bims-mitdis 2024-08-11 papers

bims-mitdis

Biomed News

on Mitochondrial disorders

Issue of 2024‒08‒11
38 papers selected by
Catalina Vasilescu, Helmholz Munich

Discovery of novel disease-causing mutation in SSBP1 and its correction using adenine base editor to improve mitochondrial function.
The interplay between mitochondria, the gut microbiome and metabolites and their therapeutic potential in primary mitochondrial disease.
SLC25A48 controls mitochondrial choline import and metabolism.
Naturally occurring hyperactive variants of human parkin.
The clinical and genetic spectrum of mitochondrial diseases in China: A multicenter retrospective cross-sectional study.
Hierarchical tricarboxylic acid cycle regulation by hepatocyte arginase 2 links the urea cycle to oxidative metabolism.
Endocrine Disorders in Children with Primary Mitochondrial Diseases: Single-Center Experience.
Leigh Syndrome due to MT-ATP6 Variants: A Case Presentation and the Review of the Literature.
A Comprehensive Review of Leber Hereditary Optic Neuropathy and Its Association with Multiple Sclerosis-Like Phenotypes Known as Harding's Disease.
Mitochondrial copper overload promotes renal fibrosis via inhibiting pyruvate dehydrogenase activity.
Assessing Neuronal Mitophagy During Development in the Nematode Caenorhabditis elegans.
Purinosomes spatially co-localize with mitochondrial transporters.
Nicotinamide riboside first alleviates symptoms but later downregulates dopamine metabolism in proteasome inhibition mouse model of Parkinson's disease.
Real time precisely tracing the fluctuations of mitochondrial SO2 in cells during ferroptosis and tissues using a mitochondrial-immobilized ratiometric fluorescent probe.
Neuro-intestinal acetylcholine signalling regulates the mitochondrial stress response in Caenorhabditis elegans.
Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ.
Activation of Mitochondria in Mesenchymal Stem Cells by Mitochondrial Delivery of Coenzyme Q10.
Methods for Monitoring Mitophagy Using mt-Keima.
Short-term starvation activates AMPK and restores mitochondrial inorganic polyphosphate, but fails to reverse associated neuronal senescence.
Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.
Acaricide Flupentiofenox Inhibits the Mitochondrial β-Oxidation Pathway of Fatty Acids.
Mitochondrial NME6: A Paradigm Change within the NME/NDP Kinase Protein Family?
Real-Time Monitoring of mtDNA Aggregation and Mitophagy Induced by a Fluorescent Platinum Complex in Living Cells.
Evolution and maintenance of mtDNA gene content across eukaryotes.
PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress.
Mitochondrial fragmentation in early differentiation of human MB135 myoblasts: Role of mitochondrial ROS production in the absence of depolarization.
Peroxiredoxin 2 Regulates DAF-16/FOXO Mediated Mitochondrial Remodelling in Response to Exercise that is Disrupted in Ageing.
A Method for Producing Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Leigh Syndrome Patients for Its Application in Disease Modeling and Drug Validation.
Preserving mitochondrial homeostasis protects against drug-induced liver injury via inducing OPTN (optineurin)-dependent Mitophagy.
Mitochondrial dysfunction and NDUFS3: Insights from a PINK1B9 Drosophila model in Parkinson's disease pathogenesis.
Protocol for the production and reconstitution of VDAC1 for functional assays.
Novel Variant of FDXR as a Molecular Etiology of Postlingual Post-Synaptic Auditory Neuropathy Spectrum Disorder via Mitochondrial Dysfunction: Reiteration of the Correlation between Genotype and Cochlear Implantation Outcomes.
The Utility of Long-Read Sequencing in Diagnosing Genetic Autosomal Recessive Parkinson's Disease: a genetic screening study.
Disease-associated mutations in C-terminus of HSP70 interacting protein (CHIP) impair its ability to negatively regulate mitophagy.
Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.
Stage-dependent phosphoproteome remodeling of Parkinson's disease blood cells.
Modeling riboflavin transporter deficiency type 2: from iPSC-derived motoneurons to iPSC-derived astrocytes.
Diagnostic utility of DNA methylation analysis in genetically unsolved pediatric epilepsies and CHD2 episignature refinement.

Mol Ther Nucleic Acids. 2024 Sep 10. 35(3): 102257

Discovery of novel disease-causing mutation in SSBP1 and its correction using adenine base editor to improve mitochondrial function.

Ju Hyuen Cha, Seok-Hoon Lee, Yejin Yun, Won Hoon Choi, Hansol Koo, Sung Ho Jung, Ho Byung Chae, Dae Hee Lee, Seok Jae Lee, Dong Hyun Jo, Jeong Hun Kim, Jae-Jin Song, Jong-Hee Chae, Jun Ho Lee, Jiho Park, Jin Young Kang, Sangsu Bae, Sang-Yeon Lee.

  Mutations in nuclear genes regulating mitochondrial DNA (mtDNA) replication are associated with mtDNA depletion syndromes. Using whole-genome sequencing, we identified a heterozygous mutation (c.272G>A:p.Arg91Gln) in single-stranded DNA-binding protein 1 (SSBP1), a crucial protein involved in mtDNA replisome. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy. This mutation impeded multimer formation and DNA-binding affinity, leading to reduced efficiency of mtDNA replication, altered mitochondria dynamics, and compromised mitochondrial function. To correct this mutation, we tested two adenine base editor (ABE) variants on patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), showed higher editing efficacy (≤30%) and enhanced mitochondrial replication and function, despite off-target editing frequencies; however, risks from bystander editing were limited due to silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (≤10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. Taken together, base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.

Keywords:  MT: RNA/ DNA editing; NG-ABE8e; NG-ABE8eWQ; NG-Cas9-based ABE8e; NG-Cas9-based ABE8eWQ; SSBP1; editing efficacy; mitochondrial diseases; myopathy; off-target effects; optic atrophy; sensorineural deafness; single-stranded binding protein 1

DOI:  https://doi.org/10.1016/j.omtn.2024.102257
Front Pharmacol. 2024 ;15 1428242

The interplay between mitochondria, the gut microbiome and metabolites and their therapeutic potential in primary mitochondrial disease.

Kassandra A Zachos, Jann Aldrin Gamboa, Aleena S Dewji, Jocelyn Lee, Sonya Brijbassi, Ana C Andreazza.

  The various roles of the mitochondria and the microbiome in health and disease have been thoroughly investigated, though they are often examined independently and in the context of chronic disease. However, the mitochondria and microbiome are closely connected, namely, through their evolution, maternal inheritance patterns, overlapping role in many diseases and their importance in the maintenance of human health. The concept known as the "mitochondria-microbiome crosstalk" is the ongoing bidirectional crosstalk between these two entities and warrants further exploration and consideration, especially in the context of primary mitochondrial disease, where mitochondrial dysfunction can be detrimental for clinical manifestation of disease, and the role and composition of the microbiome is rarely investigated. A potential mechanism underlying this crosstalk is the role of metabolites from both the mitochondria and the microbiome. During digestion, gut microbes modulate compounds found in food, which can produce metabolites with various bioactive effects. Similarly, mitochondrial metabolites are produced from substrates that undergo biochemical processes during cellular respiration. This review aims to provide an overview of current literature examining the mitochondria-microbiome crosstalk, the role of commonly studied metabolites serve in signaling and mediating these biochemical pathways, and the impact diet has on both the mitochondria and the microbiome. As a final point, this review highlights the up-to-date implications of the mitochondria-microbiome crosstalk in mitochondrial disease and its potential as a therapeutic tool or target.

Keywords:  diet; microbiome; mitochondria; mitochondria-microbiome crosstalk; mitochondrial disease

DOI:  https://doi.org/10.3389/fphar.2024.1428242
Cell Metab. 2024 Aug 01. pii: S1550-4131(24)00281-X. [Epub ahead of print]

SLC25A48 controls mitochondrial choline import and metabolism.

Anthony R P Verkerke, Xu Shi, Mark Li, Yusuke Higuchi, Tadashi Yamamuro, Daisuke Katoh, Hiroshi Nishida, Christopher Auger, Ichitaro Abe, Robert E Gerszten, Shingo Kajimura.

  Choline is an essential nutrient for the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism with a critical step being its import into mitochondria. However, the underlying mechanisms and biological significance remain poorly understood. Here, we report that SLC25A48, a previously uncharacterized mitochondrial inner-membrane carrier protein, controls mitochondrial choline transport and the synthesis of choline-derived methyl donors. We found that SLC25A48 was required for brown fat thermogenesis, mitochondrial respiration, and mitochondrial membrane integrity. Choline uptake into the mitochondrial matrix via SLC25A48 facilitated the synthesis of betaine and purine nucleotides, whereas loss of SLC25A48 resulted in increased production of mitochondrial reactive oxygen species and imbalanced mitochondrial lipids. Notably, human cells carrying a single nucleotide polymorphism on the SLC25A48 gene and cancer cells lacking SLC25A48 exhibited decreased mitochondrial choline import, increased oxidative stress, and impaired cell proliferation. Together, this study demonstrates that SLC25A48 regulates mitochondrial choline catabolism, bioenergetics, and cell survival.

Keywords:  bioenergetics; brown adipose tissue; cancer metabolism; choline; mitochondria; purine nucleotides

DOI:  https://doi.org/10.1016/j.cmet.2024.07.010
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
Clin Genet. 2024 Aug 09.

The clinical and genetic spectrum of mitochondrial diseases in China: A multicenter retrospective cross-sectional study.

Yang Zhao, Xutong Zhao, Kunqian Ji, Junling Wang, Yuying Zhao, Jie Lin, Qiang Gang, Meng Yu, Yun Yuan, Haishan Jiang, Chong Sun, Fang Fang, Chuanzhu Yan, Zhaoxia Wang.

  Mitochondrial diseases (MtDs) present diverse clinical phenotypes, yet large-scale studies are hindered by their rarity. This retrospective, multicenter study, conducted across five Chinese hospitals' neurology departments from 2009 to 2019, aimed to address this gap. Nationwide, 1351 patients were enrolled, with a median onset age of 14.0 (18.5) years. The predominant phenotype was mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) (45.0%). Mitochondrial DNA (mtDNA) mutations were prevalent (87.4%), with m.3243A>G being the most common locus (48.7%). Meanwhile, POLG mutations in nuclear DNA (nDNA) accounted for 16.5%. Comparative analysis based on age groups (with a cut-off at 14 years) revealed the highest prevalence of MELAS, with Leigh syndrome (LS) and chronic progressive external ophthalmoplegia (CPEO) being the second most common phenotypes in junior and senior groups, respectively. Notably, the most commonly mutated nuclear genes varied across age groups. In conclusion, MELAS predominated in this Chinese MtD cohort, underscored by m.3243A>G and POLG as principal mtDNA mutations and pathogenic nuclear genes. The phenotypic and genotypic disparities observed among different age cohorts highlight the complex nature of MtDs.

Keywords:  genotype; heterogeneity; mitochondrial diseases; phenotype

DOI:  https://doi.org/10.1111/cge.14605
Cell Metab. 2024 Aug 01. pii: S1550-4131(24)00278-X. [Epub ahead of print]

Hierarchical tricarboxylic acid cycle regulation by hepatocyte arginase 2 links the urea cycle to oxidative metabolism.

Yiming Zhang, Cassandra B Higgins, Stefani Tica, Joshua A Adams, Jiameng Sun, Shannon C Kelly, Xiaoyu Zong, Dennis J Dietzen, Terri Pietka, Samuel J Ballentine, Leah Shriver, Gary Patti, Yin Cao, Brian J DeBosch.

  Urea cycle impairment and its relationship to obesity and inflammation remained elusive, partly due to the dramatic clinical presentation of classical urea cycle defects. We generated mice with hepatocyte-specific arginase 2 deletion (Arg2LKO) and revealed a mild compensated urea cycle defect. Stable isotope tracing and respirometry revealed hepatocyte urea and TCA cycle flux defects, impaired mitochondrial oxidative metabolism, and glutamine anaplerosis despite normal energy and glucose homeostasis during early adulthood. Yet during middle adulthood, chow- and diet-induced obese Arg2LKO mice develop exaggerated glucose and lipid derangements, which are reversible by replacing the TCA cycle oxidative substrate nicotinamide adenine dinucleotide. Moreover, serum-based hallmarks of urea, TCA cycle, and mitochondrial derangements predict incident fibroinflammatory liver disease in 106,606 patients nearly a decade in advance. The data reveal hierarchical urea-TCA cycle control via ARG2 to drive oxidative metabolism. Moreover, perturbations in this circuit may causally link urea cycle compromise to fibroinflammatory liver disease.

Keywords:  arginase; diabetes; fasting; metabolic dysfunction-associated steatohepatitis; metabolic dysfunction-associated steatotic liver disease; nicotinamide adenine dinucleotide; nicotinamide riboside; obesity; tricarboxylic acid cycle; urea cycle

DOI:  https://doi.org/10.1016/j.cmet.2024.07.007
J Clin Res Pediatr Endocrinol. 2024 Aug 08.

Endocrine Disorders in Children with Primary Mitochondrial Diseases: Single-Center Experience.

Esra Deniz Papatya Çakır, Melike Ersıoy, Nihan Çakır Biçer, Asuman Gedikbaşı.

  Objective: Endocrine abnormalities may represent the only clinical manifestation of primary mitochondrial disorders. This study aimed to evaluate the endocrinological characteristics of mitochondrial disease in our cohort.Methods: A total of twenty-six pediatric patients diagnosed with mitochondrial disease were categorized on the basis of their specific genetic abnormalities. The auxologic data, pubertal development, and, based on their clinical symptoms, hormonal profiles were obtained.
Results: Twelve of the cohort of 26 patients (46%) were female. In 15 of the patients (57.6%), their mitochondrial disease (MD) was caused by nuclear DNA mutations (nDNA group). Four patients had Leigh syndrome, 2 patients had LHON syndrome, 2 patients had MELAS, and 1 patient had KSS clinical phenotype. The median age at diagnosis was 2.91 (0.59-16.8) years, and the median age at first endocrinologic evaluation was 4.62 (1.26-18) years. The mean height SDS was -1.34 ± 2.12, and the mean BMI SDS was -0.82 ± 1.96 for all patients. Of the 26 patients, 6 (23%) had a range of hormonal deficits. Ovarian insufficiency, central adrenal insufficiency, central hypothyroidism, diabetes mellitus, and critical illness-related adrenal insufficiency were all observed. Three of the patients were initially monitored in the endocrine clinic for hormone deficiencies but it was later determined that the hormonal abnormalities were caused by underlying mitochondrial disease.
Conclusion: Individuals diagnosed with mitochondrial disease, particularly those with specific genetic abnormalities, are considered a high-risk group for developing hormonal deficits. Endocrine diseases could be one of the primary mitochondrial disorders' early warning symptoms.

Keywords:  Endocrin abnormalities; Endocrine disorders; Primary mitochondrial disease; genotype-phenotype

DOI:  https://doi.org/10.4274/jcrpe.galenos.2024.2024-1-11
Mol Syndromol. 2024 Aug;15(4): 333-338

Leigh Syndrome due to MT-ATP6 Variants: A Case Presentation and the Review of the Literature.

Halil Tuna Akar, Esra Sayar, Özlem Sarıtaş Nakip, Esra Sönmez, Mehmet Burak Özkan, Asburçe Olgaç.

  Introduction: Leigh syndrome is a rare mitochondrial disorder characterized by subacute necrotizing encephalomyelopathy, resulting from defects in mitochondrial respiratory enzymes or pyruvate dehydrogenase complex. Symptoms can manifest in infancy, childhood, or adulthood. We present a case of a 7-month-old girl initially misdiagnosed with septic shock but was later found to have Leigh encephalomyelopathy due to MT-ATP6 deficiency.Case Presentation: A 7-month-old girl was admitted with fever, drowsiness, and wheezing, initially diagnosed with septic shock. She had a history of parental consanguinity and hypotonia. Physical examination revealed unconsciousness, miotic pupils, and respiratory distress. Initial laboratory tests showed significant metabolic acidosis and elevated lactate, creatine kinase, and ammonia levels. The patient was treated for sepsis and shock, but her condition worsened with elevated lactate and liver transaminases, eventually leading to hypertrophic cardiomyopathy and multiorgan failure. Her basic metabolic scans showed extremely low citrulline levels, whole-exome sequencing analysis did not show any pathologic change in nuclear genome, and mitochondrial genome analysis revealed an MT-ATP homoplasmic variant. She passed away on the 22nd day of hospitalization.
Discussion/Conclusion: While mitochondrial disorders are broadly acknowledged for their phenotypic diversity, it is essential to note that specific disorders, such as Leigh syndrome, display distinctive presentations with varying degrees of severity. Factors such as the percentage of homoplasmy contribute to the variability in manifestations. Notably, MT-ATP6-associated Leigh syndrome is predominantly characterized by an early onset, typically occurring before the age of 2 years. Low citrulline levels have been observed in approximately 90% of patients with MT-ATP6-related disorders, distinguishing them from other mitochondrial disorders. The exact mechanisms underlying this specific metabolic alteration are not fully understood, but it could be linked to disruptions in the mitochondrial energy production process. The mitochondria are essential for various metabolic pathways, including the urea cycle, where citrulline is involved. The association between low citrulline levels and MT-ATP6-related disorders raises the possibility of using citrulline as a potential biomarker for disease identification. MT-ATP6 defects should be kept in mind in cases with mitochondrial disease and low plasma citrulline levels.

Keywords:  Inborn errors of metabolism; Leigh’s syndrome; Mitochondrial disorders

DOI:  https://doi.org/10.1159/000536676
Eye Brain. 2024 ;16 17-24

A Comprehensive Review of Leber Hereditary Optic Neuropathy and Its Association with Multiple Sclerosis-Like Phenotypes Known as Harding's Disease.

Jehad Alorainy, Yara Alorfi, Rustum Karanjia, Nooran Badeeb.

  Leber Hereditary Optic Neuropathy (LHON) stands as a distinctive maternally inherited mitochondrial disorder marked by painless, subacute central vision loss, primarily affecting young males. This review covers the possible relationship between LHON and multiple sclerosis (MS), covering genetic mutations, clinical presentations, imaging findings, and treatment options. LHON is associated with mutations in mitochondrial DNA (mtDNA), notably m.11778G>A, m.3460G>A, and m.14484T>C, affecting complex I subunits. Beyond ocular manifestations, LHON can go beyond the eye into a multi-systemic disorder, showcasing extraocular abnormalities. Clinical presentations, varying in gender prevalence and outcomes, underscore the nature of mitochondrial optic neuropathies. Hypotheses exploring the connection between LHON and MS encompass mitochondrial DNA mutations triggering neurological diseases, immunologically mediated responses inducing demyelination, and the possibility of coincidental diseases. The research on mtDNA mutations among MS patients sheds light on potential associations with specific clinical subgroups, offering a unique perspective into the broader landscape of MS. Imaging findings, ranging from white matter alterations to cerebrospinal fluid biomarkers, further emphasize shared pathological processes between LHON-MS and classical MS. This comprehensive review contributes to the understanding of the complex relationship between LHON and MS.

Keywords:  demyelination diseases; mitochondrial DNA; neuro-ophthalmology; visual impairment

DOI:  https://doi.org/10.2147/EB.S470184
Cell Mol Life Sci. 2024 Aug 09. 81(1): 340

Mitochondrial copper overload promotes renal fibrosis via inhibiting pyruvate dehydrogenase activity.

Saiya Zhu, Yangyang Niu, Wenqian Zhou, Yuqing Liu, Jing Liu, Xi Liu, Limin Lu, Chen Yu.

  Copper is a trace element essential for numerous biological activities, whereas the mitochondria serve as both major sites of intracellular copper utilization and copper reservoir. Here, we investigated the impact of mitochondrial copper overload on the tricarboxylic acid cycle, renal senescence and fibrosis. We found that copper ion levels are significantly elevated in the mitochondria in fibrotic kidney tissues, which are accompanied by reduced pyruvate dehydrogenase (PDH) activity, mitochondrial dysfunction, cellular senescence and renal fibrosis. Conversely, lowering mitochondrial copper levels effectively restore PDH enzyme activity, improve mitochondrial function, mitigate cellular senescence and renal fibrosis. Mechanically, we found that mitochondrial copper could bind directly to lipoylated dihydrolipoamide acetyltransferase (DLAT), the E2 component of the PDH complex, thereby changing the interaction between the subunits of lipoylated DLAT, inducing lipoylated DLAT protein dimerization, and ultimately inhibiting PDH enzyme activity. Collectively, our study indicates that mitochondrial copper overload could inhibit PDH activity, subsequently leading to mitochondrial dysfunction, cellular senescence and renal fibrosis. Reducing mitochondrial copper overload might therefore serve as a strategy to rescue renal fibrosis.

Keywords:  Copper; Mitochondria; Pyruvate dehydrogenase; Renal fibrosis; Tricarboxylic acid (TCA) cycle

DOI:  https://doi.org/10.1007/s00018-024-05358-1
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
J Biol Chem. 2024 Aug 02. pii: S0021-9258(24)02121-5. [Epub ahead of print] 107620

Purinosomes spatially co-localize with mitochondrial transporters.

Zhou Sha, Stephen J Benkovic.

  In this study, we advance our understanding of the spatial relationship between the purinosome, a liquid condensate consisting of six enzymes involved in de novo purine biosynthesis, and mitochondria. Previous research has shown that purinosomes move along tubulin toward mitochondria, suggesting a direct uptake of glycine from mitochondria. Here, we propose that the purinosome is located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, facilitating the uptake of glycine, aspartate, and glutamate, essential factors for purine synthesis. We utilized the proximity ligation assay (PLA) and APEX proximity labeling to investigate the association between purinosome proteins and mitochondrial transporters. Our results indicate that purinosome assembly occurs close to the mitochondrial membrane under purine-deficient conditions, with the transporters migrating to be adjacent to the purinosome. Furthermore, both targeted and non-targeted analyses suggest that the SLC25A13-APEX2-V5 probe accurately reflects endogenous cellular status. These findings provide insights into the spatial organization of purine biosynthesis and lay the groundwork for further investigations into additional proteins involved in this pathway.

Keywords:  APEX; Purinosomes; de novo purine biosynthesis; mitochondrial transporter; proximity ligation assay (PLA)

DOI:  https://doi.org/10.1016/j.jbc.2024.107620
Heliyon. 2024 Jul 30. 10(14): e34355

Nicotinamide riboside first alleviates symptoms but later downregulates dopamine metabolism in proteasome inhibition mouse model of Parkinson's disease.

Giorgio Turconi, Farhan Alam, Tanima SenGupta, Sini Pirnes-Karhu, Soophie Olfat, Mark S Schmidt, Kärt Mätlik, Ana Montaño-Rodriguez, Vladimir Heiskanen, Daniel Garton, Petteri T Piepponen, Charles Brenner, Carina I Holmberg, Hilde Nilsen, Eija Pirinen, Jaan-Olle Andressoo.

  Parkinson's disease (PD) is associated with a reduction in 26/20S proteasome and mitochondrial function and depletion of dopamine. Activation of mitochondrial function with the NAD+ precursor nicotinamide riboside (NR) is a potential therapeutic for PD. However, despite recently started clinical trials, analysis of NR in mammalian animal PD models is lacking and data in simpler PD models is limited. We analyzed the effect of NR in C. elegans and in mouse 26/20S proteasome inhibition models of PD. In C. elegans, NR rescued α-synuclein overexpression induced phenotypes likely by activating the mitochondrial unfolded protein response. However, in a proteasome inhibitor-induced mouse model of PD, NR first partially rescued behavioural dysfunction, but later resulted in decrease in dopamine and its related gene expression in the substantia nigra. Our results suggest that reduction in 26/20S function with long term NR treatment may increase risk for developing reduced nigrostriatal DA function.

Keywords:  Dopamine; Mitochondrial activation; Nicotinamide riboside; Parkinson's disease; Proteastasis failure

DOI:  https://doi.org/10.1016/j.heliyon.2024.e34355
Talanta. 2024 Aug 05. pii: S0039-9140(24)01033-6. [Epub ahead of print]279 126654

Real time precisely tracing the fluctuations of mitochondrial SO2 in cells during ferroptosis and tissues using a mitochondrial-immobilized ratiometric fluorescent probe.

Zekun Jiang, Jingchao Wang, Minggang Tian, Lina Zhou, Xiuqi Kong, Mei Yan.

  Mitochondrial sulfur dioxide (SO2) plays important roles in physiological and pathological activities. Unfortunately, it is lack of a reliable tool to precisely visualize the mitochondrial SO2 and elaborate its complicated functions in various cytoactivities. Here we report a mitochondrial-immobilized fluorescent probe PM-Cl consisting of coumarin and benzyl chloride modified benzothiazole, which enables selective visualization of mitochondrial SO2via chemical immobilization. The spectral results demonstrated that probe PM-Cl could respond to SO2 with high selectivity and sensitivity. Co-localization and the fluorescence of cytolysis extraction verified the excellent mitochondrial targeting and anchoring abilities. Due to the chemical immobilization, probe PM-Cl could firmly retain into mitochondria after stimulation of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and H2O2. Significantly, a series of fluorescence images are indicative of capability for detecting the fluctuations of SO2 in mitochondria during ferroptosis. Furthermore, PM-Cl also could visualize SO2 in myocardium and muscle tissues after the stimulation of CCCP. Taken together, probe PM-Cl is a very potential molecular tool for precisely detecting mitochondrial SO2 to explore its complex functions in physiological and pathological activities.

Keywords:  Chemical immobilization; Fluctuations of mitochondrial SO(2); Mitochondrial SO(2); Precise imaging; Tissue imaging

DOI:  https://doi.org/10.1016/j.talanta.2024.126654
Nat Commun. 2024 Aug 03. 15(1): 6594

Neuro-intestinal acetylcholine signalling regulates the mitochondrial stress response in Caenorhabditis elegans.

Rebecca Cornell, Wei Cao, Bernie Harradine, Rasoul Godini, Ava Handley, Roger Pocock.

Neurons coordinate inter-tissue protein homeostasis to systemically manage cytotoxic stress. In response to neuronal mitochondrial stress, specific neuronal signals coordinate the systemic mitochondrial unfolded protein response (UPRmt) to promote organismal survival. Yet, whether chemical neurotransmitters are sufficient to control the UPRmt in physiological conditions is not well understood. Here, we show that gamma-aminobutyric acid (GABA) inhibits, and acetylcholine (ACh) promotes the UPRmt in the Caenorhabditis elegans intestine. GABA controls the UPRmt by regulating extra-synaptic ACh release through metabotropic GABAB receptors GBB-1/2. We find that elevated ACh levels in animals that are GABA-deficient or lack ACh-degradative enzymes induce the UPRmt through ACR-11, an intestinal nicotinic α7 receptor. This neuro-intestinal circuit is critical for non-autonomously regulating organismal survival of oxidative stress. These findings establish chemical neurotransmission as a crucial regulatory layer for nervous system control of systemic protein homeostasis and stress responses.

DOI: https://doi.org/10.1038/s41467-024-50973-y
Nucleic Acids Res. 2024 Aug 06. pii: gkae670. [Epub ahead of print]

Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ.

Amanda A Riccio, Jonathan Bouvette, Lars C Pedersen, Shruti Somai, Robert C Dutcher, Mario J Borgnia, William C Copeland.

The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Polγ, has been minimally explored. Here, we determined a 1.9-Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low-affinity site and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses a clinically relevant residue, R38, and a highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Polγ. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Polγ that mtSSB binds.

DOI: https://doi.org/10.1093/nar/gkae670
Biol Pharm Bull. 2024 ;47(8): 1415-1421

Activation of Mitochondria in Mesenchymal Stem Cells by Mitochondrial Delivery of Coenzyme Q10.

Yuji Maruo, Masahiro Shiraishi, Mitsue Hibino, Jiro Abe, Atsuhito Takeda, Yuma Yamada.

  The efficacy of mesenchymal stem cell (MSC) transplantation has been reported for various diseases. We previously developed a drug delivery system targeting mitochondria (MITO-Porter) by using a microfluidic device to encapsulate Coenzyme Q10 (CoQ10) on a large scale. The current study aimed to confirm if treatment with CoQ10 encapsulated by MITO-Porter enhanced mitochondrial functions in MSCs, with the potential to improve MSC transplantation therapy. We used highly purified human bone marrow-derived MSCs, described as rapidly expanding clones (RECs), and attempted to control and increase the amount of CoQ10 encapsulated in the MITO-Porter using microfluidic device system. We treated these RECs with CoQ10 encapsulated MITO-Porter, and evaluated its cellular uptake, co-localization with mitochondria, changes in mitochondrial respiratory capacity, and cellular toxicity. There was no significant change in mitochondrial respiratory capacity following treatment with the previous CoQ10 encapsulated MITO-Porter; however, mitochondrial respiratory capacity in RECs was significantly increased by treatment with CoQ10-rich MITO-Porter. Utilization of a microfluidic device enabled the amount of CoQ10 encapsulated in MITO-Porter to be controlled, and treatment with CoQ10-rich MITO-Porter successfully activated mitochondrial functions in MSCs. The MITO-Porter system thus provides a promising tool to improve MSC cell transplantation therapy.

Keywords:  Coenzyme Q10; drug delivery system; lipid nanoparticle; mesenchymal stem cell; microfluidics; mitochondrial delivery

DOI:  https://doi.org/10.1248/bpb.b24-00284
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
Aging Cell. 2024 Aug 05. e14289

Short-term starvation activates AMPK and restores mitochondrial inorganic polyphosphate, but fails to reverse associated neuronal senescence.

Luca Tagliafico, Renata T Da Costa, Lavinia Boccia, Sheida Kavehmoghaddam, Bryan Ramirez, Malgorzata Tokarska-Schlattner, Ernest R Scoma, Vedangi Hambardikar, Tommaso Bonfiglio, Irene Caffa, Fiammetta Monacelli, Uwe Schlattner, J Nicholas Betley, Alessio Nencioni, Maria E Solesio.

  Neuronal senescence is a major risk factor for the development of many neurodegenerative disorders. The mechanisms that drive neurons to senescence remain largely elusive; however, dysregulated mitochondrial physiology seems to play a pivotal role in this process. Consequently, strategies aimed to preserve mitochondrial function may hold promise in mitigating neuronal senescence. For example, dietary restriction has shown to reduce senescence, via a mechanism that still remains far from being totally understood, but that could be at least partially mediated by mitochondria. Here, we address the role of mitochondrial inorganic polyphosphate (polyP) in the intersection between neuronal senescence and dietary restriction. PolyP is highly present in mammalian mitochondria; and its regulatory role in mammalian bioenergetics has already been described by us and others. Our data demonstrate that depletion of mitochondrial polyP exacerbates neuronal senescence, independently of whether dietary restriction is present. However, dietary restriction in polyP-depleted cells activates AMPK, and it restores some components of mitochondrial physiology, even if this is not sufficient to revert increased senescence. The effects of dietary restriction on polyP levels and AMPK activation are conserved in differentiated SH-SY5Y cells and brain tissue of male mice. Our results identify polyP as an important component in mitochondrial physiology at the intersection of dietary restriction and senescence, and they highlight the importance of the organelle in this intersection.

Keywords:  dietary restriction; fasting; inorganic polyphosphate; metabolism; mitochondria; polyP; senescence

DOI:  https://doi.org/10.1111/acel.14289
EMBO J. 2024 Aug 05.

Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level.

Rodaria Roussou, Dirk Metzler, Francesco Padovani, Felix Thoma, Rebecca Schwarz, Boris Shraiman, Kurt M Schmoller, Christof Osman.

  Mitochondrial DNA (mtDNA) is present in multiple copies within cells and is required for mitochondrial ATP generation. Even within individual cells, mtDNA copies can differ in their sequence, a state known as heteroplasmy. The principles underlying dynamic changes in the degree of heteroplasmy remain incompletely understood, due to the inability to monitor this phenomenon in real time. Here, we employ mtDNA-based fluorescent markers, microfluidics, and automated cell tracking, to follow mtDNA variants in live heteroplasmic yeast populations at the single-cell level. This approach, in combination with direct mtDNA tracking and data-driven mathematical modeling reveals asymmetric partitioning of mtDNA copies during cell division, as well as limited mitochondrial fusion and fission frequencies, as critical driving forces for mtDNA variant segregation. Given that our approach also facilitates assessment of segregation between intact and mutant mtDNA, we anticipate that it will be instrumental in elucidating the mechanisms underlying the purifying selection of mtDNA.

Keywords:  Heteroplasmy; Mathematical Modeling; Mitochondria; Mitochondrial Fission; mtDNA

DOI:  https://doi.org/10.1038/s44318-024-00183-5
J Agric Food Chem. 2024 Aug 07.

Acaricide Flupentiofenox Inhibits the Mitochondrial β-Oxidation Pathway of Fatty Acids.

Chihiro Uchiyama, Shingo Yoshimura, Shunsuke Yamamoto, Masahiro Ogawa, Kiyoshi Kawai.

  A newly developed pesticide, flupentiofenox, has a unique trifluoroethyl phenylsulfoxide structure, and it powerfully affects spider mites, including those with resistance to multiple commercial acaricides. To clarify the mode of action of flupentiofenox, we investigated its effect on mitochondrial energy generation. We observed that flupentiofenox decreased adenosine triphosphate (ATP) levels in two-spotted spider mites (Tetranychus urticae) at a practical dose. Flupentiofenox potently inhibited mitochondrial oxygen consumption under conditions of palmitoyl-carnitine or octanoic acid supply, but not under conditions of pyruvate supply. These results show that flupentiofenox inhibits the mitochondrial fatty acid metabolic pathway between the uptake of long-chain acylcarnitine or medium-chain fatty acid and the synthesis of acetyl-CoA by β-oxidation, resulting in suppressed mitochondrial energy generation. Our investigations have led us to conclude that flupentiofenox is a pesticide with a novel mode of action.

Keywords:  Tetranychus urticae; acaricide; energy metabolism; flupentiofenox; mitochondria; novel mode of action; spider mites

DOI:  https://doi.org/10.1021/acs.jafc.4c03076
Cells. 2024 Jul 30. pii: 1278. [Epub ahead of print]13(15):

Mitochondrial NME6: A Paradigm Change within the NME/NDP Kinase Protein Family?

Bastien Proust, Maja Herak Bosnar, Helena Ćetković, Malgorzata Tokarska-Schlattner, Uwe Schlattner.

  Eukaryotic NMEs/NDP kinases are a family of 10 multifunctional proteins that occur in different cellular compartments and interact with various cellular components (proteins, membranes, and DNA). In contrast to the well-studied Group I NMEs (NME1-4), little is known about the more divergent Group II NMEs (NME5-9). Three recent publications now shed new light on NME6. First, NME6 is a third mitochondrial NME, largely localized in the matrix space, associated with the mitochondrial inner membrane. Second, while its monomeric form is inactive, NME6 gains NDP kinase activity through interaction with mitochondrial RCC1L. This challenges the current notion that mammalian NMEs require the formation of hexamers to become active. The formation of complexes between NME6 and RCC1L, likely heterodimers, seemingly obviates the necessity for hexamer formation, stabilizing a NDP kinase-competent conformation. Third, NME6 is involved in mitochondrial gene maintenance and expression by providing (d)NTPs for replication and transcription (in particular the pyrimidine nucleotides) and by a less characterized mechanism that supports mitoribosome function. This review offers an overview of NME evolution and structure and highlights the new insight into NME6. The new findings position NME6 as the most comprehensively studied protein in NME Group II and may even suggest it as a new paradigm for related family members.

Keywords:  NM23; NME; RCC1L; mitochondria; mtDNA; mtRNA; nucleoside diphosphate kinase; pyrimidine nucleotides

DOI:  https://doi.org/10.3390/cells13151278
Anal Chem. 2024 Aug 07.

Real-Time Monitoring of mtDNA Aggregation and Mitophagy Induced by a Fluorescent Platinum Complex in Living Cells.

Bing Liu, Ting Sun, Yumeng Wang, Xiao-Yu Xia, Shixian Cao, Kang-Nan Wang, Qixin Chen, Zong-Wan Mao.

Mitochondrial DNA (mtDNA) is pivotal for mitochondrial morphology and function. Upon mtDNA damage, mitochondria undergo quality control mechanisms, including fusion, fission, and mitophagy. Real-time monitoring of mtDNA enables a deeper understanding of its effect on mitochondrial function and morphology. Controllable induction and real-time tracking of mtDNA dynamics and behavior are of paramount significance for studying mitochondrial function and morphology, facilitating a deeper understanding of mitochondria-related diseases. In this work, a fluorescent platinum complex was designed and developed that not only induces mitochondrial DNA (mtDNA) aggregation but also triggers mitochondrial autophagy (mitophagy) through the MDV pathway for damaged mtDNA clearance in living cells. Additionally, this complex allows for the real-time monitoring of these processes. This complex may serve as a valuable tool for studying mitochondrial microautophagy and holds promise for broader applications in cellular imaging and disease research.

DOI: https://doi.org/10.1021/acs.analchem.4c01128
Biochem J. 2024 Aug 07. 481(15): 1015-1042

Evolution and maintenance of mtDNA gene content across eukaryotes.

Shibani Veeraragavan, Maria Johansen, Iain G Johnston.

  Across eukaryotes, most genes required for mitochondrial function have been transferred to, or otherwise acquired by, the nucleus. Encoding genes in the nucleus has many advantages. So why do mitochondria retain any genes at all? Why does the set of mtDNA genes vary so much across different species? And how do species maintain functionality in the mtDNA genes they do retain? In this review, we will discuss some possible answers to these questions, attempting a broad perspective across eukaryotes. We hope to cover some interesting features which may be less familiar from the perspective of particular species, including the ubiquity of recombination outside bilaterian animals, encrypted chainmail-like mtDNA, single genes split over multiple mtDNA chromosomes, triparental inheritance, gene transfer by grafting, gain of mtDNA recombination factors, social networks of mitochondria, and the role of mtDNA dysfunction in feeding the world. We will discuss a unifying picture where organismal ecology and gene-specific features together influence whether organism X retains mtDNA gene Y, and where ecology and development together determine which strategies, importantly including recombination, are used to maintain the mtDNA genes that are retained.

Keywords:  genome evolution; mitochondrial evolution; mtDNA

DOI:  https://doi.org/10.1042/BCJ20230415
Science. 2024 Aug 08. eadp7114

PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress.

Karinder K Brar, Daniel T Hughes, Jordan L Morris, Kelly Subramanian, Shivaani Krishna, Fei Gao, Lara-Sophie Rieder, Sebastian Uhrig, Joshua Freeman, Heather L Smith, Rebekkah Jukes-Jones, Edward Avezov, Jodi Nunnari, Julien Prudent, Adrian J Butcher, Giovanna R Mallucci.

Endoplasmic Reticulum (ER) stress induces repression of protein synthesis throughout the cell. Attempts to understand how localized stress leads to widespread repression have been limited by difficulties in resolving translation rates at the subcellular level. Here, using live-cell imaging of reporter mRNA translation, we unexpectedly found that during ER stress active translation at mitochondria was significantly protected. The mitochondrial protein, ATAD3A, interacted with PERK and mediated this effect on localized translation by competing for binding with PERK's target, eIF2. PERK-ATAD3A interactions increased during ER stress, forming mitochondria-ER contact sites. Furthermore, ATAD3A binding attenuated local PERK signaling and rescued the expression of some mitochondrial proteins. Thus, PERK-ATAD3A interactions can control translational repression at a subcellular level, mitigating the impact of ER stress on the cell.

DOI: https://doi.org/10.1126/science.adp7114
Life Sci. 2024 Aug 02. pii: S0024-3205(24)00531-9. [Epub ahead of print]354 122941

Mitochondrial fragmentation in early differentiation of human MB135 myoblasts: Role of mitochondrial ROS production in the absence of depolarization.

Daniil A Chernyavskij, Konstantin G Lyamzaev, Olga Yu Pletjushkina, Fei Chen, Anna Karpukhina, Yegor S Vassetzky, Boris V Chernyak, Ekaterina N Popova.

  AIMS: Study of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis.MAIN METHODS: Mitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria.
KEY FINDINGS: In immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation.
SIGNIFICANCE: Mitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.

Keywords:  Mitochondrial fragmentation; Mitochondrial membrane potential (ΔΨm); Mitochondrial reactive oxygen species (mtROS); Myogenesis; Myogenic differentiation

DOI:  https://doi.org/10.1016/j.lfs.2024.122941
Mol Metab. 2024 Aug 06. pii: S2212-8778(24)00134-0. [Epub ahead of print] 102003

Peroxiredoxin 2 Regulates DAF-16/FOXO Mediated Mitochondrial Remodelling in Response to Exercise that is Disrupted in Ageing.

Qin Xia, Penglin Li, José C Casas-Martinez, Antonio Miranda-Vizuete, Emma McDermott, Peter Dockery, Katarzyna Goljanek-Whysall, Brian McDonagh.

  Ageing is associated with mitochondrial dysfunction and increased oxidative stress. Exercise generates endogenous reactive oxygen species (ROS) and promotes rapid mitochondrial remodelling. We investigated the role of Peroxiredoxin 2 (PRDX-2) in mitochondrial adaptations to exercise and ageing using Caenorhabditis elegans as a model system. PRDX-2 was required for the mitochondrial remodelling in response to exercise mediated by DAF-16 transcription factor activation and regulation of mitochondrial fusion gene eat-3. Employing an acute exercise and recovery cycle, we demonstrated exercise-induced mitochondrial ER contact sites (MERCS) assembly and mitochondrial remodelling dependent on PRDX-2 and DAF-16 signalling. There was increased mitochondrial fragmentation, elevated ROS and an altered redox state of PRDX-2 concomitant with impaired DAF-16 nuclear localisation during ageing. Similarly, the prdx-2 mutant strain exhibited increased mitochondrial fragmentation and a failure to activate DAF-16 required for mitochondrial fusion. Collectively, our data highlight the critical role of PRDX-2 in orchestrating mitochondrial remodelling in response to a physiological stress by regulating DAF-16 nuclear localisation.

Keywords:  Ageing; C. elegans; DAF-16; Exercise; Mitochondrial ER Contact Sites; Peroxiredoxin 2

DOI:  https://doi.org/10.1016/j.molmet.2024.102003
Methods Mol Biol. 2024 ;2835 121-133

A Method for Producing Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Leigh Syndrome Patients for Its Application in Disease Modeling and Drug Validation.

Keshav Narayan Alagarsamy, Abhay Srivastava, Sanjiv Dhingra.

  Leigh syndrome (LS), a complex multisystemic disorder, poses significant challenges in genetic medicine due to its intricate pathogenesis and wide-ranging clinical manifestations. Notably, these arise from mutations in either nuclear genetic DNA or mitochondrial DNA, affecting ATP production and resulting in diverse clinical outcomes. The unpredictable trajectory of this disease, ranging from severe developmental delays to early mortality, underscores the need for improved therapeutic solutions. This research pivots toward the novel use of induced pluripotent stem cells (iPSCs) as a promising platform for understanding disease mechanisms and spearheading patient-specific drug discoveries. Given the past successes of iPSCs in delineating organ-specific disorders and the recent endorsement of human iPSC-derived cardiomyocytes (CMs) by the FDA for drug evaluation, our work seeks to bridge this innovation to Leigh syndrome research. We detail a methodological approach to generate iPSCs from LS patients and differentiate them into iPSCs-CMs. Using multi-electrode array (MEA) analyses, we evaluate the field potential of these cells, spotlighting the potential of hiPSC-CM in drug validation and disease modeling. This pioneering approach offers a glimpse into the future of patient-centric therapeutic interventions for Leigh/Leigh-like syndrome.

Keywords:  Cardiomyocyte differentiation; Disease modeling; Drug validation; Induced pluripotent stem cells; Leigh Syndrome; Mitochondrial disorder

DOI:  https://doi.org/10.1007/978-1-0716-3995-5_11
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
Neurosci Lett. 2024 Aug 03. pii: S0304-3940(24)00295-7. [Epub ahead of print] 137917

Mitochondrial dysfunction and NDUFS3: Insights from a PINK1B9 Drosophila model in Parkinson's disease pathogenesis.

Xueting Fan, Yafang Tang, Zaiwa Wei, Fang Shi, Yilei Cui, Qinghua Li.

  PTEN-induced kinase1 (PINK1) mutation is the main cause of autosomal recessive inheritance and early-onset Parkinson's disease. Mitochondrial respiratory chain complex I (CI) functional impairment has been considered to be an important factor in the pathogenesis of PD in recent years. In addition, NDUFS3 (nicotinamide adenine dinucleotide deoxylase iron-thionein 3) is one of the core subunits of mitochondrial CI. Therefore, this study explored the role of NDUFS3 gene in PINK1B9 transgenic Drosophila and its possible related mechanisms. In this study, the PD transgenic Drosophila model of MHC-Gal4/UAS system was selected to specifically activate the expression of PINK1B9 gene in the chest muscle tissue of Drosophila melanogaster. NDUFS3 RNAi interference was used to interfere with PINK1B9 transgenic Drosophila melanogaster and its effect on PD transgenic flies was studied. The results suggest that down-regulation of NDUFS3 gene expression may have a protective effect on PINK1B9 transgenic Drosophila melanogaster, and we speculate that down-regulation of NDUFS3 gene expression to reduce oxidative stress and restore mitochondrial function may be related to mitochondrial stress response.

Keywords:  Drosophila melanogaster; Mitochondrial dysfunction; NDUFS3 RNAi; Oxidative stress; PINK1 mutation

DOI:  https://doi.org/10.1016/j.neulet.2024.137917
STAR Protoc. 2024 Aug 07. pii: S2666-1667(24)00405-2. [Epub ahead of print]5(3): 103240

Protocol for the production and reconstitution of VDAC1 for functional assays.

Grace I Dearden, Varun Ravishankar, Ken-Taro Sakata, Anant K Menon, Lucie Bergdoll.

  The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.

Keywords:  Metabolism; Protein Biochemistry; Protein expression and purification; Single-molecule Assays

DOI:  https://doi.org/10.1016/j.xpro.2024.103240
Clin Exp Otorhinolaryngol. 2024 Aug 06.

Novel Variant of FDXR as a Molecular Etiology of Postlingual Post-Synaptic Auditory Neuropathy Spectrum Disorder via Mitochondrial Dysfunction: Reiteration of the Correlation between Genotype and Cochlear Implantation Outcomes.

Bong Jik Kim, Yujin Kim, Ju Ang Kim, Jin Hee Han, Min Young Kim, Hee Kyung Yang, Chae-Seo Rhee, Young Cheol Kang, Chun-Hyung Kim, Byung Yoon Choi.

  Objectives: FDXR encodes the mitochondrial ferredoxin reductase, which is associated with auditory neuropathy spectrum disorder (ANSD) and optic atrophy. Only two studies have described FDXRrelated hearing loss. The auditory rehabilitation outcomes of this disease entity have not been investigated, and the pathophysiologic mechanism is not well elucidated. Here we report a hearingimpaired subject with co-segregation of the FDXR variant and post-synaptic type ANSD, who underwent cochlear implantation (CI) with favorable outcomes. We suggest a possible pathophysiologic mechanism of adult-onset ANSD via mitochondrial dysfunction.Methods: A 35-year-old woman was ascertained to have ANSD. Exome sequencing identified the genetic cause of hearing loss, and functional study measuring mitochondrial activity was performed to provide molecular evidence of pathophysiology. Expression of FDXR in the mouse cochlea was evaluated by immunohistochemistry. Intraoperatively, electrically-evoked compound action potential (ECAP) responses were measured, and mapping parameters were adjusted accordingly. Audiological outcomes were monitored for over 1 year.
Results: In lymphoblastoid cell lines (LCLs) carrying a novel FDXR variant, decreased ATP and MtMP levels and increased ROS levels were observed compared to control LCLs. These dysfunctions were restored by administering mitochondria isolated from umbilical cord mesenchymal stem cells, confirming the pathogenic potential of this variant via mitochondrial dysfunction. Partial ECAP responses during CI and FDXR expression in the mouse cochlea indicate that FDXR-related ANSD is postsynaptic. By increasing the pulse width during mapping, the patient's CI outcomes showed significant improvement over 1-year post-CI.
Conclusion: Post-synaptic ANSD due to a novel FDXR variant linked to mitochondrial dysfunction was identified first in a Korean, and 1-year post CI outcomes were reported for the first time in the literature. Excellent audiologic results were obtained, and our results reiterate the correlation between genotype and CI outcomes in ANSD.

Keywords:  FDXR; auditory neuropathy; auditory rehabilitation; cochlear implantation; mitochondria; optic atrophy; post-synaptic; postlingual

DOI:  https://doi.org/10.21053/ceo.2024.00184
medRxiv. 2024 Jun 15. pii: 2024.06.14.24308784. [Epub ahead of print]

The Utility of Long-Read Sequencing in Diagnosing Genetic Autosomal Recessive Parkinson's Disease: a genetic screening study.

Kensuke Daida, Hiroyo Yoshino, Laksh Malik, Breeana Baker, Mayu Ishiguro, Rylee Genner, Kimberly Paquette, Yuanzhe Li, Kenya Nishioka, Satoshi Masuzugawa, Makito Hirano, Kenta Takahashi, Mikhail Kolmogolv, Kimberley J Billingsley, Manabu Funayama, Cornelis Blauwendraat, Nobutaka Hattori.

  Background: Mutations within the genes PRKN and PINK1 are the leading cause of early onset autosomal recessive Parkinson's disease (PD). However, the genetic cause of most early-onset PD (EOPD) cases still remains unresolved. Long-read sequencing has successfully identified many pathogenic structural variants that cause disease, but this technology has not been widely applied to PD. We recently identified the genetic cause of EOPD in a pair of monozygotic twins by uncovering a complex structural variant that spans over 7 Mb, utilizing Oxford Nanopore Technologies (ONT) long-read sequencing. In this study, we aimed to expand on this and assess whether a second variant could be detected with ONT long-read sequencing in other unresolved EOPD cases reported to carry one heterozygous variant in PRKN or PINK1.Methods: ONT long-read sequencing was performed on patients with one reported PRKN/PINK1 pathogenic variant. EOPD patients with an age at onset younger than 50 were included in this study. As a positive control, we also included EOPD patients who had already been identified to carry two known PRKN pathogenic variants. Initial genetic testing was performed using either short-read targeted panel sequencing for single nucleotide variants and multiplex ligation-dependent probe amplification (MLPA) for copy number variants.
Results: 48 patients were included in this study (PRKN "one-variant" n = 24, PINK1 "one-variant" n = 12, PRKN "two-variants" n = 12). Using ONT long-read sequencing, we detected a second pathogenic variant in six PRKN "one-variant" patients (26%, 6/23) but none in the PINK1 "one-variant" patients (0%, 0/12). Long-read sequencing identified one case with a complex inversion, two instances of structural variant overlap, and three cases of duplication. In addition, in the positive control PRKN "two-variants" group, we were able to identify both pathogenic variants in PRKN in all the patients (100%, 12/12).
Conclusions: This data highlights that ONT long-read sequencing is a powerful tool to identify a pathogenic structural variant at the PRKN locus that is often missed by conventional methods. Therefore, for cases where conventional methods fail to detect a second variant for EOPD, long-read sequencing should be considered as an alternative and complementary approach.

Keywords:  Early onset Parkinson’s disease; PINK1; PRKN; Parkinson’s disease; genetics; long-read sequencing; structural variant

DOI:  https://doi.org/10.1101/2024.06.14.24308784
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
Am J Hum Genet. 2024 Jul 31. pii: S0002-9297(24)00252-0. [Epub ahead of print]

Expanding the genetic and phenotypic landscape of replication factor C complex-related disorders: RFC4 deficiency is linked to a multisystemic disorder.

University of Washington Center for Rare Disease Research

  The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.

Keywords:  DNA replication; Mendelian disorder; gene discovery; rare disease; replication factor C complex; translational research

DOI:  https://doi.org/10.1016/j.ajhg.2024.07.008
Neurobiol Dis. 2024 Aug 02. pii: S0969-9961(24)00222-5. [Epub ahead of print]200 106622

Stage-dependent phosphoproteome remodeling of Parkinson's disease blood cells.

Giorgia Massacci, Veronica Venafra, Maximilian Zwiebel, Maria Wahle, Rocco Cerroni, Jacopo Bissacco, Livia Perfetto, Vito Michienzi, Alessandro Stefani, Nicola Biagio Mercuri, Tommaso Schirinzi, Francesca Sacco.

  The complexity and heterogeneity of PD necessitate advanced diagnostic and prognostic tools to elucidate its molecular mechanisms accurately. In this study, we addressed this challenge by conducting a pilot phospho-proteomic analysis of peripheral blood mononuclear cells (PBMCs) from idiopathic PD patients at varying disease stages to delineate the functional alterations occurring in these cells throughout the disease course and identify key molecules and pathways contributing to PD progression. By integrating clinical data with phospho-proteomic profiles across various PD stages, we identify potential stage-specific molecular signatures indicative of disease progression. This integrative approach allows for the discernment of distinct disease states and enhances our understanding of PD heterogeneity.

Keywords:  Biomarker; Parkinson's disease; Peripheral blood; Phosphoproteomic

DOI:  https://doi.org/10.1016/j.nbd.2024.106622
Front Cell Neurosci. 2024 ;18 1440555

Modeling riboflavin transporter deficiency type 2: from iPSC-derived motoneurons to iPSC-derived astrocytes.

Valentina Magliocca, Angela Lanciotti, Elena Ambrosini, Lorena Travaglini, Veronica D'Ezio, Valentina D'Oria, Stefania Petrini, Michela Catteruccia, Keith Massey, Marco Tartaglia, Enrico Bertini, Tiziana Persichini, Claudia Compagnucci.

  Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes.Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes.

Keywords:  astrocytes; in vitro disease modeling; induced pluripotent stem cells; motoneurons; neurodegenerative autosomal recessive disease; redox state; riboflavin transporter deficiency

DOI:  https://doi.org/10.3389/fncel.2024.1440555
Nat Commun. 2024 Aug 06. 15(1): 6524

Diagnostic utility of DNA methylation analysis in genetically unsolved pediatric epilepsies and CHD2 episignature refinement.

Undiagnosed Diseases Network

Sequence-based genetic testing identifies causative variants in ~ 50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. We interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 582 individuals with genetically unsolved DEEs. We identify rare differentially methylated regions (DMRs) and explanatory episignatures to uncover causative and candidate genetic etiologies in 12 individuals. Using long-read sequencing, we identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and four copy number variants. We also identify pathogenic variants associated with episignatures. Finally, we refine the CHD2 episignature using an 850 K methylation array and bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate variants as 2% (12/582) for unsolved DEE cases.

DOI: https://doi.org/10.1038/s41467-024-50159-6