bims-mitmed Biomed News
on Mitochondrial medicine
Issue of 2022‒11‒20
35 papers selected by
Dario Brunetti
Fondazione IRCCS Istituto Neurologico

  1. Proc Natl Acad Sci U S A. 2022 Nov 22. 119(47): e2210730119
      Mitochondria have their own DNA (mtDNA), which encodes essential respiratory subunits. Under live imaging, mitochondrial nucleoids, composed of several copies of mtDNA and DNA-binding proteins, such as mitochondrial transcription factor A (TFAM), actively move inside mitochondria and change the morphology, in concert with mitochondrial membrane fission. Here we found the mitochondrial inner membrane-anchored AAA-ATPase protein ATAD3A mediates the nucleoid dynamics. Its ATPase domain exposed to the matrix binds directly to TFAM and mediates nucleoid trafficking along mitochondria by ATP hydrolysis. Nucleoid trafficking also required ATAD3A oligomerization via an interaction between the coiled-coil domains in intermembrane space. In ATAD3A deficiency, impaired nucleoid trafficking repressed the clustered and enlarged nucleoids observed in mitochondrial fission-deficient cells resulted in dispersed distribution of small nucleoids observed throughout the mitochondrial network, and this enhanced respiratory complex formation. Thus, mitochondrial fission and nucleoid trafficking cooperatively determine the size, number, and distribution of nucleoids in mitochondrial network, which should modulate respiratory complex formation.
    Keywords:  ATAD3A; Drp1; mitochondrial fission; mtDNA nucleoid; respiratory complex
  2. J Cell Biol. 2023 Jan 02. pii: e202203019. [Epub ahead of print]222(1):
      Astrocytes, often considered as secondary responders to neurodegeneration, are emerging as primary drivers of brain disease. Here we show that mitochondrial DNA depletion in astrocytes affects their primary cilium, the signaling organelle of a cell. The progressive oxidative phosphorylation deficiency in astrocytes induces FOXJ1 and RFX transcription factors, known as master regulators of motile ciliogenesis. Consequently, a robust gene expression program involving motile cilia components and multiciliated cell differentiation factors are induced. While the affected astrocytes still retain a single cilium, these organelles elongate and become remarkably distorted. The data suggest that chronic activation of the mitochondrial integrated stress response (ISRmt) in astrocytes drives anabolic metabolism and promotes ciliary elongation. Collectively, our evidence indicates that an active signaling axis involving mitochondria and primary cilia exists and that ciliary signaling is part of ISRmt in astrocytes. We propose that metabolic ciliopathy is a novel pathomechanism for mitochondria-related neurodegenerative diseases.
  3. Front Physiol. 2022 ;13 1004099
      Mitochondria contain their own DNA, mitochondrial DNA, which encodes thirteen proteins. However, mitochondria require thousands of proteins encoded in the nucleus to carry out their many functions. Identifying the definitive mitochondrial proteome has been challenging as methods isolating mitochondrial proteins differ and different tissues and organisms may have specialized proteomes. Mitochondrial diseases arising from single gene mutations in nucleus encoded genes could affect the mitochondrial proteome, but deciphering which effects are due to loss of specific pathways or to accumulated general mitochondrial damage is difficult. To identify specific versus general effects, we have taken advantage of mutations in three Drosophila genes, clueless, Sod2, and Pink1, which are required for mitochondrial function through different pathways. We measured changes in each mutant's mitochondrial proteome using quantitative tandem mass tag mass spectrometry. Our analysis identified protein classes that are unique to each mutant and those shared between them, suggesting that some changes in the mitochondrial proteome are due to general mitochondrial damage whereas others are gene specific. For example, clueless mutants had the greatest number of less and more abundant mitochondrial proteins whereas loss of all three genes increased stress and metabolism proteins. This study is the first to directly compare in vivo steady state levels of mitochondrial proteins by examining loss of three pathways critical for mitochondrial function. These data could be useful to understand disease etiology, and how mutations in genes critical for mitochondrial function cause specific mitochondrial proteomic changes as opposed to changes due to generalized mitochondrial damage.
    Keywords:  Clueless; PINK1; SOD2; drosophila; mitochondria; mitochondrial proteome; respiratory chain complexes
  4. PeerJ. 2022 ;10 e14350
      Mitochondria play essential cellular roles in Adenosine triphosphate (ATP) synthesis, calcium homeostasis, and metabolism, but these vital processes have potentially deadly side effects. The production of the reactive oxygen species (ROS) and the aggregation of misfolded mitochondrial proteins can lead to severe mitochondrial damage and even cell death. The accumulation of mitochondrial damage is strongly implicated in aging and several incurable diseases, including neurodegenerative disorders and cancer. To oppose this, metazoans utilize a variety of quality control strategies, including the degradation of the damaged mitochondrial proteins by the mitochondrial-resident proteases of the ATPase Associated with the diverse cellular Activities (AAA+) family. This mini-review focuses on the quality control mediated by the mitochondrial-resident proteases of the AAA+ family used to combat the accumulation of damaged mitochondria and on how the failure of this mitochondrial quality control contributes to diseases.
    Keywords:  AAA+ Protease; Mitochondria in neurological disorders; Mitochondrial Translation; Mitochondrial Unfolded Protein Response; Mitochondrial quality control
  5. Trends Pharmacol Sci. 2022 Nov 14. pii: S0165-6147(22)00229-2. [Epub ahead of print]
      Doxorubicin (DOX) is a chemotherapeutic drug that is utilized for solid tumors and hematologic malignancies, but its clinical application is hampered by life-threatening cardiotoxicity including cardiac dilation and heart failure. Mitochondrial quality control processes, including mitochondrial proteostasis, mitophagy, and mitochondrial dynamics and biogenesis, serve to maintain mitochondrial homeostasis in the cardiovascular system. Importantly, recent advances have unveiled a major role for defective mitochondrial quality control in the etiology of DOX cardiomyopathy. Moreover, specific interventions targeting these quality control mechanisms to preserve mitochondrial function have emerged as potential therapeutic strategies to attenuate DOX cardiotoxicity. However, clinical translation is challenging because of obscure mechanisms of action and potential adverse effects. The purpose of this review is to provide new insights regarding the role of mitochondrial quality control in the pathogenesis of DOX cardiotoxicity, and to explore promising therapeutic approaches targeting these mechanisms to aid clinical management.
    Keywords:  doxorubicin-induced cardiotoxicity; mitochondria; mitochondrial biogenesis; mitochondrial dynamics; mitochondrial quality control
  6. Sci Rep. 2022 Nov 18. 12(1): 19841
      The phenomenon of intercellular mitochondrial transfer has attracted great attention in various fields of research, including stem cell biology. Elucidating the mechanism of mitochondrial transfer from healthy stem cells to cells with mitochondrial dysfunction may lead to the development of novel stem cell therapies to treat mitochondrial diseases, among other advances. To visually evaluate and analyze the mitochondrial transfer process, dual fluorescent labeling systems are often used to distinguish the mitochondria of donor and recipient cells. Although enhanced green fluorescent protein (EGFP) has been well-characterized for labeling mitochondria, other colors of fluorescent protein have been less extensively evaluated in the context of mitochondrial transfer. Here, we generated different lentiviral vectors with mitochondria-targeted red fluorescent proteins (RFPs), including DsRed, mCherry (both from Discosoma sp.) Kusabira orange (mKOκ, from Verrillofungia concinna), and TurboRFP (from Entacmaea quadricolor). Among these proteins, mitochondria-targeted DsRed and its variant mCherry often generated bright aggregates in the lysosome while other proteins did not. We further validated that TurboRFP-labeled mitochondria were successfully transferred from amniotic epithelial cells, one of the candidates for donor stem cells, to mitochondria-damaged recipient cells without losing the membrane potential. Our study provides new insight into the genetic labeling of mitochondria with red fluorescent proteins, which may be utilized to analyze the mechanism of intercellular mitochondrial transfer.
  7. Mitochondrion. 2022 Nov 09. pii: S1567-7249(22)00090-3. [Epub ahead of print]68 10-14
      Herein, we review evidence that targeting mitochondrial dysfunction with 'mitoceuticals' is an effective neuroprotective strategy following neurotrauma, and that isolated exogenous mitochondria can be effectively transplanted into host spinal cord parenchyma to increase overall cellular metabolism. We further discuss control measures to ensure greatest potential for mitochondrial transfer, notably using erodible thermogelling hydrogels to deliver respiratory competent mitochondria to the injured spinal cord.
    Keywords:  Bioenergetics; Hydrogel; Metabolism; Mitochondria; Oxidative phosphorylation; Spinal cord; Transplantation
  8. J Law Med Ethics. 2022 ;50(3): 597-602
      Mitochondrial replacement therapy (MRT), also called nuclear genome transfer and mitochondrial donation, is a new technique that can be used to prevent the transmission of mitochondrial DNA diseases. Apart from the United Kingdom, the first country to approve MRT in 2015, Australia became the second country with a clear regulatory path for the clinical applications of this technique in 2021. The rapidly evolving clinical landscape of MRT makes the elaboration and evaluation of the responsible use of this technology a pressing matter. As jurisdictions with less strict or non-existent reproductive laws are continuing to use MRT in the clinical context, the need to address the underlying ethical issues surrounding MRT's clinical translation is fundamental.
    Keywords:  Genetic Relatedness; MRT; Mitochondrial Replacement Therapy; Reproductive Autonomy
  9. Cureus. 2022 Oct;14(10): e30198
      Mitochondrial DNA (mtDNA) is responsible for encoding 13 subunits of the respiratory chain. These subunits are crucial in providing reducing equivalents for the energy-intensive intracellular processes. Leber hereditary optic neuropathy (LHON) is a mitochondrial illness that causes carcinogenesis due to oxidative stress and painless loss of central vision as a result of selective degradation of retinal ganglion cells as well as their axons. We present a case of a 23-year-old male patient who was diagnosed with subacute LHON. The mutation in our patient was found in a less commonly mutated exon sequence of MT-NDL4, which codes for NADH (nicotinamide adenine dinucleotide hydrogen, reduced) dehydrogenase subunit 4L. The MT-ND4L exon is located immediately upstream of the MTD4 exon on the human mtDNA. The take-home message is to always perform a comprehensive mitochondrial genome analysis for identifying rare mutations when LHON is suspected.
    Keywords:  blindness without neurological deficit; genetic eye diseases; leber hereditary optic neuropathy; mtdna disorder; mtdna mutation; rare genetic diseases; sudden loss of vision
  10. Sci Rep. 2022 Nov 18. 12(1): 19847
      Barth Syndrome (BTHS), a genetic disease associated with early-onset cardioskeletal myopathy, is caused by loss-of-function mutations of the TAFAZZIN gene, which is responsible for remodeling the mitochondrial phospholipid cardiolipin (CL). Deregulation of CL biosynthesis and maturation in BTHS mitochondria result in a dramatically increased monolysocardiolipin (MLCL)/CL ratio associated with bioenergetic dysfunction. One of the most promising therapeutic approaches for BTHS includes the mitochondria-targeted tetrapeptide SS-31, which interacts with CL. Here, we used TAFAZZIN knockdown (TazKD) mice to investigate for the first time whether in vivo administration of SS-31 could affect phospholipid profiles and mitochondrial dysfunction. The CL fingerprinting of TazKD cardiac mitochondria obtained by MALDI-TOF/MS revealed the typical lipid changes associated with BTHS. TazKD mitochondria showed lower respiratory rates in state 3 and 4 together with a decreased in maximal respiratory rates. Treatment of TazKD mice with SS-31 improved mitochondrial respiratory capacity and promoted supercomplex organization, without affecting the MLCL/CL ratio. We hypothesize that SS-31 exerts its effect by influencing the function of the respiratory chain rather than affecting CL directly. In conclusion, our results indicate that SS-31 have beneficial effects on improving cardiac mitochondrial dysfunction in a BTHS animal model, suggesting the peptide as future pharmacologic agent for therapy.
  11. Mol Neurobiol. 2022 Nov 15.
      Astrocytes are key glial cells for the metabolic and functional support of the brain. Mitochondrial quality control (MQC), in particular the balance between mitophagy and mitochondrial biogenesis, is a major event for the maintenance of cellular homeostasis. Carbon monoxide (CO) is an endogenous gasotransmitter that inhibits cell death and inflammation by targeting mitochondria. It is well established that CO promotes cytoprotection by increasing mitochondrial population and metabolism (oxidative phosphorylation). Thus, it is hypothesized that CO-induced cytoprotection may also be mediated by the balance between mitophagy and mitochondrial biogenesis. Herein, the carbon monoxide releasing molecule-A1 (CORM-A1) was used in primary cultures of astrocytes to assess CO role on mitochondrial turnover. PINK1/Parkin-dependent mitophagy was stimulated by CORM-A1 following 1 h of treatment. While at 24 h after treatment, CORM-A1 increased mitochondrial population, which may indicate mitochondrial biogenesis. In fact, mitochondrial biogenesis was confirmed by the enhancement of PGC-1α expression that upregulates several mitochondrial transcription factors. Furthermore, inhibition of mitophagy by knocking down PINK1 expression reverted CO-induced mitochondrial biogenesis, indicating that mitochondrial turnover is dependent on modulation of mitophagy. Finally, CORM-A1 prevented astrocytic cell death induced by oxidative stress in a mitophagy-dependent manner. In fact, whenever PINK1 was knocked down, CORM-A1-induced cytoprotection was lost. In summary, CORM-A1 stimulates mitochondrial turnover, which in turn prevents astrocytic cell death. CO cytoprotection depends on increasing mitochondrial population and on eliminating dysfunctional mitochondria.
    Keywords:  Astrocytes; Carbon monoxide; Cell death; Mitochondrial biogenesis; Mitophagy; PINK1
  12. EMBO J. 2022 Nov 18. e112006
      Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.
    Keywords:  HOIP; NEMO; OTULIN; PINK1; ubiquitin
  13. Sci Adv. 2022 Nov 16. 8(46): eabq5234
      A stop codon within the mRNA facilitates coordinated termination of protein synthesis, releasing the nascent polypeptide from the ribosome. This essential step in gene expression is impeded with transcripts lacking a stop codon, generating nonstop ribosome complexes. Here, we use deep sequencing to investigate sources of nonstop mRNAs generated from the human mitochondrial genome. We identify diverse types of nonstop mRNAs on mitochondrial ribosomes that are resistant to translation termination by canonical release factors. Failure to resolve these aberrations by the mitochondrial release factor in rescue (MTRFR) imparts a negative regulatory effect on protein synthesis that is associated with human disease. Our findings reveal a source of underlying noise in mitochondrial gene expression and the importance of responsive ribosome quality control mechanisms for cell fitness and human health.
  14. Elife. 2022 11 17. pii: e69916. [Epub ahead of print]11
      Having its genome makes the mitochondrion a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is a double-stranded closed circular molecule of about 16 kb coding for 37 genes. Mutations, including deletions in the mitochondrial genome, can culminate in different human diseases. Mapping the deletion junctions suggests that the breakpoints are generally seen at hotspots. '9-bp deletion' (8271-8281), seen in the intergenic region of cytochrome c oxidase II/tRNA<sup>Lys</sup>, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with '9-bp deletion' when it is present on a plasmid or in the mitochondrial genome. Through a series of <i>in vitro</i> and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Therefore, we uncover a new role for Endonuclease G in generating mtDNA deletions, which depends on the formation of G4 DNA within the mitochondrial genome. In summary, we identify a novel property of Endonuclease G, besides its role in apoptosis and the recently described elimination of paternal mitochondria during fertilisation.
    Keywords:  E. coli; cell biology; genetics; genomics; human; rat
  15. J Biol Chem. 2022 Nov 12. pii: S0021-9258(22)01147-4. [Epub ahead of print] 102704
      The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.
    Keywords:  MAP1LC3B/LC3B; PHB2; Parkin; mitophagy; ubiquitination
  16. Mitochondrion. 2022 Oct 29. pii: S1567-7249(22)00089-7. [Epub ahead of print]68 1-9
      TK2d is an ultrarare autosomal recessive mitochondrial DNA depletion syndrome. Nucleoside therapy improves or stabilizes disease across key outcomes including survival, ambulation, and requirement for mechanical ventilation. However, little is known about the effects of nucleoside therapy treatment of TK2d from the patient's perspective. This study sought to address this knowledge gap. Participants with TK2d and/or their parents/caregivers completed online surveys with standardized health measures and interviews. During interviews, participants rated and described TK2d's impact on 13 quality of life domains, changes since starting nucleoside therapy, and if they would recommend nucleoside therapy. Twenty-five individuals participated (17 adults with TK2d, 4 parent-participant pairs, 4 parents of children with TK2d). Adult participants with TK2d had clinically meaningfully worse scores than the general population on global physical and mental health, physical function, pain interference, fatigue, anxiety, and social function. Children's mobility and pain interference were significantly worse than the general pediatric population. Physical domains most affected by TK2d were: mobility (84%), fatigue (60%), respiratory function (56%), and hospitalizations (55%). Psychosocial domains most affected were: impact on family members (39%), mood (36%), and social life (28%). Most (77%) treated patients reported improvement; whereas, 67% in the untreated group reported worsening. All participants would recommend nucleoside therapy. In summary, TK2d has significant negative impacts on most areas of life and function. Measures of fatigue, sleep, swallowing/eating, speaking, and mood, should be considered as outcomes in clinical trials and research studies. Nucleoside therapy appears to provide meaningful improvements across many health domains affected by TK2d. SYNOPSIS: The consequences of having TK2d are devastating for both those with the disorder and their families; however, nucleoside therapy appears to provide meaningful improvements across many health domains affected by TK2d.
    Keywords:  Mitochondrial disease; Nucleoside therapy; Quality of life; Thymidine kinase 2 deficiency; Treatment effects
  17. Mol Psychiatry. 2022 Nov 15.
      Ample evidence implicate mitochondria in early brain development. However, to the best of our knowledge, there is only circumstantial data for mitochondria involvement in late brain development occurring through adolescence, a critical period in the pathogenesis of various psychiatric disorders, specifically schizophrenia. In schizophrenia, neurodevelopmental abnormalities and mitochondrial dysfunction has been repeatedly reported. Here we show a causal link between mitochondrial transplantation in adolescence and brain functioning in adulthood. We show that transplantation of allogenic healthy mitochondria into the medial prefrontal cortex of adolescent rats was beneficial in a rat model of schizophrenia, while detrimental in healthy control rats. Specifically, disparate initial changes in mitochondrial function and inflammatory response were associated with opposite long-lasting changes in proteome, neurotransmitter turnover, neuronal sprouting and behavior in adulthood. A similar inverse shift in mitochondrial function was also observed in human lymphoblastoid cells deived from schizophrenia patients and healthy subjects due to the interference of the transplanted mitochondria with their intrinsic mitochondrial state. This study provides fundamental insights into the essential role of adolescent mitochondrial homeostasis in the development of normal functioning adult brain. In addition, it supports a therapeutic potential for mitochondria manipulation in adolescence in disorders with neurodevelopmental and bioenergetic deficits, such as schizophrenia, yet emphasizes the need to monitor individuals' state including their mitochondrial function and immune response, prior to intervention.
  18. Biochim Biophys Acta Bioenerg. 2022 Nov 14. pii: S0005-2728(22)00406-6. [Epub ahead of print] 148936
      Oxidative phosphorylation is a common process to most organisms in which the main function is to generate an electrochemical gradient across the inner mitochondrial membrane (IMM) and to make energy available to the cell. However, plants, many fungi and some animals maintain non-energy conserving oxidases which serve as a bypass to coupled respiration. Namely, the alternative NADH:ubiquinone oxidoreductase NDI1, present in the complex I (CI)-lacking Saccharomyces cerevisiae, and the alternative oxidase, ubiquinol:oxygen oxidoreductase AOX, present in many organisms across different kingdoms. In the last few years, these alternative oxidases have been used to dissect previously indivisible processes in bioenergetics and have helped to discover, understand, and corroborate important processes in mitochondria. Here, we review how the use of alternative oxidases have contributed to the knowledge in CI stability, bioenergetics, redox biology, and the implications of their use in current and future research.
    Keywords:  AOX; Alternative oxidase; CoQ pool; Oxphos; ROS
  19. Mitochondrion. 2022 Nov 09. pii: S1567-7249(22)00092-7. [Epub ahead of print]
      Genome-wide deregulation contributes to mitochondrial dysfunction and impairment in oxidative phosphorylation (OXPHOS) mechanism resulting in oxidative stress, increased production of reactive oxygen species (ROS) and cell death in individuals with Down syndrome (DS). The cells, which require more energy, such as muscles, brain and heart are greatly affected. Impairment in mitochondrial network has a direct link with patho-mechanism at cellular and systemic levels at the backdrop of generalized metabolic perturbations in DS patients. Myriads of clinico-phenotypic features, including intellectual disability, early aging and neurodegeneration, and Alzheimer disease (AD)-related dementia are inevitable in DS-population where mitochondrial dysfunctions play the central role. Collectively, the mitochondrial abnormalities and altered energy metabolism perturbs several signaling pathways, particularly related to neurogenesis, which are directly associated with cognitive development and early onset of AD in individuals with DS. Therefore, therapeutic challenges for amelioration of the mitochondrial defects were perceived to improve the quality of life of DS population. A number of pharmacologically active natural compounds such as polyphenols, antioxidants and flavonoids have shown convincing outcome for reversal of the dysfunctional mitochondrial network and oxidative metabolism, and improvement in intellectual skill in mouse models of DS and DS patients.
    Keywords:  Down syndrome; antioxidants; diseases of Down syndrome; mitochondrial dysfunction; mitochondrial therapeutics; polyphenols
  20. Front Cell Dev Biol. 2022 ;10 1044672
      Mitochondrial dysfunction is strongly implicated in neurodegenerative diseases including age-related macular degeneration (AMD), which causes irreversible blindness in over 50 million older adults worldwide. A key site of insult in AMD is the retinal pigment epithelium (RPE), a monolayer of postmitotic polarized cells that performs essential functions for photoreceptor health and vision. Recent studies from our group and others have identified several features of mitochondrial dysfunction in AMD including mitochondrial fragmentation and bioenergetic defects. While these studies provide valuable insight at fixed points in time, high-resolution, high-speed live imaging is essential for following mitochondrial injury in real time and identifying disease mechanisms. Here, we demonstrate the advantages of live imaging to investigate RPE mitochondrial dynamics in cell-based and mouse models. We show that mitochondria in the RPE form extensive networks that are destroyed by fixation and discuss important live imaging considerations that can interfere with accurate evaluation of mitochondrial integrity such as RPE differentiation status and acquisition parameters. Our data demonstrate that RPE mitochondria show localized heterogeneities in membrane potential and ATP production that could reflect focal changes in metabolism and oxidative stress. Contacts between the mitochondria and organelles such as the ER and lysosomes mediate calcium flux and mitochondrial fission. Live imaging of mouse RPE flatmounts revealed a striking loss of mitochondrial integrity in albino mouse RPE compared to pigmented mice that could have significant functional consequences for cellular metabolism. Our studies lay a framework to guide experimental design and selection of model systems for evaluating mitochondrial health and function in the RPE.
    Keywords:  RPE; live imaging; mitochondria; pigmented and albino mice; retina
  21. Neurol Clin. 2023 Feb;pii: S0733-8619(22)00042-1. [Epub ahead of print]41(1): 21-44
      Cerebellar ataxia results from damage to the cerebellum and presents as movement incoordination and variability, gait impairment, and slurred speech. Patients with cerebellar ataxia can also have cognitive and mood changes. Although the identification of causes for cerebellar ataxia can be complex, age of presentation, chronicity, family history, and associated movement disorders may provide diagnostic clues. There are many genetic causes for cerebellar ataxia, and the common autosomal dominant and recessive ataxia are due to genetic repeat expansions. Step-by-step approach will lead to the identification of the causes. Symptomatic and potential disease-modifying therapies may benefit patients with cerebellar ataxia.
    Keywords:  Ataxia; Cerebellar ataxia; Genetics; Multiple system atrophy; Spinocerebellar ataxia
  22. Ann Clin Transl Neurol. 2022 Nov 16.
      The ever-increasing body of ataxia research provides opportunities for large-scale meta-analyses, systematic reviews, and data aggregation. Because multiple standardized scales are used to quantify ataxia severity, harmonization of these measures is necessary for quantitative data pooling. We applied the modified Friedreich Ataxia Rating Scale (mFARS), the Scale for the Assessment and Rating of Ataxia (SARA), and the International Cooperative Ataxia Rating Scale (ICARS) to a large cohort of people with Friedreich's ataxia. We provide regression coefficients for scale interconversion and discuss the reliability of this approach, together with insights into the differential sensitivities of mFARS and SARA to disease progression.
  23. iScience. 2022 Nov 18. 25(11): 105308
      Glaucoma is an optic neuropathy characterized by permanent visual field loss caused by the death of retinal ganglion cells (RGCs) and it is the leading cause of irreversible blindness worldwide. Consequently, there is an unmet need for the development of new strategies for its treatment. We investigated RGC replacement therapy as a treatment for ganglion cell loss. Human-induced pluripotent stem cells (hiPSCs) were differentiated into mature, functional RGCs in vitro, labeled with AAV2.7m8-SNCG-eGFP, and transplanted intravitreally in wild-type 4-month-old C57BL/6J mice. Survival of the transplanted hiPSC-RGCs was assessed by color fundus photography and histological studies confirmed the localization of the transplanted hiPSC-RGCs within the retina. Two-photon live imaging of retinal explants and electrophysiological studies confirmed that the morphology and function of the transplanted hiPSC-RGCs were similar to native RGCs. These experiments will provide key strategies to enhance the efficiency of stem cell replacement therapy for neurodegenerative diseases, including glaucoma.
    Keywords:  Bioengineering; Biological sciences; Biotechnology; Cell biology; Stem cells research; Tissue engineering
  24. Naunyn Schmiedebergs Arch Pharmacol. 2022 Nov 17.
      General anesthetic drugs have been associated with various unwanted effects including an interference with mitochondrial function. We had previously observed increases of lactate formation in the mouse brain during anesthesia with volatile anesthetic agents. In the present work, we used mitochondria that were freshly isolated from mouse brain to test mitochondrial respiration and ATP synthesis in the presence of six common anesthetic drugs. The volatile anesthetics isoflurane, halothane, and (to a lesser extent) sevoflurane caused an inhibition of complex I of the electron transport chain in a dose-dependent manner. Significant effects were seen at concentrations that are reached under clinical conditions (< 0.5 mM). Pentobarbital and propofol also inhibited complex I but at concentrations that were two-fold higher than clinical EC50 values. Only propofol caused an inhibition of complex II. Complex IV respiration was not affected by either agent. Ketamine did not affect mitochondrial respiration. Similarly, all anesthetic agents except ketamine suppressed ATP production at high concentrations. Only halothane increased cytochrome c release indicating damage of the mitochondrial membrane. In summary, volatile general anesthetic agents as well as pentobarbital and propofol dose-dependently inhibit mitochondrial respiration. This action may contribute to depressive actions of the drugs in the brain.
    Keywords:  Halothane; Isoflurane; Ketamine; Pentobarbital; Propofol; Sevoflurane
  25. iScience. 2022 Nov 18. 25(11): 105447
      An increase in permeability of the mitochondrial inner membrane, mitochondrial permeability transition (PT), is the central event responsible for cell death and tissue damage in conditions such as stroke and heart attack. PT is caused by the cyclosporin A (CSA)-dependent calcium-induced pore, the permeability transition pore (PTP). The molecular details of PTP are incompletely understood. We utilized holographic and fluorescent microscopy to assess the contribution of ATP synthase and adenine nucleotide translocator (ANT) toward PTP. In cells lacking either ATP synthase or ANT, we observed CSA-sensitive membrane depolarization, but not high-conductance PTP. In wild-type cells, calcium-induced CSA-sensitive depolarization preceded opening of PTP, which occurred only after nearly complete mitochondrial membrane depolarization. We propose that both ATP synthase and ANT are required for high-conductance PTP but not depolarization, which presumably occurs through activation of the low-conductance PT, which has a molecular nature that is different from both complexes.
    Keywords:  Cell biology; Functional aspects of cell biology; Molecular biology
  26. Methods Mol Biol. 2023 ;2587 377-386
      Herein, a method to use of mesenchymal stem cells (MSCs) to modulate immune response against rAAV transduction in a canine Duchenne muscular dystrophy (DMD) model is presented. The aim is to overcome the immune response against adeno-associated virus (AAV) capsid itself as well as against the AAV-derived transgene.AAV is currently the most used viral vector because of its relative safety and high efficiency of gene transfer to nondividing cells. Since DMD is caused by a deficiency of dystrophin protein due to mutation or deletion in the dystrophin gene, dystrophin replacement therapy using AAV vectors carrying dystrophin as a therapeutic gene is an effective treatment as shown by animal experiments and clinical trials. Because DMD is a systemic disease, the amount of AAV vector required to achieve efficacy is impractically large. MSC have been used in combination with organ transplants due to their immunomodulatory effects. By using MSCs and AAVs in combination as described below, we are able to decrease the immune response to AAV capsid and the transgene as well as to reduce the dose of AAV to approximately 1/100 of the dose used in conventional clinical trials.
    Keywords:  AAV vector; Gene therapy; Immune modulation; Mesenchymal stromal cells
  27. Orphanet J Rare Dis. 2022 Nov 12. 17(1): 415
    Clinical Management Guidelines Writing Group
      BACKGROUND: Individuals with Friedreich ataxia (FRDA) can find it difficult to access specialized clinical care. To facilitate best practice in delivering healthcare for FRDA, clinical management guidelines (CMGs) were developed in 2014. However, the lack of high-certainty evidence and the inadequacy of accepted metrics to measure health status continues to present challenges in FRDA and other rare diseases. To overcome these challenges, the Grading of Recommendations Assessment and Evaluation (GRADE) framework for rare diseases developed by the RARE-Bestpractices Working Group was adopted to update the clinical guidelines for FRDA. This approach incorporates additional strategies to the GRADE framework to support the strength of recommendations, such as review of literature in similar conditions, the systematic collection of expert opinion and patient perceptions, and use of natural history data.METHODS: A panel representing international clinical experts, stakeholders and consumer groups provided oversight to guideline development within the GRADE framework. Invited expert authors generated the Patient, Intervention, Comparison, Outcome (PICO) questions to guide the literature search (2014 to June 2020). Evidence profiles in tandem with feedback from individuals living with FRDA, natural history registry data and expert clinical observations contributed to the final recommendations. Authors also developed best practice statements for clinical care points that were considered self-evident or were not amenable to the GRADE process.
    RESULTS: Seventy clinical experts contributed to fifteen topic-specific chapters with clinical recommendations and/or best practice statements. New topics since 2014 include emergency medicine, digital and assistive technologies and a stand-alone section on mental health. Evidence was evaluated according to GRADE criteria and 130 new recommendations and 95 best practice statements were generated.
    DISCUSSION AND CONCLUSION: Evidence-based CMGs are required to ensure the best clinical care for people with FRDA. Adopting the GRADE rare-disease framework enabled the development of higher quality CMGs for FRDA and allows individual topics to be updated as new evidence emerges. While the primary goal of these guidelines is better outcomes for people living with FRDA, the process of developing the guidelines may also help inform the development of clinical guidelines in other rare diseases.
    Keywords:  Evidence; Friedreich ataxia; GRADE; Guidelines; Recommendations
  28. iScience. 2022 Nov 18. 25(11): 105410
      Deletion of genes encoding ribosomal proteins extends lifespan in yeast. This increases translation of the functionally conserved transcription factor Gcn4, and lifespan extension in these mutants is GCN4-dependent. Gcn4 is also translationally upregulated by uncharged tRNAs, as are its C aenorhabditis elegans and mammalian functional orthologs. Here, we show that cytosolic tRNA synthetase inhibitors upregulate Gcn4 translation and extend yeast lifespan in a Gcn4-dependent manner. This cytosolic tRNA synthetase inhibitor is also able to extend the lifespan of C. elegans in an atf-4-dependent manner. We show that mitochondrial tRNA synthetase inhibitors greatly extend the lifespan of C. elegans, and this depends on atf-4. This suggests that perturbations of both cytosolic and mitochondrial translation may act in part via the same downstream pathway. These findings establish GCN4 orthologs as conserved longevity factors and, as long-lived mice exhibit elevated ATF4, leave open the possibility that tRNA synthetase inhibitors could also extend lifespan in mammals.
    Keywords:  Biochemistry; Biological sciences; Molecular biology
  29. Nat Commun. 2022 Nov 12. 13(1): 6901
      Superoxide dismutase (SOD1) gene variants may cause amyotrophic lateral sclerosis, some of which are associated with a distinct phenotype. Most studies assess limited variants or sample sizes. In this international, retrospective observational study, we compare phenotypic and demographic characteristics between people with SOD1-ALS and people with ALS and no recorded SOD1 variant. We investigate which variants are associated with age at symptom onset and time from onset to death or censoring using Cox proportional-hazards regression. The SOD1-ALS dataset reports age of onset for 1122 and disease duration for 883 people; the comparator population includes 10,214 and 9010 people respectively. Eight variants are associated with younger age of onset and distinct survival trajectories; a further eight associated with younger onset only and one with distinct survival only. Here we show that onset and survival are decoupled in SOD1-ALS. Future research should characterise rarer variants and molecular mechanisms causing the observed variability.
  30. EMBO J. 2022 Nov 17. e112918
      A recent report shows that the iron chelator DFP induces both mitophagy and pexophagy in a BNIP3/NIX-dependent manner. Previously known as a mitophagy receptor, NIX was also independently localized to peroxisomes to promote pexophagy in several physiological conditions, illustrating the significance of this novel function.
  31. Mol Metab. 2022 Nov 15. pii: S2212-8778(22)00212-5. [Epub ahead of print] 101643
      OBJECTIVE: Skeletal muscle is a pivotal organ for the coordination of systemic metabolism, constituting one of the largest storage site for glucose, lipids and amino acids. Tight temporal orchestration of protein breakdown in times of fasting has to be balanced with preservation of muscle mass and function. However, the molecular mechanisms that control the fasting response in muscle are poorly understood.METHODS: We now have identified a role for the peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) in the regulation of catabolic pathways in this context in muscle-specific loss-of-function mouse models.
    RESULTS: Muscle-specific knockouts for PGC-1β experience mitigated muscle atrophy in fasting, linked to reduced expression of myostatin, atrogenes, activation of AMP-dependent protein kinase (AMPK) and other energy deprivation signaling pathways. At least in part, the muscle fasting response is modulated by a negative effect of PGC-1β on the nuclear factor of activated T-cells 1 (NFATC1).
    CONCLUSIONS: Collectively, these data highlight the complex regulation of muscle metabolism and reveal a new role for muscle PGC-1β in the control of proteostasis in fasting.
    Keywords:  PGC-1β; atrophy; fasting; myostatin; skeletal muscle; ubiquitin proteasome
  32. Exp Mol Med. 2022 Nov 15.
      The mitochondrial unfolded protein response (UPRmt) is a mitochondrial-to-nuclear signaling pathway that is activated to maintain mitochondrial function when there is an accumulation of misfolded proteins within mitochondria. Mitochondrial function is essential for chondrocyte homeostasis, and mitochondrial dysfunction is a characteristic of osteoarthritis (OA). However, the role of the UPRmt in OA remains unclear. In the present study, the level of the UPRmt was examined in primary mouse chondrocytes subjected to different stresses and in the articular cartilage of OA model mice and OA patients. The relationship between UPRmt activation and OA progression was studied. The UPRmt was induced in primary mouse chondrocytes subjected to diverse stresses and in the cartilage of OA mice. Enhancement of the UPRmt with nicotinamide riboside (NR) significantly improved mitochondrial function, reduced chondrocyte death, attenuated OA pain, and ameliorated OA progression, and the protective effects decreased significantly in chondrocyte-specific Atf5 knockout (ATF5f/fCol2a1-CreERT2) mice. UPRmt induction was also identified in the articular cartilage of OA patients and was associated with reduced chondrocyte death, less severe hip pain, and lower levels of inflammation in synovial fluid. These findings identify the induction of the UPRmt in primary mouse chondrocytes exposed to pathological stresses and in the articular cartilage of OA model mice and OA patients. Enhancement of the UPRmt ameliorates OA progression, suggesting that the UPRmt exerts a protective effect against OA and may be a potential diagnostic and therapeutic strategy for OA.
  33. Neurologia (Engl Ed). 2022 Nov 14. pii: S2173-5808(22)00174-2. [Epub ahead of print]
      Hereditary ataxia (HA) and hereditary spastic paraplegia (HSP) are rare diseases; as such, they are rarely managed in general neurology consultations. We present a set of brief, practical recommendations for the diagnosis and management of these patients, as well as a standardised procedure for comprehensive evaluation of disability. We provide definitions for HA and "HA plus," and "pure" and "complicated" HSP; describe the clinical assessment of these patients, indicating the main complementary tests and clinical scales for physical and psychological assessment of the patients; and summarise the available treatments. These recommendations are intended to facilitate daily neurological practice and to unify clinical criteria and disability assessment protocols for patients with HA and HSP.
    Keywords:  Ataxias hereditarias; Diagnostic recommendations; Disability; Discapacidad; Guia diagnóstica; Hereditary ataxia; Hereditary spastic paraplegia; Paraparesias espásticas hereditarias
  34. Sci Immunol. 2022 Nov 25. 7(77): eabm8182
      T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (TH17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. TH17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that TH17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates TH17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in TH17s. We found that TH17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS TH17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS TH17s. By contrast, glycolytic TH17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected TH17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.
  35. J Cachexia Sarcopenia Muscle. 2022 Nov 17.
      BACKGROUND: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear.METHODS: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development.
    RESULTS: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream activator of autophagosome initiation. We discovered a previously unidentified phenotype of lysosomal failure in TSC1mKO mouse muscle, in which the lysosomal defect was also conserved in the naturally aged mouse muscle, whereas 4EBP1 activation enhanced lysosomal protease activities to compensate for impaired autophagy induced by mTORC1 hyperactivity. Consequently, 4EBP1 activation relieved oxidative stress to prevent toxic aggregate accumulation (0.5-fold reduction compared with TSC1mKO, P < 0.05) in muscle and restored mitochondrial homeostasis and function.
    CONCLUSIONS: We identify 4EBP1 as a communication hub coordinating protein synthesis and degradation to protect proteostasis, revealing therapeutic potential for activating lysosomal degradation to mitigate sarcopenia.
    Keywords:  mRNA translation; mTORC1; mitochondrial dysfunction; protein degradation; sarcopenia