Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration

Ga Ram Jeong, Eun Hae Jang, Jae Ryul Bae, Soyoung Jun, Ho Chul Kang, Chi Hu Park, Joo Ho Shin, Yukio Yamamoto, Keiko Tanaka-Yamamoto, Valina Dawson, Ted M Dawson, Eun Mi Hur, Byoung Dae Lee

Research output: Contribution to journalArticle

Abstract

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. Methods: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. Results: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. Conclusions: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

Original languageEnglish (US)
Article number8
JournalMolecular Neurodegeneration
Volume13
Issue number1
DOIs
StatePublished - Feb 13 2018

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rab GTP-Binding Proteins
Leucine
Phosphotransferases
Phosphorylation
Dopaminergic Neurons
Parkinson Disease

Keywords

  • LRRK2
  • Neurodegeneration
  • Parkinson's disease
  • Phosphorylation
  • Rab GTPases

ASJC Scopus subject areas

  • Molecular Biology
  • Clinical Neurology
  • Cellular and Molecular Neuroscience

Cite this

Jeong, G. R., Jang, E. H., Bae, J. R., Jun, S., Kang, H. C., Park, C. H., ... Lee, B. D. (2018). Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration. Molecular Neurodegeneration, 13(1), [8]. https://doi.org/10.1186/s13024-018-0240-1

Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration. / Jeong, Ga Ram; Jang, Eun Hae; Bae, Jae Ryul; Jun, Soyoung; Kang, Ho Chul; Park, Chi Hu; Shin, Joo Ho; Yamamoto, Yukio; Tanaka-Yamamoto, Keiko; Dawson, Valina; Dawson, Ted M; Hur, Eun Mi; Lee, Byoung Dae.

In: Molecular Neurodegeneration, Vol. 13, No. 1, 8, 13.02.2018.

Research output: Contribution to journalArticle

Jeong, GR, Jang, EH, Bae, JR, Jun, S, Kang, HC, Park, CH, Shin, JH, Yamamoto, Y, Tanaka-Yamamoto, K, Dawson, V, Dawson, TM, Hur, EM & Lee, BD 2018, 'Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration', Molecular Neurodegeneration, vol. 13, no. 1, 8. https://doi.org/10.1186/s13024-018-0240-1
Jeong, Ga Ram ; Jang, Eun Hae ; Bae, Jae Ryul ; Jun, Soyoung ; Kang, Ho Chul ; Park, Chi Hu ; Shin, Joo Ho ; Yamamoto, Yukio ; Tanaka-Yamamoto, Keiko ; Dawson, Valina ; Dawson, Ted M ; Hur, Eun Mi ; Lee, Byoung Dae. / Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration. In: Molecular Neurodegeneration. 2018 ; Vol. 13, No. 1.
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AU - Jang, Eun Hae

AU - Bae, Jae Ryul

AU - Jun, Soyoung

AU - Kang, Ho Chul

AU - Park, Chi Hu

AU - Shin, Joo Ho

AU - Yamamoto, Yukio

AU - Tanaka-Yamamoto, Keiko

AU - Dawson, Valina

AU - Dawson, Ted M

AU - Hur, Eun Mi

AU - Lee, Byoung Dae

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N2 - Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. Methods: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. Results: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. Conclusions: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

AB - Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. Methods: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. Results: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. Conclusions: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

KW - LRRK2

KW - Neurodegeneration

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