Abstract
Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.
Highlights
Despite intense research efforts in the context of Parkinson’s disease (PD), the basic neurophysiology of Leucine-Rich Repeat Kinase-2 (LRRK2) remains largely unknown
LRRK WILD TYPE, OVEREXPRESSION AND G2019S MUTANT LEVELS IN CORTICAL NEURON CULTURES As LRRK2 is implicated in PD, a disease characterized by nigrostriatal dysfunction, we concluded it would be appropriate to study LRRK2’s synaptic activity in cortical cells (CTX) given their input into the striatum is modulated by nigrostriatal dopamine
As LRRK2 protein levels are relatively low over the first week, and because neurite phenotypes may be lost by the second week in vitro (Sepulveda et al, 2013), we decided to investigate the effects of LRRK2 manipulations upon synaptic function in neuronal networks of cortical cultures aged >21DIV
Summary
Despite intense research efforts in the context of Parkinson’s disease (PD), the basic neurophysiology of LRRK2 remains largely unknown. Progress is possibly confounded by numerous potential roles, resulting from LRRK2 being a large multi-domain protein containing ROC, COR, kinase, WD40, and leucine-rich repeats (Cookson, 2010). There is consensus between several neuronal culture studies regarding LRRK2-dependent neuritic regeneration phenotypes; axon/dendrite outgrowth are most often exaggerated by LRRK2 loss and retarded by mutant overexpression (MacLeod et al, 2006; Plowey et al, 2008; Parisiadou et al, 2009; Dachsel et al, 2010; Lee et al, 2010; Lin et al, 2010; Chan et al, 2011; Ramonet et al, 2011; Winner et al, 2011; Kawakami et al, 2012). Others have found that neurite phenotypes are robust only during the first week in vitro (Sepulveda et al, 2013). Little LRRK2 is expressed during this period, whereas LRRK2 levels ∼double between the first and second week, both in vitro and in vivo (Biskup et al, 2007; Piccoli et al, 2011), during which time glutamatergic
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