Abnormal morphology and function in retinal ganglion cells derived from patients-specific iPSCs generated from individuals with Leber's hereditary optic neuropathy.

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.

Nuclear modifier YARS2 allele correction restored retinal ganglion cells-specific deficiencies in Leber's hereditary optic neuropathy.

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.

Leber's hereditary optic neuropathy-associated ND6 14484T > C mutation caused pleiotropic effects on the complex I, RNA homeostasis, apoptosis and mitophagy.

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.

Autoinducer-2 promotes the colonization of Lactobacillus rhamnosus GG to improve the intestinal barrier function in a neonatal mouse model of antibiotic-induced intestinal dysbiosis.

BACKGROUND: Human health is seriously threatened by antibiotic-induced intestinal disorders. Herein, we aimed to determine the effects of Autoinducer-2 (AI-2) combined with Lactobacillus rhamnosus GG (LGG) on the intestinal barrier function of antibiotic-induced intestinal dysbiosis neonatal mice. METHODS: An antibiotic-induced intestinal dysbiosis neonatal mouse model was created using antibiotic cocktails, and the model mice were randomized into the control, AI-2, LGG, and LGG + AI-2 groups. Intestinal short-chain fatty acids and AI-2 concentrations were detected by mass spectrometry and chemiluminescence, respectively. The community composition of the gut microbiota was analyzed using 16S rDNA sequencing, and biofilm thickness and bacterial adhesion in the colon were assessed using scanning electron microscopy. Transcriptome RNA sequencing of intestinal tissues was performed, and the mRNA and protein levels of HCAR2 (hydroxycarboxylic acid receptor 2), claudin3, and claudin4 in intestinal tissues were determined using quantitative real-time reverse transcription PCR and western blotting. The levels of inflammatory factors in intestinal tissues were evaluated using enzyme-linked immunosorbent assays (ELISAs). D-ribose, an inhibitor of AI-2, was used to treat Caco-2 cells in vitro. RESULTS: Compared with the control, AI-2, and LGG groups, the LGG + AI-2 group showed increased levels of intestinal AI-2 and proportions of Firmicutes and Lacticaseibacillus, but a reduced fraction of Proteobacteria. Specifically, the LGG + AI-2 group had considerably more biofilms and LGG on the colon surface than those of other three groups. Meanwhile, the combination of AI-2 and LGG markedly increased the concentration of butyric acid and promoted Hcar2, claudin3 and claudin4 expression levels compared with supplementation with LGG or AI-2 alone. The ELISAs revealed a significantly higher tumor necrosis factor alpha (TNF-α) level in the control group than in the LGG and LGG + AI-2 groups, whereas the interleukin 10 (IL-10) level was significantly higher in the LGG + AI-2 group than in the other three groups. In vitro, D-ribose treatment dramatically suppressed the increased levels of Hcar2, claudin3, and claudin4 in Caco-2 cells induced by AI-2 + LGG. CONCLUSIONS: AI-2 promotes the colonization of LGG and biofilm formation to improve intestinal barrier function in an antibiotic-induced intestinal dysbiosis neonatal mouse model.

A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism.

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.

Human TRUB1 is a highly conserved pseudouridine synthase responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.

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.

Optimized allotopic expression of mitochondrial ND6 transgene restored complex I and apoptosis deficiencies caused by LHON-linked ND6 14484T > C mutation.

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.

Mechanistic insights into mitochondrial tRNAAla 3'-end metabolism deficiency.

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 tRNAAla 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 tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla 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 tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla 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.

Complex I mutations synergize to worsen the phenotypic expression of Leber's hereditary optic neuropathy.

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.

Contribution of mitochondrial ND1 3394T>C mutation to the phenotypic manifestation of Leber's hereditary optic neuropathy.

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.

Sodium Butyrate Alleviates Intestinal Inflammation in Mice with Necrotizing Enterocolitis.

OBJECTIVE: To determine the role of sodium butyrate in intestinal inflammation via regulation of high-mobility group box-1 (HMGB1), we analyzed the potential mechanism in necrotizing enterocolitis (NEC) in a neonatal mouse model. METHODS: A NEC model was created with hypoxia and cold exposure and artificial overfeeding. C57BL/6 neonatal mice were randomized into three groups: the control, untreated NEC, and sodium butyrate (150 mM)-pretreated NEC groups. Pathological variations in ileocecal intestinal tissue were observed by HE staining and scored in a double-blind manner. The mRNA expression levels of HMGB1, Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and inflammatory cytokines in intestinal tissues were determined by quantitative real-time PCR. The protein levels of HMGB1 and associated cytokines in intestinal tissues were evaluated using ELISA. The relative protein expression levels of TLR4 and NF-κB in intestinal tissues were quantified by western blot. RESULTS: Sodium butyrate administration improved the body weight and survival rate of NEC mice; relieved intestinal pathological injury; reduced the intestinal expression of HMGB1, TLR4, NF-κB, interleukin- (IL-) 1ß, IL-6, IL-8, and TNF-α; and increased the intestinal expression of IL-10 (P < 0.05). Treatment with butyrate decreased the proportion of opportunistic Clostridium_sensu_stricto_1 and Enterococcus and increased the proportion of beneficial Firmicutes and Lactobacillus in the NEC model. CONCLUSIONS: Sodium butyrate intervention relieves intestinal inflammation and partially corrects the disrupted intestinal flora in mice with NEC.

Effect of the course of treatment with broad-spectrum antibiotics on intestinal flora and short-chain fatty acids in feces of very low birth weight infants: a prospective study. / å¹¿è°±æŠ—ç”Ÿç´ ç–—ç¨‹å¯¹æžä½Žå‡ºç”Ÿä½“é‡å„¿ç²ªä¾¿è‚ é“èŒç¾¤å’ŒçŸ­é“¾è„‚è‚ªé ¸å½±å“çš„å‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To study the effect of the course of treatment with broad-spectrum antibiotics on intestinal flora and short-chain fatty acids (SCFAs) in feces of very low birth weight (VLBW) infants. METHODS: A total of 29 VLBW infants who were admitted to the Neonatal Diagnosis and Treatment Center of Children's Hospital Affiliated to Chongqing Medical University from June to December 2020 were enrolled as subjects for this prospective study. According to the course of treatment with broad-spectrum antibiotics, they were divided into two groups: ≤7 days (n=9) and >7 days (n=20). Fecal samples were collected on days 14 and 28 of hospitalization, and 16S rDNA high-throughput sequencing and gas chromatography-mass spectrometry were used to analyze the flora and SCFAs in fecal samples. RESULTS: There was a significant reduction in Chao index of the intestinal flora in the ≤7 days group and the >7 days group from week 2 to week 4 (P<0.05). In the ≤7 days group, there were significant increases in the proportions of Firmicutes and Clostridium_sensu_stricto_1 and a significant reduction in the proportion of Proteobacteria from week 2 to week 4 (P<0.05). At week 4, compared with the ≤7 days group, the >7 days group had significant reductions in the proportions of Firmicutes and Clostridium_sensu_stricto_1 and a significant increase in the proportion of Proteobacteria (P<0.05), as well as significant reductions in the content of isobutyric acid and valeric acid (P<0.05). CONCLUSIONS: The course of treatment with broad-spectrum antibiotics can affect the abundance, colonization, and evolution of intestinal flora and the content of their metabolites SCFAs in VLBW infants. The indication and treatment course for broad-spectrum antibiotics should be strictly controlled in clinical practice.

Leber's hereditary optic neuropathy (LHON)-associated ND5 12338T > C mutation altered the assembly and function of complex I, apoptosis and mitophagy.

Mutations in mitochondrial DNA (mtDNA) have been associated with Leber's hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we demonstrated that a missense mutation (m.12338T>C, p.1M>T) in the ND5 gene contributed to the pathogenesis of LHON. The m.12338T>C mutation affected the first methionine (Met1) with a threonine and shortened two amino acids of ND5. We therefore hypothesized that the mutated ND5 perturbed the structure and function of complex I. Using the cybrid cell models, generated by fusing mtDNA-less (ρ°) cells with enucleated cells from LHON patients carrying the m.12338T>C mutation and a control subject belonging to the same mtDNA haplogroup, we demonstrated that the m.12338T>C mutation caused the reduction of ND5 polypeptide, perturbed assemble and activity of complex I. Furthermore, the m.12338T>C mutation caused respiratory deficiency, diminished mitochondrial adenosine triphosphate levels and membrane potential and increased the production of reactive oxygen species. The m.12338T>C mutation promoted apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and Poly ADP ribose polymerase in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. Moreover, we also document the involvement of m.12338T>C mutation in decreased mitophagy, as showed by reduced levels of autophagy protein light chain 3 and accumulation of autophagic substrate p62 in the in mutant cybrids as compared with control cybrids. These data demonstrated the direct link between mitochondrial dysfunction caused by complex I mutation and apoptosis or mitophagy. Our findings may provide new insights into the pathophysiology of LHON.

Biochemical evidence for a mitochondrial genetic modifier in the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease. Mitochondrial modifiers are proposed to modify the phenotypic expression of primary LHON-associated mitochondrial DNA (mtDNA) mutations. In this study, we demonstrated that the LHON susceptibility allele (m.14502T > C, p. 58I > V) in the ND6 gene modulated the phenotypic expression of primary LHON-associated m.11778G > A mutation. Twenty-two Han Chinese pedigrees carrying m.14502T > C and m.11778G > A mutations exhibited significantly higher penetrance of optic neuropathy than those carrying only m.11778G > A mutation. We performed functional assays using the cybrid cell models, generated by fusing mtDNA-less ρo cells with enucleated cells from LHON patients carrying both m.11778G > A and m.14502T > C mutations, only m.14502T > C or m.11778G > A mutation and a control belonging to the same mtDNA haplogroup. These cybrids cell lines bearing m.14502T > C mutation exhibited mild effects on mitochondrial functions compared with those carrying only m.11778G > A mutation. However, more severe mitochondrial dysfunctions were observed in cell lines bearing both m.14502T > C and m.11778G > A mutations than those carrying only m.11778G > A or m.14502T > C mutation. In particular, the m.14502T > C mutation altered assemble of complex I, thereby aggravating the respiratory phenotypes associated with m.11778G > A mutation, resulted in a more defective complex I. Furthermore, more reductions in the levels of mitochondrial ATP and increasing production of reactive oxygen species were also observed in mutant cells bearing both m.14502T > C and m.11778G > A mutation than those carrying only 11778G > A mutation. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between primary and secondary mtDNA mutations.

The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

[Progress in research on pathogenic genes and gene therapy for inherited retinal diseases].

Inherited retinal diseases (IRDs), including retinitis pigmentosa, Usher syndrome, Cone-Rod degenerations, inherited macular dystrophy, Leber's congenital amaurosis, Leber's hereditary optic neuropathy are the most common and severe types of hereditary ocular diseases. So far more than 200 pathogenic genes have been identified. With the growing knowledge of the genetics and mechanisms of IRDs, a number of gene therapeutic strategies have been developed in the laboratory or even entered clinical trials. Here the progress of IRD research on the pathogenic genes and therapeutic strategies, particularly gene therapy, are reviewed.

[The role of MT-ND1 m.3635G>A mutation in Leber's hereditary optic neuropathy].

OBJECTIVE: To investigate the role of MT-ND1 m.3635G>A mutation in the pathogenesis of Leber's hereditary optic neuropathy (LHON). METHODS: Biochemical characteristics including the activity of complex Ⅰ, ATP production and oxygen consumption rate among lymphoblastoid cell lines derived from 3 carriers, 3 affected matrilineal relatives of the families and 3 controls were compared. RESULTS: Comparison of mitochondrial functions in lymphoblastoid cell lines of the carriers, patients and controls showed a 51.0% decrease in the activity of complex Ⅰ in patients compared with controls (P<0.05). The m.3635G>A mutation has resulted in decreased efficiency of ATP synthesis (P<0.05). Comparison of oxygen consumption rate showed that the basal OCR (P<0.05), ATP-linked OCR (P<0.05) and the maximum OCR (P<0.05) have all reduced to some extent compared with the controls. CONCLUSION: These results showed that m.3635G>A, as a LHON-associated mutation, can lead to mitochondrial dysfunction.

Cellular models for mitochondrial DNA-based diseases: lymphoblastoid cell lines and transmitochondrial cybrids.

Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial DNA-based diseases which have been studied using Lymphoblastoid cell lines (LCLs) and transmitochondrial cybrids. Individual genetic information is preserved permanently in LCLs while the development of transmitochondrial cybrids provide ex-vivo cellular platform to study molecular mechanism of mitochondrial DNA-based diseases. The cytoplasmic donor cells for previous transmitochondrial cybrids come from patient's tissue or platelet directly. Here, we depicted in details the principle, methods and techniques to establish LCLs from frozen peripheral bloods harboring mitochondrial 4401G > A mutation by infection of Epstein Barr virus, and then to generate cybrids using ρ0 206 and LCLs. The process of establishing these two cellular models was summarized into four steps as follows: (1) Generation of LCLs; (2) Transformation; (3) Selection; (4) Verification. To faithfully represent the function of mtDNA mutation, we analyzed and identified the sites of mtDNA mutations and copy numbers of each cellular models as well as the karyotype of transmitochondrial cybrids. Those clones with consistent parameters were selected for preservation and future analysis of the function of point mutations of mtDNA. Although these two cellular models play important roles in understanding molecular mechanism of mitochondrial DNA-based diseases on the cellular level, their limitations should be considered when elucidating the character of tissue specificity of mitochondrial DNA-based diseases.

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families.

To investigate the pathophysiology of Leber's hereditary optic neuropathy (LHON), a cohort of 1164 Han Chinese subjects with LHON were screened for ND1 G3460A mutation. A total of 295 subjects from 16 Han Chinese families carrying the G3460A mutation underwent a clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA. The incidence of G3460A mutation was 1.4% in this cohort of Chinese subjects with LHON. Twenty-seven (20 males/7 females) of 109 matrilineal relatives among 10 Chinese pedigrees carrying this mutation exhibited a wide range of severity and age-at-onset in visual impairment. Penetrances of optic neuropathy ranged from 7.1% to 50%, with the average of 24.5%. The age-at-onset of 27 affected matrilineal relatives varied from 10 to 40 years, with the average of 22 years. Molecular analysis identified the homoplasmic G3460A mutation and distinct sets of variants belonging to eight haplogroups. Haplogroup M with G3460A mutation was of higher frequency than those in controls. The penetrances of visual loss in families carrying mitochondrial DNA haplogroups A, B and M were higher than those in other families. Furthermore, haplogroup-specific variants tRNA(Ser(AGY)) A12223G, tRNA(Thr) G15927A and tRNA(Glu) A14693G may enhance the penetrance of visual loss in these families. The G3460A mutation occurred through recurrent origins and founder events in Chinese population. Mitochondrial modifiers may modulate the penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the G3460A mutation. Thus, our findings may provide new insights into the understanding of pathophysiology and valuable information on the management of LHON.

Exogenous autoinducer-2 alleviates intestinal damage in necrotizing enterocolitis via PAR2/MMP3 signaling pathway.

BACKGROUND: Imbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation. OBJECTIVE: This study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells. METHODS: Pathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1ß (IL-1ß), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence. RESULTS: AI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1ß and IL-6 mRNA expression (P < 0.05). CONCLUSION: AI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.