Regulation of DNA repair by parkin

Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson’s disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.     

ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation

Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.     

Estrogen receptor β‐mediated nuclear interaction between IRS‐1 and Rad51 inhibits homologous recombination directed DNA repair in medulloblastoma

In medulloblastomas, which are highly malignant cerebellar tumors of the childhood genotoxic treatments such as cisplatin or γ‐irradiation are frequently associated with DNA damage, which often associates with unfaithful DNA repair, selection of new adaptations and possibly tumor recurrences. Therefore, better understanding of molecular mechanisms which control DNA repair fidelity upon DNA damage is a critical task. Here we demonstrate for the first time that estrogen receptor beta (ERβ) can contribute to the development of genomic instability in medulloblastomas. Specifically, ERβ was found highly expressed and active in mouse and human medulloblastoma cell lines. Nuclear ERβ was also present in human medulloblastoma clinical samples. Expression of ERβ coincided with nuclear translocation of insulin receptor substrate 1 (IRS‐1), which was previously reported to interfere with the faithful component of DNA repair when translocated to the nucleus. We demonstrated that ERβ and IRS‐1 bind each other, and the interaction involves C‐terminal domain of IRS‐1 (aa 931–1233). Following cisplatin‐induced DNA damage, nuclear IRS‐1 localized at the sites of damaged DNA, and interacted with Rad51—an enzymatic component of homologous recombination directed DNA repair (HRR). In medulloblastoma cells, engineered to express HRR‐DNA reporter plasmid, ER antagonist, ICI 182,780, or IRS mutant (931–1233) significantly increased DNA repair fidelity. These data strongly suggest that both molecular and pharmacological interventions are capable of preventing ERβ‐mediated IRS‐1 nuclear translocation, which in turn improves DNA repair fidelity and possibly counteracts accumulation of malignant mutations in actively growing medulloblastomas. J. Cell. Physiol. 219: 392–401, 2009. © 2008 Wiley‐Liss, Inc.     

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long‐lived and cancer‐resistant subterranean wild rodent,Spalax

The blind mole rat (Spalax) is a wild, long‐lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence‐associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response. Spalax fibroblasts undergo replicative senescence (RS) and etoposide‐induced senescence (EIS), evidenced by an increased activity of senescence‐associated beta‐galactosidase (SA‐β‐Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, RS and EIS Spalax cells showed undetectable or decreased expression of the well‐known SASP factors: interleukin‐6 (IL6), IL8, IL1α, growth‐related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM‐1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation. Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro‐inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA‐MB‐231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF‐κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory‐related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age‐related chronic inflammation supporting healthy aging and longevity.     

Chromatin PTEN is involved in DNA damage response partly through regulating Rad52 sumoylation

A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN’s role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3β are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN’s association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as γH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.     

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50?mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10?Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.     

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage rapir in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.     

DNA repair in organelles: Pathways, organization, regulation, relevance in disease and aging

Both endogenous processes and exogenous physical and chemical sources generate deoxyribonucleic acid (DNA) damage in the nucleus and organelles of living cells. To prevent deleterious effects, damage is balanced by repair pathways. DNA repair was first documented for the nuclear compartment but evidence was subsequently extended to the organelles. Mitochondria and chloroplasts possess their own repair processes. These share a number of factors with the nucleus but also rely on original mechanisms. Base excision repair remains the best characterized. Repair is organized with the other DNA metabolism pathways in the organelle membrane-associated nucleoids. DNA repair in mitochondria is a regulated, stress-responsive process. Organelle genomes do not encode DNA repair enzymes and translocation of nuclear-encoded repair proteins from the cytosol seems to be a major control mechanism. Finally, changes in the fidelity and efficiency of mitochondrial DNA repair are likely to be involved in DNA damage accumulation, disease and aging. The present review successively addresses these different issues.     

Sequestration of Mammalian Rad51-Recombination Protein into Micronuclei

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after γ irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.     

PDCD5-regulated cell fate decision after ultraviolet-irradiation-induced DNA damage

Programmed cell death 5 (PDCD5) is a human apoptosis-related molecule that is involved in both the cytoplasmic caspase-3 activity pathway (by regulating Bax translocation from cytoplasm to mitochondria) and the nuclear pathway (by interacting with Tip60). In this study, we developed a mathematical model of the PDCD5-regulated switching of the cell response from DNA repair to apoptosis after ultraviolet irradiation-induced DNA damage. We established the model by combining several hypotheses with experimental observations. Our simulations indicate that the ultimate cell response to DNA damage is dependent on a signal threshold mechanism, and the PDCD5 promotion of Bax translocation plays an essential role in PDCD5-regulated cell apoptosis. Furthermore, the model simulations revealed that PDCD5 nuclear translocation can attenuate cell apoptosis, and PDCD5 interactions with Tip60 can accelerate DNA damage-induced apoptosis, but the final cell fate decision is insensitive to the PDCD5-Tip60 interaction. These results are consistent with experimental observations. The effect of recombinant human PDCD5 was also investigated and shown to sensitize cells to DNA damage by promoting caspase-3 activity.     

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild‐type but not PD‐linked mutant parkin supports the biogenesis of a population of mitochondria‐derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin‐ and PINK1‐dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.     

The 3′–5′ DNA Exonuclease TREX1 Directly Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) during the DNA Damage Response

The main function of the 3′–5′ DNA exonuclease TREX1 is to digest cytosolic single-stranded DNA to prevent activation of cell-intrinsic responses to immunostimulatory DNA. TREX1 translocates to the nucleus following DNA damage with its nuclear activities being less well defined. Although mutations in human TREX1 have been linked to autoimmune/inflammatory diseases, the mechanisms contributing to the pathogenesis of these diseases remain incompletely understood. Here, using mass spectrometry and co-immunoprecipitation assays and in vivo overexpression models, we show that TREX1 interacts with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear enzyme involved in the DNA damage response. Two zinc finger domains at the amino terminus of PARP1 were required for the interaction with TREX1 that occurs after nuclear translocation of TREX1 in response to DNA damage. Functional studies suggested that TREX1 may contribute to stabilization of PARP1 levels in the DNA damage response and its activity. These results provide new insights into the mechanisms of single-stranded DNA repair following DNA damage and alterations induced by gene mutations.     

Inhibition of apoptotic Bax translocation to the mitochondria is a central function of parkin

Parkinson''s disease (PD) is the second most prevalent neurodegenerative disorder, affecting 1–3% of the population over 65. Mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. The parkin protein possesses potent cell-protective properties and has been mechanistically linked to both the regulation of apoptosis and the turnover of damaged mitochondria. Here, we explored these two functions of parkin and the relative scale of these processes in various cell types. While biochemical analyses and subcellular fractionation were sufficient to observe robust parkin-dependent mitophagy in immortalized cells, higher resolution techniques appear to be required for primary culture systems. These approaches, however, did affirm a critical role for parkin in the regulation of apoptosis in primary cultured neurons and all other cells studied. Our prior work demonstrated that parkin-dependent ubiquitination of endogenous Bax inhibits its mitochondrial translocation and can account for the anti-apoptotic effects of parkin. Having found a central role for parkin in the regulation of apoptosis, we further investigated the parkin-Bax interaction. We observed that the BH3 domain of Bax is critical for its recognition by parkin, and identified two lysines that are crucial for parkin-dependent regulation of Bax translocation. Last, a disease-linked mutation in parkin failed to influence Bax translocation to mitochondria after apoptotic stress. Taken together, our data suggest that regulation of apoptosis by the inhibition of Bax translocation is a prevalent physiological function of parkin regardless of the kind of cell stress, preventing overt cell death and supporting cell viability during mitochondrial injury and repair.Loss-of-function mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive Parkinson''s disease (PD).¹ Multiple functions have been ascribed to parkin, most notably the inhibition of apoptosis^{2, 3, 4, 5, 6, 7} and the induction of autophagic mitochondrial turnover (mitophagy).^{8, 9} However, the relative scale of these effects mediated by endogenous parkin and whether these processes can occur concomitantly or are mutually exclusive, is not known.Bax is a primary effector of cell death that translocates from the cytosol to the mitochondria upon stress, where it facilitates cytochrome c release and the subsequent caspase cascade.¹⁰ We previously identified Bax as a parkin substrate, and found that the anti-apoptotic effects of parkin can be directly linked to the parkin-dependent ubiquitination of Bax and inhibition of its mitochondrial translocation.³ Recent corroborative evidence showed that primary cultured neurons from parkin knock-out (KO) mice accumulate greater levels of activated Bax at the mitochondria than wild-type (WT) neurons after apoptotic stimulation,¹¹ while a separate report showed the parkin-dependent ubiquitination of Bax during mitophagy.¹²In addition to its anti-apoptotic function, parkin facilitates a depolarization-induced and autophagy-dependent turnover of mitochondria. This process is robustly observed in immortalized cell lines expressing human parkin, where exposure to the mitochondrial depolarizing agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) causes rapid recruitment of parkin from the cytosol to the mitochondrial outer membrane and a coordinated proteasome and autophagosome-mediated turnover of the entire organelle.^{8, 13, 14, 15} Examination of this process in primary neuronal cultures with endogenous parkin expression, however, has been challenging,^{16, 17, 18, 19} and a cooperative role for inhibition of mitochondria-dependent cell death has not been investigated in the context of mitophagy.In this study, we sought further insight into the biological functions of parkin across multiple cell types. Our data showed that whole-cell biochemical techniques were not sufficient to observe the participation of endogenous parkin in mitochondrial turnover but were able to confirm the parkin-dependent regulation of apoptosis. Further examination of the parkin-dependent regulation of apoptosis identified two specific lysines of Bax that are critical for recognition and inhibition of its translocation to the mitochondria by parkin. In addition, the BH3 domain of Bax was critical for its association with parkin. Importantly, we observed parkin-dependent mitophagy and inhibition of apoptotic Bax translocation in the same cell culture systems, suggesting that these two pathways coexist and likely cooperate within neurons. Taken together, our data indicate that the parkin-dependent regulation of Bax is critical for cell survival, irrespective of the nature of cell stress involved.     

Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative pentose phosphate cycle, regulates the NADPH/NADP(+) ratio in eukaryotic cells. G6PD deficiency is one of the most common mutations in humans and is known to cause health problems for hundreds of millions worldwide. Although it is known that decreased G6PD functionality can result in increased susceptibility to oxidative stress, the molecular targets of this stress are not known. Using a Chinese hamster ovary G6PD-null mutant, we previously demonstrated that exposure to a thiol-specific oxidant, hydroxyethyldisulfide, caused enhanced radiation sensitivity and an inability to repair DNA double strand breaks. We now demonstrate a molecular mechanism for these observations: the direct inhibition of DNA end binding activity of the Ku heterodimer, a DNA repair protein, by oxidation of its cysteine residues. Inhibition of Ku DNA end binding was found to be reversible by treatment of the nuclear extract with dithiothreitol, suggesting that the homeostatic regulation of reduced cysteine residues in Ku is a critical function of G6PD and the oxidative pentose cycle. In summary, we have discovered a new layer of DNA damage repair, that of the functional maintenance of repair proteins themselves. In view of the rapidly escalating number of roles ascribed to Ku, these results may have widespread ramifications.     

alpha-Synuclein aggregates interfere with Parkin solubility and distribution: role in the pathogenesis of Parkinson disease

Parkinson disease (PD) belongs to a heterogeneous group of neurodegenerative disorders with movement alterations, cognitive impairment, and alpha-synuclein accumulation in cortical and subcortical regions. Jointly, these disorders are denominated Lewy body disease. Mutations in the parkin gene are the most common cause of familial parkinsonism, and a growing number of studies have shown that stress factors associated with sporadic PD promote parkin accumulation in the insoluble fraction. alpha-Synuclein and parkin accumulation and mutations in these genes have been associated with familial PD. To investigate whether alpha-synuclein accumulation might be involved in the pathogenesis of these disorders by interfering with parkin solubility, synuclein-transfected neuronal cells were transduced with lentiviral vectors expressing parkin. Challenging neurons with proteasome inhibitors or amyloid-beta resulted in accumulation of insoluble parkin and, to a lesser extent, alpha-tubulin. Similarly to neurons in the brains of patients with Lewy body disease, in co-transduced cells alpha-synuclein and parkin colocalized and co-immunoprecipitated. These effects resulted in decreased parkin and alpha-tubulin ubiquitination, accumulation of insoluble parkin, and cytoskeletal alterations with reduced neurite outgrowth. Taken together, accumulation of alpha-synuclein might contribute to the pathogenesis of PD and other Lewy body diseases by promoting alterations in parkin and tubulin solubility, which in turn might compromise neural function by damaging the neuronal cytoskeleton. These studies provide a new perspective on the potential nature of pathogenic alpha-synuclein and parkin interactions in Parkinson disease.     

The regulatory role of α-synuclein and parkin in neuronal cell apoptosis; possible implications for the pathogenesis of Parkinson’s disease

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder beyond Alzheimer’s disease, affecting approximately 1% of people over the age of 65. The major pathological hallmarks of PD are significant loss of nigrostriatal dopaminergic (DA) neurons and the presence of intraneuronal protein inclusions termed Lewy bodies. Sporadic cases represent more than 90% of total patients with PD, while there exist several inherited forms caused by mutations in single genes. Identification and characterization of these causative genes and their products can help us understand the molecular mechanisms of DA neuronal cell death and design new approaches to treat both the inherited and sporadic forms of PD. Based on the finding that a point mutation in the gene encoding α-synuclein (αSyn) protein causes a rare familial form of PD, PARK1, it is now confirmed that αSyn is a major component of Lewy bodies in patients with sporadic PD. Abnormal accumulation of αSyn protein is considered a neurotoxic event in the development of PD. PARK4, another dominantly inherited form of familial PD, is caused by duplication or triplication of the αSyn gene locus. This genetic mutation results in the production of large amounts of wild-type αSyn protein, supporting the αSyn-induced neurodegeneration hypothesis. On the other hand, the recessively inherited early-onset Parkinsonism is caused in about half of the cases with loss-of-function mutations in PARK2, which encodes E3 ubiquitin ligase parkin in the ubiquitin–proteasome system. These findings have shed light on DA neurodegeneration caused by accumulation of toxic protein species that can be degraded and/or detoxicated through parkin activity. In this review, we will focus on the regulatory roles of αSyn and parkin proteins in DA neuronal cell apoptosis and provide evidence for the possible therapeutic action of parkin in sporadic patients with PD.