HiPSC-derived 3D neural models reveal neurodevelopmental pathomechanisms of the Cockayne Syndrome B.

Cockayne Syndrome B (CSB) is a hereditary multiorgan syndrome which-through largely unknown mechanisms-can affect the brain where it clinically presents with microcephaly, intellectual disability and demyelination. Using human induced pluripotent stem cell (hiPSC)-derived neural 3D models generated from CSB patient-derived and isogenic control lines, we here provide explanations for these three major neuropathological phenotypes. In our models, CSB deficiency is associated with (i) impaired cellular migration due to defective autophagy as an explanation for clinical microcephaly; (ii) altered neuronal network functionality and neurotransmitter GABA levels, which is suggestive of a disturbed GABA switch that likely impairs brain circuit formation and ultimately causes intellectual disability; and (iii) impaired oligodendrocyte maturation as a possible cause of the demyelination observed in children with CSB. Of note, the impaired migration and oligodendrocyte maturation could both be partially rescued by pharmacological HDAC inhibition.

Cockayne Syndrome Linked to Elevated R-Loops Induced by Stalled RNA Polymerase II during Transcription Elongation.

Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.

CS proteins and ubiquitination: orchestrating DNA repair with transcription and cell division.

To face genotoxic stress, eukaryotic cells evolved extremely refined mechanisms. Defects in counteracting the threat imposed by DNA damage underlie the rare disease Cockayne syndrome (CS), which arises from mutations in the CSA and CSB genes. Although initially defined as DNA repair proteins, recent work shows that CSA and CSB act instead as master regulators of the integrated response to genomic stress by coordinating DNA repair with transcription and cell division. CSA and CSB exert this function through the ubiquitination of target proteins, which are effectors/regulators of these processes. This review describes how the ubiquitination of target substrates is a common denominator by which CSA and CSB participate in different aspects of cellular life and how their mutation gives rise to the complex disease CS.

Spectrum of ERCC6-Related Cockayne Syndrome (Type B): From Mild to Severe Forms.

(1) Background: Cockayne syndrome (CS) is an ultra-rare multisystem disorder, classically subdivided into three forms and characterized by a clinical spectrum without a clear genotype-phenotype correlation for both the two causative genes ERCC6 (CS type B) and ERCC8 (CS type A). We assessed this, presenting a series of patients with genetically confirmed CSB. (2) Materials and Methods: We retrospectively collected demographic, clinical, genetic, neuroimaging, and serum neurofilament light-chain (sNFL) data about CSB patients; diagnostic and severity scores were also determined. (3) Results: Data of eight ERCC6/CSB patients are presented. Four patients had CS I, three patients CS II, and one patient CS III. Various degrees of ataxia and spasticity were cardinal neurologic features, with variably combined systemic characteristics. Mean age at diagnosis was lower in the type II form, in which classic CS signs were more evident. Interestingly, sNFL determination appeared to reflect clinical classification. Two novel premature stop codon and one novel missense variants were identified. All CS I subjects harbored the p.Arg735Ter variant; the milder CS III subject carried the p.Leu764Ser missense change. (4) Conclusion: Our work confirms clinical variability also in the ERCC6/CSB type, where manifestations may range from severe involvement with prenatal or neonatal onset to normal psychomotor development followed by progressive ataxia. We propose, for the first time in CS, sNFL as a useful peripheral biomarker, with increased levels compared to currently available reference values and with the potential ability to reflect disease severity.

Transcription-coupled repair of DNA-protein cross-links depends on CSA and CSB.

Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

Hepatotoxicity of metronidazole in Cockayne syndrome: A clinical report.

Cockayne syndrome (CS) is a rare autosomal recessive genetic disorder characterized by growth failure and progressive multisystem dysfunction caused by deficient nucleotide excision repair. Whereas metronidazole (MTZ) hepatotoxicity is quite rare in the general population, cases of severe hepatic reaction to MTZ have been reported in CS patients. We report here the case of a 21-year-old CS patient who presented with jaundice following one week of treatment with MTZ combined with spiramycin for dental care. This case is the first one documented with a liver biopsy. Histopathological analysis revealed portal and lobular inflammation with predominance of neutrophils, ballooning degeneration and severe cholestasis without bile duct damage. The outcome was marked by regression of jaundice over 6 weeks. Analysis of this case highlights the probable responsibility of MTZ and adds support to the recommendation to strictly avoid the prescription of this drug in CS patients.

Aldehyde-driven transcriptional stress triggers an anorexic DNA damage response.

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.

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.

Cockayne syndrome group A and ferrochelatase finely tune ribosomal gene transcription and its response to UV irradiation.

CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.

Cockayne Syndrome-Associated CSA and CSB Mutations Impair Ribosome Biogenesis, Ribosomal Protein Stability, and Global Protein Folding.

Cockayne syndrome (CS) is a developmental disorder with symptoms that are typical for the aging body, including subcutaneous fat loss, alopecia, and cataracts. Here, we show that in the cells of CS patients, RNA polymerase I transcription and the processing of the pre-rRNA are disturbed, leading to an accumulation of the 18S-E intermediate. The mature 18S rRNA level is reduced, and isolated ribosomes lack specific ribosomal proteins of the small 40S subunit. Ribosomal proteins are susceptible to unfolding and the CS cell proteome is heat-sensitive, indicating misfolded proteins and an error-prone translation process in CS cells. Pharmaceutical chaperones restored impaired cellular proliferation. Therefore, we provide evidence for severe protein synthesis malfunction, which together with a loss of proteostasis constitutes the underlying pathophysiology in CS.

Skin Abnormalities in Disorders with DNA Repair Defects, Premature Aging, and Mitochondrial Dysfunction.

Defects in DNA repair pathways and alterations of mitochondrial energy metabolism have been reported in multiple skin disorders. More than 10% of patients with primary mitochondrial dysfunction exhibit dermatological features including rashes and hair and pigmentation abnormalities. Accumulation of oxidative DNA damage and dysfunctional mitochondria affect cellular homeostasis leading to increased apoptosis. Emerging evidence demonstrates that genetic disorders of premature aging that alter DNA repair pathways and cause mitochondrial dysfunction, such as Rothmund-Thomson syndrome, Werner syndrome, and Cockayne syndrome, also exhibit skin disease. This article summarizes recent advances in the research pertaining to these syndromes and molecular mechanisms underlying their skin pathologies.

COFS type 3 in an Indian family with antenatally detected arthrogryposis.

Fetal akinesia and contractures can be caused by mutations in various genes that lead to overlapping phenotypes with contractures, rocker bottom feet, cerebellar hypoplasia, ventriculomegaly, growth retardation, pulmonary hypoplasia, cystic hygroma and cleft palate in various combinations. Cerebro-oculo-facio-skeletal (COFS) syndrome is a condition resulting from defects in DNA repair pathway, and genes involved include ERCC1 (COFS), ERCC2 (XPD), ERCC5(XPG), and ERCC6 (CSB). It is a severe disorder presenting in fetal or neonatal period with microcephaly, arthrogryposis, prominent nose, and kyphoscoliosis, and leads to early death in childhood. We report a baby with antenatally identified arthrogryposis in which the homozygous pathogenic variant in exon 8 was identified in ERCC5 gene, by targeted next generation sequencing. This was predicted to cause premature chain termination in the protein. ERCC5 gene is mainly implicated in xeroderma pigmentosum, sometimes in COFS syndrome.

Review of How Genetic Research on Segmental Progeroid Syndromes Has Documented Genomic Instability as a Hallmark of Aging But Let Us Now Pursue Antigeroid Syndromes!

The purpose of this early contribution to the new Fellows Forum of this pioneering journal for what is now called Geroscience is to provide an example of how the author's interest in using the emerging tools of human genetics has led to strong support for one of the hallmarks of aging-Genomic Instability. We shall also briefly review our emerging interests in the genetic analysis of what we have called Antigeroid Syndromes. While there has been significant progress in that direction via genetic studies of centenarians, the search for genetic pathways that make individuals unusually resistant or resilient to the ravages of specific geriatric disorders has been comparatively neglected. We refer to these disorders as Unimodal Antigeroid Syndromes. It is our hope that our young colleagues will consider research efforts in that direction.

Cockayne syndrome proteins CSA and CSB maintain mitochondrial homeostasis through NAD+ signaling.

Cockayne syndrome (CS) is a rare premature aging disease, most commonly caused by mutations of the genes encoding the CSA or CSB proteins. CS patients display cachectic dwarfism and severe neurological manifestations and have an average life expectancy of 12 years. The CS proteins are involved in transcription and DNA repair, with the latter including transcription-coupled nucleotide excision repair (TC-NER). However, there is also evidence for mitochondrial dysfunction in CS, which likely contributes to the severe premature aging phenotype of this disease. While damaged mitochondria and impaired mitophagy were characterized in mice with CSB deficiency, such changes in the CS nematode model and CS patients are not fully known. Our cross-species transcriptomic analysis in CS postmortem brain tissue, CS mouse, and nematode models shows that mitochondrial dysfunction is indeed a common feature in CS. Restoration of mitochondrial dysfunction through NAD+ supplementation significantly improved lifespan and healthspan in the CS nematodes, highlighting mitochondrial dysfunction as a major driver of the aging features of CS. In cerebellar samples from CS patients, we found molecular signatures of dysfunctional mitochondrial dynamics and impaired mitophagy/autophagy. In primary cells depleted for CSA or CSB, this dysfunction can be corrected with supplementation of NAD+ precursors. Our study provides support for the interconnection between major causative aging theories, DNA damage accumulation, mitochondrial dysfunction, and compromised mitophagy/autophagy. Together, these three agents contribute to an accelerated aging program that can be averted by cellular NAD+ restoration.

Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells.

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5',8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3-6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

Atypical features and de novo heterozygous mutations in two siblings with Cockayne syndrome.

BACKGROUND: Cockayne syndrome (CS) is a rare autosomal recessive disorder which displays multiorgan dysfunction, especially within the nervous system including psychomotor retardation, cerebral atrophy, microcephaly, cognitive dysfunction, mental retardation, and seizures. Many genetic variations reported were related to this syndrome, but splicing mutations with cardiac anomalies have not been found in previous studies. METHODS: Herein, we described a pair of brothers and sisters who present essential manifestations of CS including premature feature, developmental delay, growth failure, microcephaly, and characteristic facial features, such as sunken eyes and a beaked nose. Interestingly, the brother also presented with atypical features which included cardiac anomalies such as left atrioventricular enlargement and cardiac dysfunction such as dilated cardiomyopathy. In addition, whole exome sequencing and RNA sequencing were employed to analyze their genetic landscape. RESULTS: WES analysis showed that these two cases carried double unreported heterozygous spliced mutations in the excision repair cross-complementing group 8 (ERCC8, also known as CSA, NM_000082) gene, which were c.78-2 (IVS1) A>T and c.1042-1 (IVS10) G>A, respectively. Moreover, transcript sequencing analysis validated these mutation sites. In this study, Gene Ontology enrichment and KEGG pathway analyses from RNA sequencing demonstrated similarities but some differences when compared with previous studies. CONCLUSION: For patients with Cockayne syndrome, cardiac changes need to be monitored carefully, especially for cases with splicing mutations of the ERCC8 gene.

Prenatal diagnosis of cerebro-oculo-facio-skeletal syndrome: Report of three fetuses and review of the literature.

Cerebro-oculo-facio-skeletal syndrome (COFS) is a rare autosomal recessive neurodegenerative disease belonging to the family of DNA repair disorders, characterized by microcephaly, congenital cataracts, facial dysmorphism and arthrogryposis. Here, we describe the detailed morphological and microscopic phenotype of three fetuses from two families harboring ERCC5/XPG likely pathogenic variants, and review the five previously reported fetal cases. In addition to the classical features of COFS, the fetuses display thymus hyperplasia, splenomegaly and increased hematopoiesis. Microencephaly is present in the three fetuses with delayed development of the gyri, but normal microscopic anatomy at the supratentorial level. Microscopic anomalies reminiscent of pontocerebellar hypoplasia are present at the infratentorial level. In conclusion, COFS syndrome should be considered in fetuses when intrauterine growth retardation is associated with microcephaly, arthrogryposis and ocular anomalies. Further studies are needed to better understand XPG functions during human development.

Heterogeneity and overlaps in nucleotide excision repair disorders.

Nucleotide excision repair (NER) is an essential DNA repair pathway devoted to the removal of bulky lesions such as photoproducts induced by the ultraviolet (UV) component of solar radiation. Deficiencies in NER typically result in a group of heterogeneous distinct disorders ranging from the mild UV sensitive syndrome to the cancer-prone xeroderma pigmentosum and the neurodevelopmental/progeroid conditions trichothiodystrophy, Cockayne syndrome and cerebro-oculo-facio-skeletal-syndrome. A complicated genetic scenario underlines these disorders with the same gene linked to different clinical entities as well as different genes associated with the same disease. Overlap syndromes with combined hallmark features of different NER disorders can occur and sporadic presentations showing extra features of the hematological disorder Fanconi Anemia or neurological manifestations mimicking Hungtinton disease-like syndromes have been described. Here, we discuss the multiple functions of the five major pleiotropic NER genes (ERCC3/XPB, ERCC2/XPD, ERCC5/XPG, ERCC1 and ERCC4/XPF) and their relevance in phenotypic complexity. We provide an update of mutational spectra and examine genotype-phenotype relationships. Finally, the molecular defects that could explain the puzzling overlap syndromes are discussed.

Renal disease in Cockayne syndrome.

BACKGROUND: Cockayne Syndrome (CS) is a rare autosomal recessive multi-systemic disorder, characterized; by developmental delay, microcephaly, severe growth failure and sensorial impairment. Renal complications have been reported but remain underinvestigated. The objective of this study was to perform a review of renal disease in a cohort of CS patients. METHODS: We retrospectively collected relevant clinical, biochemical and genetic data from a cohort of 136 genetically confirmed CS patients. Blood pressure (BP), proteinuria, albuminemia, uric acid, creatinine clearance, renal ultrasounds and renal biopsy result were analysed. RESULTS: Thirty-two patients had a renal investigation. We found that 69% of investigated patients had a renal disorder and/or an elevated BP. Fifteen out of 21 patients (71% of investigated patients) had an increased BP, 10 out of 16 patients (62% of investigated patients) presented with proteinuria and 4 of them had a nephrotic syndrome. Thirteen patients out of 29 (45%) had a decreased Glomerular Filtration Rate (GFR), 18 out of 25 patients (72%) had a hyperuricemia. No correlation with the genetic background or clinical types of CS was found, except for the renal clearance. CONCLUSIONS: Renal disease, increased blood pressure and hyperuricemia were highly prevalent in our study. We believe that CS patients should benefit from a nephrological follow-up and that anti-uric acid drug and Angiotensin-converting enzyme (ACE) inhibitor should be discussed in these patients.

Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction.

Cockayne syndrome (CS) is a rare autosomal recessive inherited disorder characterized by a variety of clinical features, including increased sensitivity to sunlight, progressive neurological abnormalities, and the appearance of premature aging. However, the pathogenesis of CS remains unclear due to the limitations of current disease models. Here, we generate integration-free induced pluripotent stem cells (iPSCs) from fibroblasts from a CS patient bearing mutations in CSB/ERCC6 gene and further derive isogenic gene-corrected CS-iPSCs (GC-iPSCs) using the CRISPR/Cas9 system. CS-associated phenotypic defects are recapitulated in CS-iPSC-derived mesenchymal stem cells (MSCs) and neural stem cells (NSCs), both of which display increased susceptibility to DNA damage stress. Premature aging defects in CS-MSCs are rescued by the targeted correction of mutant ERCC6. We next map the transcriptomic landscapes in CS-iPSCs and GC-iPSCs and their somatic stem cell derivatives (MSCs and NSCs) in the absence or presence of ultraviolet (UV) and replicative stresses, revealing that defects in DNA repair account for CS pathologies. Moreover, we generate autologous GC-MSCs free of pathogenic mutation under a cGMP (Current Good Manufacturing Practice)-compliant condition, which hold potential for use as improved biomaterials for future stem cell replacement therapy for CS. Collectively, our models demonstrate novel disease features and molecular mechanisms and lay a foundation for the development of novel therapeutic strategies to treat CS.