The LRRK2 N-terminal domain influences vesicle trafficking: impact of the E193K variant

Abstract

The LRRK2 protein consists of multiple functional domains, including protein-binding domains at its N and C-terminus. Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) have been linked to familial and sporadic Parkinson's disease (PD). We have recently described a novel variant falling within the N-terminal armadillo repeats, E193K. Herein, our aim is to investigate the functional impact of LRRK2 N-terminal domain and the E193K variant on vesicle trafficking. By combining Total Internal Reflection Fluorescence (TIRF) microscopy and a synaptopHluorin assay, we found that expression of a construct lacking the N-terminal domain increases the frequency and amplitude of spontaneous synaptic events. Complementary biochemical approaches showed that the E193K variant alters the binding properties of LRRK2, decreases LRRK2 binding to synaptic vesicles, and promotes vesicle fusion. Our results confirm the physiological and pathological relevance of the nature of the LRRK2-associated macro-molecular complex solidifying the idea that different pathological mutations critically alter the scaffolding function of LRRK2 resulting in a perturbation of the vesicular trafficking as a common denominator.

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

“…They show specifically how this LRRK2 mutant forms periodically repeating dimers, which then polymerize in a helical array onto MTs. The cellular phenotypes associated with G2019S, I2020T, and R1441C/Y1699C and other PD-associated mutations include perturbation of MT-related processes such as vesicular trafficking, autophagy, cilia formation, and nuclear/mitochondria morphology, so it is very likely that LRRK2 dysfunction physiologically interferes globally with dynamic cross-talk with MTs ( 9 , 29 – 31 ).…”

Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

“…They show speci cally how the I2020T LRRK2 mutant forms periodically repeating dimers, which then polymerize in a helical array onto MTs. The cellular phenotypes associated with G2019S, I2020T, and R1441C/Y1699C and other PD associated mutations include perturbation of MT-related processes such as vesicular tra cking, autophagy, cilia formation, and nuclear/mitochondria morphology, so it is very likely that LRRK2 dysfunction physiologically interferes globally with dynamic cross talk with MTs (20,32,(65)(66)(67)(68)(69)(70)(71)(72).…”

A Critical LRRK at the Synapse? The Neurobiological Function and Pathophysiological Dysfunction of LRRK2

“…(iv) SV trafficking and recycling may involve LRRK2 in concert with VPS35 (Inoshita et al, 2017 ; Mir et al, 2018 ), Rab29 (aka Rab7L1; MacLeod et al, 2013 ), Rab10, Rab11, and Rab35 (Steger et al, 2016 ) which regulate cargo and membrane recycling from sorting endosomes back into the cycle or the endolysosomal pathway for degradation (reviewed in Taylor and Alessi, 2020 ). LRRK2 is also implicated in SV storage and mobilization through its phosphorylation/binding of synapsin-I (Beccano-Kelly et al, 2014 ; Cirnaru et al, 2014 ; Carrion et al, 2017 ; Marte et al, 2019 ; Marku et al, 2020 ). (B) Cartoon of a generalized postsynaptic structure showing processes in which LRRK2 has been implicated, and which are also regulated by numerous Rab GTPases (reviewed in Hausser and Schlett, 2019 ).…”

“…Phosphorylation at tyrosine and serine sites on synapsin-I impart opposite effects on its association to actin and SVs (Cesca et al, 2010 ). It is noteworthy that LRRK2’s C-terminal WD40 domain was previously identified as binding synapsin-I and other SV-associated proteins (Piccoli et al, 2014 ), whereas recent work by the same group shows that the armadillo repeats at the N-terminus also affect LRRK2’s regulation of SV trafficking; these findings may seem contradictory at first, but the complex architecture resulting from LRRK2 dimerization may allow for both terminals to work together in shaping SV dynamics (Marku et al, 2020 ). Overall, altered synapsin-I phosphorylation, and other functional interactions with LRRK2, may contribute to the increased glutamate release observed in G2019S-LRRK2 neurons (Beccano-Kelly et al, 2014 ; Matikainen-Ankney et al, 2016 ; Volta et al, 2017 ), although an exact mechanism remains to be determined.…”