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.

Mitochondrial complex I deficiency stratifies idiopathic Parkinson's disease.

Idiopathic Parkinson's disease (iPD) is believed to have a heterogeneous pathophysiology, but molecular disease subtypes have not been identified. Here, we show that iPD can be stratified according to the severity of neuronal respiratory complex I (CI) deficiency, and identify two emerging disease subtypes with distinct molecular and clinical profiles. The CI deficient (CI-PD) subtype accounts for approximately a fourth of all cases, and is characterized by anatomically widespread neuronal CI deficiency, a distinct cell type-specific gene expression profile, increased load of neuronal mtDNA deletions, and a predilection for non-tremor dominant motor phenotypes. In contrast, the non-CI deficient (nCI-PD) subtype exhibits no evidence of mitochondrial impairment outside the dopaminergic substantia nigra and has a predilection for a tremor dominant phenotype. These findings constitute a step towards resolving the biological heterogeneity of iPD with implications for both mechanistic understanding and treatment strategies.

From powerhouse to regulator: The role of mitoepigenetics in mitochondrion-related cellular functions and human diseases.

Beyond their crucial role in energy production, mitochondria harbor a distinct genome subject to epigenetic regulation akin to that of nuclear DNA. This paper delves into the nascent but rapidly evolving fields of mitoepigenetics and mitoepigenomics, exploring the sophisticated regulatory mechanisms governing mitochondrial DNA (mtDNA). These mechanisms encompass mtDNA methylation, the influence of non-coding RNAs (ncRNAs), and post-translational modifications of mitochondrial proteins. Together, these epigenetic modifications meticulously coordinate mitochondrial gene transcription, replication, and metabolism, thereby calibrating mitochondrial function in response to the dynamic interplay of intracellular needs and environmental stimuli. Notably, the dysregulation of mitoepigenetic pathways is increasingly implicated in mitochondrial dysfunction and a spectrum of human pathologies, including neurodegenerative diseases, cancer, metabolic disorders, and cardiovascular conditions. This comprehensive review synthesizes the current state of knowledge, emphasizing recent breakthroughs and innovations in the field. It discusses the potential of high-resolution mitochondrial epigenome mapping, the diagnostic and prognostic utility of blood or tissue mtDNA epigenetic markers, and the promising horizon of mitochondrial epigenetic drugs. Furthermore, it explores the transformative potential of mitoepigenetics and mitoepigenomics in precision medicine. Exploiting a theragnostic approach to maintaining mitochondrial allostasis, this paper underscores the pivotal role of mitochondrial epigenetics in charting new frontiers in medical science.

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.

Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene.

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.

Intracranial calcifications simulating Aicardi-Goutières syndrome in PARS2-related mitochondrial disease.

PARS2 encodes an aminoacyl-tRNA synthetase that catalyzes the ligation of proline to mitochondrial prolyl-tRNA molecules. Diseases associated with PARS2 primarily affect the central nervous system, causing early infantile developmental epileptic encephalopathies (EIDEE; DEE75; MIM #618437) with infantile-onset neurodegeneration. Dilated cardiomyopathy has also been reported in the affected individuals. About 10 individuals to date have been described with pathogenic biallelic variants in PARS2. While many of the reported individuals succumbed to the disease in the first two decades of life, autopsy findings have not yet been reported. Here, we describe neuropathological findings in a deceased male with evidence of intracranial calcifications in the basal ganglia, thalamus, cerebellum, and white matter, similar to Aicardi-Goutières syndrome. This report describes detailed autopsy findings in a child with PARS2-related mitochondrial disease and provides plausible evidence that intracranial calcifications may be a previously unrecognized feature of this disorder.

Mitochondria in biology and medicine - 2023.

Mitochondria are an indispensable part of the cell that plays a crucial role in regulating various signaling pathways, energy metabolism, cell differentiation, proliferation, and cell death. Since mitochondria have their own genetic material, they differ from their nuclear counterparts, and dysregulation is responsible for a broad spectrum of diseases. Mitochondrial dysfunction is associated with several disorders, including neuro-muscular disorders, cancer, and premature aging, among others. The intricacy of the field is due to the cross-talk between nuclear and mitochondrial genes, which has also improved our knowledge of mitochondrial functions and their pathogenesis. Therefore, interdisciplinary research and communication are crucial for mitochondrial biology and medicine due to the challenges they pose for diagnosis and treatment. The ninth annual conference of the Society for Mitochondria Research and Medicine (SMRM)- India, titled "Mitochondria in Biology and Medicine" was organized at the Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India, on June 21-23, 2023. The latest advancements in the field of mitochondrial biology and medicine were discussed at the conference. In this article, we summarize the entire event for the benefit of researchers working in the field of mitochondrial biology and medicine.

FARS2 Deficiency Causes Cardiomyopathy by Disrupting Mitochondrial Homeostasis and the Mitochondrial Quality Control System.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common heritable heart disease. Although HCM has been reported to be associated with many variants of genes involved in sarcomeric protein biomechanics, pathogenic genes have not been identified in patients with partial HCM. FARS2 (the mitochondrial phenylalanyl-tRNA synthetase), a type of mitochondrial aminoacyl-tRNA synthetase, plays a role in the mitochondrial translation machinery. Several variants of FARS2 have been suggested to cause neurological disorders; however, FARS2-associated diseases involving other organs have not been reported. We identified FARS2 as a potential novel pathogenic gene in cardiomyopathy and investigated its effects on mitochondrial homeostasis and the cardiomyopathy phenotype. METHODS: FARS2 variants in patients with HCM were identified using whole-exome sequencing, Sanger sequencing, molecular docking analyses, and cell model investigation. Fars2 conditional mutant (p.R415L) or knockout mice, fars2-knockdown zebrafish, and Fars2-knockdown neonatal rat ventricular myocytes were engineered to construct FARS2 deficiency models both in vivo and in vitro. The effects of FARS2 and its role in mitochondrial homeostasis were subsequently evaluated using RNA sequencing and mitochondrial functional analyses. Myocardial tissues from patients were used for further verification. RESULTS: We identified 7 unreported FARS2 variants in patients with HCM. Heart-specific Fars2-deficient mice presented cardiac hypertrophy, left ventricular dilation, progressive heart failure accompanied by myocardial and mitochondrial dysfunction, and a short life span. Heterozygous cardiac-specific Fars2R415L mice displayed a tendency to cardiac hypertrophy at age 4 weeks, accompanied by myocardial dysfunction. In addition, fars2-knockdown zebrafish presented pericardial edema and heart failure. FARS2 deficiency impaired mitochondrial homeostasis by directly blocking the aminoacylation of mt-tRNAPhe and inhibiting the synthesis of mitochondrial proteins, ultimately contributing to an imbalanced mitochondrial quality control system by accelerating mitochondrial hyperfragmentation and disrupting mitochondrion-related autophagy. Interfering with the mitochondrial quality control system using adeno-associated virus 9 or specific inhibitors mitigated the cardiac and mitochondrial dysfunction triggered by FARS2 deficiency by restoring mitochondrial homeostasis. CONCLUSIONS: Our findings unveil the previously unrecognized role of FARS2 in heart and mitochondrial homeostasis. This study may provide new insights into the molecular diagnosis and prevention of heritable cardiomyopathy as well as therapeutic options for FARS2-associated cardiomyopathy.

Variants in Human ATP Synthase Mitochondrial Genes: Biochemical Dysfunctions, Associated Diseases, and Therapies.

Mitochondrial ATP synthase (Complex V) catalyzes the last step of oxidative phosphorylation and provides most of the energy (ATP) required by human cells. The mitochondrial genes MT-ATP6 and MT-ATP8 encode two subunits of the multi-subunit Complex V. Since the discovery of the first MT-ATP6 variant in the year 1990 as the cause of Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome, a large and continuously increasing number of inborn variants in the MT-ATP6 and MT-ATP8 genes have been identified as pathogenic. Variants in these genes correlate with various clinical phenotypes, which include several neurodegenerative and multisystemic disorders. In the present review, we report the pathogenic variants in mitochondrial ATP synthase genes and highlight the molecular mechanisms underlying ATP synthase deficiency that promote biochemical dysfunctions. We discuss the possible structural changes induced by the most common variants found in patients by considering the recent cryo-electron microscopy structure of human ATP synthase. Finally, we provide the state-of-the-art of all therapeutic proposals reported in the literature, including drug interventions targeting mitochondrial dysfunctions, allotopic gene expression- and nuclease-based strategies, and discuss their potential translation into clinical trials.

CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering.

Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base editors and more recently guanosine base editors. Because of having low off-target and indel rates, there is a growing interest in developing and evolving this research field. Here, we provide a detailed update on DNA base editors. While base editing has widely been used for nuclear genome engineering, the growing interest in applying this technology to mitochondrial DNA has been faced with several challenges. While Cas9 protein has been shown to enter mitochondria, use of smaller Cas proteins, such as Cas12a, has higher import efficiency. However, sgRNA transfer into mitochondria is the most challenging step. sgRNA structure and ratio of Cas protein to sgRNA are both important factors for efficient sgRNA entry into mitochondria. In conclusion, while there are still several challenges to be addressed, ongoing research in this field holds the potential for new treatments and therapies for mitochondrial disorders.

Gene therapy for mitochondrial disorders.

In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno-associated virus-based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator-like effector nucleases, zinc-finger nucleases, and meganucleases (mitoARCUS). rAAV-based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR-Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR-free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design.

Challenges and opportunities to bridge translational to clinical research for personalized mitochondrial medicine.

Mitochondrial disorders are a group of rare and heterogeneous genetic diseases characterized by dysfunctional mitochondria leading to deficient adenosine triphosphate synthesis and chronic energy deficit in patients. The majority of these patients exhibit a wide range of phenotypic manifestations targeting several organ systems, making their clinical diagnosis and management challenging. Bridging translational to clinical research is crucial for improving the early diagnosis and prognosis of these intractable mitochondrial disorders and for discovering novel therapeutic drug candidates and modalities. This review provides the current state of clinical testing in mitochondrial disorders, discusses the challenges and opportunities for converting basic discoveries into clinical settings, explores the most suited patient-centric approaches to harness the extraordinary heterogeneity among patients affected by the same primary mitochondrial disorder, and describes the current outlook of clinical trials.

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated.

The cold-inducible RNA-binding protein-Thioredoxin 1 pathway ameliorates mitochondrial dysfunction and mitochondrial dynamin-related protein 1 level in the hippocampus of aged mice with perioperative neurocognitive dysfunction.

BACKGROUND: As a multi-disease model, neuroinflammation, mitochondrial dysfunction, and oxidative stress might be involved in the pathogenic process of perioperative neurocognitive dysfunction (PND). Dynamin-related protein 1 (Drp1) could mediate mitochondrial fission and play important roles in mitochondrial dynamic homeostasis and mitochondria function. The Drp1 may be involved in PND development. The cold-inducible RNA-binding protein (Cirbp) could bind to the 3'-UTR of the thioredoxin 1 (Trx1) mRNA, control oxidative stress, and improve mitochondrial function. In this study, we hypothesized that the Cirbp-Trx1 pathway could ameliorate mitochondrial dysfunction and Drp1 levels in PND mice. METHODS: Differentially expressed genes were screened using the Gene Expression Omnibus (GEO) database GSE95426 and validated using PCR. Eighteen-month-old C57BL/6 mice were subjected to tibial fracture surgery to generate a PND model. Cirbp was upregulated by hippocampal stereotaxic injections of over-Cirbp plasmid according to the manufacturer's instructions for the in vivo DNA transfection reagent. Cirbp expression was measured using western blot (WB) and immunofluorescence (IF). The Morris water maze (MWM) was used to assess cognitive function. After behavioral testing, the hippocampal tissue was extracted to examine changes in mitochondrial Drp1, mitochondrial function, neuroinflammation, and oxidative stress. RESULTS: Differential gene screening showed that Cirbp expression was significantly downregulated (fold change >1.5, p = 0.003272) in the PND model. In this study, we also found that Cirbp protein levels were downregulated, accompanied by an impairment of cognition, a decrease in superoxide dismutase (SOD) activity, and an increase in malondialdehyde (MDA) content, mitochondrial Drp1 levels, neuroinflammation, and apoptosis. Cirbp overexpression increased Trx1 protein levels and reversed the damage. However, this protective effect was abolished by PX-12 treatment with a Trx1 inhibitor. CONCLUSIONS: The Cirbp-Trx1 pathway may regulate mitochondrial dysfunction and mitochondrial Drp1 expression in the hippocampus of PND mice to ameliorate cognitive dysfunction.

Atypical presentation of Pearson syndrome in an infant with suspected myelodysplastic syndrome.

BACKGROUND: Anemia exhibits complex causation mechanisms and genetic heterogeneity. Some cases result in poor outcomes with multisystemic dysfunction, including renal tubulopathy. Early diagnosis is crucial to improve management. CASE-DIAGNOSIS/TREATMENT: A 21-month-old female patient was admitted with severe anemia. Persistent neutropenia and dysplastic signs suggested myelodysplastic syndrome, but targeted gene panel results were negative. After multiple transfusions, spontaneous hematologic recovery was observed. At 4 years old, she presented failure to thrive, renal Fanconi syndrome, and severe metabolic acidosis. Differential diagnosis included Pearson syndrome (PS), a life-threatening condition associated with mitochondrial DNA (mtDNA), featuring anemia and pancreatic insufficiency. Further analysis revealed a ~ 7.5 kb mtDNA deletion. Until the age of 5, supportive care has been provided, without pancreatic insufficiency. CONCLUSIONS: This PS case highlights the importance of genetic testing, even in the absence of typical features. Understanding the nature of mitochondrial disorders enables treatment tailoring and counseling about the prognosis.

Excessive BNIP3- and BNIP3L-dependent mitophagy underlies the pathogenesis of FBXL4-mutated mitochondrial DNA depletion syndrome.

Mitophagy, the process of removing damaged mitochondria to promote cell survival, plays a crucial role in cellular functionality. However, excessive, or uncontrolled mitophagy can lead to reduced mitochondrial content that burdens the remaining organelles, triggering mitophagy-mediated cell death. FBXL4 mutations, which affect the substrate-binding adaptor of the CUL1 (cullin 1)-RING ubiquitin ligase complex (CRL1), have been linked to mitochondrial DNA depletion syndrome type 13 (MTDPS13) characterized by reduced mtDNA content and impaired energy production in affected organs. However, the mechanism behind FBXL4 mutation-driven MTDPS13 remain poorly understood. In a recent study, we demonstrate that the CRL1-FBXL4 complex promotes the degradation of BNIP3 and BNIP3L, two key mitophagy cargo receptors. Deficiency of FBXL4 results in a strong accumulation of BNIP3 and BNIP3L proteins and triggers high levels of BNIP3- and BNIP3L-dependent mitophagy. Patient-derived FBXL4 mutations do not affect its interaction with BNIP3 and BNIP3L but impair the assembly of an active CRL1-FBXL4 complex. Furthermore, excessive mitophagy is observed in knockin mice carrying a patient-derived FBXL4 mutation, and in cortical neurons generated from human patient induced pluripotent stem cells (hiPSCs). These findings support the model that the CRL1-FBXL4 complex tightly restricts basal mitophagy, and its dysregulation leads to severe symptoms of MTDPS13.

Bufalin targeting CAMKK2 inhibits the occurrence and development of intrahepatic cholangiocarcinoma through Wnt/ß-catenin signal pathway.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) accounts for about 15% of primary liver cancer, and the incidence rate has been rising in recent years. Surgical resection is the best treatment for ICC, but the 5-year survival rate is less than 30%. ICC signature genes are crucial for the early diagnosis of ICC, so it is especially important to find its signature genes and therapeutic drug. Here, we studied that bufalin targeting CAMKK2 promotes mitochondrial dysfunction and inhibits the occurrence and metastasis of intrahepatic cholangiocarcinoma through Wnt/ß-catenin signal pathway. METHODS: IC50 of bufalin in ICC cells was determined by CCK8 and invasive and migratory abilities were verified by wound healing, cell cloning, transwell and Western blot. IF and IHC verified the expression of CAMKK2 between ICC patients and normal subjects. BLI and pull-down demonstrated the binding ability of bufalin and CAMKK2. Bioinformatics predicted whether CAMKK2 was related to the Wnt/ß-catenin pathway. SKL2001, an activator of ß-catenin, verified whether bufalin acted through this pathway. In vitro and in vivo experiments verified whether overexpression of CAMKK2 affects the proliferative and migratory effects of ICC. Transmission electron microscopy verified mitochondrial integrity. Associated Ca2+ levels verified the biological effects of ANXA2 on ICC. RESULTS: It was found that bufalin inhibited the proliferation and migration of ICC, and CAMKK2 was highly expressed in ICC, and its high expression was positively correlated with poor prognosis.CAMKK2 is a direct target of bufalin, and is associated with the Wnt/ß-catenin signaling pathway, which was dose-dependently decreased after bufalin treatment. In vitro and in vivo experiments verified that CAMKK2 overexpression promoted ICC proliferation and migration, and bufalin reversed this effect. CAMKK2 was associated with Ca2+, and changes in Ca2+ content induced changes in the protein content of ANXA2, which was dose-dependently decreasing in cytoplasmic ANXA2 and dose-dependently increasing in mitochondrial ANXA2 after bufalin treatment. In CAMKK2 overexpressing cells, ANXA2 was knocked down, and we found that reversal of CAMKK2 overexpression-induced enhancement of ICC proliferation and migration after siANXA2. CONCLUSIONS: Our results suggest that bufalin targeting CAMKK2 promotes mitochondrial dysfunction and inhibits the proliferation and migration of intrahepatic cholangiocarcinoma through Wnt/ß-catenin signal pathway. Thus, bufalin, as a drug, may also be used for cancer therapy in ICC in the future.

Specifications of the ACMG/AMP guidelines for ACADVL variant interpretation.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a relatively common inborn error of metabolism, but due to difficulty in accurately predicting affected status through newborn screening, molecular confirmation of the causative variants by sequencing of the ACADVL gene is necessary. Although the ACMG/AMP guidelines have helped standardize variant classification, ACADVL variant classification remains disparate due to a phenotype that can be nonspecific, the possibility of variants that produce late-onset disease, and relatively high carrier frequency, amongst other challenges. Therefore, an ACADVL-specific variant curation expert panel (VCEP) was created to facilitate the specification of the ACMG/AMP guidelines for VLCADD. We expect these guidelines to help streamline, increase concordance, and expedite the classification of ACADVL variants.

Mitochondrial encephalomyopathy.

Mitochondrial dysfunction, especially perturbation of oxidative phosphorylation and adenosine triphosphate (ATP) generation, disrupts cellular homeostasis and is a surprisingly frequent cause of central and peripheral nervous system pathology. Mitochondrial disease is an umbrella term that encompasses a host of clinical syndromes and features caused by in excess of 300 different genetic defects affecting the mitochondrial and nuclear genomes. Patients with mitochondrial disease can present at any age, ranging from neonatal onset to late adult life, with variable organ involvement and neurological manifestations including neurodevelopmental delay, seizures, stroke-like episodes, movement disorders, optic neuropathy, myopathy, and neuropathy. Until relatively recently, analysis of skeletal muscle biopsy was the focus of diagnostic algorithms, but step-changes in the scope and availability of next-generation sequencing technology and multiomics analysis have revolutionized mitochondrial disease diagnosis. Currently, there is no specific therapy for most types of mitochondrial disease, although clinical trials research in the field is gathering momentum. In that context, active management of epilepsy, stroke-like episodes, dystonia, brainstem dysfunction, and Parkinsonism are all the more important in improving patient quality of life and reducing mortality.

Gene Editing Technologies Targeting TFAM and Its Relation to Mitochondrial Diseases.

Mitochondria are organelles present in the cytoplasm of eukaryotic cells; they play a key role in adenosine triphosphate (ATP) synthesis and oxidative phosphorylation. Mitochondria have their own DNA, mitochondrial DNA (mtDNA), keeping the function of the mitochondria. Mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters is and considered essential in mtDNA replication and transcription. More recently, TFAM has been shown to play a central role in the maintenance and regulation of mitochondrial copy number, inflammatory response, expression regulation, and mitochondrial genome activity. Gene editing tools such as the CRISPR-Cas 9 technique, TALENs, and other gene editing tools have been used to investigate the role of TFAM in mitochondrial mechanics and biogenesis as well as its correlation to mitochondrial disorders. Thus this chapter brings a summary of mitochondria function, dysfunction, the importance of TFAM in the maintenance of mitochondria, and state of the art of gene editing tools involving TFAM and mtDNA.