Methyl-CpG binding protein 2 (MeCP2) and histone H1 are important chromatin associated proteins. Both exhibit their own extent of complexity as MeCP2 is an intrinsically disordered protein (IDP) that interacts with many different partners involved in several cellular processes and histone H1 consists of 11 different subtypes each of them associated with different posttranslational modifications (PTMs). An interesting avenue for the study of these proteins is in neurons where MeCP2 is very abundant and histone H1 level is half that observed in other somatic tissues. Several reports in the past have proposed that this lower level of histone H1 is due to the abundance of MeCP2 which displaces histone H1. However, this hypothesis has been debated and there is no clear consensus. In an attempt to study this controversy, a cell model system ReNCell WT and MeCP2-KO was used that can be induced to differentiate into neurons. The protein levels, transcript levels and localization of histone H1 subtypes in these cells were analyzed using HPLC, RT-qPCR and immunofluorescence, respectively. The results show that ReNCell WT and MeCP2-KO do not exhibit significant differences in their relative amount of histone H1 protein and transcript level neither at the proliferative nor at the later differentiated stages. However, HPLC analyses show that the histone H1 subtypes of these two cell types exhibit significant elution differences probably resulting from differences in their PTM content. Immunofluorescence analyses show that WT ReNCell differentiation as determined by extension of dendritic or axonic processes can be seen to occur over the course of one week and there is a significant difference in the nuclear area of these two cells at 8 DIV. This study provides important preliminary data for future research in MeCP2 and histone H1 using this cell model system and show that MeCP2 may have a bearing on histone H1 PTMs. / Graduate

Parkinson's disease (PD) is a neurodegenerative disorder that is characterised by uncontrollable shaking, muscular rigidity and cognitive impairment, due to low levels of dopamine caused by loss of dopamine containing neurons (DCN). The loss of DCN has been strongly associated with Caspase mediated apoptotic death. At present there are many studies that indicate exercise is beneficial in PD treatment, but there is a lack of research exploring the potential pathways, which exercise can activate and suppress to provide such positive and even negative effects. This study is the first to explore the effect of treadmill exercise on the level of Caspases, along with CAMK-IV protein in different brain regions of MPTP-treated rat model, using WB analysis. The results of this research has demonstrated reduction or completely suppression of some active Caspases, as well as, elevated amount of CAMK-IV in different brain regions of exercised PD animal model. To determine how exercise is reducing and inhibiting activation of Caspases, the first step was to identify how Caspases are stimulated, using ReNcell VM stem cell line that had been differentiated and treated with 6OHDA. The results of the study demonstrated 6OHDA triggered Caspase mediated apoptotic death of dDCN via PERK ER stress and NFκB classical pathway. IF, WB and cell viability analysis, using a wide range of inhibitors, showed that Caspase-2 is activated by the PERK pathway of ER stress and NFB classical pathway in 6OHDA treated dDCN. 6OHDA triggered activation of Caspase- 8 by the classical pathway in NFB mediated death of dDCN. 6OHDA triggered Caspase-4 activation but the exact mechanism involved remains to be identified. Only through understanding the molecular pathways regulating death of DCN in PD, new potential targets for therapy may be identified, which may ultimately reduce further death of DCN and slow PD progression. This proposed study has the potential to seek for more efficient drugs, which can suppress Caspase activation by targeting key targets in the pathways that the Caspases follow. These new specific targeted drugs could be used with treadmill exercise to achieve maximum effect, by slowing down or inhibiting further death of DCN.