Neuronal differentiation in an induced pluripotent neural stem cell model derived from PD patients with LRRK2 G2019S mutation or synuclein gene triplication.

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

Leipzig

Simposio; Fraunhofer Life Science Symposium on Stem Cell and Clinical Applications; 2012

German Society for Stem Cell Research

Objective: To differentiate iPSCs derived from PD patients and healthy controls into dopaminergic neurons (DAn), and to investigate the pathogenic mechanisms induced by the G2019S LRRK2 mutation or overproduction of alpha-synuclein (aSyn) in the generation of PD. Parkinson’s disease (PD) is the second most common neurodegenerative disease, and its pathologic hallmark is the loss of dopaminergic neurons (DAn). Although the majority of PD cases are sporadic, some familial mutations are known. Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) have been associated with late-onset familial PD, with clinical and pathological features indistinguishable from the common sporadic form of PD. LRRK2, a large multi-domain protein with serine/threonine protein kinase and GTPase activities, is localized in the cytoplasm and is associated with various intracellular components. The most common alteration in the LRRK2 protein is an autosomal dominant missense mutation, G2019S, leading to an increase in kinase activity. aSyn is the main protein component present in the substantia nigra of affected individuals, and is believed to play a major role in the genesis of PD. Families with an extra copy of the aSyn gene develop early onset disease. The ability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) provides a valuable tool to study disease relevant cell types, especially when animal models cannot mimic the same pathogenic mechanisms, as in PD. Materials and methods: Neuronal Stem Cells (NSCs) were produced from iPSCs of patients with familial PD with LRRK2 G2019S mutations or an aSyn triplication and from age-matched controls. After a two-step differentiation protocol, the differentiated cells were analyzed for the expression of neuron markers by immunofluorescence and western blotting. Physiological functions were assessed. Zinc-finger nuclease (ZFN) technology was used to correct the LRRK2 G2019S mutation in one patient-derived iPSC line. Results: The two-step differentiation process generated neurons, as shown by the upregulation of MAP2 and the neuronal marker β III tubulin (Tuj), ~ 20% of which were tyrosine hydroxylase (TH) positive and thus indicative of DAn. The cells with the aSyn triplication and LRRK2 mutations differentiated more slowly and produced fewer TH positive neurons when compared with the controls. No astrocyte markers were detected. Mitochondria function was assessed in control, aSyn triplication, ZFN corrected and LRRK2 mutated cell lines using a Seahorse XF24 analyzer that measures mitochondrial respiration and glycolysis. Preliminary results show a reduction in the aSyn neuronal iPSCs relative to the control, but no significant difference between the ZFN corrected and LRRK2 mutation lines in the mitochondrial bioenergetic profile of the cells prior to differentiation. Conclusions: Dopaminergic neurons were successfully produced from iPSCs generated from patients with PD familial mutations, LRRK2 G2019S and aSyn triplication and from age-matched controls. Interestingly the number of TH positive cells was significantly less in the cell lines derived from PD patients than in the control or ZFN corrected lines, indicating an increase in DAn cell death or impairment in the differentiation process leading to DAn. The aSyn triplication line showed mitochondrial dysfunction. Other experiments analyzing mitochondrial movement and degradation in all lines are being conducted.