Introduction. Neonatal jaundice is a common and benign condition usually resolved during the first week of life. Neonates with very high level of unconjugated bilirubin (UCB), with an increase of the free bilirubin (Bf), may develop encephalopathy, with different grade of severity. Typical symptoms include motor disorder, auditory dysfunction, memory and learning deficits, reflecting selective damage respectively for cerebellum and basal ganglia, inferior colliculus, and hippocampus. Several studies reported that bilirubin may activate signal cascades that culminate to cell survival or death, as well as modulation of mRNA expression of genes involved in. Histone acetylation, an epigenetic mechanism responsible for the gene expression regulation via opening/closing of the chromatin, was never investigated in bilirubin-induced central nervous system (CNS) damage until now. The involvement of histone acetylation in common and rare neuropathologies was widely described, and also the potential neuroprotective action of drugs that regulate this epigenetic process. Aims. By the use of the animal model for hyperbilirubinemia (Gunn rat), we purpose to investigate the modulation of the acetyl-histone H3 (lys14) (H3K14ac) in five brain regions (Cortex: Cx; Cerebellum: Cll; Superior Colliculi: SC; Inferior Colliculi: IC; Hippocampus: Hip) of hyperbilirubinemic animals (jj) vs. non hyperbilirubinemic littermates at four post natal ages (P2, P9, P17 and old). Moreover, we would like to individuate the genes regulated by H3K14ac in jj cerebella vs. controls at P9 and eventually interpret the biological effects of this epigenetic modulation performing histological stainings. Results. Western blot analysis of H3K14ac level in all the brain regions and ages considered revealed a modulation of this protein that is region- and age-dependent. A significant increase of H3K14ac was observed in Cll, IC, and Hip in P9 jj animals, while a significant decrease was present in Cll and IC at P17 and Cx in older jj rats. ChIP-seq was performed in order to link the effect of hyperbilirubinemia on the H3K14ac with the genes controlled by this epigenetic mechanism in cerebella of P9 animals. Our data on H3K14ac in Cll and the fact that the cerebellar hypoplasia is the hallmark of hyperbilirubinemic Gunn rat supported this choice. Our results revealed that a very high number of genes regulated by H3K14ac was silenced in jj rats, while other few genes seemed to be activated in hyperbilirubinemic subjects. The gene ontology analysis showed that cellular development and differentiation, proliferation and migration, as well as apoptosis were the most modulated biological processes in jj rats. These ChIP-Seq data were confirmed by our histological findings, that revealed higher cell density with reduction of the fibrillary component and a possible impairment of cell differentiation. Conclusions. This work reported for the first time the involvement of histone acetylation alterations in the brain of hyperbilirubinemic animals with a strong regulation of the gene expression. The complete panel of genes we obtained seemed to well-correlate with the bilirubin-induced damage that evidenced the development impairment. These data contribute to better understand the bio-molecular mechanisms that cause bilirubin toxicity but also might open to new therapeutical strategies in kernicterus, testing the neuroprotective properties of drugs that regulate the histone acetylation.

In vivo evaluation of biomolecular mechanisms of bilirubin toxicity and pre-clinical therapies.

VIANELLO, ELEONORA
2017-05-09

Abstract

Introduction. Neonatal jaundice is a common and benign condition usually resolved during the first week of life. Neonates with very high level of unconjugated bilirubin (UCB), with an increase of the free bilirubin (Bf), may develop encephalopathy, with different grade of severity. Typical symptoms include motor disorder, auditory dysfunction, memory and learning deficits, reflecting selective damage respectively for cerebellum and basal ganglia, inferior colliculus, and hippocampus. Several studies reported that bilirubin may activate signal cascades that culminate to cell survival or death, as well as modulation of mRNA expression of genes involved in. Histone acetylation, an epigenetic mechanism responsible for the gene expression regulation via opening/closing of the chromatin, was never investigated in bilirubin-induced central nervous system (CNS) damage until now. The involvement of histone acetylation in common and rare neuropathologies was widely described, and also the potential neuroprotective action of drugs that regulate this epigenetic process. Aims. By the use of the animal model for hyperbilirubinemia (Gunn rat), we purpose to investigate the modulation of the acetyl-histone H3 (lys14) (H3K14ac) in five brain regions (Cortex: Cx; Cerebellum: Cll; Superior Colliculi: SC; Inferior Colliculi: IC; Hippocampus: Hip) of hyperbilirubinemic animals (jj) vs. non hyperbilirubinemic littermates at four post natal ages (P2, P9, P17 and old). Moreover, we would like to individuate the genes regulated by H3K14ac in jj cerebella vs. controls at P9 and eventually interpret the biological effects of this epigenetic modulation performing histological stainings. Results. Western blot analysis of H3K14ac level in all the brain regions and ages considered revealed a modulation of this protein that is region- and age-dependent. A significant increase of H3K14ac was observed in Cll, IC, and Hip in P9 jj animals, while a significant decrease was present in Cll and IC at P17 and Cx in older jj rats. ChIP-seq was performed in order to link the effect of hyperbilirubinemia on the H3K14ac with the genes controlled by this epigenetic mechanism in cerebella of P9 animals. Our data on H3K14ac in Cll and the fact that the cerebellar hypoplasia is the hallmark of hyperbilirubinemic Gunn rat supported this choice. Our results revealed that a very high number of genes regulated by H3K14ac was silenced in jj rats, while other few genes seemed to be activated in hyperbilirubinemic subjects. The gene ontology analysis showed that cellular development and differentiation, proliferation and migration, as well as apoptosis were the most modulated biological processes in jj rats. These ChIP-Seq data were confirmed by our histological findings, that revealed higher cell density with reduction of the fibrillary component and a possible impairment of cell differentiation. Conclusions. This work reported for the first time the involvement of histone acetylation alterations in the brain of hyperbilirubinemic animals with a strong regulation of the gene expression. The complete panel of genes we obtained seemed to well-correlate with the bilirubin-induced damage that evidenced the development impairment. These data contribute to better understand the bio-molecular mechanisms that cause bilirubin toxicity but also might open to new therapeutical strategies in kernicterus, testing the neuroprotective properties of drugs that regulate the histone acetylation.
GAZZIN, SILVIA
TIRIBELLI, CLAUDIO
29
2015/2016
Settore BIO/10 - Biochimica
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2908155
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