ATR (ATM and Rad-3 related) is a kinase protein of approximately 301 KDa, mainly known for its nuclear function played in proliferating cells. Indeed, in these cells it is recruited and activated at the site of DNA damage, promoting the phosphorylation of numbers of substrates directly involved in the cell cycle regulation and DNA damage repair, such as CHK1, p53 and BRCA1. Mutations in the Atr gene are responsible for the onset of the Seckel Syndrome (SS), an autosomal recessive disorder mainly characterized by intrauterine growth retardation, craniofacial abnormalities, dwarfism, chromosomal instability and mental retardation. Recent studies have revealed a much wider role of ATR, in particular it is present into the neuronal cytoplasm participating in the regulation of the synaptic vesicles trafficking; also, genetic deletion of the protein seems to be responsible for an increased excitatory activity and subsequently for the onset of sporadic non-lethal epileptic seizures in 12 months-old mice. Therefore, the abnormal synaptic transmission due to ATR mutations could partially explain the neurological dysfunctions found in SS patients. Despite the evidence related to the genetic deletion of ATR, up to now, no data regarding the pharmacological inhibition of ATR kinase activity have been provided. Thus, in this thesis, we aimed at filling this scientific gap investigating the impact mediated by a specific ATR kinase activity blocker on hippocampal function and neuronal transmission. This drug can penetrate inside the brain and it is currently involved in oncological pre- clinical studies, indeed it prevents the ATR-mediated signaling and inhibit the DNA damage checkpoint activation, promoting cancer cells apoptosis. Hence, hippocampal neuronal cultures have been treated with the ATR inhibitor in the attempt to block the ATR pathway and study related consequences at different stages of neuronal maturation. The treatment was administered both in acute, a single day treatment, and in chronic, one-week protocol. First of all, Ca2+ imaging experiments in 7 DIV neurons demonstrated that reduced ATR activity in immature neurons has no impact on the GABAergic development. Subsequently, electrophysiological and immunofluorescence experiments in mature hippocampal neurons revealed that both acute and chronic ATR inhibition are responsible for neuronal transmission defects, inducing a marked imbalance between excitation and inhibition, thus favoring the establishment of a more inhibited tone. Also, synaptic plasticity was found affected, resulting in defective long term potentiation and depression. Real time PCR experiments performed in vitro and RNA sequencing in vivo were useful to investigate the molecular mechanism at the basis of the defects here described. In particular, data collected by both these approaches suggest the immediate early gene Egr1 as one of the main factors deregulated by ATR inhibition. Importantly, several findings describe Egr1 as a key transcriptional regulator of genes contributing to the development of hyperexcitability during epileptogenesis. Also, increased Egr1 expression levels have been detected in hippocampal biopsies collected from epileptic individuals. Thus, we evaluated the effect mediated by pharmacological ATR inhibition in a mouse model of acute seizures and, coherently, we found a strong anticonvulsant effect. These results highlight ATR as a novel target in acute seizures onset and the ATR inhibitor as an innovative therapeutic tool in neurological disorders characterized by increased excitability. As a matter of fact, RNA profiling also unveiled similarity with other conventional antiepileptic drugs. Altogether, our data demonstrating that ATR kinase inhibition prevents the hyperactivity-induced Egr1 expression nicely fall with the recent theory of “master regulators candidates” in epilepsy, given that among the transcriptional activators and repressors suggested there is Egr1. On the other hand, we performed biochemical investigations of the active ATR levels in the attempt to monitor changes of ATR activity along life. Data obtained from mouse and human hippocampal tissues collected at increasing age display a reduction of the phosphorylated ATR (the active form of the protein) suggesting that ATR levels variations participate in the establishment of cognitive defects during the physiological aging. It remains to investigate the role of Egr1 in this field. Overall, the results collected in this thesis suggest a novel role of ATR in the maintenance of the proper neuronal transmission and plasticity in mature hippocampal neurons and highlight ATR inhibition as an important therapeutic tool for neurological disorders characterized by hyperexcitability.

ATR CONTROLS THE EXCITATORY/INHIBITORY RATIO IN THE CENTRAL NERVOUS SYSTEM AND ITS INHIBITION LEADS TO ANTICONVULSANT EFFECTS / C.m. Cambria ; tutor: F. ANTONUCCI ; PhD coordinator: M. LOCATI, N. LANDSBERGER. - : . Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, 2022 Apr 08. ((34. ciclo, Anno Accademico 2021.

ATR CONTROLS THE EXCITATORY/INHIBITORY RATIO IN THE CENTRAL NERVOUS SYSTEM AND ITS INHIBITION LEADS TO ANTICONVULSANT EFFECTS

C.M. Cambria
2022-04-08

Abstract

ATR (ATM and Rad-3 related) is a kinase protein of approximately 301 KDa, mainly known for its nuclear function played in proliferating cells. Indeed, in these cells it is recruited and activated at the site of DNA damage, promoting the phosphorylation of numbers of substrates directly involved in the cell cycle regulation and DNA damage repair, such as CHK1, p53 and BRCA1. Mutations in the Atr gene are responsible for the onset of the Seckel Syndrome (SS), an autosomal recessive disorder mainly characterized by intrauterine growth retardation, craniofacial abnormalities, dwarfism, chromosomal instability and mental retardation. Recent studies have revealed a much wider role of ATR, in particular it is present into the neuronal cytoplasm participating in the regulation of the synaptic vesicles trafficking; also, genetic deletion of the protein seems to be responsible for an increased excitatory activity and subsequently for the onset of sporadic non-lethal epileptic seizures in 12 months-old mice. Therefore, the abnormal synaptic transmission due to ATR mutations could partially explain the neurological dysfunctions found in SS patients. Despite the evidence related to the genetic deletion of ATR, up to now, no data regarding the pharmacological inhibition of ATR kinase activity have been provided. Thus, in this thesis, we aimed at filling this scientific gap investigating the impact mediated by a specific ATR kinase activity blocker on hippocampal function and neuronal transmission. This drug can penetrate inside the brain and it is currently involved in oncological pre- clinical studies, indeed it prevents the ATR-mediated signaling and inhibit the DNA damage checkpoint activation, promoting cancer cells apoptosis. Hence, hippocampal neuronal cultures have been treated with the ATR inhibitor in the attempt to block the ATR pathway and study related consequences at different stages of neuronal maturation. The treatment was administered both in acute, a single day treatment, and in chronic, one-week protocol. First of all, Ca2+ imaging experiments in 7 DIV neurons demonstrated that reduced ATR activity in immature neurons has no impact on the GABAergic development. Subsequently, electrophysiological and immunofluorescence experiments in mature hippocampal neurons revealed that both acute and chronic ATR inhibition are responsible for neuronal transmission defects, inducing a marked imbalance between excitation and inhibition, thus favoring the establishment of a more inhibited tone. Also, synaptic plasticity was found affected, resulting in defective long term potentiation and depression. Real time PCR experiments performed in vitro and RNA sequencing in vivo were useful to investigate the molecular mechanism at the basis of the defects here described. In particular, data collected by both these approaches suggest the immediate early gene Egr1 as one of the main factors deregulated by ATR inhibition. Importantly, several findings describe Egr1 as a key transcriptional regulator of genes contributing to the development of hyperexcitability during epileptogenesis. Also, increased Egr1 expression levels have been detected in hippocampal biopsies collected from epileptic individuals. Thus, we evaluated the effect mediated by pharmacological ATR inhibition in a mouse model of acute seizures and, coherently, we found a strong anticonvulsant effect. These results highlight ATR as a novel target in acute seizures onset and the ATR inhibitor as an innovative therapeutic tool in neurological disorders characterized by increased excitability. As a matter of fact, RNA profiling also unveiled similarity with other conventional antiepileptic drugs. Altogether, our data demonstrating that ATR kinase inhibition prevents the hyperactivity-induced Egr1 expression nicely fall with the recent theory of “master regulators candidates” in epilepsy, given that among the transcriptional activators and repressors suggested there is Egr1. On the other hand, we performed biochemical investigations of the active ATR levels in the attempt to monitor changes of ATR activity along life. Data obtained from mouse and human hippocampal tissues collected at increasing age display a reduction of the phosphorylated ATR (the active form of the protein) suggesting that ATR levels variations participate in the establishment of cognitive defects during the physiological aging. It remains to investigate the role of Egr1 in this field. Overall, the results collected in this thesis suggest a novel role of ATR in the maintenance of the proper neuronal transmission and plasticity in mature hippocampal neurons and highlight ATR inhibition as an important therapeutic tool for neurological disorders characterized by hyperexcitability.
ANTONUCCI, FLAVIA
LANDSBERGER, NICOLETTA
LOCATI, MASSIMO
ATR; epilepsy; E/I balance; neuronal plasticity; hyperexcitability;
Settore BIO/14 - Farmacologia
ATR CONTROLS THE EXCITATORY/INHIBITORY RATIO IN THE CENTRAL NERVOUS SYSTEM AND ITS INHIBITION LEADS TO ANTICONVULSANT EFFECTS / C.m. Cambria ; tutor: F. ANTONUCCI ; PhD coordinator: M. LOCATI, N. LANDSBERGER. - : . Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, 2022 Apr 08. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/920095
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