NEURONAL DIFFERENTIATION CHARACTERIZATION OF iPS CELLS DERIVED FROM PARKINSON´S DISEASE PATIENTS

FALOMIR LOCKHART LJ; OLIVEIRA LMA; BOTELHO MG; MAK S; ARNDT-JOVIN DJ; SCHUELE B; JOVIN TM

Maastricht

Congreso; Focus on Microscopy 2013; 2013

Focus on Microscopy Network - University of Maastricht

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Its pathologic hallmark is the functional loss of dopaminergic neurons and the appearance of Lewy Bodies, intracellular amyloid aggregates constituted mainly by the protein α-Synuclein (aSyn). Although the majority of PD cases are sporadic, some familial mutations are known and are usually correlated with early onset forms of PD. In this context, we studied the neuronal differentiation of iPS cell lines [1], developed at The Parkinson’s Institute, that were obtained from patients with a triplication event in the chromosome 4 that contains the SNCA gene (aSyn), a LRKK2 point mutation (G2019S) or age matched healthy controls. The iPS cell lines derived from fibroblasts were first committed into a neuronal phenotype and then differentiated into neurons combining recombinant differentiation factors and small molecules [2]. In addition, astrocyte assisted differentiation was also tested since its presence better approximates the cellular complexity of the central nervous system and reduces the requirements for exogenous growth and differentiation factors. In order to identify affected metabolic pathways or cellular processes, antibodies against different neuronal markers were used to compare the differentiation process of all lines by immunohistochemistry. Oxidative stress susceptibility was tested using the ROS sensors H2DCFDA and MitoTracker CM-X-H2-Ros, while mitochondrial fitness was assayed with JC-1 probe. Preliminary results suggest aSyn triplication lines have difficulties to differentiate, but astrocyte-conditioned media is beneficial for neuronal differentiation and partially compensates this effect. However, supplementation with specific factors or drugs may be required in order to achieve a high yield of dopaminergic neurons, particularly in the case of the lines with PD associated genetic alterations that exhibit difficulties differentiating. Also, the lines with genetic alterations exhibit larger oxidative stress levels. To complement these results, new tools are being developed to infect differentiated cells with Lentivirus that would induce the expression of fluorescent proteins, free or targeted to specific subcellular locations, which will help to take a closer look at specific cellular processes like mitochondrial ROS stress response, synaptic vesicles and mitochondrial dynamics, cell to cell contacts, etc. We hope that this work will help unraveling the initial molecular triggers leading to neurodegeneration associated with these genetic alterations. [1] B. Byres et al.,”SNCA triplication Parkinson´s patient´s iPSC-derived DA neurons accumulate α synuclein and are susceptible to oxidative stress,” PLoS One 6, e26159 (2011). [2] S.K. Mak et al., “Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage,” Stem Cells Int. Article ID 140427 (2012).