Objective: To estimate SHH responsiveness & signaling of Floor Plate Cells (FPCs) derived from LRRK2 I1371V PD patient-iPSCs along with its link with LRRK2 I1371V mutation through membrane fluidity & Rab8A phosphorylation.

Background: A loss of 50-60% of midbrain dopaminergic neurons (mDAn) manifests in motor symptoms in PD, suggesting that a lower number of mDAn increases susceptibility of individuals to PD. To date, many LRRK2 mutant variants have been identified, & it shows differences in geographical distributions, disease risk, age of onset, brain pathology, drug response etc. Experimental embryology studies indicate that mDAn depend on SHH morphogen from ventral neural tube & floor plate.

Method: FPCs were characterized for SHH receptors Patched1 (Ptch1), Smoothened (Smo) & Glioma-Associated Oncogene Homolog 1 (Gli1) by FACS & immunofluorescence. Functional characterization of the FPCs was performed to assess intracellular Ca²⁺ response by live cell imaging & estimation of cellular cyclic-AMP (cAMP) by ELISA. Neurite & axon degeneration was noted using fluorescence microscopy.

Results: LRRK2 I1371V PD iPSCs gave rise to reduced yield of mDANs.  Total expression of SHH receptors Ptch1 & Smo was comparable between HC & LRRK2 I1371V FPCs however, distinct reduced cell-surface expression of Ptch1 & Smo. A corresponding decreased nuclear translocation of transcription factor Gli1 was observed. PD FPCs had compromised intracellular Ca²⁺ response on exposure to SHH along with elevated levels of cAMP. LRRK2 I1371V FPCs showed high substrate phosphorylation of Rab8A. A concurrent increase in membrane fluidity was observed in LRRK2 I1371V FPCs along with reduced cell-surface expression of Caveolin1(lipid raft marker). Reduced neurite extension & axon degeneration was detected.

Conclusion: The impaired SHH responsiveness of LRRK2 I1371V PD FPCs imparts to the lesser yield of mDAn during ontology.  The reduced cell surface expression of the SHH receptors is influenced by the alteration in the membrane fluidity rendered by increased substrate phosphorylation of Rab8a by LRRK2. The mutation alters membrane fluidity & membrane related morphology such as neurite length & axon degeneration.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/impairment-of-shh-signaling-in-pd-lrrk2-i1371v-ipsc-derived-floor-plate-cells-contribute-to-ontogenic-origin-of-lower-dopaminergic-neuron-yield/