bims-mitdis 2022-07-17 papers

bims-mitdis

Biomed News

on Mitochondrial disorders

Issue of 2022‒07‒17
27 papers selected by
Catalina Vasilescu
University of Helsinki

Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations.
Mitochondrial heterogeneity and homeostasis through the lens of a neuron.
Mitochondrial Transfusion Improves Mitochondrial Function Through Up-regulation of Mitochondrial Complex II Protein Subunit SDHB in the Hippocampus of Aged Mice.
Novel TOP3A Variant Associated With Mitochondrial Disease: Expanding the Clinical Spectrum of Topoisomerase III Alpha-Related Diseases.
Regulation of Heme Synthesis by Mitochondrial Homeostasis Proteins.
The MitoAging Project: Single nucleotide polymorphisms (SNPs) in mitochondrial genes and their association to longevity.
Decreasing pdzd8-mediated mito-ER contacts improves organismal fitness and mitigates Aβ42 toxicity.
Treatment and Prevention of Pathological Mitochondrial Dysfunction in Retinal Degeneration and in Photoreceptor Injury.
Age-related mitochondrial dysfunction in Parkinson's disease: new insights into the disease pathology.
Feedforward activation of PRKN/parkin.
Mitochondria in neurodegeneration.
The nuclear encoded Cox7c mRNA co-transport with mitochondria along axons via coding-region dependent mechanism.
Base editing in human cells with monomeric DddA-TALE fusion deaminases.
Fluctuating ataxia caused by mitochondrial tRNA (Lys) gene m.8363G > A variant.
Functional analysis of missense DARS2 variants in siblings with leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation.
Characterization of mitochondrial dysfunction due to laser damage by 2-photon FLIM microscopy.
Structure of the nutrient-sensing hub GATOR2.
In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing.
mTORC1 Crosstalk With Stress Granules in Aging and Age-Related Diseases.
Comparative Analysis of Single-Cell RNA Sequencing Platforms and Methods.
A conceptual disease model for quality of life in mitochondrial disease.
A novel TYMP mutation in a family with MNGIE syndrome: Molecular docking, dynamic simulation and computational investigations.
Cardiac-specific overexpression of Ndufs1 ameliorates cardiac dysfunction after myocardial infarction by alleviating mitochondrial dysfunction and apoptosis.
Parkinson's disease risk protein TMEM175 keeps lysosomes running on a proton leak.
Automated design of CRISPR prime editors for 56,000 human pathogenic variants.
Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.
Shackled ribosomes unleashed.

Cell Cycle. 2022 Jul 10. 1-16

Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations.

Cecilie Katrin Kristiansen, Anbin Chen, Lena Elise Høyland, Mathias Ziegler, Gareth John Sullivan, Laurence A Bindoff, Kristina Xiao Liang.

  Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have distinct origins: ESCs are derived from pre-implanted embryos while iPSCs are reprogrammed somatic cells. Both have their own characteristics and lineage specificity, and both are valuable tools for studying human neurological development and disease. Thus far, few studies have analyzed how differences between stem cell types influence mitochondrial function and mitochondrial DNA (mtDNA) homeostasis during differentiation into neural and glial lineages. In this study, we compared mitochondrial function and mtDNA replication in human ESCs and iPSCs at three different stages - pluripotent, neural progenitor and astrocyte. We found that while ESCs and iPSCs have a similar mitochondrial signature, neural and astrocyte derivations manifested differences. At the neural stem cell (NSC) stage, iPSC-NSCs displayed decreased ATP production and a reduction in mitochondrial respiratory chain (MRC) complex IV expression compared to ESC-NSCs. IPSC-astrocytes showed increased mitochondrial activity including elevated ATP production, MRC complex IV expression, mtDNA copy number and mitochondrial biogenesis relative to those derived from ESCs. These findings show that while ESCs and iPSCs are similar at the pluripotent stage, differences in mitochondrial function may develop during differentiation and must be taken into account when extrapolating results from different cell types.Abbreviation: BSA: Bovine serum albumin; DCFDA: 2',7'-dichlorodihydrofluorescein diacetate; DCX: Doublecortin; EAAT-1: Excitatory amino acid transporter 1; ESCs: Embryonic stem cells; GFAP: Glial fibrillary acidic protein; GS: Glutamine synthetase; iPSCs: Induced pluripotent stem cells; LC3B: Microtubule-associated protein 1 light chain 3β; LC-MS: Liquid chromatography-mass spectrometry; mito-ROS: Mitochondrial ROS; MMP: Mitochondrial membrane potential; MRC: Mitochondrial respiratory chain; mtDNA: Mitochondrial DNA; MTDR: MitoTracker Deep Red; MTG: MitoTracker Green; NSCs: Neural stem cells; PDL: Poly-D-lysine; PFA: Paraformaldehyde; PGC-1α: PPAR-γ coactivator-1 alpha; PPAR-γ: Peroxisome proliferator-activated receptor-gamma; p-SIRT1: Phosphorylated sirtuin 1; p-ULK1: Phosphorylated unc-51 like autophagy activating kinase 1; qPCR: Quantitative PCR; RT: Room temperature; RT-qPCR: Quantitative reverse transcription PCR; SEM: Standard error of the mean; TFAM: Mitochondrial transcription factor A; TMRE: Tetramethylrhodamine ethyl ester; TOMM20: Translocase of outer mitochondrial membrane 20.

Keywords:  ESCs; IPSCs; NSCs; astrocytes; mitochondrial biogenesis; mitochondrial function

DOI:  https://doi.org/10.1080/15384101.2022.2092185
Nat Metab. 2022 Jul 11.

Mitochondrial heterogeneity and homeostasis through the lens of a neuron.

Gulcin Pekkurnaz, Xinnan Wang.

Mitochondria are vital organelles with distinct morphological features and functional properties. The dynamic network of mitochondria undergoes structural and functional adaptations in response to cell-type-specific metabolic demands. Even within the same cell, mitochondria can display wide diversity and separate into functionally distinct subpopulations. Mitochondrial heterogeneity supports unique subcellular functions and is crucial to polarized cells, such as neurons. The spatiotemporal metabolic burden within the complex shape of a neuron requires precisely localized mitochondria. By travelling great lengths throughout neurons and experiencing bouts of immobility, mitochondria meet distant local fuel demands. Understanding mitochondrial heterogeneity and homeostasis mechanisms in neurons provides a framework to probe their significance to many other cell types. Here, we put forth an outline of the multifaceted role of mitochondria in regulating neuronal physiology and cellular functions more broadly.

DOI: https://doi.org/10.1038/s42255-022-00594-w
Mol Neurobiol. 2022 Jul 14.

Mitochondrial Transfusion Improves Mitochondrial Function Through Up-regulation of Mitochondrial Complex II Protein Subunit SDHB in the Hippocampus of Aged Mice.

A Adlimoghaddam, T Benson, B C Albensi.

  The mitochondrial theory of aging is characterized by mitochondrial electron transport chain dysfunction. As a hallmark of aging, an increasing number of investigations have attempted to improve mitochondrial function in both aging and age-related disease. Emerging from these attempts, methods involving mitochondrial isolation, transfusion, and transplantation have taken center stage. In particular, mitochondrial transfusion refers to the administration of mitochondria from healthy tissue into the bloodstream or into tissues affected by injury, disease, or aging. In this study, methods of mitochondrial isolation and transfusion were developed and utilized. First, we found a significant decrease (p < 0.05) in the expression of mitochondrial complex proteins (I-V) in aged (12 months old) mouse brain tissue (C57BL/6 mice) in comparison to healthy young brain tissue (1 month old). To investigate whether healthy young mitochondria taken from the liver could improve mitochondrial function in older animals, we intravenously injected mitochondria isolated from young C57BL/6 mice into aged mice from the same strain. This study, for the first time, demonstrates that mitochondrial transfusion significantly (p < 0.05) improves mitochondrial function via the up-regulation of the mitochondrial complex II protein subunit SDHB in the hippocampus of aged mice. This result has identified a role for mitochondrial complex II in the aging process. Therefore, mitochondrial complex II could serve as a putative target for therapeutic interventions against aging. However, more importantly, methods of mitochondrial transfusion should be further tested to treat a variety of human diseases or disorders and to slow down or reverse processes of aging.

Keywords:  Age-related disease; Aging; Bioenergetics; Brain; Complex II; Mitochondrial dysfunction; Mitochondrial transfusion; Neuroscience

DOI:  https://doi.org/10.1007/s12035-022-02937-w
Neurol Genet. 2022 Aug;8(4): e200007

Novel TOP3A Variant Associated With Mitochondrial Disease: Expanding the Clinical Spectrum of Topoisomerase III Alpha-Related Diseases.

Guido Primiano, Alessandra Torraco, Daniela Verrigni, Andrea Sabino, Enrico Bertini, Rosalba Carrozzo, Gabriella Silvestri, Serenella Servidei.

Objectives: Topoisomerase III alpha plays a key role in the dissolution of double Holliday junctions and is required for mitochondrial DNA (mtDNA) replication and maintenance. Sequence variants in the TOP3A gene have been associated with the Bloom syndrome-like disorder and described in an adult patient with progressive external ophthalmoplegia. The purpose of this report is to expand the clinical phenotype of the TOP3A-related diseases and clarify the role of this gene in primary mitochondrial disorders.Methods: A 44-year-old woman was referred to our hospital because of exercise intolerance and creatine kinase increase. Muscle biopsy and a targeted next-generation sequencing (NGS) analysis were performed.
Results: A histopathologic assessment documented a mitochondrial myopathy, and a molecular analysis revealed a novel homozygous variant in the TOP3A gene associated with multiple mtDNA deletions.
Discussion: This case suggests that TOP3A is one of the several nuclear genes associated with mtDNA maintenance disorder and expands the spectrum of its associated phenotypes, ranging from a clinical condition defined Bloom syndrome-like disorder to canonical mitochondrial syndromes.

DOI: https://doi.org/10.1212/NXG.0000000000200007
Front Cell Dev Biol. 2022 ;10 895521

Regulation of Heme Synthesis by Mitochondrial Homeostasis Proteins.

Yvette Y Yien, Mark Perfetto.

  Heme plays a central role in diverse, life-essential processes that range from ubiquitous, housekeeping pathways such as respiration, to highly cell-specific ones such as oxygen transport by hemoglobin. The regulation of heme synthesis and its utilization is highly regulated and cell-specific. In this review, we have attempted to describe how the heme synthesis machinery is regulated by mitochondrial homeostasis as a means of coupling heme synthesis to its utilization and to the metabolic requirements of the cell. We have focused on discussing the regulation of mitochondrial heme synthesis enzymes by housekeeping proteins, transport of heme intermediates, and regulation of heme synthesis by macromolecular complex formation and mitochondrial metabolism. Recently discovered mechanisms are discussed in the context of the model organisms in which they were identified, while more established work is discussed in light of technological advancements.

Keywords:  erythroid; heme; housekeeping protein; mice; mitochondria; model organisms; yeast; zebrafish

DOI:  https://doi.org/10.3389/fcell.2022.895521
Mitochondrion. 2022 Jul 08. pii: S1567-7249(22)00056-3. [Epub ahead of print]

The MitoAging Project: Single nucleotide polymorphisms (SNPs) in mitochondrial genes and their association to longevity.

Verónica Castañedaa, Alissen Haro-Vinueza, Ivonne Salinas, Andrés Caicedod, Miguel Angel Mendez.

  Mitochondrial dysfunction is a major hallmark of aging. Mitochondrial DNA (mtDNA) mutations (inherited or acquired) may cause a malfunction of the respiratory chain (RC), and thus negatively affect cell metabolism and function. In contrast, certain mtDNA single nucleotide polymorphisms (SNPs) may be beneficial to mitochondrial electron transport chain function and the extension of cellular health as well as lifespan. The goal of the MitoAging project is to detect key physiological characteristics and mechanisms that improve mitochondrial function and use them to develop therapies to increase longevity and a healthy lifespan. We chose to perform a systematic literature review (SLR) as a tool to collect key mtDNA SNPs associated with an increase in lifespan. Then validated our results by comparing them to the MitoMap database. Next, we assessed the effect of relevant SNPs on protein stability. A total of 28 SNPs were found in protein coding regions. These SNPs were reported in Japan, China, Turkey, and India. Among the studied SNPs, the C5178A mutation in the ND2 gene of Complex I of the RC was detected in all the reviewed reports except in Uygur Chinese centenarians. Then, we found that G9055A (ATP6 gene) and A10398G (ND3 gene) polymorphisms have been associated with a protective effect against Parkinson's disease (PD). Additionally, C8414T in ATP8 was significantly associated with longevity in three Japanese reports. Interestingly, using MitoMap we found that G9055A (ATP6 gene) was the only SNP promoting longevity not associated with any pathology. The identification of SNPs associated with an increase in lifespan opens the possibility to better understand individual differences regarding a decrease in illness susceptibility and find strategies that contribute to healthy aging.

Keywords:  Mitochondria; SNP; aging; longevity; mitochondrial DNA; mitochondrial protein

DOI:  https://doi.org/10.1016/j.mito.2022.06.008
Life Sci Alliance. 2022 Nov;pii: e202201531. [Epub ahead of print]5(11):

Decreasing pdzd8-mediated mito-ER contacts improves organismal fitness and mitigates Aβ42 toxicity.

Victoria L Hewitt, Leonor Miller-Fleming, Madeleine J Twyning, Simonetta Andreazza, Francesca Mattedi, Julien Prudent, Franck Polleux, Alessio Vagnoni, Alexander J Whitworth.

Mitochondria-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. Here, we identify and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid β42 (Aβ42). Together, our results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.

DOI: https://doi.org/10.26508/lsa.202201531
Biochem Pharmacol. 2022 Jul 11. pii: S0006-2952(22)00262-3. [Epub ahead of print] 115168

Treatment and Prevention of Pathological Mitochondrial Dysfunction in Retinal Degeneration and in Photoreceptor Injury.

Walter H Moos, Douglas V Faller, Ioannis P Glavas, David N Harpp, Natalia Kamperi, Iphigenia Kanara, Krishna Kodukula, Anastasios N Mavrakis, Julie Pernokas, Mark Pernokas, Carl A Pinkert, Whitney R Powers, Konstantina Sampani, Kosta Steliou, Constantin Tamvakopoulos, Demetrios G Vavvas, Robert J Zamboni, Xiaohong Chen.

  Pathological deterioration of mitochondrial function is increasingly linked with multiple degenerative illnesses as a mediator of a wide range of neurologic and age-related chronic diseases, including those of genetic origin. Several of these diseases are rare, typically defined in the United States as an illness affecting fewer than 200,000 people in the U.S. population, or about one in 1,600 individuals. Vision impairment due to mitochondrial dysfunction in the eye is a prominent feature evident in numerous primary mitochondrial diseases and is common to the pathophysiology of many of the familiar ophthalmic disorders, including age-related macular degeneration, diabetic retinopathy, glaucoma and retinopathy of prematurity - a collection of syndromes, diseases and disorders with significant unmet medical needs. Focusing on metabolic mitochondrial pathway mechanisms, including the possible roles of cuproptosis and ferroptosis in retinal mitochondrial dysfunction, we shed light on the potential of α-lipoyl-L-carnitine in treating eye diseases. α-Lipoyl-L-carnitine is a bioavailable mitochondria-targeting lipoic acid prodrug that has shown potential in protecting against retinal degeneration and photoreceptor cell loss in ophthalmic indications.

Keywords:  Cuproptosis; ferroptosis; macular degeneration; mitochondrial disease; ophthalmology; photoreceptor cells; rare diseases; retinal disease; therapeutics; α-lipoic acid

DOI:  https://doi.org/10.1016/j.bcp.2022.115168
Neuroscience. 2022 Jul 12. pii: S0306-4522(22)00350-5. [Epub ahead of print]

Age-related mitochondrial dysfunction in Parkinson's disease: new insights into the disease pathology.

Linchi Rani, Manas Ranjan Sahu, Amal Chandra Mondal.

  Aging is a progressive loss of physiological function that increases risk of disease and death. Among the many factors that contribute to human aging, mitochondrial dysfunction has emerged as one of the most prominent features of the aging process. It has been linked to the development of various age-related pathologies, including Parkinson's disease (PD). Mitochondria has a complex quality control system that ensures mitochondrial integrity and function. Perturbations in these mitochondrial mechanisms have long been linked to various age-related neurological disorders. Even though research has shed light on several aspects of the disease pathology, the underlying mechanism of age-related factors responsible for individuals developing this disease is still unknown. This review article aims to discuss the role of mitochondria in the transition from normal brain aging to pathological brain aging, which leads to the progression of PD. We have discussed the emerging evidence on how age-related disruption of mitochondrial quality control mechanisms contributes to the development of PD-related pathophysiology.

Keywords:  Aging; Mitochondrial aberrations; Mitochondrial quality control mechanism; Parkinson’s disease

DOI:  https://doi.org/10.1016/j.neuroscience.2022.07.007
Autophagy. 2022 Jul 15. 1-2

Feedforward activation of PRKN/parkin.

Rayan Fakih, Véronique Sauvé, Kalle Gehring.

  Parkinson disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the midbrain. The majority of early onset forms of Parkinson disease are a result of autosomal mutations in PRKN (parkin RBR E3 ubiquitin protein ligase) and PINK1 (PTEN induced kinase 1), which together regulate the clearance of damaged mitochondria from cells through selective autophagy of mitochondria (mitophagy). In a pair of recent papers, we characterized a secondary mechanism of activation of PRKN by PINK1 that is responsible for approximately a quarter of mitophagy in a cellular model. Our deepening understanding of PRKN-PINK1 signaling affords hope for the development of small molecule therapeutics for the treatment of Parkinson disease.

Keywords:  Autophagy; Parkinson disease; kinase; mitochondria; neurodegenerative disease; protein phosphorylation; ubiquitin

DOI:  https://doi.org/10.1080/15548627.2022.2100615
Curr Opin Physiol. 2022 Apr;pii: 100532. [Epub ahead of print]26

Mitochondria in neurodegeneration.

Charleen T Chu.

  The brain is one of the most energetically demanding tissues in the human body, and mitochondrial pathology is strongly implicated in chronic neurodegenerative diseases. In contrast to acute brain injuries in which bioenergetics and cell death play dominant roles, studies modeling familial neurodegeneration implicate a more complex and nuanced relationship involving the entire mitochondrial life cycle. Recent literature on mitochondrial mechanisms in Parkinson's disease, Alzheimer's disease, frontotemporal dementia, Huntington's disease, and amyotrophic lateral sclerosis is reviewed with an emphasis on mitochondrial quality control, transport and synaptodendritic calcium homeostasis. Potential neuroprotective interventions include targeting the mitochondrial kinase PTEN-induced kinase 1 (PINK1), which plays a role in regulating not only multiple facets of mitochondrial biology, but also neuronal morphogenesis and dendritic arborization.

Keywords:  Alzheimer disease; PTEN-induced kinase 1 (PINK1); Parkinson disease; drug discovery; mitochondrial calcium; mitochondrial proteases; mitophagy; synaptic degeneration

DOI:  https://doi.org/10.1016/j.cophys.2022.100532
J Cell Sci. 2022 Jul 14. pii: jcs.259436. [Epub ahead of print]

The nuclear encoded Cox7c mRNA co-transport with mitochondria along axons via coding-region dependent mechanism.

Bar Cohen, Topaz Altman, Adi Golani-Armon, Anca F Savulescu, Amjd Ibraheem, Musa M Mhlanga, Eran Perlson, Yoav S Arava.

  Nuclear encoded mitochondrial protein mRNAs have been found to be localized and locally translated within neuronal processes. However, the transport mechanism of those mRNAs to distal locations is not fully understood. Here, we describe axonal co-transport of Cox7c with mitochondria. Fractionation analysis and smFISH assay revealed that endogenous mRNA encoding Cox7c is preferentially associated with mitochondria from a neuronal cell line and within primary motor neuron axons, while other mRNAs, which do not encode mitochondrial protein are much less associated. Live cell imaging of MS2-tagged Cox7c mRNA further confirmed the preferential colocalization and co-transport of Cox7c mRNA with mitochondria in motor neuron axons. Intriguingly, the coding region, rather than the 3' UTR, was the key domain for the cotransport. Our results reveal that Cox7c mRNA can be transported with mitochondria along significant distances and its coding region is a major recognition feature. This is consistent with the idea that mitochondria can play a vital role in spatial regulation of the axonal transcriptome at distant neuronal sites.

Keywords:  Axonal transport; Cox7c; Mitochondria; mRNA localization; mRNA transport

DOI:  https://doi.org/10.1242/jcs.259436
Nat Commun. 2022 Jul 12. 13(1): 4038

Base editing in human cells with monomeric DddA-TALE fusion deaminases.

Young Geun Mok, Ji Min Lee, Eugene Chung, Jaesuk Lee, Kayeong Lim, Sung-Ik Cho, Jin-Soo Kim.

Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddAtox, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddAtox variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.

DOI: https://doi.org/10.1038/s41467-022-31745-y
Neurol Sci. 2022 Jul 12.

Fluctuating ataxia caused by mitochondrial tRNA (Lys) gene m.8363G > A variant.

Ying Zhao, Bing Zhao, Kunqian Ji, Chuanzhu Yan.

DOI: https://doi.org/10.1007/s10072-022-06246-x
Mol Genet Metab. 2022 Jul 05. pii: S1096-7192(22)00361-4. [Epub ahead of print]136(4): 260-267

Functional analysis of missense DARS2 variants in siblings with leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation.

Parith Wongkittichote, Martina Magistrati, Joshua S Shimony, Christopher D Smyser, Seyed Ali Fatemi, Amena S Fine, Emanuele Bellacchio, Cristina Dallabona, Marwan Shinawi.

  Biallelic pathogenic variants in the nuclear gene DARS2 (MIM# 610956), encoding the mitochondrial enzyme aspartyl-tRNA synthetase (MT-ASPRS) cause leukoencephalopathy with Brain Stem and Spinal Cord Involvement and Lactate Elevation (LBSL) (MIM# 611105), a neurometabolic disorder characterized by progressive ataxia, spasticity, developmental arrest or regression and characteristic brain MRI findings. Most patients exhibit a slowly progressive disease course with motor deterirartion that begins in childhood or adolescence, but can also occasionaly occur in adulthood. More severe LBSL presentations with atypical brain MRI findings have been recently described. Baker's yeast orthologue of DARS2, MSD1, is required for growth on oxidative carbon sources. A yeast with MSD1 knockout (msd1Δ) demonstrated a complete lack of oxidative growth which could be rescued by wild-type MSD1 but not MSD1 with pathogenic variants. Here we reported two siblings who exhibited developmental regression and ataxia with different age of onset and phenotypic severity. Exome sequencing revealed 2 compound heterozygous missense variants in DARS2: c.473A>T (p.Glu158Val) and c.829G>A (p.Glu277Lys); this variant combination has not been previously reported. The msd1Δ yeast transformed with plasmids expressing p.Glu259Lys, equivalent to human p.Glu277Lys, showed complete loss of oxidative growth and oxygen consumption, while the strain carrying p.Gln137Val, equivalent to human p.Glu158Val, showed a significant reduction of oxidative growth, but a residual ability to grow was retained. Structural analysis indicated that p.Glu158Val may interfere with protein binding of tRNAAsp, while p.Glu277Lys may impact both homodimerization and catalysis of MT-ASPRS. Our data illustrate the utility of yeast model and in silico analysis to determine pathogenicity of DARS2 variants, expand the genotypic spectrum and suggest intrafamilial variability in LBSL.

Keywords:  Aspartyl-tRNA synthetase; DARS2; Leukodystrophy; Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation; Mitochondrial disorder

DOI:  https://doi.org/10.1016/j.ymgme.2022.07.002
Sci Rep. 2022 Jul 13. 12(1): 11938

Characterization of mitochondrial dysfunction due to laser damage by 2-photon FLIM microscopy.

Shagufta Rehman Alam, Horst Wallrabe, Kathryn G Christopher, Karsten H Siller, Ammasi Periasamy.

Mitochondria are the central organelles in cellular bio-energetics with key roles to play in energy metabolism and cell fate decisions. Fluorescence Lifetime Imaging microscopy (FLIM) is used to track metabolic changes by following the intrinsic co-enzymes NAD(P)H and FAD, present in metabolic pathways. FLIM records-lifetimes and the relative fractions of free (unbound) and bound states of NAD(P)H and FAD are achieved by multiphoton excitation of a pulsed femto-second infra-red laser. Optimization of multiphoton laser power levels is critical to achieve sufficient photon counts for correct lifetime fitting while avoiding phototoxic effects. We have characterized two photon (2p) laser induced changes at the intra-cellular level, specifically in the mitochondria, where damage was assessed at rising 2p laser average power excitation. Our results show that NAD(P)H-a2%-the lifetime-based enzyme bound fraction, an indicator of mitochondrial OXPHOS activity is increased by rising average power, while inducing changes in the mitochondria at higher power levels, quantified by different probes. Treatment response tracked by means of NAD(P)H-a2% can be confounded by laser-induced damage producing the same effect. Our study demonstrates that 2p-laser power optimization is critical by characterizing changes in the mitochondria at increasing laser average power.

DOI: https://doi.org/10.1038/s41598-022-15639-z
Nature. 2022 Jul 13.

Structure of the nutrient-sensing hub GATOR2.

Max L Valenstein, Kacper B Rogala, Pranav V Lalgudi, Edward J Brignole, Xin Gu, Robert A Saxton, Lynne Chantranupong, Jonas Kolibius, Jan-Philipp Quast, David M Sabatini.

Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients1,2. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively3-5. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes6. The scaffold orients the WD40 β-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.

DOI: https://doi.org/10.1038/s41586-022-04939-z
Nucleic Acids Res. 2022 Jul 12. pii: gkac581. [Epub ahead of print]

In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing.

Anna Karlowicz, Andrzej B Dubiel, Jolanta Czerwinska, Adela Bledea, Piotr Purzycki, Marta Grzelewska, Ryan J McAuley, Roman J Szczesny, Gabriela Brzuska, Ewelina Krol, Bartosz Szczesny, Michal R Szymanski.

The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5'-3' exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria.

DOI: https://doi.org/10.1093/nar/gkac581
Front Aging. 2021 ;2 761333

mTORC1 Crosstalk With Stress Granules in Aging and Age-Related Diseases.

Marti Cadena Sandoval, Alexander Martin Heberle, Ulrike Rehbein, Cecilia Barile, José Miguel Ramos Pittol, Kathrin Thedieck.

  The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a master regulator of metabolism and aging. A complex signaling network converges on mTORC1 and integrates growth factor, nutrient and stress signals. Aging is a dynamic process characterized by declining cellular survival, renewal, and fertility. Stressors elicited by aging hallmarks such as mitochondrial malfunction, loss of proteostasis, genomic instability and telomere shortening impinge on mTORC1 thereby contributing to age-related processes. Stress granules (SGs) constitute a cytoplasmic non-membranous compartment formed by RNA-protein aggregates, which control RNA metabolism, signaling, and survival under stress. Increasing evidence reveals complex crosstalk between the mTORC1 network and SGs. In this review, we cover stressors elicited by aging hallmarks that impinge on mTORC1 and SGs. We discuss their interplay, and we highlight possible links in the context of aging and age-related diseases.

Keywords:  MTOR; aging hallmarks; amino acids; autophagy; cellular signaling; insulin; stress; stress granules (SGs)

DOI:  https://doi.org/10.3389/fragi.2021.761333
J Biomol Tech. 2021 Dec 15. pii: 3fc1f5fe.3eccea01. [Epub ahead of print]32(4):

Comparative Analysis of Single-Cell RNA Sequencing Platforms and Methods.

John M Ashton, Hubert Rehrauer, Jason Myers, Jacqueline Myers, Michelle Zanche, Malene Balys, Jonathan Foox, Chistopher E Mason, Robert Steen, Marcy Kuentzel, Catharine Aquino, Natàlia Garcia-Reyero, Sridar V Chittur.

  Single-cell RNA sequencing (scRNA-seq) offers great new opportunities for increasing our understanding of complex biological processes. In particular, development of an accurate Human Cell Atlas is largely dependent on the rapidly advancing technologies and molecular chemistries employed in scRNA-seq. These advances have already allowed an increase in throughput for scRNA-seq from 96 to 80,000 cells on a single instrument run by capturing cells within nanoliter droplets. Although this increase in throughput is critical for many experimental questions, a thorough comparison between microfluidic-based, plate-based, and droplet-based technologies or between multiple available platforms utilizing these technologies is largely lacking. Here, we report scRNA-seq data from SUM149PT cells treated with the histone deacetylase inhibitor trichostatin A versus untreated controls across several scRNA-seq platforms (Fluidigm C1, WaferGen iCell8, 10x Genomics Chromium Controller, and Illumina/BioRad ddSEQ). The primary goal of this project was to demonstrate RNA sequencing methods for profiling the ultra-low amounts of RNA present in individual cells, and this report discusses the results of the study, as well as technical challenges and lessons learned and present general guidelines for best practices in sample preparation and analysis.

Keywords:  RNA-seq; platforms; single cell

DOI:  https://doi.org/10.7171/3fc1f5fe.3eccea01
Orphanet J Rare Dis. 2022 Jul 15. 17(1): 263

A conceptual disease model for quality of life in mitochondrial disease.

Kim F E van de Loo, Nander T van Zeijl, José A E Custers, Mirian C H Janssen, Christianne M Verhaak.

  BACKGROUND: Previous studies in patients with a mitochondrial disease (MD) highlight the high prevalence of cognitive impairments, fatigue, depression, and a lower quality of life (QoL). The relationship with biological and physiological factors remains complex. The aim of this study is to investigate the status of and interrelationships between biological and physiological functioning, cognitive functioning as well as fatigue, depression, societal participation, health perceptions, and QoL, by using the Wilson and Cleary conceptual disease model, adapted to MD.METHODS: Patients with a genetically confirmed MD were included. The following health concepts in MD were investigated according to the conceptual model: (1) Biological and physiological: disease manifestation (Newcastle Mitochondrial Disease Adult Scale), (2) Symptom status: cognitive functioning, patient reported fatigue and depressive symptoms, (3) Functional health: societal participation, (4) Patient reported health perceptions, and (5) Overall QoL. Data were compared to healthy normative data and/or data from other patient groups. Correlations as well as a hierarchical regression analysis were performed to assess the relations between the different levels of health concepts in the conceptual model.
RESULTS: Of the 95 included patients, 42% had a severe disease manifestation. Comparable or worse than normative data and other patient groups, 35% reported cognitive impairments, 80% severe fatigue, and 27% depressive symptoms. Patients experienced impairments in societal participation and QoL. Disease manifestation was significantly correlated with cognitive functioning, societal participation, physical functioning and overall QoL, but not with fatigue or depressive symptoms. Almost all outcome measures regarding functional health, health perceptions and QoL were correlated with symptom status variables. Overall QoL was significantly predicted by fatigue and physical functioning.
CONCLUSIONS: Symptom status is related to the functional health, health perceptions and QoL in patients with MD. Moreover, fatigue and physical functioning are important contributors to the overall QoL of MD patients. In order to provide adequate patient care it is important to have a broad view on patients' functioning, not only by providing a proper clinical assessment, but also to screen for symptom status; cognitive functioning, fatigue and depression.

Keywords:  Cognitive functioning; Depressive symptoms; Disease manifestation; Fatigue; Mental health; Mitochondrial disease; Quality of life; Societal participation; Wilson and Cleary model

DOI:  https://doi.org/10.1186/s13023-022-02411-9
Int J Dev Neurosci. 2022 Jul 15.

A novel TYMP mutation in a family with MNGIE syndrome: Molecular docking, dynamic simulation and computational investigations.

Marwa Ammar, Wajdi Safi, Abdelaziz Tlili, Olfa Alila-Fersi, Fakher Frikha, Jihen Chouchen, Fatma Mnif, Marwa Kharrat, Marwa Maalej, Rahma Felhi, Mohamed Abid, Mouna Mnif-Feki, Faten Hadj Kacem, Faiza Fakhfakh, Emna Mkaouar-Rebai.

  Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM 603041) is a rare inherited metabolic disorder mostly caused by mutations in TYMP gene encoding thymidine phosphorylase (TP) protein that affects the mitochondrial nucleotide metabolism. TP, functionally active as a homodimer, is involved in the salvage pathway of pyrimidine nucleosides. MNGIE-like syndrome having an overlapping phenotype of MNGIE was also described and has been associated with mutations in POLG and RRM2B genes. In the present study, we report the molecular investigation of a consanguineous family including two patients with clinical features suggestive of MNGIE syndrome. Bioinformatics analyses were carried out in addition to mtDNA deletion screening and copy number quantification in the blood of the two patients. Whole exome sequencing and Sanger sequencing analyses revealed the segregation in the affected family a novel mutation c.1205T>A (p.L402Q) within the exon 9 of the TYMP gene. In addition, mtDNA analysis revealed the absence of mtDNA deletions and a decrease of the copy number in the blood of the two patients of the studied family. The p.Leu402Gln mutation was located in a conserved amino acid within the α/β domain of the TP protein and several software supported its pathogenicity. In addition, and based on docking and molecular dynamic simulation analyses, results revealed that L402Q caused a conformational change in TP mutated structure and could therefore alter its flexibility and stability. These changes prevent also the formation of stable homodimer leading to non-functional protein with partial or complete loss of its catalytic activity.

Keywords:  MNGIE syndrome; TYMP gene; WES; molecular docking; molecular dynamic simulation; mtDNA depletion

DOI:  https://doi.org/10.1002/jdn.10215
Exp Mol Med. 2022 Jul 11.

Cardiac-specific overexpression of Ndufs1 ameliorates cardiac dysfunction after myocardial infarction by alleviating mitochondrial dysfunction and apoptosis.

Bingchao Qi, Liqiang Song, Lang Hu, Dong Guo, Gaotong Ren, Tingwei Peng, Mingchuan Liu, Yexian Fang, Chunyu Li, Mingming Zhang, Yan Li.

Myocardial infarction (MI) is the leading cause of premature death among adults. Cardiomyocyte death and dysfunction of the remaining viable cardiomyocytes are the main pathological factors of heart failure after MI. Mitochondrial complexes are emerging as critical mediators for the regulation of cardiomyocyte function. However, the precise roles of mitochondrial complex subunits in heart failure after MI remain unclear. Here, we show that NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) expression is decreased in the hearts of heart failure patients and mice with myocardial infarction. Furthermore, we found that cardiac-specific Ndufs1 overexpression alleviates cardiac dysfunction and myocardial fibrosis in the healing phase of MI. Our results demonstrated that Ndufs1 overexpression alleviates MI/hypoxia-induced ROS production and ROS-related apoptosis. Moreover, upregulation of Ndufs1 expression improved the reduced activity of complex I and impaired mitochondrial respiratory function caused by MI/hypoxia. Given that mitochondrial function and cardiomyocyte apoptosis are closely related to heart failure after MI, the results of this study suggest that targeting Ndufs1 may be a potential therapeutic strategy to improve cardiac function in patients with heart failure.

DOI: https://doi.org/10.1038/s12276-022-00800-5
Nat Struct Mol Biol. 2022 Jul;29(7): 626

Parkinson's disease risk protein TMEM175 keeps lysosomes running on a proton leak.

Carolina N Perdigoto.

DOI: https://doi.org/10.1038/s41594-022-00809-4
iScience. 2021 Nov 19. 24(11): 103380

Automated design of CRISPR prime editors for 56,000 human pathogenic variants.

John A Morris, Jahan A Rahman, Xinyi Guo, Neville E Sanjana.

  Prime editors (PEs) are clustered regularly interspaced short palindromic repeats (CRISPR)-based genome engineering tools that can introduce precise base-pair edits. We developed an automated pipeline to correct (therapeutic editing) or introduce (disease modeling) human pathogenic variants from ClinVar that optimizes the design of several RNA constructs required for prime editing and avoids predicted off-targets in the human genome. However, using optimal PE design criteria, we find that only a small fraction of these pathogenic variants can be targeted. Through the use of alternative Cas9 enzymes and extended templates, we increase the number of targetable pathogenic variants from 32,000 to 56,000 variants and make these pre-designed PE constructs accessible through a web-based portal (http://primeedit.nygenome.org). Given the tremendous potential for therapeutic gene editing, we also assessed the possibility of developing universal PE constructs, finding that common genetic variants impact only a small minority of designed PEs.

Keywords:  Biotechnology; Computational bioinformatics

DOI:  https://doi.org/10.1016/j.isci.2021.103380
CRISPR J. 2022 Jul 12.

Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.

Taylor L Mighell, Andrew Nishida, Brendan L O'Connell, Caitlin V Miller, Sally Grindstaff, Casey A Thornton, Andrew C Adey, Daniel Doherty, Brian J O'Roak.

Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.

DOI: https://doi.org/10.1089/crispr.2021.0140
Nat Chem Biol. 2022 Jul 14.

Shackled ribosomes unleashed.

Aleksandra Filipovska, Oliver Rackham.

DOI: https://doi.org/10.1038/s41589-022-01020-8