Lopes, Amanda et al. (2021) International Worm Meeting "A C. elegans model to study the molecular pathogenesis of Cockayne Syndrome progeria"

Lopes, Amanda, Rieckher, Matthias, Schumacher, Bjorn

[International Worm Meeting, 2021]

Cockayne Syndrome (CS) is a rare congenital disease that causes photosensitivity, growth and mental retardation, impaired nervous system development and premature ageing. The syndrome displays a complex array of symptoms that are caused by mutations in the CSB, or CSA gene, respectively, which facilitate DNA damage recognition in transcription-coupled nucleotide excision repair (TC-NER). While CS mouse models are photosensitive, and have been important for understanding some of the clinical features observed in humans, they lack concrete evidence for neurodegeneration. Here, we present evidence of neuronal and mitochondrial aberrations in csb-1(ok2335) mutant worms, which can be rescued by transgenic expression of pcsb-1CSB-1::GFP. Neurodegeneration manifests progressively and is paralleled by neuro-muscular functional decline substantiated by reduced pharyngeal pumping, locomotion, touch sensation and chemosensation abilities, which are further enhanced upon UVB-induced DNA damage or transcription-blocking lesions caused by the cytotoxin Illudin M. The csb-1 mutant shows the accumulation of dysfunctional mitochondria, and increased hyperfusion, which is exacerbated upon exposure to UVB, resulting in reduced respiratory activity. Our data support the causal role of endogenous DNA damage for neurodegeneration and mitochondrial dysfunction in CS, and warrants the use of the model for identifying pharmacological interventions to improve CS and ageing.

Rieckher, Matthias et al. (2015) International Worm Meeting "Investigations on cell- and tissue-specific activity of NER upon UVB irradiation in development and ageing of C. elegans."

Werthenbach, Paul, Lopes, Amanda, Rieckher, Matthias, Schumacher, Bjorn, Anton, Vincent

[International Worm Meeting, 2015]

DNA damage and genome instability are hallmarks of ageing. In humans, congenital defects in DNA repair mechanisms such as nucleotide excision repair (NER) lead to increased cancer risk, accelerated ageing including neurodegeneration and developmental growth defects. NER activity is highly conserved in C. elegans and NER deficient animals are incapable of repairing DNA damage inflicted by UV irradiation, resulting in growth arrest and decreased life span. We investigate the tissue-specific and age-dependent activity of NER in maintaining genome stability in C. elegans. To this end, we established an in vivo imaging system to monitor NER activity in C. elegans: We generated transgenic animals expressing the central NER factors XPA-1::GFP or CSB-1::GFP under control of the respective endogenous promoters. In vivo expression analysis reveals a progressive decline of XPA-1::GFP during ageing in all tissues, except the neuronal system. Further, we are performing tissue-specific rescue studies of the highly UVB sensitive xpa-1 mutant animals by constitutively driving expression of XPA-1::GFP in muscles, intestine, cuticle, and the neuronal system. We apply a UVC laser system for precise single-cell DNA damage induction in cuticle tissue, and observe rapid recruitment of XPA-1::GFP to the site of irradiation. This system allows investigating cell type-specific dynamics of DNA damage response factors during ageing and in the context of a whole organism. To further understand tissue-specific relevance of DNA repair, we created transgenic animals that ectopically express the A. thaliana photolyase PHR1, which specifically repairs cyclobutane pyrimidine dimers (CPDs), which comprise the main lesion type induced by UV. Ubiquitous and constitutive expression of PHR1::GFP enhances UV resistance of wild type and NER deficient animals. Understanding tissue-specific DNA damage responses and genome maintenance in C. elegans will lead to a better understanding of the complex human disorders that are caused by DNA repair defects and allow the development of intervention strategies to counteract age-related tissue-decline and promote longevity.

H Van Luenen et al. (1999) International C. elegans Meeting "World-wide worm SNPs: Dissecting the molecular evolution of the species using a high density SNP map"

R Plasterk, R Koch, H Van Luenen

[International C. elegans Meeting, 1999]

We would like to reconstruct the history of the C. elegans species at the genome level, therefore we sampled the genomes of four natural isolates (strain CB4857 isolated in Claremont, California, RC301 from Freiburg, Germany, TR403 from Madison, Wisconsin and AB1 from Adelaide, Australia) for single nucleotide polymorphisms (SNPs). Random genomic DNA fragments from the 4 strains were shotgun cloned and sequenced. There was no selection for transcribed or non-transcribed regions of the genome. In total we sequenced 1572 clones resulting in over 1 Mb of sequence information. The sequences are compared to the canonical Bristol N2 sequence to ask the question whether the clone maps to a unique sequence, and -if so- whether it contains polymorphisms. Once a SNP is identified we check other strains for the presence of the same polymorphism by PCR amplification and sequence analysis. In an initial experiment we found approximately one SNP per 3000 bp sequenced. The SNPs are randomly spread over the genome. Based on these observations we expect to find approximately 500 SNPs, one in every 200 kb. In the initial experiment we found, as expected, that several SNPs initially detected in one strain were also present in some but not all other strains. For example: a T in the Australian AB1, is a G at the same position in Bristol N2 in cosmid K10D2 at position 27946, and we found it to be like AB1 in the Californian CB4857 strain and the German RC301 strain, while the TR403 strain from Wisconsin resembles the Bristol N2 strain. Thus different patches of the genome have different ancestors. With our high density SNP map we will generate a genome map for each isolate which will show how each genome is patched together from a limited set of parental strains. The SNP's will be added to ACeDB, and can also be used as markers on the genetic map. They can be recognised by PCR followed by sequencing, but we also found that the SNPs we looked at could be visualised by SSCP analysis. We thank Jane Rogers and Amanda McMurray for their assistance in sequencing the clones.