Overexpression, characterisation, and encapsulation of LRP/LR for treatment of neurodegenerative disorders
The neurodegenerative diseases of Parkinson's disease (PD) and Alzheimer’s disease (AD) are debilitating conditions affecting millions of people worldwide. Both diseases pose a significant economic burden and require therapeutic strategies. AD is identified by intracellular neurofibrillary tangles and the accumulation of amyloid beta (Aβ) plaques, while PD is characterised by the loss of dopamine-producing neurons and mutations in PINK1, parkin, and α-synuclein proteins. The 37 kDa/67 kDa laminin receptor (LRP/LR) is a multifunctional receptor found to impede the progression of AD and PD. In-vitro studies have revealed that the LRP::FLAG overexpression increases hTERT expression and telomerase activity while rescuing cells from Aβ-mediated cytotoxicity. Cuttler et al. 2019 also revealed that LRP::FLAG overexpression decreased phosphorylated tau levels and tauopathy-related proteins. Within PD, in vitro studies demonstrated that LRP::FLAG overexpression plays a protective role, through the degradation of α-synuclein and rescuing cells from MPP+ cytotoxicity. Thus, the current study comprised of three parts. The first part aimed at investigating the effect of overexpressing LRP::FLAG in HEK293 cells by assessing LRP/LR and PINK1 protein levels, as well as cell viability after treatment with MPP+, TBHP, and Aβ!", in vitro AD and PD models. The results demonstrated that the overexpression of LRP::FLAG rescued cells from MPP+, TBHP, and Aβ!" induced cytotoxicity, and increased LRP/LR and PINK1 protein levels. The second part of the study focussed on overexpressing, purifying, and structurally characterizing the 37 kDa LRP protein. The protein was successfully overexpressed and purified through Co2+-IMAC while structural characterisation indicated that protein was correctly folded and predominantly αhelical, as expected. The purified LRP protein was then encapsulated in PLGA nanoparticles to develop an efficient in vivo delivery system. Thus, the third part of the study investigated the effect of empty and LRP-encapsulated PLGA nanoparticles on cell viability, LRP levels, and telomerase activity in SH-SY5Y and HEK 293 cells. The results demonstrated that LRPencapsulated PLGA nanoparticles successfully delivered the therapeutic protein and increased exogenous LRP protein levels. Additionally, SH-SY5Y cells treated with LRPencapsulated PLGA nanoparticles exhibited increased cellular viability and telomerase activity. Therefore, the LRP-encapsulated PLGA nanoparticles could be a possible therapeutic for conditions such as ageing and age-related diseases including AD and PD.
A research report submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023
Neurodegenerative diseases, Parkinson's disease (PD), Alzheimer’s disease (AD)