Distinct DNA repair mechanisms prevent formaldehyde toxicity during development, reproduction and aging.

Formaldehyde (FA) is a recognized environmental and metabolic toxin implicated in cancer development and aging. Inherited mutations in the FA-detoxifying enzymes ADH5 and ALDH2 genes lead to FA overload in the severe multisystem AMeD syndrome. FA accumulation causes genome damage including DNA-protein-, inter- and intra-strand crosslinks and oxidative lesions. However, the influence of distinct DNA repair systems on organismal FA resistance remains elusive. We have here investigated the consequence of a range of DNA repair mutants in a model of endogenous FA overload generated by downregulating the orthologs of human ADH5 and ALDH2 in C. elegans. We have focused on the distinct components of nucleotide excision repair (NER) during developmental growth, reproduction and aging. Our results reveal three distinct modes of repair of FA-induced DNA damage: Transcription-coupled repair (TCR) operating NER-independently during developmental growth or through NER during adulthood, and, in concert with global-genome (GG-) NER, in the germline and early embryonic development. Additionally, we show that the Cockayne syndrome B (CSB) factor is involved in the resolution of FA-induced DNA-protein crosslinks, and that the antioxidant and FA quencher N-acetyl-l-cysteine (NAC) reverses the sensitivity of detoxification and DNA repair defects during development, suggesting a therapeutic intervention to revert FA-pathogenic consequences.

A C. elegans model for neurodegeneration in Cockayne syndrome.

Cockayne syndrome (CS) is a congenital syndrome characterized by growth and mental retardation, and premature ageing. The complexity of CS and mammalian models warrants simpler metazoan models that display CS-like phenotypes that could be studied in the context of a live organism. Here, we provide a characterization of neuronal and mitochondrial aberrations caused by a mutation in the csb-1 gene in Caenorhabditis elegans. We report a progressive neurodegeneration in adult animals that is enhanced upon UV-induced DNA damage. The csb-1 mutants show dysfunctional hyperfused mitochondria that degrade upon DNA damage, resulting in diminished respiratory activity. Our data support the role of endogenous DNA damage as a driving factor of CS-related neuropathology and underline the role of mitochondrial dysfunction in the disease.

Improved Biocompatibility of Amino-Functionalized Graphene Oxide in Caenorhabditis elegans.

Graphene oxide (GO) holds high promise for diagnostic and therapeutic applications in nanomedicine but reportedly displays immunotoxicity, underlining the need for developing functionalized GO with improved biocompatibility. This study describes adverse effects of GO and amino-functionalized GO (GONH2 ) during Caenorhabditis elegans development and ageing upon acute or chronic exposure. Chronic GO treatment throughout the C. elegans development causes decreased fecundity and a reduction of animal size, while acute treatment does not lead to any measurable physiological decline. However, RNA-Sequencing data reveal that acute GO exposure induces innate immune gene expression. The p38 MAP kinase, PMK-1, which is a well-established master regulator of innate immunity, protects C. elegans from chronic GO toxicity, as pmk-1 mutants show reduced tissue-functionality and facultative vivipary. In a direct comparison, GONH2 exposure does not cause detrimental effects in the wild type or in pmk-1 mutants, and the innate immune response is considerably less pronounced. This work establishes enhanced biocompatibility of amino-functionalized GO in a whole-organism, emphasizing its potential as a biomedical nanomaterial.

Systematic analysis of DNA crosslink repair pathways during development and aging in Caenorhabditis elegans.

DNA interstrand crosslinks (ICLs) are generated by endogenous sources and chemotherapeutics, and pose a threat to genome stability and cell survival. Using Caenorhabditis elegans mutants, we identify DNA repair factors that protect against the genotoxicity of ICLs generated by trioxsalen/ultraviolet A (TMP/UVA) during development and aging. Mutations in nucleotide excision repair (NER) components (e.g. XPA-1 and XPF-1) imparted extreme sensitivity to TMP/UVA relative to wild-type animals, manifested as developmental arrest, defects in adult tissue morphology and functionality, and shortened lifespan. Compensatory roles for global-genome (XPC-1) and transcription-coupled (CSB-1) NER in ICL sensing were exposed. The analysis also revealed contributions of homologous recombination (BRC-1/BRCA1), the MUS-81, EXO-1, SLX-1 and FAN-1 nucleases, and the DOG-1 (FANCJ) helicase in ICL resolution, influenced by the replicative-status of the cell/tissue. No obvious or critical role in ICL repair was seen for non-homologous end-joining (cku-80) or base excision repair (nth-1, exo-3), the Fanconi-related proteins BRC-2 (BRCA2/FANCD1) and FCD-2 (FANCD2), the WRN-1 or HIM-6 (BLM) helicases, or the GEN-1 or MRT-1 (SNM1) nucleases. Our efforts uncover replication-dependent and -independent ICL repair networks, and establish nematodes as a model for investigating the repair and consequences of DNA crosslinks in metazoan development and in adult post-mitotic and proliferative germ cells.

A simple answer to complex questions: Caenorhabditis elegans as an experimental model for examining the DNA damage response and disease genes.

The genetic information is constantly challenged by genotoxic attacks. DNA repair mechanisms evolved early in evolution and recognize and remove the various lesions. A complex network of DNA damage responses (DDR) orchestrates a variety of physiological adaptations to the presence of genome instability. Erroneous repair or malfunctioning of the DDR causes cancer development and the accumulation of DNA lesions drives the aging process. For understanding the complex DNA repair and DDR mechanisms it is pivotal to employ simple metazoan as model systems. The nematode Caenorhabditis elegans has become a well-established and popular experimental organism that allows dissecting genome stability mechanisms in dynamic and differentiated tissues and under physiological conditions. We provide an overview of the distinct advantages of the nematode system for studying DDR and provide a range of currently applied methodologies.

Light Sheet Microscopy to Measure Protein Dynamics.

Visualizing protein dynamics is the key to a quantitative understanding of molecular mechanisms in biological systems. Recent developments in fluorescent microscopy techniques allow for novel experimental approaches to study stochastic localization, distribution, and movement of fluorescently labeled molecules in high resolution as they occur over time in the context of living cells and whole organisms. Particularly suitable for such studies is the application of light sheet microscopy, as it enables rapid in vivo imaging of a wide range and size of specimen, from whole organisms and organs, down to the dynamics of subcellular structures and single molecules. This article summarizes the principles of light sheet microscopy and its advantages over other optical imaging techniques, such as light microscopy and confocal microscopy, and highlights recent innovations that significantly enhance spatio-temporal resolution. Also, this manuscript contains basic guidelines for the implementation of light sheet microscopy in the laboratory and presents thus far unpublished light sheet microscopy applications demonstrating the ability of this technology to measure protein dynamics in a whole-organism context. J. Cell. Physiol. 232: 27-35, 2017. © 2016 Wiley Periodicals, Inc.

Endogenous formaldehyde scavenges cellular glutathione resulting in redox disruption and cytotoxicity.

Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans. Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine.

Molecular pathology of rare progeroid diseases.

Progeroid syndromes (PSs) are characterized by the premature onset of age-related pathologies. The genetic mutations underlying PSs are functionally linked to genome maintenance and repair, supporting the causative role of DNA damage accumulation in aging. Recent advances from studies in animal models of PSs have provided new insight into the role of DNA repair mechanisms in human disease and the physiological adaptations to accumulating DNA damage during aging. The molecular pathology of PSs is reminiscent of the natural aging process, highlighting the relevance for a wide range of age-related diseases. Recent progress has led to the development of novel therapeutic strategies against age-related diseases that are relevant to rare diseases as well as the general aging population.

Transgenesis in Caenorhabditis elegans.

Two efficient strategies have been developed and are widely used for the genetic transformation of the nematode Caenorhabditis elegans, DNA microinjection, and DNA-coated microparticle bombardment. Both methodologies facilitate the delivery of exogenous DNA into the developing oocytes of adult hermaphrodite animals, which then generate transgenic worms among their progeny. Although both approaches share the common underlying principle of introducing foreign DNA into the germline of C. elegans, they offer distinct transformation outcomes. In this chapter, we present DNA microinjection and bombardment methods for transgenesis in C. elegans and provide time-tested procedures for their implementation. We also discuss their relative advantages as well as their limitations and evaluate their potential for a range of applications.

Restoration of Proteostasis in the Endoplasmic Reticulum Reverses an Inflammation-Like Response to Cytoplasmic DNA in Caenorhabditis elegans.

Innate immune responses protect organisms against various insults, but may lead to tissue damage when aberrantly activated. In higher organisms, cytoplasmic DNA can trigger inflammatory responses that can lead to tissue degeneration. Simpler metazoan models could shed new mechanistic light on how inflammatory responses to cytoplasmic DNA lead to pathologies. Here, we show that in a DNase II-defective Caenorhabditis elegans strain, persistent cytoplasmic DNA leads to systemic tissue degeneration and loss of tissue functionality due to impaired proteostasis. These pathological outcomes can be therapeutically alleviated by restoring protein homeostasis, either via ectopic induction of the ER unfolded protein response or N-acetylglucosamine treatment. Our results establish C. elegans as an ancestral metazoan model for studying the outcomes of inflammation-like conditions caused by persistent cytoplasmic DNA and provide insight into potential therapies for human conditions involving chronic inflammation.

Maintenance of Proteostasis by P Body-Mediated Regulation of eIF4E Availability during Aging in Caenorhabditis elegans.

Aging is accompanied by a pervasive collapse of proteostasis, while reducing general protein synthesis promotes longevity across taxa. Here, we show that the eIF4E isoform IFE-2 is increasingly sequestered in mRNA processing (P) bodies during aging and upon stress in Caenorhabditis elegans. Loss of the enhancer of mRNA decapping EDC-3 causes further entrapment of IFE-2 in P bodies and lowers protein synthesis rates in somatic tissues. Animals lacking EDC-3 are long lived and stress resistant, congruent with IFE-2-deficient mutants. Notably, neuron-specific expression of EDC-3 is sufficient to reverse lifespan extension, while sequestration of IFE-2 in neuronal P bodies counteracts age-related neuronal decline. The effects of mRNA decapping deficiency on stress resistance and longevity are orchestrated by a multimodal stress response involving the transcription factor SKN-1, which mediates lifespan extension upon reduced protein synthesis. Our findings elucidate a mechanism of proteostasis control during aging through P body-mediated regulation of protein synthesis in the soma.

Demonstrating Improved Multiple Transport-Mean-Free-Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics.

Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids. To this end, we apply LSFM to quantify the spatio-temporal localization of the GFP-tagged aging and stress response factor DAF-16/FOXO in transgenic C. elegans. Our analysis reveals a linear nuclear localization of DAF-16::GFP across tissues in response to heat stress, using a system that outperforms confocal scanning fluorescent microscopy in imaging speed, 3D resolution and reduced photo-toxicity. Furthermore, we present how PRT can improve the depth-to-resolution-ratio when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in a T47D cancer cell spheroid recorded with a customized OPT/LSFM system. Our studies demonstrate that LSFM combined with our novel approaches enables higher resolution and more accurate 3D quantification than previously applied technologies, proving its advance as new gold standard for fluorescence microscopy.

P-body and Stress Granule Quantification in Caenorhabditis elegans.

Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules are Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA degradation, translational repression and cellular mRNA transport (Sheth and Parker, 2003). Another type of RNP granules, the stress granules (SGs), majorly contain mRNAs associated with translation initiation factors and are formed upon stress-induced translational stalling (Kedersha et al., 2000 and 1999). Multiple evidence obtained from studies in unicellular organisms supports a model in which P bodies and SGs physically interact during cellular stress to direct mRNAs for transport, decay, temporal storage or reentry into translation (Anderson and Kedersha, 2008; Decker and Parker, 2012). The quantification, distribution and colocalization of P bodies and/or SGs are essential tools to study the composition of RNP granules and their contribution to fundamental cellular processes, such as stress response and translational regulation. In this protocol we describe a method to quantify P bodies and SGs in somatic tissues of the nematode Caenorhabditis elegans.

Caenorhabditis elegans Microinjection.

Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.

A customized light sheet microscope to measure spatio-temporal protein dynamics in small model organisms.

We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.

Automated motion correction for in vivo optical projection tomography.

In in vivo optical projection tomography (OPT), object motion will significantly reduce the quality and resolution of the reconstructed image. Based on the well-known Helgason-Ludwig consistency condition (HLCC), we propose a novel method for motion correction in OPT under parallel beam illumination. The method estimates object motion from projection data directly and does not require any other additional information, which results in a straightforward implementation. We decompose object movement into translation and rotation, and discuss how to correct for both translation and general motion simultaneously. Since finding the center of rotation accurately is critical in OPT, we also point out that the system's geometrical offset can be considered as object translation and therefore also calibrated through the translation estimation method. In order to verify the algorithm effectiveness, both simulated and in vivo OPT experiments are performed. Our results demonstrate that the proposed approach is capable of decreasing movement artifacts significantly thus providing high quality reconstructed images in the presence of object motion.

Microscopic optical projection tomography in vivo.

We describe a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. Our tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms.

Correction for specimen movement and rotation errors for in-vivo Optical Projection Tomography.

The application of optical projection tomography to in-vivo experiments is limited by specimen movement during the acquisition. We present a set of mathematical correction methods applied to the acquired data stacks to correct for movement in both directions of the image plane. These methods have been applied to correct experimental data taken from in-vivo optical projection tomography experiments in Caenorhabditis elegans. Successful reconstructions for both fluorescence and white light (absorption) measurements are shown. Since no difference between movement of the animal and movement of the rotation axis is made, this approach at the same time removes artifacts due to mechanical drifts and errors in the assumed center of rotation.

Suppression of polyglutamine-induced toxicity in cell and animal models of Huntington's disease by ubiquilin.

Expanded polyglutamine (polyQ) tracts are associated with the induction of protein aggregation and cause cytotoxicity in nine different neurodegenerative disorders. Here, we report that ubiquilin suppresses polyQ-induced protein aggregation and toxicity in cells and in an animal model of Huntington's disease. Overexpression of ubiquilin in HeLa cells and primary neurons reduced aggregation of polyQ-containing proteins and cell death induced by overexpression of a green fluorescent protein (GFP)-huntingtin fusion protein containing 74 polyQ repeats [GFP-Htt(Q74)], in a dose-dependent manner. Moreover, overexpression of ubiquilin suppressed oxidative stress-induced cell death in HeLa cell lines stably expressing GFP-Htt(Q74). In contrast, knockdown of ubiquilin expression in these cell lines was associated with increases in DNA fragmentation, caspase activation, GFP-fusion protein aggregation, and cell death. Caenorhabditis elegans lines expressing GFP-Htt fusion proteins in body wall muscle displayed a polyQ repeat length-dependent decrease in body movement compared with wild-type animals. RNA interference of the C. elegans ubiquilin gene exacerbated the motility defect, whereas overexpression of ubiquilin prevented, and could rescue, loss of worm movement induced by overexpression of GFP-Htt(Q55). These results suggest that ubiquilin might be a novel therapeutic target for treating polyQ diseases.