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Elżbieta Kania1,2,3, Jaclyn S Long1,2, David G McEwan1,2

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Leucine-rich repeat kinase 2 (LRRK2) phosphorylation sites regulate autophagy. Impaired LRRK2 phosphorylation disrupts cellular waste removal, contributing to Parkinson's disease pathology by increasing kinase activity.

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Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Neuroscience

Background:

  • Mutations in leucine-rich repeat kinase 2 (LRRK2) are a leading genetic cause of Parkinson's disease (PD).
  • LRRK2 protein possesses GTPase and kinase activity, influenced by its phosphorylation status.
  • The role of constitutive phosphorylation sites (S910/S935/S955/S973) in LRRK2 function is largely unknown.

Purpose of the Study:

  • To investigate the role of LRRK2 phosphorylation at S910/S935/S955/S973 sites in regulating autophagy.
  • To determine the impact of altered LRRK2 phosphorylation on cellular lysosomal function and autophagic flux.
  • To elucidate the mechanistic link between LRRK2 kinase activity, phosphorylation status, and autophagy impairment in Parkinson's disease models.

Main Methods:

  • Generated quadruple LRRK2 phosphomutant cells (4xSA) to mimic reduced phosphorylation.
  • Utilized LRRK2 kinase inhibitors (MLi-2, PF-06447475) to assess the effect of kinase activity modulation on autophagy.
  • Analyzed downstream LRRK2 phosphorylation targets (Rab8a, Rab10) and their role in autophagy.
  • Examined cells expressing the pathogenic LRRK2 R1441C mutant.

Main Results:

  • Cells with quadruple LRRK2 phosphomutant (4xSA) exhibited impaired lysosomal function and failed to induce autophagy during starvation.
  • LRRK2 kinase inhibitors, despite reducing phosphorylation at S910/S935/S955/S973, did not affect autophagy.
  • Autophagy impairment in 4xSA cells was attributed to enhanced LRRK2 kinase activity, evidenced by increased phosphorylation of Rab8a and Rab10.
  • Expression of phosphorylation-deficient Rab8a and Rab10 mutants rescued the autophagy defect.
  • Pathological LRRK2 R1441C mutant cells showed reduced autophagy and decreased constitutive phosphorylation, correlating with increased kinase activity.

Conclusions:

  • LRRK2 phosphorylation at S910/S935/S955/S973 sites is critical for regulating autophagy under basal and starvation conditions.
  • Enhanced LRRK2 kinase activity, rather than direct phosphorylation status at these sites, drives autophagy impairment in LRRK2-associated Parkinson's disease.
  • These findings highlight the intricate relationship between LRRK2 phosphorylation, kinase activity, and autophagy, offering insights into Parkinson's disease pathogenesis.