LRRK2 and neuroinflammation: Partners in crime in Parkinson's disease?

3noopenopenRusso, Isabella*; Bubacco, Luigi; Greggio, ElisaRusso, Isabella; Bubacco, Luigi; Greggio, Elis

Secretion-Positive LGI1 Mutations Linked to Lateral Temporal Epilepsy Impair Binding to ADAM22 and ADAM23 Receptors

Autosomal dominant lateral temporal epilepsy (ADTLE) is a focal epilepsy syndrome caused by mutations in the LGI1 gene, which encodes a secreted protein. Most ADLTE-causing mutations inhibit LGI1 protein secretion, and only a few secretion-positive missense mutations have been reported. Here we describe the effects of four disease-causing nonsynonymous LGI1 mutations, T380A, R407C, S473L, and R474Q, on protein secretion and extracellular interactions. Expression of LGI1 mutant proteins in cultured cells shows that these mutations do not inhibit protein secretion. This finding likely results from the lack of effects of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair interaction of LGI1 with the ADAM22 and ADAM23 receptors on the cell surface. These results support the existence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing LGI1 mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE

Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation

Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson’s disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies

Leucine-rich repeat kinase 2 and alpha-synuclein: intersecting pathways in the pathogenesis of Parkinson's disease?

Although Parkinson's disease (PD) is generally a sporadic neurological disorder, the discovery of monogenic, hereditable forms of the disease has been crucial in delineating the molecular pathways that lead to this pathology. Genes responsible for familial PD can be ascribed to two categories based both on their mode of inheritance and their suggested biological function. Mutations in parkin, PINK1 and DJ-1 cause of recessive Parkinsonism, with a variable pathology often lacking the characteristic Lewy bodies (LBs) in the surviving neurons. Intriguingly, recent findings highlight a converging role of all these genes in mitochondria function, suggesting a common molecular pathway for recessive Parkinsonism. Mutations in a second group of genes, encoding alpha-synuclein (α-syn) and LRRK2, are transmitted in a dominant fashion and generally lead to LB pathology, with α-syn being the major component of these proteinaceous aggregates. In experimental systems, overexpression of mutant proteins is toxic, as predicted for dominant mutations, but the normal function of both proteins is still elusive. The fact that α-syn is heavily phosphorylated in LBs and that LRRK2 is a protein kinase, suggests that a link, not necessarily direct, exists between the two. What are the experimental data supporting a common molecular pathway for dominant PD genes? Do α-syn and LRRK2 target common molecules? Does LRRK2 act upstream of α-syn? In this review we will try to address these of questions based on the recent findings available in the literature

Leucine-rich repeat kinase 2 (LRRK2): an update on the potential therapeutic target for Parkinson’s disease

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are a risk factor for and a cause of sporadic and familial Parkinson’s disease (PD), respectively. These mutations are some of the most common genetic contributors to PD and render the kinase hyperactive. Increasingly within the past decade, there has been substantial effort investigating LRRK2 as a target for therapeutics in preclinical studies, and currently, small-molecule inhibitors and antisense oligonucleotides are being assessed in clinical trials as therapies to reduce the toxic hyperactivity of its kinase and/or reduce total levels of the protein in healthy individuals and people with PD

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field

Genetic, structural, and molecular insights into the function of ras of complex proteins domains

Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored

Editorial:LRRK2-Fifteen Years From Cloning to the Clinic

none4noneRideout, Hardy; Greggio, Elisa; Kortholt, Arjan; Nichols, R JeremyRideout, Hardy; Greggio, Elisa; Kortholt, Arjan; Nichols, R Jerem

Simulations of ELT-GMCAO performance for deep field observations

The Global-Multi Conjugated Adaptive Optics (GMCAO) approach offers an alternative way to correct an adequate scientific Field of View (FoV) using only natural guide stars (NGSs) to extremely large ground-based telescopes. Thus, even in the absence of laser guide stars, a GMCAO-equipped ELT-like telescope can achieve optimal performance in terms of Strehl Ratio (SR), retrieving impressive results in studying star-poor fields, as in the cases of the deep field observations. The benefits and usability of GMCAO have been demonstrated by studying 6000 mock high redshift galaxies in the Chandra Deep Field South region. However, a systematic study simulating observations in several portions of the sky is mandatory to have a robust statistic of the GMCAO performance. Technical, tomographic and astrophysical parameters, discussed here, are given as inputs to GIUSTO, an IDL-based code that estimates the SR over the considered field, and the results are analyzed with statistical considerations. The best performance is obtained using stars that are relatively close to the Scientific FoV; therefore, the SR correlates with the mean off-axis position of NGSs, as expected, while their magnitude plays a secondary role. This study concludes that the SRs correlate linearly with the galactic latitude, as also expected. Because of the lack of natural guide stars needed for low-order aberration sensing, the GMCAO confirms as a promising technique to observe regions that can not be studied without the use of laser beacons. It represents a robust alternative way or a risk mitigation strategy for laser approaches on the ELTs.Comment: 18 pages, 10 figures, accepted for publication on PAS

Leucine-rich repeat kinase 2 positively regulates inflammation and down-regulates NF-κB p50 signaling in cultured microglia cells

7openopenRusso, Isabella; Berti, Giulia; Plotegher, Nicoletta; Bernardo, Greta; Filograna, Roberta; Bubacco, Luigi; Greggio, Elisa*Russo, Isabella; Berti, Giulia; Plotegher, Nicoletta; Bernardo, Greta; Filograna, Roberta; Bubacco, Luigi; Greggio, Elis