General loss of proteostasis links Huntington disease to Cockayne syndrome.

Cockayne syndrome (CS) is an autosomal recessive disorder of developmental delay, multiple organ system degeneration and signs of premature ageing. We show here, using the RNA-seq data from two CS mutant cell lines, that the CS key transcriptional signature displays significant enrichment of neurodegeneration terms, including genes relevant in Huntington disease (HD). By using deep learning approaches and two published RNA-Seq datasets, the CS transcriptional signature highly significantly classified and predicted HD and control samples. Neurodegeneration is one hallmark of CS disease, and fibroblasts from CS patients with different causative mutations display disturbed ribosomal biogenesis and a consecutive loss of protein homeostasis - proteostasis. Encouraged by the transcriptomic data, we asked whether this pathomechanism is also active in HD. In different HD cell-culture models, we showed that mutant Huntingtin impacts ribosomal biogenesis and function. This led to an error-prone protein synthesis and, as shown in different mouse models and human tissue, whole proteome instability, and a general loss of proteostasis.

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.

A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function.

Cockayne syndrome (CS) is a premature ageing condition characterized by microcephaly, growth failure, and neurodegeneration. It is caused by mutations in ERCC6 or ERCC8 encoding for Cockayne syndrome B (CSB) and A (CSA) proteins, respectively. CSA and CSB have well-characterized roles in transcription-coupled nucleotide excision repair, responsible for removing bulky DNA lesions, including those caused by UV irradiation. Here, we report that CSA dysfunction causes defects in the nuclear envelope (NE) integrity. NE dysfunction is characteristic of progeroid disorders caused by a mutation in NE proteins, such as Hutchinson-Gilford progeria syndrome. However, it has never been reported in Cockayne syndrome. We observed CSA dysfunction affected LEMD2 incorporation at the NE and increased actin stress fibers that contributed to enhanced mechanical stress to the NE. Altogether, these led to NE abnormalities associated with the activation of the cGAS/STING pathway. Targeting the linker of the nucleoskeleton and cytoskeleton complex was sufficient to rescue these phenotypes. This work reveals NE dysfunction in a progeroid syndrome caused by mutations in a DNA damage repair protein, reinforcing the connection between NE deregulation and ageing.

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.

Differential processing of RNA polymerase II at DNA damage correlates with transcription-coupled repair syndrome severity.

DNA damage severely impedes gene transcription by RNA polymerase II (Pol II), causing cellular dysfunction. Transcription-Coupled Nucleotide Excision Repair (TC-NER) specifically removes such transcription-blocking damage. TC-NER initiation relies on the CSB, CSA and UVSSA proteins; loss of any results in complete TC-NER deficiency. Strikingly, UVSSA deficiency results in UV-Sensitive Syndrome (UVSS), with mild cutaneous symptoms, while loss of CSA or CSB activity results in the severe Cockayne Syndrome (CS), characterized by neurodegeneration and premature aging. Thus far the underlying mechanism for these contrasting phenotypes remains unclear. Live-cell imaging approaches reveal that in TC-NER proficient cells, lesion-stalled Pol II is swiftly resolved, while in CSA and CSB knockout (KO) cells, elongating Pol II remains damage-bound, likely obstructing other DNA transacting processes and shielding the damage from alternative repair pathways. In contrast, in UVSSA KO cells, Pol II is cleared from the damage via VCP-mediated proteasomal degradation which is fully dependent on the CRL4CSA ubiquitin ligase activity. This Pol II degradation might provide access for alternative repair mechanisms, such as GG-NER, to remove the damage. Collectively, our data indicate that the inability to clear lesion-stalled Pol II from the chromatin, rather than TC-NER deficiency, causes the severe phenotypes observed in CS.

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.

The ARK2N-CK2 complex initiates transcription-coupled repair through enhancing the interaction of CSB with lesion-stalled RNAPII.

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.

Cockayne Syndrome Patient iPSC-Derived Brain Organoids and Neurospheres Show Early Transcriptional Dysregulation of Biological Processes Associated with Brain Development and Metabolism.

Cockayne syndrome (CS) is a rare hereditary autosomal recessive disorder primarily caused by mutations in Cockayne syndrome protein A (CSA) or B (CSB). While many of the functions of CSB have been at least partially elucidated, little is known about the actual developmental dysregulation in this devasting disorder. Of particular interest is the regulation of cerebral development as the most debilitating symptoms are of neurological nature. We generated neurospheres and cerebral organoids utilizing Cockayne syndrome B protein (CSB)-deficient induced pluripotent stem cells derived from two patients with distinct severity levels of CS and healthy controls. The transcriptome of both developmental timepoints was explored using RNA-Seq and bioinformatic analysis to identify dysregulated biological processes common to both patients with CS in comparison to the control. CSB-deficient neurospheres displayed upregulation of the VEGFA-VEGFR2 signalling pathway, vesicle-mediated transport and head development. CSB-deficient cerebral organoids exhibited downregulation of brain development, neuron projection development and synaptic signalling. We further identified the upregulation of steroid biosynthesis as common to both timepoints, in particular the upregulation of the cholesterol biosynthesis branch. Our results provide insights into the neurodevelopmental dysregulation in patients with CS and strengthen the theory that CS is not only a neurodegenerative but also a neurodevelopmental disorder.

DNA repair deficiencies and neurodegeneration.

Neurodegenerative diseases are the second most prevalent cause of death in industrialized countries. Alzheimer's Disease is the most widespread and also most acknowledged form of dementia today. Together with Parkinson's Disease they account for over 90 % cases of neurodegenerative disorders caused by proteopathies. Far less known are the neurodegenerative pathologies in DNA repair deficiency syndromes. Such diseases like Cockayne - or Werner Syndrome are described as progeroid syndromes - diseases that cause the premature ageing of the affected persons, and there are clear implications of such diseases in neurologic dysfunction and degeneration. In this review, we aim to draw the attention on commonalities between proteopathy-associated neurodegeneration and neurodegeneration caused by DNA repair defects and discuss how mitochondria are implicated in the development of both disorder classes. Furthermore, we highlight how nematodes are a valuable and indispensable model organism to study conserved neurodegenerative processes in a fast-forward manner.

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.

Epigenomic signature of accelerated ageing in progeroid Cockayne syndrome.

Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS) are rare genetic disorders caused by mutation of the DNA repair and multifunctional CSA or CSB protein, but only CS patients display a progeroid and neurodegenerative phenotype, providing a unique conceptual and experimental paradigm. As DNA methylation (DNAm) remodelling is a major ageing marker, we performed genome-wide analysis of DNAm of fibroblasts from healthy, UVSS and CS individuals. Differential analysis highlighted a CS-specific epigenomic signature (progeroid-related; not present in UVSS) enriched in three categories: developmental transcription factors, ion/neurotransmitter membrane transporters and synaptic neuro-developmental genes. A large fraction of CS-specific DNAm changes were associated with expression changes in CS samples, including in previously reported post-mortem cerebella. The progeroid phenotype of CS was further supported by epigenomic hallmarks of ageing: the prediction of DNAm of repetitive elements suggested an hypomethylation of Alu sequences in CS, and the epigenetic clock returned a marked increase in CS biological age respect to healthy and UVSS cells. The epigenomic remodelling of accelerated ageing in CS displayed both commonalities and differences with other progeroid diseases and regular ageing. CS shared DNAm changes with normal ageing more than other progeroid diseases do, and included genes functionally validated for regular ageing. Collectively, our results support the existence of an epigenomic basis of accelerated ageing in CS and unveil new genes and pathways that are potentially associated with the progeroid/degenerative phenotype.

The nucleotide excision repair proteins through the lens of molecular dynamics simulations.

Mutations that affect the proteins responsible for the nucleotide excision repair (NER) pathway can lead to diseases such as xeroderma pigmentosum, trichothiodystrophy, Cockayne syndrome, and Cerebro-oculo-facio-skeletal syndrome. Hence, understanding their molecular behavior is needed to elucidate these diseases' phenotypes and how the NER pathway is organized and coordinated. Molecular dynamics techniques enable the study of different protein conformations, adaptable to any research question, shedding light on the dynamics of biomolecules. However, as important as they are, molecular dynamics studies focused on DNA repair pathways are still becoming more widespread. Currently, there are no review articles compiling the advancements made in molecular dynamics approaches applied to NER and discussing: (i) how this technique is currently employed in the field of DNA repair, focusing on NER proteins; (ii) which technical setups are being employed, their strengths and limitations; (iii) which insights or information are they providing to understand the NER pathway or NER-associated proteins; (iv) which open questions would be suited for this technique to answer; and (v) where can we go from here. These questions become even more crucial considering the numerous 3D structures published regarding the NER pathway's proteins in recent years. In this work, we tackle each one of these questions, revising and critically discussing the results published in the context of the NER pathway.

Cockayne syndrome group A protein localizes at centrosomes during mitosis and regulates Cyclin B1 ubiquitination.

Mutations in CSA and CSB proteins cause Cockayne syndrome, a rare genetic neurodevelopment disorder. Alongside their demonstrated roles in DNA repair and transcription, these two proteins have recently been discovered to regulate cytokinesis, the final stage of the cell division. This last finding allowed, for the first time, to highlight an extranuclear localization of CS proteins, beyond the one already known at mitochondria. In this study, we demonstrated an additional role for CSA protein being recruited at centrosomes in a strictly determined step of mitosis, which ranges from pro-metaphase until metaphase exit. Centrosomal CSA exerts its function in specifically targeting the pool of centrosomal Cyclin B1 for ubiquitination and proteasomal degradation. Interestingly, a lack of CSA recruitment at centrosomes does not affect Cyclin B1 centrosomal localization but, instead, it causes its lasting centrosomal permanence, thus inducing Caspase 3 activation and apoptosis. The discovery of this unveiled before CSA recruitment at centrosomes opens a new and promising scenario for the understanding of some of the complex and different clinical aspects of Cockayne Syndrome.

Transcription-Coupled Nucleotide Excision Repair and the Transcriptional Response to UV-Induced DNA Damage.

Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main repair pathways to remove such lesions: global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER). The modes by which these subpathways recognize DNA lesions are distinct, but they converge onto the same downstream steps for DNA repair. Here, we first summarize the current understanding of these repair mechanisms, specifically focusing on the roles of stalled RNA polymerase II, Cockayne syndrome protein B (CSB), CSA and UV-stimulated scaffold protein A (UVSSA) in TC-NER. We also discuss the intriguing role of protein ubiquitylation in this process. Additionally, we highlight key aspects of the effect of UV irradiation on transcription and describe the role of signaling cascades in orchestrating this response. Finally, we describe the pathogenic mechanisms underlying xeroderma pigmentosum and Cockayne syndrome, the two main diseases linked to mutations in NER factors.

Identification and characterization of Necdin as a target for the Cockayne syndrome B protein in promoting neuronal differentiation and maintenance.

Cockayne syndrome (CS) is a devastating autosomal recessive genetic disorder, mainly characterized by photosensitivity, growth failure, neurological abnormalities, and premature aging. Mutations in CSB (ERCC6) are associated with almost all clinical phenotypes resembling classic CS. Using RNA-seq approach in multiple cell types, we identified Necdin (NDN) as a target of the CSB protein. Supportive of the RNA-seq results, CSB directly binds to NDN and manipulates the remodeling of active histone marks and DNA 5mC methylation on the regulatory elements of the NDN gene. Intriguingly, hyperactivation of NDN due to CSB deficiency does not interfere with nucleotide excision repair (1), but greatly affects neuronal cell differentiation. Inhibition of NDN can partially rescue the motor neuron defects in CSB mouse models. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for intervention, these data substantiate a reciprocal communication between CSB and NDN in the context of general transcription regulation.

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.

DRP1 Inhibition Rescues Mitochondrial Integrity and Excessive Apoptosis in CS-A Disease Cell Models.

Cockayne syndrome group A (CS-A) is a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. Cells derived from CS-A patients present as pathological hallmarks excessive oxidative stress, mitochondrial fragmentation and apoptosis associated with hyperactivation of the mitochondrial fission dynamin related protein 1 (DRP1). In this study, by using human cell models we further investigated the interplay between DRP1 and CSA and we determined whether pharmacological or genetic inhibition of DRP1 affects disease progression. Both reactive oxygen and nitrogen species are in excess in CS-A cells and when the mitochondrial translocation of DRP1 is inhibited a reduction of these species is observed together with a recovery of mitochondrial integrity and a significant decrease of apoptosis. This study indicates that the CSA-driven modulation of DRP1 pathway is key to control mitochondrial homeostasis and apoptosis and suggests DRP1 as a potential target in the treatment of CS patients.

Cockayne Syndrome Group B (CSB): The Regulatory Framework Governing the Multifunctional Protein and Its Plausible Role in Cancer.

Cockayne syndrome (CS) is a DNA repair syndrome characterized by a broad spectrum of clinical manifestations such as neurodegeneration, premature aging, developmental impairment, photosensitivity and other symptoms. Mutations in Cockayne syndrome protein B (CSB) are present in the vast majority of CS patients and in other DNA repair-related pathologies. In the literature, the role of CSB in different DNA repair pathways has been highlighted, however, new CSB functions have been identified in DNA transcription, mitochondrial biology, telomere maintenance and p53 regulation. Herein, we present an overview of identified structural elements and processes that impact on CSB activity and its post-translational modifications, known to balance the different roles of the protein not only during normal conditions but most importantly in stress situations. Moreover, since CSB has been found to be overexpressed in a number of different tumors, its role in cancer is presented and possible therapeutic targeting is discussed.

The emerging role of Cockayne group A and B proteins in ubiquitin/proteasome-directed protein degradation.

When mutated, csa and csb genes are responsible of the complex phenotype of the premature aging Cockayne Syndrome (CS). Our working hypothesis is to reconcile the multiple cellular and molecular phenotypes associated to CS within the unifying molecular function of CSA and CSB proteins in the cascade of events leading to ubiquitin/proteasome-directed protein degradation, which occurs in processes as DNA repair, transcription and cell division. This achievement may reasonably explain the plethora of cellular UPS-regulated functions that result impaired when either CSA or CSB are mutated and suggestively explains part of their pleiotropic effect. This review is aimed to solicit the interest of the scientific community in further investigating this aspect, since we believe that the identification of the ubiquitin-proteasome machinery as a new potential therapeutic target, able to comprehensively face the different molecular aspects of CS, whether confirmed and corroborated by in vivo studies, would open a promising avenue to design effective therapeutic intervention.

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.