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      Leucine-rich repeat kinase 2 controls protein kinase A activation state through phosphodiesterase 4

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          Abstract

          Background

          Evidence indicates a cross-regulation between two kinases, leucine-rich repeat kinase 2 (LRRK2) and protein kinase A (PKA). In neurons, LRRK2 negatively regulates PKA activity in spiny projecting neurons during synaptogenesis and in response to dopamine D1 receptor activation acting as an A-anchoring kinase protein (AKAP). In microglia cells, we showed that LRRK2 kinase activity negatively regulates PKA, impacting NF-κB p50 signaling and the inflammatory response. Here, we explore the molecular mechanism underlying the functional interaction between LRRK2 and PKA in microglia.

          Methods

          To understand which step of PKA signaling is modulated by LRRK2, we used a combination of in vitro and ex vivo systems with hyperactive or inactive LRRK2 as well as different readouts of PKA signaling.

          Results

          We confirmed that LRRK2 kinase activity acts as a negative regulator of PKA activation state in microglia. Specifically, we found that LRRK2 controls PKA by affecting phosphodiesterase 4 (PDE4) activity, modulating cAMP degradation, content, and its dependent signaling. Moreover, we showed that LRRK2 carrying the G2019S pathological mutation downregulates PKA activation causing a reduction of PKA-mediated NF-κB inhibitory signaling, which results, in turn, in increased inflammation in LRRK2 G2019S primary microglia upon α-synuclein pre-formed fibrils priming.

          Conclusions

          Overall, our findings indicate that LRRK2 kinase activity is a key regulator of PKA signaling and suggest PDE4 as a putative LRRK2 effector in microglia. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD.

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          Most cited references28

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          Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents.

          Cyclic nucleotide phosphodiesterases (PDEs), which are ubiquitously distributed in mammalian tissues, play a major role in cell signaling by hydrolyzing cAMP and cGMP. Due to their diversity, which allows specific distribution at cellular and subcellular levels, PDEs can selectively regulate various cellular functions. Their critical role in intracellular signaling has recently designated them as new therapeutic targets for inflammation. The PDE superfamily represents 11 gene families (PDE1 to PDE11). Each family encompasses 1 to 4 distinct genes, to give more than 20 genes in mammals encoding the more than 50 different PDE proteins probably produced in mammalian cells. Although PDE1 to PDE6 were the first well-characterized isoforms because of their predominance in various tissues and cells, their specific contribution to tissue function and their regulation in pathophysiology remain open research fields. This concerns particularly the newly discovered families, PDE7 to PDE11, for which roles are not yet established. In many pathologies, such as inflammation, neurodegeneration, and cancer, alterations in intracellular signaling related to PDE deregulation may explain the difficulties observed in the prevention and treatment of these pathologies. By inhibiting specifically the up-regulated PDE isozyme(s) with newly synthesized potent and isozyme-selective PDE inhibitors, it may be potentially possible to restore normal intracellular signaling selectively, providing therapy with reduced adverse effects.
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            Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.

            Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.
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              Localized effects of cAMP mediated by distinct routes of protein kinase A.

              More than 20% of the human genome encodes proteins involved in transmembrane and intracellular signaling pathways. The cAMP-protein kinase A (PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells and is involved in regulation of cellular functions in almost all tissues in mammals. Various extracellular signals converge on this signal pathway through ligand binding to G protein-coupled receptors, and the cAMP-PKA pathway is therefore tightly regulated at several levels to maintain specificity in the multitude of signal inputs. Ligand-induced changes in cAMP concentration vary in duration, amplitude, and extension into the cell, and cAMP microdomains are shaped by adenylyl cyclases that form cAMP as well as phosphodiesterases that degrade cAMP. Different PKA isozymes with distinct biochemical properties and cell-specific expression contribute to cell and organ specificity. A kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP-PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphatases and cAMP-specific phosphodiesterases as well as components of other signaling pathways into multiprotein signaling complexes that serve as crossroads for different paths of cell signaling. Targeting of PKA and integration of a wide repertoire of proteins involved in signal transduction into complex signal networks further increase the specificity required for the precise regulation of numerous cellular and physiological processes.
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                Author and article information

                Contributors
                +390302717255 , isabella.russo@unibs.it
                giuli@bio.unipd.it
                kaganovichal@grc.nia.nih.gov
                dingj@mail.nih.gov
                mrcdnl@unife.it
                mri@unife.it
                cookson@mail.nih.gov
                luigi.bubacco@unipd.it
                elisa.greggio@unipd.it
                Journal
                J Neuroinflammation
                J Neuroinflammation
                Journal of Neuroinflammation
                BioMed Central (London )
                1742-2094
                27 October 2018
                27 October 2018
                2018
                : 15
                : 297
                Affiliations
                [1 ]ISNI 0000 0004 1757 3470, GRID grid.5608.b, Department of Biology, , University of Padova, ; Padua, Italy
                [2 ]ISNI 0000000417571846, GRID grid.7637.5, Present Address: Department of Molecular and Translational Medicine, , University of Brescia, ; Viale Europa 11, 25123 Brescia, Italy
                [3 ]ISNI 0000 0001 1940 4177, GRID grid.5326.2, Neuroscience Institute, Italian National Research Council, ; Padua, Italy
                [4 ]ISNI 0000 0001 2297 5165, GRID grid.94365.3d, Laboratory of Neurogenetics, National Institute on Aging, , National Institutes of Health, ; Bethesda, MD USA
                [5 ]ISNI 0000 0004 1757 2064, GRID grid.8484.0, Department of Medical Sciences, National Institute for Neuroscience, , University of Ferrara, ; Ferrara, Italy
                Author information
                http://orcid.org/0000-0002-6151-2451
                Article
                1337
                10.1186/s12974-018-1337-8
                6204045
                30368241
                5a4386fd-a161-4e23-994d-9e3a7c24424c
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 9 June 2018
                : 17 October 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000864, Michael J. Fox Foundation for Parkinson's Research;
                Funded by: FundRef http://dx.doi.org/10.13039/501100002803, Fondazione Cariplo;
                Award ID: 2016-0428
                Award Recipient :
                Funded by: Intramural Research Program
                Funded by: FundRef http://dx.doi.org/10.13039/501100004710, Fondazione Umberto Veronesi;
                Award ID: 1395
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Neurosciences
                lrrk2,pka,microglia,neuroinflammation,parkinson’s disease,pde4
                Neurosciences
                lrrk2, pka, microglia, neuroinflammation, parkinson’s disease, pde4

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