The role of lysine(K)-deacetylases on mitochondrial function, dynamics and degradation, and post-translation modifications of misfolded proteins: relevance for neuroprotection

Abstract

The aim of this project is to characterize the role of Lysine(K)-deacetylases (KDACs) on mitochondrial function, dynamics and clearance, as well as on post-translation modifications (acetylation and phosphorylation) of misfolded proteins, since KDACs, including classical histone deacetylases (HDACs), are able to regulate several mechanisms named gene transcription, signal transduction and metabolism. Because KDACs also have as targets proteins related to autophagy pathway, it will be further investigated the levels of autophagy and mitophagy in different Huntington Disease (HD) models expressing full-length mutant huntingtin (mHtt).Although it is hard to predict the sequence of cell-damaging events occurring in any neurodegenerative disorder, several pathological mechanisms have been proposed including mitochondrial dysfunction. Mitochondria are susceptible to changes in nutrient factors, toxic agents, misfolded proteins and changes in cytoplasmic clearance. Alterations in the mechanisms regulating mitochondrial fission/fusion, axonal transport (dynamics), biogenesis and degradation may occur in parallel, promoting each other, converging to cellular dysfunction. Moreover, the central role of mitochondria in cell metabolism requires the integration and coordination of their functions with those of other organelles, namely the nucleus. Thus, mitochondrial function can be directly affected by changes in transcription regulation both at the level of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). During this project, three different models will be investigated: immortalized neurons (SH-SY5Y) and astrocytes (1321N1) transfected with full-length mHtt and fibroblasts from HD patients and control subjects. In task 1, it will be investigated the role of KDACs on mitochondrial function. Changes in KDACs levels, after treatment with different KDACs inhibitors (KDACis) or siRNA transfection against specific KDACs, will be correlated with mitochondrial function (membrane potential, oxygen consumption and calcium buffering), levels of reactive oxygen species (ROS), mtDNA copy number and mitochondrial fission and fusion using antibodies against Fis1, Mfn1, Mfn2, Drp1, PINK1 and Parkin. In addition, it will be tested the presence of mHtt acetylated and phosphorylated in these processes. To counter prove the effect of KDACi or siRNA in mHtt post-translational modification, cells will be transfected with Ac-mHtt-K444, Ac-mHtt-K444(R) and P-mHtt-S13/S16/S431, acetylated and resistant mHtt forms to acetylation and phosphorylation, respectively. In task 2, it will be evaluated the influence of different KDACs, after KDACis treatment or siRNA transfection, on autophagy and mitophagy induction. For that, autophagy and mitophagy will be evaluated by western blot (WB) for several hallmarks proteins of this pathway, including LC3, LAMP-1 and -2 and autophagosome markers, and by fluorescence, with Hoechst, Lysotracker Red and Autophagy LC3B-GFP. Experiments will be also performed after transfection with Ac-mHtt-K444(R) and P-mHtt-S13/S16/S431. In task 3, it will be studied the impact of different KDACs on transcription regulation of autophagic and metabolism genes (mitochondrial-related genes). Transcription will be analyzed through PCR-Array (autophagy, mitochondrial, mTOR signaling) in addition to Real-Time(q) PCR and WB in the presence of KDACis, siRNA, mHtt, Ac-mHtt-K444(R) and P-mHtt-S13/S16/S431.Altogether, it is expected that this project can provide breakthroughs regarding new mechanisms of neuroprotection involving epigenetic and post-translational modification; data generated herein can be potential relevant for future studies addressing the role of KDACs on other neurodegenerative and neuropsychiatric disorders. (AU)