Neuronal Differentiation of iPSCs Derived from Parkinson?s Disease Patients

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

Baden Sooden

Workshop; Cluster of Excellence for Microscopy and Physiology of the Brain (CMPB) Retreat 2012; 2012

MPIbpc-MPIem

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. For example, mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) have been associated with lateonset familial PD, with clinical and pathological features indistinguishable from the common sporadic form of PD. Early onset PD develops in families with extra copies of the SCNA gene that codes for αSynuclein (aSyn), the main component present in the aggregates of the substantia nigra of affected individuals. Due to the dominant character of this genetic alterations, LRRK2 and aSyn are believed to play a major role in the etiology of PD. The ability to reprogram human somatic cells through induced pluripotent stem cells (iPSCs) into neurons provides a valuable tool to study disease relevant cell types, especially when animal models cannot mimic the same pathogenic mechanisms, as in PD. Neuronal Stem Cells were produced from iPSCs (NiPSCs) of patients with familial PD with LRRK2 G2019S mutations, a triplication that includes SNCA gene and from age-matched controls. The NiPSCs were characterized and differentiated into neurons in a two-step protocol. Cells were analyzed for the expression of neuronal markers by immunofluorescence and western blotting. Mitochondrial function was assessed in control and aSyn triplication NiPSCs using a Seahorse XF24 analyzer that measures O2 consumption and H+ production. Preliminary results show a reduction in the aSyn neuronal iPSCs relative to the control. Differentiated cells resembled neurons, morphologically and as shown by the upregulation of MAP2 and the neuronal marker βIII 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.