Giovanni Piccoli

LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the single most common cause of inherited Parkinsons disease. Little is known about its involvement in the pathogenesis of Parkinsons disease mainly because of the lack of knowledge about the physiological role of LRRK2. To determine the function of LRRK2, we studied the impact of short hairpin RNA-mediated silencing of LRRK2 expression in cortical neurons. Paired recording indicated that LRRK2 silencing affects evoked postsynaptic currents. Furthermore, LRRK2 silencing induces at the presynaptic site a redistribution of vesicles within the bouton, altered recycling dynamics, and increased vesicle kinetics. Accordingly, LRRK2 protein is present in the synaptosomal compartment of cortical neurons in which it interacts with several proteins involved in vesicular recycling. Our results suggest that LRRK2 modulates synaptic vesicle trafficking and distribution in neurons and in consequence participates in regulating the dynamics between vesicle pools inside the presynaptic bouton.

The fragile X mental retardation protein-RNP granules show an mGluR-dependent localization in the post-synaptic spines

The localization of RNA/mRNA in dendrites plays a role in both local and temporal regulation of protein synthesis, which is required for certain forms of synaptic plasticity. A key molecule in these processes is the fragile X mental retardation protein (FMRP). Using in situ hybridization coupled to immunofluorescence confocal microscopy, we find that the FMRP-RNP particle contains alphaCaMKII and BC1 RNAs as well as Staufen and CPEB proteins. Furthermore, following mGluR activation, the FMRP-mRNP complex moves into spines as shown by co-localization with the PSD-95 and Shank proteins. This study shows, for the first time, that the translationally inactive FMRP-mRNP complex relocates into neuronal spines after stimulation and that de novo protein synthesis mainly contributes to the pool of FMRP at synapses.

Synaptic Activity Controls Dendritic Spine Morphology by Modulating eEF2-Dependent BDNF Synthesis

Activity-dependent changes in synaptic structure and spine morphology are required for learning and memory, and depend on protein translation. We show that the kinase for eukaryotic elongation factor 2 (eEF2K) regulates dendritic spine stability and synaptic structure by modulating activity-dependent dendritic BDNF synthesis. Specifically RNAi knockdown of eEF2K reduces dendritic spine stability and inhibits dendritic BDNF protein expression; whereas overexpression of a constitutively activated eEF2K induces spine maturation and increases expression of dendritic BDNF. Furthermore, BDNF overexpression rescues the spine stability reduced by RNAi knockdown of eEF2K. We also show that synaptic activity-dependent spine maturation and dendritic BDNF protein expression depend on mGluR/EF2K-induced eEF2 phosphorylation. We propose that the eEF2K/eEF2 pathway is a key biochemical sensor that couple neuronal activity to spine plasticity, by controlling the dendritic translation of BDNF.

A Functional Role of Postsynaptic Density-95-Guanylate Kinase-Associated Protein Complex in Regulating Shank Assembly and Stability to Synapses

Postsynaptic density (PSD) proteins include scaffold, cytoskeletal, and signaling proteins that structurally and functionally interact with glutamate receptors and other postsynaptic membrane proteins. The molecular mechanisms regulating the assembly of PSD proteins and their associations with synapses are still widely unknown. We investigated the molecular mechanisms of Shank1 targeting and synapse assembly by looking at the function of guanylate kinase-associated protein (GKAP) and PSD-95 interactions. Shank1 when it is not associated to GKAP, which binds to the Shank PSD-95-Discs Large-zona occludens-1 domain, forms filamentous and fusiform structures in which the Src homology 3 domain specifically interacts with the ankyrin repeat domain, thus allowing its multimerization via a novel form of intermolecular interaction. Surprisingly, in both COS-7 cells and hippocampal neurons, GKAP forms insoluble aggregates with Shank that colocalize with heat shock protein 70 and neurofilaments, two markers of the aggresomes in which misfolded proteins accumulate. However, the two proteins are organized in clusters in COS cells and synaptic clusters in neurons when both are overexpressed and associated with wild-type PSD-95, but not with palmitoylation-deficient PSD-95. Synaptic activity in neurons induces the formation of Shank and GKAP intracellular aggregation and degradation. Similarly, the overexpression of a GKAP mutant that is incapable of binding PSD-95 induces Shank aggregation and degradation in neurons. Our data suggest a possible functional and structural role of the PSD-95-GKAP complex in Shank and PSD protein assembly and stability to synapses.

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinsons disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex.

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

BackgroundLrrk2, a gene linked to Parkinson’s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins.ResultsHere, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity.ConclusionsGiven that the most common Parkinson’s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

ABSTRACT Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinsons disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.

Clearance of Rhodopsin(P23H) aggregates requires the ERAD effector VCP.

Dominant mutations in the visual pigment Rhodopsin (Rh) cause retinitis pigmentosa (RP) characterized by progressive blindness and retinal degeneration. The most common Rh mutation, Rh(P23H) forms aggregates in the endoplasmic reticulum (ER) and impairs the proteasome; however, the mechanisms linking Rh aggregate formation to proteasome dysfunction and photoreceptor cell loss remain unclear. Using mammalian cell cultures, we provide the first evidence that misfolded Rh(P23H) is a substrate of the ERAD effector VCP, an ATP-dependent chaperone that extracts misfolded proteins from the ER and escorts them for proteasomal degradation. VCP co-localizes with misfolded Rh(P23H) in retinal cells and requires functional N-terminal and D1 ATPase domains to form a complex with Rh(P23H) aggregates. Furthermore, VCP uses its D2 ATPase activity to promote Rh(P23H) aggregate retrotranslocation and proteasomal delivery. Our results raise the possibility that modulation of VCP and ERAD activity might have potential therapeutic significance for RP.

Presynaptic dysfunction in Parkinson's disease: a focus on LRRK2.

PD (Parkinsons disease) is a common neurodegenerative disease clinically characterized by bradykinesia, rigidity and resting tremor. Recent studies have proposed that synaptic dysfunction, implicated in numerous studies of animal models of PD, might be a key factor in PD. The molecular defects that lead to PD progression might be hidden at the presynaptic neuron: in fact accumulating evidence has shown that the majority of the genes linked to PD play a critical role at the presynaptic site. In the present paper, we focus on the presynaptic function of LRRK2 (leucine-rich repeat kinase 2), a protein that mutated represents the main genetic cause of familial PD described to date. Neurotransmission relies on proper presynaptic vesicle trafficking; defects in this process, variation in dopamine flow and alteration of presynaptic plasticity have been reported in several animal models of LRRK2 mutations. Furthermore, impaired dopamine turnover has been described in presymptomatic LRRK2 PD patients. Thus, given the pathological events occurring at the synapses of PD patients, the presynaptic site may represent a promising target for early diagnostic therapeutic intervention.

A cell surface biotinylation assay to reveal membrane associated neuronal cues: Negr1 regulates dendritic arborization.

A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.