Crosstalk between Different DNA repair pathways contributes to neurodegenerative diseases

Genomic integrity is maintained by DNA repair and the DNA damage response (DDR). Defects in certain DNA repair genes give rise to many rare progressive neurodegenerative diseases (NDDs), such as ocular motor ataxia, Huntington disease (HD), and spinocerebellar ataxias (SCA). Dysregulation or dysfunction of DDR is also proposed to contribute to more common NDDs, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Here, we present mechanisms that link DDR with neurodegeneration in rare NDDs caused by defects in the DDR and discuss the relevance for more common age-related neurodegenerative diseases. Moreover, we highlight recent insight into the crosstalk between the DDR and other cellular processes known to be disturbed during NDDs. We compare the strengths and limitations of established model systems to model human NDDs, ranging from C. elegans and mouse models towards advanced stem cell-based 3D models

Cross-Species Functional Genomic Analysis Identifies Resistance Genes of the Histone Deacetylase Inhibitor Valproic Acid

The mechanisms of successful epigenetic reprogramming in cancer are not well characterized as they involve coordinated removal of repressive marks and deposition of activating marks by a large number of histone and DNA modification enzymes. Here, we have used a cross-species functional genomic approach to identify conserved genetic interactions to improve therapeutic effect of the histone deacetylase inhibitor (HDACi) valproic acid, which increases survival in more than 20% of patients with advanced acute myeloid leukemia (AML). Using a bidirectional synthetic lethality screen revealing genes that increased or decreased VPA sensitivity in C. elegans, we identified novel conserved sensitizers and synthetic lethal interactors of VPA. One sensitizer identified as a conserved determinant of therapeutic success of HDACi was UTX (KDM6A), which demonstrates a functional relationship between protein acetylation and lysine-specific methylation. The synthetic lethal screen identified resistance programs that compensated for the HDACi-induced global hyper-acetylation, and confirmed MAPKAPK2, HSP90AA1, HSP90AB1 and ACTB as conserved hubs in a resistance program for HDACi that are drugable in human AML cell lines. Hence, these resistance hubs represent promising novel targets for refinement of combinatorial epigenetic anti-cancer therapy

Effects of fatty acid esters on mechanical, thermal, microbial, and moisture barrier properties of carboxymethyl cellulose-based edible films

Fatty acid esters being biodegradable and environment friendly has been a sought-after class of molecule for various food grade applications. This work involves the incorporation of fatty acid esters namely cetyl-caprylate and cetyl-caprate in edible Carboxymethyl cellulose -based films. The esters were enzymatically synthesized by esterification of caprylic acid and capric acid respectively with cetyl alcohol at a molar ratio of 1:1, using Candida antarctica lipase B which was immobilized (10 % w/w) at 65 °C. Carboxymethyl cellulose films were prepared. To it, glycerol and by emulsification, cetyl-caprylate or cetyl-caprate esters were amalgamated. Film characterizations involved analysis of surface morphology, mechanical properties, and thermal properties. It was further characterized by X-Ray diffraction analysis, water vapor permeability, and moisture uptake. Barrier property carboxymethyl cellulose films showed significant improvement due to the incorporation of cetyl-caprylate or cetyl-caprate esters. However, when the film's melting point was measured, it was seen that glycerol influenced the thermal properties more prominently than cetyl-caprylate and cetyl-caprate esters. Thus, the addition of an optimized amount of glycerol and cetyl-caprylate or cetyl-caprate esters to the carboxymethyl cellulose films is required for improved mechanical strength and better thermal properties. Further, an antimicrobial well diffusion assay of both the esters established the antimicrobial property of the same, which thereby recommends the addition of the wax esters even more

Effects of fatty acid esters on mechanical, thermal, microbial, and moisture barrier properties of carboxymethyl cellulose-based edible films

Fatty acid esters being biodegradable and environment friendly has been a sought-after class of molecule for various food grade applications. This work involves the incorporation of fatty acid esters namely cetyl-caprylate and cetyl-caprate in edible Carboxymethyl cellulose -based films. The esters were enzymatically synthesized by esterification of caprylic acid and capric acid respectively with cetyl alcohol at a molar ratio of 1:1, using Candida antarctica lipase B which was immobilized (10 % w/w) at 65 ◦C. Carboxymethyl cellulose films were prepared. To it, glycerol and by emulsification, cetyl-caprylate or cetyl-caprate esters were amalgamated. Film charac?terizations involved analysis of surface morphology, mechanical properties, and thermal properties. It was further characterized by X-Ray diffraction analysis, water vapor permeability, and moisture uptake. Barrier property carboxymethyl cellulose films showed significant improvement due to the incorporation of cetylcaprylate or cetyl-caprate esters. However, when the film’s melting point was measured, it was seen that glycerol influenced the thermal properties more prominently than cetyl-caprylate and cetyl-caprate esters. Thus, the addition of an optimized amount of glycerol and cetyl-caprylate or cetyl-caprate esters to the carboxymethyl cellulose films is required for improved mechanical strength and better thermal properties. Further, an antimicrobial well diffusion assay of both the esters established the antimicrobial property of the same, which thereby recommends the addition of the wax esters even more.</p

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.publishe

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

SummaryMitochondrial 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

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

Targeted Central Nervous System Irradiation with Proton Microbeam Induces Mitochondrial Changes in <i>Caenorhabditis elegans</i>

Fifty percent of all patients with cancer worldwide require radiotherapy. In the case of brain tumors, despite the improvement in the precision of radiation delivery with proton therapy, studies have shown structural and functional changes in the brains of treated patients with protons. The molecular pathways involved in generating these effects are not completely understood. In this context, we analyzed the impact of proton exposure in the central nervous system area of Caenorhabditis elegans with a focus on mitochondrial function, which is potentially implicated in the occurrence of radiation-induced damage. To achieve this objective, the nematode C. elegans were micro-irradiated with 220 Gy of protons (4 MeV) in the nerve ring (head region) using the proton microbeam, MIRCOM. Our results show that protons induce mitochondrial dysfunction, characterized by an immediate dose-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with oxidative stress 24 h after irradiation, which is itself characterized by the induction of the antioxidant proteins in the targeted region, observed using SOD-1::GFP and SOD-3::GFP strains. Moreover, we demonstrated a two-fold increase in the mtDNA copy number in the targeted region 24 h after irradiation. In addition, using the GFP::LGG-1 strain, an induction of autophagy in the irradiated region was observed 6 h following the irradiation, which is associated with the up-regulation of the gene expression of pink-1 (PTEN-induced kinase) and pdr-1 (C. elegans parkin homolog). Furthermore, our data showed that micro-irradiation of the nerve ring region did not impact the whole-body oxygen consumption 24 h following the irradiation. These results indicate a global mitochondrial dysfunction in the irradiated region following proton exposure. This provides a better understanding of the molecular pathways involved in radiation-induced side effects and may help in finding new therapies

Histone methylation capacity affects basal histone acetylation in <i>C. elegans</i> embryos.

<p>VPA-treatment induced global acetylation in 100-cell stage <i>C. elegans</i> embryos. The strain AZ212, expressing GFP in fusion with H2B, was fed the empty vector L4440 (RNAi), the VPA-sensitizers <i>lex-1</i> (RNAi), <i>utx-1</i> (RNAi) (histone demethylase), and <i>set-12</i> (RNAi) (histone methyltransferase), and exposed to 15 mM VPA at L4 larval stage for 24 hours at 20°C. The embryos were fixed with acetone and methanol prior to staining with an Acetyl-Histone H4 (Lys8) antibody. At the 100-cell stage, baseline levels of acetylation were seen in untreated control worms. Global hyperacetylation was observed after treatment with VPA by all. Depletion of <i>lex-1</i>, <i>utx-1</i> and <i>set-12</i> gives increased baseline acetylation in the absence of VPA. Scale bar = 10 µm.</p

UTX-1 is required for VPA action in <i>C. elegans</i> embryos and in human cells.

<p><b>A</b>) Two <i>UTX</i> wild type cell lines (MV4-11 and NB4) as well as the <i>UTX</i> mutant cell line THP-1 were treated with 1 mM VPA for 48 hours and analyzed for H3K27me3 and H2BK120ac expression. The mean intensity on one representative Western blot was calculated and normalized to beta-actin. The numbers shown are in arbitrary units compared to MV4-11 control. Blots show the increase of H3K27 trimethylation as well as an increase in the degree of H2BK120 acetylation except THP-1, where the level of methylation is unchanged and level of acetylation is decreased. <b>B</b>) AML cell lines with mutated <i>UTX</i> are resistant to VPA. MV4-11 and NB4 as well as two <i>UTX</i> mutant cell lines (MONO-MAC-1 and THP-1) were subjected to 2 mM VPA for 48 hours and scored for abnormal nuclei by Hoechst staining. * indicates t-test versus MV4-11, x indicates versus NB4. ***/xxx <i>p</i><0.0004. Error bars represent SEM. <b>C</b>) The mutant cell lines show decreased apoptosis, determined by Hoechst staining compared to the wild type cells. Arrows indicate fragmented and condensed nuclei. Scale bar = 10 µm. <b>D</b>) MV4-11 or NB4 cells were subjected to 600 nM UTX siRNA or negative control siRNA for 18 hours prior to 48 hours treatment with 2 mM VPA. Cells were scored for abnormal nuclei by Hoechst staining, showing UTX siRNA to reduce the effect of VPA on cell death. Values are normalized against untreated cells. ** indicates t-test of UTX siRNA versus negative control siRNA, <i>p</i> = 0.014. Error bars represent SEM. <b>E</b>) Western blot of MV4-11 or NB4 cells treated with 600 nM negative control siRNA or UTX siRNA for 24 hours. Numbers shown are arbitrary units compared to negative control siRNA. The blot shows a 60% decrease in UTX by siRNA treatment, confirming the efficacy of transfection. <b>F</b>) The <i>C. elegans</i> strain AZ212 was fed the empty vector L4440 or <i>utx-1</i> (RNAi), and the <i>utx-1(3136)</i> mutant was fed <i>E. coli</i> OP50 and exposed to 15 mM VPA at L4 larval stage for 24 hours at 20°C. The embryos were immunostained using antibodies recognizing H4K8ac. In the 100-cell stage embryos, baseline levels of this activating acetylation mark were observed in untreated control worms, whereas the <i>utx-1</i> (RNAi) and <i>utx-1(3136)</i> mutant worms showed highly increased acetylation levels (100% and 76% acetylation, respectively). Error bars represent SEM. <b>G</b>) Worms were treated as in F) and stained with a H3K36me2 antibody. At the 100-cell stage, control embryos showed 73% methylation while both <i>utx-1</i> (RNAi) and the <i>utx-1(3136)</i> mutant showed baseline methylation. By VPA treatment, control worms show baseline methylation whereas both <i>utx-1</i> (RNAi) and <i>utx-1(3136)</i> mutant shows 86% and 67% methylation respectively. Error bars represent SEM.</p