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[Preprint]. 2024 Jan 18:2024.01.17.24300927.

doi: 10.1101/2024.01.17.24300927.

A pathogenic variant in RAB32 causes autosomal dominant Parkinson's disease and activates LRRK2 kinase

Emil K Gustavsson^{1

2

3}, Jordan Follett^{4

2}, Joanne Trinh^{5

2}, Sandeep K Barodia⁶, Raquel Real^{7

8

3}, Zhiyong Liu⁶, Melissa Grant-Peters^{1

9}, Jesse D Fox², Silke Appel-Cresswell¹⁰, A Jon Stoessl¹⁰, Alex Rajput¹¹, Ali H Rajput¹¹, Roland Auer¹², Russel Tilney^{7

8}, Marc Sturm¹³, Tobias B Haack¹³, Suzanne Lesage¹⁴, Christelle Tesson¹⁴, Alexis Brice^{14

15}, Carles Vilariño-Güell¹⁶, Mina Ryten^{1

17

3}, Matthew S Goldberg⁶, Andrew B West¹⁸, Michele T Hu¹⁹, Huw R Morris^{7

8

3}, Manu Sharma²⁰, Ziv Gan-Or^{21

22

23}, Bedia Samanci²⁴, Pawel Lis^{9

3}, Teresa Tocino²⁵, Rim Amouri²⁶, Samia Ben Sassi²⁶, Faycel Hentati²⁶; Global Parkinson’s Genetics Program (GP2); Francesca Tonelli^{9

3}, Dario R Alessi^{9

3}, Matthew J Farrer^{4

2}

Affiliations

¹ Department of Genetics and Genomic Medicine, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK.
² Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
³ Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA.
⁴ McKnight Brain Institute, Department of Neurology, University of Florida, Gainesville, Florida, USA.
⁵ Institute of Neurogenetics, University of Lübeck, Ratzeburger Allee 160, Lübeck 23538, Germany.
⁶ Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, AL, USA.
⁷ Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.
⁸ UCL Movement Disorders Centre, University College London, London WC1N 3BG, UK.
⁹ MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
¹⁰ Pacific Parkinson's Research Centre, Djavad Mowafaghian Centre for Brain Health, Division of Neurology, University of British Columbia, Vancouver, BC, Canada.
¹¹ Movement Disorders Program, Division of Neurology, University of Saskatchewan and Saskatchewan Health Authority, Saskatoon, SK, Canada.
¹² Department of Pathology, University of Saskatchewan and Saskatchewan Health Authority, Saskatoon, SK, Canada.
¹³ Institute for Medical Genetics and Applied Genomics, University of Tübingen, Germany.
¹⁴ Sorbonne Université, Institut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, Paris, France.
¹⁵ Assistance Publique Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Département de Neurologie, Centre d'Investigation Clinique Neurosciences, DMU Neuroscience, Paris, France.
¹⁶ Department of Medical Genetics, University of British Columbia, Vancouver V6T 1Z3, Canada.
¹⁷ NIHR Great Ormond Street Hospital Biomedical Research Centre, University College London, London WC1N 1EH, UK.
¹⁸ Duke Center for Neurodegeneration and Neurotherapeutics, Department of Pharmacology and Cancer Biology, Duke University, 3 Genome Court, Durham 27710, North Carolina, USA.
¹⁹ Division of Neurology, Nuffield Department of Clinical Neurosciences, University of Oxford, UK.
²⁰ Centre for Genetic Epidemiology, Institute for Clinical Epidemiology and Applied Biometry, University of Tübingen, Germany.
²¹ The Neuro (Montreal Neurological Institute-Hospital), Montreal, Quebec, Canada.
²² Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada.
²³ Department of Human Genetics, McGill University, Montreal, Quebec, Canada.
²⁴ Behavioural Neurology and Movement Disorders Unit, Department of Neurology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey.
²⁵ Queen Mary College, University of London, UK.
²⁶ Service de Neurologie, Institut National de Neurologie, La Rabta, Tunis 1007, Tunisia.

PMID: 38293014
PMCID: PMC10827257
DOI: 10.1101/2024.01.17.24300927

A pathogenic variant in RAB32 causes autosomal dominant Parkinson's disease and activates LRRK2 kinase

Emil K Gustavsson et al. medRxiv. 2024.

[Preprint]. 2024 Jan 18:2024.01.17.24300927.

doi: 10.1101/2024.01.17.24300927.

Authors

Affiliations

¹ Department of Genetics and Genomic Medicine, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK.
² Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
³ Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA.
⁴ McKnight Brain Institute, Department of Neurology, University of Florida, Gainesville, Florida, USA.
⁵ Institute of Neurogenetics, University of Lübeck, Ratzeburger Allee 160, Lübeck 23538, Germany.
⁶ Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, Birmingham, AL, USA.
⁷ Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.
⁸ UCL Movement Disorders Centre, University College London, London WC1N 3BG, UK.
⁹ MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
¹⁰ Pacific Parkinson's Research Centre, Djavad Mowafaghian Centre for Brain Health, Division of Neurology, University of British Columbia, Vancouver, BC, Canada.
¹¹ Movement Disorders Program, Division of Neurology, University of Saskatchewan and Saskatchewan Health Authority, Saskatoon, SK, Canada.
¹² Department of Pathology, University of Saskatchewan and Saskatchewan Health Authority, Saskatoon, SK, Canada.
¹³ Institute for Medical Genetics and Applied Genomics, University of Tübingen, Germany.
¹⁴ Sorbonne Université, Institut du Cerveau-Paris Brain Institute-ICM, Inserm, CNRS, Paris, France.
¹⁵ Assistance Publique Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Département de Neurologie, Centre d'Investigation Clinique Neurosciences, DMU Neuroscience, Paris, France.
¹⁶ Department of Medical Genetics, University of British Columbia, Vancouver V6T 1Z3, Canada.
¹⁷ NIHR Great Ormond Street Hospital Biomedical Research Centre, University College London, London WC1N 1EH, UK.
¹⁸ Duke Center for Neurodegeneration and Neurotherapeutics, Department of Pharmacology and Cancer Biology, Duke University, 3 Genome Court, Durham 27710, North Carolina, USA.
¹⁹ Division of Neurology, Nuffield Department of Clinical Neurosciences, University of Oxford, UK.
²⁰ Centre for Genetic Epidemiology, Institute for Clinical Epidemiology and Applied Biometry, University of Tübingen, Germany.
²¹ The Neuro (Montreal Neurological Institute-Hospital), Montreal, Quebec, Canada.
²² Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada.
²³ Department of Human Genetics, McGill University, Montreal, Quebec, Canada.
²⁴ Behavioural Neurology and Movement Disorders Unit, Department of Neurology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey.
²⁵ Queen Mary College, University of London, UK.
²⁶ Service de Neurologie, Institut National de Neurologie, La Rabta, Tunis 1007, Tunisia.

PMID: 38293014
PMCID: PMC10827257
DOI: 10.1101/2024.01.17.24300927

Update in

RAB32 Ser71Arg in autosomal dominant Parkinson's disease: linkage, association, and functional analyses.
Gustavsson EK, Follett J, Trinh J, Barodia SK, Real R, Liu Z, Grant-Peters M, Fox JD, Appel-Cresswell S, Stoessl AJ, Rajput A, Rajput AH, Auer R, Tilney R, Sturm M, Haack TB, Lesage S, Tesson C, Brice A, Vilariño-Güell C, Ryten M, Goldberg MS, West AB, Hu MT, Morris HR, Sharma M, Gan-Or Z, Samanci B, Lis P, Periñan MT, Amouri R, Ben Sassi S, Hentati F; Global Parkinson's Genetics Program (GP2); Tonelli F, Alessi DR, Farrer MJ. Gustavsson EK, et al. Lancet Neurol. 2024 Jun;23(6):603-614. doi: 10.1016/S1474-4422(24)00121-2. Epub 2024 Apr 10. Lancet Neurol. 2024. PMID: 38614108 Free PMC article.

Abstract

Background: Parkinson's disease (PD) is a progressive neurodegenerative disorder. Mendelian forms have revealed multiple genes, with a notable emphasis on membrane trafficking; RAB GTPases play an important role in PD as a subset are both regulators and substrates of LRRK2 protein kinase. To explore the role of RAB GTPases in PD, we undertook a comprehensive examination of their genetic variability in familial PD.

Methods: Affected probands from 130 multi-incident PD families underwent whole-exome sequencing and genotyping, Potential pathogenic variants in 61 RAB GTPases were genotyped in relatives to assess disease segregation. These variants were also genotyped in a larger case-control series, totaling 3,078 individuals (2,734 with PD). The single most significant finding was subsequently validated within genetic data (6,043 with PD). Clinical and pathologic findings were summarized for gene-identified patients, and haplotypes were constructed. In parallel, wild-type and mutant RAB GTPase structural variation, protein interactions, and resultant enzyme activities were assessed.

Findings: We found RAB32 c.213C>G (Ser71Arg) to co-segregate with autosomal dominant parkinsonism in three multi-incident families. RAB32 Ser71Arg was also significantly associated with PD in case-control samples: genotyping and database searches identified thirteen more patients with the same variant that was absent in unaffected controls. Notably, RAB32 Ser71Arg heterozygotes share a common haplotype. At autopsy, one patient had sparse neurofibrillary tangle pathology in the midbrain and thalamus, without Lewy body pathology. In transfected cells the RAB32 Arg71 was twice as potent as Ser71 wild type to activate LRRK2 kinase.

Interpretation: Our study provides unequivocal evidence to implicate RAB32 Ser71Arg in PD. Functional analysis demonstrates LRRK2 kinase activation. We provide a mechanistic explanation to expand and unify the etiopathogenesis of monogenic PD.

Funding: National Institutes of Health, the Canada Excellence Research Chairs program, Aligning Science Across Parkinson's, the Michael J. Fox Foundation for Parkinson's Research, and the UK Medical Research Council.

Keywords: LRRK2; PINK1; Parkinson’s disease; RAB32.

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Conflict of interest statement

AR receives unrestricted research support from the Dr. Ali Rajput Endowment for Parkinson’s Disease and Movement Disorders; in the past two years AR has received honoraria from CQDM/Brain Canada and Ipsen Biopharmaceuticals Canada. MSG reports grants from NIH/NINDS and the Michael J. Fox Foundation for Parkinson’s Research. AJS has received fees from Neurocrine (Chair, DSMB), AskBio (Member, DSMB) and Capsida (advisor), receives a stipend from the International Parkinsons and Movement Disorders Society (Editor-in-Chief, Movement Disorders) and grant funding from Michael J. Fox Foundation, Weston Brain Institute and Brain Canada. ZGO received consultancy fees from Bial Biotec, Bial, Capsida, Handl Therapeutics, Idorsia, Neuron23, Ono Therapeutics, Prevail Therapeutics, UCB and Vanqua. He reports grants from the Michael J. Fox Foundation for Parkinson’s Research, The Weston Family Foundation, The Silverstein Foundation, NIH and the Canadian Consortium on Neurodegeneration in Aging (CCNA). MJF reports US patents associated with LRRK2 mutations and mouse models (8409809, 8455243), and methods of treating neurodegenerative disease (20110092565). SAC has received honoraria from Merz, and grant funding from the Pacific Parkinson’s Research Institute, the Weston Family Foundation, Parkinson Canada, Canadian Institutes of Health Research, the VGH and UBC Hospital Foundation, Rick’s Heart Foundation and the Jack and Darlene Poole Foundation.

Figures

**Figure 1.. RAB32 Ser71Arg co-segregates with Parkinson’s disease.**
A) Simplified pedigrees for three families presenting the RAB32 Ser71Arg mutation. Probands are represented by an arrow and a diagonal line indicates deceased subjects. Patients diagnosed with Parkinson’s disease have black filled symbols, patients with a grey filled symbols had tremors but no diagnosis. Heterozygote mutation carriers (M) and wild-type (wt) genotypes are indicated with corresponding age (* age at death) and age at onset of disease.

**Figure 2.**
Overview of the AlphaFold model of Rab32 (Ser71Arg) and LRRK2(350–550) protein complex (Rab32 Arg71 marked in red) **(A)**, with detailed view of electrostatic **(B)** and hydrophobic **(C)** interactions predicted to be crucial for the protein-protein binding. **(D)** Overlay of 2 different rotamers of the Rab32 Arg71 residue, showing the rotamer (in brown) with a possible electrostatic interaction between Arg71 and LRRK2 Asp478. All models showed a binding interface consistent with interfaces identified for RAB29 and RAB38, with a hydrophobic patch formed by Met402, Leu403, Leu406 and Met407 of LRRK2, interacting with Phe62, Val60, Trp80 and Val94 of RAB32 (B). Electrostatic interactions (C) were predicted to form between the following pairs of residues (LRRK2 – RAB32): Arg399 – Asp61, Lys451 – Glu98 and Asp478 – Arg76, with additional salt bridges predicted between Arg361 – Glu86, Asp392 – Arg55 and Lys439 – Asp61.

**Figure 3:**
Quantitative immunoblotting analysis of the cellular kinase activity of wild-type LRRK2 in the presence of Rab32 (Ser71 wild-type or Arg71 mutant) or Rab29. HEK293 cells were co-transfected with wild-type LRRK2 and either HA-empty vector (−) or the indicated variants of HA-tagged Rab32 or HA-tagged Rab29, and treated +/− MLi-2 (200 nM, 2 hrs) or DMSO control (0.1% v/v) prior to lysis. Quantitation of phosphorylated Rab10-T73, phosphorylated LRRK2-S1292 and phosphorylated LRRK2-S935 normalized to total Rab10 and total LRRK2 respectively is shown. Error bars indicate mean with SD from two independent experiments, each performed in duplicate.

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References

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This is a preprint.

A pathogenic variant in RAB32 causes autosomal dominant Parkinson's disease and activates LRRK2 kinase

Affiliations

A pathogenic variant in RAB32 causes autosomal dominant Parkinson's disease and activates LRRK2 kinase

Authors

Affiliations

Update in

Abstract

Conflict of interest statement

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