Impaired complex I repair causes recessive Leber’s hereditary optic neuropathy

Leber’s hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit–encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.     

Restoration of the Activated Rig-I Pathway in Hepatitis C Virus (HCV) Replicon Cells by HCV Protease,Polymerase, and NS5A Inhibitors In Vitro at Clinically Relevant Concentrations

Development of persistent hepatitis C virus (HCV) infection may be mediated by HCV NS3 · 4A protease-dependent inhibition of host innate immunity. When double-stranded RNA (dsRNA) is detected in virus-infected cells, host innate immunity mounts an antiviral response by upregulating production of type I interferons (α/β interferon [IFN-α/β]); HCV counters by cleaving the IFN-β stimulator 1 (IPS-1) adaptor protein, decreasing synthesis of IFN-α/β. We evaluated HCV protease (telaprevir, boceprevir, and TMC435350), polymerase (HCV-796 and VX-222), and NS5A (BMS-790052) inhibitors for the ability to restore IPS-1-mediated Rig-I signaling by measuring Sendai virus-induced IFN-β promoter activation in HCV replicon cells after various exposure durations. All direct-acting HCV antivirals tested restored mitochondrial localization of IPS-1 and rescued Sendai virus-induced IRF3 signaling after 7 days by inhibiting HCV replication, thereby reducing the abundance of HCV NS3 · 4A protease. With 4-day treatment, HCV protease inhibitors, but not polymerase inhibitors, restored mitochondrial localization of IPS-1 and rescued IFN-β promoter activation in the presence of equivalent levels of NS3 protein in protease or polymerase inhibitor-treated cells. The concentrations of HCV protease and polymerase inhibitors needed to rescue IRF3-mediated signaling in vitro were in the range of those observed in vivo in the plasma of treated HCV patients. These findings suggest that (i) HCV protease, polymerase, and NS5A inhibitors can restore virus-induced IRF3 signaling by inhibiting viral replication, thereby reducing NS3 protease levels, and (ii) HCV protease inhibitors can restore innate immunity by directly inhibiting NS3 protease-mediated cleavage of IPS-1 at clinically achievable concentrations.     

Late onset of type 2 diabetes is associated with mitochondrial tRNATrp A5514G and tRNASer(AGY) C12237T mutations

BackgroundMitochondrial dysfunctions caused by mitochondrial DNA (mtDNA) pathogenic mutations play putative roles in type 2 diabetes mellitus (T2DM) progression. But the underlying mechanism remains poorly understood.MethodsA large Chinese family with maternally inherited diabetes and deafness (MIDD) underwent clinical, genetic, and molecular assessment. PCR and sequence analysis are carried out to detect mtDNA variants in affected family members, in addition, phylogenetic conservation analysis, haplogroup classification, and pathogenicity scoring system are performed. Moreover, the GJB2, GJB3, GJB6, and TRMU genes mutations are screened by PCR‐Sanger sequencing.ResultsSix of 18 matrilineal subjects manifested different clinical phenotypes of diabetes. The average age at onset of diabetic patients is 52 years. Screening for the entire mitochondrial genomes suggests the co‐existence of two possibly pathogenic mutations: tRNA^Trp A5514G and tRNA^Ser(AGY) C12237T, which belongs to East Asia haplogroup G2a. By molecular level, m.A5514G mutation resides at acceptor stem of tRNA^Trp (position 3), which is critical for steady‐state level of tRNA^Trp. Conversely, m.C12237T mutation occurs in the variable region of tRNA^Ser(AGY) (position 31), which creates a novel base‐pairing (11A‐31T). Thus, the mitochondrial dysfunctions caused by tRNA^Trp A5514G and tRNA^Ser(AGY) C12237T mutations, may be associated with T2DM in this pedigree. But we do not find any functional mutations in those nuclear genes.ConclusionOur findings suggest that m.A5514G and m.C12337T mutations are associated with T2DM, screening for mt‐tRNA mutations is useful for molecular diagnosis and prevention of mitochondrial diabetes.     

DNAJC30 biallelic mutations extend mitochondrial complex I–deficient phenotypes to include recessive Leber’s hereditary optic neuropathy

Leber’s hereditary optic neuropathy (LHON) is the most common mitochondrial disease and in most cases is caused by mutations in mitochondrial DNA–encoded (mtDNA-encoded) respiratory complex I subunit ND1, ND4, or ND6. In this issue of the JCI, Stenton et al. describe biallelic mutations in a nuclear DNA–encoded gene, DNAJC30, establishing recessively inherited LHON (arLHON). Functional studies suggest that DNAJC30 is a protein chaperone required for exchange of damaged complex I subunits. Hallmark mtDNA LHON features were also found in arLHON, including incomplete penetrance, male predominance, and positive response to idebenone therapy. These results extend complex I–deficient phenotypes to include recessively inherited optic neuropathy, with important clinical implications for genetic counseling and therapeutic considerations.     

TLR7-dependent and FcγR-independent production of type I interferon in experimental mouse lupus

Increased type I interferon (IFN-I) production and IFN-stimulated gene (ISG) expression are linked to the pathogenesis of systemic lupus erythematosus (SLE). Although the mechanisms responsible for dysregulated IFN-I production in SLE remain unclear, autoantibody-mediated uptake of endogenous nucleic acids is thought to play a role. 2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane) induces a lupus-like disease in mice characterized by immune complex nephritis with autoantibodies to DNA and ribonucleoproteins. We recently reported that TMPD also causes increased ISG expression and that the development of the lupus is completely dependent on IFN-I signaling (Nacionales, D.C., K.M. Kelly-Scumpia, P.Y. Lee, J.S. Weinstein, R. Lyons, E. Sobel, M. Satoh, and W.H. Reeves. 2007. Arthritis Rheum. 56:3770–3783). We show that TMPD elicits IFN-I production, monocyte recruitment, and autoantibody production exclusively through a Toll-like receptor (TLR) 7– and myeloid differentiation factor 88 (MyD88)–dependent pathway. In vitro studies revealed that TMPD augments the effect of TLR7 ligands but does not directly activate TLR7 itself. The effects of TMPD were amplified by the Y-linked autoimmune acceleration cluster, which carries a duplication of the TLR7 gene. In contrast, deficiency of Fcγ receptors (FcγRs) did not affect the production of IFN-I. Collectively, the data demonstrate that TMPD-stimulated IFN-I production requires TLR7/MyD88 signaling and is independent of autoantibody-mediated uptake of ribonucleoproteins by FcγRs.     

IFNβ Treatment Inhibits Nerve Injury-induced Mechanical Allodynia and MAPK Signaling By Activating ISG15 in Mouse Spinal Cord

Neuropathic pain is difficult to treat and remains a major clinical challenge worldwide. While the mechanisms which underlie the development of neuropathic pain are incompletely understood, interferon signaling by the immune system is known to play a role. Here, we demonstrate a role for interferon β (IFNβ) in attenuating mechanical allodynia induced by the spared nerve injury in mice. The results show that intrathecal administration of IFNβ (dosages up to 5,000 U) produces significant, transient, and dose-dependent attenuation of mechanical allodynia without observable effects on motor activity or feeding behavior, as is common with IFN administration. This analgesic effect is mediated by the ubiquitin-like protein interferon-stimulated gene 15 (ISG15), which is potently induced within the spinal cord following intrathecal delivery of IFNβ. Both free and conjugated ISG15 are elevated following IFNβ treatment, and this effect is increased in UBP43^−/− mice lacking a key deconjugating enzyme. The IFNβ-mediated analgesia reduces MAPK signaling activation following nerve injury, and this effect requires induction of ISG15. These findings highlight a new role for IFNβ, ISG15, and MAPK signaling in immunomodulation of neuropathic pain and may lead to new therapeutic possibilities.PerspectiveNeuropathic pain is frequently intractable in a clinical setting, and new treatment options are needed. Characterizing the antinociceptive potential of IFNβ and the associated downstream signaling pathways in preclinical models may lead to the development of new therapeutic options for debilitating neuropathies.     

Maternally transmitted nonsyndromic hearing impairment may be associated with mitochondrial tRNAAla 5601C>T and tRNALeu(CUN) 12311T>C mutations

BackgroundSequence alternations in mitochondrial genomes, especially in genes encoding mitochondrial tRNA (mt‐tRNA), were the important contributors to nonsyndromic hearing loss (NSHL); however, the molecular mechanisms remained largely undetermined.MethodsA maternally transmitted Chinese pedigree with NSHL underwent clinical, genetic, and biochemical assessment. PCR and direct sequence analyses were performed to detect mitochondrial DNA (mtDNA), GJB2, and SLC26A4 gene mutations from matrilineal relatives of this family. Mitochondrial functions including mitochondrial membrane potential (MMP), ATP, and ROS were evaluated in polymononuclear leukocytes (PMNs) derived from three deaf patients and three controls from this pedigree.ResultsFour of nine matrilineal relatives developed hearing loss at the variable age of onset. Two putative pathogenic mutations, m.5601C>T in tRNA^Ala and m.12311T>C in tRNA^Leu(CUN), were identified via PCR‐Sanger sequencing, as well as 34 variants that belonged to mtDNA haplogroup G2b2. Intriguingly, m.5601C>T mutation resided at very conserved nucleotide in the TψC loop of tRNA^Ala (position 59), while the T‐to‐C substitution at position 12311 located at position 48 in the variable stem of tRNA^Leu(CUN) and was believed to alter the aminoacylation and the steady‐state level of tRNA. Biochemical analysis revealed the impairment of mitochondrial functions including the significant reductions of ATP and MMP, whereas markedly increased ROS levels were found in PMNs derived from NSHL patients with m.5601C>T and m.12311T>C mutations. However, we did not detect any mutations in GJB2 and SLC26A4 genes.ConclusionOur data indicated that mt‐tRNA^Ala m.5601C>T and tRNA^Leu(CUN) 12311T>C mutations were associated with NSHL.     

Screening the three LHON primary mutations in the general Chinese population by using an optimized multiplex allele-specific PCR

BackgroundLeber hereditary optic neuropathy (LHON) is one of the most common mitochondrial diseases, which is mainly caused by three mitochondrial DNA (mtDNA) mutations (m.3460G> A, m.11778G> A and m.14484T> C). Incomplete penetrance suggests that there might be asymptomatic carriers in general populations. These asymptomatic carriers are clinically important as they are potential future patients and the female carriers could transfer the pathogenic mutations to their offspring. Thus, screening the three LHON primary mutations in general populations is important for genetic counseling.MethodsWe optimized a multiplex allele-specific PCR method based on previous studies, and the sensitivity was evaluated. The three LHON primary mutations were screened by using this MAS-PCR method in 1571 subjects from general Chinese populations that are without symptoms or family history of optic neuropathy.ResultsThe optimized MAS-PCR approach can detect a heteroplasmy level at 5%, 5%, and 20% for m.3460G> A, m.11778G> A and m.14484T> C, respectively. None of the three LHON primary mutations was detected in the 1571 subjects.ConclusionThe three LHON primary mutations are rare in general Chinese populations. The optimized MAS-PCR assay provides an easier, faster and more cost-effective method for detection of the three LHON primary mutations, making it practical for clinical diagnosis.     

Catenin α 1 mutations cause familial exudative vitreoretinopathy by overactivating Norrin/β-catenin signaling

Familial exudative vitreoretinopathy (FEVR) is a severe retinal vascular disease that causes blindness. FEVR has been linked to mutations in several genes associated with inactivation of the Norrin/β-catenin signaling pathway, but these account for only approximately 50% of cases. We report that mutations in α-catenin (CTNNA1) cause FEVR by overactivating the β-catenin pathway and disrupting cell adherens junctions. We identified 3 heterozygous mutations in CTNNA1 (p.F72S, p.R376Cfs*27, and p.P893L) by exome sequencing and further demonstrated that FEVR-associated mutations led to overactivation of Norrin/β-catenin signaling as a result of impaired protein interactions within the cadherin-catenin complex. The clinical features of FEVR were reproduced in mice lacking Ctnna1 in vascular endothelial cells (ECs) or with overactivated β-catenin signaling by an EC-specific gain-of-function allele of Ctnnb1. In isolated mouse lung ECs, both CTNNA1-P893L and F72S mutants failed to rescue either the disrupted F-actin arrangement or the VE-cadherin and CTNNB1 distribution. Moreover, we discovered that compound heterozygous Ctnna1 F72S and a deletion allele could cause a similar phenotype. Furthermore, in a FEVR family, we identified a mutation of LRP5, which activates Norrin/β-catenin signaling, and the corresponding knockin mice exhibited a partial FEVR-like phenotype. Our study demonstrates that the precise regulation of β-catenin activation is critical for retinal vascular development and provides new insights into the pathogenesis of FEVR.     

Natriuretic peptides enhance the oxidative capacity of human skeletal muscle

Cardiac natriuretic peptides (NP) are major activators of human fat cell lipolysis and have recently been shown to control brown fat thermogenesis. Here, we investigated the physiological role of NP on the oxidative metabolism of human skeletal muscle. NP receptor type A (NPRA) gene expression was positively correlated to mRNA levels of PPARγ coactivator-1α (PGC1A) and several oxidative phosphorylation (OXPHOS) genes in human skeletal muscle. Further, the expression of NPRA, PGC1A, and OXPHOS genes was coordinately upregulated in response to aerobic exercise training in human skeletal muscle. In human myotubes, NP induced PGC-1α and mitochondrial OXPHOS gene expression in a cyclic GMP–dependent manner. NP treatment increased OXPHOS protein expression, fat oxidation, and maximal respiration independent of substantial changes in mitochondrial proliferation and mass. Treatment of myotubes with NP recapitulated the effect of exercise training on muscle fat oxidative capacity in vivo. Collectively, these data show that activation of NP signaling in human skeletal muscle enhances mitochondrial oxidative metabolism and fat oxidation. We propose that NP could contribute to exercise training–induced improvement in skeletal muscle fat oxidative capacity in humans.     

Zidovudine Induces Downregulation of Mitochondrial Deoxynucleoside Kinases: Implications for Mitochondrial Toxicity of Antiviral Nucleoside Analogs

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK. In organello studies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs.     

Dosage Effects of Cohesin Regulatory Factor PDS5 on Mammalian Development: Implications for Cohesinopathies

Cornelia de Lange syndrome (CdLS), a disorder caused by mutations in cohesion proteins, is characterized by multisystem developmental abnormalities. PDS5, a cohesion protein, is important for proper chromosome segregation in lower organisms and has two homologues in vertebrates (PDS5A and PDS5B). Pds5B mutant mice have developmental abnormalities resembling CdLS; however the role of Pds5A in mammals and the association of PDS5 proteins with CdLS are unknown. To delineate genetic interactions between Pds5A and Pds5B and explore mechanisms underlying phenotypic variability, we generated Pds5A-deficient mice. Curiously, these mice exhibit multiple abnormalities that were previously observed in Pds5B-deficient mice, including cleft palate, skeletal patterning defects, growth retardation, congenital heart defects and delayed migration of enteric neuron precursors. They also frequently display renal agenesis, an abnormality not observed in Pds5B^−/− mice. While Pds5A^−/− and Pds5B^−/− mice die at birth, embryos harboring 3 mutant Pds5 alleles die between E11.5 and E12.5 most likely of heart failure, indicating that total Pds5 gene dosage is critical for normal development. In addition, characterization of these compound homozygous-heterozygous mice revealed a severe abnormality in lens formation that does not occur in either Pds5A^−/− or Pds5B^−/− mice. We further identified a functional missense mutation (R1292Q) in the PDS5B DNA-binding domain in a familial case of CdLS, in which affected individuals also develop megacolon. This study shows that PDS5A and PDS5B functions other than those involving chromosomal dynamics are important for normal development, highlights the sensitivity of key developmental processes on PDS5 signaling, and provides mechanistic insights into how PDS5 mutations may lead to CdLS.     

Immunomodulatory effects of voriconazole on monocytes challenged with Aspergillus fumigatus: differential role of Toll-like receptors

Voriconazole (VRC) has activity against Aspergillus fumigatus, the most frequent cause of invasive aspergillosis in immunocompromised patients. The combination of VRC and A. fumigatus hyphae induced a more pronounced profile of expression of genes encoding inflammatory molecules in human monocytes than Aspergillus alone did. Herein, we provide further evidence of the potential mechanism underlying this immunomodulatory effect of VRC on human monocytes in response to A. fumigatus hyphae. A significant additive antifungal effect was shown when VRC was combined with monocytes against A. fumigatus hyphae. Both A. fumigatus hyphae and VRC induced pronounced profiles of mRNA and protein expression of Toll-like receptor 2 (TLR2) as well as tumor necrosis factor alpha (TNF-α) in THP-1 monocytic cells compared to untreated cells. The VRC-induced increase was greater than that induced by hyphae. The combination of VRC and hyphae increased mRNA and protein expression of TLR2 and TNF-α to even higher levels than did either VRC or hyphae alone. In contrast, TLR4 expression, both at the mRNA and protein levels, was not increased by either VRC or hyphae or their combination. In addition, significantly more NF-κB was translocated to the nuclei of THP-1 cells treated with VRC than untreated cells. While VRC induced more NF-κB than hyphae did, treatment with the combination of the two factors induced the greatest NF-κB expression. The pronounced profile of TLR2 signaling, TNF-α expression, and NF-κB activation in the presence of VRC suggests an immunomodulatory effect leading to a more efficient response to A. fumigatus.     

Modulation of energy metabolism and mitochondrial biogenesis by a novel proteoglycan from Ganoderma lucidum

In this study we first focused on the effects of a novel proteoglycan extracted from Ganoderma lucidum (FYGL) on mitochondrial biogenesis, because mitochondrial dysfunction is highly related to insulin resistance. We found that FYGL can decrease ROS levels and increase ATP content in rat skeletal muscle L6 cells. In PGC-1α silent cells, FYGL increased expression of PGC-1α and positively modulated the Sirt1/PGC-1α pathway. Moreover, FYGL orally administered up-regulated the mitochondrial DNA (mtDNA) copy number and the related gene expressions that control mitochondrial biogenesis in ob/ob mice. Our work well elucidated the mechanism of FYGL ameliorating insulin resistance in the aspect of energy metabolism.

In this study we first focused on the effects of a novel proteoglycan extracted from Ganoderma lucidum (FYGL) on mitochondrial biogenesis, because mitochondrial dysfunction is highly related to insulin resistance.     

PML-RARA requires DNA methyltransferase 3A to initiate acute promyelocytic leukemia

The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA–driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1– and MLL-AF9–driven self-renewal. Furthermore, both the PML-RARA–driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice.     

A novel unstable mutation in mitochondrial DNA responsible for maternally inherited diabetes and deafness

OBJECTIVE

The m.3243A>G mutation in mitochondrial DNA (mtDNA) is responsible for maternally inherited diabetes and deafness (MIDD). Other mtDNA mutations are extremely rare.

RESEARCH DESIGN AND METHODS

We studied a patient presenting with diabetes and deafness who does not carry the m.3243A>G mutation.

RESULTS

We identified a deficiency of respiratory chain complex I in the patient’s fibroblasts. mtDNA sequencing revealed a novel mutation that corresponds to an insertion of one or two cytosine residues in the coding region of the MT-ND6 gene (m.14535_14536insC or CC), leading to premature stop codons. This heteroplasmic mutation is unstable in the patient’s somatic tissues.

CONCLUSIONS

We describe for the first time an unstable mutation in a mitochondrial gene coding for a complex I subunit, which is responsible for the MIDD phenotype. This mutation is likely favored by the m.14530T>C polymorphism, which is homoplasmic and leads to the formation of an 8-bp polyC tract responsible for genetic instability.The most common form of maternally inherited diabetes and deafness (MIDD) is associated with the m.3243A>G mutation in mitochondrial DNA (mtDNA), which is located in the tRNA^Leu gene (1). The mutation that affects up to 1% of diabetic patients leads to both impaired glucose-induced insulin secretion (2) and progressive β-cell loss (3). However, in some rare cases characterized by a highly suggestive phenotype but without m.3243A>G mutation, geneticists should look for other diabetes-prone variants (4). Here, we describe a patient presenting an MIDD phenotype who carries a novel unstable mutation in the mitochondrial MT-ND6 gene responsible for a deficiency in the respiratory chain complex I.