Lrrk2, 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. Here, 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. Given 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.

Background: Neuroinflammation is associated with virtually all major neurodegenerative disorders, including Alzheimer’s disease (AD). Although it remains unclear whether neuroinflammation is the driving force behind these disorders, compelling evidence implicates its role in exacerbating disease progression, with a key player being the potent proinflammatory cytokine TNF-α. Elevated TNF-α levels are commonly detected in the clinic and animal models of AD. Methods: The potential benefits of a novel TNF-α-lowering agent, 3,6′-dithiothalidomide, were investigated in cellular and rodent models of neuroinflammation with a specific focus on AD. These included central and systemic inflammation induced by lipopolysaccharide (LPS) and Aβ_1–42 challenge, and biochemical and behavioral assessment of 3xTg-AD mice following chronic 3,6′-dithiothaliodmide. Results: 3,6′-Dithiothaliodmide lowered TNF-α, nitrite (an indicator of oxidative damage) and secreted amyloid precursor protein (sAPP) levels in LPS-activated macrophage-like cells (RAW 264.7 cells). This translated into reduced central and systemic TNF-α production in acute LPS-challenged rats, and to a reduction of neuroinflammatory markers and restoration of neuronal plasticity following chronic central challenge of LPS. In mice centrally challenged with Aβ_1–42 peptide, prior systemic 3,6′-dithiothalidomide suppressed Aβ-induced memory dysfunction, microglial activation and neuronal degeneration. Chronic 3,6′-dithiothalidomide administration to an elderly symptomatic cohort of 3xTg-AD mice reduced multiple hallmark features of AD, including phosphorylated tau protein, APP, Aβ peptide and Aβ-plaque number along with deficits in memory function to levels present in younger adult cognitively unimpaired 3xTg-AD mice. Levels of the synaptic proteins, SNAP25 and synaptophysin, were found to be elevated in older symptomatic drug-treated 3xTg-AD mice compared to vehicle-treated ones, indicative of a preservation of synaptic function during drug treatment. Conclusions: Our data suggest a strong beneficial effect of 3,6′-dithiothalidomide in the setting of neuroinflammation and AD, supporting a role for neuroinflammation and TNF-α in disease progression and their targeting as a means of clinical management.

It is now well established that chronic inflammation is a prominent feature of several neurodegenerative disorders including Parkinson’s disease (PD). Growing evidence indicates that neuroinflammation can contribute greatly to dopaminergic neuron degeneration and progression of the disease. Recent literature highlights that leucine-rich repeat kinase 2 (LRRK2), a kinase mutated in both autosomal-dominantly inherited and sporadic PD cases, modulates inflammation in response to different pathological stimuli. In this review, we outline the state of the art of LRRK2 functions in microglia cells and in neuroinflammation. Furthermore, we discuss the potential role of LRRK2 in cytoskeleton remodeling and vesicle trafficking in microglia cells under physiological and pathological conditions. We also hypothesize that LRRK2 mutations might sensitize microglia cells toward a pro-inflammatory state, which in turn results in exacerbated inflammation with consequent neurodegeneration.

J. Neurochem. (2011) 116, 947–956. In the adult brain, neurogenesis under physiological conditions occurs in the subventricular zone and in the dentate gyrus. Although the exact molecular mechanisms that regulate neural stem cell proliferation and differentiation are largely unknown, several factors have been shown to affect neurogenesis. Decreased neurogenesis in the hippocampus has been recognized as one of the mechanisms of age‐related brain dysfunction. Furthermore, in pathological conditions of the central nervous system associated with neuroinflammation, inflammatory mediators such as cytokines and chemokines can affect the capacity of brain stem cells and alter neurogenesis. In this review, we summarize the state of the art on the effects of neuroinflammation on adult neurogenesis and discuss the use of the lipopolysaccharide‐model to study the effects of inflammation and reactive‐microglia on brain stem cells and neurogenesis. Furthermore, we discuss the possible causes underlying reduced neurogenesis with normal aging and potential anti‐inflammatory, pro‐neurogenic interventions aimed at improving memory deficits in normal and pathological aging and in neurodegenerative diseases.

Over-activated microglia and chronic neuroinflammation contribute to dopaminergic neuron degeneration and progression of Parkinson’s disease (PD). Leucine-rich repeat kinase 2 (LRRK2), a kinase mutated in autosomal dominantly inherited and sporadic PD cases, is highly expressed in immune cells, in which it regulates inflammation through a yet unclear mechanism. Here, using pharmacological inhibition and cultured Lrrk2 −/− primary microglia cells, we validated LRRK2 as a positive modulator of inflammation and we investigated its specific function in microglia cells. Inhibition or genetic deletion of LRRK2 causes reduction of interleukin-1β and cyclooxygenase-2 expression upon lipopolysaccharide-mediated inflammation. LRRK2 also takes part of the signaling trigged by α-synuclein fibrils, which culminates in induction of inflammatory mediators. At the molecular level, loss of LRRK2 or inhibition of its kinase activity results in increased phosphorylation of nuclear factor kappa-B (NF-κB) inhibitory subunit p50 at S337, a protein kinase A (PKA)-specific phosphorylation site, with consequent accumulation of p50 in the nucleus. Taken together, these findings point to a role of LRRK2 in microglia activation and sustainment of neuroinflammation and in controlling of NF-κB p50 inhibitory signaling. Understanding the molecular pathways coordinated by LRRK2 in activated microglia cells after pathological stimuli such us fibrillar α-synuclein holds the potential to provide novel targets for PD therapeutics.

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson’s 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.