The Parkinson's Institute and Clinical Center Research Grants 2013


The Parkinson's Institute and Clinical Center is using its 2013 distribution to fund:

What Does Alpha-synuclein Accumulation in Glia Have to do with Parkinsonism?

PROJECT TITLE:  What Does Alpha-synuclein Accumulation in Glia Have to do with Parkinsonism?

Investigators/Authors: Birgitt Schuele, MD, Director for Gene Discovery and Stem Cell Modeling; J. William Langston, MD, CEO and Founder

Objective: The goal of this study is to derive induced pluripotent stem cell (iPSC) lines from patients with multiple system atrophy (MSA) and assess expression and accumulation of alpha-synuclein in patient-specific differentiated oligodendrocytes.

Background: MSA is a form of atypical parkinsonism that presents as a more severe and rapidly progressing form of Parkinson’s Disease (PD) with autonomic failure.  A distinctive feature of the disease (in addition to the degeneration of certain neuronal populations) is the presence of glial cytoplasmic inclusion bodies (GCIs). In MSA, these inclusions are observed in a type of glia known as oligodendrocytes and importantly contain alpha-synuclein aggregates. At the moment, their role in MSA is a complete mystery. Although several iPSC-based model systems have been reported for genetically linked forms of PD, MSA iPSC models have not been established to date.  

Methods/Design: In Aim 1, we will derive three MSA iPSC lines from patients with the disease and matched controls. We will use established protocols from our laboratory with Sendaivirus for footprint-free reprogamming. All derived clones will be characterized based on standards set forward by the International Society for Stem Cell Research. In Aim 2, we will differentiate the iPSC lines into precursor cells and then further differentiate them into oligodendrocytes using a newly established protocol by Tesar et al. 2011 which will be adapted to human. In the last Aim, we will assess alpha-synuclein in oligodendrocytes by measuring expression of total alpha-synuclein, alpha-synuclein aggregation, and post-translational modifications using immunoblotting and immunocytochemistry techniques. We will further test, if knockdown by gene engineering (TALEN) of alpha-synuclein in these iPSCs will reverse any observed phenotype(s).

Relevance to Diagnosis/Treatment of Parkinson’s disease: The lack of established animal models that accurately recapitulate MSA or PD, and access to the cell types that are most affected in these disorders - human neurons/glia- is extremely limiting. The promise of stem cell technologies, such as the ability to create relevant cell types, to manipulate, engineer, and study these cells and tissues, opens new avenues for disease research and drug discovery.  Importantly, protein misfolding and aggregation of alpha-synuclein is a common feature across synucleinopathies and an attractive target for drug development. Thus this novel project could provide vital insights into the role of alpha-synuclein for all forms of parkinsonism that involve alpha-synuclein aggregation, including typical Parkinson’s disease.

September 2014 Project Update:

We have successfully completed Aim 1 by deriving  iPSC clones from the skin cell of two individuals clinically diagnosed with MSA (Patient 1: Female, 62 years and Patient 2: Female, 79 years) and matched healthy controls.
Our next step was to test different protocols for differentiating these new patient-specific induced stem cells to the cell type that is key to MSA.  As noted in the above proposal, this cell type is a form of glial (supportive) brain cells that are affected in MSA known as oligodendrocytes.  Importantly these glia have synuclein positive aggregates (inclusion bodies) that are the hallmark pathological finding in MSA.  To be sure that these newly born glia were indeed oligodentrocytes, we use a custom designed approach, and were eventually able to find markers that were specific enough to show that we have indeed recreated the cell type that is affected in MSA “in a dish”.  These markers (MOG, MBP, and NKX2.2) distinguish between these oligodendrocytes and neurons.

Utilizing these markers, we are now in the process of developing an approach that will allow us to assess alpha-synuclein (a key protein in PD) in oligodendrocytes by measuring expression of total alpha-synuclein, alpha-synuclein aggregation, and a variety of other techniques to characterize these cells.  If we do not observe changes of alpha-synuclein expression, the presence of GCI- like structures, or any other abnormalities in the MSA cell lines, we will stress the cells with environmental toxins. Overall, if  this project is successful it will open up a myriad  of exciting new avenues to study MSA which have never before been possible, ranging from directly investigating disease mechanism to screening for new drugs that might halt or even reverse the disease process.  Our results would likely lead to new insights for Parkinson’s disease and other synucleinopathies as well.

September 2015 Project Update:

Over the course of the PUW project, we developed a human stem cell model from patient-derived skin cells. Using reprogramming techniques, we turned patient skin cells into pluripotent stem cells. These two patients were clinically diagnosed with multiple system atrophy (MSA). MSA is a neurodegenerative disorder that resembles in some aspects Parkinson’s disease, however, there are some key differences that make this disorder distinct and it is critical to better understand disease mechanisms of MSA for the development of therapeutic approaches.

One key difference in MSA is that there are cellular alpha-synuclein protein inclusions in the supporting cells of the brain, called glia. This is in contrast to Parkinson’s disease, where the protein inclusions are localized in nerve cells.

We have successfully derived tissue cultures of glia-like cells from patient-derived stem cells and have created a genetically engineered panel of these stem cells with different expression levels of alpha-synuclein expression to assess how alpha-synuclein affects the development of cellular deficiencies and ultimately cell death. We are currently working on the differentiation of these MSA stem cells into dopaminergic neurons and will assay gene expression during the differentiation process, neuronal makers and alpha-synuclein expression to start understanding the disease process in neurons.

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