Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

  1. Shahzad S Khan
  2. Yuriko Sobu
  3. Herschel S Dhekne
  4. Francesca Tonelli
  5. Kerryn Berndsen
  6. Dario R Alessi
  7. Suzanne R Pfeffer  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. University of Dundee, United Kingdom

Abstract

Activating LRRK2 mutations cause Parkinson's disease, and pathogenic LRRK2 kinase interferes with ciliogenesis. Previously, we showed that cholinergic interneurons of the dorsal striatum lose their cilia in R1441C LRRK2 mutant mice (Dhekne et al., 2018). Here, we show that cilia loss is seen as early as 10 weeks of age in these mice and also in two other mouse strains carrying the most common human G2019S LRRK2 mutation. Loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases yields the same cilia loss phenotype seen in mice expressing pathogenic LRRK2 kinase, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. Moreover, astrocytes throughout the striatum show a ciliation defect in all LRRK2 and PPM1H mutant models examined. Hedgehog signaling requires cilia, and loss of cilia in LRRK2 mutant rodents correlates with dysregulation of Hedgehog signaling as monitored by in situ hybridization of Gli1 and Gdnf transcripts. Dopaminergic neurons of the substantia nigra secrete a Hedgehog signal that is sensed in the striatum to trigger neuroprotection; our data support a model in which LRRK2 and PPM1H mutant mice show altered responses to critical Hedgehog signals in the nigrostriatal pathway.

Data availability

All primary data associated with each figure has been deposited in the Dryad repository and can be found at https://doi.org/10.5061/dryad.76hdr7sxx.

The following data sets were generated

Article and author information

Author details

  1. Shahzad S Khan

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3962-0226
  2. Yuriko Sobu

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Herschel S Dhekne

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2240-1230
  4. Francesca Tonelli

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4600-6630
  5. Kerryn Berndsen

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9353-7565
  6. Dario R Alessi

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2140-9185
  7. Suzanne R Pfeffer

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    For correspondence
    pfeffer@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

Funding

Michael J. Fox Foundation for Parkinson's Research (17298 & 6986)

  • Shahzad S Khan
  • Yuriko Sobu
  • Herschel S Dhekne
  • Francesca Tonelli
  • Kerryn Berndsen
  • Dario R Alessi
  • Suzanne R Pfeffer

Aligning Science Across Parkinson's (ASAP-000463)

  • Shahzad S Khan
  • Yuriko Sobu
  • Herschel S Dhekne
  • Francesca Tonelli
  • Kerryn Berndsen
  • Dario R Alessi
  • Suzanne R Pfeffer

National Institutes of Health (DK37332)

  • Suzanne R Pfeffer

Medical Research Council (MC_UU_12016/2)

  • Dario R Alessi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal studies were performed in accordance with the Animals (Scientific Procedures) Act of 1986 and regulations set by the University of Dundee, the U.K. Home Office, and the Administrative Panel on Laboratory Animal Care at Stanford University.

Reviewing Editor

  1. Wade Harper, Harvard Medical School, United States

Publication history

  1. Preprint posted: March 2, 2021 (view preprint)
  2. Received: March 2, 2021
  3. Accepted: October 17, 2021
  4. Accepted Manuscript published: October 18, 2021 (version 1)
  5. Version of Record published: October 27, 2021 (version 2)
  6. Version of Record updated: November 2, 2021 (version 3)

Copyright

© 2021, Khan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Shahzad S Khan
  2. Yuriko Sobu
  3. Herschel S Dhekne
  4. Francesca Tonelli
  5. Kerryn Berndsen
  6. Dario R Alessi
  7. Suzanne R Pfeffer
(2021)
Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain
eLife 10:e67900.
https://doi.org/10.7554/eLife.67900

Further reading

    1. Cell Biology
    Herschel S Dhekne et al.

    Parkinson’s disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases, including Rab8A and Rab10. We show here that LRRK2 kinase interferes with primary cilia formation in cultured cells, human LRRK2 G2019S iPS cells and in the cortex of LRRK2 R1441C mice. Rab10 phosphorylation strengthens its intrinsic ability to block ciliogenesis by enhancing binding to RILPL1. Importantly, the ability of LRRK2 to interfere with ciliogenesis requires both Rab10 and RILPL1 proteins. Pathogenic LRRK2 influences the ability of cells to respond to cilia-dependent, Hedgehog signaling as monitored by Gli1 transcriptional activation. Moreover, cholinergic neurons in the striatum of LRRK2 R1441C mice show decreased ciliation, which will decrease their ability to sense Sonic hedgehog in a neuro-protective circuit that supports dopaminergic neurons. These data reveal a molecular pathway for regulating cilia function that likely contributes to Parkinson’s disease-specific pathology.

    Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

    1. Cell Biology
    Pranidhi Sood et al.
    Research Article Updated

    The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half regenerates an intact cell. We used RNA sequencing (RNAseq) to assay the dynamic changes in Stentor’s transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA -binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the RNA-binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi-mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.