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

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Shahzad S Khan ◽  
Yuriko Sobu ◽  
Herschel S Dhekne ◽  
Francesca Tonelli ◽  
Kerryn Berndsen ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Dorsal Striatum ◽  
Mutant Mice ◽  
Mouse Strains ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Nigrostriatal Pathway ◽  
Cholinergic Interneurons ◽  
Lrrk2 Kinase

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.

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

2021 ◽  
Author(s):  
Shahzad S. Khan ◽  
Yuriko Sobu ◽  
Herschel S. Dhekne ◽  
Francesca Tonelli ◽  
Kerryn Berndsen ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Dorsal Striatum ◽  
Mouse Strains ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Lrrk2 Mutation ◽  
Cholinergic Interneurons ◽  
To Receive

AbstractPreviously, we showed that cholinergic interneurons of the dorsal striatum lose cilia in mice harboring the Parkinson’s disease associated, kinase activating, R1441C LRRK2 mutation (Dhekne et al., 2018). Here we show that this phenotype is also seen in two mouse strains carrying the most common human G2019S LRRK2 mutation. Heterozygous loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases (Berndsen et al., 2019) yields the same cilia loss phenotype, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. In addition, astrocytes throughout the striatum show a ciliation defect in LRRK2 and PPM1H-/+ mutant models. Hedgehog signaling requires cilia, and loss of cilia correlates here with a loss in induction of Hedgehog signaling as monitored by in situ hybridization of Gli1 transcripts. These data support a model in which LRRK2 and PPM1H mutant mice struggle to receive and respond to critical Hedgehog signals in the nigral-striatal pathway.

scholarly journals A pathway for Parkinson’s Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Herschel S Dhekne ◽  
Izumi Yanatori ◽  
Rachel C Gomez ◽  
Francesca Tonelli ◽  
Federico Diez ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Sonic Hedgehog ◽  
Transcriptional Activation ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Cultured Cells ◽  
Editorial Note ◽  
Rab Gtpases ◽  
Lrrk2 Kinase

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 (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

scholarly journals Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Martin Steger ◽  
Federico Diez ◽  
Herschel S Dhekne ◽  
Pawel Lis ◽  
Raja S Nirujogi ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Protein Transport ◽  
Serum Starvation ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Rab Protein ◽  
Affinity Enrichment ◽  
Fold Reduction ◽  
Cilia Formation ◽  
Conserved Residue

We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

scholarly journals Distinct profiles of LRRK2 activation and Rab GTPase phosphorylation in clinical samples from different PD cohorts

2021 ◽  
Author(s):  
Lilian Peptropoulou-Vathi ◽  
Athina M Simitsi ◽  
Politymi-Eleni Valkimadi ◽  
Maria Kedariti ◽  
Lampros Dimitrakopoulos ◽  
...  
Keyword(s):  
Outcome Measures ◽  
Kinase Activity ◽  
Disease Status ◽  
Clinical Samples ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Comprehensive Picture ◽  
Lrrk2 Kinase ◽  
A53t Mutation ◽  
Lrrk2 Kinase Activity

Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself (levels, phosphorylation), or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in 2 separate idiopathic PD cohorts, but not in carriers of the A53T mutation in α-synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease.

scholarly journals Functional dissection of Rab GTPases involved in primary cilium formation

2007 ◽  
Vol 178 (3) ◽  
pp. 363-369 ◽  
Author(s):  
Shin-ichiro Yoshimura ◽  
Johannes Egerer ◽  
Evelyn Fuchs ◽  
Alexander K. Haas ◽  
Francis A. Barr
Keyword(s):  
Membrane Trafficking ◽  
Primary Cilia ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Cytoskeleton ◽  
Gtpase Activating Proteins ◽  
Cilia Formation ◽  
Sensory Structures ◽  
And Function ◽  
Specific Membrane

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.

scholarly journals Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

2019 ◽  
Vol 116 (12) ◽  
pp. 5550-5557 ◽  
Author(s):  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Joshua Yoon ◽  
Tim Stearns ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Transmembrane Protein ◽  
Cellular Level ◽  
Live Cells ◽  
Cholesterol Depletion ◽  
Motional Dynamics ◽  
Directional Transport ◽  
Bulk Behavior

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

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