Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domaindomain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.

J. Neurochem. (2011) 116, 304–315. Abstract Mutations in the leucine‐rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson’s disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C‐terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC‐COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra‐molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra‐molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC‐COR tandem of LRRK2, showing that Y1699 is positioned at the intra‐molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra‐molecular ROC : COR interface, strengthens the intra‐molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC‐COR tandem domain resulting in decreased GTPase activity.

␣-Synuclein (␣-SYN) is a key player in the pathogenesis of Parkinson's disease (PD).

Padsevonil is an antiepileptic drug (AED) candidate synthesized in a medicinal chemistry program initiated to rationally design compounds with high affinity for synaptic vesicle 2 (SV2) proteins and low-to-moderate affinity for the benzodiazepine binding site on GABA A receptors. The pharmacological profile of padsevonil was characterized in binding and electrophysiological experiments. At recombinant SV2 proteins, padsevonil's affinity for SV2A was greater than that of levetiracetam and brivaracetam (pKi 8.5, 5.2, and 6.6, respectively). Unlike the latter AEDs, both selective SV2A ligands, padsevonil also displayed high affinity for the SV2B and SV2C isoforms (pKi 7.9 and 8.5, respectively). Padsevonil's interaction with SV2A differed from that of levetiracetam and brivaracetam; it exhibited slower binding kinetics: dissociation t 1/2 30 minutes from the human protein at 37°C compared with ,0.5 minute for levetiracetam and brivaracetam. In addition, its binding was not potentiated by the allosteric modulator UCB1244283. At recombinant GABA A receptors, padsevonil displayed low to moderate affinity (pIC 50 #6.1) for the benzodiazepine site, and in electrophysiological studies, its relative efficacy compared with zolpidem (full-agonist reference drug) was 40%, indicating partial agonist properties. In in vivo (mice) receptor occupancy studies, padsevonil exhibited SV2A occupancy at low ED 50 (0.2 mg/kg) and benzodiazepine site occupancy at higher doses (ED 50 36 mg/kg), supporting in vitro results. Padsevonil's selectivity for its intended targets was confirmed in profiling studies, where it lacked significant effects on a wide variety of ion channels, receptors, transporters, and enzymes. Padsevonil is a first-in-class AED candidate with a unique target profile allowing for presynaptic and postsynaptic activity. SIGNIFICANCE STATEMENTPadsevonil is an antiepileptic drug candidate developed as a single molecular entity interacting with both presynaptic and postsynaptic targets. Results of in vitro and in vivo radioligand binding assays confirmed this target profile: padsevonil displayed nanomolar affinity for the three synaptic vesicle 2 protein isoforms (SV2A, B, and C) and micromolar affinity for the benzodiazepine binding site on GABA A receptors. Furthermore, padsevonil showed greater affinity for and slower binding kinetics at SV2A than the selective SV2A ligands, levetiracetam, and brivaracetam.All studies described in this report were funded by UCB Pharma.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Although PD has long been considered a purely sporadic disorder, genetic research has revealed an underlying genetic cause in at least 10% of all PD cases. To date, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD. Moreover, given the strong clinical and neuropathological similarities between LRRK2 PD and the sporadic forms of the disease, the notion is supported that the unravelling of the molecular pathways underlying LRRK2 PD will greatly contribute to our general understanding of PD. Therefore, intense research efforts have been focused on the understanding of the physiological function of LRRK2 and its relation to PD. To date, progress has been made in these fields based on the study of LRRK2 cell culture models, the identification of LRRK2 interaction partners and kinase substrates and the generation of LRRK2 animal models. In this review, the current insights into the cellular role of LRRK2 are discussed. The overview reveals a potential involvement of LRRK2 in major cell signalling pathways including apoptosis, cytoskeleton dynamics, protein translation, mitogen-activated protein kinase signalling and specific dopaminergic functions, consistent with its proposed role as a signal transduction protein.

BACKGROUND AND PURPOSESynaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. EXPERIMENTAL APPROACHUCB1244283 was characterized in vitro using radioligand binding assays with [ 3 H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. KEY RESULTSSaturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of CONCLUSIONS AND IMPLICATIONSThese results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. AbbreviationsLBS, levetiracetam binding site; LEV, levetiracetam; SV2A, synaptic vesicle protein 2A

The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters). We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD). By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds.

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca(2+) -dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca(2+) responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca(2+) channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A-siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca(2+) current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.