bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2021‒08‒01
sixty papers selected by
Catalina Vasilescu
University of Helsinki

  1. Neurology. 2021 Jul 27. pii: 10.1212/WNL.0000000000012559. [Epub ahead of print]
  2. Dev Cell. 2021 Jul 26. pii: S1534-5807(21)00546-3. [Epub ahead of print]56(14): 2014-2015
      Mechanisms by which cells remove damaged mitochondria extracellularly are unclear. Recent work by Jiao and colleagues in Cell shows that migrating cells expel dysfunctional mitochondria in membrane-bound structures called migrasomes to maintain mitochondrial homeostasis.
  3. J Cell Sci. 2021 07 01. pii: jcs252197. [Epub ahead of print]134(13):
      The mitochondrial inner membrane is a protein-rich environment containing large multimeric complexes, including complexes of the mitochondrial electron transport chain, mitochondrial translocases and quality control machineries. Although the inner membrane is highly proteinaceous, with 40-60% of all mitochondrial proteins localised to this compartment, little is known about the spatial distribution and organisation of complexes in this environment. We set out to survey the arrangement of inner membrane complexes using stochastic optical reconstruction microscopy (STORM). We reveal that subunits of the TIM23 complex, TIM23 and TIM44 (also known as TIMM23 and TIMM44, respectively), and the complex IV subunit COXIV, form organised clusters and show properties distinct from the outer membrane protein TOM20 (also known as TOMM20). Density based cluster analysis indicated a bimodal distribution of TIM44 that is distinct from TIM23, suggesting distinct TIM23 subcomplexes. COXIV is arranged in larger clusters that are disrupted upon disruption of complex IV assembly. Thus, STORM super-resolution microscopy is a powerful tool for examining the nanoscale distribution of mitochondrial inner membrane complexes, providing a 'visual' approach for obtaining pivotal information on how mitochondrial complexes exist in a cellular context.
    Keywords:  COXIV; Mitochondria; Mitochondrial complexes; Nanoscopy; Protein import; STORM; TIM23
  4. Neuromuscul Disord. 2021 Jun 04. pii: S0960-8966(21)00139-5. [Epub ahead of print]
      Pathogenic variants in mitochondrial DNA (mtDNA) are associated with significant clinical heterogeneity with neuromuscular involvement commonly reported. Non-syndromic presentations of mtDNA disease continue to pose a diagnostic challenge and with genomic testing still necessitating a muscle biopsy in many cases. Here we describe an adult patient who presented with progressive ataxia, neuropathy and exercise intolerance in whom the application of numerous Mendelian gene panels had failed to make a genetic diagnosis. Muscle biopsy revealed characteristic mitochondrial pathology (cytochrome c oxidase deficient, ragged-red fibers) prompting a thorough investigation of the mitochondrial genome. Two heteroplasmic MT-CO2 gene variants (NC_012920.1: m.7887G>A and m.8250G>A) were identified, necessitating single fiber segregation and familial studies - including the biopsy of the patient's clinically-unaffected mother - to demonstrate pathogenicity of the novel m.7887G>A p.(Gly101Asp) variant and establishing this as the cause of the mitochondrial biochemical defects and clinical presentation. In the era of high throughput whole exome and genome sequencing, muscle biopsy remains a key investigation in the diagnosis of patients with non-syndromic presentations of adult-onset mitochondrial disease and fully defining the pathogenicity of novel mtDNA variants.
    Keywords:  Mitochondrial DNA; Muscle biopsy; Segregation study
  5. J Biol Chem. 2021 Jul 24. pii: S0021-9258(21)00807-3. [Epub ahead of print] 101005
      Barth syndrome (BTHS) is an X-linked disorder of mitochondrial phospholipid metabolism caused by pathogenic variants in the gene TAFFAZIN (TAZ), which results in abnormal cardiolipin (CL) content in the inner mitochondrial membrane. To identify unappreciated pathways of mitochondrial dysfunction in BTHS, we utilized an unbiased proteomics strategy and identified that complex I of the mitochondrial respiratory chain and the mitochondrial quality control protease PARL are altered in a new HEK293-based TAZ-deficiency model. Follow-up studies confirmed decreased steady state levels of specific complex I subunits and an assembly factor in the absence of TAZ; this decrease is in part based on decreased transcription, and results in reduced complex I assembly and function. PARL, a rhomboid protease associated with the inner mitochondrial membrane with a role in the mitochondrial response to stress such as mitochondrial membrane depolarization, is increased in TAZ-deficient cells. The increased abundance of PARL correlates with augmented processing of a downstream target, PGAM5, both at baseline and in response to mitochondrial depolarization. To clarify the relationship between abnormal CL content, complex I levels, and increased PARL expression that occurs when TAZ is missing, we used blue-native page and gene expression analysis to determine that these defects are remediated by SS-31 and bromoenol lactone, pharmacologic agents that bind CL or inhibit CL deacylation, respectively. These findings have the potential to enhance our understanding of the cardiac pathology of BTHS, where defective mitochondrial quality control and complex I dysfunction have well-recognized roles in the pathology of diverse forms of cardiac dysfunction.
    Keywords:  Barth Syndrome; Cardiolipin; Mitochondrial metabolism
  6. eNeuro. 2021 Jul 26. pii: ENEURO.0232-21.2021. [Epub ahead of print]
      Mitochondrial composition varies by organ and their constituent cell types. This mitochondrial diversity likely determines variations in mitochondrial function. However, the heterogeneity of mitochondria in the brain remains underexplored despite the large diversity of cell types in neuronal tissue. Here, we used molecular systems biology tools to address whether mitochondrial composition varies by brain region and neuronal cell type in mice. We reasoned that proteomics and transcriptomics of microdissected brain regions combined with analysis of single cell mRNA sequencing could reveal the extent of mitochondrial compositional diversity. We selected nuclear encoded gene products forming complexes of fixed stoichiometry, such as the respiratory chain complexes and the mitochondrial ribosome, as well as molecules likely to perform their function as monomers, such as the family of SLC25 transporters. We found that the proteome encompassing these nuclear-encoded mitochondrial genes and obtained from microdissected brain tissue segregated the hippocampus, striatum, and cortex from each other. Nuclear-encoded mitochondrial transcripts could only segregate cell types and brain regions when the analysis was performed at the single cell level. In fact, single cell mitochondrial transcriptomes were able to distinguish glutamatergic and distinct types of GABAergic neurons from one another. Within these cell categories, unique SLC25A transporters were able to identify distinct cell subpopulations. Our results demonstrate heterogeneous mitochondrial composition across brain regions and cell types. We postulate that mitochondrial heterogeneity influences regional and cell type specific mechanisms in health and disease.Significance StatementMitochondria are important organelles for maintaining brain health. The composition of proteins making up mitochondria is essential for their function. Disturbances to mitochondria are thought to contribute to neurodegeneration and neurodevelopmental disorders. These conditions typically affect specific brain regions or cell types. Despite the link between mitochondria and diseases with distinct anatomical and cellular patterns, how mitochondrial composition varies across brain regions and cell types remains poorly explored. Here, we analyze mitochondrial composition in different brain regions and cell types in adult mice, showing composition differs by region and cell lineage. Our work provides a resource of genes enriched in certain cell types or regions that improves our understanding of how mitochondrial composition influences brain function in health and disease.
    Keywords:  GABA; Glutamate; Mitochondria; Mitochondrial Ribosome; Respiratory Chain; Solute transporter
  7. Stem Cell Reports. 2021 Jul 15. pii: S2213-6711(21)00324-6. [Epub ahead of print]
      The generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.
    Keywords:  human iPSCs; iPSCs quality control; mtDNA deep sequencing; neuronal precursor cells
  8. Front Genet. 2021 ;12 685035
      Objective: The cytochrome c oxidase assembly factor 7 (COA7) gene encodes a protein localized to mitochondria that is involved in the assembly of mitochondrial respiratory chain complex IV. Here, we report the clinical, genetic and biochemical analysis of a female patient with suspected mitochondrial disorder and novel variants in COA7, that presented with a considerably different phenotype and age of onset than the five COA7 patients reported to date.Methods: We performed trio-exome sequencing in the affected patient and both parents. To verify the pathogenicity of the detected variants in COA7, mitochondrial enzyme activities and oxygen consumption rate were investigated in fibroblasts of the patient and her parents.
    Results: A Chinese girl was referred at 9 months of age with a history of developmental delay and regression since 3 months of age. In the following months, she lost previously acquired skills and developed progressive spasticity of the lower extremities. Trio-exome sequencing revealed compound heterzygous variants in COA7 (c.511G > A/p.Ala171Thr and c.566A > G/p.Asn189Ser). Functional validation experiments revealed isolated complex IV deficiency and a significantly reduced mitochondrial respiration rate in patient-derived fibroblasts.
    Interpretation: Hitherto, characteristic features of COA7 patients were described as slowly progressing neuropathy and spinocerebellar ataxia, starting at the toddler age and progressing into adulthood. In contrast, our patient was reported to show developmental delay from 3 months of age, which was found to be due to a rapidly progressive encephalopathy and brain atrophy seen at 9 months of age. Unexpectedly, the genetic investigation revealed a COA7-associated mitochondrial disease, which was confirmed functionally. Thus, this report broadens the genetic and clinical spectrum of this heterogeneous mitochondriopathy and highlights the value of the presented unbiased approach.
    Keywords:  COA7/RESA1; COX assembly factor; biallelic variant; mitochondrial disease; nervous system disorder
  9. Exp Anim. 2021 Jul 28.
      Focal segmental glomerulosclerosis (FSGS) is a major renal complication of human mitochondrial disease. However, its pathogenesis has not been fully explained. In this study, we focused on the glomerular injury of mito-miceΔ and investigated the pathogenesis of their renal involvement. We analyzed biochemical data and histology in mito-miceΔ. The proteinuria began to show in some mito-miceΔ with around 80% of mitochondrial DNA deletion, then proteinuria developed dependent with higher mitochondrial DNA deletion, more than 90% deletion. Mito-miceΔ with proteinuria histologically revealed FSGS. Immunohistochemistry demonstrated extensive distal tubular casts due to abundant glomerular proteinuria. Additionally, the loss of podocyte-related protein and podocyte's number were found. Therefore, the podocyte injuries and its depletion had a temporal relationship with the development of proteinuria. This study suggested mitochondrial DNA deletion-dependent podocyte injuries as the pathogenesis of renal involvement in mito-miceΔ. The podocytes are the main target of mitochondrial dysfunction originated from the accumulation of mitochondrial DNA abnormality in the kidney.
    Keywords:  focal segmental glomerulosclerosis; mitochondrial DNA deletion; mitochondrial disease model; podocyte injury
  10. Am J Physiol Cell Physiol. 2021 07 28.
      Mitochondria are recognized as signaling organelles because, under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased post-translational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel crosstalk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.
    Keywords:  acetylation; acylations; energy; heart; mitochondria
  11. Invest Ophthalmol Vis Sci. 2021 Jul 01. 62(9): 38
      Purpose: To investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation.Methods: Cybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis.
    Results: Nineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy.
    Conclusions: Our findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.
  12. Front Physiol. 2021 ;12 707634
      Diabetic cardiomyopathy has been associated with mitochondrial damage. Mitochondria-endoplasmic reticulum (ER) contact is an important determinant of mitochondrial function and ER homeostasis. We therefore investigated whether hyperglycemia can damage the mitochondria by increasing their contact with the ER in cardiomyocytes. We found that hyperglycemia induced mitochondria-ER contact in cardiomyocytes, as evidenced by the increased MMM1, MDM34, and BAP31 expressions. Interestingly, the silencing of Mfn2 reduced the cooperation between the mitochondria and the ER in cardiomyocytes. Mfn2 silencing improved cardiomyocyte viability and function under hyperglycemic conditions. Additionally, the silencing of Mfn2 markedly attenuated the release of calcium from the ER to the mitochondria, thereby preserving mitochondrial metabolism in cardiomyocytes under hyperglycemic conditions. Mfn2 silencing reduced mitochondrial reactive oxygen species production, which reduced mitochondria-dependent apoptosis in hyperglycemia-treated cardiomyocytes. Finally, Mfn2 silencing attenuated ER stress in cardiomyocytes subjected to high-glucose stress. These results demonstrate that Mfn2 promotes mitochondria-ER contact in hyperglycemia-treated cardiomyocytes. The silencing of Mfn2 sustained mitochondrial function, suppressed mitochondrial calcium overload, prevented mitochondrial apoptosis, and reduced ER stress, thereby enhancing cardiomyocyte survival under hyperglycemic conditions.
    Keywords:  ER; Mfn2; apoptosis; mitochondria; mitochondria-ER contact
  13. Front Neurosci. 2021 ;15 680572
      Pathogenic variants in SPG11 are the most frequent cause of autosomal recessive complicated hereditary spastic paraplegia (HSP). In addition to spastic paraplegia caused by corticospinal degeneration, most patients are significantly affected by progressive weakness and muscle wasting due to alpha motor neuron (MN) degeneration. Mitochondria play a crucial role in neuronal health, and mitochondrial deficits were reported in other types of HSPs. To investigate whether mitochondrial pathology is present in SPG11, we differentiated MNs from induced pluripotent stem cells derived from SPG11 patients and controls. MN derived from human embryonic stem cells and an isogenic SPG11 knockout line were also included in the study. Morphological analysis of mitochondria in the MN soma versus neurites revealed specific alterations of mitochondrial morphology within SPG11 neurites, but not within the soma. In addition, impaired mitochondrial membrane potential was indicative of mitochondrial dysfunction. Moreover, we reveal neuritic aggregates further supporting neurite pathology in SPG11. Correspondingly, using a microfluidic-based MN culture system, we demonstrate that axonal mitochondrial transport was significantly impaired in SPG11. Overall, our data demonstrate that alterations in morphology, function, and transport of mitochondria are an important feature of axonal dysfunction in SPG11 MNs.
    Keywords:  SPG11; alpha motor neuron; axonal transport; hereditary spastic paraplegia; induced pluripotent stem cells; mitochondria
  14. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jul 20. pii: S1570-0232(21)00344-5. [Epub ahead of print]1179 122863
      Mitochondria play an essential role in various biochemical processes that maintain cellular homeostasis. Minor defects in the mitochondrial genome can lead to aversive behavioral responses in an organism. Nevertheless, little is known about the impact of mitochondrial mutations on the metabolome of Caenorhabditis elegans (C. elegans). In this study, an untargeted metabolomics approach was employed to elucidate the metabolic aberrant caused by mitochondrial DNA mutations in C. elegans. Specifically, three mutant strains of C. elegans, including clk-1, mev-1, and phb-2, were adopted to study corresponding metabolic signatures. Adult worms were collected, and metabolites were extracted and analyzed by gas chromatography-mass spectrometry. Uni- and multivariate analyses were performed to elucidate perturbed metabolism between wildtype worms and mutant strains, and metabolic differences among the mutants. The tricarboxylic acid cycle intermediates, amino acids, and sugars were significantly affected in the mitochondrial mutants. Overall, each mitochondrial DNA mutation exhibited a different pattern of metabolic alterations. The shift of metabolome appeared to be associated with the lifespan of C. elegans. In particular, clk-1 and mev-1 strains, which had the opposite phenotypes of lifespan, had apparently different metabolomes. Our findings set light on the metabolic consequences of mitochondrial genetic variants, which may help better understand mitochondrial disease mechanisms.
    Keywords:  Caenorhabditis elegans; Clk-1; Metabolomics; Mev-1; Mitochondria; Phb-2
  15. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30193-X. [Epub ahead of print]165 153-161
      Selective elimination of damaged mitochondria via macroautophagy (mitophagy) is a conserved cellular process that plays an important role in organismal health. In recent years mitophagy has been studied in parallel to the more general, non-selective autophagy pathway induced in response to amino acid starvation with important similarities and differences noted between the two. The elaborate sequence of membrane rearrangements that give rise to autophagosomes in the non-selective pathway have their counterpart in mitophagy, but with the addition of other factors, such as a ubiquitin mark and mitophagy receptors, which mediate cargo recognition. In some types of mitophagy such as the one induced by ivermectin, the forming autophagosomal structure contains six different elements: the targeted mitochondrial fragment, a section of endoplasmic reticulum that provides a cradle, a ubiquitin layer, the mitophagy receptors and the early and late autophagosomal proteins/membranes. Super-resolution microscopy is ideally suited to investigate the spatial relationships between these elements that converge together but retain some distinctive localization, and we provide here a general protocol that can be used for mammalian cells.
    Keywords:  Autophagy; Endoplasmic reticulum; Ivermectin; Mitochondria; Mitophagy; Structured illumination microscopy
  16. Stem Cell Reports. 2021 Jul 16. pii: S2213-6711(21)00327-1. [Epub ahead of print]
      Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is often caused by an adenine to guanine variant at m.3243 (m.3243A>G) of the MT-TL1 gene. To understand how this pathogenic variant affects the nervous system, we differentiated human induced pluripotent stem cells (iPSCs) into excitatory neurons with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function from MELAS patients with the m.3243A>G pathogenic variant. We combined micro-electrode array (MEA) measurements with RNA sequencing (MEA-seq) and found reduced expression of genes involved in mitochondrial respiration and presynaptic function, as well as non-cell autonomous processes in co-cultured astrocytes. Finally, we show that the clinical phase II drug sonlicromanol can improve neuronal network activity when treatment is initiated early in development. This was intricately linked with changes in the neuronal transcriptome. Overall, we provide insight in transcriptomic changes in iPSC-derived neurons with high m.3243A>G heteroplasmy, and show the pathology is partially reversible by sonlicromanol.
    Keywords:  MELAS; iPSC-derived neurons; micro-electrode arrays; mitochondria; neurons; sonlicromanol
  17. FASEB J. 2021 Aug;35(8): e21796
      Polycystin-1 (PC1) is a transmembrane protein found in different cell types, including cardiomyocytes. Alterations in PC1 expression have been linked to mitochondrial damage in renal tubule cells and in patients with autosomal dominant polycystic kidney disease. However, to date, the regulatory role of PC1 in cardiomyocyte mitochondria is not well understood. The analysis of mitochondrial morphology from cardiomyocytes of heterozygous PC1 mice (PDK1+/- ) using transmission electron microscopy showed that cardiomyocyte mitochondria were smaller with increased mitochondria density and circularity. These parameters were consistent with mitochondrial fission. We knocked-down PC1 in cultured rat cardiomyocytes and human-induced pluripotent stem cells (iPSC)-derived cardiomyocytes to evaluate mitochondrial function and morphology. The results showed that downregulation of PC1 expression results in reduced protein levels of sub-units of the OXPHOS complexes and less functional mitochondria (reduction of mitochondrial membrane potential, mitochondrial respiration, and ATP production). This mitochondrial dysfunction activates the elimination of defective mitochondria by mitophagy, assessed by an increase of autophagosome adapter protein LC3B and the recruitment of the Parkin protein to the mitochondria. siRNA-mediated PC1 knockdown leads to a loss of the connectivity of the mitochondrial network and a greater number of mitochondria per cell, but of smaller sizes, which characterizes mitochondrial fission. PC1 silencing also deregulates the AKT-FoxO1 signaling pathway, which is involved in the regulation of mitochondrial metabolism, mitochondrial morphology, and processes that are part of cell quality control, such as mitophagy. Together, these data provide new insights about the controls that PC1 exerts on mitochondrial morphology and function in cultured cardiomyocytes dependent on the AKT-FoxO1 signaling pathway.
    Keywords:  FoxO1; cardiomyocyte; mitochondrial dynamics; mitochondrial metabolism; mitophagy; polycystin-1
  18. J Cell Biol. 2021 Sep 06. pii: e202105043. [Epub ahead of print]220(9):
      Ferroptosis is a form of iron-dependent regulated cell death driven by uncontrolled lipid peroxidation. Mitochondria are double-membrane organelles that have essential roles in energy production, cellular metabolism, and cell death regulation. However, their role in ferroptosis has been unclear and somewhat controversial. In this Perspective, I summarize the diverse metabolic processes in mitochondria that actively drive ferroptosis, discuss recently discovered mitochondria-localized defense systems that detoxify mitochondrial lipid peroxides and protect against ferroptosis, present new evidence for the roles of mitochondria in regulating ferroptosis, and outline outstanding questions on this fascinating topic for future investigations. An in-depth understanding of mitochondria functions in ferroptosis will have important implications for both fundamental cell biology and disease treatment.
  19. Sci Rep. 2021 Jul 30. 11(1): 15510
      Ischemia is a major cause of kidney damage. Proximal tubular epithelial cells (PTECs) are highly susceptible to ischemic insults that frequently cause acute kidney injury (AKI), a potentially life-threatening condition with high mortality. Accumulating evidence has identified altered mitochondrial function as a central pathologic feature of AKI. The mitochondrial NAD+-dependent enzyme sirtuin 5 (SIRT5) is a key regulator of mitochondrial form and function, but its role in ischemic renal injury (IRI) is unknown. SIRT5 expression was increased in murine PTECs after IRI in vivo and in human PTECs (hPTECs) exposed to an oxygen/nutrient deprivation (OND) model of IRI in vitro. SIRT5-depletion impaired ATP production, reduced mitochondrial membrane potential, and provoked mitochondrial fragmentation in hPTECs. Moreover, SIRT5 RNAi exacerbated OND-induced mitochondrial bioenergetic dysfunction and swelling, and increased degradation by mitophagy. These findings suggest SIRT5 is required for normal mitochondrial function in hPTECs and indicate a potentially important role for the enzyme in the regulation of mitochondrial biology in ischemia.
  20. FEBS J. 2021 Jul 26.
      Mitochondria form a branched tubular network in many types of cells, depending on a balance between mitochondrial fusion and fission. How mitochondrial fusion and fission are involved in regulating mitochondrial function and cell proliferation is not well understood. Here, we dissected the roles of mitochondrial fusion and fission in mitochondrial function and cell proliferation in fission yeast. We examined mitochondrial membrane potential by staining cells with DiOC6 and assessed mitochondrial respiration by directly measuring oxygen consumption of cells with a dissolved oxygen respirometer. We found that defects in mitochondrial fission or fusion reduce mitochondrial membrane potential and compromise mitochondrial respiration while the absence of both mitochondrial fusion and fission restores wild-type-like respiration, normal membrane potential, and tubular networks of mitochondria. Moreover, we found that the absence of either mitochondrial fission or fusion prolongs the cell cycle and that the absence of both mitochondrial fusion and fission significantly delays cell cycle progression after nitrogen replenishment. The prolonged/delayed cell cycle is likely due to the deregulation of Cdc2 activation. Hence, our work not only establishes an intimate link between mitochondrial morphology and function but also underscores the importance of mitochondrial dynamics in regulating the cell cycle.
    Keywords:  Cell cycle; Dnm1; Fzo1; Mitochondria; Mitochondrial dynamics
  21. Methods Mol Biol. 2021 ;2319 51-60
      Cardiovascular disease is a worldwide health issue that affects millions of lives every year, and thus, researchers are in need of high-throughput model systems with which to investigate mechanisms of disease and to develop and test potential therapies. The use of human-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes aims to address this need. While cardiac differentiation protocols have been established previously in iPSCs, optimization of cardiac differentiation remains crucial to obtaining high quality cardiomyocytes for future experimental analyses. Important factors to consider include cell density and rate of proliferation, temporal regulation of media changes throughout the differentiation process, and the concentration of the chemicals utilized. In this chapter, we present a detailed protocol to outline the process of differentiating cardiomyocytes from human iPSCs via modulation of Wnt signaling, characterization of cardiomyocytes by immunofluorescence, as well as guidelines for troubleshooting and optimizing these techniques.
    Keywords:  Cardiac; Cardiac myocytes; Cardiomyocyte; Cardiomyocyte characterization; Disease modeling; Drug discovery; In vitro; Induced pluripotent stem cells; Wnt inhibition; iPSC
  22. Nat Commun. 2021 07 28. 12(1): 4578
      Mitochondria are transported along microtubules by opposing kinesin and dynein motors. Kinesin-1 and dynein-dynactin are linked to mitochondria by TRAK proteins, but it is unclear how TRAKs coordinate these motors. We used single-molecule imaging of cell lysates to show that TRAK2 robustly activates kinesin-1 for transport toward the microtubule plus-end. TRAK2 is also a novel dynein activating adaptor that utilizes a conserved coiled-coil motif to interact with dynein to promote motility toward the microtubule minus-end. However, dynein-mediated TRAK2 transport is minimal unless the dynein-binding protein LIS1 is present at a sufficient level. Using co-immunoprecipitation and co-localization experiments, we demonstrate that TRAK2 forms a complex containing both kinesin-1 and dynein-dynactin. These motors are functionally linked by TRAK2 as knockdown of either kinesin-1 or dynein-dynactin reduces the initiation of TRAK2 transport toward either microtubule end. We propose that TRAK2 coordinates kinesin-1 and dynein-dynactin as an interdependent motor complex, providing integrated control of opposing motors for the proper transport of mitochondria.
  23. Mitochondrion. 2021 Jul 21. pii: S1567-7249(21)00097-0. [Epub ahead of print]
      We investigated the concordance of mitochondrial DNA heteroplasmic mutations (heteroplasmies) in 6,745 maternal pairs of European (EA, n=4,718 pairs) and African (AA, n=2,027 pairs) Americans in whole blood. Mother-offspring pairs displayed the highest concordance rate, followed by sibling-sibling and more distantly-related maternal pairs. The allele fractions of concordant heteroplasmies exhibited high correlation (R2=0.8) between paired individuals. Discordant heteroplasmies were more likely to be in coding regions, be nonsynonymous or nonsynonymous-deleterious (p<0.001). The number of deleterious heteroplasmies was significantly correlated with advancing age (20-44, 45-64, and ≥65 years, p-trend=0.01). One standard deviation increase in heteroplasmic burden (i.e., the number of heteroplasmies carried by an individual) was associated with 0.17 to 0.26 (p<1e-23) standard deviation decrease in mtDNA copy number, independent of age. White blood cell count and differential count jointly explained 0.5% to 1.3% (p≤0.001) variance in heteroplasmic burden. A genome-wide association and meta-analysis identified a region at 11p11.12 (top signal rs779031139, p=2.0e-18, minor allele frequency=0.38) associated with the heteroplasmic burden. However, the 11p11.12 region is adjacent to a nuclear mitochondrial DNA (NUMT) corresponding to a 542 bp area of the D-loop. This region was no longer significant after removing heteroplasmic mutations within the 542 bp from the heteroplasmic burden. The discovery that blood mtDNA heteroplasmic mutations were both inherited and somatic origins and that an increase in heteroplasmic burden was strongly associated with a decrease in average number of mtDNA copy number in blood are important findings to be considered in association studies of mtDNA with disease traits.
  24. Methods Mol Biol. 2021 ;2369 27-40
      We present a detailed method for extraction of high-molecular weight genomic DNA suitable for numerous DNA sequencing applications, and a straightforward in silico approach for reconstructing novel mitochondrial (mt) genomes directly from total genomic DNA extracts derived from next-generation sequencing (NGS) data sets. The in silico post-sequencing pipeline described is fast, accurate, and highly efficient, with modest memory requirements that can be performed using a standard desktop computer. The approach is particularly effective for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information currently available and overcomes many of the limitations of traditional strategies. The described methodologies are also applicable for metagenomics sequencing from mixed or pooled samples containing multiple species and subsequent specific assembly of specific mitochondrial genomes.
    Keywords:  DNA extraction; Genomics; Helminth; Mitochondrial genome; Parasite
  25. JCI Insight. 2021 Jul 27. pii: 147692. [Epub ahead of print]
      Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than one thousand nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here we show that histone demethylase LSD1 knockout from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in nucleus. Lsd1 knockout reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.
    Keywords:  Diabetes; Endocrinology; Obesity
  26. Mech Ageing Dev. 2021 Jul 21. pii: S0047-6374(21)00117-2. [Epub ahead of print] 111545
      Strategies to correct declining nicotinamide adenine dinucleotide (NAD+) levels in neurological disease and biological ageing are promising therapeutic candidates. These strategies include supplementing with NAD+ precursors, small molecule activation of NAD+ biosynthetic enzymes, and treatment with small molecule inhibitors of NAD+ consuming enzymes such as CD38, SARM1 or members of the PARP family. While these strategies have shown efficacy in animal models of neurological disease, each of these has the mechanistic potential for adverse events that could preclude their preclinical use. Here, we discuss the implications of these strategies for treating neurological diseases, including potential off-target effects that may be unique to the brain.
    Keywords:  CD38; Neurodegeneration; SARM1; Therapeutics; nicotinamide adenine dinucleotide (NAD+)
  27. Autophagy. 2021 Jul 27. 1-3
      ATG7 drives macroautophagy, hereafter "autophagy", by generating ATG12-ATG5 conjugates and lipidating Atg8 homologs including LC3. A pioneering body of work has defined the requirement of ATG7 for survival in mice and shown that neural-specific atg7 deletion causes neurodegeneration, but it has not been ascertained whether human life is compatible with ATG7 dysfunction. Recently, we defined the importance of ATG7 in human physiology by identifying twelve patients from five families harboring pathogenic, biallelic ATG7 variants causing a neurodevelopmental disorder. Patient fibroblasts show undetectable or severely diminished ATG7 protein levels, and biochemical assessment via autophagic flux and long-lived protein degradation assays demonstrated that attenuated autophagy underpins the pathology. Confirming the pathogenicity of patient variants, mouse cells expressing mutated ATG7 are unable to rescue LC3/Atg8 lipidation to wild-type levels. Our work defines mutated ATG7 as an important cause of human neurological disease and expands our understanding of autophagy in longevity and human health. We demonstrated that in certain circumstances, human survival with relatively mild phenotypes is possible even with undetectable levels of a nonredundant core autophagy protein.
    Keywords:  Autophagy; atg7; cell biology; disease; macroautophagy; molecular genetics; neurodegeneration
  28. PLoS One. 2021 ;16(7): e0255355
      Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.
  29. Eur J Hum Genet. 2021 Jul 26.
      The aetiology of dystonia disorders is complex, and next-generation sequencing has become a useful tool in elucidating the variable genetic background of these diseases. Here we report a deleterious heterozygous truncating variant in the inosine monophosphate dehydrogenase gene (IMPDH2) by whole-exome sequencing, co-segregating with a dominantly inherited dystonia-tremor disease in a large Finnish family. We show that the defect results in degradation of the gene product, causing IMPDH2 deficiency in patient cells. IMPDH2 is the first and rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides, a dopamine synthetic pathway previously linked to childhood or adolescence-onset dystonia disorders. We report IMPDH2 as a new gene to the dystonia disease entity. The evidence underlines the important link between guanine metabolism, dopamine biosynthesis and dystonia.
  30. Front Cell Dev Biol. 2021 ;9 698658
      Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.
    Keywords:  GFP; SNAP-tag; fluorescence microscopy; microinjection; mitochondria; mitochondrial protein import
  31. Dis Model Mech. 2021 Jul 27. pii: dmm.048995. [Epub ahead of print]
      Mitochondrial dysfunction in different cell types is associated to several pathological processes and potentially contributes to chronic inflammatory and ageing-related diseases. Mitochondrial Transcription Factor A (TFAM) plays a critical role in maintaining mtDNA integrity and function. Taking advantage of the Tfamfl/fl UBC-Cre/ERT2+/+ mice, we sought to develop a cellular in vitro system to investigate the role of mitochondrial dysfunction in the stromal cell component. We describe an inducible model of mitochondrial dysfunction by stable depletion of TFAM in primary mouse skin fibroblast (SK-FB) after 4-hydroxytamoxifen (4-OHT) administration. Tfam gene deletion caused a sustained reduction of Tfam and mtDNA-encoded mRNA expression in Cre(+) cultured for low (LP) and high passages (HP). Ultimately, Tfam knockout translated into a loss of TFAM protein. TFAM depletion led to a substantial reduction of the mitochondrial respiratory chain (MRC) complexes that was exacerbated in HP SK-FB cultures. The assembly pattern showed that the respiratory complexes fail to reach the respirasome in 4-OHT Cre(+) SK-FB. Functionally, we determined the mitochondrial function and the glycolytic activity by mito-stress and glycolysis-stress test respectively. These analysis showed that mitochondrial dysfunction was developed after long-term 4-OHT treatment in HP Cre(+) SK-FB and was compensated by an increase in the glycolytic capacity. Finally, expression analysis revealed that 4-OHT-treated HP Cre(+) SK-FB showed a senescent and pro-inflammatory phenotype. In conclusion, we have generated and validated the first ex vivo model of fibroblast mitochondrial dysfunction that results in a pro-inflammatory phenotype applicable to explore this process in other cell types in a variety of pathological conditions.
    Keywords:  Cellular senescence; Fibroblasts; Inflammation; Mitochondrial dysfunction; TFAM
  32. Neurochem Int. 2021 Jul 27. pii: S0197-0186(21)00193-5. [Epub ahead of print] 105147
      Huntington's disease (HD), as well as Parkinson's disease and Alzheimer's disease, belong to a group of neurodegenerative diseases characterized by common features, such as the progressive loss of neurons and the presence of pathogenic forms of misfolded protein aggregates. A quality control system such as autophagy is crucial for the clearance of protein aggregates and dysfunctional organelles and thus essential for the maintenance of neuronal homeostasis. The constant high energy demand of neuronal tissue links neurodegeneration to mitochondria. Inefficient removal of damaged mitochondria is thought to contribute to the pathogenesis of neurodegenerative diseases such as HD. In addition, direct involvement of the huntingtin protein in the autophagic machinery has been described. In this review, we focus on mitophagy, a selective form of autophagy responsible for mitochondrial turnover. We also discuss the relevance of pharmacological regulation of mitophagy in the future therapeutic approach to neurodegenerations, including HD.
    Keywords:  Huntington's disease; Mitochondria; Mitophagy; Mitophagy adaptors; Pharmacological induction of mitophagy
  33. Front Cell Dev Biol. 2021 ;9 673839
      Cardiovascular disease (CVD) is the main cause of death worldwide. Atherosclerosis is the underlying pathological basis of CVD. Mitochondrial homeostasis is maintained through the dynamic processes of fusion and fission. Mitochondria are involved in many cellular processes, such as steroid biosynthesis, calcium homeostasis, immune cell activation, redox signaling, apoptosis, and inflammation, among others. Under stress conditions, mitochondrial dynamics, mitochondrial cristae remodeling, and mitochondrial ROS (mitoROS) production increase, mitochondrial membrane potential (MMP) decreases, calcium homeostasis is imbalanced, and mitochondrial permeability transition pore open (mPTP) and release of mitochondrial DNA (mtDNA) are activated. mtDNA recognized by TLR9 can lead to NF-κB pathway activation and pro-inflammatory factor expression. At the same time, TLR9 can also activate NLRP3 inflammasomes and release interleukin, an event that eventually leads to tissue damage and inflammatory responses. In addition, mitochondrial dysfunction may amplify the activation of NLRP3 through the production of mitochondrial ROS, which together aggravate accumulating mitochondrial damage. In addition, mtDNA defects or gene mutation can lead to mitochondrial oxidative stress. Finally, obesity, diabetes, hypertension and aging are risk factors for the progression of CVD, which are closely related to mitochondrial dynamics. Mitochondrial dynamics may represent a new target in the treatment of atherosclerosis. Antioxidants, mitochondrial inhibitors, and various new therapies to correct mitochondrial dysfunction represent a few directions for future research on therapeutic intervention and amelioration of atherosclerosis.
    Keywords:  cytoskeleton; fission; fusion; mitochondria; morphology; transport
  34. Front Pediatr. 2021 ;9 626657
      Background: Mitochondrial dynamics, including mitochondrial fission and fusion, transport and distribution, biogenesis and degradation, are critical to neuronal function. The dynamin-1 like (DNM1L) gene encodes dynamin-related protein 1 (DRP1/DLP1), which is an evolutionarily conserved member of the dynamin family and is responsible for mitochondrial division. DNM1L variants can lead to mitochondrial fission dysfunction and neurological disorders. Methods: We report a case of DNM1L-related mitochondrial disease admitted to Tianjin Children's Hospital. We searched for similar reported cases in the PubMed database using the terms "DNM1L" and "mitochondrial," reviewed recent literature to summarize the clinical and genetic characteristics, and analyzed genotype-phenotype correlations. Results: The patient presented with psychomotor retardation, motor disturbance (muscle weakness with paroxysmal hypermyotonia), and a de novo variant (c.116G>A, g.22229G>A, p.S39N) in the GTPase domain of DNM1L (reference sequence NM_012062), which has not previously been reported in the literature. This case was combined with an additional 35 cases identified in 20 relevant references in order to analyze a total of 36 patients. The male-to-female ratio was 1:1.06, and the median age of onset was 6 months (range, neonatal period to 9 years). The cardinal symptoms included psychomotor retardation in 77.8% (28/36), limb paralysis in 66.7% (18/27), dystonia in 82.8% (24/29), and epilepsy in 59.4% (19/32). The clinical manifestations of variants in the GTPase domain of DRP1 were milder than those identified in the middle domain. Conclusion: This case report describes a new variant of the DNM1L gene, and summarizes previously reported cases. Furthermore, the clinical phenotype and the genotype of DNM1L gene-associated mitochondrial disease was analyzed to improve the understanding of this disease.
    Keywords:  DNM1L; DRP1; dyskinesia; follow-up study; hypertonia; mitochondrial disease; psychomotor retardation
  35. J Cell Biochem. 2021 Jul 28.
      Mitochondria and peroxisomes are metabolically interconnected and functionally active subcellular organelles. These two dynamic organelles, share a number of common biochemical functions such as β-oxidation of fatty acids and detoxification of peroxides. The biogenesis and morphology of both these organelles in the mammalian cells is controlled by common transcription factors like PGC1α, and by a common fission machinery comprising of fission proteins like DRP1, Mff, and hFis1, respectively. In addition, the outer membrane mitochondria-anchored protein ligase (MAPL), the first mitochondrial SUMO E3 ligase with a RING-finger domain, also regulates mitochondrial morphology inducing mitochondrial fragmentation upon its overexpression. This fragmentation is dependent on both the RING domain of MAPL and the presence of the mitochondrial fission GTPase dynamin-related protein-1 (DRP1). Earlier studies have demonstrated that mitochondrial-derived vesicles are formed independently of the known mitochondrial fission GTPase, DRP1 are enriched for MAPL and are targeted to peroxisomes. The current study shows that MAPL regulates morphology of peroxisomes in a cell-type specific manner. Fascinatingly, the peroxisome elongation caused either due to silencing of DRP1 or by addition of polyunsaturated fatty acid, docosahexaenoic acid was blocked by overexpressing MAPL in mammalian cell lines. Furthermore, the transfection and colocalisation studies of MAPL with peroxisome membrane marker, PMP70, in different cell lines clearly revealed a cell-type specificity of transport of MAPL to peroxisomes. Previous work has placed the Vps35 (retromer component) as vital for delivery of MAPL to peroxisomes, placing the retromer as critical for the formation of MAPL-positive mitochondrial-derived vesicles. The results of polyethylene glycol-based cell-cell fusion assay signified that the enrichment of MAPL in peroxisomes is through vesicles and a retromer dependent phenomenon. Thus, a novel function for MAPL in peroxisomes is established to regulate peroxisome elongation and morphology under growth conditions and thus possibly modulate peroxisome fission.
    Keywords:  Vps35; mitochondrial anchored protein ligase; mitochondrial-derived vesicles; peroxisome fission; retromer complex; vesicular transport
  36. Methods Mol Biol. 2021 ;2352 149-170
      Oligodendrocytes are the main glial cell type in the central nervous system supporting the axonal part of neurons via myelin and lactate delivery. Both the conductive myelin formation and the energy support via lactate can be affected in diseases, such as multiple sclerosis and amyotrophic lateral sclerosis, respectively. Therefore, human disease modeling is needed to gain more mechanistic insights to drive drug discovery research. Here, patient-derived induced pluripotent stem cells (iPSCs) serve as a necessary tool providing an infinite cell source for patient-specific disease modeling, which allows investigation of oligodendrocyte involvement in human disease.Small molecule-based differentiation protocols to generate oligodendrocytes from pluripotent stem cells can last more than 90 days. Here, we provide a transcription factor-based, fast and efficient protocol for generating O4+ oligodendrocytes in just 20-24 days. After a neural induction phase of 8-12 days, SOX10 is overexpressed either with the use of lentiviral vectors or via engineered iPSCs, which inducibly overexpress SOX10 after doxycycline addition. Using this last method, a pure O4+ cell population is achieved after keeping the SOX10-overexpressing neural stem cells in culture for an additional 10 days. Furthermore, these O4+ cells can be co-cultured with iPSC-derived cortical neurons in 384-well format, allowing pro-myelinating drug screens. In conclusion, we provide a fast and efficient oligodendrocyte differentiation protocol allowing both in vitro human disease modeling and a high-throughput co-culture system for drug discovery.
    Keywords:  Differentiation protocol; Induced pluripotent stem cells; Neuron-oligodendrocyte co-culture; Oligodendrocytes; Recombinase-mediated cassette exchange; SOX10
  37. Neurobiol Aging. 2021 Jul 04. pii: S0197-4580(21)00217-7. [Epub ahead of print]
      With the aging population and increasing life expectancy, Parkinson's disease (PD), a neurological disorder rapidly increasing in morbidity and mortality, is causing a huge burden on society and the economy. Several studies have suggested that one-carbon metabolites, including homocysteine, vitamin B6, vitamin B12 and folate acid, are associated with PD risk. However, the results remain inconsistent and controversial. Thus, we performed a two-sample Mendelian randomization (MR) study to detect the causality between one-carbon metabolites and PD susceptibility as well as age at PD onset. We collected several genetic variants as instrumental variables from large genome-wide association studies of one-carbon metabolites (homocysteine: N = 14, vitamin B6: N = 1, vitamin B12: N = 10, folate acid: N = 2). We then conducted MR analyses using the inverse variance-weighted (IVW) approach and additional MR-Egger regression, weighted median and MR-pleiotropy residual sum and outlier (MR-PRESSO) methods to further test causality. The results showed no causal association between circulating homocysteine levels and PD risk (p = 0.868) or age at PD onset (p = 0.222) with the IVW method. Meanwhile, similar results were obtained by three complementary analyses. In addition, we did not observe any evidence that the circulating levels of vitamin B6, vitamin B12 and folate acid affected the risk of PD or age at onset of PD. Our findings implied that lowering homocysteine levels through vitamin B6, vitamin B12 or folate acid supplementation may not be clinically helpful in preventing PD or delaying the age at PD onset.
    Keywords:  Homocysteine; Mendelian randomization study; One-carbon metabolism; Parkinson's disease
  38. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30198-9. [Epub ahead of print]165 103-110
      Mitophagy is an autophagic mechanism for targeting damaged or unnecessary mitochondria and responsible for mitochondria quality control. Emerging evidence revealed that mitophagy is associated with many physiological processes and cellular activities. Therefore, the determination of mitophagy may provide insights into human physiological and pathological processes. Electron microscopy, one of the best approaches, can directly provide the ultrastructure evidence for mitophagy. Here, we detail an experiment protocol for electron microscopy preparation, so as to detect mitophagy in biological samples. Compare with other biochemical techmology, conventional electron microscopy are still essential for strengthening or replacing biochemical methods, and a better understanding of this method could be important to investigate mitophagy.
    Keywords:  Biological sample preparation; Detection; Electron microscope; Method; Mitophagy
  39. Front Endocrinol (Lausanne). 2021 ;12 675581
      Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder worldwide. We aimed to examine the associations of two mitochondrial DNA (mtDNA) biomarkers in the peripheral blood, mtDNA copy number (CN), and mtDNA4977 deletion rate (DR), with PCOS in a clinical setting.Methods: We performed a study involving 263 women with PCOS and 326 age-matched controls between June 2015 and June 2019. The mtDNA CN and mtDNA4977 DR were measured using multiplex probe-based qPCR. The associations of the mtDNA CN and mtDNA4977 DR with the risk of PCOS were estimated using logistic regression.
    Results: Analysis of the associations between mtDNA biomarkers and PCOS indicate that the mtDNA CN (P = 0.003) and mtDNA4977 DR (P < 0.001) in PCOS patients were significantly higher than those in the controls. After adjusting for the body mass index, luteinizing hormone/follicle-stimulating hormone ratio, and testosterone level, only higher mtDNA4977 DR was associated with PCOS (odds ratio 1.053, 95% confidence interval 1.024 to 1.083; P < 0.001). The linear dose-response trends of the mtDNA4977 DR were also supported by the quartile analysis.
    Conclusion: Multivariable models suggest that mtDNA4977 DR levels are strongly associated with PCOS and represent an independent risk factor for PCOS. Further investigation of the utility of mtDNA as a biomarker for PCOS is warranted.
    Keywords:  infertility; mitochondria; mitochondrial DNA copy number; mitochondrial DNA deletion; polycystic ovary syndrome
  40. Trends Genet. 2021 Jul 24. pii: S0168-9525(21)00195-5. [Epub ahead of print]
      Human large-scale genetic association studies have identified sequence variations at thousands of genetic risk loci that are more common in patients with diverse metabolic disease compared with healthy controls. While these genetic associations have been replicated in multiple large cohorts and sometimes can explain up to 50% of heritability, the molecular and cellular mechanisms affected by common genetic variation associated with metabolic disease remains mostly unknown. A variety of new genome-wide data types, in conjunction with novel biostatistical and computational analytical methodologies and foundational experimental technologies, are paving the way for a principled approach to systematic variant-to-function (V2F) studies for metabolic diseases, turning associated regions into causal variants, cell types and states of action, effector genes, and cellular and physiological mechanisms. Identification of new target genes and cellular programs for metabolic risk loci will improve mechanistic understanding of disease biology and identification of novel therapeutic strategies.
    Keywords:  GWAS; common variants; epigenetics; functional annotation; genetic variants
  41. Nat Commun. 2021 07 27. 12(1): 4542
      Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.
  42. Cardiovasc Res. 2021 Jul 27. 117(9): e106-e109
    Keywords:  Ageing; Cardiovascular disease; Diabetes; NMN; NR; Niacin; Nicotinamide; Obesity
  43. Nat Commun. 2021 07 28. 12(1): 4583
      Voltage dependent anion channel 2 (VDAC2) is an outer mitochondrial membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. However, the specific role of VDAC2 in intracellular calcium dynamics and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac ventricular myocyte-specific developmental deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium causes severe impairment in excitation-contraction coupling by altering both intracellular and mitochondrial calcium signaling. We also observed adverse cardiac remodeling which progressed to severe cardiomyopathy and death. Reintroduction of VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype. Activation of VDAC2 by efsevin increased cardiac contractile force in a mouse model of pressure-overload induced heart failure. In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing cellular calcium signaling. Through this unique role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.
  44. Nucleic Acids Res. 2021 Jul 28. pii: gkab637. [Epub ahead of print]
      Whole genome bisulphite sequencing (WGBS) permits the genome-wide study of single molecule methylation patterns. One of the key goals of mammalian cell-type identity studies, in both normal differentiation and disease, is to locate differential methylation patterns across the genome. We discuss the most desirable characteristics for DML (differentially methylated locus) and DMR (differentially methylated region) detection tools in a genome-wide context and choose a set of statistical methods that fully or partially satisfy these considerations to compare for benchmarking. Our data simulation strategy is both biologically informed-employing distribution parameters derived from large-scale consortium datasets-and thorough. We report DML detection ability with respect to coverage, group methylation difference, sample size, variability and covariate size, both marginally and jointly, and exhaustively with respect to parameter combination. We also benchmark these methods on FDR control and computational time. We use this result to backend and introduce an expanded version of DMRcate: an existing DMR detection tool for microarray data that we have extended to now call DMRs from WGBS data. We compare DMRcate to a set of alternative DMR callers using a similarly realistic simulation strategy. We find DMRcate and RADmeth are the best predictors of DMRs, and conclusively find DMRcate the fastest.
  45. Acta Neuropathol. 2021 Jul 26.
      Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) are three clinically, genetically and neuropathologically overlapping neurodegenerative diseases collectively known as the Lewy body diseases (LBDs). A variety of molecular mechanisms have been implicated in PD pathogenesis, but the mechanisms underlying PDD and DLB remain largely unknown, a knowledge gap that presents an impediment to the discovery of disease-modifying therapies. Transcriptomic profiling can contribute to addressing this gap, but remains limited in the LBDs. Here, we applied paired bulk-tissue and single-nucleus RNA-sequencing to anterior cingulate cortex samples derived from 28 individuals, including healthy controls, PD, PDD and DLB cases (n = 7 per group), to transcriptomically profile the LBDs. Using this approach, we (i) found transcriptional alterations in multiple cell types across the LBDs; (ii) discovered evidence for widespread dysregulation of RNA splicing, particularly in PDD and DLB; (iii) identified potential splicing factors, with links to other dementia-related neurodegenerative diseases, coordinating this dysregulation; and (iv) identified transcriptomic commonalities and distinctions between the LBDs that inform understanding of the relationships between these three clinical disorders. Together, these findings have important implications for the design of RNA-targeted therapies for these diseases and highlight a potential molecular "window" of therapeutic opportunity between the initial onset of PD and subsequent development of Lewy body dementia.
    Keywords:  Alternative splicing; Human brain; Lewy body diseases; Parkinson’s disease; Single-nucleus RNA-sequencing
  46. Methods Mol Biol. 2021 ;2352 127-132
      Human motor neurons are important materials for the research of the pathogenesis and drug discovery of motor neuron diseases. Various methods to generate motor neurons (MNs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by the addition of signaling molecules have been reported. However, they require multiple steps and complicated processes. Here we describe an approach for generating human MNs from ESCs/iPSCs using a single Sendai virus vector encoding three transcription factors-Lhx3, Ngn2, and Isl1. This approach enabled us to generate MNs in one step, adding Sendai virus vector in culture medium. This simple method significantly reduces the efforts to generate MNs, and it provides a useful tool for motor neuron disease research.
    Keywords:  Direct conversion; ESC; Motor neuron; Sendai virus; iPSC
  47. Methods Mol Biol. 2021 ;2352 97-115
      Since the first demonstration of direct dopaminergic neuronal reprogramming, over a dozen methods have been developed to generate induced dopaminergic neurons from various sources of cells. Here, we first present an overview of the different methods to generate induced neurons of a generic type and of different subtypes, with a particular focus on induced dopaminergic neurons generated from human fibroblasts. We then describe a protocol to generate induced dopaminergic neurons from commercially available human fetal lung fibroblasts. These cells could serve for various biomedical application, including regenerative medicine for conditions such as Parkinson's disease.
    Keywords:  Direct reprogramming; Dopaminergic neurons; Human fetal fibroblasts; Induced neurons; Transcription factors; Viral vectors
  48. Methods Mol Biol. 2021 ;2350 69-76
      Super Resolution (SR) microscopy has become a powerful tool to study cellular architecture at the nanometer scale. Single molecule localization microscopy (SMLM) is a method in which fluorophore labels repeatedly switch On and Off ("blink"). Their exact locations are estimated by computing the centers of individual blinks. Therefore, the image quality depends on the density of the detected labels, as well as the accuracy of the estimation of their location. Both are influenced by several factors. Here we present a step-by-step method that optimizes many of these factors to facilitate multicolor imaging.
    Keywords:  Fluorescence; Imaging buffer; Microscopy; Multicolor; OxEA; SMLM; Super resolution
  49. Nat Commun. 2021 07 27. 12(1): 4544
      Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.
  50. Methods Mol Biol. 2021 ;2352 45-55
      Astrocytes play important roles in neurodevelopment and diseases. Previous studies described ways to derive astrocytes from somatic cells by going through iPSC or iNSC/iNPC intermediates. Here we describe a method to directly convert mouse fibroblasts into functional astrocytes using small molecules without transgenes or viral transduction. The direct chemical reprogramming method described in this study provides a more rapid way to derive astrocytes from fibroblasts.
    Keywords:  Chemical reprogramming; Direct conversion; Induced astrocytes; Small molecules; iPSC-derived astrocytes; iPSCs
  51. Methods Mol Biol. 2021 ;2352 73-96
      Progressive aging is a physiological process that represents a central risk factor for the development of several human age-associated chronic diseases, including neurodegenerative diseases. A major focus in biomedical research is the pursuit for appropriate model systems to better model the biology of human aging and the interface between aging and disease mechanisms. Direct conversion of human fibroblasts into induced neurons (iNs) has emerged as a novel technology for the in vitro modeling of age-dependent neurological diseases. Similar to other cellular reprogramming techniques, e.g., iPSC-based cellular reprograming, direct conversion relies on the ectopic overexpression of transcription factors, typically including well-known pioneer factors. However, in contrast to alternative technologies to generate neurons, the entire process of direct conversion bypasses any proliferative or stem cell-like stage, which in fact renders it the unique aptitude of preserving age-associated hallmarks from the initial fibroblast source. In this chapter, we introduce direct conversion as a practical and easy-to-approach disease model for aging and neurodegenerative disease research. A focus here is to provide a stepwise protocol for the efficient and highly reproducible generation of iNs from adult dermal fibroblasts from human donors.
    Keywords:  Aging; Cell reprogramming; Direct conversion; Induced neurons (iNs); Neurodegeneration; Neurological diseases
  52. Methods Mol Biol. 2021 ;2352 133-148
      Astrocytes are essential cells for normal brain functionality and have recently emerged as key players in many neurological diseases. However, the limited availability of human primary astrocytes for cell culture studies hinders our understanding of their physiology and precise role in disease development and progression. Here, we describe a detailed step-by-step protocol to rapidly and efficiently generate functionally mature induced astrocytes (iAs) from human embryonic and induced pluripotent stem cells (hES/iPSCs). Astrocyte induction is accomplished by ectopic lentiviral expression of two gliogenic transcription factors, Sox9 and Nfib. iAs exhibit morphology features as well as gene and protein expression similar to human mature astrocytes and display important astrocytic functions, such as glutamate uptake, propagation of calcium waves, expression of various cytokines after stimulation, and support of synapse formation and function, making them suitable models for studying the role of astrocytes in health and disease. Moreover, we describe a procedure for cryopreservation of iAs for long-term storage or shipping. Finally, we provide the required information needed to set up cocultures with human induced neurons (iNs, also described in this book), generated from hES/iPSCs, to generate cocultures, allowing studies on astrocyte-neuron interactions and providing new insights in astrocyte-associated disease mechanisms.
    Keywords:  Astrocyte-neuron cocultures; Astrocytes; Direct conversion; Reprogramming; Stem cells
  53. FEBS J. 2021 Jul 26.
      Cytoplasmic microbial and host aberrant DNAs act as danger signals and trigger host immune responses. Upon recognition, the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) catalyzes the production of a second messenger 2'3'-cGAMP, which activates endoplasmic reticulum (ER)-associated stimulator of interferon genes (STING) and ultimately leads to the induction of type I interferons and inflammatory genes that collectively initiate host immune defense against microbial invasion. Inappropriate activation or suppression of this signaling pathway has been implicated in the development of some autoimmune diseases, sterile inflammation and cancers. In this review, we describe how the activity of cGAS and STING is regulated by host post-translational modifications and summarize the recent advances of cell-specific cGAS-STING activation and its association in sterile inflammatory diseases. We also discuss key outstanding questions in the field, including how our knowledge of cGAS-STING pathway could be translated into clinical applications.
    Keywords:  STING; autoimmune disease; cGAS; post translational modification; sterile inflammatory disease
  54. Front Mol Biosci. 2021 ;8 703532
      Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.
    Keywords:  NAD; NMNAT2; Sarm1; axon degeneration; innate immunity
  55. J Neuromuscul Dis. 2021 Jul 20.
      BACKGROUND: Leigh syndrome (LS) is the most frequent paediatric clinical presentation of mitochondrial disease. The clinical phenotype of LS is highly heterogeneous. Though historically the treatment for LS is largely supportive, new treatments are on the horizon. Due to the rarity of LS, large-scale interventional studies are scarce, limiting dissemination of information of therapeutic options to the wider scientific and clinical community.OBJECTIVE: We conducted a systematic review of pharmacological therapies of LS following the guidelines for FAIR-compliant datasets.
    METHODS: We searched for interventional studies within and European Clinical trials databases. Randomised controlled trials, observational studies, case reports and case series formed part of a wider MEDLINE search.
    RESULTS: Of the 1,193 studies initially identified, 157 met our inclusion criteria, of which 104 were carried over into our final analysis.Treatments for LS included very few interventional trials using EPI-743 and cysteamine bitartrate. Wider literature searches identified case series and reports of treatments repleting glutathione stores, reduction of oxidative stress and restoration of oxidative phosphorylation.
    CONCLUSIONS: Though interventional randomised controlled trials have begun for LS, the majority of evidence remains in case reports and case series for a number of treatable genes, encoding cofactors or transporter proteins of the mitochondria. Our findings will form part of the international expert-led Solve-RD efforts to assist clinicians initiating treatments in patients with treatable variants of LS.
  56. Front Genet. 2021 ;12 659287
      Most single-nucleotide polymorphisms (SNPs) are located in non-coding regions, but the fraction usually studied is harbored in protein-coding regions because potential impacts on proteins are relatively easy to predict by popular tools such as the Variant Effect Predictor. These tools annotate variants independently without considering the potential effect of grouped or haplotypic variations, often called "multi-nucleotide variants" (MNVs). Here, we used a large RNA-seq dataset to survey MNVs, comprising 382 chicken samples originating from 11 populations analyzed in the companion paper in which 9.5M SNPs- including 3.3M SNPs with reliable genotypes-were detected. We focused our study on in-codon MNVs and evaluate their potential mis-annotation. Using GATK HaplotypeCaller read-based phasing results, we identified 2,965 MNVs observed in at least five individuals located in 1,792 genes. We found 41.1% of them showing a novel impact when compared to the effect of their constituent SNPs analyzed separately. The biggest impact variation flux concerns the originally annotated stop-gained consequences, for which around 95% were rescued; this flux is followed by the missense consequences for which 37% were reannotated with a different amino acid. We then present in more depth the rescued stop-gained MNVs and give an illustration in the SLC27A4 gene. As previously shown in human datasets, our results in chicken demonstrate the value of haplotype-aware variant annotation, and the interest to consider MNVs in the coding region, particularly when searching for severe functional consequence such as stop-gained variants.
    Keywords:  FATP4; MNV; SLC27A4; SNP; rescued stop-gained; variation
  57. Muscle Nerve. 2021 Jul 30.
      Recent development of novel therapies has improved mobility and quality of life for people suffering from inheritable neuromuscular disorders. Despite this progress, the majority of neuromuscular disorders are still incurable, in part due to a lack of predictive models of neuromuscular junction (NMJ) breakdown. Improvement of predictive models of a human NMJ would be transformative in terms of expanding our understanding of the mechanisms that underpin development, maintenance, and disease, and as a testbed with which to evaluate novel therapeutics. Induced pluripotent stem cells (iPSCs) are emerging as a clinically relevant and non-invasive cell source to create human NMJs to study synaptic development and maturation, as well as disease modeling and drug discovery. This review will highlight the recent advances and remaining challenges to generating an NMJ capable of eliciting contraction of stem cell-derived skeletal muscle in vitro. We explore the advantages and shortcomings of traditional NMJ culturing platforms, as well as the pioneering technologies and novel, biomimetic culturing systems currently in use to guide development and maturation of the neuromuscular synapse and extracellular microenvironment. Then, we will explore how this NMJ-in-a-dish can be used to study normal assembly and function of the efferent portion of the neuromuscular arc, and how neuromuscular disease-causing mutations disrupt structure, signaling, and function.
    Keywords:  NMJ; disease modeling; drug discovery; hiPSC; neuromuscular junction; stem cells
  58. Methods Cell Biol. 2021 ;pii: S0091-679X(21)00019-4. [Epub ahead of print]165 187-197
      Exosomes are bi-layered vesicles secreted by the cells in physiological and pathological conditions. They are involved in cell-cell communication facilitating the transfer of functional macromolecules, including DNA, RNA, proteins and lipids. In this chapter, we will focus on specific class of RNA, the microRNAs, that are shuttled from the exosome-producing cells to the recipient cells where they affect biological processes. We will describe the recent methodologies developed to detect and isolate exosomal microRNAs providing a suitable workflow that contributes to quickly expand the field of exosomes-derived microRNAs and their potential use as biomarkers.
    Keywords:  Biomarkers; Cargo; Detection; Exosomes; Extracellular vesicles; Isolation; Methods; Nanovesicles; RNA; exomiR; microRNA
  59. Methods Mol Biol. 2021 ;2319 143-152
      Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.
    Keywords:  Adult cardiomyocytes isolation; Langendorff; Trypsin
  60. Methods Mol Biol. 2021 ;2350 95-104
      In multicellular organisms, most physiological and pathological processes involve an interplay between various cells and molecules that act both locally and systemically. To understand how these complex and dynamic processes occur in time and space, imaging techniques are key. Advances in tissue processing techniques and microscopy now allow us to probe these processes at a large scale and at the same time at a level of detail previously unachievable. Indeed, it is now possible to reliably quantify multiple protein expression levels at single-cell resolution in whole organs using three-dimensional fluorescence imaging techniques. Here we describe a method to prepare adult mouse bone tissue for multiplexed confocal imaging of thick tissue sections. Up to eight different fluorophores can be multiplexed using this technique and spectrally resolved using standard confocal microscopy. The optical clearing method described allows detection of these fluorophores up to a depth of >700 μm in the far-red. Although the method was initially developed for bone tissue imaging, we have successfully applied it to several other tissue types.
    Keywords:  3D imaging; Bone marrow; Confocal; Fluorescence; In situ; Multicolor; Quantitative microscopy; Single cell; Tissue clearing