bims-tofagi 2024-08-11 papers

on Mitophagy

Issue of 2024–08–11
ten papers selected by
Michele Frison, University of Cambridge and Aitor Martínez Zarate, Euskal Herriko Unibertsitatea

Naturally occurring hyperactive variants of human parkin.
Methods for Monitoring Mitophagy Using mt-Keima.
Disease-associated mutations in C-terminus of HSP70 interacting protein (CHIP) impair its ability to negatively regulate mitophagy.
Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.
Assessing Neuronal Mitophagy During Development in the Nematode Caenorhabditis elegans.
Assays to Study Mitophagy in Plants.
Monitoring Mitophagy Dynamics in Live Cells.
Analysis of Mitophagy Reporters in Drosophila.
Role of AMBRA1 in mitophagy regulation: emerging evidence in aging-related diseases.
Preserving mitochondrial homeostasis protects against drug-induced liver injury via inducing OPTN (optineurin)-dependent Mitophagy.

Commun Biol. 2024 Aug 08. 7(1): 961

Naturally occurring hyperactive variants of human parkin.

Tahrima Saiha Huq, Jean Luo, Rayan Fakih, Véronique Sauvé, Kalle Gehring.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Although most cases are sporadic and occur later in life, 10-15% of cases are genetic. Loss-of-function mutations in the ring-between-ring E3 ubiquitin ligase parkin, encoded by the PRKN gene, cause autosomal recessive forms of early onset PD. Together with the kinase PINK1, parkin forms a mitochondrial quality control pathway that tags damaged mitochondria for clearance. Under basal conditions, parkin is inhibited and compounds that increase its activity have been proposed as a therapy for PD. Recently, several naturally occurring hyperactive parkin variants were identified, which increased mitophagy in cultured cells. Here, we validate the hyperactivities of these variants in vitro and compare the levels of activity of the variants to those of the wild-type and the well-characterized hyperactive variant, W403A. We also study the effects of mutating the parkin ACT (activating element) on parkin activity in vitro. This work advances our understanding of the pathogenicity of parkin variants and is an important first step in the design of molecules to increase parkin activity.

DOI: https://doi.org/10.1038/s42003-024-06656-x
Methods Mol Biol. 2024 ;2845 151-160

Methods for Monitoring Mitophagy Using mt-Keima.

Alexandra J Gilsrud, Derek P Narendra.

  Mitochondria-targeted Keima (mt-Keima) is a pH-sensitive, acid-stable fluorescent protein used for the quantification of mitophagy. Mt-Keima contains a mitochondrial matrix targeting sequence and has bimodal excitation with peaks at 440 nM in neutral environments and 586 nM in acidic environments. From this bimodal excitation, a ratiometric signal may be calculated to quantify mitophagy in live cells. This chapter describes procedures for measuring mitophagy by flow cytometry and live cell confocal microscopy with mt-Keima.

Keywords:  Mitochondria; Mitophagy; PINK1; Parkin; Selective autophagy

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_12
Neurobiol Dis. 2024 Aug 06. pii: S0969-9961(24)00225-0. [Epub ahead of print] 106625

Disease-associated mutations in C-terminus of HSP70 interacting protein (CHIP) impair its ability to negatively regulate mitophagy.

Rebecca Earnshaw, Yu Tong Zhang, Gregory Heymann, Kazuko Fujisawa, Sarah Hui, Minesh Kapadia, Lorraine V Kalia, Suneil K Kalia.

  C-terminus of HSP70 interacting protein (CHIP) is an E3 ubiquitin ligase and HSP70 cochaperone. Mutations in the CHIP encoding gene are the cause of two forms of neurodegenerative conditions: spinocerebellar ataxia autosomal dominant type 48 (SCA48) and autosomal recessive type 16 (SCAR16). The mechanisms underlying CHIP-associated diseases are currently unknown. Mitochondrial dysfunction, specifically dysfunction in mitochondrial autophagy (mitophagy), is increasingly being implicated in neurodegenerative diseases and loss of CHIP has been demonstrated to result in mitochondrial dysfunction in multiple animal models, although how CHIP is involved in mitophagy regulation has been previously unknown. Here, we demonstrate that CHIP acts as a negative regulator of the PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy pathway, promoting the degradation of PINK, impairing Parkin translocation to the mitochondria, and suppressing mitophagy in response to mitochondrial stress. We also show that loss of CHIP enhances neuronal mitophagy in a PINK1 and Parkin dependent manner in Caenorhabditis elegans. Furthermore, we find that multiple disease-associated mutations in CHIP dysregulate mitophagy both in vitro and in vivo in C. elegans neurons, a finding which could implicate mitophagy dysregulation in CHIP-associated diseases.

Keywords:  Ataxia; Mitophagy; Neurodegeneration; SCA48; STUB1

DOI:  https://doi.org/10.1016/j.nbd.2024.106625
Methods Mol Biol. 2024 ;2845 1-14

Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.

Yuki Nakayama, Koji Okamoto.

  Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.

Keywords:  Atg32; Budding yeast; Fluorescence microscopy; Immunoblotting; Mitochondria

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_1
Methods Mol Biol. 2024 ;2845 55-66

Assessing Neuronal Mitophagy During Development in the Nematode Caenorhabditis elegans.

Christina Ploumi, Antonis Roussos, Konstantinos Palikaras.

  Preserving mitochondrial homeostasis is vital, particularly for the energetically demanding and metabolically active nerve cells. Mitophagy, the selective autophagic removal of mitochondria, stands out as a prominent mechanism for efficient mitochondrial turnover, which is crucial for proper neuronal development and function. Dysfunctional mitochondria and disrupted mitophagy pathways have been linked to a diverse array of neurological disorders. The nematode Caenorhabditis elegans, with its well-defined nervous system, serves as an excellent model to unravel the intricate involvement of mitophagy in developing neurons. This chapter describes the use of Rosella biosensor in C. elegans to monitor neuronal mitophagy, providing a user-friendly platform for screening genes and drugs affecting mitophagic pathways under physiological conditions or in the context of neurodevelopmental pathologies.

Keywords:  Caenorhabditis elegans; Development; Mitochondria; Mitophagy; Neurodevelopmental diseases; Neurons; Rosella biosensor

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_5
Methods Mol Biol. 2024 ;2845 39-53

Assays to Study Mitophagy in Plants.

Chengyang Li, Juncai Ma, Tian Zhen, Byung-Ho Kang, Pengwei Wang.

  Like most eukaryotic cells, mitophagy is essential in plant development and stress response. Several recent studies have revealed proteins that regulate this process, such as Friendly (FMT) and TraB family proteins (TRB), which are plant-unique mitophagy regulators so far. Here, we describe methods for studying mitophagy activity in plants through conventional microscopy and the use of loss-of-function mutants, such as using transgenic mitochondrial marker lines followed by image analysis, chemical inhibitor treatment, and plant phenotype studies. These methods can be used in combination to identify the putative mitophagy regulators and understand their functions in mitochondrial-related activities in plants.

Keywords:  Arabidopsis thaliana; De-etiolation; Mitochondria; Mitophagy; Nicotiana benthamiana

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_4
Methods Mol Biol. 2024 ;2845 161-175

Monitoring Mitophagy Dynamics in Live Cells.

Christina Karantanou, Sofia-Iris Bibli.

  The purpose of this protocol is to provide a comprehensive, stepwise guide for assessing mitophagy flux utilizing a live-cell mt-KEIMA approach. The proposed protocol is sensitive, reproducible, quantitative, and easy to perform. While mitophagy has been extensively studied, current methodologies primarily focus on terminal measurements, neglecting the dynamic aspect of this process. Hence, the introduction of this straightforward live-cell mitophagy tracing protocol enables real-time monitoring of the dynamics of mitochondrial selective autophagy, thereby enhancing the ability to draw conclusions regarding key regulators and the reversibility of the process. The assay employs a lentiviral approach to induce mt-KEIMA expression in primary or immortalized cell lines. Subsequently, the respective mitophagy reporter cells are observed using a live-cell imaging system at specific time intervals, and further quantification allows the detection of mitophagy flux. This protocol has proven efficacious in investigating mitophagy flux, including responses to chemical inducers or genetically modified cells over time. Notably, this approach is well-suited for large throughput screening of chemicals or appropriate gene-editing libraries that may influence mitophagy responses in cells.

Keywords:  Flux; Live-cell imaging; Mitophagy; Screening; mt-Keima

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_13
Methods Mol Biol. 2024 ;2845 79-93

Analysis of Mitophagy Reporters in Drosophila.

Alvaro Sanchez-Martinez, Aitor Martinez, Alexander J Whitworth.

  Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.

Keywords:  Brain; Drosophila; Light microscopy; Mitochondria; Mitophagy; Muscle; Neurodegeneration; Reporter; mito-QC; mtx-QC

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_7
Autophagy. 2024 Aug 08.

Role of AMBRA1 in mitophagy regulation: emerging evidence in aging-related diseases.

Martina Di Rienzo, Alessandra Romagnoli, Giulia Refolo, Tiziana Vescovo, Fabiola Ciccosanti, Candida Zuchegna, Francesca Lozzi, Luca Occhigrossi, Mauro Piacentini, Gian Maria Fimia.

  Aging is a gradual and irreversible physiological process that significantly increases the risks of developing a variety of pathologies, including neurodegenerative, cardiovascular, metabolic, musculoskeletal, and immune system diseases. Mitochondria are the energy-producing organelles, and their proper functioning is crucial for overall cellular health. Over time, mitochondrial function declines causing an increased release of harmful reactive oxygen species (ROS) and DNA, which leads to oxidative stress, inflammation and cellular damage, common features associated with various age-related pathologies. The impairment of mitophagy, the selective removal of damaged or dysfunctional mitochondria by autophagy, is relevant to the development and progression of age-related diseases. The molecular mechanisms that regulates mitophagy levels in aging remain largely uncharacterized. AMBRA1 is an intrinsically disordered scaffold protein with a unique property of regulating the activity of both proliferation and autophagy core machineries. While the role of AMBRA1 during embryonic development and neoplastic transformation has been extensively investigated, its functions in post-mitotic cells of adult tissues have been limited due to the embryonic lethality caused by AMBRA1 deficiency. Recently, a key role of AMBRA1 in selectively regulating mitophagy in post-mitotic cells has emerged. Here we summarize and discuss these results with the aim of providing a comprehensive view of the mitochondrial roles of AMBRA1, and how defective activity of AMBRA1 has been functionally linked to mitophagy alterations observed in age-related degenerative disorders, including muscular dystrophy/sarcopenia, Parkinson diseases, Alzheimer diseases and age-related macular degeneration.

Keywords:  AMBRA1; Aging; aging-related diseases; autophagy; mitochondria; mitophagy

DOI:  https://doi.org/10.1080/15548627.2024.2389474
Autophagy. 2024 Aug 04. 1-20

Preserving mitochondrial homeostasis protects against drug-induced liver injury via inducing OPTN (optineurin)-dependent Mitophagy.

Jiajia Wang, Yueping Qiu, Lijun Yang, Jincheng Wang, Jie He, Chengwu Tang, Zhaoxu Yang, Wenxiang Hong, Bo Yang, Qiaojun He, Qinjie Weng.

  Disruption of mitochondrial function is observed in multiple drug-induced liver injuries (DILIs), a significant global health threat. However, how the mitochondrial dysfunction occurs and whether maintain mitochondrial homeostasis is beneficial for DILIs remains unclear. Here, we show that defective mitophagy by OPTN (optineurin) ablation causes disrupted mitochondrial homeostasis and aggravates hepatocytes necrosis in DILIs, while OPTN overexpression protects against DILI depending on its mitophagic function. Notably, mass spectrometry analysis identifies a new mitochondrial substrate, GCDH (glutaryl-CoA dehydrogenase), which can be selectively recruited by OPTN for mitophagic degradation, and a new cofactor, VCP (valosin containing protein) that interacts with OPTN to stabilize BECN1 during phagophore assembly, thus boosting OPTN-mediated mitophagy initiation to clear damaged mitochondria and preserve mitochondrial homeostasis in DILIs. Then, the accumulation of OPTN in different DILIs is further validated with a protective effect, and pyridoxine is screened and established to alleviate DILIs by inducing OPTN-mediated mitophagy. Collectively, our findings uncover a dual role of OPTN in mitophagy initiation and implicate the preservation of mitochondrial homeostasis via inducing OPTN-mediated mitophagy as a potential therapeutic approach for DILIs.Abbreviation: AILI: acetaminophen-induced liver injury; ALS: amyotrophic lateral sclerosis; APAP: acetaminophen; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DILI: drug-induced liver injury; FL: full length; GCDH: glutaryl-CoA dehydrogenase; GOT1/AST: glutamic-oxaloacetic transaminase 1; GO: gene ontology; GSEA: gene set enrichment analysis; GPT/ALT: glutamic - pyruvic transaminase; INH: isoniazid; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMP: mitochondrial membrane potential; MST: microscale thermophoresis; MT-CO2/COX-II: mitochondrially encoded cytochrome c oxidase II; OPTN: optineurin; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; TSN: toosendanin; VCP: valosin containing protein, WIPI2: WD repeat domain, phosphoinositide interacting 2.

Keywords:  Drug-induced liver injury; mitochondrial homeostasis; mitophagy; optineurin; phagophore formation

DOI:  https://doi.org/10.1080/15548627.2024.2384348