Objective: To evaluate the pathophysiology of the midbrain dopaminergic neurons (mDAN) derived from LRRK2 I1371V PD patient-iPSCs leading to susceptibility to oxidative stress.

Background: Studies suggest that the LRRK2 I1371V mutation is pathogenic and leads to a lower yield of mDANs with functional impairments in vesicular dopamine release and synapse density. Reports on PD patient iPSC derived mDANs indicate kinase domain mutations lead to ER stress, Ca²⁺ dysregulation and membrane-associated neurite collapse and axon degeneration. The impact of the I1371V mutation on membrane associated properties and oxidative stress susceptibility of mDANs remains unexplored.

Method: We subjected mDANs to sub-lethal 6-OHDA oxidative stress and assessed cell survival using the MTT assay, Reactive Oxygen Species (ROS) levels using the H2-DCFDA assay, and apoptotic population using FACS with Annexin V. Fluorescence microscopy was used to check axon degeneration while live-cell imaging was used to determine neurite collapse, ER stress and ER Ca²⁺ homeostasis. Changes in ER proteins responsible for Ca²⁺ release and refiling were checked by FACS and PCR.

Results: LRRK2 I1371V mDANs showed higher ER stress, reduced ER Ca²⁺ release and refiling. A concurrent decrease in expression of proteins STIM, ORAI, iP3 and SERCA involved in ER Ca²⁺ homeostasis was observed. Neurite collapse and axon degeneration was also higher in case of PD mDANs. PD mDANs exhibited higher cell death, increased apoptotic population and elevated ROS levels in response to 6-OHDA stress.

Conclusion: The dysregulation of endoplasmic reticulum (ER) calcium ion (Ca2+) homeostasis, attributed to decreased expression levels of STIM, ORAI, iP3, and SERCA, is a contributing factor to heightened ER stress in LRRK2 I1371V midbrain dopaminergic neurons (mDANs). This dysregulation is particularly significant due to the close proximity of the ER organelle to the cell membrane. As a result, neurite collapse and axon degeneration, both membrane-associated phenomena, were observed. The accumulation of such intrinsic stress renders PD mDANs more susceptible to extrinsic oxidative stress, such as that induced by 6-OHDA.

« Back to 2023 International Congress

MDS Abstracts - https://www.mdsabstracts.org/abstract/membrane-associated-properties-and-er-stress-alterations-in-lrrk2-i1371v-patient-ipsc-derived-neurons-increase-susceptibility-to-oxidative-stress/