LRRK2 and RAB8A regulate cell death after lysosomal damage in macrophages through cholesterol-related pathways

Area of Science:

• Cell Biology
• Neuroscience
• Genetics

Background:

• Activating mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are a common genetic cause of Parkinson's disease (PD).
• The precise mechanisms linking LRRK2 mutations to PD pathogenesis remain unclear, but evidence suggests a role for LRRK2 in modulating lysosomal function.
• Previous studies indicate LRRK2 is recruited to lysosomes during damage, increasing phosphorylation of RAB GTPase substrates.

Purpose of the Study:

• To investigate the functional interaction between lysosomal damage and LRRK2 signaling.
• To determine if LRRK2 kinase inhibition can protect against lysosomal stress-induced cell death.
• To elucidate downstream pathways affected by LLOMe-induced lysosomal damage and LRRK2 inhibition, focusing on cholesterol biosynthesis.

Main Methods:

• Utilized RAW 264.7 macrophage cells treated with the lysosomotropic compound LLOMe to induce lysosomal damage.
• Assessed cell death, lysosomal permeabilization (Lysotracker), and LRRK2 signaling pathway activity.
• Employed siRNA-mediated knockdown of LRRK2 and RAB GTPases, alongside RNA sequencing and cholesterol metabolism modulation (U18666A).

Main Results:

• LRRK2 kinase inhibition significantly reduced cell death induced by LLOMe, demonstrating a bidirectional interaction between lysosomal damage and LRRK2.
• LRRK2 inhibition specifically attenuated cell death caused by lysosomal stressors, not other cell death inducers, and modulated the cellular response post-permeabilization.
• Knockdown of LRRK2 and RAB8A, but not other RABs, attenuated LLOMe-induced cell death. RNA sequencing revealed downregulation of cholesterol biosynthesis genes by LLOMe, which was reversed by LRRK2 inhibition.
• Lysosomal cholesterol accumulation (via U18666A) reduced LLOMe-induced cell death, and this effect overlapped with LRRK2 inhibition, suggesting shared mechanisms.

Conclusions:

• This study demonstrates a LRRK2- and RAB8A-dependent mechanism that attenuates cell death following lysosomal damage in RAW 264.7 cells.
• Lysosomal cholesterol levels play a crucial role in modulating this protective pathway, indicating a link between LRRK2, lysosomal function, and lipid metabolism in cell survival.
• These findings offer new insights into Parkinson's disease pathogenesis and suggest potential therapeutic strategies targeting LRRK2 and lysosomal cholesterol homeostasis.