Spinal Müsküler Atrofi'li Drosophıla Melanogaster Modelinde Rpd3 Proteini ve Histon Asetilasyon Düzeylerinin Araştırılması

Abstract

Histone acetylation causes relaxation of the DNA-histone interactions and transcriptional activation. It is controlled by the balance between histone acetyl transferase (HAT) enzymes which add acetyl groups to lysine amino acids on the N- terminal tail regions of histone proteins, and histone deacetylase enzymes (HDAC) that work reversibly to it. Disruption of this balance has been associated with the pathophysiology of neurodegenerative diseases and compounds that inhibit HDAC enzymes have been developed. In our previous studies, several HDAC inhibitors were synthesized and their HDAC1 specific inhibition activities were determined in vitro, however, in vivo effects of these compounds were needed to be investigated. This study aims to determine the appropriate stages and treatment conditions which can be used in HDAC inhibitor research in the Drosophila melanogaster model of SMA. In our study, the infrastructure to investigate in vivo effects of previously synthesized HDAC inhibitors were developed by using phenylbutyric acid as an HDAC inhibitor. The expression level of Rpd3 protein, -the Drosophila ortholog of human HDAC1- was investigated at larval and adult stages of wild type and SMA flies. Rpd3 protein level was found to be higher in adult stage than larval, however, the difference was not statistically significant, therefore, further studies were carried out with the adult stage. Flies were treated with 5 mM and 10 mM phenylbutyric acid for 5 days and then H3K27 and H3K18 acetylation levels analyzed by Western blot. Our results showed a significant increase in H3K27, but not H3K18 acetylation levels at 5 mM and 10 mM treatment. Besides, flies were treated with the same concentrations of phenylbutyric acid for 10 days and acetylation levels of H3K27 and H3K18 were analyzed. No significant change was observed neither in H3K27 nor in H3K18 acetylation levels. In conclusion, in vivo drug treatment conditions on HDAC inhibition were identified through phenylbutyric acid application and it has been shown for the first time that it increases the H3K27 acetylation level of SMA Drosophila melanogaster model.