Genetic deletion of Polo-like kinase 2 reduces alpha-synuclein serine-129 phosphorylation in presynaptic terminals but not Lewy bodies
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100273
ISSN1083-351X
AutoresLeah J. Weston, Teresa L. Stackhouse, Kateri J. Spinelli, Sydney Weber Boutros, Elizabeth P. Rose, Valerie R. Osterberg, Kelvin C. Luk, Jacob Raber, Tamily A. Weissman, Vivek K. Unni,
Tópico(s)Alzheimer's disease research and treatments
ResumoPhosphorylation of alpha-synuclein at serine-129 is an important marker of pathologically relevant, aggregated forms of the protein in several important human diseases, including Parkinson's disease, Dementia with Lewy bodies, and Multiple system atrophy. Although several kinases have been shown to be capable of phosphorylating alpha-synuclein in various model systems, the identity of the kinase that phosphorylates alpha-synuclein in the Lewy body remains unknown. One member of the Polo-like kinase family, PLK2, is a strong candidate for being the Lewy body kinase. To examine this possibility, we have used a combination of approaches, including biochemical, immunohistochemical, and in vivo multiphoton imaging techniques to study the consequences of PLK2 genetic deletion on alpha-synuclein phosphorylation in both the presynaptic terminal and preformed fibril-induced Lewy body pathology in mouse cortex. We find that PLK2 deletion reduces presynaptic terminal alpha-synuclein serine-129 phosphorylation, but has no effect on Lewy body phosphorylation levels. Serine-129 mutation to the phosphomimetic alanine or the unphosphorylatable analog aspartate does not change the rate of cell death of Lewy inclusion-bearing neurons in our in vivo multiphoton imaging paradigm, but PLK2 deletion does slow the rate of neuronal death. Our data indicate that inhibition of PLK2 represents a promising avenue for developing new therapeutics, but that the mechanism of neuroprotection by PLK2 inhibition is not likely due to reducing alpha-synuclein serine-129 phosphorylation and that the true Lewy body kinase still awaits discovery. Phosphorylation of alpha-synuclein at serine-129 is an important marker of pathologically relevant, aggregated forms of the protein in several important human diseases, including Parkinson's disease, Dementia with Lewy bodies, and Multiple system atrophy. Although several kinases have been shown to be capable of phosphorylating alpha-synuclein in various model systems, the identity of the kinase that phosphorylates alpha-synuclein in the Lewy body remains unknown. One member of the Polo-like kinase family, PLK2, is a strong candidate for being the Lewy body kinase. To examine this possibility, we have used a combination of approaches, including biochemical, immunohistochemical, and in vivo multiphoton imaging techniques to study the consequences of PLK2 genetic deletion on alpha-synuclein phosphorylation in both the presynaptic terminal and preformed fibril-induced Lewy body pathology in mouse cortex. We find that PLK2 deletion reduces presynaptic terminal alpha-synuclein serine-129 phosphorylation, but has no effect on Lewy body phosphorylation levels. Serine-129 mutation to the phosphomimetic alanine or the unphosphorylatable analog aspartate does not change the rate of cell death of Lewy inclusion-bearing neurons in our in vivo multiphoton imaging paradigm, but PLK2 deletion does slow the rate of neuronal death. Our data indicate that inhibition of PLK2 represents a promising avenue for developing new therapeutics, but that the mechanism of neuroprotection by PLK2 inhibition is not likely due to reducing alpha-synuclein serine-129 phosphorylation and that the true Lewy body kinase still awaits discovery. Parkinson's disease (PD) is a progressive neurological disorder with a variety of motor and nonmotor symptoms affecting approximately 10 million people worldwide (1Ball N. Teo W.P. Chandra S. Chapman J. Parkinson's disease and the Environment.Front Neurol. 2019; 10: 218Crossref PubMed Scopus (96) Google Scholar). The motor symptoms are due to dopamine neuron loss in the substantia nigra of the midbrain (2Del Tredici K. Braak H. Review: Sporadic Parkinson's disease: Development and distribution of alpha-synuclein pathology.Neuropathol. Appl. Neurobiol. 2016; 42: 33-50Crossref PubMed Scopus (204) Google Scholar). Cytoplasmic inclusions called Lewy bodies are observed in surviving dopamine neurons, and Lewy bodies in other peripheral and central nervous system regions correlate with many of the nonmotor symptoms of PD (3Adler C.H. Beach T.G. Neuropathological basis of nonmotor manifestations of Parkinson's disease.Mov Disord. 2016; 31: 1114-1119Crossref PubMed Scopus (117) Google Scholar). In addition, a group of related disorders termed "synucleinopathies" are characterized by Lewy body presence in a variety of nondopaminergic neuron types that is also associated with neurological dysfunction, such as cortical Lewy bodies and cognitive dysfunction in Dementia with Lewy bodies (DLB), or autonomic system Lewy bodies and orthostatic hypotension in pure autonomic failure. Lewy bodies are somatic structures comprised of misfolded and aggregated alpha-synuclein, which is normally localized primarily to the presynaptic terminal and nucleus (4Lee V.M. Trojanowski J.Q. Mechanisms of Parkinson's disease linked to pathological alpha-synuclein: New targets for drug discovery.Neuron. 2006; 52: 33-38Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 5Surguchov A. Intracellular dynamics of synucleins: "Here, there and Everywhere".Int. Rev. Cell Mol Biol. 2015; 320: 103-169Crossref PubMed Scopus (43) Google Scholar). One of the characteristic hallmarks of aggregated alpha-synuclein within Lewy bodies is a high level of specific posttranslational modifications of the protein, including phosphorylation, ubiquitination, nitrosylation, and C-terminal truncation (6Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. 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Characterization of kinases involved in the phosphorylation of aggregated alpha-synuclein.J. Neurosci. Res. 2011; 89: 231-247Crossref PubMed Scopus (48) Google Scholar). Furthermore, in mouse and rat brain PLK family members, including PLK2, play a major role in serine-129 phosphorylation (23Inglis K.J. Chereau D. Brigham E.F. Chiou S.S. Schobel S. Frigon N.L. Yu M. Caccavello R.J. Nelson S. Motter R. Wright S. Chian D. Santiago P. Soriano F. Ramos C. et al.Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.J. Biol. Chem. 2009; 284: 2598-2602Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 26Mbefo M.K. Paleologou K.E. Boucharaba A. Oueslati A. Schell H. Fournier M. Olschewski D. Yin G. Zweckstetter M. Masliah E. Kahle P.J. Hirling H. Lashuel H.A. Phosphorylation of synucleins by members of the Polo-like kinase family.J. Biol. 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By using point mutations to study the effect of serine-129 phosphorylation, we show that this protective effect of PLK2 deletion is likely not due to specific effects on this phosphorylation, but rather to other consequences of PLK2 deletion. To test the role of PLK family members in the phosphorylation of alpha-synuclein at serine-129 in both presynaptic terminal and other cytosolic subcellular compartments within cortical neurons, we treated heterozygous Syn-GFP transgenic animals with a single dose of the selective small-molecule PLK inhibitor BI 2536 (50 mg/kg) and measured phosphorylated and total Syn-GFP levels in cortical tissue using western blot analysis after biochemical fractionation. We determined the time course of the resulting phosphorylation changes by sacrificing animals at various times following BI 2536 injection. Both synaptosomal and cytosolic pools of Syn-GFP showed significant decreases in phosphorylated and total levels starting at 4 h after BI 2536 administration, which then returned to baseline levels by 12 h and remained there at 24 and 48 h (Fig. 1A). An analysis of Syn-GFP animals similarly treated with BI 2536 using immunohistochemistry (IHC) for phosphorylated Syn-GFP and GFP fluorescence signal for measuring total levels from cortical sections revealed similar results. Significant decreases in presynaptic and somatic levels were again observed 4 h after BI 2536 administration (Fig. 1B). Phosphorylated Syn-GFP levels remained reduced at 12 h but returned to baseline by 24 h, while total levels recovered to baseline in terminals and remained reduced in the cell body at 12 h (Fig. 1B). At 24 h post BI 2536 administration, both terminal and somatic total Syn-GFP levels were transiently increased before returning to baseline at 48 h (Fig. 1B). Since BI2536 is known to inhibit PLK1, PLK2, and PLK3 (38Kothe M. Kohls D. Low S. Coli R. Rennie G.R. Feru F. Kuhn C. Ding Y.H. Selectivity-determining residues in Plk1.Chem. Biol. Drug Des. 2007; 70: 540-546Crossref PubMed Scopus (91) Google Scholar, 39Steegmaier M. Hoffmann M. Baum A. Lenart P. Petronczki M. Krssak M. Gurtler U. Garin-Chesa P. Lieb S. Quant J. Grauert M. Adolf G.R. Kraut N. Peters J.M. Rettig W.J. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo.Curr. Biol. 2007; 17: 316-322Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 40Johnson E.F. Stewart K.D. Woods K.W. Giranda V.L. Luo Y. Pharmacological and functional comparison of the polo-like kinase family: Insight into inhibitor and substrate specificity.Biochemistry. 2007; 46: 9551-9563Crossref PubMed Scopus (93) Google Scholar), these experiments suggest that endogenous mouse PLK constitutively phosphorylates cortical Syn-GFP in this transgenic line. To be able to more directly test the role of PLK in the phosphorylation of alpha-synuclein in mouse brain, we bred our Syn-GFP line to PLK2 knockout (PLK2 KO) animals to generate heterozygous Syn-GFP mice that were on a PLK2 wild-type (WT, +/+), heterozygous KO (+/-), or homozygous KO (−/−) background. We chose the PLK2 family member because several independent reports suggest that PLK2 is a major synuclein kinase in vitro and in mouse, rat, and human Lewy body disease brain (23Inglis K.J. Chereau D. Brigham E.F. Chiou S.S. Schobel S. Frigon N.L. Yu M. Caccavello R.J. Nelson S. Motter R. Wright S. Chian D. Santiago P. Soriano F. Ramos C. et al.Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.J. Biol. Chem. 2009; 284: 2598-2602Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 26Mbefo M.K. Paleologou K.E. Boucharaba A. Oueslati A. Schell H. Fournier M. Olschewski D. Yin G. Zweckstetter M. Masliah E. Kahle P.J. Hirling H. Lashuel H.A. Phosphorylation of synucleins by members of the Polo-like kinase family.J. Biol. Chem. 2010; 285: 2807-2822Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 28Waxman E.A. Giasson B.I. Characterization of kinases involved in the phosphorylation of aggregated alpha-synuclein.J. Neurosci. Res. 2011; 89: 231-247Crossref PubMed Scopus (48) Google Scholar, 30Wang Y. Shi M. Chung K.A. Zabetian C.P. Leverenz J.B. Berg D. Srulijes K. Trojanowski J.Q. Lee V.M. Siderowf A.D. Hurtig H. Litvan I. Schiess M.C. Peskind E.R. Masuda M. et al.Phosphorylated alpha-synuclein in Parkinson's disease.Sci. Transl Med. 2012; 4: 121ra120Crossref Scopus (176) Google Scholar, 31Aubele D.L. Hom R.K. Adler M. Galemmo Jr., R.A. Bowers S. Truong A.P. Pan H. Beroza P. Neitz R.J. Yao N. Lin M. Tonn G. Zhang H. Bova M.P. 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A parallel analysis of cortical tissue by IHC demonstrated a significant decrease in phosphorylated, but not total, Syn-GFP in the cortical presynaptic terminals of PLK2 KO mice (Fig. 2B). These data suggest that PLK2 is important for phosphorylation of alpha-synuclein at terminals and in the cytosol and that this may regulate total alpha-synuclein levels, as has been suggested previously (41Oueslati A. Schneider B.L. Aebischer P. Lashuel H.A. Polo-like kinase 2 regulates selective autophagic alpha-synuclein clearance and suppresses its toxicity in vivo.Proc. Natl. Acad. Sci. U S A. 2013; 110: E3945-3954Crossref PubMed Scopus (118) Google Scholar, 42Kofoed R.H. Zheng J. Ferreira N. Lykke-Andersen S. Salvi M. Betzer C. Reimer L. Jensen T.H. Fog K. Jensen P.H. Polo-like kinase 2 modulates alpha-synuclein protein levels by regulating its mRNA production.Neurobiol. Dis. 2017; 106: 49-62Crossref PubMed Scopus (13) Google Scholar). The exact mechanism by which this regulation of levels occurs, however, is subjec
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