Search

Chunyan Wang,1,2,* Zhengyun Chen,1,* Xianlei Zhao,2,* Cuicui Lin,2 Shenghui Hong,3 Yuhan Lou,2 Xiaomeng Shi,1 Mengdan Zhao,1 Xiaohang Yang,1,2 Min-Xin Guan,2 Yongmei Xi1,2 1The Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310006, People’s Republic of China; 2Institute of Genetics, Zhejiang University; Department of Human Genetics, Zhejiang University School of Medicine, Zhejiang Provincial Key Laboratory of Genetic & Developmental Disorders, Hangzhou, Zhejiang, 310058, People’s Republic of China; 3Laboratory Animal Center of Zhejiang University, Hangzhou, Zhejiang, 310001, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yongmei Xi; Min-Xin Guan Tel +86-0571-88206623; +86-0571-88981371Email xyyongm@zju.edu.cn; gminxin88@zju.edu.cnIntroduction: Endometriosis (EMs) is associated with severe chronic pelvic pain and infertility and the development of improved EMs treatment options is an ongoing focus. In this study, we investigated the effects of resveratrol on EMs and analyzed transcriptional changes in the lesions of model rats before and after resveratrol treatment.Methods: We established arat model of endometriosis through the trans-implantation of endometrial fragments to the peritoneal wall and then used resveratrol as treatment. We then analyzed the results using RNA sequencing of the lesion tissues of each of the model rats before resveratrol treatment and the reduced lesion tissues after the treatment. Examinations of anatomy, biochemistry, immunohistochemical staining and flow cytometry examinations were also conducted. Other trans-implanted rats were also given sham treatments as sham-treatment control and other untrans-implanted rats served as sham-operation controls.Results: In addition to the obvious lesions observed in the model rats, there were significant differences in the glucose tolerance, macrophage M1/M2 polarization, and adipocyte sizes between the treated model rats and sham (control) rats. Resveratrol treatment in the model rats showed significant efficacy and positive therapeutic effect. Transcriptional analysis showed that the effects of resveratrol on the endometriosis model rats were manifested by alterations in the PPAR, insulin resistance, MAPK and PI3K/Akt signaling pathways. Correspondingly, changes in PPARγ activation, M1/M2 polarization and lipid metabolism were also detected after resveratrol treatment.Discussion: Our study revealed that resveratrol treatment displayed efficient therapeutic effects for EMs model rats, probably through its important roles in anti-inflammation, immunoregulation and lipid-related metabolism regulation.Keywords: endometriosis, resveratrol, RNA-seq, glucose tolerance, PPAR&#x03B3

The degeneration of retinal ganglion cells (RGC) due to mitochondrial dysfunctions manifests optic neuropathy. However, the molecular components of RGC linked to optic neuropathy manifestations remain largely unknown. Here, we identified a novel optic atrophy-causative CRYAB gene encoding a highly conserved major lens protein acting as mitochondrial chaperone and possessing anti-apoptotic activities. The heterozygous CRYAB mutation (c.313G>A, p. Glu105Lys) was cosegregated with autosomal dominant inheritance of optic atrophy in 3 Chinese families. The p.E105K mutation altered the structure and function of CRYAB, including decreased stability, reduced formation of oligomers and decreasing chaperone activity. Coimmunoprecipitation indicated that the p.E105K mutation reduced the interaction of CRYAB with apoptosis-associated cytochrome c and VDAC. The cell lines carrying the p.E105K mutation displayed promoting apoptosis, defective assembly, stability and activities of oxidative phosphorylation system and imbalance of mitochondrial dynamics. Involvement of CRYAB in optic atrophy was confirmed by phenotypic evaluations of Cryabp.E105K knock-in mice. These mutant mice exhibited ocular lesions including changing intra-retina layers, degeneration of RGCs, photoreceptor deficits and abnormal retinal vasculature. Furthermore, Cryab-deficient mice displayed elevated apoptosis and mitochondrial dysfunctions. Our findings provide new insight of pathophysiology of optic atrophy arising from RGC degeneration caused by CRYAB deficiency-induced elevated apoptosis and mitochondrial dysfunctions.

Leber's hereditary optic neuropathy (LHON), a maternally inherited ocular disease, is predominantly caused by mitochondrial DNA (mtDNA) mutations. Mitochondrial tRNA variants are hypothesized to amplify the pathogenic impact of three primary mutations. However, the exact mechanisms remained unclear. In the present study, the synergistic effect of the tRNA Glu 14693A > G and ND6 14484T > C mutations in three Chinese families affected by LHON is investigated. The m.14693A > G mutation nearly abolishes the pseudouridinylation at position 55 of tRNA Glu , leading to structural abnormalities, decreased stability, aberrant mitochondrial protein synthesis, and increased autophagy. In contrast, the ND6 14484T > C mutation predominantly impairs complex I function, resulting in heightened apoptosis and virtually no induction of mitochondrial autophagy compared to control cell lines. The presence of dual mutations in the same cell lines exhibited a coexistence of both upregulated cellular stress responses to mitochondrial damage, indicating a scenario of autophagy and mutation dysregulation within these dual-mutant cell lines. The data proposes a novel hypothesis that mitochondrial tRNA gene mutations generally lead to increased mitochondrial autophagy, while mutations in genes encoding mitochondrial proteins typically induce apoptosis, shedding light on the intricate interplay between different genetic factors in the manifestation of LHON., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

We present SpliceTransformer (SpTransformer), a deep-learning framework that predicts tissue-specific RNA splicing alterations linked to human diseases based on genomic sequence. SpTransformer outperforms all previous methods on splicing prediction. Application to approximately 1.3 million genetic variants in the ClinVar database reveals that splicing alterations account for 60% of intronic and synonymous pathogenic mutations, and occur at different frequencies across tissue types. Importantly, tissue-specific splicing alterations match their clinical manifestations independent of gene expression variation. We validate the enrichment in three brain disease datasets involving over 164,000 individuals. Additionally, we identify single nucleotide variations that cause brain-specific splicing alterations, and find disease-associated genes harboring these single nucleotide variations with distinct expression patterns involved in diverse biological processes. Finally, SpTransformer analysis of whole exon sequencing data from blood samples of patients with diabetic nephropathy predicts kidney-specific RNA splicing alterations with 83% accuracy, demonstrating the potential to infer disease-causing tissue-specific splicing events. SpTransformer provides a powerful tool to guide biological and clinical interpretations of human diseases., (© 2024. The Author(s).)

Defects in mitochondrial RNA metabolism have been linked to sensorineural deafness that often occurs as a consequence of damaged or deficient inner ear hair cells. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNA Phe 593T > C mutation that changed a highly conserved uracil to cytosine at position 17 of the DHU-loop. The m.593T > C mutation altered tRNA Phe structure and function, including increased melting temperature, resistance to S1 nuclease-mediated digestion, and conformational changes. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced by decreases in levels of ND1, ND5, CYTB, CO1, and CO3 harboring higher numbers of phenylalanine. These alterations resulted in aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, III, IV, and intact supercomplexes overall. Furthermore, we found that the m.593T > C mutation caused markedly diminished membrane potential, and increased the production of reactive oxygen species in the mutant cell lines carrying the m.593T > C mutation. These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance via increasing fission with abnormal mitochondrial morphology. Excessive fission impaired the process of autophagy including the initiation phase, formation, and maturation of the autophagosome. In particular, the m.593T > C mutation upregulated the PARKIN-dependent mitophagy pathway. These alterations promoted an intrinsic apoptotic process for the removal of damaged cells. Our findings provide critical insights into the pathophysiology of maternally inherited deafness arising from tRNA mutation-induced defects in mitochondrial and cellular integrity., Competing Interests: Conflict of interest All authors declare that they have no conflicts of interest with contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria are dynamic organelles in cellular metabolism and physiology. Mitochondrial DNA (mtDNA) mutations are associated with a broad spectrum of clinical abnormalities. However, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we demonstrated that mt-tRNA Met 4435A>G mutation-induced nucleotide modification deficiency dysregulated the expression of nuclear genes involved in cytosolic proteins involved in oxidative phosphorylation system (OXPHOS) and impaired the assemble and integrity of OXPHOS complexes. These dysfunctions caused mitochondrial dynamic imbalance, thereby increasing fission and decreasing fusion. Excessive fission impaired the process of autophagy including initiation phase, formation, and maturation of autophagosome. Strikingly, the m.4435A>G mutation upregulated the PARKIN dependent mitophagy pathways but downregulated the ubiquitination-independent mitophagy. These alterations promoted intrinsic apoptotic process for the removal of damaged cells. Our findings provide new insights into mechanism underlying deficient tRNA posttranscription modification regulated intracellular signaling related to the mitochondrial and cellular integrity., Competing Interests: All authors declare no competing interests., (© 2024 The Author(s).)

Mitochondria are essential organelles for cellular metabolism and physiology in eukaryotic cells. Human mitochondria have their own genome (mtDNA), which is maternally inherited with 37 genes, encoding 13 polypeptides for oxidative phosphorylation, and 22 tRNAs and 2 rRNAs for translation. mtDNA mutations are associated with a wide spectrum of degenerative and neuromuscular diseases. However, the pathophysiology of mitochondrial diseases, especially for threshold effect and tissue specificity, is not well understood and there is no effective treatment for these disorders. Especially, the lack of appropriate cell and animal disease models has been significant obstacles for deep elucidating the pathophysiology of maternally transmitted diseases and developing the effective therapy approach. The use of human induced pluripotent stem cells (iPSCs) derived from patients to obtain terminally differentiated specific lineages such as inner ear hair cells is a revolutionary approach to deeply understand pathogenic mechanisms and develop the therapeutic interventions of mitochondrial disorders. Here, we review the recent advances in patients-derived iPSCs as ex vivo models for mitochondrial diseases. Those patients-derived iPSCs have been differentiated into specific targeting cells such as retinal ganglion cells and eventually organoid for the disease modeling. These disease models have advanced our understanding of the pathophysiology of maternally inherited diseases and stepped toward therapeutic interventions for these diseases., (© 2023. National Science Council of the Republic of China (Taiwan).)

Polaron formation is ubiquitous in polarized materials, but severely hampers carrier transport for which effective controlling methods are urgently needed. Here, we show that laser-controlled coherent phonon excitation enables orders of magnitude enhancement of carrier mobility via accelerating polaron transport in a prototypical material, lithium peroxide (Li 2 O 2 ). The selective excitation of specific phonon modes, whose vibrational pattern directly overlap with the polaronic lattice deformation, can remarkably reduce the energy barrier for polaron hopping. The strong nonadiabatic couplings between the electronic and ionic subsystem play a key role in triggering the migration of polaron, via promoting phonon-phonon scattering in q space within sub-picoseconds. These results extend our understanding of polaron transport dynamics to the nonequilibrium regime and allow for optoelectronic devices with ultrahigh on-off ratio and ultrafast responsibility, competitive with those of state-of-the-art devices fabricated based on free electron transport.

Background: Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mutations in mitochondrial DNA. However, there is no effective treatment for this disease. LHON-linked ND6 14484T > C (p.M64V) mutation caused complex I deficiency, diminished ATP production, increased production of reactive oxygen species (ROS), elevated apoptosis, and impaired mitophagy. Here, we investigated if the allotopic expression of human mitochondrial ND6 transgene corrected the mitochondrial dysfunctions due to LHON-associated m.14484T > C mutation., Methods: Nucleus-versions of ND6 was generated by changing 6 non-universal codons with universal codons and added to mitochondrial targeting sequence of COX8. Stable transfectants were generated by transferring human ND6 cDNA expressed in a pCDH-puro vector into mutant cybrids carrying the m.14484T > C mutation and control cybrids. The effect of allotopic expression of ND6 on oxidative phosphorylation (OXPHOS) was evaluated using Blue Native gel electrophoresis and extracellular flux analyzer. Assessment of ROS production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analyses for apoptosis and mitophagy were undertaken via flow cytometry, TUNEL and immunofluorescence assays., Results: The transfer of human ND6 into the cybrids carrying the m.14484T > C mutation raised the levels of ND6, ND1 and ND4L but did not change the levels of other mitochondrial proteins. The overexpression of ND6 led to 20~23% increases in the assembly and activity of complex I, and ~ 53% and ~ 33% increases in the levels of mitochondrial ATP and ΔΨm in the mutant cybrids bearing m.14484T > C mutation. Furthermore, mutant cybrids with overexpression of ND6 exhibited marked reductions in the levels of mitochondrial ROS. Strikingly, ND6 overexpression markedly inhibited the apoptosis process and restored impaired mitophagy in the cells carrying m.14484T > C mutation. However, overexpression of ND6 did not affect the ND6 level and mitochondrial functions in the wild-type cybrids, indicating that this ND6 level appeared to be the maximum threshold level to maintain the normal cell function., Conclusion: We demonstrated that allotopic expression of nucleus-versions of ND6 restored complex I, apoptosis and mitophagy deficiencies caused by the m.14484T > C mutation. The restoration of m.14484T > C mutation-induced mitochondrial dysfunctions by overexpression of ND6 is a step toward therapeutic interventions for LHON and mitochondrial diseases., (© 2023. National Science Council of the Republic of China (Taiwan).)

Introduction: The pathogenic gene CDH23 plays a pivotal role in tip links, which is indispensable for mechanoelectrical transduction in the hair cells. However, the underlying molecular mechanism and signal regulatory networks that influence deafness is still largely unknown., Methods: In this study, a congenital deafness family, whole exome sequencing revealed a new mutation in the pathogenic gene CDH23 , subsequently; the mutation has been validated using Sanger sequencing method. Then CRISPR/Cas9 technology was employed to knockout zebrafish cdh23 gene. Startle response experiment was used to compare with wide-type, the response to sound stimulation between wide-type and cdh23 -/- . To further illustrate the molecular mechanisms underlying congenital deafness, comparative transcriptomic profiling and multiple bioinformatics analyses were performed., Results: The YO-PRO-1 assay result showed that in cdh23 deficient embryos, the YO-PRO-1 signal in inner ear and lateral line neuromast hair cells were completely lost. Startle response experiment showed that compared with wide-type, the response to sound stimulation decreased significantly in cdh23 mutant larvae. Comparative transcriptomic showed that the candidate genes such as atp1b2b and myof could affect hearing by regulating ATP production and purine metabolism in a synergetic way with cdh23 . RT-qPCR results further confirmed the transcriptomics results. Further compensatory experiment showed that ATP treated cdh23 -/- embryos can partially recover the mutant phenotype., Conclusion: In conclusion, our study may shed light on deciphering the principal mechanism and provide a potential therapeutic method for congenital hearing loss under the condition of CDH23 mutation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JW declared a shared affiliation with the author M-xG to the handling editor at the time of review., (Copyright © 2023 Yang, Xie, Dong, Wang, Zhu, Wang, Wu, Lai, Tao, Guan, Chen, Tan, Deng, Xie, Zeng, Xiao and Xie.)

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted eye disease due to the degeneration of retinal ganglion cells (RGCs). Mitochondrial 11778G > A mutation is the most common LHON-associated mitochondrial DNA (mtDNA) mutation. Our recent studies demonstrated some LHON families manifested by synergic interaction between m.11778G > A mutation and YARS2 allele (c.572G > T, p.Gly191Val) encoding mitochondrial tyrosyl-tRNA synthetase. However, the RGC-specific effects of LHON-associated mtDNA mutations remain elusive and there is no highly effective therapy for LHON. Here, we generated patients-derived induced pluripotent stem cells (iPSCs) from fibroblasts derived from a Chinese LHON family (both m.11778G > A and c.572G > T mutations, only m.11778G > A mutation, and control subject). The c.572G > T mutation in iPSC lines from a syndromic individual was corrected by CRISPR/Cas9. Those iPSCs were differentiated into neural progenitor cells and subsequently induced RGC-like cells using a stepwise differentiation procedure. Those RGC-like cells derived from symptomatic individual harboring both m.11778G > A and c.572G > T mutations exhibited greater defects in neuronal differentiation, morphology including reduced area of soma, numbers of neurites and shortened length of axons, electrophysiological properties than those in cells bearing only m.11778G > A mutation. Furthermore, these RGC-like cells revealed more drastic reductions in oxygen consumption rates, levels of mitochondrial ATP and increasing productions of reactive oxygen species than those in other cell models. These mitochondrial dysfunctions promoted the apoptotic process for RGC degenerations. Correction of YARS2 c.572G > T mutation rescued deficiencies of patient-derived RGC-like cells. These findings provide new insights into pathophysiology of LHON arising from RGC-specific mitochondrial dysfunctions and step toward therapeutic intervention for this disease., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease that results from degeneration of retinal ganglion cells (RGC). Mitochondrial ND4 11778G > A mutation, which affects structural components of complex I, is the most prevalent LHON-associated mitochondrial DNA (mtDNA) mutation worldwide. The m.11778G > A mutation is the primary contributor underlying the development of LHON and X-linked PRICKLE3 allele (c.157C > T, p.Arg53Trp) linked to biogenesis of ATPase interacts with m.11778G > A mutation to cause LHON. However, the lack of appropriate cell and animal models of LHON has been significant obstacles for deep elucidation of disease pathophysiology, specifically the tissue-specific effects. Using RGC-like cells differentiated from induced pluripotent stem cells (iPSCs) from members of one Chinese family (asymptomatic subjects carrying only m.11778G > A mutation or PRICKLE3 p.Arg53Trp mutation, symptomatic individuals bearing both m.11778G > A and PRICKLE3 p.Arg53Trp mutations and control lacking these mutations), we demonstrated the deleterious effects of mitochondrial dysfunctions on the morphology and functions of RGCs. Notably, iPSCs bearing only m.11778G > A or p.Arg53Trp mutation exhibited mild defects in differentiation to RGC-like cells. The RGC-like cells carrying only m.11778G > A or p.Arg53Trp mutation displayed mild defects in RGC morphology, including the area of soma and numbers of neurites, electrophysiological properties, ATP contents and apoptosis. Strikingly, those RGC-like cells derived from symptomatic individuals harboring both m.11778G > A and p.Arg53Trp mutations displayed greater defects in the development, morphology and functions than those in cells bearing single mutation. These findings provide new insights into pathophysiology of LHON arising from RGC deficiencies caused by synergy between m.11778G > A and PRICKLE3 p.Arg53Trp mutation., (© The Author(s) 2022. Published by Oxford University Press.)

Methods to probe and understand the dynamic response of materials following impulsive excitation are important for many fields, from materials and energy sciences to chemical and neuroscience. To design more efficient nano, energy, and quantum devices, new methods are needed to uncover the dominant excitations and reaction pathways. In this work, we implement a newly-developed superlet transform-a super-resolution time-frequency analytical method-to analyze and extract phonon dynamics in a laser-excited two-dimensional (2D) quantum material. This quasi-2D system, 1T-TaSe 2 , supports both equilibrium and metastable light-induced charge density wave (CDW) phases mediated by strongly coupled phonons. We compare the effectiveness of the superlet transform to standard time-frequency techniques. We find that the superlet transform is superior in both time and frequency resolution, and use it to observe and validate novel physics. In particular, we show fluence-dependent changes in the coupled dynamics of three phonon modes that are similar in frequency, including the CDW amplitude mode, that clearly demonstrate a change in the dominant charge-phonon couplings. More interestingly, the frequencies of the three phonon modes, including the strongly-coupled CDW amplitude mode, remain time- and fluence-independent, which is unusual compared to previously investigated materials. Our study opens a new avenue for capturing the coherent evolution and couplings of strongly-coupled materials and quantum systems., (© 2022. The Author(s).)

Numerous RNAs are exported from the nucleus, abnormalities of which lead to cellular complications and diseases. How thousands of circular RNAs (circRNAs) are exported from the nucleus remains elusive. Here, we provide lines of evidence to demonstrate a link between the conserved Exportin 4 (XPO4) and nuclear export of a subset of circRNAs in metazoans. Exonic circRNAs (ecircRNAs) with higher expression levels, larger length, and lower GC content are more sensitive to XPO4 deficiency. Cellular insufficiency of XPO4 leads to nuclear circRNA accumulation, circRNA:DNA (ciR-loop) formation, linear RNA:DNA (liR-loop) buildup, and DNA damage. DDX39 known to modulate circRNA export can resolve ciR-loop, and splicing factors involved in the biogenesis of circRNAs can also affect the levels of ciR-loop. Testis and brain are two organs with high abundance of circRNAs, and insufficient XPO4 levels are detrimental, as Xpo4 heterozygous mice display male infertility and neural phenotypes. Increased levels of ciR-loop, R-loop, and DNA damage along with decreased cell numbers are observed in testis and hippocampus of Xpo4 heterozygotes. This study sheds light on the understandings of mechanism of circRNA export and reveals the significance of efficient nuclear export of circRNAs in cellular physiology., (© 2022. The Author(s).)

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14484T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484T > C mutation in the pathogenesis of LHON., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Sensorineural hearing loss often results from damaged or deficient inner ear hair cells. Mitochondrial 12S rRNA 1555A>G mutation has been associated with hearing loss in many families. The m.1555A>G mutation is a primary factor underlying the development of hearing loss and TRMU allele (c.28G>T, p.Ala10Sser) encoding tRNA thiouridylase interact with m.1555A>G mutation to cause hearing loss. However, the tissue specificity of mitochondrial dysfunction remains elusive and there is no highly effective therapy for mitochondrial deafness. We report here the generation of induced pluripotent stem cells (iPSCs) from lymphoblastoid cell lines derived from members of an Arab-Israeli family (asymptomatic individual carrying only m.1555A>G mutation, symptomatic individual bearing both m.1555A>G and c.28G>T mutations, and control subject). The c.28G>T mutation in iPSC lines from a hearing-impaired subject was corrected by CRISPR/Cas9. These iPSCs were differentiated into otic epithelial progenitor (OEP) cells and subsequent inner ear hair cell (HC)-like cells. The iPSCs bearing m.1555A>G mutation exhibited mildly deficient differentiation into OEP and resultant HC-like cells displayed mild defects in morphology and electrophysiological properties. Strikingly, those HC-like cells harboring m.1555A>G and TRMU c.28G>T mutations displayed greater defects in the development, morphology and functions than those in cells bearing only m.1555A>G mutation. Transcriptome analysis of patients-derived HC-like cells revealed altered expressions of genes vital for mechanotransduction of hair cells. Genetic correction of TRMU c.28G>T mutation yielded morphologic and functional recovery of patient derived HC-like cells. These findings provide new insights into pathophysiology of maternally inherited hearing loss and a step toward therapeutic interventions for this disease., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Pseudouridine (Ψ) at position 55 in tRNAs plays an important role in their structure and function. This modification is catalyzed by TruB/Pus4/Cbf5 family of pseudouridine synthases in bacteria and yeast. However, the mechanism of TRUB family underlying the formation of Ψ55 in the mammalian tRNAs is largely unknown. In this report, the CMC/reverse transcription assays demonstrated the presence of Ψ55 in the human mitochondrial tRNAAsn, tRNAGln, tRNAGlu, tRNAPro, tRNAMet, tRNALeu(UUR) and tRNASer(UCN). TRUB1 knockout (KO) cell lines generated by CRISPR/Cas9 technology exhibited the loss of Ψ55 modification in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro but did not affect other 18 mitochondrial tRNAs. An in vitro assay revealed that recombinant TRUB1 protein can catalyze the efficient formation of Ψ55 in tRNAAsn and tRNAGln, but not in tRNAMet and tRNAArg. Notably, the overexpression of TRUB1 cDNA reversed the deficient Ψ55 modifications in these tRNAs in TRUB1KO HeLa cells. TRUB1 deficiency affected the base-pairing (18A/G-Ψ55), conformation and stability but not aminoacylation capacity of these tRNAs. Furthermore, TRUB1 deficiency impacted mitochondrial translation and biogenesis of oxidative phosphorylation system. Our findings demonstrated that human TRUB1 is a highly conserved mitochondrial pseudouridine synthase responsible for the Ψ55 modification in the mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this report, we investigated the molecular mechanism underlying a deafness-associated m.5783C > T mutation that affects the canonical C50-G63 base-pairing of TΨC stem of tRNACys and immediately adjacent to 5' end of light-strand origin of mitochondrial DNA (mtDNA) replication (OriL). Two dimensional agarose gel electrophoresis revealed marked decreases in the replication intermediates including ascending arm of Y-fork arcs spanning OriL in the mutant cybrids bearing m.5783C > T mutation. mtDNA replication alterations were further evidenced by decreased levels of PolγA, Twinkle and SSBP1, newly synthesized mtDNA and mtDNA contents in the mutant cybrids. The m.5783C > T mutation altered tRNACys structure and function, including decreased melting temperature, conformational changes, instability and deficient aminoacylation of mutated tRNACys. The m.5783C > T mutation impaired the 5' end processing efficiency of tRNACys precursors and reduced the levels of tRNACys and downstream tRNATyr. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced effects in the polypeptides harboring higher numbers of cysteine and tyrosine codons. These alterations led to deficient oxidative phosphorylation including instability and reduced activities of the respiratory chain enzyme complexes I, III, IV and intact supercomplexes overall. Our findings highlight the impact of mitochondrial dysfunction on deafness arising from defects in mitochondrial DNA replication and tRNA metabolism., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Inherited kidney diseases are the fifth most common cause of end-stage renal disease (ESRD). Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mitochondrial-transfer RNA (mt-tRNA) variants and their pathogenic contributions to kidney disease remain largely unclear. In this study, we identified the pathogenic mt-tRNAPhe 616T>C mutation in 3 families and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, 1 proband with homoplamic m.616T>C presented ESRD as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m.616T>C exhibited swollen mitochondria, underwent active mitophagy, and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial ROS. Pathogenic m.616T>C abolished a highly conserved base pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mtDNA-encoded polypeptides. Collectively, these findings provide potentially new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.

The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we focus on more conserved m-gate network in the c-state AAC. All-atom molecular dynamics (MD) simulations on a variety of mutants and the CATR-AAC complex have revealed that: (1) without involvement of other positive residues, the charged residues from the three Px[DE]xx[KR] motifs only are prone to form symmetrical inter-helical network; (2) R235 plays a determinant role for the asymmetry in m-gate network of AAC; (3) R235 significantly strengthens the interactions between H3 and H5; (4) R79 exhibits more significant impact on m-gate than R279; (5) CATR promotes symmetry in m-gate mainly through separating R234 from D231 and fixing R79; (6) vulnerability of the H2-H3 interface near matrix side could be functionally important. Our results provide new insights into the highly conserved yet variable m-gate network in the big mitochondrial carrier family.

Mutations in genes encoding mitochondrial aminoacyl-tRNA synthetases are linked to diverse diseases. However, the precise mechanisms by which these mutations affect mitochondrial function and disease development are not fully understood. Here, we develop a Drosophila model to study the function of dFARS2, the Drosophila homologue of the mitochondrial phenylalanyl-tRNA synthetase, and further characterize human disease-associated FARS2 variants. Inactivation of dFARS2 in Drosophila leads to developmental delay and seizure. Biochemical studies reveal that dFARS2 is required for mitochondrial tRNA aminoacylation, mitochondrial protein stability, and assembly and enzyme activities of OXPHOS complexes. Interestingly, by modeling FARS2 mutations associated with human disease in Drosophila, we provide evidence that expression of two human FARS2 variants, p.G309S and p.D142Y, induces seizure behaviors and locomotion defects, respectively. Together, our results not only show the relationship between dysfunction of mitochondrial aminoacylation system and pathologies, but also illustrate the application of Drosophila model for functional analysis of human disease-causing variants., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mammalian mitochondrial tRNA (mt-tRNA) plays a central role in the synthesis of the 13 subunits of the oxidative phosphorylation complex system (OXPHOS). However, many aspects of the context-dependent expression of mt-tRNAs in mammals remain unknown. To investigate the tissue-specific effects of mt-tRNAs, we performed a comprehensive analysis of mitochondrial tRNA expression across five mice tissues (brain, heart, liver, skeletal muscle, and kidney) using Northern blot analysis. Striking differences in the tissue-specific expression of 22 mt-tRNAs were observed, in some cases differing by as much as tenfold from lowest to highest expression levels among these five tissues. Overall, the heart exhibited the highest levels of mt-tRNAs, while the liver displayed markedly lower levels. Variations in the levels of mt-tRNAs showed significant correlations with total mitochondrial DNA (mtDNA) contents in these tissues. However, there were no significant differences observed in the 2-thiouridylation levels of tRNA Lys , tRNA Glu , and tRNA Gln among these tissues. A wide range of aminoacylation levels for 15 mt-tRNAs occurred among these five tissues, with skeletal muscle and kidneys most notably displaying the highest and lowest tRNA aminoacylation levels, respectively. Among these tissues, there was a negative correlation between variations in mt-tRNA aminoacylation levels and corresponding variations in mitochondrial tRNA synthetases (mt-aaRS) expression levels. Furthermore, the variable levels of OXPHOS subunits, as encoded by mtDNA or nuclear genes, may reflect differences in relative functional emphasis for mitochondria in each tissue. Our findings provide new insight into the mechanism of mt-tRNA tissue-specific effects on oxidative phosphorylation., Competing Interests: Conflict of interest All authors declare that they have no conflict of interest with contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNA Ala 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNA Ala 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNA Ala precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNA Ala aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNA Ala in these cells. The impact of m.5587A>G mutation on tRNA Ala function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNA Ala metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency., Competing Interests: Conflict of interest All authors declare that they have no conflict of interest with contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the mechanism underlying the synergic interaction between Leber's hereditary optic neuropathy (LHON)-associated ND1 and mitochondrial tyrosyl-tRNA synthetase (YARS2) mutations., Methods: Molecular dynamics simulation and differential scanning fluorimetry were used to evaluate the structure and stability of proteins. The impact of ND1 3635G>A and YARS2 p.G191V mutations on the oxidative phosphorylation machinery was evaluated using blue native gel electrophoresis and enzymatic activities assays. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Analysis of effect of mutations on autophagy was undertaken via flow cytometry for autophagic flux., Results: Members of one Chinese family bearing both the YARS2 p.191Gly>Val and m.3635G>A mutations exhibited much higher penetrance of optic neuropathy than those pedigrees carrying only the m.3635G>A mutation. The m.3635G>A (p.Ser110Asn) mutation altered the ND1 structure and function, whereas the p.191Gly>Val mutation affected the stability of YARS2. Lymphoblastoid cell lines harboring both m.3635G>A and p.191Gly>Val mutations revealed more reductions in the levels of mitochondrion-encoding ND1 and CO2 than cells bearing only the m.3635G>A mutation. Strikingly, both m.3635G>A and p.191Gly>Val mutations exhibited decreases in the nucleus-encoding subunits of complex I and IV. These deficiencies manifested greater defects in the stability and activities of complex I and complex IV and overproduction of ROS and promoted greater autophagy in cell lines harboring both m.3635G>A and p.191Gly>Val mutations compared with cells bearing only the m.3635G>A mutation., Conclusions: Our findings provide new insights into the pathophysiology of LHON arising from the synergy between ND1 3635G>A mutation and mitochondrial YARS2 mutations.

Deficient maturations of mitochondrial transcripts are linked to clinical abnormalities but their pathophysiology remains elusive. Previous investigations showed that pathogenic variants in MTO1 for the biosynthesis of τm5U of tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR) were associated with hypertrophic cardiomyopathy (HCM). Using mto1 knock-out(KO) zebrafish generated by CRISPR/Cas9 system, we demonstrated the pleiotropic effects of Mto1 deficiency on mitochondrial RNA maturations. The perturbed structure and stability of tRNAs caused by mto1 deletion were evidenced by conformation changes and sensitivity to S1-mediated digestion of tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). Notably, mto1KO zebrafish exhibited the global decreases in the aminoacylation of mitochondrial tRNAs with the taurine modification. Strikingly, ablated mto1 mediated the expression of MTPAP and caused the altered polyadenylation of cox1, cox3, and nd1 mRNAs. Immunoprecipitation assay indicated the interaction of MTO1 with MTPAP related to mRNA polyadenylation. These alterations impaired mitochondrial translation and reduced activities of oxidative phosphorylation complexes. These mitochondria dysfunctions caused heart development defects and hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the mto1KO zebrafish recapitulated the clinical phenotypes in HCM patients carrying the MTO1 mutation(s). Our findings highlighted the critical role of MTO1 in mitochondrial transcript maturation and their pathological consequences in hypertrophic cardiomyopathy., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Optical control of structural and electronic properties of Weyl semimetals allows development of switchable and dissipationless topological devices at the ultrafast scale. An unexpected orbital-selective photoexcitation in type-II Weyl material WTe 2 is reported under linearly polarized light (LPL), inducing striking transitions among several topologically-distinct phases mediated by effective electron-phonon couplings. The symmetry features of atomic orbitals comprising the Weyl bands result in asymmetric electronic transitions near the Weyl points, and in turn a switchable interlayer shear motion with respect to linear light polarization, when a near-infrared laser pulse is applied. Consequently, not only annihilation of Weyl quasiparticle pairs, but also increasing separation of Weyl points can be achieved, complementing existing experimental observations. In this work, we provide a new perspective on manipulating the Weyl node singularity and coherent control of electron and lattice quantum dynamics simultaneously.

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondria maintain a distinct pool of ribosomal machinery, including tRNAs and tRNAs activating enzymes, such as mitochondrial tyrosyl-tRNA synthetase (YARS2). Mutations in YARS2, which typically lead to the impairment of mitochondrial protein synthesis, have been linked to an array of human diseases including optic neuropathy. However, the lack of YARS2 mutation animal model makes us difficult to elucidate the pathophysiology underlying YARS2 deficiency. To explore this system, we generated YARS2 knockout (KO) HeLa cells and zebrafish using CRISPR/Cas9 technology. We observed the aberrant tRNA Tyr aminoacylation overall and reductions in the levels in mitochondrion- and nucleus-encoding subunits of oxidative phosphorylation system (OXPHOS), which were especially pronounced effects in the subunits of complex I and complex IV. These deficiencies manifested the decreased levels of intact supercomplexes overall. Immunoprecipitation assays showed that YARS2 bound to specific subunits of complex I and complex IV, suggesting the posttranslational stabilization of OXPHOS. Furthermore, YARS2 ablation caused defects in the stability and activities of OXPHOS complexes. These biochemical defects could be rescued by the overexpression of YARS2 cDNA in the YARS2 KO cells. In zebrafish, the yars2 KO larva conferred deficient COX activities in the retina, abnormal mitochondrial morphology, and numbers in the photoreceptor and retinal ganglion cells. The zebrafish further exhibited the retinal defects affecting both rods and cones. Vision defects in yars2 KO zebrafish recapitulated the clinical phenotypes in the optic neuropathy patients carrying the YARS2 mutations. Our findings highlighted the critical role of YARS2 in the stability and activity of OXPHOS and its pathological consequence in vision impairments., Competing Interests: Conflict of interest All the authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The pathophysiology underlying spiral ganglion cell defect-induced deafness remains elusive. Using the whole exome sequencing approach, in combination with functional assays and a mouse disease model, we identified the potentially novel deafness-causative MAP1B gene encoding a highly conserved microtubule-associated protein. Three novel heterozygous MAP1B mutations (c.4198A>G, p.1400S>G; c.2768T>C, p.923I>T; c.5512T>C, p.1838F>L) were cosegregated with autosomal dominant inheritance of nonsyndromic sensorineural hearing loss in 3 unrelated Chinese families. Here, we show that MAP1B is highly expressed in the spiral ganglion neurons in the mouse cochlea. Using otic sensory neuron-like cells, generated by pluripotent stem cells from patients carrying the MAP1B mutation and control subject, we demonstrated that the p.1400S>G mutation caused the reduced levels and deficient phosphorylation of MAP1B, which are involved in the microtubule stability and dynamics. Strikingly, otic sensory neuron-like cells exhibited disturbed dynamics of microtubules, axonal elongation, and defects in electrophysiological properties. Dysfunctions of these derived otic sensory neuron-like cells were rescued by genetically correcting MAP1B mutation using CRISPR/Cas9 technology. Involvement of MAP1B in hearing was confirmed by audiometric evaluation of Map1b heterozygous KO mice. These mutant mice displayed late-onset progressive sensorineural hearing loss that was more pronounced in the high frequencies. The spiral ganglion neurons isolated from Map1b mutant mice exhibited the deficient phosphorylation and disturbed dynamics of microtubules. Map1b deficiency yielded defects in the morphology and electrophysiology of spiral ganglion neurons, but it did not affect the morphologies of cochlea in mice. Therefore, our data demonstrate that dysfunctions of spiral ganglion neurons induced by MAP1B deficiency caused hearing loss.

Tyrosine kinase A (TrkA) is a membrane receptor which, upon ligand binding, activates several pathways including MAPK/ERK signaling, implicated in a spectrum of human pathologies; thus, TrkA is an emerging therapeutic target in treatment of neuronal diseases and cancer. However, mechanistic insights into TrKA signaling are lacking due to lack of site-dependent phosphorylation control. Here we engineer two light-sensitive tyrosine analogues, namely p-azido-L-phenylalanine (AzF) and the caged-tyrosine (ONB), through amber codon suppression to optically manipulate the phosphorylation state of individual intracellular tyrosines in TrkA. We identify TrkA-AzF and ONB mutants, which can activate the ERK pathway in the absence of NGF ligand binding through light control. Our results not only reveal how TrkA site-dependent phosphorylation controls the defined signaling process, but also extend the genetic code expansion technology to enable regulation of receptor-type kinase activation by optical control at the precision of a single phosphorylation site. It paves the way for comprehensive analysis of kinase-associated pathways as well as screening of compounds intervening in a site-directed phosphorylation pathway for targeted therapy.

In this report, we investigated the molecular mechanism underlying a deafness-associated m.7516delA mutation affecting the 5' end processing sites of mitochondrial tRNAAsp and tRNASer(UCN). An in vitro processing experiment demonstrated that m.7516delA mutation caused the aberrant 5' end processing of tRNASer(UCN) and tRNAAsp precursors, catalyzed by RNase P. Using cytoplasmic hybrids (cybrids) derived from one hearing-impaired Chinese family bearing the m.7516delA mutation and control, we demonstrated the asymmetrical effects of m.7516delA mutation on the processing of tRNAs in the heavy (H)-strand and light (L)-strand polycistronic transcripts. Specially, the m.7516delA mutation caused the decreased levels of tRNASer(UCN) and downstream five tRNAs, including tRNATyr from the L-strand transcripts and tRNAAsp from the H-strand transcripts. Strikingly, mutant cybrids exhibited the lower level of COX2 mRNA and accumulation of longer and uncleaved precursors of COX2 from the H-strand transcripts. Aberrant RNA metabolisms yielded variable reductions in the mitochondrial proteins, especially marked reductions in the levels of ND4, ND5, CO1, CO2 and CO3. The impairment of mitochondrial translation caused the proteostasis stress and respiratory deficiency, diminished ATP production and membrane potential, increased production of reactive oxygen species and promoted apoptosis. Our findings provide new insights into the pathophysiology of deafness arising from mitochondrial tRNA processing defects., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Ji et al.)

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease. X-linked nuclear modifiers were proposed to modify the phenotypic manifestation of LHON-associated mitochondrial DNA (mtDNA) mutations. By whole-exome sequencing, we identified the X-linked LHON modifier (c.157C>T, p.Arg53Trp) in PRICKLE3 encoding a mitochondrial protein linked to biogenesis of ATPase in 3 Chinese families. All affected individuals carried both ND4 11778G>A and p.Arg53Trp mutations, while subjects bearing only a single mutation exhibited normal vision. The cells carrying the p.Arg53Trp mutation exhibited defective assembly, stability, and function of ATP synthase, verified by PRICKLE3-knockdown cells. Coimmunoprecipitation indicated the direct interaction of PRICKLE3 with ATP synthase via ATP8. Strikingly, cells bearing both p.Arg53Trp and m.11778G>A mutations displayed greater mitochondrial dysfunction than those carrying only a single mutation. This finding indicated that the p.Arg53Trp mutation acted in synergy with the m.11778G>A mutation and deteriorated mitochondrial dysfunctions necessary for the expression of LHON. Furthermore, we demonstrated that Prickle3-deficient mice exhibited pronounced ATPase deficiencies. Prickle3-knockout mice recapitulated LHON phenotypes with retinal deficiencies, including degeneration of retinal ganglion cells and abnormal vasculature. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutations and X-linked nuclear modifiers.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNA His The primary defect in this mutation is an alteration in tRNA His aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNA His 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene ( HARS2 ) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNA His structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNA His causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNA His from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNA His and of noncognate tRNAs, including tRNA Ala , tRNA Gln , tRNA Glu , tRNA Leu(UUR) , tRNA Lys , and tRNA Met , in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNA His mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders., (© 2020 Gong et al.)

The origin of charge density waves (CDWs) in TiSe[Formula: see text] has long been debated, mainly due to the difficulties in identifying the timescales of the excitonic pairing and electron-phonon coupling (EPC). Without a time-resolved and microscopic mechanism, one has to assume simultaneous appearance of CDW and periodic lattice distortions (PLD). Here, we accomplish a complete separation of ultrafast exciton and PLD dynamics and unravel their interplay in our real-time time-dependent density functional theory simulations. We find that laser pulses knock off the exciton order and induce a homogeneous bonding-antibonding transition in the initial 20 fs, then the weakened electronic order triggers ionic movements antiparallel to the original PLD. The EPC comes into play after the initial 20 fs, and the two processes mutually amplify each other leading to a complete inversion of CDW ordering. The self-amplified dynamics reproduces the evolution of band structures in agreement with photoemission experiments. Hence we resolve the key processes in the initial dynamics of CDWs that help elucidate the underlying mechanism.

Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations. Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNA Ser(UCN) 7511A>G mutation in causing deafness. Strikingly, members of a Chinese family bearing both the YARS2 p.191Gly>Val and m.7511A>G mutations displayed much higher penetrance of deafness than those pedigrees carrying only the m.7511A>G mutation. The m.7511A>G mutation changed the A4:U69 base-pairing to G4:U69 pairing at the aminoacyl acceptor stem of tRNA Ser(UCN) and perturbed tRNA Ser(UCN) structure and function, including an increased melting temperature, altered conformation, instability, and aberrant aminoacylation of mutant tRNA. Using lymphoblastoid cell lines derived from symptomatic and asymptomatic members of these Chinese families and control subjects, we show that cell lines harboring only the m.7511A>G or p.191Gly>Val mutation revealed relatively mild defects in tRNA Ser(UCN) or tRNA Tyr metabolism, respectively. However, cell lines harboring both m.7511A>G and p.191Gly>Val mutations displayed more severe defective aminoacylations and lower tRNA Ser(UCN) and tRNA Tyr levels, aberrant aminoacylation, and lower levels of other tRNAs, including tRNA Thr , tRNA Lys , tRNA Leu(UUR) , and tRNA Ser(AGY) , than those in the cell lines carrying only the m.7511A>G or p.191Gly>Val mutation. Furthermore, mutant cell lines harboring both m.7511A>G and p.191Gly>Val mutations exhibited greater decreases in the levels of mitochondrial translation, respiration, and mitochondrial ATP and membrane potentials, along with increased production of reactive oxygen species. Our findings provide molecular-level insights into the pathophysiology of maternally transmitted deafness arising from the synergy between tRNA Ser(UCN) and mitochondrial YARS mutations., (© 2019 Fan et al.)

Introduction: Methylenetetrahydrofolate reductase (MTHFR) is essential in mediating folate metabolism, and thus plays an important role in diabetes and diabetic complications. MTHFR C677T (rs1801133 C>T) polymorphism has been proposed to be linked with type 2 diabetes mellitus (T2DM) susceptibility. However, the conclusions are inconsistent. Therefore, we rechecked their linkage aiming to obtain a more reliable estimation by performing an updated meta-analysis., Methods: We searched electronic databases PubMed, EMBASE, CNKI, and Wanfang to obtain studies updated to October 2019., Results: After carefully screening, we finally incorporated 68 studies with 10,812 cases and 8,745 controls. The genotype frequency of C677T polymorphism was analyzed pooled to generate odds ratios (ORs) and 95% confidence intervals (CIs). Pooled results presented that MTHFR C677T polymorphism was significantly associated with T2DM under homozygous (OR = 1.64, 95% CI = 1.39-1.94), heterozygous (OR = 1.38, 95% CI = 1.20-1.59), recessive (OR = 1.41, 95% CI = 1.23-1.61), dominant (OR = 1.47, 95% CI = 1.27-1.70), and allele (OR = 1.37, 95% CI = 1.23-1.52) genetic models. Stratified analysis demonstrated that C677T genotype was associated with T2DM in Asian populations, but not Caucasian and African populations., Conclusion: Our results indicated that MTHFR C677T polymorphism confers to T2DM, especially in Asian populations. Much more large-scale case-control studies are needed to strengthen such conclusion in the future., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Mitochondrial tRNA processing defects were associated with human diseases but their pathophysiology remains elusively. The hypertension-associated m.4401A>G mutation resided at a spacer between mitochondrial tRNAMet and tRNAGln genes. An in vitro processing experiment revealed that the m.4401A>G mutation caused 59% and 69% decreases in the 5' end processing efficiency of tRNAGln and tRNAMet precursors, catalyzed by RNase P, respectively. Using human umbilical vein endothelial cells-derived cybrids, we demonstrated that the m.4401A>G mutation caused the decreases of all 8 tRNAs and ND6 and increases of longer and uncleaved precursors from the Light-strand transcript. Conversely, the m.4401A>G mutation yielded the reduced levels of tRNAMet level but did not change the levels of other 13 tRNAs, 12 mRNAs including ND1, 12S rRNA and 16S rRNA from the Heavy-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects play the determined roles in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease. In our previous investigations, we have reported the spectrum and frequency of mitochondrial MT-ND1 , MT-ND4 and MT-ND6 gene in Chinese LHON population. This study aimed to assess the molecular epidemiology of MT-TT mutations in Chinese families with LHON. A cohort of 352 Chinese Han probands lacking the known LHON-associated mtDNA mutations and 376 control subjects underwent molecular analysis of mtDNA. All variants were evaluated for evolutionary conservation, structural and functional consequences. Fifteen variants were identified in the MT-TT gene by mitochondrial genome analysis of LHON pedigrees, which was substantially higher than that of individuals from general Chinese populations. The incidences of the two known LHON-associated mutations, m.15927G > A and m.15951A > G, were 2.27% and 1.14%, respectively. Nine putative LHON-associated variants were identified in 20 probands, translated into 2.1% cases of this cohort. Moreover, mtDNAs in 41 probands carrying the MT-TT mutation(s) were widely dispersed among nine Eastern Asian haplogroups. Our results suggest that the MT-TT gene is a mutational hotspot for these 352 Chinese families lacking the known LHON-associated mutations. These data further showed the molecular epidemiology of MT-TT mutations in Chinese Han LHON pedigrees., Competing Interests: All authors have no proprietary or commercial interest in any of materials discussed in this article., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

GTPBP3 is a highly conserved tRNA modifying enzyme for the biosynthesis of τm5U at the wobble position of mitochondrial tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). The previous investigations showed that GTPBP3 mutations were associated with hypertrophic cardiomyopathy (HCM). However, the pathophysiology of GTPBP3 deficiency remains elusively. Using the gtpbp3 knockout zebrafish generated by CRISPR/Cas9 system, we demonstrated the aberrant mitochondrial tRNA metabolism in gtpbp3 knock-out zebrafish. The deletion of gtpbp3 may alter functional folding of tRNA, indicated by conformation changes and sensitivity to S1-mediated digestion of tRNAGlu, tRNALys, tRNATrp and tRNALeu(UUR). Strikingly, gtpbp3 knock-out zebrafish displayed the global increases in the aminoacylated efficiencies of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms impaired mitochondrial translation, produced proteostasis stress and altered activities of respiratory chain complexes. These mitochondria dysfunctions caused the alterations in the embryonic heart development and reduced fractional shortening of ventricles in mutant zebrafish. Notably, the gtpbp3 knock-out zebrafish exhibited hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the gtpbp3 knock-out zebrafish recapitulated the clinical phenotypes in HCM patients carrying the GTPBP3 mutation(s). Our findings highlight the fundamental role of defective nucleotide modifications of tRNAs in mitochondrial biogenesis and their pathological consequences in hypertrophic cardiomyopathy., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondrial DNA (mtDNA) mutations have been associated with Leber's hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we investigated the pathophysiology of a LHON susceptibility allele (m.3394T>C, p.30Y>H) in the Mitochondrial (MT)-ND1 gene. The incidence of m.3394T>C mutation was 2.7% in the cohort of 1741 probands with LHON. Extremely low penetrances of LHON were observed in 26 pedigrees carrying only m.3394T>C mutation, while 21 families bearing m.3394T>C, together with m.11778G>A or m.14484T>C mutation, exhibited higher penetrance of LHON than those in families carrying single mtDNA mutation(s). The m.3394T>C mutation disrupted the specific electrostatic interactions between Y30 of p.MT-ND1 with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 1) of complex I, thereby altering the structure and function of complex I. We demonstrated that these cybrids bearing only m.3394T>C mutation caused mild mitochondrial dysfunctions and those harboring both m.3394T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions than cybrids carrying only m.11778G>A mutation. In particular, the m.3394T>C mutation altered the stability of p.MT-ND1 and complex I assembly. Furthermore, the m.3394T>C mutation decreased the activities of mitochondrial complexes I, diminished mitochondrial ATP levels and membrane potential and increased the production of reactive oxygen species in the cybrids. These m.3394T>C mutation-induced alterations aggravated mitochondrial dysfunctions associated with the m.11778G>A mutation. These resultant biochemical defects contributed to higher penetrance of LHON in these families carrying both mtDNA mutations. Our findings provide new insights into the pathophysiology of LHON arising from the synergy between mitochondrial ND1 and ND4 mutations., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNAThr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNAThr. Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNAThr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr. The aberrant tRNAThr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNAThr mutation-induced alterations., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Abnormal activation of neddylation modification and dysregulated energy metabolism are frequently seen in many types of cancer cells. Whether and how neddylation modification affects cellular metabolism remains largely unknown. Here, we showed that MLN4924, a small-molecule inhibitor of neddylation modification, induces mitochondrial fission-to-fusion conversion in breast cancer cells via inhibiting ubiquitylation and degradation of fusion-promoting protein mitofusin 1 (MFN1) by SCFβ-TrCP E3 ligase and blocking the mitochondrial translocation of fusion-inhibiting protein DRP1. Importantly, MLN4924-induced mitochondrial fusion is independent of cell cycle progression, but confers cellular survival. Mass-spectrometry-based metabolic profiling and mitochondrial functional assays reveal that MLN4924 inhibits the TCA cycle but promotes mitochondrial OXPHOS. MLN4924 also increases glycolysis by activating PKM2 via promoting its tetramerization. Biologically, MLN4924 coupled with the OXPHOS inhibitor metformin, or the glycolysis inhibitor shikonin, significantly inhibits cancer cell growth both in vitro and in vivo. Together, our study links neddylation modification and energy metabolism, and provides sound strategies for effective combined cancer therapies.

Auditory function in vertebrates depends on the transduction of sound vibrations into electrical signals by inner ear hair cells. In general, hearing loss resulting from hair cell damage is irreversible because the human ear has been considered to be incapable of regenerating or repairing these sensory elements following severe injury. Therefore, regeneration and protection of inner ear hair cells have become an exciting, rapidly evolving field of research during the last decade. However, mammalian auditory hair cells are few in number, experimentally inaccessible, and barely proliferate postnatally in vitro. Various in vitro primary culture systems of inner ear hair cells have been established by different groups, although many challenges remain unresolved. Here, we briefly explain the structure of the inner ear, summarize the published methods of in vitro hair cell cultures, and propose a feasible protocol for culturing these cells, which gave satisfactory results in our study. A better understanding of in vitro hair cell cultures will substantially facilitate research involving auditory functions, drug development, and the isolation of critical molecules involved in hair cell biology.

Non-syndromic hearing loss (NSHL) is a common defect in humans. Variants of MARVELD2 at the DFNB49 locus have been shown to cause bilateral, moderate to profound NSHL. However, the role of MARVELD2 in NSHL susceptibility in the Chinese population has not been studied. Here we conducted a case-control study in an eastern Chinese population to profile the spectrum and frequency of MARVELD2 variants, as well as the association of MARVELD2 gene variants with NSHL. Our results showed that variants identified in the Chinese population are significantly different from those reported in Slovak, Hungarian, and Czech Roma, as well as Pakistani families. We identified 11 variants in a cohort of 283 NSHL cases. Through Sanger sequencing and bioinformatics analysis, we found that c.730G>A variant has detrimental effects in the eastern Chinese population, and may have relatively high correlation with NSHL pathogenicity.

Mtu1(Trmu) is a highly conserved tRNA modifying enzyme responsible for the biosynthesis of τm5s2U at the wobble position of tRNAGln, tRNAGlu and tRNALys. Our previous investigations showed that MTU1 mutation modulated the phenotypic manifestation of deafness-associated mitochondrial 12S rRNA mutation. However, the pathophysiology of MTU1 deficiency remains poorly understood. Using the mtu1 knock-out zebrafish generated by CRISPR/Cas9 system, we demonstrated the abolished 2-thiouridine modification of U34 of mitochondrial tRNALys, tRNAGlu and tRNAGln in the mtu1 knock-out zebrafish. The elimination of this post-transcriptional modification mediated mitochondrial tRNA metabolisms, causing the global decreases in the levels of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms led to the impairment of mitochondrial translation, respiratory deficiencies and reductions of mitochondrial ATP production. These mitochondria dysfunctions caused the defects in hearing organs. Strikingly, mtu1-/- mutant zebrafish displayed the abnormal startle response and swimming behaviors, significant decreases in the sizes of saccular otolith and numbers of hair cells in the auditory and vestibular organs. Furthermore, mtu1-/- mutant zebrafish exhibited the significant reductions in the hair bundle densities in utricle, saccule and lagena. Therefore, our findings may provide new insights into the pathophysiology of deafness, which was manifested by the deficient modifications at wobble position of mitochondrial tRNAs.