Henok Kassahun

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.

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.

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.

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.

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.

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

Significance In this paper we describe a possible pathogenesis for the accelerated aging disease Cockayne syndrome that entails defective transcription through DNA secondary structures leading to activation of the DNA damage response enzyme poly-ADP-ribose polymerase 1 and downstream mitochondrial derangement. These findings are important because they signify a possible new role of transcription in the resolution of DNA structures that form spontaneously and suggest a possible pathogenesis for this accelerated aging disease. 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.

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.

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.

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.

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.