Impaired SHH responsiveness of induced pluripotent stem cell-derived floor plate cells carrying the LRRK2 I1371V mutation contributes to the ontogenic origin of lower dopaminergic-neuron yield.

Abstract

Lower population of dopaminergic (DA)-neurons is known to increase susceptibility to PD, and our earlier study showed a lower yield of DA-neurons in LRRK2-I1371V mutation-carrying PD patient-iPSCs. While the role of SHH in DA-neurogenesis of Floor-Plate Cells (FPCs) is known, effect of LRRK2 mutations on SHH-responsiveness of FPCs impacting DA-neuronal yield has not been studied. We investigated SHH-responsiveness of FPCs derived from LRRK2-I1371V PD patient-derived iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 expression, intracellular Ca2+ rise, and cytosolic cAMP levels upon SHH induction. Additionally, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane-fluidity and Rab8A & Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC)-FPCs overexpressing LRRK2-I1371V as well as FPCs. While total expression of Ptch1 and Smo was comparable, receptor expression on cell-surface was significantly lower in LRRK2-I1371V FPCs than in HC, with distinctly lower nuclear-expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2 I1371V exhibited a similarly reduced cell-surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared to HC upon SHH-stimulation. Both LRRK2-I1371V mutant FPCs and LRRK2-I1371V transfected SH-SY5Y and HC-FPCs further exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A & Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker Caveolin1 were also observed in them. These findings suggest that impaired SHH-responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA-neurons during ontogeny. Reduced cell-surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.