Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2.

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Clinical and Genetic Characterization of Brazilian Patients with Ataxia and Oculomotor Apraxia.

BACKGROUND: Ataxia with oculomotor apraxia (AOA) is characterized by early-onset cerebellar ataxia associated with oculomotor apraxia. AOA1, AOA2, AOA3, and AOA4 subtypes may present pathogenic variants in APTX, SETX, PIK3R5, and PNKP genes, respectively. Mutations in XRCC1 have been found to cause autosomal recessive spinocerebellar ataxia-26 (SCAR26) now considered AOA5. OBJECTIVES: To examine a cohort of Brazilians with autosomal recessive cerebellar ataxia plus oculomotor apraxia and determine the frequencies of AOA subtypes through genetic investigation. METHODS: We evaluated clinical, biomarkers, electrophysiological, and radiological findings of 52 patients with AOA phenotype and performed a genetic panel including APTX, SETX, PIK3R5, PNKP, and XRCC1. RESULTS: We found pathogenic variants in SETX (15 patients), PNKP (12), and APTX (5). No mutations in PIK3R5 or XRCC1 were identified. CONCLUSIONS: AOA2 and AOA4 were the most common forms of AOA in Brazil. Mutations in PIK3R5 and XRCC1 were not part of this genetic spectrum. © 2022 International Parkinson and Movement Disorder Society.

Identification of novel senataxin mutations in Chinese patients with autosomal recessive cerebellar ataxias by targeted next-generation sequencing.

BACKGROUND: Autosomal recessive cerebellar ataxias (ARCA) are a group of neurodegenerative disorders characterized by early onset of gait impairment, disturbed limb coordination, dysarthria, and eye movement abnormalities, most likely due to the degeneration of cerebellum, brainstem, and spinal cord. Despite of the rarity, ARCA are both clinically and genetically heterogeneous. To date, more than 30 culprit genes have been identified in ARCA. Unraveling the specific causative mutation in cases with ARCA remains challenging so far. METHODS: Three ARCA pedigrees of Chinese ancestry were recruited. Clinical features were evaluated and peripheral blood was collected after obtaining the written inform. Laboratory examinations, brain MRI, and EMG were performed for all the affected individuals. Genomic DNA was extracted, followed by the screening of GAA repeat expansion in FXN gene to exclude Friedreich's ataxia. Targeted next-generation sequencing combining Sanger sequencing was performed in each proband of these families. RESULTS: Compound heterozygous mutations, c.3190G > T (p.E1064X) and c.4883C > G (p.S1628X) of senataxin (SETX) gene were identified in one family with two affected cases. Both of the patients presented with early onset of unsteady walk, dysarthria, and diplopia. EMG test revealed decreased conduction velocity and evoked potential of both motor and sensory nerve. Moreover, elevated serum alpha-fetoprotein (AFP) and apparent cerebellar atrophy were observed. These features were typical features of ataxia with oculomotor apraxia type 2 (AOA2) and in line with the genetic results. However, no specific mutation was identified in the other two pedigrees. CONCLUSIONS: We identified novel compound heterozygous mutations of SETX in Chinese AOA2 pedigree, which broaden the mutation spectrum of SETX. To our knowledge, this is the first report concerning Chinese AOA2 cases with SETX mutations.

Ataxia with oculomotor apraxia type 1 associated with mutation in the APTX gene: A case study and literature review.

Cerebellar ataxia is a disorder characterized by a broad spectrum of phenotypes. Ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease presenting with early-onset and slowly progressing cerebellar ataxia, areflexia and peripheral axonal neuropathy. Mutations in the APTX gene c.751C>T p.(His251Tyr) were detected with probable homozygosity in the APTX gene (chromosome 9) that encodes a nuclear protein called aprataxin that is involved in DNA repair. AOA1 also contributes to neuronal development and function. Ocular apraxia is most prominent in the early stages of the disease, while hypoalbuminemia, hypercholesterolemia and cognitive impairment are common symptoms in the adult stage. The present study reported the clinical features of an 8-year-old female patient with mutations in the APTX gene and discussed the differential diagnosis from other forms of hereditary ataxia.

Case report: A novel APTX p.Ser168GlufsTer19 mutation in a Chinese family with ataxia with oculomotor apraxia type 1.

Ataxia with oculomotor apraxia type 1 (AOA1) is a rare genetic disorder and is inherited in an autosomal recessive manner. It is mainly characterized by childhood-onset progressive cerebellar ataxia, with dysarthria and gait disturbance being the two most common and typical manifestations. Axonal sensorimotor peripheral neuropathy, dystonia, chorea, and cognitive impairment are common associated symptoms, as are hypoalbuminemia and hypercholesterolemia. Oculomotor apraxia (OMA)has been reported to be a feature often, although not exclusively, associated with AOA1. The Aprataxin gene, APTX, is ubiquitously expressed, and numerous APTX mutations are associated with different clinical phenotypes have been found. In the present study, we enrolled a 14-year-old boy who developed ataxia with staggering gait from the age of 4 years. Early-onset cerebellar ataxia, peripheral axonal neuropathy, cognitive impairment and hypoalbuminemia, hypercholesterolemia were presented in this patient, except for OMA. We applied ataxia-related genes filtering strategies and whole-exome sequencing (WES) to discover the genetic factors in a Chinese family. Sanger sequencing was used in the co segregation analysis in the family members. A compound heterozygous mutation in APTX gene (c.739C>T and c.501dupG) was identified. This is the first description of a genetically confirmed patient of AOA1 in a Chinese family in addition to a novel mutation of c.501dupG in APTX.

Ataxia with oculomotor apraxia type 2 caused by a novel homozygous mutation in SETX gene, and literature review.

Objectives: Autosomal recessive inherited ataxia with oculomotor apraxia type 2 (AOA2), caused by SETX gene mutations, is characterized by early-onset, progressive cerebellar ataxia, peripheral neuropathy, oculomotor apraxia and elevated serum α-fetoprotein (AFP). This study aimed to expand and summarize the clinical and genetic characteristics of SETX variants related to AOA2. Methods: The biochemical parameters, electromyogram and radiological findings of the patient were evaluated. Whole-exome sequencing (WES) was performed on the patient using next-generation sequencing (NGS), the variants were confirmed by Sanger sequencing and the pathogenicity of the variants was classified according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. We reviewed 57 studies of AOA2 patients with SETX mutations and collected clinical and genetic information. Results: The patient was a 40-year-old Chinese woman who primarily presented with numbness and weakness of the lower limbs in her teenage years. She had elevated AFP, increased serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and decreased anti-Müllerian hormone (AMH) levels. We identified a novel homozygous missense mutation of the SETX gene, c.7118 C>T (p. Thr2373Ile), in the patient via Whole-exome and Sanger sequencing. The variant was located in the DNA/RNA helicase domain and is highly conserved. The protein prediction analysis verified the SETX variant as a damaging alteration and ACMG/AMP guidelines classified it as likely pathogenic. Through a literature review, we identified 229 AOA2 cases with SETX variants, and among the variants, 156 SETX variants were exonic. We found that 107 (46.7%) patients were European, 50 (21.8%) were African and 48 (21.0%) were Asian. Among the Asian patients, five from two families were Mainland Chinese. The main clinical features were cerebellar ataxia (100%), peripheral neuropathy (94.6%), cerebellar atrophy (95.3%) and elevated AFP concentration (92.0%). Most reported SETX mutations in AOA2 patients were missense, frameshift and nonsense mutations. Conclusion: We discovered a novel homozygous variant of the SETX gene as a cause of AOA2 in the current patient and expanded the genotypic spectrum of AOA2. Moreover, the clinical features of AOA2 and genetic findings in SETX were assessed in reported cohorts and are summarized in the present study.

Complex Movement Disorders in Ataxia with Oculomotor Apraxia Type 1: Beyond the Cerebellar Syndrome.

Background: Ataxia with oculomotor apraxia (AOA1) is characterized by early-onset progressive cerebellar ataxia with peripheral neuropathy, oculomotor apraxia and hypoalbuminemia and hypercholesterolemia. Case Report: A 23-year-old previously healthy woman presented with slowly-progressive gait impairment since the age of six years. Neurological examination revealed profound areflexia, chorea, generalized dystonia and oculomotor apraxia. Brain MRI revealed mild cerebellar atrophy and needle EMG showed axonal sensorimotor neuropathy. Whole exome sequencing revealed a mutation in the aprataxin gene. Discussion: AOA1 can present with choreoathetosis mixed with dystonic features, resembling ataxia-telangiectasia. This case is instructive since mixed and complex movement disorders is not very common in AOA1. Highlights: Ataxia with oculomotor apraxia type 1 (AOA1) is characterized by early-onset ataxia and oculomotor apraxia caused by variants in the APTX gene.Ataxia is usually not the sole movement abnormality in AOA1.Hyperkinetic movement disorders, especially chorea and dystonia, may occur.Mixed and complex movement disorders is not very common in AOA1.Patients with early-onset ataxia associated with mixed movement disorders should also be investigated for AOA1.

The Phenotypic Spectrum of PNKP-Associated Disease and the Absence of Immunodeficiency and Cancer Predisposition in a Dutch Cohort.

BACKGROUND: We aimed to expand the number of currently known pathogenic PNKP mutations, to study the phenotypic spectrum, including radiological characteristics and genotype-phenotype correlations, and to assess whether immunodeficiency and increased cancer risk are part of the DNA repair disorder caused by mutations in the PNKP gene. METHODS: We evaluated nine patients with PNKP mutations. A neurological history and examination was obtained. All patients had undergone neuroimaging and genetic testing as part of the prior diagnostic process. Laboratory measurements included potential biomarkers, and, in the context of a DNA repair disorder, we performed a detailed immunologic evaluation, including B cell repertoire analysis. RESULTS: We identified three new mutations in the PNKP gene and confirm the phenotypic spectrum of PNKP-associated disease, ranging from microcephaly, seizures, and developmental delay to ataxia with oculomotor apraxia type 4. Irrespective of the phenotype, alpha-fetoprotein is a biochemical marker and increases with age and progression of the disease. On neuroimaging, (progressive) cerebellar atrophy was a universal feature. No clinical signs of immunodeficiency were present, and immunologic assessment was unremarkable. One patient developed cancer, but this was attributed to a concurrent von Hippel-Lindau mutation. CONCLUSIONS: Immunodeficiency and cancer predisposition do not appear to be part of PNKP-associated disease, contrasting many other DNA repair disorders. Furthermore, our study illustrates that the previously described syndromes microcephaly, seizures, and developmental delay, and ataxia with oculomotor apraxia type 4, represent the extremes of an overlapping spectrum of disease. Cerebellar atrophy and elevated serum alpha-fetoprotein levels are early diagnostic findings across the entire phenotypical spectrum.

Ataxia with Oculomotor Apraxia Type 4 with PNKP Common "Portuguese" and Novel Mutations in Two Belarusian Families.

Ataxia with oculomotor apraxia type 4 (AOA4) is a rare autosomal recessive, PNKP -related disorder delineated in 2015 in Portugal. We diagnosed AOA4 by next generation sequencing (NGS) followed by Sanger's sequencing in three boys from two unrelated Belarusian families. In both families, one of the heterozygous PNKP mutations was c.1123G>T, common in Portuguese patients; biallelic mutations, c.1270_1283dup14 and c.1029+2T>C, respectively, were novel. These are the first reported AOA4 Slavic cases and the first with a "Portuguese" PNKP mutation outside Portugal. Distinction in two brothers was microcephaly but their disease was not severe in contrast to PNKP -related "microcephaly, seizures, and developmental delay" and reported cases with features of both phenotypes.

A Novel Homozygous Variant in the Fork-Head-Associated Domain of Polynucleotide Kinase Phosphatase in a Patient Affected by Late-Onset Ataxia With Oculomotor Apraxia Type 4.

Ataxia with oculomotor apraxia (AOA) is a clinical syndrome featuring a group of genetic diseases including at least four separate autosomal-recessive cerebellar ataxias. All these disorders are due to altered genes involved in DNA repair. AOA type 4 (AOA4) is caused by mutations in DNA repair factor polynucleotide kinase phosphatase (PNKP), which encodes for a DNA processing enzyme also involved in other syndromes featured by microcephaly or neurodegeneration. To date, only a few AOA4 patients have been reported worldwide. All these patients are homozygous or compound heterozygous carriers for mutations in the kinase domain of PNKP. In this report, we describe a 56 years old patient affected by AOA4 characterized by ataxia, polyneuropathy, oculomotor apraxia, and cognitive impairment with the absence of dystonia. The disease is characterized by a very late onset (50 years) when compared with other AOA4 patients described so far (median age of onset at 4 years). In this proband, Clinical Exome Analysis through Next Generation Sequencing (NGS) consisting of 4,800 genes, identified the PNKP homozygous mutation p.Gln50Glu. This variant, classified as a likely pathogenic variant according to American College of Medical Genetics (ACMG) guidelines, does not involve the kinase domain but falls in the fork-head-associated (FHA) domain. So far, mutations in such a domain were reported to associate only with a pure seizure syndrome without the classic AOA4 features. Therefore, this is the first report of patients carrying a mutation of the FHA domain within the PNKP gene which expresses the clinical phenotype known as the AOA4 syndrome and the lack of any seizure activity. Further studies are required to investigate specifically the significance of various mutations within the FHA domain, and it would be worth to correlate these variants with the age of onset of the AOA4 syndrome.

An algorithm as a diagnostic tool for central ocular motor disorders, also to diagnose rare disorders.

BACKGROUND: Recently an increasing number of digital tools to aid clinical work have been published. This study's aim was to create an algorithm which can assist physicians as a "digital expert" with the differential diagnosis of central ocular motor disorders, in particular in rare diseases. RESULTS: The algorithm's input consists of a maximum of 60 neurological and oculomotor signs and symptoms. The output is a list of the most probable diagnoses out of 14 alternatives and the most likely topographical anatomical localizations out of eight alternatives. Positive points are given for disease-associated symptoms, negative points for symptoms unlikely to occur with a disease. The accuracy of the algorithm was evaluated using the two diagnoses and two brain zones with the highest scores. In a first step, a dataset of 102 patients (56 males, 48.0 ± 22 yrs) with various central ocular motor disorders and underlying diseases, with a particular focus on rare diseases, was used as the basis for developing the algorithm iteratively. In a second step, the algorithm was validated with a dataset of 104 patients (59 males, 46.0 ± 23 yrs). For 12/14 diseases, the algorithm showed a sensitivity of between 80 and 100% and the specificity of 9/14 diseases was between 82 and 95% (e.g., 100% sensitivity and 75.5% specificity for Niemann Pick type C, and 80% specificity and 91.5% sensitivity for Gaucher's disease). In terms of a topographic anatomical diagnosis, the sensitivity was between 77 and 100% for 4/8 brain zones, and the specificity of 5/8 zones ranged between 79 and 99%. CONCLUSION: This algorithm using our knowledge of the functional anatomy of the ocular motor system and possible underlying diseases is a useful tool, in particular for the diagnosis of rare diseases associated with typical central ocular motor disorders, which are often overlooked.

[Ataxia with oculomotor apraxia type 4 detected by next-generation sequencing]. / Ataksiia s okulomotornoi apraksiei 4-go tipa, diagnostirovannaia metodom panel'nogo ékzomnogo sekvenirovaniia.

Ataxias with oculomotor apraxia (AOA) belong to autosomal recessive ataxias. Their common feature is oculomotor apraxia: inability to coordinate eye movements not due to muscle weakness. Next-generation sequencing (NGS) gives unique opportunities of rare disorders diagnostics and discovering of new forms, including AOA. In 2015, AOA type 4 produced by PNKP mutations was delineated in a group of Portuguese patients. We diagnosed AOA4 in a 9-year-old boy from Byelorussian family. He presented with ataxia since 2 years and deterioration in 8 years, oculomotor apraxia, dystonic hyperkinesia, dysarthria, polyneuropathy, borderline/mildly impaired intelligence, cerebellar atrophy on MRI and moderate hypercholesterolemia. Panel NGS detected two PNKP mutations: c.1123G>T (p.Gly375Trp) common in Portuguese patients, and novel c.1270_1283dupACAAACCCAGACGC (p.Ala429fs). This is one of a few world AOA4 cases and first non-Portuguese case with 'Portuguese' common mutation. The case illustrates NGS diagnostic value, particularly in rare heterogeneous disorders like AOA.

Recessive ataxias.

Recessive ataxias (spinocerebellar ataxias, recessive or SCARs) are a heterogeneous group of rare, mostly neurodegenerative genetic disorders which usually start in childhood or early adult life. They can be subdivided into two major groups: predominant sensory or afferent ataxias, which are disorders mainly of the peripheral input to the cerebellum, and predominant cerebellar ataxias, in which the cerebellum is primarily affected. Next-generation sequencing technology has enabled the identification of >100 novel SCAR genes in the last 5 years, although most of them are ultrarare. To guide clinical workup and management in SCARs, we provide an up-to-date overview of the most frequent SCARs and their phenotypic features. These include Friedreich ataxia, spastic paraplegia type 7-related ataxia, autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) and spectrin repeat-containing nuclear envelope protein (SYNE)-related ataxia. In some restricted populations ARSACS or ataxia with vitamin E deficiency (AVED) is most common. All require a high index of suspicion in patients who present with an early-onset disorder of balance, especially children, in whom normal development and the lack of typical clinical characteristics seen in later stages of the respective SCARs can confuse the clinical picture. We summarize the diagnostic features which can help guide diagnosis, the natural history for common SCARs, and the approach to therapy, both in current use and in ongoing clinical trials. We also provide a summary table for other clinically relevant SCARs. Based on the frequency data, phenotypes, and the cost-effectiveness of recent next-generation sequencing approaches, we conclude with a diagnostic algorithm for the workup of patients with unexplained SCAR.

Current and Promising Therapies in Autosomal Recessive Ataxias.

BACKGROUND & OBJECTIVE: Ataxia is clinically characterized by unsteady gait and imbalance. Cerebellar disorders may arise from many causes such as metabolic diseases, stroke or genetic mutations. The genetic causes are classified by mode of inheritance and include autosomal dominant, X-linked and autosomal recessive ataxias. Many years have passed since the description of the Friedreich's ataxia, the most common autosomal recessive ataxia, and mutations in many other genes have now been described. The genetic mutations mostly result in the accumulation of toxic metabolites which causes Purkinje neuron lost and eventual cerebellar dysfunction. Unfortunately, the recessive ataxias remain a poorly known group of diseases and most of them are yet untreatable. CONCLUSION: The aim of this review is to provide a comprehensive clinical profile and to review the currently available therapies. We overview the physiopathology, neurological features and diagnostic approach of the common recessive ataxias. The emphasis is also made on potential drugs currently or soon-to-be in clinical trials. For instance, promising gene therapies raise the possibility of treating differently Friedreich's ataxia, Ataxia-telangiectasia, Wilson's disease and Niemann-Pick disease in the next few years.

More Than Ataxia: Hyperkinetic Movement Disorders in Childhood Autosomal Recessive Ataxia Syndromes.

BACKGROUND: The autosomal recessive ataxias are a heterogeneous group of disorders that are characterized by complex neurological features in addition to progressive ataxia. Hyperkinetic movement disorders occur in a significant proportion of patients, and may sometimes be the presenting motor symptom. Presentations with involuntary movements rather than ataxia are diagnostically challenging, and are likely under-recognized. METHODS: A PubMed literature search was performed in October 2015 utilizing pairwise combinations of disease-related terms (autosomal recessive ataxia, ataxia-telangiectasia, ataxia with oculomotor apraxia type 1 (AOA1), ataxia with oculomotor apraxia type 2 (AOA2), Friedreich ataxia, ataxia with vitamin E deficiency), and symptom-related terms (movement disorder, dystonia, chorea, choreoathetosis, myoclonus). RESULTS: Involuntary movements occur in the majority of patients with ataxia-telangiectasia and AOA1, and less frequently in patients with AOA2, Friedreich ataxia, and ataxia with vitamin E deficiency. Clinical presentations with an isolated hyperkinetic movement disorder in the absence of ataxia include dystonia or dystonia with myoclonus with predominant upper limb and cervical involvement (ataxia-telangiectasia, ataxia with vitamin E deficiency), and generalized chorea (ataxia with oculomotor apraxia type 1, ataxia-telangiectasia). DISCUSSION: An awareness of atypical presentations facilitates early and accurate diagnosis in these challenging cases. Recognition of involuntary movements is important not only for diagnosis, but also because of the potential for effective targeted symptomatic treatment.

Emerging role of RNA•DNA hybrids in C9orf72-linked neurodegeneration.

RNA plays an active role in structural polymorphism of the genome through the formation of stable RNA•DNA hybrids (R-loops). R-loops can modulate normal physiological processes and are also associated with pathological conditions, such as those related to nucleotide repeat expansions. A guanine-rich hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been linked to a spectrum of neurological conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we discuss the possible roles, both locally and genome-wide, of R-loops that may arise from the C9orf72 hexanucleotide repeat. R-loops have the potential to influence the pathological processes identified in many repeat expansion diseases, such as repeat instability, transcriptional dysregulation, epigenetic modification, and antisense-mediated gene regulation. We propose that, given the wide-ranging consequences of R-loops in the cell, these structures could underlie multiple pathological processes in C9orf72-linked neurodegeneration.

Alpha-fetoprotein, a fascinating protein and biomarker in neurology.

Alpha-fetoprotein (AFP) is present in fetal serum in concentrations up to 5,000,000 µg/l. After birth, AFP gene expression is turned down with a subsequent fall of the serum concentrations of this albumin-like protein to 'adult values' of circa 0.5-15 µg/l from the age of 2 years onwards. Irrespective of its assumed important functions, individuals with AFP deficiency appear fully healthy. The other way around, the presence of AFP in the circulation after the first years of life doesn't seem to harm, since individuals with 'hereditary persistence of AFP' are also without clinical abnormalities. During pregnancy, AFP (in maternal serum) has long been recognized as a marker for congenital anomalies of the fetus. Equally well known is AFP as biomarker for hepatocellular carcinoma and some other malignancies. There are at least four neurodegenerative disorders, all inherited as autosomal recessive traits and characterized by the presence of cerebellar ataxia, abnormal ocular movements, and neuropathy, for which an elevated concentration of serum AFP is an important diagnostic biomarker. The availability of a reliable biomarker is not only important during screening or diagnostic processes, but is also relevant for objective follow-up during (future) therapeutic interventions.

Studying the cerebellar DNA damage response in the tissue culture dish.

The cerebellum is exquisitely sensitive to deficiencies in the cellular response to specific DNA lesions. Genetic disorders caused by such deficiencies involve relentless, progressive cerebellar atrophy with striking loss of Purkinje and granule neurons. The reason for the extreme sensitivity of these cells to defective response to certain DNA lesions is unclear. This is particularly true for ataxia-telangiectasia (A-T) - a genomic instability syndrome whose major symptom is cerebellar atrophy. It is important to understand whether the DNA damage response in the cerebellum, particularly in Purkinje neurons, has special characteristics that stem from the unique features of these cells. Murine cerebellar organotypic cultures provide a valuable experimental system for this purpose since they retain the tissue organization for several weeks in culture and appear to provide the delicate Purkinje neurons with a physiological environment close to that in vivo. We have optimized this system and are using it to examine the Atm-mediated DNA damage response (DDR) in the cerebellum, with special emphasis on Purkinje cells. Our results to date, which indicate special chromatin organization in Purkinje cells that affects certain pathways of the DDR, demonstrate the usefulness of cerebellar organotypic cultures for addressing the above questions.