Mitochondrial Disease Research Articles

Distinct roles for the domains of the mitochondrial aspartate/glutamate carrier citrin in organellar localization and substrate transport

ObjectiveCitrin, the mitochondrial aspartate/glutamate carrier isoform 2, is structurally and mechanistically the most complex SLC25 family member, because it consists of three-domains and forms a homodimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. MethodsWe have employed structural modelling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knock-out of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. ResultsUsing 33 pathogenic variants of citrin we clarify determinants of sub-cellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. ConclusionsOur work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency.

525P Management of seizures in patients with primary mitochondrial diseases: consensus statement from the inter-ERNs mitochondrial working group

525P Management of seizures in patients with primary mitochondrial diseases: consensus statement from the inter-ERNs mitochondrial working group

Classic ketogenic diet-induced ketoacidosis in the treatment of pyruvate dehydrogenase deficiency: a case report and literature review

BackgroundAs a rare mitochondrial disorder, the pyruvate dehydrogenase complex (PDC) deficiency is a rare inborn disease characterized with glucose metabolism defects, which leads to neurological dysfunction, serum lactic acid buildup and a resultant trend of metabolic acidosis. Although the ketogenic diet (KD) is the first-line treatment for PDC deficiency, there is currently no widely accepted consensus on specific implementation of KD for this condition. Due to the combined effect of pre-existing hyperlactacidemia and KD-induced ketoacidosis that can further exacerbate metabolic disturbances, maintaining metabolic homeostasis should be prioritized during the implementation of KD.Case presentationHerein, the authors present a 6-year-old boy with lactic acidosis, ataxia, hypotonia and neuromotor development retardation. The KD was started after the patient was diagnosed with PDC deficiency based on genetic testing. The initiation with classic KD resulted in severe non-diabetic ketoacidosis with elevated anion gap, which was promptly alleviated by dextrose supplementation and dietary modification to a less-restrictive KD. Long-term supervision demonstrated the efficacy of a modified KD in improving both clinical course and metabolic acidosis of the patient.ConclusionsThis rare case adds to the limited evidence of KD application in PDC deficiency, and provides valuable insights into the importance of reasonably lowering the ketogenic ratio of KD at the start of treatment to reduce the risk of metabolic acidosis.

Selection promotes age-dependent degeneration of the mitochondrial genome.

Somatic mutations in mitochondrial genomes (mtDNA) accumulate exponentially during aging. Using single cell sequencing, we characterize the spectrum of age-accumulated mtDNA mutations in mouse and human liver and identify directional forces that accelerate the accumulation of mutations beyond the rate predicted by a neutral model. "Driver" mutations that give genomes a replicative advantage rose to high cellular abundance and carried along "passenger" mutations, some of which are deleterious. In addition, alleles that alter mtDNA-encoded proteins selectively increased in abundance overtime, strongly supporting the idea of a "destructive" selection that favors genomes lacking function. Overall, this combination of selective forces acting in hepatocytes promotes somatic accumulation of mutations in coding regions of mtDNA that are otherwise conserved in evolution. We propose that these selective processes could contribute to the population prevalence of mtDNA mutations, accelerate the course of heteroplasmic mitochondrial diseases and promote age-associated erosion of the mitochondrial genome.

Somatic mitochondrial DNA mutations are a source of heterogeneity among primary leukemic cells.

Somatic mitochondrial DNA (mtDNA) mutations are prevalent in tumors, yet defining their biological significance remains challenging due to the intricate interplay between selective pressure, heteroplasmy, and cell state. Utilizing bulk whole-genome sequencing data from matched tumor and normal samples from two cohorts of pediatric cancer patients, we uncover differences in the accumulation of synonymous and nonsynonymous mtDNA mutations in pediatric leukemias, indicating distinct selective pressures. By integrating single-cell sequencing (SCS) with mathematical modeling and network-based systems biology approaches, we identify a correlation between the extent of cell-state changes associated with tumor-enriched mtDNA mutations and the selective pressures shaping their distribution among individual leukemic cells. Our findings also reveal an association between specific heteroplasmic mtDNA mutations and cellular responses that may contribute to functional heterogeneity among leukemic cells and influence their fitness. This study highlights the potential of SCS strategies for distinguishing between pathogenic and passenger somatic mtDNA mutations in cancer.

SENP1 prevents high fat diet-induced non-alcoholic fatty liver diseases by regulating mitochondrial dynamics

Mitochondrial dynamics plays a crucial role in the occurrence and development of non-alcoholic fatty liver diseases (NAFLD). SENP1, a SUMO-specific protease, catalyzes protein de-SUMOylation and involves in various physiological and pathological processes. However, the exact role of SENP1 in NAFLD remains unclear. Therefore, we investigated the regulatory role of SENP1 in mitochondrial dynamics during the progression of NAFLD. In the study, the NAFLD in vivo model induced by high fat diet (HFD) and in vitro model induced by free fatty acids (FFA) were established to investigate the role and underlying mechanism of SENP1 through detecting mitochondrial morphology and dynamics. Our results showed that the down-regulation of SENP1 expression and the mitochondrial dynamics dysregulation occurred in the NAFLD, evidenced as mitochondrial fragmentation, up-regulation of p-Drp1 ser616 and down-regulation of MFN2, OPA1. However, over-expression of SENP1 significantly alleviated the NAFLD, rectified the mitochondrial dynamics disorder, reduced Cyt-c release and ROS levels induced by FFA or HFD; moreover, the over-expression of SENP1 also reduced the SUMOylation levels of Drp1 and prevented the Drp1 translocation to mitochondria. Our findings suggest that the possible mechanisms of SENP1 were through rectifying the mitochondrial dynamics disorder, reducing Cyt-c release and ROS-mediated oxidative stress. The findings would provide a novel target for the prevention and treatment of NALFD.

Interferon Stimulated Gene Expression Is a Biomarker for Primary Mitochondrial Disease.

Mitochondria are implicated in regulation of the innate immune response. We hypothesized that abnormalities in interferon signaling may contribute to pathophysiology in patients with primary mitochondrial disease (PMD). Expression of interferon stimulated genes (ISGs) was measured by real-time polymerase chain reaction (PCR) in whole blood samples from a cohort of patients with PMD. Upregulated ISG expression was observed in a high proportion (41/55, 75%) of patients with PMD on at least 1 occasion, most frequently IFI27 upregulation, seen in 50% of the samples. Some patients had extremely high IFI27 levels, similar to those seen in patients with primary interferonopathies. A statistically significant correlation was observed between elevated IFI27 gene expression and PMD, but not between IFI27 and secondary mitochondrial dysfunction, suggesting that ISG upregulation is a biomarker of PMD. In some patients with PMD, ISG abnormalities persisted on repeat measurement over several years, indicative of ongoing chronic inflammation. Subgroup analyses suggested common ISG signatures in patients with similar mitochondrial disease mechanisms and positive correlations with disease severity among patients with identical genetic diagnoses. Dysregulated interferon signaling is frequently seen in patients with PMD suggesting that interferon dysregulation is a contributor to pathophysiology. This may indicate a role for repurposing of immunomodulatory therapies for the treatment of PMDs by targeting interferon signaling. ANN NEUROL 2024.

Polyendocrinopathies in an infant with a mitochondrial disorder

Polyendocrinopathies in an infant with a mitochondrial disorder

Assessment of amino acid charge states based on cryo-electron microscopy and molecular dynamics simulations of respiratory complex I

The charge states of titratable amino acid residues play a key role in the function of membrane-bound bioenergetic proteins. However, determination of these charge states both through experimental and computational approaches is extremely challenging. Cryo-EM density maps can provide insights on the charge states of titratable amino acid residues. By performing classical atomistic molecular dynamics simulations on the high resolution cryo-EM structures of respiratory complex I from Yarrowia lipolytica, we analyze the conformational and charge states of a key acidic residue in its ND1 subunit, aspartic acid D203, which is also a mitochondrial disease mutation locus. We suggest that in the native state of respiratory complex I, D203 is negatively charged and maintains a stable hydrogen bond to a conserved arginine residue. Alternatively, upon conformational change in the turnover state of the enzyme, its sidechain attains a charge-neutral status. We discuss the implications of this analysis on the molecular mechanism of respiratory complex I.

Age-induced changes in skeletal muscle mitochondrial DNA synthesis, quantity, and quality in genetically unique rats

AbstractMitochondrial genomic integrity is a key element of physiological processes and health. Changes in the half-life of the mitochondrial genome are implicated in the generation and accumulation of age-induced mitochondrial DNA (mtDNA) mutations, which are implicated in skeletal muscle aging and sarcopenia. There are conflicting data on the half-life of mtDNA, and there is limited information on how aging affects half-life in skeletal muscle. We hypothesized that skeletal muscle mtDNA synthesis rates would decrease with age in both female and male rats concomitant with changes in mtDNA integrity reflected in mtDNA copy number and mutation frequency. We measured mitochondrial genome half-life using stable isotope labeling over a period of 14 days and assessed mtDNA copy number and deletion mutation frequency using digital PCR in the quadriceps muscle of 9-month-old and 26-month-old male and female OKC-HET rats. We found a significant age-related increase in mtDNA half-life, from 132 days at 9 months to 216 days at 26 months of age in OKC-HET quadriceps. Concomitant with the increase in mtDNA half-life, we found an age-related increase in mtDNA deletion mutation frequency in both male and female rats. Notably, 26-month-old female rats had a lower mutation frequency than male rats, and there were no changes in mtDNA copy number with sex, age, or mitochondrial genotype. These data reveal several key findings: (1) mtDNA turnover in rat skeletal muscle decreases with age, (2) mtDNA half-lives in skeletal muscle are approximately an order of magnitude longer than what is reported for other tissues, and (3) muscle mtDNA turnover differs significantly from the turnover of other mitochondrial macromolecules including components of the mitochondrial nucleoid. These findings provide insight into the factors driving age-induced mtDNA mutation accumulation, which contribute to losses of mitochondrial genomic integrity and may play a role in skeletal muscle dysfunction.

Recognizing Leber's Hereditary Optic Neuropathy to avoid delayed diagnosis and misdiagnosis.

Leber's Hereditary Optic Neuropathy (LHON) is a maternally inherited optic nerve disease primarily caused by mutations in mitochondrial DNA (mtDNA). The peak of onset is typically between 15 and 30 years, but variability exists. Misdiagnosis, often as inflammatory optic neuritis, delays treatment, compounded by challenges in timely genetic diagnosis. Given the availability of a specific treatment for LHON, its early diagnosis is imperative to ensure therapeutic appropriateness. This work gives an updated guidance about LHON differential diagnosis to clinicians dealing also with multiple sclerosi and neuromyelitis optica spectrtum disorders-related optic neuritis. LHON diagnosis relies on clinical signs and paraclinical evaluations. Differential diagnosis in the acute phase primarily involves distinguishing inflammatory optic neuropathies, considering clinical clues such as ocular pain, fundus appearance and visual recovery. Imaging analysis obtained with Optical Coherence Tomography (OCT) assists clinicians in early recognition of LHON and help avoiding misdiagnosis. Genetic testing for the three most common LHON mutations is recommended initially, followed by comprehensive mtDNA sequencing if suspicion persists despite negative results. We present and discuss crucial strategies for accurate diagnosis and management of LHON cases.

The Role and Research Progress of Mitochondria in Sensorineural Hearing Loss.

Hearing loss is one of the most common human diseases, seriously affecting everyday lives. Mitochondria, as the energy metabolism center in cells, are also involved in regulating active oxygen metabolism and mediating the occurrence of inflammation and apoptosis. Mitochondrial defects are closely related to hearing diseases. Studies have shown that mitochondrial DNA mutations are one of the causes of hereditary hearing loss. In addition, changes in mitochondrial homeostasis are directly related to noise-induced hearing loss and presbycusis. This review mainly summarizes and discusses the effects of mitochondrial dysfunction and mitophagy on hearing loss. Subsequently, we introduce the recent research progress of targeted mitochondria therapy in the hearing system.

Research advances on CaMKs-mediated neurodevelopmental injury.

Calcium/calmodulin-dependent protein kinases (CaMKs) are important proteins in the calcium signaling cascade response pathway, which can broadly regulate biological functions in vivo. Multifunctional CaMKs play key roles in neural development, including neuronal circuit building, synaptic plasticity establishment, and neurotrophic factor secretion. Currently, four familial proteins, calcium/calmodulin-dependent protein kinase I (CaMKI), calcium/calmodulin-dependent protein kinase II (CaMKII), eukaryotic elongation factor 2 kinase (eEF2K, popularly known as CaMKIII) and calcium/calmodulin-dependent protein kinase IV (CaMKIV), are thought to have been the most extensively studied during neurodevelopment. Although their spatial structures are extremely similar, as well as the initial starting point of activation, both require the activation of calcium and calmodulin (CaM) complexes to be involved in the process, and the phosphorylation sites and modes of each member are different. Furthermore, due to the high structural similarity of CaMKs, their members may play synergistic roles in the regulation of neural development, but different CaMKs also have their own means of regulating neural development. In this review, we first describe the visualized protein structural forms of CaMKI, CaMKII, eEF2K and CaMKIV, and then describe the functions of each kinase in neurodevelopment. After that, we focus on four main mechanisms of neurodevelopmental damage caused by CaMKs: CaMKI/ERK/CREB pathway inhibition leading to dendritic spine structural damage; Ca2+/CaM/CaMKII through induction of mitochondrial kinetic disorders leading to neurodevelopmental damage; CaMKIII/eEF2 hyperphosphorylation affects the establishment of synaptic plasticity; and CaMKIV/JNK/NF-κB through induction of an inflammatory response leading to neurodevelopmental damage. In conclusion, we briefly discuss the pathophysiological significance of aberrant CaMK family expression in neurodevelopmental disorders, as well as the protective effects of conventional CaMKII and CaMKIII antagonists against neurodevelopmental injury.

The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1

It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases.

Quantitative proteomics of patient fibroblasts reveal biomarkers and diagnostic signatures of mitochondrial disease.

BACKGROUNDMitochondrial diseases belong to the group of inborn errors of metabolism (IEM), with a prevalence of 1 in 2,000-5,000 individuals. They are the most common form of IEM, but, despite advances in next-generation sequencing technologies, almost half of the patients are left genetically undiagnosed.METHODSWe investigated a cohort of 61 patients with defined mitochondrial disease to improve diagnostics, identify biomarkers, and correlate metabolic pathways to specific disease groups. Clinical presentations were structured using human phenotype ontology terms, and mass spectrometry-based proteomics was performed on primary fibroblasts. Additionally, we integrated 6 patients carrying variants of uncertain significance (VUS) to test proteomics as a diagnostic expansion.RESULTSProteomic profiles from patient samples could be classified according to their biochemical and genetic characteristics, with the expression of 5 proteins (GPX4, MORF4L1, MOXD1, MSRA, and TMED9) correlating with the disease cohort, thus acting as putative biomarkers. Pathway analysis showed a deregulation of inflammatory and mitochondrial stress responses. This included the upregulation of glycosphingolipid metabolism and mitochondrial protein import, as well as the downregulation of arachidonic acid metabolism. Furthermore, we could assign pathogenicity to a VUS in MRPS23 by demonstrating the loss of associated mitochondrial ribosome subunits.CONCLUSIONWe established mass spectrometry-based proteomics on patient fibroblasts as a viable and versatile tool for diagnosing patients with mitochondrial disease.FUNDINGThe NovoNordisk Foundation, Knut and Alice Wallenberg Foundation, Wellcome Centre for Mitochondrial Research, UK Medical Research Council, and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children.

Epicatechin ameliorates palmitate-induced insulin resistance in C2C12 myogenic cells by alleviating oxidative stress and activating the AMPK/ACC pathway

ABSTRACT Epicatechin (EC) is a water-soluble natural organic compound belonging to the flavanol class of compounds. It exhibits various physiological activities such as anti-inflammatory, antioxidant, treatment of mitochondrial diseases, and prevention of cardiovascular diseases.To examineEC’s impact and mechanism on adult myoblasts’ insulin resistance. The effects of various EC concentrations on the viability of C2C12 adult myoblasts were measured, as were glucose consumption and glycogen content; in vitro antioxidant activity of EC was measured; cellular oxidative marker content and apoptosis were measured; and protein expression was detected by Western blot. A model of insulin resistance in C2C12 myogenic cells was established using palmitic acid. EC significantly increased the glucose consumption and glycogen content; EC increased the levels of CAT, SOD and GSH and decreased the levels of NO, MDA and ROS; EC decreased the apoptosis rate of cells; EC treatment upregulated the levels of GLUT4, p-AMPK, p-ACC, NRF2, and HO-1 proteins. These results suggest that EC can regulate the NRF2/HO-1 signaling pathway, enhance GLUT4 transport, activate AMPK/ACC pathway, enhance cellular uptake of glucose, thus effectively improving insulin resistance in C2C12 cells.

Effective management of chronic intestinal pseudo-obstruction in MELAS using acotiamide: A case report

Background: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is reported to be a secondary cause of chronic intestinal pseudo-obstruction (CIPO). Although few case reports have described CIPO in MELAS, effective treatment for CIPO has not been established. Here, we present a case report of amelioration of CIPO symptoms using acotiamide in a patient with MELAS. Case Presentation: A 51-year-old Japanese female with a mitochondrial disorder with m.3243A>G mutation and a history of anorexia for two years presented to our hospital with a left temporal headache and acute paraphasia. A stroke-like episode of MELAS was suspected, and combined therapy with arginine, edaravone, and levetiracetam was initiated. Although her symptoms improved, she presented with nausea and vomiting and abdominal distension six days following admission. Abdominal contrast-enhanced computed tomography revealed dilatation from the stomach to the intestine, particularly marked the stomach, with neither obstruction nor impaired blood flow in the intestine. CIPO exacerbation with MELAS was suspected, and the patient’s symptoms gradually improved with acotiamide. Conclusion: Patients with MELAS could possibly experience stroke-like episodes during CIPO. Since acetylcholine possibly plays an important role in the pathophysiology of CIPO and acotiamide possesses prokinetic activity by inhibiting acetylcholinesterase, acotiamide could possibly improve CIPO symptoms.

Overactive mitochondrial DNA replication disrupts perinatal cardiac maturation

High mitochondrial DNA (mtDNA) amount has been reported to be beneficial for resistance and recovery of metabolic stress, while increased mtDNA synthesis activity can drive aging signs. The intriguing contrast of these two mtDNA boosting outcomes prompted us to jointly elevate mtDNA amount and frequency of replication in mice. We report that high activity of mtDNA synthesis inhibits perinatal metabolic maturation of the heart. The offspring of the asymptomatic parental lines are born healthy but manifest dilated cardiomyopathy and cardiac collapse during the first days of life. The pathogenesis, further enhanced by mtDNA mutagenesis, involves prenatal upregulation of mitochondrial integrated stress response and the ferroptosis-inducer MESH1, leading to cardiac fibrosis and cardiomyocyte death after birth. Our evidence indicates that the tight control of mtDNA replication is critical for early cardiac homeostasis. Importantly, ferroptosis sensitivity is a potential targetable mechanism for infantile-onset cardiomyopathy, a common manifestation of mitochondrial diseases.

Cascade testing in mitochondrial diseases: a cross-sectional retrospective study

BackgroundCascade testing can offer improved surveillance and timely introduction of clinical management for the at-risk biological relatives. Data on cascade testing and costs in mitochondrial diseases are lacking. To address this gap, we performed a cross-sectional retrospective study to provide a framework for cascade testing in mitochondrial diseases, to estimate the eligibility versus real-time uptake of cascade testing and to evaluate the cost of the genetic diagnosis of index cases and the cost of predictive cascade testing.MethodsData was collected through retrospective chart review. The variant inheritance pattern guided the identification of eligible first-degree relatives: (i) Males with mitochondrial DNA (mtDNA) single nucleotide variants (SNVs) – siblings and mothers. (ii) Females with mtDNA SNVs – siblings, mothers and offspring. (iii) Autosomal Dominant (AD) nuclear DNA (nDNA) variants – siblings, offspring and both parents. (iv) Autosomal Recessive (AR) nDNA variants – siblings.ResultsWe recruited 99 participants from the Adult Mitochondrial Disease Clinic in Sydney. The uptake of cascade testing was 55.2% in the mtDNA group, 55.8% in the AD nDNA group and 0% in AR nDNA group. Of the relatives in mtDNA group who underwent cascade testing, 65.4% were symptomatic, 20.5% were oligosymptomatic and 14.1% were asymptomatic. The mean cost of cascade testing for eligible first-degree relatives (mtDNA group: $694.7; AD nDNA group: $899.1) was lower than the corresponding index case (mtDNA group: $4578.4; AD nDNA group: $5715.1) (p < 0.001).ConclusionThe demand for cascade testing in mitochondrial diseases varies according to the genotype and inheritance pattern. The real-time uptake of cascade testing can be influenced by multiple factors. Early diagnosis of at-risk biological relatives of index cases through cascade testing, confirms the diagnosis in those who are symptomatic and facilitates implementation of surveillance strategies and clinical care at an early stage of the disease.

Role of Mitochondrial Dysfunctions in Neurodegenerative Disorders: Advances in Mitochondrial Biology.

Mitochondria, essential organelles responsible for cellular energy production, emerge as a key factor in the pathogenesis of neurodegenerative disorders. This review explores advancements in mitochondrial biology studies that highlight the pivotal connection between mitochondrial dysfunctions and neurological conditions such as Alzheimer's, Parkinson's, Huntington's, ischemic stroke, and vascular dementia. Mitochondrial DNA mutations, impaired dynamics, and disruptions in the ETC contribute to compromised energy production and heightened oxidative stress. These factors, in turn, lead to neuronal damage and cell death. Recent research has unveiled potential therapeutic strategies targeting mitochondrial dysfunction, including mitochondria targeted therapies and antioxidants. Furthermore, the identification of reliable biomarkers for assessing mitochondrial dysfunction opens new avenues for early diagnosis and monitoring of disease progression. By delving into these advancements, this review underscores the significance of understanding mitochondrial biology in unraveling the mechanisms underlying neurodegenerative disorders. It lays the groundwork for developing targeted treatments to combat these devastating neurological conditions.