Abrey J. Yeo

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx−/− revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation

Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.

R-Loops in Proliferating Cells but Not in the Brain: Implications for AOA2 and Other Autosomal Recessive Ataxias

Disruption of the Setx gene, defective in ataxia oculomotor apraxia type 2 (AOA2) leads to the accumulation of DNA/RNA hybrids (R-loops), failure of meiotic recombination and infertility in mice. We report here the presence of R-loops in the testes from other autosomal recessive ataxia mouse models, which correlate with fertility in these disorders. R-loops were coincident in cells showing high basal levels of DNA double strand breaks and in those cells undergoing apoptosis. Depletion of Setx led to high basal levels of R-loops and these were enhanced further by DNA damage both in vitro and in vivo in tissues with proliferating cells. There was no evidence for accumulation of R-loops in the brains of mice where Setx, Atm, Tdp1 or Aptx genes were disrupted. These data provide further evidence for genome destabilization as a consequence of disrupted transcription in the presence of DNA double strand breaks arising during DNA replication or recombination. They also suggest that R-loop accumulation does not contribute to the neurodegenerative phenotype in these autosomal recessive ataxias.

Histologic and phenotypic factors and MC1R status associated with BRAF(V600E), BRAF(V600K), and NRAS mutations in a community-based sample of 414 cutaneous melanomas

Cutaneous melanomas arise through causal pathways involving interplay between exposure to UV radiation and host factors, resulting in characteristic patterns of driver mutations in BRAF, NRAS, and other genes. To gain clearer insights into the factors contributing to somatic mutation genotypes in melanoma, we collected clinical and epidemiologic data, performed skin examinations, and collected saliva and tumor samples from a community-based series of 414 patients aged 18 to 79, newly diagnosed with cutaneous melanoma. We assessed constitutional DNA for nine common polymorphisms in melanocortin-1 receptor gene (MC1R). Tumor DNA was assessed for somatic mutations in 25 different genes. We observed mutually exclusive mutations in BRAF(V600E) (26%), BRAF(V600K) (8%), BRAF(other) (5%), and NRAS (9%). Compared to patients with BRAF wild-type melanomas, those with BRAF(V600E) mutants were significantly younger, had more nevi but fewer actinic keratoses, were more likely to report a family history of melanoma, and had tumors that were more likely to harbor neval remnants. BRAF(V600K) mutations were also associated with high nevus counts. Both BRAF(V600K) and NRAS mutants were associated with older age but not with high sun exposure. We also found no association between MC1R status and any somatic mutations in this community sample of cutaneous melanomas, contrary to earlier reports.

A new model to study neurodegeneration in ataxia oculomotor apraxia type 2

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia. Recent evidence suggests that the protein defective in this syndrome, senataxin (SETX), functions in RNA processing to protect the integrity of the genome. To date, only patient-derived lymphoblastoid cells, fibroblasts and SETX knockdown cells were available to investigate AOA2. Recent disruption of the Setx gene in mice did not lead to neurobehavioral defects or neurodegeneration, making it difficult to study the etiology of AOA2. To develop a more relevant neuronal model to study neurodegeneration in AOA2, we derived neural progenitors from a patient with AOA2 and a control by induced pluripotent stem cell (iPSC) reprogramming of fibroblasts. AOA2 iPSC and neural progenitors exhibit increased levels of oxidative damage, DNA double-strand breaks, increased DNA damage-induced cell death and R-loop accumulation. Genome-wide expression and weighted gene co-expression network analysis in these neural progenitors identified both previously reported and novel affected genes and cellular pathways associated with senataxin dysfunction and the pathophysiology of AOA2, providing further insight into the role of senataxin in regulating gene expression on a genome-wide scale. These data show that iPSCs can be generated from patients with the autosomal recessive ataxia, AOA2, differentiated into neurons, and that both cell types recapitulate the AOA2 cellular phenotype. This represents a novel and appropriate model system to investigate neurodegeneration in this syndrome.

Senataxin protects the genome: implications for neurodegeneration and other abnormalities

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive disorder characterized by cerebellar atrophy, peripheral neuropathy, loss of Purkinje cells and elevated α-fetoprotein. AOA2 is caused by mutations in the SETX gene that codes for the high molecular weight protein senataxin. Mutations in this gene also cause dominant neurodegenerative disorders. Similar to that observed for other autosomal recessive ataxias, this protein protects the integrity of the genome against oxidative and other forms of DNA damage to reduce the risk of neurodegeneration. Senataxin functions in transcription termination and RNA splicing and it has been shown to resolve RNA/DNA hybrids (R-loops) that arise at transcription pause sites or when transcription is blocked. Recent data suggest that this protein functions at the interface between transcription and DNA replication to minimise the risk of collision and maintain genome stability. Our recent data using SETX gene-disrupted mice revealed that male mice were defective in spermatogenesis and were infertile. DNA double strand-breaks persisted throughout meiosis and crossing-over failed in SETX mutant mice. These changes can be explained by the accumulation of R-loops, which interfere with Holiday junctions and crossing-over. We also showed that senataxin was localized to the XY body in pachytene cells and was involved in transcriptional silencing of these chromosomes. While the defect in meiotic recombination was striking in these animals, there was no evidence of neurodegeneration as observed in AOA2 patients. We discuss here potentially different roles for senataxin in proliferating and post-mitotic cells.

Loss of ATM in Airway Epithelial Cells Is Associated with Susceptibility to Oxidative Stress

Ataxia-telangiectasia (A-T) is characterized by chromosomal instability, immunodeficiency, cancer susceptibility, neurodegeneration, and pulmonary disease (1). One of the cardinal features of A-T is recurrent sinopulmonary infection, which is associated with the development of bronchiectasis and interstitial lung disease (2). Respiratory disease causes significant morbidity and mortality in patients with A-T, being responsible for up to 40% of deaths (3). Respiratory infections with Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, and Haemophilus influenzae have been reported (4). A recent report showed that S. pneumoniae caused DNA damage and apoptosis in lung cells by secretion of H2O2, an agent known to cause oxidative stress (5). In this context, there is accumulating evidence that oxidative stress is associated with A-T disease, including increased sensitivity to oxidative damage, elevated levels of reactive oxygen species in A-T cells, protection against damage by antioxidants, and the observation that ataxia-telangiectasia mutated (ATM), the protein defective in A-T, is activated by oxidative stress (6–9). On the basis of these observations, the aim of this pilot study was to determine whether airway epithelial cells from patients with A-T show increased susceptibility to oxidative stress. Some of the results of these studies have been previously reported in the form of an abstract (10).

Therapeutic targets and investigated treatments for Ataxia-Telangiectasia

ABSTRACT Introduction: Ataxia-Telangiectasia (A-T) is an autosomal recessive multisystem disease affecting the brain, immune system, lungs, liver and also characterised by an enhanced risk of lymphoid and other tumours. At present there is no cure for A-T with management relying on supportive care using symptom-specific medications. Identification of the gene defective in this syndrome, ATM, and further characterization of the disorder together with greater insight into the function of the ATM protein has provided greater opportunity for the development of potential therapies. Areas covered: Here we review conventional as well as more recently developed approaches to manage the symptoms of patients with A-T. In addition we explore ongoing and potential strategies for therapy involving gene correction, stem cells and use of antioxidants and anti-inflammatory agents. Expert opinion: Prevention or arrest of the progressive neurodegeneration, the most debilitating feature of A-T, represents a major goal in the development of a cure for this disorder. However, since lung disease and increased risk of cancer are responsible for the majority of mortality in A-T, a greater understanding of these pathologies together with more effective approaches to treatment is required in the overall management of patients.