Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation
Identifieur interne : 000041 ( Pmc/Checkpoint ); précédent : 000040; suivant : 000042Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation
Auteurs : E. Lobbestael [Belgique] ; L. Civiero [Italie] ; T. De Wit [Belgique] ; J.-M. Taymans [France] ; E. Greggio [Italie] ; V. Baekelandt [Belgique]Source :
- Scientific Reports [ 2045-2322 ] ; 2016.
Abstract
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.
Url:
DOI: 10.1038/srep33897
PubMed: 27658356
PubMed Central: 5034242
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Civiero</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Biology, University of Padova</institution>, 35131 Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="De Wit, T" sort="De Wit, T" uniqKey="De Wit T" first="T." last="De Wit">T. De Wit</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven</institution>, Kapucijnenvoer 33, 3000 Leuven,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Taymans, J M" sort="Taymans, J M" uniqKey="Taymans J" first="J.-M." last="Taymans">J.-M. 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Lobbestael</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven</institution>, Kapucijnenvoer 33, 3000 Leuven,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Civiero, L" sort="Civiero, L" uniqKey="Civiero L" first="L." last="Civiero">L. Civiero</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Biology, University of Padova</institution>, 35131 Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="De Wit, T" sort="De Wit, T" uniqKey="De Wit T" first="T." last="De Wit">T. De Wit</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven</institution>, Kapucijnenvoer 33, 3000 Leuven,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Taymans, J M" sort="Taymans, J M" uniqKey="Taymans J" first="J.-M." last="Taymans">J.-M. Taymans</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>UMR-S1172 Jean-Pierre Aubert Research Center – (INSERM - CHRU de Lille - Université de Lille), Early Stages of Parkinson’s Disease Team</institution>, Lille,<country>France</country></nlm:aff><country xml:lang="fr">France</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Greggio, E" sort="Greggio, E" uniqKey="Greggio E" first="E." last="Greggio">E. Greggio</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Biology, University of Padova</institution>, 35131 Padova,<country>Italy</country></nlm:aff><country xml:lang="fr">Italie</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Baekelandt, V" sort="Baekelandt, V" uniqKey="Baekelandt V" first="V." last="Baekelandt">V. Baekelandt</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven</institution>, Kapucijnenvoer 33, 3000 Leuven,<country>Belgium</country></nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>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.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Taymans, J M" uniqKey="Taymans J">J. M. Taymans</name></author><author><name sortKey="Greggio, E" uniqKey="Greggio E">E. Greggio</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gasser, T" uniqKey="Gasser T">T. Gasser</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Paisan Ruiz, C" uniqKey="Paisan Ruiz C">C. Paisan-Ruiz</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lill, C M" uniqKey="Lill C">C. M. Lill</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ross, O A" uniqKey="Ross O">O. A. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27658356</article-id><article-id pub-id-type="pmc">5034242</article-id><article-id pub-id-type="pii">srep33897</article-id><article-id pub-id-type="doi">10.1038/srep33897</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lobbestael</surname><given-names>E.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Civiero</surname><given-names>L.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>De Wit</surname><given-names>T.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Taymans</surname><given-names>J.-M.</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Greggio</surname><given-names>E.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Baekelandt</surname><given-names>V.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven</institution>, Kapucijnenvoer 33, 3000 Leuven,<country>Belgium</country></aff><aff id="a2"><label>2</label><institution>Department of Biology, University of Padova</institution>, 35131 Padova,<country>Italy</country></aff><aff id="a3"><label>3</label><institution>UMR-S1172 Jean-Pierre Aubert Research Center – (INSERM - CHRU de Lille - Université de Lille), Early Stages of Parkinson’s Disease Team</institution>, Lille,<country>France</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>veerle.baekelandt@kuleuven.be</email></corresp></author-notes><pub-date pub-type="epub"><day>23</day><month>09</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>6</volume><elocation-id>33897</elocation-id><history><date date-type="received"><day>12</day><month>08</month><year>2015</year></date><date date-type="accepted"><day>06</day><month>09</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2016, The Author(s)</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>The Author(s)</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>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.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>LRRK2 kinase inhibition reduces LRRK2 protein levels.</title><p>SH-SY5Y overexpressing 3flag-LRRK2 WT (<bold>a</bold>) A2016T (<bold>b</bold>) K1906M (<bold>c</bold>) G2019S (<bold>d</bold>) S910A (<bold>e</bold>) or S935A (<bold>f</bold>) were treated according to different time schedules with LRRK2-IN1 (L2-IN1, 1 μM), CZC-25146 (CZC, 200 nM), PF-06447475 (PF, 150 nM), GSK2578215A (GSK, 1 μM), MLi-2 (10 nM) or HG 10-102-01 (HG, 1 μM) or DMSO. Cell lysates were analyzed with immunoblotting using FlagM2 antibody for LRRK2 detection, anti-LRRK2 P-S935 and anti-α- or β-tubulin, anti-GAPDH or anti-vinculin for equal loading. Shown are representative blots. Graphs show the quantification of blots representing the ratio of phosphorylation at S935 over total LRRK2 signal or total LRRK2 over housekeeping protein signal. Error bars indicate s.e.m. with N ≥ 3. Statistical significance was tested using a two-way ANOVA test with Bonferroni post-tests. ***p < 0.001, **p < 0.01, *p < 0.05.</p></caption><graphic xlink:href="srep33897-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>LRRK2 kinase inhibitor-induced reduction of LRRK2 protein levels is caused by proteasomal degradation.</title><p>(<bold>a</bold>) QPCR analysis was performed on mRNA derived from SH-SY5Y cells overexpressing 3flag-LRRK2 WT, treated with CZC-25146 (CZC, 200 nM) or DMSO for different periods of time. Immunoblotting analysis was performed on cells cultured and treated in parallel with cells for QPCR analysis, using FlagM2 antibody for LRRK2 detection, anti-LRRK2 P-S935 and anti-β-tubulin for equal loading. Shown are representative blots. Quantification of LRRK2 mRNA shows LRRK2 mRNA levels normalized to β-actin mRNA levels with error bars indicating s.e.m., N = 3. (<bold>b</bold>) SH-SY5Y cells overexpressing 3flag-LRRK2 WT were treated for 17 h with LRRK2-IN1 (L2-IN1, 1 μM) and/or MG132 (1 μM), DMSO was used as negative control. Cell lysates were analyzed with immunoblotting using FlagM2 antibody for LRRK2 detection, anti-LRRK2 P-S935 and anti-β-tubulin for equal loading, N ≥ 4. Statistical significance was assessed by column statistics (one-sample t-test) or a nonparametric t-test (Mann-Whitney). **p < 0.01; *p < 0.05. (<bold>c</bold>) SH-SY5Y cells overexpressing 3flag-LRRK2 WT were treated for 48 h with LRRK2-IN1 (L2-IN1, 1 μM) and/or chloroquine (CQ) (10 μM), DMSO was used as negative control. Cell lysates were analyzed with immunoblotting using FlagM2 antibody for LRRK2 detection, anti-LRRK2 P-S935, anti-LC3 antibody and anti-α tubulin for equal loading, N ≥ 4. Statistical significance was assessed by column statistics (one-sample t-test) with Bonferroni correction or a nonparametric t-test (Mann-Whitney). **p < 0.01; *p < 0.05.</p></caption><graphic xlink:href="srep33897-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>LRRK2 kinase inhibition <italic>in vivo</italic> induces LRRK2 protein destabilization in brain, lung and kidney.</title><p>C57BL/6J mice received four intraperitoneal injections with 10 mg/kg MLi-2 or DMSO over 30 h. Brain, lung and kidney extracts were taken 2 h after the last injection and analyzed with immunoblotting using MJFF-2 anti-LRRK2 antibody, anti-LRRK2 P-S935 and anti-vinculin or α-tubulin for equal loading. Shown are representative blots with each lane representing a separate animal. Quantification of the blots is represented as a histogram and shows the ratio of total LRRK2 over vinculin or α-tubulin signal, error bars indicate s.e.m. with N ≥ 3, statistical significance was assessed using column statistics. **p < 0.01; *p < 0.05.</p></caption><graphic xlink:href="srep33897-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>LRRK2 kinase inhibition in primary astrocytes.</title><p>(<bold>a</bold>) Primary astrocytes were treated with GSK2578215A (GSK, 2 μM), CZC-25146 (CZC, 200 nM), HG 10-102-01 (HG, 1 μM) or PF-06447475 (PF, 150 nM) for 90 min or 48 h or with a tenfold dilution of the concentrations mentioned for 5 or 10 days with fresh compound every two days. Immunoblotting was done with MJFF-2 anti-LRRK2 antibody, anti-LRRK2 P-S935 and anti-β-tubulin for equal loading. Representative blots are shown. (<bold>b</bold>) Quantifications of the blots represent total LRRK2 signal over β-tubulin. Statistical significance was assessed by column statistics (one-sample t-test) with Bonferroni correction for multiple comparisons. *p < 0.05.</p></caption><graphic xlink:href="srep33897-f4"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Belgique</li><li>France</li><li>Italie</li></country></list><tree><country name="Belgique"><noRegion><name sortKey="Lobbestael, E" sort="Lobbestael, E" uniqKey="Lobbestael E" first="E." last="Lobbestael">E. Lobbestael</name></noRegion><name sortKey="Baekelandt, V" sort="Baekelandt, V" uniqKey="Baekelandt V" first="V." last="Baekelandt">V. Baekelandt</name><name sortKey="De Wit, T" sort="De Wit, T" uniqKey="De Wit T" first="T." last="De Wit">T. De Wit</name></country><country name="Italie"><noRegion><name sortKey="Civiero, L" sort="Civiero, L" uniqKey="Civiero L" first="L." last="Civiero">L. Civiero</name></noRegion><name sortKey="Greggio, E" sort="Greggio, E" uniqKey="Greggio E" first="E." last="Greggio">E. Greggio</name></country><country name="France"><noRegion><name sortKey="Taymans, J M" sort="Taymans, J M" uniqKey="Taymans J" first="J.-M." last="Taymans">J.-M. Taymans</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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