LRRK2 (Leucine-rich repeat kinase 2) is a large multidomain scaffolding protein with kinase and GTPase activities involved in synaptic vesicle (SV) dynamics. While its role in Parkinson's disease has been largely investigated, little is known about LRRK2 physiological role and until now few proteins have been described as substrates. We have previously demonstrated that LRRK2 through its WD40 domain interacts with synapsin I, an important SV-associated phosphoprotein involved in neuronal development and in the regulation of neurotransmitter release. To test whether synapsin I is substrate for LRRK2 and characterize the properties of its phosphorylation, we used in vitro kinase and binding assays as well as cellular model and site-direct mutagenesis. By using synaptosomes in superfusion, patch-clamp recordings in autaptic WT and synapsin I KO cortical neurons and SypHy assay on primary cortical culture from wild-type and BAC human LRRK2 G2019S mice we characterize the role of LRRK2 kinase activity on glutamate release and SV trafficking. Here we reported that synapsin I is phosphorylated by LRRK2 and demonstrated that the interaction between LRRK2 WD40 domain and synapsin I is crucial for this phosphorylation. Moreover, we showed that LRRK2 phosphorylation of synapsin I at threonine 337 and 339 significantly reduces synapsin I-SV/actin interactions. By using complementary experimental approaches, we demonstrated that LRRK2 controls glutamate release and SV dynamics in a kinase activity and synapsin I-dependent manner. Our findings show that synapsin I is a LRRK2 substrate and describe a novel mechanisms of regulation of glutamate release by LRRK2 kinase activity. This article is protected by copyright. All rights reserved.

LRRK2 phosphorylation on synapsin I regulates glutamate release at presynaptic sites / Marte, Antonella; Russo, Isabella; Rebosio, Claudia; Valente, Pierluigi; Belluzzi, Elisa; Pischedda, Francesca; Montani, Caterina; Lavarello, Chiara; Petretto, Andrea; Fedele, Ernesto; Baldelli, Pietro; Benfenati, Fabio; Piccoli, Giovanni; Greggio, Elisa; Onofri, Franco. - In: JOURNAL OF NEUROCHEMISTRY. - ISSN 0022-3042. - 2019:(2019). [10.1111/jnc.14778]

LRRK2 phosphorylation on synapsin I regulates glutamate release at presynaptic sites

Pischedda, Francesca;Montani, Caterina;Piccoli, Giovanni;
2019

Abstract

LRRK2 (Leucine-rich repeat kinase 2) is a large multidomain scaffolding protein with kinase and GTPase activities involved in synaptic vesicle (SV) dynamics. While its role in Parkinson's disease has been largely investigated, little is known about LRRK2 physiological role and until now few proteins have been described as substrates. We have previously demonstrated that LRRK2 through its WD40 domain interacts with synapsin I, an important SV-associated phosphoprotein involved in neuronal development and in the regulation of neurotransmitter release. To test whether synapsin I is substrate for LRRK2 and characterize the properties of its phosphorylation, we used in vitro kinase and binding assays as well as cellular model and site-direct mutagenesis. By using synaptosomes in superfusion, patch-clamp recordings in autaptic WT and synapsin I KO cortical neurons and SypHy assay on primary cortical culture from wild-type and BAC human LRRK2 G2019S mice we characterize the role of LRRK2 kinase activity on glutamate release and SV trafficking. Here we reported that synapsin I is phosphorylated by LRRK2 and demonstrated that the interaction between LRRK2 WD40 domain and synapsin I is crucial for this phosphorylation. Moreover, we showed that LRRK2 phosphorylation of synapsin I at threonine 337 and 339 significantly reduces synapsin I-SV/actin interactions. By using complementary experimental approaches, we demonstrated that LRRK2 controls glutamate release and SV dynamics in a kinase activity and synapsin I-dependent manner. Our findings show that synapsin I is a LRRK2 substrate and describe a novel mechanisms of regulation of glutamate release by LRRK2 kinase activity. This article is protected by copyright. All rights reserved.
Marte, Antonella; Russo, Isabella; Rebosio, Claudia; Valente, Pierluigi; Belluzzi, Elisa; Pischedda, Francesca; Montani, Caterina; Lavarello, Chiara; Petretto, Andrea; Fedele, Ernesto; Baldelli, Pietro; Benfenati, Fabio; Piccoli, Giovanni; Greggio, Elisa; Onofri, Franco
LRRK2 phosphorylation on synapsin I regulates glutamate release at presynaptic sites / Marte, Antonella; Russo, Isabella; Rebosio, Claudia; Valente, Pierluigi; Belluzzi, Elisa; Pischedda, Francesca; Montani, Caterina; Lavarello, Chiara; Petretto, Andrea; Fedele, Ernesto; Baldelli, Pietro; Benfenati, Fabio; Piccoli, Giovanni; Greggio, Elisa; Onofri, Franco. - In: JOURNAL OF NEUROCHEMISTRY. - ISSN 0022-3042. - 2019:(2019). [10.1111/jnc.14778]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11572/237116
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