Polydatin and Nicotinamide Rescue the Cellular Phenotype of Mitochondrial Diseases by Mitochondrial Unfolded Protein Response (mtUPR) Activation.

Primary mitochondrial diseases result from mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) genes, encoding proteins crucial for mitochondrial structure or function. Given that few disease-specific therapies are available for mitochondrial diseases, novel treatments to reverse mitochondrial dysfunction are necessary. In this work, we explored new therapeutic options in mitochondrial diseases using fibroblasts and induced neurons derived from patients with mutations in the GFM1 gene. This gene encodes the essential mitochondrial translation elongation factor G1 involved in mitochondrial protein synthesis. Due to the severe mitochondrial defect, mutant GFM1 fibroblasts cannot survive in galactose medium, making them an ideal screening model to test the effectiveness of pharmacological compounds. We found that the combination of polydatin and nicotinamide enabled the survival of mutant GFM1 fibroblasts in stress medium. We also demonstrated that polydatin and nicotinamide upregulated the mitochondrial Unfolded Protein Response (mtUPR), especially the SIRT3 pathway. Activation of mtUPR partially restored mitochondrial protein synthesis and expression, as well as improved cellular bioenergetics. Furthermore, we confirmed the positive effect of the treatment in GFM1 mutant induced neurons obtained by direct reprogramming from patient fibroblasts. Overall, we provide compelling evidence that mtUPR activation is a promising therapeutic strategy for GFM1 mutations.

Illuminating mitochondrial translation through mouse models.

Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development.

Origins of tissue and cell-type specificity in mitochondrial DNA (mtDNA) disease.

Mutations of mitochondrial (mt)DNA are a major cause of morbidity and mortality in humans, accounting for approximately two thirds of diagnosed mitochondrial disease. However, despite significant advances in technology since the discovery of the first disease-causing mtDNA mutations in 1988, the comprehensive diagnosis and treatment of mtDNA disease remains challenging. This is partly due to the highly variable clinical presentation linked to tissue-specific vulnerability that determines which organs are affected. Organ involvement can vary between different mtDNA mutations, and also between patients carrying the same disease-causing variant. The clinical features frequently overlap with other non-mitochondrial diseases, both rare and common, adding to the diagnostic challenge. Building on previous findings, recent technological advances have cast further light on the mechanisms which underpin the organ vulnerability in mtDNA diseases, but our understanding is far from complete. In this review we explore the origins, current knowledge, and future directions of research in this area.

Mitochondrial dysfunction: mechanisms and advances in therapy.

Mitochondria, with their intricate networks of functions and information processing, are pivotal in both health regulation and disease progression. Particularly, mitochondrial dysfunctions are identified in many common pathologies, including cardiovascular diseases, neurodegeneration, metabolic syndrome, and cancer. However, the multifaceted nature and elusive phenotypic threshold of mitochondrial dysfunction complicate our understanding of their contributions to diseases. Nonetheless, these complexities do not prevent mitochondria from being among the most important therapeutic targets. In recent years, strategies targeting mitochondrial dysfunction have continuously emerged and transitioned to clinical trials. Advanced intervention such as using healthy mitochondria to replenish or replace damaged mitochondria, has shown promise in preclinical trials of various diseases. Mitochondrial components, including mtDNA, mitochondria-located microRNA, and associated proteins can be potential therapeutic agents to augment mitochondrial function in immunometabolic diseases and tissue injuries. Here, we review current knowledge of mitochondrial pathophysiology in concrete examples of common diseases. We also summarize current strategies to treat mitochondrial dysfunction from the perspective of dietary supplements and targeted therapies, as well as the clinical translational situation of related pharmacology agents. Finally, this review discusses the innovations and potential applications of mitochondrial transplantation as an advanced and promising treatment.

Digenic Inheritance in Rare Disorders and Mitochondrial Disease-Crossing the Frontier to a More Comprehensive Understanding of Etiology.

Our understanding of rare disease genetics has been shaped by a monogenic disease model. While the traditional monogenic disease model has been successful in identifying numerous disease-associated genes and significantly enlarged our knowledge in the field of human genetics, it has limitations in explaining phenomena like phenotypic variability and reduced penetrance. Widening the perspective beyond Mendelian inheritance has the potential to enable a better understanding of disease complexity in rare disorders. Digenic inheritance is the simplest instance of a non-Mendelian disorder, characterized by the functional interplay of variants in two disease-contributing genes. Known digenic disease causes show a range of pathomechanisms underlying digenic interplay, including direct and indirect gene product interactions as well as epigenetic modifications. This review aims to systematically explore the background of digenic inheritance in rare disorders, the approaches and challenges when investigating digenic inheritance, and the current evidence for digenic inheritance in mitochondrial disorders.

Mitochondrial Biomarkers in the Omics Era: A Clinical-Pathophysiological Perspective.

Mitochondrial diseases (MDs) affect 4300 individuals, with different ages of presentation and manifestation in any organ. How defects in mitochondria can cause such a diverse range of human diseases remains poorly understood. In recent years, several published research articles regarding the metabolic and protein profiles of these neurogenetic disorders have helped shed light on the pathogenetic mechanisms. By investigating different pathways in MDs, often with the aim of identifying disease biomarkers, it is possible to identify molecular processes underlying the disease. In this perspective, omics technologies such as proteomics and metabolomics considered in this review, can support unresolved mitochondrial questions, helping to improve outcomes for patients.

Identification of a Novel Indel Variant in the DARS2 Gene in Russian Patients with Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is an inherited disease caused by pathogenic biallelic variants in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. This disease is characterized by slowly progressive spastic gait, cerebellar symptoms, and leukoencephalopathy with brainstem and spinal cord involvement. CASE PRESENTATION: Peripheral blood samples were collected from four patients from four unrelated families to extract genomic DNA. All patients underwent partial exon analysis of the DARS2 gene using Sanger sequencing, which detected the c.228-21_228-20delinsC variant in a heterozygous state. Further DNA from three patients was analyzed using a next-generation sequencing-based custom AmpliSeq™ panel for 59 genes associated with leukodystrophies, and one of the patients underwent whole genome sequencing. We identified a novel pathogenic variant c.1675-1256_*115delinsGCAACATTTCGGCAACATTCCAACC in the DARS2 gene. Three patients (patients 1, 2, and 4) had slowly progressive cerebellar ataxia, and two patients (patients 1 and 2) had spasticity. In addition, two patients (patients 2 and 4) showed signs of axonal neuropathy, such as decreased tendon reflexes and loss of distal sensitivity. Three patients (patients 1, 2, and 3) also had learning difficulties. It should be noted the persistent presence of characteristic changes in brain MRI in all patients, which emphasizes its importance as the main diagnostic tool for suspicion and subsequent confirmation of LBSL. Conclusions: We found a novel indel variant in the DARS2 gene in four patients with LBSL and described their clinical and genetic characteristics. These results expand the mutational spectrum of LBSL and aim to improve the laboratory diagnosis of this form of leukodystrophy.

Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.

Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.

Echocardiographic manifestations of mitochondrial disease with GTPBP3 gene mutations: A case report.

RATIONALE: Mitochondrial diseases are a group of disorders in which mutations in mitochondrial DNA or nuclear DNA lead to dysfunctional oxidative phosphorylation of cells, with mutations in mitochondrial DNA being the most common cause of mitochondrial disease, and mutations in nuclear genes being rarely reported. The echocardiographic findings of mitochondrial diseases with nuclear gene mutations in children's hearts are even rarer. Even more valuable is that we followed up the patient for 4 years and dynamically observed the cardiac echocardiographic manifestations of mitochondrial disease. Provide ideas for the clinical diagnosis and prognosis of mitochondrial diseases. PATIENT CONCERNS: The patient was seen in the pediatric outpatient clinic for poor strength and mental retardation. echocardiography: mild left ventricular (LV) enlargement and LV wall thickening. Nuclear genetic testing: uanosine triphosphate binding protein 3 (GTPBP3) gene mutation. Diagnosis of mitochondrial disease. DIAGNOSES: Mitochondrial disease with GTPBP3 gene mutations. OUTCOMES: After receiving drug treatment, the patient exhibited a reduction in lactate levels, an enhanced physical condition compared to prior assessments, and demonstrated average intellectual development. LESSONS SUBSECTIONS: For echocardiographic indications of LV wall thickening and LV enlargement, one needs to be alert to the possibility of hereditary cardiomyopathy, especially in children.

Attenuating mitochondrial dysfunction and morphological disruption with PT320 delays dopamine degeneration in MitoPark mice.

BACKGROUND: Mitochondria are essential organelles involved in cellular energy production. Changes in mitochondrial function can lead to dysfunction and cell death in aging and age-related disorders. Recent research suggests that mitochondrial dysfunction is closely linked to neurodegenerative diseases. Glucagon-like peptide-1 receptor (GLP-1R) agonist has gained interest as a potential treatment for Parkinson's disease (PD). However, the exact mechanisms responsible for the therapeutic effects of GLP-1R-related agonists are not yet fully understood. METHODS: In this study, we explores the effects of early treatment with PT320, a sustained release formulation of the GLP-1R agonist Exenatide, on mitochondrial functions and morphology in a progressive PD mouse model, the MitoPark (MP) mouse. RESULTS: Our findings demonstrate that administration of a clinically translatable dose of PT320 ameliorates the reduction in tyrosine hydroxylase expression, lowers reactive oxygen species (ROS) levels, and inhibits mitochondrial cytochrome c release during nigrostriatal dopaminergic denervation in MP mice. PT320 treatment significantly preserved mitochondrial function and morphology but did not influence the reduction in mitochondria numbers during PD progression in MP mice. Genetic analysis indicated that the cytoprotective effect of PT320 is attributed to a reduction in the expression of mitochondrial fission protein 1 (Fis1) and an increase in the expression of optic atrophy type 1 (Opa1), which is known to play a role in maintaining mitochondrial homeostasis and decreasing cytochrome c release through remodeling of the cristae. CONCLUSION: Our findings suggest that the early administration of PT320 shows potential as a neuroprotective treatment for PD, as it can preserve mitochondrial function. Through enhancing mitochondrial health by regulating Opa1 and Fis1, PT320 presents a new neuroprotective therapy in PD.

The mechanisms of mitochondrial abnormalities that contribute to sleep disorders and related neurodegenerative diseases.

Sleep is a highly intricate biological phenomenon, and its disorders play a pivotal role in numerous diseases. However, the specific regulatory mechanisms remain elusive. In recent years, the role of mitochondria in sleep disorders has gained considerable attention. Sleep deprivation not only impairs mitochondrial morphology but also decreases the number of mitochondria and triggers mitochondrial dysfunction. Furthermore, mitochondrial dysfunction has been implicated in the onset and progression of various sleep disorder-related neurological diseases, especially neurodegenerative conditions. Therefore, a greater understanding of the impact of sleep disorders on mitochondrial dysfunction may reveal new therapeutic targets for neurodegenerative diseases. In this review, we comprehensively summarize the recent key findings on the mechanisms underlying mitochondrial dysfunction caused by sleep disorders and their role in initiating or exacerbating common neurodegenerative diseases. In addition, we provide fresh insights into the diagnosis and treatment of sleep disorder-related diseases.

Oculomasticatory rhythmic movements, insomnia and stroke-like episodes in a patient with POLG mutation.

The POLG mutation, a leading cause of mitochondrial diseases, exhibits a wide-ranging age of onset and a complex clinical presentation. We encountered an atypical clinical profile in an elderly man with a POLG mutation, characterised by a stroke-like episode, chronic insomnia and transient oculomasticatory rhythmic movement. History revealed chronic constipation since his 50s and progressive bilateral ophthalmoplegia since his early 60s. Subsequently, he had experienced acute encephalopathy and later developed chronic insomnia. The present neurological examination showed bilateral complete ophthalmoplegia, ptosis, and rhythmic ocular and jaw movements. Imaging indicated findings suggestive of a stroke-like episode and eventual genetic analysis revealed a homozygous missense mutation in the POLG gene. This case expands the clinical spectrum of POLG mutations in individuals over 60 years, showcasing the rare combination of a stroke-like episode, chronic insomnia and oculomasticatory rhythmic movement.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

NDUFV1-Related Mitochondrial Complex-1 Disorders: A Retrospective Case Series and Literature Review.

BACKGROUND: Pathogenic variants in the NDUFV1 gene disrupt mitochondrial complex I, leading to neuroregression with leukoencephalopathy and basal ganglia involvement on neuroimaging. This study aims to provide a concise review on NDUFV1-related disorders while adding the largest cohort from a single center to the existing literature. METHODS: We retrospectively collected genetically proven cases of NDUFV1 pathogenic variants from our center over the last decade and explored reported instances in existing literature. Magnetic resonance imaging (MRI) patterns observed in these patients were split into three types-Leigh (putamen, basal ganglia, thalamus, and brainstem involvement), mitochondrial leukodystrophy (ML) (cerebral white matter involvement with cystic cavitations), and mixed (both). RESULTS: Analysis included 44 children (seven from our center and 37 from literature). The most prevalent comorbidities were hypertonia, ocular abnormalities, feeding issues, and hypotonia at onset. Children with the Leigh-type MRI pattern exhibited significantly higher rates of breathing difficulties, whereas those with a mixed phenotype had a higher prevalence of dystonia. The c.1156C>T variant in exon 8 of the NDUFV1 gene was the most common variant among individuals of Asian ethnicity and is predominantly associated with irritability and dystonia. Seizures and Leigh pattern of MRI of the brain was found to be less commonly associated with this variant. Higher rate of mortality was observed in children with Leigh-type pattern on brain MRI and those who did not receive mitochondrial cocktail. CONCLUSIONS: MRI phenotyping might help predict outcome. Appropriate and timely treatment with mitochondrial cocktail may reduce the probability of death and may positively impact the long-term outcomes, regardless of the genetic variant or age of onset.

Hydrogen sulfide supplementation as a potential treatment for primary mitochondrial diseases.

Primary mitochondrial diseases (PMD) are amongst the most common inborn errors of metabolism causing fatal outcomes within the first decade of life. With marked heterogeneity in both inheritance patterns and physiological manifestations, these conditions present distinct challenges for targeted drug therapy, where effective therapeutic countermeasures remain elusive within the clinic. Hydrogen sulfide (H2S)-based therapeutics may offer a new option for patient treatment, having been proposed as a conserved mitochondrial substrate and post-translational regulator across species, displaying therapeutic effects in age-related mitochondrial dysfunction and neurodegenerative models of mitochondrial disease. H2S can stimulate mitochondrial respiration at sites downstream of common PMD-defective subunits, augmenting energy production, mitochondrial function and reducing cell death. Here, we highlight the primary signalling mechanisms of H2S in mitochondria relevant for PMD and outline key cytoprotective proteins/pathways amenable to post-translational restoration via H2S-mediated persulfidation. The mechanisms proposed here, combined with the advent of potent mitochondria-targeted sulfide delivery molecules, could provide a framework for H2S as a countermeasure for PMD disease progression.

A PCR-independent approach for mtDNA enrichment and next-generation sequencing: comprehensive evaluation and clinical application.

BACKGROUND: Sequencing the mitochondrial genome has been increasingly important for the investigation of primary mitochondrial diseases (PMD) and mitochondrial genetics. To overcome the limitations originating from PCR-based mtDNA enrichment, we set out to develop and evaluate a PCR-independent approach in this study, named Pime-Seq (PCR-independent mtDNA enrichment and next generation Sequencing). RESULTS: By using the optimized mtDNA enrichment procedure, the mtDNA reads ratio reached 88.0 ± 7.9% in the sequencing library when applied on human PBMC samples. We found the variants called by Pime-Seq were highly consistent among technical repeats. To evaluate the accuracy and reliability of this method, we compared Pime-Seq with lrPCR based NGS by performing both methods simultaneously on 45 samples, yielding 1677 concordant variants, as well as 146 discordant variants with low-level heteroplasmic fraction, in which Pime-Seq showed higher reliability. Furthermore, we applied Pime-Seq on 4 samples of PMD patients retrospectively, and successfully detected all the pathogenic mtDNA variants. In addition, we performed a prospective study on 192 apparently healthy pregnant women during prenatal screening, in which Pime-Seq identified pathogenic mtDNA variants in 4 samples, providing extra information for better health monitoring in these cases. CONCLUSIONS: Pime-Seq can obtain highly enriched mtDNA in a PCR-independent manner for high quality and reliable mtDNA deep-sequencing, which provides us an effective and promising tool for detecting mtDNA variants for both clinical and research purposes.

Elevating the significance of legume intake: A novel strategy to counter aging-related mitochondrial dysfunction and physical decline.

Mitochondrial dysfunction increasingly becomes a target for promoting healthy aging and longevity. The dysfunction of mitochondria with age ultimately leads to a decline in physical functions. Among them, biogenesis dysfunction and the imbalances in the metabolism of reactive oxygen species and mitochondria as signaling organelles in the aging process have aroused our attention. Dietary intervention in mitochondrial dysfunction and physical decline during aging processes is essential, and greater attention should be directed toward healthful legume intake. Legumes are constantly under investigation for their nutritional and bioactive properties, and their consumption may yield antiaging and mitochondria-protecting benefits. This review summarizes mitochondrial dysfunction with age, discusses the benefits of legumes on mitochondrial function, and introduces the potential role of legumes in managing aging-related physical decline. Additionally, it reveals the benefits of legume intake for the elderly and offers a viable approach to developing legume-based functional food.

Remarkable clinical improvement with oral nucleoside treatment in a patient with adult-onset TK2 deficiency: A case report.

OBJECTIVES: Thymidine kinase 2 deficiency (TK2d) is a rare autosomal recessive mitochondrial disorder. It manifests as a continuous clinical spectrum, from fatal infantile mitochondrial DNA depletion syndromes to adult-onset mitochondrial myopathies characterized by ophthalmoplegia-plus phenotypes with early respiratory involvement. Treatment with pyrimidine nucleosides has recently shown striking effects on survival and motor outcomes in the more severe infantile-onset clinical forms. We present the response to treatment in a patient with adult-onset TK2d. METHODS: An adult with ptosis, ophthalmoplegia, facial, neck, and proximal muscle weakness, non-invasive nocturnal mechanical ventilation, and dysphagia due to biallelic pathogenic variants in TK2 received treatment with 260 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT) under a Compassionate Use Program. Prospective motor and respiratory assessments are presented. RESULTS: After 27 months of follow-up, the North Star Ambulatory Assessment improved by 11 points, he walked 195 m more in the 6 Minute-Walking-Test, ran 10 s faster in the 100-meter time velocity test, and the Forced Vital Capacity stabilized. Growth Differentiation Factor-15 (GDF15) levels, a biomarker of respiratory chain dysfunction, normalized. The only reported side effect was dose-dependent diarrhea. DISCUSSION: Treatment with dC and dT can significantly improve motor performance and stabilize respiratory function safely in patients with adult-onset TK2d.