Cockayne Syndrome: The many challenges and approaches to understand a multifaceted disease

The striking and complex phenotype of Cockayne syndrome (CS) patients combines progeria-like features with developmental deficits. Since the establishment of the in vitro culture of skin fibroblasts derived from patients with CS in the 1970s, significant progress has been made in the understanding of the genetic alterations associated with the disease and their impact on molecular, cellular, and organismal functions. In this review, we provide a historic perspective on the research into CS by revisiting seminal papers in this field. We highlighted the great contributions of several researchers in the last decades, ranging from the cloning and characterization of CS genes to the molecular dissection of their roles in DNA repair, transcription, redox processes and metabolism control. We also provide a detailed description of all pathological mutations in genes ERCC6 and ERCC8 reported to date and their impact on CS-related proteins. Finally, we review the contributions (and limitations) of many genetic animal models to the study of CS and how cutting-edge technologies, such as cell reprogramming and state-of-the-art genome editing, are helping us to address unanswered questions

In vivo effects of Nucleotide Excision Repair related DNA lesions

A molécula de DNA, responsável por carregar informações genéticas, está sob constante estresse químico e físico, proveniente de fontes endógenas e exógenas, que pode levar à formação de lesões no DNA. Para lidar com esses danos, células dispõem de mecanismos de reparo, sendo as lesões que distorcem a molécula de DNA reparadas pela via de Reparo por Excisão de Nucleotídeos (NER). Deficiências em genes da via NER podem levar à doenças humanas, como o Xeroderma Pigmentosum (XP) e a Síndrome de Cockayne (CS), caracterizadas principalmente por um grande aumento na incidência de câncer de pele e por neurodegeneração relacionada à um fenótipo de envelhecimento precoce, respectivamente. Para melhor compreendermos dessas doenças, assim como os efeitos sistêmicos das lesões de DNA relacionadas à via NER, são utilizados modelos de camundongos nocaute (KO), assim como fontes exógenas geradoras de danos no DNA, como a radiação ultravioleta (UVR). Neste trabalho, usamos dois modelos deficientes em NER para estudar os efeitos in vivo de lesões relacionadas à via NER, sendo o primeiro um modelo que mimetiza XP e o segundo CS. No primeiro modelo, XPA KO, estudamos o efeito das principais lesões geradas por UVR, os dímeros de pirimidina ciclobutano (CPDs) e os pirimidina (6-4) pirimidona fotoprodutos (6-4PPs) em queratinócitos. Para tanto, utilizamos fotoliases, enzimas capazes de reparar especificamente ou lesões do tipo CPD (CPD-phl) ou 6-4PP (6-4PP-phl). Observamos que em camundongos XPA KO, a remoção de CPDs foi capaz de inibir completamente a proliferação de células epidermais induzidas por UVR, enquanto a remoção de 6-4PPs reduziu, porém não impediu esse efeito. A remoção de lesões do tipo CPD ou 6-4PP foi capaz de diminuir os efeitos de morte celular e extravasamento de leucócitos na pele induzida por UVR em níveis similares, indicando que CPDs têm um maior impacto que 6-4PP sobre o efeito de hiperplasia, enquanto ambos tipos de lesão possuem efeitos similares na indução de apoptose e inflamação por UVR em camundongos XPA KO, tendo os queratinócitos um papel central na regulação desses efeitos. No segundo modelo, estudamos os efeitos de lesões relacionadas ao envelhecimento em camundongos duplo nocaute para os genes CSA/XPA (CX), previamente descrito como tendo morte prematura e neurodegeneração. Apesar de termos encontrado evidências de falhas na barreira hematoencefálica (BBB) nesses animais, não encontramos indícios de disfunção nas células endoteliais. Descobrimos, no entanto, um aumento significativo de marcadores de neuroinflamação, assim como ativação de astrócitos e microglia, os dois principais tipos celulares relacionados à ativação de inflamação no cérebro, indicando que a neuroinflamação pode estar relacionada à neurodegeneração e defeitos da BBB encontrados neste modelo. As descobertas nesses modelos deficientes em NER podem ajudar a elucidar o papel in vivo das lesões de DNA em relação à resposta de morte e proliferação celular, assim como demonstra novos impactos da DDR sobre a indução de inflamação, com esses efeitos tendo implicações na etiologia de XP e CS, assim como fenômenos associados a danos no DNA como câncer e envelhecimento.The DNA molecule, responsible for carrying genetic information, is under constant chemical and physical stress, both by endogenous and exogenous sources, which may lead to the formation of DNA lesions. These damages are dealt with using several different DNA repair mechanisms, with lesions that distort the DNA molecule are repaired by the Nucleotide Excision Repair (NER) pathway. Deficiencies in the genes related to NER may lead to human syndromes, such as Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS), characterized mainly by a severely increased skin cancer incidence and premature aging like (progeroid) neurodegeneration, respectively. In order to further study these diseases, as well as the role of NER-related DNA lesions in generating cellular and systemic effects, knockout (KO) mice models are often used, as well as exogenous DNA damaging sources, such as ultraviolet radiation (UVR). In this work, we used two NER deficient models to study in vivo effects of NER-related lesions, the first KO model mimicking XP and the second one CS. In the first model, we studied the effect of the main UVR generated photolesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) on keratinocytes, one of the main cell types in the epidermis. In order to study the effect of each lesions, we used photolyases, enzymes that specifically repair either CPD (CPD-phl) or 6-4PP (6-4PP-phl) lesions. We observed that in XPA KO mice, CPD removal in keratinocytes was able to completely inhibit UV induced epidermal cell proliferation and hyperplasia, while the removal of 6-4PPs in keratinocytes reduced, but not abolished these effects. The removal of either CPDs or 6-4PPs in keratinocytes was able to reduce UV induced cell death and leukocyte extravasation on similar levels, indicating that CPDs have a greater impact than 6-4PPs regarding the hyperplasia effect of UV irradiation, and that both types of DNA lesions have similar effects on promoting apoptosis and inflammation in XPA KO mice, with keratinocytes having a central role in regulating these effects. In the second model, we studied the effect of aging related lesions on CSA/XPA double knockout (CX) mice, previously established as exhibiting premature death and neurodegeneration. Although we found evidence of blood brain barrier (BBB) defects in CX mice, we did not find cell autonomous vascular dysfunction. However, we discovered a significant increase of neuroinflammation markers, as well as activation of astrocytes and microglia, the two main endogenous inflammation related cell types of the brain, which indicates that neuroinflammation could play a role in the neurodegenerative and BBB phenotype observed in this model. The findings in these two NER deficient models help elucidate the in vivo role of DNA lesions regarding cell death and proliferation response, as well demonstrating novel impacts of DDR on inflammation induction, with these effects having implications on the etiology of XP and CS, as well as DNA damage associated biological phenomena such as cancer and aging

Transcription blockage by DNA damage in nucleotide excision repair-related neurological dysfunctions

Human genetic syndromes deficient in nucleotide excision repair (NER), such as xeroderma pigmentosum and Cockayne syndrome, may present neurological abnormalities and premature aging symptoms. Unrepaired endogenously generated DNA damage that hampers transcription is a strong candidate that contributes to the development of these severe effects in neuronal tissue. Endogenous lesions include those generated due to byproducts of cellular metabolisms, such as reactive oxygen species. This review presents much of the evidence on the mechanisms related to neurodegenerative processes associated with DNA damage responses. The primary focus is on the effects of the transcription machinery, including the accumulation of DNA•RNA hybrids (R-loops) that, in turn, influence DNA damage and repair metabolism. Moreover, several neuronal tissues present higher expression of long genes, a genomic subset more affected by DNA lesions, which may explain part of the neurological abnormalities in these patients. Also, neuronal tissues have different DNA repair capabilities that might result in different neurological consequences, as observed in patients and NER deficient animal models. The better understanding of how the accumulation of transcription blocking lesions can lead to neurological abnormalities and premature aging-like phenotypes may assist us in finding potential biomarkers and therapeutic targets that might improve the lives of these patients, as well as other neurological disorders in the general population.This work was supported under the International Collaboration Research Funding from São Paulo Research Foundation (FAPESP, SP, Brazil) and the Netherlands Organization for Scientific Research – NWO (Grant #2019/19435-3). Financial support was also received from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant #308868/2018-8) and Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES, Brasília, DF, Brazil, financial code 001)

Neurovascular dysfunction and neuroinflammation in a Cockayne syndrome mouse model

Cockayne syndrome (CS) is a rare, autosomal genetic disorder characterized by premature aging-like features, such as cachectic dwarfism, retinal atrophy, and progressive neurodegeneration. The molecular defect in CS lies in genes associated with the transcription-coupled branch of the nucleotide excision DNA repair (NER) pathway, though it is not yet clear how DNA repair deficiency leads to the multiorgan dysfunction symptoms of CS. In this work, we used a mouse model of severe CS with complete loss of NER (Csa−/−/Xpa−/−), which recapitulates several CS-related phenotypes, resulting in premature death of these mice at approximately 20 weeks of age. Although this CS model exhibits a severe progeroid phenotype, we found no evidence of in vitro endothelial cell dysfunction, as assessed by measuring population doubling time, migration capacity, and ICAM-1 expression. Furthermore, aortas from CX mice did not exhibit early senescence nor reduced angiogenesis capacity. Despite these observations, CX mice presented blood brain barrier disruption and increased senescence of brain endothelial cells. This was accompanied by an upregulation of inflammatory markers in the brains of CX mice, such as ICAM-1, TNFα, p-p65, and glial cell activation. Inhibition of neovascularization did not exacerbate neither astro- nor microgliosis, suggesting that the pro-inflammatory phenotype is independent of the neurovascular dysfunction present in CX mice. These findings have implications for the etiology of this disease and could contribute to the study of novel therapeutic targets for treating Cockayne syndrome patients.ISSN:1945-458

Biomass burning in the Amazon region causes DNA damage and cell death in human lung cells

Abstract Most of the studies on air pollution focus on emissions from fossil fuel burning in urban centers. However, approximately half of the world's population is exposed to air pollution caused by biomass burning emissions. In the Brazilian Amazon population, over 10 million people are directly exposed to high levels of pollutants resulting from deforestation and agricultural fires. This work is the first study to present an integrated view of the effects of inhalable particles present in emissions of biomass burning. Exposing human lung cells to particulate matter smaller than 10 µm (PM10), significantly increased the level of reactive oxygen species (ROS), inflammatory cytokines, autophagy, and DNA damage. Continued PM10 exposure activated apoptosis and necrosis. Interestingly, retene, a polycyclic aromatic hydrocarbon present in PM10, is a potential compound for the effects of PM10, causing DNA damage and cell death. The PM10 concentrations observed during Amazon biomass burning were sufficient to induce severe adverse effects in human lung cells. Our study provides new data that will help elucidate the mechanism of PM10-mediated lung cancer development. In addition, the results of this study support the establishment of new guidelines for human health protection in regions strongly impacted by biomass burning