# Leucine-Rich Repeat Kinase 1 Signaling Targets Proteins Critical for Endosome/Lysosome Sorting and Trafficking in Osteoclasts

## TL;DR

This study shows that LRRK1 regulates osteoclast function by controlling proteins involved in endosome/lysosome trafficking and cytoskeleton changes, which affects bone resorption.

## Contribution

The paper identifies specific phosphoproteins regulated by LRRK1 in osteoclasts, linking them to endosome/lysosome trafficking and bone resorption.

## Key findings

- LRRK1-deficient osteoclasts show altered phosphorylation of proteins like VPS35 and CFL1.
- Six phosphoproteins are involved in endosome/lysosome sorting and trafficking.
- Phosphorylation changes in VPS35 and CFL1 are critical for osteoclast function.

## Abstract

Two types of cells located in the bone regulate bone mass. Osteoclasts cause bone destruction whereas osteoblasts produce new bone matrix. Bone loss occurs with age partially because the rate of bone formation cannot compensate for the increase in bone degradation because of sex hormone deficiency and other causes. Our previous studies demonstrated that LRRK1 played a critical role in regulating osteoclast-mediated bone resorption. Mice lacking LRRK1 manifested high bone mass because of the failure of osteoclasts to resorb bone. The molecular mechanism of LRRK1 regulation of osteoclast function is elusive. To identify potential LRRK1 targets in osteoclasts, we performed a 2D phosphor-proteomics analysis with lysates extracted from osteoclasts derived from Lrrk1 gene knockout and wild-type mice. Differentially phosphorylated peptide spots between the two types of cells were collected, digested with trypsin, and identified by mass spectrometry. A total of 6 out of 17 differentially expressed phosphoproteins are known to be implicated in endosome/lysosome sorting, vacuolar protection, and trafficking. SNX2, VPS35, VTA1, CFL1, and CTSA were highly hypophosphorylated whereas SNX3 was hyperphosphorylated in LRRK1-deficient osteoclasts. Further Phos-tag SDS PAGE analyses confirmed the downregulation of VPS35 and CFL1 phosphorylation in LRRK1-depleted osteoclasts. Our findings suggest that LRRK1 signaling regulates osteoclast function via phosphorylating VPS35 and CFL1 and modulating endosome/lysosome distribution and F-actin remodeling in osteoclasts.

Global knockout (KO) of the Lrrk1 gene in mice causes severe osteopetrosis because of the failure of osteoclasts to resorb bone. The molecular mechanism of LRRK1 regulation of osteoclast function is not fully understood. Here, we performed a 2D DIGE phosphor-proteomics analysis to identify potential LRRK1 targets in osteoclasts. Splenocytes from Lrrk1 KO and wild-type (WT) mice were differentiated into osteoclasts for protein extraction. Lysates from Lrrk1 KO and WT cells were labeled with Cy3- and Cy5-dye, respectively. Labeled proteins were mixed and analyzed on the same 2D SDS PAGE for protein profiling. The same amounts of cellular protein were also labeled with Cy3-dye and ran on a 2D SDS PAGE. The gels were then stained using Pro-Q® Diamond Phosphoprotein Gel Stain for phosphoprotein profiling. Differentially phosphorylated protein spots between the two types of cells were collected, digested with trypsin, and identified by mass spectrometry. Seventeen phosphoproteins were identified, six of which are known to be involved in endosome/lysosome sorting, vacuolar protection, and trafficking. While five of these proteins (SNX2, VPS35, VTA1, CFL1, and CTSA) were significantly hypophosphorylated, SNX3 was hyperphosphorylated in LRRK1-deficient osteoclasts. The downregulation of VSP35 and CFL1 phosphorylation in LRRK1-deficient cells was validated by Phos-tag SDS PAGE analysis. Our results indicate that LRRK1 signaling regulates osteoclast function via modulating VPS35 and CFL1 phosphorylation critical for endosome/lysosome trafficking and dynamic cytoskeleton arrangement in osteoclasts.