Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction.

Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA-deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD(+)-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP-1. This phenotype is rescued by PARP-1 inhibition or by supplementation with NAD(+) precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a nuclear-mitochondrial crosstalk that is critical for the maintenance of mitochondrial health.

Reduction of mRNA export unmasks different tissue sensitivities to low mRNA levels during Caenorhabditis elegans development.

Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes.

BRCA1 and BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in Caenorhabditis elegans.

Interventions that promote healthy aging are typically associated with increased stress resistance. Paradoxically, reducing the activity of core biological processes such as mitochondrial or insulin metabolism promotes the expression of adaptive responses, which in turn increase animal longevity and resistance to stress. In this study, we investigated the relation between the extended Caenorhabditis elegans lifespan elicited by reduction in mitochondrial functionality and resistance to genotoxic stress. We find that reducing mitochondrial activity during development confers germline resistance to DNA damage-induced cell cycle arrest and apoptosis in a cell-non-autonomous manner. We identified the C. elegans homologs of the BRCA1/BARD1 tumor suppressor genes, brc-1/brd-1, as mediators of the anti-apoptotic effect but dispensable for lifespan extension upon mitochondrial stress. Unexpectedly, while reduced mitochondrial activity only in the soma was not sufficient to promote longevity, its reduction only in the germline or in germline-less strains still prolonged lifespan. Thus, in animals with partial reduction in mitochondrial functionality, the mechanisms activated during development to safeguard the germline against genotoxic stress are uncoupled from those required for somatic robustness and animal longevity.

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA.

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Quantitative proteome analysis reveals RNA processing factors as modulators of ionizing radiation-induced apoptosis in the C. elegans germline.

The nematode Caenorhabditis elegans is an organism most recognized for forward and reverse genetic and functional genomic approaches. Proteomic analyses of DNA damage-induced apoptosis have not been shown because of a limited number of cells undergoing apoptosis. We applied mass spectrometry-based quantitative proteomics to evaluate protein changes induced by ionizing radiation (IR) in isolated C. elegans germlines. For this purpose, we used isobaric peptide termini labeling (IPTL) combined with the data analysis tool IsobariQ, which utilizes MS/MS spectra for relative quantification of peak pairs formed during fragmentation. Using stringent statistical critera, we identified 48 proteins to be significantly up- or down-regulated, most of which are part of a highly interconnected protein-protein interaction network dominated by proteins involved in translational control. RNA-mediated depletion of a selection of the IR-regulated proteins revealed that the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel negative regulator of DNA-damage induced apoptosis. Finally, a central role of nucleolar proteins in orchestrating these responses was confirmed as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis in the C. elegans germline.

Base excision repair causes age-dependent accumulation of single-stranded DNA breaks that contribute to Parkinson disease pathology.

Aging, genomic stress, and mitochondrial dysfunction are risk factors for neurodegenerative pathologies, such as Parkinson disease (PD). Although genomic instability is associated with aging and mitochondrial impairment, the underlying mechanisms are poorly understood. Here, we show that base excision repair generates genomic stress, promoting age-related neurodegeneration in a Caenorhabditis elegans PD model. A physiological level of NTH-1 DNA glycosylase mediates mitochondrial and nuclear genomic instability, which promote degeneration of dopaminergic neurons in older nematodes. Conversely, NTH-1 deficiency protects against α-synuclein-induced neurotoxicity, maintaining neuronal function with age. This apparent paradox is caused by modulation of mitochondrial transcription in NTH-1-deficient cells, and this modulation activates LMD-3, JNK-1, and SKN-1 and induces mitohormesis. The dependance of neuroprotection on mitochondrial transcription highlights the integration of BER and transcription regulation during physiological aging. Finally, whole-exome sequencing of genomic DNA from patients with idiopathic PD suggests that base excision repair might modulate susceptibility to PD in humans.

NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome.

Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.

Publisher Correction: Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy.

This corrects the article DOI: 10.1038/ncomms3674.

Constitutive MAP-kinase activation suppresses germline apoptosis in NTH-1 DNA glycosylase deficient C. elegans.

Oxidation of DNA bases, an inevitable consequence of oxidative stress, requires the base excision repair (BER) pathway for repair. Caenorhabditis elegans is a well-established model to study phenotypic consequences and cellular responses to oxidative stress. To better understand how BER affects phenotypes associated with oxidative stress, we characterised the C. elegans nth-1 mutant, which lack the only DNA glycosylase dedicated to repair of oxidative DNA base damage, the NTH-1 DNA glycosylase. We show that nth-1 mutants have mitochondrial dysfunction characterised by lower mitochondrial DNA copy number, reduced mitochondrial membrane potential, and increased steady-state levels of reactive oxygen species. Consistently, nth-1 mutants express markers of chronic oxidative stress with high basal phosphorylation of MAP-kinases (MAPK) but further activation of MAPK in response to the superoxide generator paraquat is attenuated. Surprisingly, nth-1 mutants also failed to induce apoptosis in response to paraquat. The ability to induce apoptosis in response to paraquat was regained when basal MAPK activation was restored to wild type levels. In conclusion, the failure of nth-1 mutants to induce apoptosis in response to paraquat is not a direct effect of the DNA repair deficiency but an indirect consequence of the compensatory cellular stress response that includes MAPK activation.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway.

Aging is a major international concern that brings formidable socioeconomic and healthcare challenges. Small molecules capable of improving the health of older individuals are being explored. Small molecules that enhance cellular stress resistance are a promising avenue to alleviate declines seen in human aging. Tomatidine, a natural compound abundant in unripe tomatoes, inhibits age-related skeletal muscle atrophy in mice. Here we show that tomatidine extends lifespan and healthspan in C. elegans, an animal model of aging which shares many major longevity pathways with mammals. Tomatidine improves many C. elegans behaviors related to healthspan and muscle health, including increased pharyngeal pumping, swimming movement, and reduced percentage of severely damaged muscle cells. Microarray, imaging, and behavioral analyses reveal that tomatidine maintains mitochondrial homeostasis by modulating mitochondrial biogenesis and PINK-1/DCT-1-dependent mitophagy. Mechanistically, tomatidine induces mitochondrial hormesis by mildly inducing ROS production, which in turn activates the SKN-1/Nrf2 pathway and possibly other cellular antioxidant response pathways, followed by increased mitophagy. This mechanism occurs in C. elegans, primary rat neurons, and human cells. Our data suggest that tomatidine may delay some physiological aspects of aging, and points to new approaches for pharmacological interventions for diseases of aging.

NAD+ Replenishment Improves Lifespan and Healthspan in Ataxia Telangiectasia Models via Mitophagy and DNA Repair.

Ataxia telangiectasia (A-T) is a rare autosomal recessive disease characterized by progressive neurodegeneration and cerebellar ataxia. A-T is causally linked to defects in ATM, a master regulator of the response to and repair of DNA double-strand breaks. The molecular basis of cerebellar atrophy and neurodegeneration in A-T patients is unclear. Here we report and examine the significance of increased PARylation, low NAD+, and mitochondrial dysfunction in ATM-deficient neurons, mice, and worms. Treatments that replenish intracellular NAD+ reduce the severity of A-T neuropathology, normalize neuromuscular function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD+ also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy. This work links two major theories on aging, DNA damage accumulation, and mitochondrial dysfunction through nuclear DNA damage-induced nuclear-mitochondrial signaling, and demonstrates that they are important pathophysiological determinants in premature aging of A-T, pointing to therapeutic interventions.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

Oxidative stress promotes human aging and contributes to common neurodegenerative diseases. Endogenous DNA damage induced by oxidative stress is believed to be an important promoter of neurodegenerative diseases. Although a large amount of evidence correlates a reduced DNA repair capacity with aging and neurodegenerative disease, there is little direct evidence of causality. Moreover, the contribution of oxidative DNA damage to the aging process is poorly understood. We have used the nematode Caenorhabditis elegans to study the contribution of oxidative DNA damage and repair to aging. C. elegans is particularly well suited to tackle this problem because it has a minimum complexity DNA repair system, which enables us to circumvent the important limitation presented by the extensive redundancy of DNA repair enzymes in mammals.

Base excision repair AP endonucleases and mismatch repair act together to induce checkpoint-mediated autophagy.

Cellular responses to DNA damage involve distinct DNA repair pathways, such as mismatch repair (MMR) and base excision repair (BER). Using Caenorhabditis elegans as a model system, we present genetic and molecular evidence of a mechanistic link between processing of DNA damage and activation of autophagy. Here we show that the BER AP endonucleases APN-1 and EXO-3 function in the same pathway as MMR, to elicit DNA-directed toxicity in response to 5-fluorouracil, a mainstay of systemic adjuvant treatment of solid cancers. Immunohistochemical analyses suggest that EXO-3 generates the DNA nicks required for MMR activation. Processing of DNA damage via this pathway, in which both BER and MMR enzymes are required, leads to induction of autophagy in C. elegans and human cells. Hence, our data show that MMR- and AP endonuclease-dependent processing of 5-fluorouracil-induced DNA damage leads to checkpoint activation and induction of autophagy, whose hyperactivation contributes to cell death.

Caenorhabditis elegans NDX-4 is a MutT-type enzyme that contributes to genomic stability.

MutT enzymes prevent DNA damage by hydrolysis of 8-oxodGTP, an oxidized substrate for DNA synthesis and antimutagenic, anticarcinogenic, and antineurodegenerative functions of MutT enzymes are well established. MutT has been found in almost all kingdoms of life, including many bacterial species, yeasts, plants and mammals. However, a Caenorhabditis elegans MutT homologue was not previously identified. Here, we demonstrate that NDX-4 exhibits both hallmarks of a MutT-type enzyme with an ability to hydrolyze 8-oxodGTP and suppress the Escherichia coli mutT mutator phenotype. Moreover, we show that NDX-4 contributes to genomic stability in vivo in C. elegans. Phenotypic analyses of an ndx-4 mutant reveal that loss of NDX-4 leads to upregulation of key stress responsive genes that likely compensate for the in vivo role of NDX-4 in protection against deleterious consequences of oxidative stress. This discovery will enable us to use this extremely robust genetic model for further research into the contribution of oxidative DNA damage to phenotypes associated with oxidative stress.

A two-tiered compensatory response to loss of DNA repair modulates aging and stress response pathways.

Activation of oxidative stress-responses and downregulation of insulin-like signaling (ILS) is seen in Nucleotide Excision Repair (NER) deficient segmental progeroid mice. Evidence suggests that this is a survival response to persistent transcription-blocking DNA damage, although the relevant lesions have not been identified. Here we show that loss of NTH-1, the only Base Excision Repair (BER) enzyme known to initiate repair of oxidative DNA damage inC. elegans, restores normal lifespan of the short-lived NER deficient xpa-1 mutant. Loss of NTH-1 leads to oxidative stress and global expression profile changes that involve upregulation of genes responding to endogenous stress and downregulation of ILS. A similar, but more extensive, transcriptomic shift is observed in the xpa-1 mutant whereas loss of both NTH-1 and XPA-1 elicits a different profile with downregulation of Aurora-B and Polo-like kinase 1 signaling networks as well as DNA repair and DNA damage response genes. The restoration of normal lifespan and absence oxidative stress responses in nth-1;xpa-1 indicate that BER contributes to generate transcription blocking lesions from oxidative DNA damage. Hence, our data strongly suggests that the DNA lesions relevant for aging are repair intermediates resulting from aberrant or attempted processing by BER of lesions normally repaired by NER.

Loss of Caenorhabditis elegans UNG-1 uracil-DNA glycosylase affects apoptosis in response to DNA damaging agents.

The nematode Caenorhabditis elegans has been used extensively to study responses to DNA damage. In contrast, little is known about DNA repair in this organism. C. elegans is unusual in that it encodes few DNA glycosylases and the uracil-DNA glycosylase (UDG) encoded by the ung-1 gene is the only known UDG. C. elegans could therefore become a valuable model organism for studies of the genetic interaction networks involving base excision repair (BER). As a first step towards characterization of BER in C. elegans, we show that the UNG-1 protein is an active uracil-DNA glycosylase. We demonstrate that an ung-1 mutant has reduced ability to repair uracil-containing DNA but that an alternative Ugi-inhibited activity is present in ung-1 nuclear extracts. Finally, we demonstrate that ung-1 mutants show altered levels of apoptotic cell corpses formed in response to DNA damaging agents. Increased apoptosis in the ung-1 mutant in response to ionizing radiation (IR) suggests that UNG-1 contributes to repair of IR-induced DNA base damage in vivo. Following treatment with paraquat however, the apoptotic corpse-formation was reduced. Gene expression profiling suggests that this phenotype is a consequence of compensatory transcriptomic shifts that modulate oxidative stress responses in the mutant and not an effect of reduced DNA damage signaling.

Caenorhabditis elegans APN-1 plays a vital role in maintaining genome stability.

We previously showed that Caenorhabditis elegans APN-1, the only metazoan apurinic/apyrimidinc (AP) endonuclease belonging to the endonuclease IV family, can functionally rescue the DNA repair defects of Saccharomyces cerevisiae mutants completely lacking AP endonuclease/3'-diesterase activities. While this complementation study provided the first evidence that APN-1 possesses the ability to act on DNA lesions that are processed by AP endonucleases/3'-diesterase activities, no former studies were conducted to examine its biological importance in vivo. Herein, we show that C. elegans knockdown for apn-1 by RNAi displayed phenotypes that are directly linked with a defect in maintaining the integrity of the genome. apn-1(RNAi) animals exhibited a 5-fold increase in the frequency of mutations at a gfp-lacZ reporter and showed sensitivities to DNA damaging agents such as methyl methane sulfonate and hydrogen peroxide that produce AP site lesions and strand breaks with blocked 3'-ends. The apn-1(RNAi) worms also displayed a delay in the division of the P1 blastomere, a defect that is consistent with the accumulation of unrepaired lesions. Longevity was only compromised, if the apn-1(RNAi) animals were challenged with the DNA damaging agents. We showed that apn-1(RNAi) knockdown suppressed formation of apoptotic corpses in the germline caused by an overburden of AP sites generated from uracil DNA glycosylase mediated removal of misincorporated uracil. Finally, we showed that depletion of APN-1 by RNAi partially rescued the lethality resulting from uracil misincorporation, suggesting that APN-1 is an important AP endonuclease for repair of misincorporated uracil.