DNA damage and transcriptional regulation in iPSC-derived neurons from Ataxia Telangiectasia patients

Abstract

Ataxia Telangiectasia (A-T) is neurodegenerative syndrome caused by inherited mutations inactivating the ATM kinase, a master regulator of the DNA damage response (DDR). What makes neurons vulnerable to ATM loss remains unclear. In this study we assessed on human iPSC-derived neurons whether the abnormal accumulation of DNA-Topoisomerase 1 adducts (Top1ccs) found in A-T impairs transcription elongation, thus favoring neurodegeneration. Furthermore, whether neuronal activity-induced immediate early genes (IEGs), a process involving the formation of DNA breaks, is affected by ATM deficiency. We found that Top1cc trapping by CPT induces an ATM-dependent DDR as well as an ATM-independent induction of IEGs and repression especially of long genes. As revealed by nascent RNA sequencing, transcriptional elongation and recovery were found to proceed with the same rate, irrespective of gene length and ATM status. Neuronal activity induced by glutamate receptors stimulation, or membrane depolarization with KCl, triggered a DDR and expression of IEGs, the latter independent of ATM. In unperturbed A-T neurons a set of genes (FN1, DCN, RASGRF1, FZD1, EOMES, SHH, NR2E1) implicated in the development, maintenance and physiology of central nervous system was specifically downregulated, underscoring their potential involvement in the neurodegenerative process in A-T patients.

Immune-tolerance to human iPS-derived neural progenitors xenografted into the immature cerebellum is overridden by species-specific differences in differentiation timing

“…hiPSdNP cells were differentiated in vitro according to the protocol of Yan et al 49 . During this in vitro differentiation protocol hiPSdNP stop dividing and become post-mitotic 32 . hiPSdNP were seeded at a 5 × 10 4 cells/cm 2 on twofold concentrated Geltrex (Thermo Fisher Scientific) pre-coated plates and kept in Neural Differentiation Medium (Neurobasal medium, 2% B27 supplement serum free, 2 mM GlutaMAX supplement, from Thermo Fisher Scientific, 10 ng/μl brain derived neurotrophic factor and 10 ng/μl glial cell-derived neurotrophic factor, both from Immunotools, 200 μM L-ascorbic Acid from Merck, 0.1 mM non essential amino acids, 100 units/ml penicillin and 100 μg/ml streptomycin from Lonza) up to 55 days in vitro.…”

“…Cells used for transplantations. hiPSdNP cells were previously described and indicated as healthy donorderived Neural Precursor Cells (WT1-NPCs) 32 . Briefly they were obtained from hiPS clones generated from integration-free reprogrammed fibroblasts (Coriell Institute Biobank, Camden, New Jersey, USA).…”

“…hiPSdNP and DhiPSdNP are both immunogenic in vivo. To test hiPSdNP immunogenicity in vivo 9 naïve adult CD1 Swiss mice were immunized either with proliferating hiPSdNP or with the same cells submitted to an in vitro differentiation protocol that makes them post-mitotic (DhiPSdNP) 32 . After completing the in vitro differentiation treatment total protein extracts of DhiPSdNP contained higher levels of both neuronal maturation markers like β-III tubulin and MAP2 and SCG10 and glial markers as GFAP (Fig.…”

“…Human antigens that were not expressed by the xeno-grafted cells during the maturation of the host immune system but started to be expressed later, were recognized by the host immune system as not-self triggering the immune-rejection of the xeno-grafted cells. To test this hypothesis, we co-xeno-grafted in the newborn (P3) cerebellum eGFP labeled hiPSdNP mixed with an equal amount of eGFP negative DhiPSdNP www.nature.com/scientificreports/ previously submitted to the same in vitro differentiation protocol used to generate DhiPSdNP for immunogenic testing 32 . In vitro differentiated DhiPSdNP became post-mitotic and expressed mature neural and glial antigens ( Fig.…”

Stress-sensing in the human greying hair follicle: Ataxia Telangiectasia Mutated (ATM) depletion in hair bulb melanocytes in canities-prone scalp

“…The importance of ATM in the cellular response to oxidative stress can be seen by investigating its role in the autosomal recessive disorder ataxia telangiectasia (A-T), after which ATM is named 55 . A-T patients lack fully functioning ATM and are predisposed to a range of malignancies, radiation sensitivity, immunological and neurological disorders 56 caused by abnormalities in the cells response to DNA repair and elevated ROS levels particularly in neurons 57 . Studies in ATM-deficient mouse models have also shown similar abnormalities 58 .…”