Developing therapeutics against oxidative stress –related neurodegeneration.

The rapid development of technology worldwide makes modern medicine available for more patients every year. This quick and easy access to medicine prolongs human life thus provoking the inevitable development of aging disorders. Neurodegenerative diseases are defined as a range of conditions that drive progressive loss of function or structure in neurons which may eventually lead to cell death due to the inability to reproduce and replace themselves (Bredesen et al., 2006). One of the abnormal states that are highlighted regarding neuronal damage is oxidative stress. It is not usually defined as a primary cause of neurodegenerative diseases, but it is highly enhanced when brain homeostasis is disrupted (Uttara et al., 2009). Nevertheless, successful targeting of abnormal ROS production could slow down the progression of neuronal damage. This thesis focused on functional analysis of proteins that could reverse or decrease oxidative insults as well as finding compounds that could offset the damaging effects of ROS generation.

First, thein vitro models focusing on oxidative stress-induced damage by different factors were optimized. Short exposures to damaging compounds like menadione sodium bisulfite (15μM MSB), as well as longer lasting injury caused by hypoxia and/or inflammation (Tα and INF-γ),were applied to three different cell lines 1321N1, THP-1,and SH-SY5Y after which ROS, calcium, and cell viability were assessed. Besides, the responses of all cell lines to MSB-induced damage were verified by other known compounds that cause ROS generation. Furthermore, all three cell lines were exposed to 15μM MSB to show their responses to a damaging factor by measuring ROS, calcium, and cellular viability and shed a light on possible mechanisms of an oxidative stress injury in different cellular phenotypes. The expression of SOD, CAT, NQO1, GPX1, GSR was assessed and the genes showing the highest change were further analysed on protein level. 1321N1 and THP-1 cell lines showed low cell loss(resistance) when exposed to MSB whereasSH-SY5Y cell line experienced high cell death. Moreover, co-culture models were tested, where1321N1 or THP-1 cell lines were shown to offsetSH-SY5Y cell loss. The observed SH-SY5Y cell line protection by co-culturing with other cell lines was verified by N-acetylcystine (NAC)and glutathione (GSH), known to offset menadione toxicity. This thesis focused also on prohibitins which are involved in protection against oxidative stress in most cell types. Altered expression of the prohibitins could affect ROS homeostasis in the cells and were thus quantified in brains of MS patients(IHC)and in in vitro setting. The previously optimized in vitro models of oxidative stress damage were applied to three cell lines U-251 MG, THP-1,and SH-SY5Y cell lines after which PHBs expression was measured. Additionally, PHBs were shown to be present in all cellular compartments, taking part in different signalling pathways. U-251 MG cell line exposed to hypoxia and inflammation showed increased levels of PHBs in mitochondrial and nuclear fractions. Moreover, computational targeting of PHBs was undertaken to find potential compounds that could affect their function. The enhancement of PHBs involvement in protection against oxidative stress could be beneficial for patients with neurodegenerative diseases.

This study showed that optimization of in vitro models focusing on oxidative stress-induced injury depends on the damaging factor used. In most cases, compounds used in this study triggered ROS generation where cellular damage was rapidly observed, whereas hypoxia and inflammatory cytokines caused prolonged oxidative stress after 24 and 48H with lower cell loss. Additionally, 1321N1 and THP-1 cell lines showed lower cell death when exposed to MSB than SH-SY5Y cell line suggesting a different mechanism of protection against MSB induced damage. The gene and protein analysis showed the highest differences in GSR in all three cell lines after MSB exposure, indicating an involvement of the GSH pathway in the protection against MSB. Additionally, only the SH-SY5Y cell line experienced lower GSH levels after the exposure to MSB. Furthermore, NAC and GSH treatments showed similar cellular protection against MSB in the SH-SY5Y cell line giving further support to the involvement of the GSH pathway in protection against MSB-induced damage. Moreover, PHBs levels varied in brain regions of MS patients, showing induced expression of prohibitins in the NAGM and NAWM but not WM lesions. Further verification on a larger MS cohort should be performed. PHBs levels increased in all cellular compartments except membrane fraction in the U-251 MG cell line after the exposure to hypoxia and inflammation. The in silico approach resulted in eight compounds that were tested in a cellular assay. Four of the eight compounds were further applied as possible treatments to oxidative stress-induced models; however, further verification of the results should be carried out in the future.