bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒01‒16
thirty-nine papers selected by
Catalina Vasilescu
University of Helsinki

  1. Cell Metab. 2022 Jan 07. pii: S1550-4131(21)00636-7. [Epub ahead of print]
      Mitophagy is a quality control mechanism that eliminates damaged mitochondria, yet its significance in mammalian pathophysiology and aging has remained unclear. Here, we report that mitophagy contributes to mitochondrial dysfunction in skeletal muscle of aged mice and human patients. The early disease stage is characterized by muscle fibers with central nuclei, with enhanced mitophagy around these nuclei. However, progressive mitochondrial dysfunction halts mitophagy and disrupts lysosomal homeostasis. Interestingly, activated or halted mitophagy occur in a mosaic manner even in adjacent muscle fibers, indicating cell-autonomous regulation. Rapamycin restores mitochondrial turnover, indicating mTOR-dependence of mitochondrial recycling in advanced disease stage. Our evidence suggests that (1) mitophagy is a hallmark of age-related mitochondrial pathology in mammalian muscle, (2) mosaic halting of mitophagy is a mechanism explaining mosaic respiratory chain deficiency and accumulation of pathogenic mtDNA variants in adult-onset mitochondrial diseases and normal aging, and (3) augmenting mitophagy is a promising therapeutic approach for muscle mitochondrial dysfunction.
    Keywords:  SBFSEM; centrally nucleated fibers; lysosome; mito-QC; mitochondrial disease; mitochondrial myopathy; mitophagy; patient; ragged-red fibers
  2. Int J Mol Sci. 2021 Dec 25. pii: 210. [Epub ahead of print]23(1):
      The retina is an exquisite target for defects of oxidative phosphorylation (OXPHOS) associated with mitochondrial impairment. Retinal involvement occurs in two ways, retinal dystrophy (retinitis pigmentosa) and subacute or chronic optic atrophy, which are the most common clinical entities. Both can present as isolated or virtually exclusive conditions, or as part of more complex, frequently multisystem syndromes. In most cases, mutations of mtDNA have been found in association with mitochondrial retinopathy. The main genetic abnormalities of mtDNA include mutations associated with neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) sometimes with earlier onset and increased severity (maternally inherited Leigh syndrome, MILS), single large-scale deletions determining Kearns-Sayre syndrome (KSS, of which retinal dystrophy is a cardinal symptom), and mutations, particularly in mtDNA-encoded ND genes, associated with Leber hereditary optic neuropathy (LHON). However, mutations in nuclear genes can also cause mitochondrial retinopathy, including autosomal recessive phenocopies of LHON, and slowly progressive optic atrophy caused by dominant or, more rarely, recessive, mutations in the fusion/mitochondrial shaping protein OPA1, encoded by a nuclear gene on chromosome 3q29.
    Keywords:  Kearns-Sayre syndrome; Leber’s hereditary optic neuropathy (LHON); ataxia and retinitis pigmentosa (NARP); autosomal dominant optic atrophy (ADOA); mitochondrial DNA; mitochondrial disorders; mtDNA heteroplasmic deletions; neurogenic muscle weakness; optic atrophy; retina; retinitis pigmentosa
  3. Hum Mol Genet. 2022 Jan 13. pii: ddac002. [Epub ahead of print]
      The SLC25A26 gene encodes a mitochondrial inner membrane carrier that transports S-adenosylmethionine (SAM) into the mitochondrial matrix in exchange for S-adenosylhomocysteine (SAH). SAM is the predominant methyl-group donor for most cellular methylation processes, of which SAH is produced as a by-product. Pathogenic, bi-allelic SLC25A26 variants are a recognised cause of mitochondrial disease in children, with a severe neonatal-onset caused by decreased SAM transport activity. Here, we describe two, unrelated adult cases, one of whom presented with recurrent episodes of severe abdominal pain and metabolic decompensation with lactic acidosis. Both patients had exercise intolerance and mitochondrial myopathy associated with bi-allelic variants in SLC25A26 which led to marked respiratory chain deficiencies and mitochondrial histopathological abnormalities in skeletal muscle that are comparable to those previously described in early-onset cases. We demonstrate using both mouse and fruit fly models that impairment of SAH, rather than SAM, transport across the mitochondrial membrane is likely the cause of this milder, late-onset phenotype. Our findings associate a novel pathomechanism with a known disease-causing protein and highlight the quests of precision medicine in optimising diagnosis, therapeutic intervention, and prognosis.
  4. Front Cell Dev Biol. 2021 ;9 803205
      Mitochondrial membrane proteins play an essential role in all major mitochondrial functions. The respiratory complexes of the inner membrane are key for the generation of energy. The carrier proteins for the influx/efflux of essential metabolites to/from the matrix. Many other inner membrane proteins play critical roles in the import and processing of nuclear encoded proteins (∼99% of all mitochondrial proteins). The outer membrane provides another lipidic barrier to nuclear-encoded protein translocation and is home to many proteins involved in the import process, maintenance of ionic balance, as well as the assembly of outer membrane components. While many aspects of the import and assembly pathways of mitochondrial membrane proteins have been elucidated, many open questions remain, especially surrounding the assembly of the respiratory complexes where certain highly hydrophobic subunits are encoded by the mitochondrial DNA and synthesised and inserted into the membrane from the matrix side. This review will examine the various assembly pathways for inner and outer mitochondrial membrane proteins while discussing the most recent structural and biochemical data examining the biogenesis process.
    Keywords:  assembly; membrane proteins; mitochondria; mitochondrial chaperones; translocons
  5. Nat Commun. 2022 Jan 11. 13(1): 209
      Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs.
  6. Nucleic Acids Res. 2022 Jan 08. pii: gkab1251. [Epub ahead of print]
      Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.
  7. Biochimie. 2022 Jan 10. pii: S0300-9084(22)00002-5. [Epub ahead of print]
      This review aims to make a framework of exogenous healthy mitochondrial transplantation and to assemble present information for improving new therapeutic applications in a variety of diseases. Recently, the significance of mitochondrial transplantation has been emphasized in a variety of mitochondrial dysfunction-related diseases such as neurodegenerative diseases, toxic injury, ischemia, cardiovascular diseases. We describe the natural mitochondrial transfer mechanisms (ie. TNT, EVs, mitochondrial dynamics), mitochondrial isolation process for transplantation (ie. source of mitochondria, requirements for successful isolation), mitochondrial transplantation methods (in vivo, in vitro), the effects and limitations of mitochondrial transplantation. Since mitochondrial transplantation is seen as an innovative potential treatment for diseases that can not be treated at the desired level, we expect to represent how the mitochondrial transplantation methods can be used in different diseases.
    Keywords:  Mitochondria dysfunction; Mitochondrial dynamics; Mitochondrial isolation; Mitochondrial transplantation
  8. Front Mol Biosci. 2021 ;8 769135
      Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.
    Keywords:  OPA1; cardiolipin; membrane asymmetry; membrane heterogeneity; mitochondrial fusion
  9. Int J Mol Sci. 2021 Dec 21. pii: 7. [Epub ahead of print]23(1):
      With few exceptions, proteins that constitute the proteome of mitochondria originate outside of this organelle in precursor forms. Such protein precursors follow dedicated transportation paths to reach specific parts of mitochondria, where they complete their maturation and perform their functions. Mitochondrial precursor targeting and import pathways are essential to maintain proper mitochondrial function and cell survival, thus are tightly controlled at each stage. Mechanisms that sustain protein homeostasis of the cytosol play a vital role in the quality control of proteins targeted to the organelle. Starting from their synthesis, precursors are constantly chaperoned and guided to reduce the risk of premature folding, erroneous interactions, or protein damage. The ubiquitin-proteasome system provides proteolytic control that is not restricted to defective proteins but also regulates the supply of precursors to the organelle. Recent discoveries provide evidence that stress caused by the mislocalization of mitochondrial proteins may contribute to disease development. Precursors are not only subject to regulation but also modulate cytosolic machinery. Here we provide an overview of the cellular pathways that are involved in precursor maintenance and guidance at the early cytosolic stages of mitochondrial biogenesis. Moreover, we follow the circumstances in which mitochondrial protein import deregulation disturbs the cellular balance, carefully looking for rescue paths that can restore proteostasis.
    Keywords:  mitochondrial biogenesis; molecular chaperone; proteasome; protein degradation; protein precursor; protein transport; proteostasis; quality control; ubiquitin
  10. J Pediatr Neurosci. 2021 Apr-Jun;16(2):16(2): 153-155
      MTHFR enzyme deficiency is an autosomal-recessive inborn error of folate metabolism. The deficiency cause defect in the remethylation of homocysteine to methionine leading to increased blood levels of homocysteine. Hyperhomocysteinemia in infants cause seizures, hypotonia, apnoea, microcephaly, progressing to coma and death if untreated whereas in childhood onset it causes developmental delay, seizures, psychiatric disturbances, spastic gait, and ataxia. We report a 10-year-old girl with rapidly progressive spastic paraplegia requiring wheelchair ambulation within 3 months of symptom onset with behavioral disturbances. Plasma homocysteine and plasma lactate were high with normal vitamin B12 levels. Clinical exome sequencing showed homozygous missense mutation in MTHFR gene which was likely pathogenic variant. Respiratory chain complex assay from muscle sample showed reduced complex 1 deficiency (<20%).
    Keywords:  Complex 1; MTHFR gene; mitochondria; spastic paraplegia
  11. Free Radic Biol Med. 2022 Jan 11. pii: S0891-5849(22)00016-8. [Epub ahead of print]
      Acetylation is one of the most important methods of modification that lead to a change in the function of proteins. In humans, metabolic enzymes commonly undergo acetylation, which regulates the activities of metabolic enzymes and metabolic pathways. Sirtuin 3 (SIRT3) is a prominent deacetylase that participates in mitochondrial metabolism, redox balance, and mitochondrial dynamics by regulating mitochondrial protein acetylation, thereby protecting mitochondria from damage. Normal mitochondrial function is essential for maintaining the metabolism and function of the heart. Therefore, mitochondrial dysfunction caused by SIRT3 consumption and defects leads to the development of a variety of cardiovascular diseases. A comprehensive understanding of the role of SIRT3 in cardiovascular disease is critical for developing new therapeutic strategies. Herein, we summarize the function of SIRT3 in mitochondria, the complex mechanisms mediating cardiovascular diseases, and the potential value of SIRT3 small-molecule agonists in future clinical treatments.
    Keywords:  Acetylation; Cardiovascular diseases; Mitochondria; Sirtuin 3
  12. Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01717-4. [Epub ahead of print]38(2): 110213
      Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.
    Keywords:  ATP production; Adipocyte; Calcium handling; ER-LD contact sites; Krebs cycle metabolites; MAMs; Mitochondria dysfunction; lipid droplet; lipodystrophy; seipin
  13. J Inherit Metab Dis. 2022 Jan 13.
      SUPV3L1 encodes a helicase that is mainly localised in the mitochondria. It has been shown in vitro to possess both double-stranded RNA and DNA unwinding activity that is ATP-dependent. Here we report the first two patients for this gene who presented with a homozygous preliminary stop codon in the C-terminus of SUPV3L1. They presented with a characteristic phenotype of neurodegenerative nature with progressive spastic paraparesis, growth restriction, hypopigmentation, and predisposition to autoimmune disease. Ophthalmological examination showed severe photophobia with corneal erosions, optic atrophy, and pigmentary retinopathy, while neuroimaging showed atrophy of the optic chiasm and the pons with calcification of putamina, with intermittent and mild elevation of lactate. We show that the amino acids that are eliminated by the preliminary stop codon are highly conserved and are predicted to form an amphipathic helix. To investigate if the mutation causes mitochondrial dysfunction, we examined fibroblasts of the proband. We observed very low expression of the truncated protein, a reduction in the mature ND6 mRNA species as well as the accumulation of double stranded RNA. Lentiviral complementation with the full-length SUPV3L1 cDNA partly restored the observed RNA phenotypes, supporting that the SUPV3L1 mutation in these patients is pathogenic and the cause of the disease. This article is protected by copyright. All rights reserved.
    Keywords:  SUPV3L1; degradosome; mitochondrial RNA processing; mitochondrial disease; mtDNA; neurodegenerative syndrome
  14. iScience. 2022 Jan 21. 25(1): 103635
      Nicotinamide riboside supplements (NRS) have been touted as a nutraceutical that promotes cardiometabolic and musculoskeletal health by enhancing nicotinamide adenine dinucleotide (NAD+) biosynthesis, mitochondrial function, and/or the activities of NAD-dependent sirtuin deacetylase enzymes. This investigation examined the impact of NRS on whole body energy homeostasis, skeletal muscle mitochondrial function, and corresponding shifts in the acetyl-lysine proteome, in the context of diet-induced obesity using C57BL/6NJ mice. The study also included a genetically modified mouse model that imposes greater demand on sirtuin flux and associated NAD+ consumption, specifically within muscle tissues. In general, whole body glucose control was marginally improved by NRS when administered at the midpoint of a chronic high-fat diet, but not when given as a preventative therapy upon initiation of the diet. Contrary to anticipated outcomes, the study produced little evidence that NRS increases tissue NAD+ levels, augments mitochondrial function, and/or mitigates diet-induced hyperacetylation of the skeletal muscle proteome.
    Keywords:  Nutrition; Physiology; Proteomics
  15. Brain. 2022 Jan 12. pii: awab243. [Epub ahead of print]
      Mitochondrial defects are a hallmark of early pathophysiology in Alzheimer's disease, with pathologically phosphorylated tau reported to induce mitochondrial toxicity. Mitophagy constitutes a key pathway in mitochondrial quality control by which damaged mitochondria are targeted for autophagy. However, few details are known regarding the intersection of mitophagy and pathologies in tauopathy. Here, by applying biochemical and cell biological approaches including time-lapse confocal imaging in live tauopathy neurons, combined with gene rescue experiments via stereotactic injections of adeno-associated virus particles into tauopathy mouse brains, electrophysiological recordings and behavioural tests, we demonstrate for the first time that mitochondrial distribution deficits at presynaptic terminals are an early pathological feature in tauopathy brains. Furthermore, Parkin-mediated mitophagy is extensively activated in tauopathy neurons, which accelerates mitochondrial Rho GTPase 1 (Miro1) turnover and consequently halts Miro1-mediated mitochondrial anterograde movement towards synaptic terminals. As a result, mitochondrial supply at tauopathy synapses is disrupted, impairing synaptic function. Strikingly, increasing Miro1 levels restores the synaptic mitochondrial population by enhancing mitochondrial anterograde movement and thus reverses tauopathy-associated synaptic failure. In tauopathy mouse brains, overexpression of Miro1 markedly elevates synaptic distribution of mitochondria and protects against synaptic damage and neurodegeneration, thereby counteracting impairments in learning and memory as well as synaptic plasticity. Taken together, our study reveals that activation of the Parkin pathway triggers an unexpected effect-depletion of mitochondria from synaptic terminals, a characteristic feature of early tauopathy. We further provide new mechanistic insights into how parkin activation-enhanced Miro1 degradation and impaired mitochondrial anterograde transport drive tauopathy-linked synaptic pathogenesis and establish a foundation for future investigations into new therapeutic strategies to prevent synaptic deterioration in Alzheimer's disease and other tauopathies.
    Keywords:  Alzheimer’s disease; Parkin-mediated mitophagy; mitochondrial anterograde transport; synaptic mitochondrial deficits; tauopathy
  16. Hum Mutat. 2022 Jan 13.
      Iron-sulfur cluster proteins are involved in critical functions for gene expression regulation and mitochondrial bioenergetics including the oxidative phosphorylation system. The c.215G>A p.(Arg72Gln) variant in NFS1 has been previously reported to cause infantile mitochondrial complex II and III deficiency. We describe three additional unrelated patients with the same missense variant. Two infants with the same homozygous variant presented with hypotonia, weakness and lactic acidosis, and one patient with compound heterozygous p.(Arg72Gln) and p.(Arg412His) variants presented as a young adult with gastrointestinal symptoms and fatigue. Skeletal muscle biopsy from patients 1 and 3 showed abnormal mitochondrial morphology, and functional analyses demonstrated decreased activity in respiratory chain complex II and variably in complexes I and III. We found decreased mitochondrial and cytosolic aconitase activities but only mildly affected lipoylation of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase enzymes. Our studies expand the phenotypic spectrum and provide further evidence for the pathogenicity and functional sequelae of NFS1-related disorders with disturbances in both mitochondrial and cytosolic iron-sulfur cluster containing enzymes. This article is protected by copyright. All rights reserved.
    Keywords:  NFS1; iron-sulfur clusteropathies; lactic acidosis; mitochondrial; pediatric
  17. J Clin Med. 2021 Dec 22. pii: 22. [Epub ahead of print]11(1):
      Autosomal dominant mutations in the TWNK gene, which encodes a mitochondrial DNA helicase, cause adult-onset progressive external ophthalmoplegia (PEO) and PEO-plus presentations. In this retrospective observational study, we describe clinical and complementary data from 25 PEO patients with mutations in TWNK recruited from the Hospital 12 de Octubre Mitochondrial Disorders Laboratory Database. The mean ages of onset and diagnosis were 43 and 63 years, respectively. Family history was positive in 22 patients. Ptosis and PEO (92% and 80%) were the most common findings. Weakness was present in 48%, affecting proximal limbs, neck, and bulbar muscles. Exercise intolerance was present in 28%. Less frequent manifestations were cardiac (24%) and respiratory (4%) involvement, neuropathy (8%), ataxia (4%), and parkinsonism (4%). Only 28% had mild hyperCKemia. All 19 available muscle biopsies showed signs of mitochondrial dysfunction. Ten different TWNK mutations were identified, with c.1361T>G (p.Val454Gly) and c.1070G>C (p.Arg357Pro) being the most common. Before definitive genetic confirmation, 56% of patients were misdiagnosed (36% with myasthenia, 20% with oculopharyngeal muscle dystrophy). Accurate differential diagnosis and early confirmation with appropriately chosen complementary studies allow genetic counseling and the avoidance of unnecessary treatments. Thus, mitochondrial myopathies must be considered in PEO/PEO-plus presentations, and particularly, TWNK is an important cause when positive family history is present.
    Keywords:  TWNK gene; mitochondrial dysfunction; mtDNA maintenance defects; progressive external ophthalmoplegia
  18. EMBO J. 2022 Jan 13. e108587
      The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region.
    Keywords:  BCL-2 proteins; fluorescence correlation spectroscopy; membrane protein complex; mitochondrial division; super-resolution microscopy
  19. Int J Mol Sci. 2022 Jan 04. pii: 528. [Epub ahead of print]23(1):
      AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named "early infantile epileptic encephalopathy 39" (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.
    Keywords:  AGC1/Aralar deficiency; malate-aspartate shuttle; mitochondrial aspartate-glutamate carrier; mitochondrial disorders; mitochondrial function
  20. BMC Neurol. 2022 Jan 10. 22(1): 20
      BACKGROUND: Pontocerebellar hypoplasia (PCH) is increasingly known as a degenerative disease rather than simple "hypoplasia". At least 21 disease-causing genes have been identified for PCH so far. Because PCH is very heterogenous, prognostic prediction based solely on clinical or radiologic findings is not feasible.CASE PRESENTATION: Here, we report two siblings who had a fulminant neonatal course. The documentation of pontocerebellar hypoplasia by postmortem brain CT imaging in one of the siblings and a subsequent complex and comprehensive whole genome analysis established that both siblings had bi-allelic compound heterozygous variants (a splicing variant and a deletion) in the SLC25A46 gene which encodes a solute carrier protein essential for mitochondrial function. Long-read whole genome sequencing was required to confirm the presence of the deletion. The fulminant courses suggest that SLC25A46-related PCH is an acutely progressive degenerative condition starting in utero, rather than a simple static hypoplasia.
    CONCLUSION: The genomic analysis was instrumental and essential to solving the enigma of the unexplained neonatal deaths of these two siblings and to provide accurate genetic counseling.
    Keywords:  Genomic analysis; Pontocerebellar hypoplasia; SLC25A46; Whole genome sequencing
  21. Brain. 2022 Jan 13. pii: awab488. [Epub ahead of print]
      Hereditary spastic paraplegias (HSPs) are characterized by lower limb spasticity resulting from degeneration of long corticospinal axons. SPG11 is one of the most common autosomal recessive HSPs, and the SPG11 protein spatacsin forms a complex with the SPG15 protein spastizin and heterotetrameric AP5 adaptor protein complex, which includes the SPG48 protein AP5Z1. Using the integration-free episomal method, we established SPG11 patient-specific induced pluripotent stem cells (iPSCs) from patient fibroblasts. We differentiated SPG11 iPSCs, as well as SPG48 iPSCs previously established, into cortical projection neurons (PNs) and examined protective effects by targeting mitochondrial dynamics using P110, a peptide that selectively inhibits mitochondrial fission GTPase Drp1. P110 treatment mitigates mitochondrial fragmentation, improves mitochondrial motility, and restores mitochondrial health and ATP levels in SPG11 and SPG48 neurons. Neurofilament (NF) aggregations are increased in SPG11 and SPG48 axons, and these are also suppressed by P110. Similarly, P110 mitigates NF disruption in both SPG11 and SPG48 knockdown cortical PNs, confirming the contribution of HSP gene deficiency to subsequent NF and mitochondrial defects. Strikingly, NF aggregations in SPG11 and SPG48 deficient neurons double stain with ubiquitin and autophagy related proteins, resembling the pathological hallmark observed in SPG11 autopsy brain sections. To confirm the cause-effect relationship between the SPG11 mutations and disease phenotypes, we knocked-in SPG11 disease mutations to human embryonic stem cells (hESCs) and differentiated these stem cells into cortical PNs. Reduced ATP levels and accumulated NF aggregations along axons are observed, and both are mitigated by P110. Furthermore, rescue experiment with expression of wildtype SPG11 in cortical PNs derived from both SPG11 patient iPSCs and SPG11 disease mutation knock-in hESCs leads to rescue of mitochondrial dysfunction and NF aggregations in these SPG11 neurons. Finally, in SPG11 and SPG48 long-term cultures, increased release of phosphoNF-H, a biomarker for nerve degeneration, is significantly reduced by inhibiting mitochondrial fission pharmacologically using P110 and genetically using Drp1 shRNA. Taken together, our results demonstrate that impaired mitochondrial dynamics underlie both cytoskeletal disorganization and axonal degeneration in SPG11 and SPG48 neurons, highlighting the importance of targeting these pathologies therapeutically.
    Keywords:  axonal degeneration; cortical projection neuron; cytoskeletal organization; hereditary spastic paraplegias; mitochondrial dynamics
  22. Autophagy. 2022 Jan 13. 1-2
      Mitochondrial dysfunction is a key determinant of the development of cardiomyopathy in patients with obesity and diabetes. We recently reported that mitophagy is activated in the mouse heart during the chronic phase of high-fat diet (HFD) consumption, despite downregulation of general macroautophagy/autophagy. This form of mitophagy is mediated by a mechanism distinct from that of conventional autophagy and is termed alternative mitophagy. We here discuss the underlying mechanisms of alternative mitophagy and its functional significance in heart disease.
    Keywords:  Mitophagy; Rab9; cardiomyopathy; diabetes; heart; obesity
  23. Cells. 2021 Dec 23. pii: 30. [Epub ahead of print]11(1):
      Mitochondria are multifunctional subcellular organelles essential for cellular energy homeostasis and apoptotic cell death. It is, therefore, crucial to maintain mitochondrial fitness. Mitophagy, the selective removal of dysfunctional mitochondria by autophagy, is critical for regulating mitochondrial quality control in many physiological processes, including cell development and differentiation. On the other hand, both impaired and excessive mitophagy are involved in the pathogenesis of different ageing-associated diseases such as neurodegeneration, cancer, myocardial injury, liver disease, sarcopenia and diabetes. The best-characterized mitophagy pathway is the PTEN-induced putative kinase 1 (PINK1)/Parkin-dependent pathway. However, other Parkin-independent pathways are also reported to mediate the tethering of mitochondria to the autophagy apparatuses, directly activating mitophagy (mitophagy receptors and other E3 ligases). In addition, the existence of molecular mechanisms other than PINK1-mediated phosphorylation for Parkin activation was proposed. The adenosine5'-monophosphate (AMP)-activated protein kinase (AMPK) is emerging as a key player in mitochondrial metabolism and mitophagy. Beyond its involvement in mitochondrial fission and autophagosomal engulfment, its interplay with the PINK1-Parkin pathway is also reported. Here, we review the recent advances in elucidating the canonical molecular mechanisms and signaling pathways that regulate mitophagy, focusing on the early role and spatial specificity of the AMPK/ULK1 axis.
    Keywords:  AMPK; E3 ligases; PINK1–Parkin pathway; Parkin activation; ULK1; mitochondria; mitophagy; mitophagy receptors; ubiquitin
  24. Cells. 2021 Dec 23. pii: 38. [Epub ahead of print]11(1):
      Mitochondria in the cell are the center for energy production, essential biomolecule synthesis, and cell fate determination. Moreover, the mitochondrial functional versatility enables cells to adapt to the changes in cellular environment and various stresses. In the process of discharging its cellular duties, mitochondria face multiple types of challenges, such as oxidative stress, protein-related challenges (import, folding, and degradation) and mitochondrial DNA damage. They mitigate all these challenges with robust quality control mechanisms which include antioxidant defenses, proteostasis systems (chaperones and proteases) and mitochondrial biogenesis. Failure of these quality control mechanisms leaves mitochondria as terminally damaged, which then have to be promptly cleared from the cells before they become a threat to cell survival. Such damaged mitochondria are degraded by a selective form of autophagy called mitophagy. Rigorous research in the field has identified multiple types of mitophagy processes based on targeting signals on damaged or superfluous mitochondria. In this review, we provide an in-depth overview of mammalian mitophagy and its importance in human health and diseases. We also attempted to highlight the future area of investigation in the field of mitophagy.
    Keywords:  BNIP3; FUNDC1; PARKIN; PINK1; Parkinson’s disease; autophagy; cardiolipin; mitophagy; quality control
  25. ACS Appl Bio Mater. 2021 May 17. 4(5): 4361-4372
      Environment-sensitive molecular probes offer the potential for a comprehensive mapping of the complex cellular milieu. We present here a radically new strategy of multiplexing highly sensitive, spectrally tuned fluorescent dyes for sensing cellular microenvironment. To achieve this multicolor, ratiometric cellular imaging, we first developed a series of highly sensitive, tunable molecular rotors for mitochondrial imaging, with emission wavelengths spanning the visible spectrum. These fluorogenic merocyanine dyes are all sensitive to solvent viscosity despite distinctive photophysical features. Our results show that merocyanine dyes can show a rotor-like behavior despite significant changes to the conventional donor-acceptor or push-pull scaffolds, thereby revealing conserved features of rotor dye chemistry. Developing closely related but spectrally separated dyes that have distinct response functions allows us to do ″two-color, two-dye″ imaging of the mitochondrial microenvironment. Our results with multidye, combinatorial imaging provide a direct visualization of the intrinsic heterogeneity of the mitochondrial microenvironment. The overall mitochondrial microenvironment (including contributions from local membrane order) as reported through two-color fluorescence ″ratio″ changes of multiplexed rotor dyes shows dynamic heterogeneity with distinct spatiotemporal signatures that evolve over time and respond to chemical perturbations. Our results offer a powerful illustration of how multiplexed dye imaging allows the quantitative imaging of mitochondrial membrane order and cellular microenvironment.
    Keywords:  microviscosity; mitochondrial heterogeneity; pH; ratio imaging; rotor dyes
  26. Comp Biochem Physiol B Biochem Mol Biol. 2022 Jan 10. pii: S1096-4959(22)00001-X. [Epub ahead of print] 110713
      Mitochondria are key cellular sources of reactive oxygen species (ROS) and contain at least 12 known sites on multiple enzymes that convert molecular oxygen to superoxide and hydrogen peroxide (H2O2). Quantitation of site-specific ROS emission is critical to understand the relative contribution of different sites and the pathophysiologic importance of mitochondrial ROS. However, factors that affect mitochondrial ROS emission are not well understood. We characterized and optimized conditions for maximal total and site-specific H2O2 emission during oxidation of standard substrates and probed the source of the high H2O2 emission in unenergized rainbow trout liver mitochondria. We found that mitochondrial H2O2 emission capacity depended on the substrate being oxidized, mitochondrial protein concentration, and composition of the ROS detection system. Contrary to our expectation, addition of exogenous superoxide dismutase reduced H2O2 emission. Titration of conventional mitochondrial electron transfer system (ETS) inhibitors over a range of conditions revealed that one size does not fit all; inhibitor concentrations evoking maximal responses varied with substrate and were moderated by the presence of other inhibitors. Moreover, the efficacy of suppressors of electron leak (S1QEL1.1 and S3QEL2) was low and depended on the substrate being oxidized. We found that H2O2 emission in unenergized rainbow trout liver mitochondria was suppressed by GKT136901 suggesting that it is associated with NADPH oxidase activity. We conclude that optimization of assay conditions is critical for quantitation of maximal H2O2 emission and would facilitate more valid comparisons of mitochondrial total and site-specific H2O2 emission capacities between studies, tissues, and species.
    Keywords:  Amplex UltraRed; Liver mitochondria; Optimal conditions; ROS; Site-specific H(2)O(2) emission
  27. Commun Biol. 2022 Jan 13. 5(1): 49
      Parkinson's disease (PD) is the second-most prevalent neurodegenerative disorder, characterized by the loss of dopaminergic neurons (mDA) in the midbrain. The underlying mechanisms are only partly understood and there is no treatment to reverse PD progression. Here, we investigated the disease mechanism using mDA neurons differentiated from human induced pluripotent stem cells (hiPSCs) carrying the ILE368ASN mutation within the PINK1 gene, which is strongly associated with PD. Single-cell RNA sequencing (RNAseq) and gene expression analysis of a PINK1-ILE368ASN and a control cell line identified genes differentially expressed during mDA neuron differentiation. Network analysis revealed that these genes form a core network, members of which interact with all known 19 protein-coding Parkinson's disease-associated genes. This core network encompasses key PD-associated pathways, including ubiquitination, mitochondrial function, protein processing, RNA metabolism, and vesicular transport. Proteomics analysis showed a consistent alteration in proteins of dopamine metabolism, indicating a defect of dopaminergic metabolism in PINK1-ILE368ASN neurons. Our findings suggest the existence of a network onto which pathways associated with PD pathology converge, and offers an inclusive interpretation of the phenotypic heterogeneity of PD.
  28. Cells. 2021 Dec 27. pii: 65. [Epub ahead of print]11(1):
      High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.
    Keywords:  3D imaging; 3D reconstruction; Amira; FIB-SEM; SBF-SEM; mitochondrial imaging; organelles; segmentation; volume analysis
  29. Nat Biotechnol. 2022 Jan 13.
      Spatial transcriptomic technologies promise to resolve cellular wiring diagrams of tissues in health and disease, but comprehensive mapping of cell types in situ remains a challenge. Here we present сell2location, a Bayesian model that can resolve fine-grained cell types in spatial transcriptomic data and create comprehensive cellular maps of diverse tissues. Cell2location accounts for technical sources of variation and borrows statistical strength across locations, thereby enabling the integration of single-cell and spatial transcriptomics with higher sensitivity and resolution than existing tools. We assessed cell2location in three different tissues and show improved mapping of fine-grained cell types. In the mouse brain, we discovered fine regional astrocyte subtypes across the thalamus and hypothalamus. In the human lymph node, we spatially mapped a rare pre-germinal center B cell population. In the human gut, we resolved fine immune cell populations in lymphoid follicles. Collectively, our results present сell2location as a versatile analysis tool for mapping tissue architectures in a comprehensive manner.
  30. Mol Biomed. 2020 Sep 20. 1(1): 7
      Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor and innate immune response initiator. Binding with exogenous or endogenous nucleic acids, cGAS activates its downstream adaptor, stimulator of interferon genes (STING). STING then triggers protective immune to enable the elimination of the pathogens and the clearance of cancerous cells. Apparently, aberrantly activated by self-DNA, cGAS/STING pathway is threatening to cause autoimmune and inflammatory diseases. The effects of cGAS/STING in defenses against infection and autoimmune diseases have been well studied, still it is worthwhile to discuss the roles of cGAS/STING pathway beyond the "classical" realm of innate immunity. Recent studies have revealed its involvement in non-canonical inflammasome formation, calcium hemostasis regulation, endoplasmic reticulum (ER) stress response, perception of leaking mitochondrial DNA (mtDNA), autophagy induction, cellular senescence and senescence-associated secretory phenotype (SASP) production, providing an exciting area for future exploration. Previous studies generally focused on the function of cGAS/STING pathway in cytoplasm and immune response. In this review, we summarize the latest research of this pathway on the regulation of other physiological process and STING independent reactions to DNA in micronuclei and nuclei. Together, these studies provide a new perspective of cGAS/STING pathway in human diseases.
    Keywords:  DNA sensor; Immune; Micronuclei; Nuclei; STING; Senescence; Tumor; cGAS
  31. Int J Mol Sci. 2021 Dec 23. pii: 128. [Epub ahead of print]23(1):
      Coenzyme Q (CoQ) is a key component of the respiratory chain of all eukaryotic cells. Its function is closely related to mitochondrial respiration, where it acts as an electron transporter. However, the cellular functions of coenzyme Q are multiple: it is present in all cell membranes, limiting the toxic effect of free radicals, it is a component of LDL, it is involved in the aging process, and its deficiency is linked to several diseases. Recently, it has been proposed that coenzyme Q contributes to suppressing ferroptosis, a type of iron-dependent programmed cell death characterized by lipid peroxidation. In this review, we report the latest hypotheses and theories analyzing the multiple functions of coenzyme Q. The complete knowledge of the various cellular CoQ functions is essential to provide a rational basis for its possible therapeutic use, not only in diseases characterized by primary CoQ deficiency, but also in large number of diseases in which its secondary deficiency has been found.
    Keywords:  LDL; OxPhos; age-related diseases; coenzyme Q10; mitochondria; statins; ubiquinol-10; ubiquinone-10
  32. Nat Struct Mol Biol. 2022 Jan 10.
      Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.
  33. Nat Commun. 2022 Jan 10. 13(1): 139
      Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.
  34. Am J Hum Genet. 2022 Jan 10. pii: S0002-9297(21)00461-4. [Epub ahead of print]
    Pediatric Cardiomyopathy Registry Study Group
      To understand the genetic contribution to primary pediatric cardiomyopathy, we performed exome sequencing in a large cohort of 528 children with cardiomyopathy. Using clinical interpretation guidelines and targeting genes implicated in cardiomyopathy, we identified a genetic cause in 32% of affected individuals. Cardiomyopathy sub-phenotypes differed by ancestry, age at diagnosis, and family history. Infants < 1 year were less likely to have a molecular diagnosis (p < 0.001). Using a discovery set of 1,703 candidate genes and informatic tools, we identified rare and damaging variants in 56% of affected individuals. We see an excess burden of damaging variants in affected individuals as compared to two independent control sets, 1000 Genomes Project (p < 0.001) and SPARK parental controls (p < 1 × 10-16). Cardiomyopathy variant burden remained enriched when stratified by ancestry, variant type, and sub-phenotype, emphasizing the importance of understanding the contribution of these factors to genetic architecture. Enrichment in this discovery candidate gene set suggests multigenic mechanisms underlie sub-phenotype-specific causes and presentations of cardiomyopathy. These results identify important information about the genetic architecture of pediatric cardiomyopathy and support recommendations for clinical genetic testing in children while illustrating differences in genetic architecture by age, ancestry, and sub-phenotype and providing rationale for larger studies to investigate multigenic contributions.
    Keywords:  ancestry; bioinformatics; clinical interpretation; exome; heart; infant; molecular diagnosis; variant
  35. Nat Protoc. 2022 Jan 12.
      Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >106 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.
  36. JIMD Rep. 2022 Jan;63(1): 3-10
      Objective: To report an adolescent with infantile-onset carnitine palmitoyltransferase 2 (CPT2) deficiency and cerebral malformations and to review the occurrence of brain malformations in CPT2 deficiency. The patient presented clinically at age 5 months with dehydration and hepatomegaly. He also has an unrelated condition, X-linked nephrogenic diabetes insipidus. He had recurrent rhabdomyolysis but normal psychomotor development. At age 17 years, he developed spontaneous focal seizures. Cerebral magnetic resonance imaging revealed extensive left temporo-parieto-occipital polymicrogyria, white matter heterotopias, and schizencephaly. Neuronal migration defects were previously reported in lethal neonatal CPT2 deficiency but not in later-onset forms.Design and Methods: We searched PubMed, Google Scholar, and the bibliographies of the articles found by these searches, for cerebral malformations in CPT2 deficiency. All antenatal, neonatal, infantile, and adult-onset cases were included. Exclusion criteria included insufficient information about age of clinical onset and lack of confirmation of CPT2 deficiency by enzymatic assay or genetic testing. For each report, we noted the presence of cerebral malformations on brain imaging or pathological examination.
    Results: Of 26 neonatal-onset CPT2-deficient patients who met the inclusion criteria, brain malformations were reported in 16 (61.5%). In 19 infantile-onset cases, brain malformations were not reported, but only 3 of the 19 reports (15.8%) include brain imaging or neuropathology data. In 276 adult-onset cases, no brain malformations were reported.
    Conclusion: To the best of our knowledge, this is the first report of cerebral malformations in an infantile onset CPT2-deficient patient. Brain imaging should be considered in patients with CPTII deficiency and neurological manifestations, even in those with later clinical onset.
    Keywords:  CPT2; carnitine; cerebral; heterotopias; infantile; malformation; palmitoyltransferase; polymicrogyria
  37. Eur J Med Chem. 2021 Dec 31. pii: S0223-5234(21)00936-3. [Epub ahead of print]229 114087
      STING mediates innate immune responses that are triggered by the presence of cytosolic DNA. Activation of STING to boost antigen recognition is a therapeutic modality that is currently being tested in cancer patients using nucleic-acid based macrocyclic STING ligands. We describe here the discovery of 3,4-dihydroquinazolin-2(1H)-one based 6,6-bicyclic heterocyclic agonists of human STING that activate all known human variants of STING with high potency.
    Keywords:  Immune oncology; Innate immunity; Interferon; STING; cGAS