drug.

Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis.

ß-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain iron accumulation. Mutations in the WD repeat domain 45 (WDR45) gene encoding WIPI4 lead to loss of function in BPAN but the cellular mechanisms of how these trigger pathology are unclear. The prevailing view in the literature is that BPAN is simply the consequence of autophagy deficiency given that WIPI4 functions in this degradation pathway. However, our data indicate that WIPI4 depletion causes ferroptosis-a type of cell death induced by lipid peroxidation-via an autophagy-independent mechanism, as demonstrated both in cell culture and in zebrafish. WIPI4 depletion increases ATG2A localization at endoplasmic reticulum-mitochondrial contact sites, which enhances phosphatidylserine import into mitochondria. This results in increased mitochondrial synthesis of phosphatidylethanolamine, a major lipid prone to peroxidation, thus enabling ferroptosis. This mechanism has minimal overlap with classical ferroptosis stimuli but provides insights into the causes of neurodegeneration in BPAN and may provide clues for therapeutic strategies.

Subacute Progressive Hearing Loss and Lower Body Parkinsonism in Primary Sjögren's Syndrome.

In primary Sjögren's syndrome, it is extremely rare to observe subacute progressive lower-body parkinsonism with severe sensory hearing loss responsive to corticosteroid therapy. Sjögren's syndrome can cause heterogeneous symptoms; therefore, its diagnosis and introduction of treatment are prone to be delayed, particularly in cases without sicca symptoms or seronegative cases, which are more likely to be seen in patients with neurological complications. This report may help clinicians identify atypical early neurological symptoms in primary Sjögren's syndrome.

Systemic Metabolic Alteration Dependent on the Thyroid-Liver Axis in Early PD.

OBJECTIVE: Parkinson's disease (PD) is a common neurodegenerative disease characterized by initial involvement of the olfactory bulb/amygdala or autonomic nerves followed by nigral degeneration. Although autonomic innervation strictly regulates multiorgan systems, including endocrine functions, circulation, and digestion, how dysautonomia in PD affects systemic metabolism has not been identified. In this study, we tried to estimate the pathogenic linkage of PD by nuclear medicine techniques, trans-omic analysis of blood samples, and cultured cell experiments. METHODS: Thyroid mediastinum ratio of 123 I-metaiodobenzylguanidine (MIBG) scintigraphy was measured in 1,158 patients with PD. Furthermore, serum exosome miRNA transcriptome analysis and plasma metabolome analysis followed by trans-omic analysis were performed in patients with de novo PD and age-matched healthy control persons. Additionally, thyroid hormone was administered to skeletal muscle and liver derived cells to evaluate the effect of hypothyroidism for these organs. RESULTS: Sympathetic denervation of thyroid correlating with its cardiac denervation was confirmed in 1,158 patients with PD by MIBG scintigraphy. Among patients with drug-naïve PD, comprehensive metabolome analysis revealed decreased levels of thyroxine and insufficient fatty acid ß-oxidation, which positively correlate with one another. Likewise, both plasma metabolome data and transcriptome data of circulating exosomal miRNAs, revealed specific enrichment of the peroxisome proliferator-activated receptor (PPARα) axis. Finally, association of thyroid hormone with PPARα-dependent ß-oxidation regulation was confirmed by in vitro experiments. INTERPRETATION: Our findings suggest that interorgan communications between the thyroid and liver are disorganized in the early stage of PD, which would be a sensitive diagnostic biomarker for PD. ANN NEUROL 2023;93:303-316.

Reversible Pisa syndrome caused by chronic subdural hematoma in a patient with Parkinson's disease: a case report.

BACKGROUND: Pisa syndrome (PS), characterized by lateral trunk flexion, is quite common in patients with Parkinson's disease (PD). Patients with PS are older and have a significantly longer disease duration, more severe motor phenotype, ongoing combined treatment with levodopa and dopamine agonists, and higher levodopa equivalent daily dose. We describe here, to the best of our knowledge, the first case of a woman with PD who developed acute-onset PS caused by chronic subdural hematoma (CSDH). CASE PRESENTATION: A 70-year-old woman developed acute-onset lateral flexion of her trunk to the left side while standing, and she was admitted to our hospital. One month before, she had a mild head trauma with loss of consciousness. At 65 years of age, she noticed difficulty with walking and clumsiness with her hands. She was diagnosed as having PD (Hoehn and Yahr stage 2) and levodopa was initiated. Her symptoms were markedly improved. At 67 years of age, she developed orthostatic hypotension and was treated sequentially with fluids, compression stockings, and midodrine. Urgently performed brain computed tomography (CT) showed a CSDH in the right hemisphere resulting in a marked compression of the hemisphere. After surgical evacuation, her PS disappeared. She has fully recovered to her preoperative level of function. CONCLUSION: The present case provides a valuable insight, that is, the mesial frontal lobe and its connections from the posterior parietal cortex play crucial roles in maintaining the body schema and in the pathophysiology of PS. This case suggests that CSDH should be considered when clinicians examine acute-onset PS, even in patients with neurodegenerative disorders such as PD. Appropriate patient triage and timely neurosurgical intervention should be considered.

The different autophagy degradation pathways and neurodegeneration.

The term autophagy encompasses different pathways that route cytoplasmic material to lysosomes for degradation and includes macroautophagy, chaperone-mediated autophagy, and microautophagy. Since these pathways are crucial for degradation of aggregate-prone proteins and dysfunctional organelles such as mitochondria, they help to maintain cellular homeostasis. As post-mitotic neurons cannot dilute unwanted protein and organelle accumulation by cell division, the nervous system is particularly dependent on autophagic pathways. This dependence may be a vulnerability as people age and these processes become less effective in the brain. Here, we will review how the different autophagic pathways may protect against neurodegeneration, giving examples of both polygenic and monogenic diseases. We have considered how autophagy may have roles in normal CNS functions and the relationships between these degradative pathways and different types of programmed cell death. Finally, we will provide an overview of recently described strategies for upregulating autophagic pathways for therapeutic purposes.

Non-invasive diagnostic tool for Parkinson's disease by sebum RNA profile with machine learning.

Parkinson's disease (PD) is a progressive neurodegenerative disease presenting with motor and non-motor symptoms, including skin disorders (seborrheic dermatitis, bullous pemphigoid, and rosacea), skin pathological changes (decreased nerve endings and alpha-synuclein deposition), and metabolic changes of sebum. Recently, a transcriptome method using RNA in skin surface lipids (SSL-RNAs) which can be obtained non-invasively with an oil-blotting film was reported as a novel analytic method of sebum. Here we report transcriptome analyses using SSL-RNAs and the potential of these expression profiles with machine learning as diagnostic biomarkers for PD in double cohorts (PD [n = 15, 50], controls [n = 15, 50]). Differential expression analysis between the patients with PD and healthy controls identified more than 100 differentially expressed genes in the two cohorts. In each cohort, several genes related to oxidative phosphorylation were upregulated, and gene ontology analysis using differentially expressed genes revealed functional processes associated with PD. Furthermore, machine learning using the expression information obtained from the SSL-RNAs was able to efficiently discriminate patients with PD from healthy controls, with an area under the receiver operating characteristic curve of 0.806. This non-invasive gene expression profile of SSL-RNAs may contribute to early PD diagnosis based on the neurodegeneration background.

Huntingtin-lowering strategies for Huntington's disease.

INTRODUCTION: Huntington's disease (HD) is an incurable, autosomal dominant neurodegenerative disease caused by an abnormally long polyglutamine tract in the huntingtin protein. Because this mutation causes disease via gain-of-function, lowering huntingtin levels represents a rational therapeutic strategy. AREAS COVERED: We searched MEDLINE, CENTRAL, and other trial databases, and relevant company and HD funding websites for press releases until April 2020 to review strategies for huntingtin lowering, including autophagy and PROTACs, which have been studied in preclinical models. We focussed our analyses on oligonucleotide (ASOs) and miRNA approaches, which have entered or are about to enter clinical trials. EXPERT OPINION: ASO and mRNA approaches for lowering mutant huntingtin protein production and strategies for increasing mutant huntingtin clearance are attractive because they target the cause of disease. However, questions concerning the optimal mode of delivery and associated safety issues remain. It is unclear if the human CNS coverage with intrathecal or intraparenchymal delivery will be sufficient for efficacy. The extent that one must lower mutant huntingtin levels for it to be therapeutic is uncertain and the extent to which CNS lowering of wild-type huntingtin is safe is unclear. Polypharmacy may be an effective approach for ameliorating signs and symptoms and for preventing/delaying onset and progression.

Erratum: Author Correction: Mendelian neurodegenerative disease genes involved in autophagy.

[This corrects the article DOI: 10.1038/s41421-020-0158-y.].

Shared Metabolic Profile of Caffeine in Parkinsonian Disorders.

OBJECTIVE: The objective of this study was to determine comprehensive metabolic changes of caffeine in the serum of patients with parkinsonian disorders including Parkinson's disease (PD), progressive supranuclear palsy (PSP), and multiple system atrophy (MSA) and to compare this with healthy control serum. METHODS: Serum levels of caffeine and its 11 downstream metabolites from independent double cohorts consisting of PD (n = 111, 160), PSP (n = 30, 19), MSA (n = 23, 17), and healthy controls (n = 43, 31) were examined by liquid chromatography-mass spectrometry. The association of each metabolite with clinical parameters and medication was investigated. Mutations in caffeine-associated genes were investigated by direct sequencing. RESULTS: A total of 9 metabolites detected in more than 50% of participants in both cohorts were decreased in 3 parkinsonian disorders compared with healthy controls without any significant association with age at sampling, sex, or disease severity (Hoehn and Yahr stage and Unified Parkinson's Disease Rating Scale motor section) in PD, and levodopa dose or levodopa equivalent dose in PSP and MSA. Of the 9 detected metabolites, 8 in PD, 5 in PSP, and 3 in MSA were significantly decreased in both cohorts even after normalizing to daily caffeine consumption. No significant genetic variations in CYP1A2 or CYP2E1 were detected when compared with controls. CONCLUSION: Serum caffeine metabolic profiles in 3 parkinsonian diseases show a high level of overlap, indicative of a common potential mechanism such as caffeine malabsorption from the small intestine, hypermetabolism, increased clearance of caffeine, and/or reduced caffeine consumption. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Mendelian neurodegenerative disease genes involved in autophagy.

The lysosomal degradation pathway of macroautophagy (herein referred to as autophagy) plays a crucial role in cellular physiology by regulating the removal of unwanted cargoes such as protein aggregates and damaged organelles. Over the last five decades, significant progress has been made in understanding the molecular mechanisms that regulate autophagy and its roles in human physiology and diseases. These advances, together with discoveries in human genetics linking autophagy-related gene mutations to specific diseases, provide a better understanding of the mechanisms by which autophagy-dependent pathways can be potentially targeted for treating human diseases. Here, we review mutations that have been identified in genes involved in autophagy and their associations with neurodegenerative diseases.

Nonmercaptalbumin as an oxidative stress marker in Parkinson's and PARK2 disease.

OBJECTIVE: To investigate the oxidized albumin ratio, which is the redox ratio of human nonmercaptalbumin (HNA) to serum albumin (%HNA), as a biomarker in idiopathic Parkinson's disease (iPD) and related neurodegenerative disorders. METHODS: This prospective study enrolled 216 iPD patients, 15 patients with autosomal recessive familial PD due to parkin mutations (PARK2), 30 multiple system atrophy (MSA) patients, 32 progressive nuclear palsy (PSP) patients, and 143 healthy controls. HNA was analyzed using modified high-performance liquid chromatography and was evaluated alongside other parameters. RESULTS: iPD and PARK2 patients had a higher %HNA than controls (iPD vs. controls: odds ratio (OR) 1.325, P < 0.001; PARK2 vs. controls: OR 1.712, P < 0.001). Even iPD patients at an early Hoehn & Yahr stage (I and II) showed a higher %HNA than controls. iPD patients had a higher %HNA than MSA and PSP patients (iPD vs. MSA: OR 1.249, P < 0.001, iPD vs. PSP: OR 1.288, P < 0.05). When discriminating iPD patients from controls, %HNA corrected by age achieved an AUC of 0.750; when discriminating iPD patients from MSA and PSP patients, an AUC of 0.747 was achieved. Furthermore, uric acid, an antioxidant compound, was decreased in iPD patients, similar to the change in %HNA. INTERPRETATION: %HNA was significantly increased in iPD and PARK2 patients compared with controls, regardless of disease course and severity. Oxidative stress might be increased from the early stages of iPD and PARK2 and play an important role in their pathomechanisms.

Plasma metabolite biomarkers for multiple system atrophy and progressive supranuclear palsy.

Radiological biomarkers have been reported for multiple system atrophy and progressive supranuclear palsy, but serum/plasma biomarkers for each disorder have not been established. In this context, we performed a pilot study to identify disease-specific plasma biomarkers for multiple system atrophy and progressive supranuclear palsy. Plasma samples collected from 20 progressive supranuclear palsy patients, 16 multiple system atrophy patients and 20 controls were investigated by comprehensive metabolome analysis using capillary electrophoresis mass spectrometry and liquid chromatography mass spectrometry. Medication data were obtained from patients with multiple system atrophy and progressive supranuclear palsy, and correlations with associated metabolites were examined. Receiver operating characteristics curve analyses were used to investigate diagnostic values for each disorder. The levels of 15 and eight metabolites were significantly changed in multiple system atrophy and progressive supranuclear palsy, respectively. Multiple system atrophy was mainly characterized by elevation of long-chain fatty acids and neurosteroids, whereas progressive supranuclear palsy was characterized by changes in the level of oxidative stress-associated metabolites. Receiver operating characteristic curve analyses revealed that patients with multiple system atrophy or progressive supranuclear palsy were effectively differentiated from controls by 15 or 7 metabolites, respectively. Disease-specific metabolic changes of multiple system atrophy and progressive supranuclear palsy were identified. These biomarker sets should be replicated in a larger sample.

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.

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.

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.

Metabolomics-based identification of metabolic alterations in PARK2.

Objective: Parkin is the causative gene for autosomal recessive familial Parkinson's disease (PD), although it remains unclear how parkin dysfunction is involved with the general condition. Recently, serum and/or plasma metabolomics revealed alterations in metabolic pathways that might reflect pathomechanisms of idiopathic PD (iPD). Thus, we hypothesized that serum metabolomics of patients with homozygous or compound heterozygous parkin mutations (namely, PARK2) might reflect metabolic alterations due to parkin dysfunction. Methods: We enrolled 15 PARK2 patients (52 ± 17.6 years) confirmed with homozygous (seven cases) and compound heterozygous (eight cases) parkin mutations, along with 19 healthy age-matched controls (51 ± 11.5 years). We analyzed 830 metabolites from participants' serum using well-established metabolomics technologies, including ultra-high performance liquid chromatography/tandem mass spectroscopy. Results: Based on metabolic profiles, hierarchical matrix analysis can divide samples between control and PARK2 subjects. Profiles from PARK2 patients showed significantly higher levels of fatty acid (FA) metabolites and oxidized lipids, and significantly lower levels of antioxidant, caffeine, and benzoate-related metabolites. Interpretation: Metabolomics can identify specific metabolic alterations in PARK2 patients compared with controls. Alterations in FA metabolites suggest a relationship between parkin function and lipid metabolism. The elevation of oxidized lipids in combination with decreasing antioxidants may reflect general hyperoxidative stress. Decreasing benzoate-related metabolites might be due to the alteration in gut microbiota. Consequently, caffeine and its metabolites may be decreased due to malabsorption. These findings are similar to metabolic alterations in iPD. Thus, serum/plasma metabolomics may reflect the association between parkin dysfunction and parkinsonism.

Serum caffeine and metabolites are reliable biomarkers of early Parkinson disease.

OBJECTIVE: To investigate the kinetics and metabolism of caffeine in serum from patients with Parkinson disease (PD) and controls using liquid chromatography-mass spectrometry. METHODS: Levels of caffeine and its 11 metabolites in serum from 108 patients with PD and 31 age-matched healthy controls were examined by liquid chromatography-mass spectrometry. Mutations in caffeine-associated genes were screened by direct sequencing. RESULTS: Serum levels of caffeine and 9 of its downstream metabolites were significantly decreased even in patients with early PD, unrelated to total caffeine intake or disease severity. No significant genetic variations in CYP1A2 or CYP2E1, encoding cytochrome P450 enzymes primarily involved in metabolizing caffeine in humans, were detected compared with controls. Likewise, caffeine concentrations in patients with PD with motor complications were significantly decreased compared with those without motor complications. No associations between disease severity and single nucleotide variants of the ADORA2A gene encoding adenosine 2A receptor were detected, implying a dissociation of receptor sensitivity changes and phenotype. The profile of serum caffeine and metabolite levels was identified as a potential diagnostic biomarker by receiver operating characteristic curve analysis. CONCLUSION: Absolute lower levels of caffeine and caffeine metabolite profiles are promising diagnostic biomarkers for early PD. This is consistent with the neuroprotective effect of caffeine previously revealed by epidemiologic and experimental studies. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that decreased serum levels of caffeine and its metabolites identify patients with PD.

Zonisamide Administration Improves Fatty Acid ß-Oxidation in Parkinson's Disease.

Although many experimental studies have shown the favorable effects of zonisamide on mitochondria using models of Parkinson's disease (PD), the influence of zonisamide on metabolism in PD patients remains unclear. To assess metabolic status under zonisamide treatment in PD, we performed a pilot study using a comprehensive metabolome analysis. Plasma samples were collected for at least one year from 30 patients with PD: 10 without zonisamide medication and 20 with zonisamide medication. We performed comprehensive metabolome analyses of plasma with capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography time-of-flight mass spectrometry. We also measured disease severity using Hoehn and Yahr (H&Y) staging and the Unified Parkinson's Disease Rating Scale (UPDRS) motor section, and analyzed blood chemistry. In PD with zonisamide treatment, 15 long-chain acylcarnitines (LCACs) tended to be increased, of which four (AC(12:0), AC(12:1)-1, AC(16:1), and AC(16:2)) showed statistical significance. Of these, two LCACs (AC(16:1) and AC(16:2)) were also identified by partial least squares analysis. There was no association of any LCAC with age, disease severity, levodopa daily dose, or levodopa equivalent dose. Because an upregulation of LCACs implies improvement of mitochondrial ß-oxidation, zonisamide might be beneficial for mitochondrial ß-oxidation, which is suppressed in PD.

Immunocytochemical Monitoring of PINK1/Parkin-Mediated Mitophagy in Cultured Cells.

Both PINK1 and parkin are the responsible genes (PARK6 and PARK2, respectively) for familial early-onset Parkinson's disease (PD). Several lines of evidences have suggested that mitochondrial dysfunction would be associated with PD pathogenesis. Lewy body, one of PD pathological hallmarks, contains alpha-synuclein, a familial PD (PARK1/4)-gene product, which is eliminated by macroautophagy, while PINK1 and parkin coordinately mediate mitophagy (hereafter called as PINK1/parkin-mediated mitophagy) reported firstly by Youle's group. The mitochondrial quality control system is specific for elimination of damaged mitochondria especially in the loss of mitochondrial membrane potential induced by treatment with mitochondrial uncoupler like CCCP or FCCP. In this chapter, we summarized immunocytochemical methods to monitor the PINK1/parkin-mediated mitophagy using cultured cells.