The development of an alpha synuclein overexpressing In vitro model for parkinson's disease research

Abstract

Abstract
Parkinson’s disease (PD) is characterized by selective loss of dopaminergic neurons and progressive accumulation of intracellular alpha synuclein protein aggregates known as Lewy bodies. Due to pathological links to both sporadic and inherited cases of PD, alpha synuclein (asyn) has become a focal point for study, although the exact mechanisms of its toxicity are not known. In contrast to previous toxin-induced PD models, recent evidence from asyn overexpressing models has elucidated a role for asyn in the impairment of signaling by the neuroprotective glial cell line-derived neurotrophic factor (GDNF), which has become a promising candidate for the treatment of PD. This project sought to develop an asyn overexpressing model of PD in a dopaminergic neuroblastoma (TGW) cell line to study the toxic effects of asyn on GDNF signaling within the cell; specifically Tyrosine Hydroxylase (TH) upregulation induced by GDNF, and expression of RET which is an essential part of the receptor complex for GDNF signaling.
While the results from this asyn overexpressing TGW model cell line do not indicate an effect on TH protein regulation, there is evidence a downregulation of the GDNF receptor RET in the presence of increased asyn and furthermore a significant decrease in cell growth rate.

Popular science summary:

Modelling Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative affecting the world today. PD pathogenesis is characterized by a focused degeneration of a particular central nervous system cell type known as dopaminergic neurons (DA). Alpha-synuclein (asyn) protein whose exact function remains elusive, accumulates as aggregates in the surviving cells, and is strongly linked to the pathogenic process. Cellular or in vitro laboratory models that mimic disease hallmarks, allow a detailed and simplified study in an isolated environment. This project was focused on developing such a model of PD in dopaminergic cells to allow a study of its pathogenesis and the examination of possible future therapies.
Presently, treatment for PD patients focuses on alleviating symptoms of motor dysfunction, but now, a new increasingly promising treatment option is centred on a group of growth factors called neurotrophic factors (NTF) that aim to halt the progression of the disease. One such NTF known as GDNF, has demonstrated this so-called neuroprotective ability in classical models of PD. These models mirror the disease-relevant DA cell loss by use of targeted neurotoxin insults. However, conversely, GDNF has failed to provide a similar protection in recently developed rat animal models that more accurately recreate clinical disease. The DA cells in these models express increased cellular asyn levels due to virus-mediated gene transfer and can effectively recreate the temporal degeneration and symptomatic PD. This project focused on creating a DA cell culture laboratory model that expresses increased levels of asyn protein and studying the resulting effects, in particular on the cells ability to use GDNF.
Results and implications of this project
In general, the methods of this project analyzed cellular protein changes and cell growth rate alterations as a result of increasing the level of asyn in the cultured model cells. Asyn overexpression was mediated by viral vector mediated transfer of the asyn gene to the proposed model cell line TGW. We observed a significant decrease in cell growth rate of asyn-overexpressing cells in comparison to control cells, indicating an overt toxicity of increased asyn protein. Then, specifically, we examined two appropriate markers of cellular GDNF machinery and signalling, Tyrosine Hydroxlase (TH) and the GDNF target cell surface receptor, RET. TH protein abundance is increased when GDNF is available to the cell, but this induction is known to be reduced when asyn is expressed at above endogenous levels. By using western blot (WB), a method used to separate and examine cellular protein amounts; we observed no conclusive changes in TH or RET protein levels in our cultured cells in response to increased asyn protein. We then used an intracellular staining method to analyze RET protein expression. RET expression has been shown to be reduced in response to raised asyn levels in a rat model. If less RET is available for GDNF signalling, then less of a protective effect can be mediated in turn, and therefore is a vital factor in terms of GDNFs therapeutic application. Intracellular staining employs a fluorescent antibody to detect RET that allows the quantification of RET-specific signal in terms of fluorescence in detergent permeablized cells. By this method, we saw an indication that total cellular RET is reduced at increased asyn levels. This result holds some significant promise for the continuation of developing this relevant cellular model of PD. It may, in future, provide a platform for testing potential therapeutic possibilities. As such, if RET is decreased in asyn- overexpressing diseased cells, then an appropriate and controlled increase of RET to counteract this may allow GDNF to afford its protective ability to affected DA neurons and halt the progression of disease.

Supervisors: Luis Quintino PhD, Professor Cecilia Lundberg
Master Degree project 30 credits in Molecular Biology (Molecular genetics and Biotechnology) 2012
CNS Gene Therapy Group, Department of Experimental Medical Science, Biomedicine Centre, Lund University

• author: Stephenson, Nicholas
• supervisor: Luis Quintino; Cecilia Lundberg
• organization: Degree Projects in Molecular Biology
• course: MOBM19 20122
• year: 2013
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 3513753
• date added to LUP: 2013-02-20 11:48:58
• date last changed: 2013-03-26 14:58:03