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Primarydata associated withthe manuscript "Structural basis of LRRK2 membrane recruitment and activation" (Hanwen Zhu*, Francesca Tonelli*, Mansoore Esmaili, Martin Turk, Alan Prescott, Dario R. Alessi, Ji Sun). These include immunoblotting data and their quantitation, representative confocal microscopy images, and quantitation and statistical analysis of confocal microscopy images. &nbsp

Mutations enhancing the kinase activity of LRRK2 cause Parkinson’s disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared to a biochemical kinase assay, as only a minority of activating variants [CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)], enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppressed microtubule association in the presence of a Type I kinase inhibitor [ARM(M712V), LRR(R1320S), ROC (A1442P, K1468E, S1508R), CORA(A1589S), CORB (Y1699C, R1728H/L) and WD40(R2143M, S2350I, G2385R)]. Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2 dependent and PPM1H phosphatase reversible; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain and we show that residues predicted to be essential for Rab12 interaction at this site influence overall phosphoRab levels in a manner distinct from Rab29 activation of LRRK2. Our data support a model in which Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.

Primary data associated with the manuscript "Genome wide screen reveals Rab12 GTPase as a critical activator of pathogenic LRRK2 kinase" (Herschel S. Dhekne, Francesca Tonelli, Wondwossen M. Yeshaw,Claire Y. Chiang, Charles Limouse, Ebsy Jaimon, Elena Purlyte,Dario Alessi, and Suzanne Pfeffer).These include raw immunoblotting data (annotated .tiff exports of Image Studio Lite Licor scans) and their quantitation (.xlsx files) and graphs obtained using GraphPad Prism(.pzf files). The new version (published 09/06/2023) includes the primary data added for paper revision.

Mouse embryo fibroblasts (MEFs) isolated from genetically altered mouse strains are a powerful resource for investigating the consequences of defined genetic mutations at the cellular level. Here we describe a protocol for the isolation of MEFs from mouse embryos collected at E12.5. MEFs obtained by this procedure are suitable for use in biochemical assays and for further genetic manipulation (e.g. viral transduction). This protocol was adapted from: Durkin ME, Qian X, Popescu NC, Lowy DR. Isolation of Mouse Embryo Fibroblasts. Bio Protoc. 2013 Sep 20;3(18):e908. doi: 10.21769/bioprotoc.908. PMID: 27376106; PMCID: PMC4928858.

Previous studies using confocal fluorescence microscopy showed that transient over-expression of Rab29 recruits the bulk of cellular LRRK2 to the Golgi, which results in its activation (PMID: 29212815). Here we describe our confocal immunofluorescence microscopy method for measuring the co-localization of LRRK2 and Rab29 in a cell-based assay. This method can be used to screen the impact that LRRK2 mutations or Rab29 mutations have on LRRK2 recruitment to the Golgi, as well as the effect of any compound on Rab29-induced LRRK2 recruitment to the Golgi. Note: This protocol can be used for HEK293 cells and HeLa cells. For other cell types, experimental conditions may need optimising. In parallel with the preparation of samples for immunofluorescence microscopy, we recommend preparing samples for quantitative immunoblotting analysis (as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6) to confirm successful transfection.

Raw data files used for the manuscript "Impact of 100LRRK2 variants linked to Parkinson’s Disease on kinase activity and microtubule binding". This is a revised versionof the manuscript"Impact of 98 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding". The Word document (File description v2.docx) contains a description of each file.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) increase its activity leading to increased phosphorylation of Rab proteins. Here we introduce a sensitive and accurate assay to measure increased phospho Rab levels using synthetic stable isotope-labeled analogues for both phosphorylated and non-phosphorylated tryptic peptides surrounding Rab10-Thr73. Compared to healthy controls, carriers of mutated LRRK2 had 1.9-fold and those with VPS35 mutation 3.7-fold increased pRab10 levels in their neutrophils. Our generic MS-based assay helps to stratify PD patient and determine LRRK2 inhibitor efficiency in clinical trials., Graphical Abstract Highlights • MS-based clinical assay that accurately determines phospho Rab10 occupancy. • Stable isotope labeled phosphopeptide injected as a standard with endogenous tryptic phospho Rab peptide for accurate ratio determination. • Determination of pRab levels in neutrophils of Parkinson disease patients. • Relevance of pRab levels as marker of PD., Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.

SummaryMutations in LRRK2 are the most common genetic cause of late-onset Parkinson’s disease (PD). LRRK2 encodes the leucine-rich repeat kinase 2 (LRRK2), whose kinase activity is regulated by Rab29, a membrane-anchored GTPase. However, molecular mechanisms underlying Rab29-dependent recruitment and activation of LRRK2 remain unclear. Here we report cryo-EM structures of LRRK2–Rab29 complexes in three oligomeric states, illustrating snapshots of key steps during LRRK2 membrane recruitment and activation. Rab29 binds to the ARM domain of LRRK2, and disruption at the interface abrogates LRRK2 kinase activity. Activation of LRRK2 is underpinned by the formation of an unexpected Rab29-induced super-assembly containing two central kinase-active and two peripheral kinase-inactive LRRK2 protomers. Central protomers undergo pronounced oligomerization-associated rearrangements and adopt an active conformation. Our work reveals the structural mechanism for LRRK2’s spatial regulation controlled by Rab GTPases, provides mechanistic insights into pathogenic mutations and identifies new opportunities to design LRRK2 inhibitors for PD treatment.

Raw data files used for the manuscript "Impact of 98 LRRK2 variants linked to Parkinson’s Disease on kinase activity and microtubule binding". The Word document (File description.docx) contains a description of each file. Mutations enhancing the kinase activity of LRRK2 cause Parkinson's disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 98 LRRK2 variants linked to PD, including previously described pathogenic mutations, and identify 22 variants that robustly stimulate LRRK2 kinase activity. These include variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)] as well as variants residing within the GTPase domain, predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORB (S1761R, L1795F)] and CORB:CORB dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer910/pSer935), consistent with the notion that the active kinase conformation blocks their phosphorylation. We observe that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation at Thr73 compared to an in vitro kinase assay, as only a minority of activating variants enhance the activity of immunoprecipitated LRRK2, comprising the CORB (Y1699C, R1728H, S1761R) and kinase (G2019S, I2020T, T2031S) variants. Twelve variants including some that activated LRRK2, suppressed microtubule association in the presence of a Type 1 kinase inhibitor [ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)]. Our findings will aid in understanding the impact of these variants on LRRK2 kinase activation, stimulate work to reveal mechanisms by which variants impact biology, and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 clinical trials.

Accurate, quantitative analysis of protein expression and modifications (such as phosphorylation) is critical when studying cell signalling. Here we describe our method for efficient immunoblotting analysis of the LRRK1 signalling pathway components in cell and mouse tissue extracts. Specifically, we immunoblot using rigorously validated and characterized antibodies for LRRK1-total, Rab7A and pS72 Rab7A, pT202/Y204 ERK1/2, phosphor(Ser)-PKC Substrates and GAPDH or Tubulin (loading controls), although the protocol described here can also be applied to different cell components. Included are procedures for sample preparation from cultured cells and mouse tissue, gel electrophoresis, protein transfer, and antibody incubation.

Previous studies using confocal fluorescence microscopy and cryo-electron microscopy reported that most pathogenic LRRK2 mutants, as well as pharmacological inhibition of LRRK2 kinase activity with Type-I inhibitors, cause relocalization of LRRK2 to filamentous structures that colocalize with microtubules (PMID: 22080837; PMID: 28453723; PMID: 32783917; PMID: 32814344). Here we describe our confocal immunofluorescence microscopy method for measuring LRRK2 association with microtubules in a cell-based assay. This method can be used to screen the impact that LRRK2 mutations have on LRRK2 microtubule binding, as well as the effect of any compound on LRRK2 association with microtubules.

Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation‐positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient‐derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p

Several studies attest to the long-term consequences of COVID-19 infection on survivors' mental illness, especially in terms of high prevalence of post-traumatic stress disorder (PTSD) 1-3 months after hospitalization. Aims of the present study were (1) to jointly evaluate PTSD and positive mental health among COVID-19 survivors and family members after hospital discharge, and (2) to investigate the relationship between perceived healthcare staff's relational empathy during hospitalization and survivors' post-traumatic stress levels. In this cross-sectional study, 60 survivors (M

We describe a non-radioactive assay that we deploy for analysing LRRK2 protein kinase activity in vitro using Rab proteins as a substrate. This assay can be used to measure the intrinsic activity of LRRK2 immunoprecipitated from cells as well as to assess the impact of mutations on LRRK2 activity. Protocol Overview: 1) Transient transfection of HEK293 cells for analysis of over-expressed LRRK2 activity. 2) Preparation and quantification of cell lysates (from HEK293 cells over-expressing LRRK2 or MEFs). 3) Immunoprecipitation of over-expressed FLAG-tagged LRRK2 from cell lysates for analysis of LRRK2 activity in vitro. 4) Immunoprecipitation of endogenous LRRK2 from MEFs for analysis of LRRK2 activity in vitro. 5) In vitro kinase assay using LRRK2 immunoprecipitated from cells. 6) Analysis of kinase reaction products by quantitative immunoblotting analysis.

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.

Accurate, quantitative analysis of protein expression and modifications (such as phosphorylation) is critical when studying cell signalling. Here we describe our method for efficient immunoblotting analysis of the LRRK2 signalling pathway components in cell and mouse tissue extracts. Specifically, we immunoblot using rigorously validated and characterized antibodies for LRRK2-total and LRRK2-pSer935 (multiplexed), Rab10-total and Rab10-pThr73 (multiplexed), Rab12-total and Rab12-pSer105 (multiplexed) and GAPDH or tubulin (loading control), although the protocol described here can also be applied to different cell components. Included are procedures for sample preparation from cultured cells and mouse tissue, gel electrophoresis, protein transfer, and antibody incubation. Note: If analysing cells isolated from human blood (neutrophils, monocytes or PBMCs), please refer to the specific protocols deposited in Protocols.io on how to isolate these cells. These lysates can then be analysed by immunoblotting as described here ( See Section "Preparation of samples for immunoblot analysis" to Section "Image acquisition and Analysis").

Introduction Among the multiple complex pathophysiological mechanisms underlying COVID‐19 pneumonia, immunothrombosis has been shown to play a key role. One of the most dangerous consequences of the prothrombotic imbalance is the increased incidence of micro‐ and macrothrombotic phenomena, especially deep vein thrombosis (DVT) and pulmonary embolism (PE). Methods We investigated the correlation between radiological and clinical–biochemical characteristics in a cohort of hospitalised COVID‐19 patients. Results PE was confirmed in 14/61 (23%) patients, five (35.7%) had DVT. The radiographic findings, quantified by Qanadli score calculated on CT angiography, correlated with the clinical score and biochemical markers. The ratio between the right and left ventricle diameter measured at CT angiography correlated with the length of hospital stay. Conclusion In our cohort radiological parameters showed a significant correlation with clinical prognostic indices and scores, thus suggesting that a multidisciplinary approach is advisable in the evaluation of PE in COVID‐19 patients.

Pathogenic LRRK2 phosphorylation of Rab10 GTPase dramatically redistributes Myosin Va and RILPL2 proteins to the mother centriole and sequesters Myosin Va at that location in a manner that likely interferes with its role in ciliogenesis., Activating mutations in LRRK2 kinase causes Parkinson’s disease. Pathogenic LRRK2 phosphorylates a subset of Rab GTPases and blocks ciliogenesis. Thus, defining novel phospho-Rab interacting partners is critical to our understanding of the molecular basis of LRRK2 pathogenesis. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 and MyoVa relocalize to the peri-centriolar region in a phosphoRab10-dependent manner. PhosphoRab10 retains Myosin Va over pericentriolar membranes as determined by fluorescence loss in photobleaching microscopy. Without pathogenic LRRK2, RILPL2 is not essential for ciliogenesis but RILPL2 over-expression blocks ciliogenesis in RPE cells independent of tau tubulin kinase recruitment to the mother centriole. These experiments show that LRRK2 generated-phosphoRab10 dramatically redistributes a significant fraction of Myosin Va and RILPL2 to the mother centriole in a manner that likely interferes with Myosin Va’s role in ciliogenesis.

Much attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinson’s disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson’s mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson’s pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson’s patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

We describe here our method for immunostaining of primary cilia in brain sections from wild type and LRRK2 or other mutant mice. Included are procedures for perfusion, tissue harvesting, sectioning and staining, image acquisition and analysis.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson' disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Excessive skeletal deformations and brittle fractures in the vast majority of patients suffering from osteogenesis imperfecta (OI) are a result of substantially reduced bone quality. Because the mechanical competence of bone is dependent on the tissue characteristics at small length scales, it is of crucial importance to assess how OI manifests at the micro- and nanoscale of bone. In this context, the Chihuahua (Chi/+) zebrafish, carrying a heterozygous glycine substitution in the α1 chain of collagen type I, has recently been proposed as a suitable animal model of classical dominant OI, showing skeletal deformities, altered mineralization patterns, and a smaller body size. This study assessed the bone quality properties of Chi/+ at multiple length scales using micro-computed tomography (micro-CT), histomorphometry, quantitative back-scattered electron imaging, Fourier-transform infrared spectroscopy, nanoindentation, and X-ray microscopy. At the skeletal level, the Chi/+ displays smaller body size, deformities, and fracture calli in the ribs. Morphological changes at the whole bone level showed that the vertebrae in Chi/+ had a smaller size, smaller thickness, and distorted shape. At the tissue level, Chi/+ displayed a higher degree of mineralization, lower collagen maturity, lower mineral maturity, altered osteoblast morphology, and lower osteocyte lacunar density compared to wild-type zebrafish. The alterations in the cellular, compositional, and structural properties of Chi/+ bones bear an explanation for the impaired local mechanical properties, which promote an increase in overall bone fragility in Chi/+. The quantitative assessment of bone quality in Chi/+ thus further validates this mutant as an important model reflecting osseous characteristics associated with human classical dominant OI. © 2018 American Society for Bone and Mineral Research.

Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector‐binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans‐Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild‐type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29‐mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2‐mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.