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• W2799684901 abstract "•Gsk3b kinase activity and inflammation are increased in mice models of Rett syndrome•Specific inhibition of Gsk3b reduces motor deficits and improves general well-being•Neuronal morphology is restored in vitro and in vivo and excitatory synapses altered•Gsk3b is a potential therapeutic target in Rett syndrome treatment Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG-binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763. Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG-binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763. Rett syndrome (RTT; OMIM #312750) is a rare neurodevelopmental pathology. It was first described in 1966 as a cognitive impairment disorder affecting 1 in 10,000 female births (Rett, 1966Rett A. [On a unusual brain atrophy syndrome in hyperammonemia in childhood].Wien. Med. Wochenschr. 1966; 116: 723-726PubMed Google Scholar, Hagberg et al., 1983Hagberg B. Aicardi J. Dias K. Ramos O. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett’s syndrome: report of 35 cases.Ann. Neurol. 1983; 14: 471-479Crossref PubMed Scopus (1065) Google Scholar). In 1999, mutations in the MeCP2 gene were found to be the genetic basis of most cases of RTT (Amir et al., 1999Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3493) Google Scholar). MeCP2 is a nuclear protein, expressed widely in different tissues but most abundantly in neurons of the mature nervous system, which had been identified a few years earlier by Bird and coworkers as a new protein that bound to the methylated CpG dinucleotides (Lewis et al., 1992Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1017) Google Scholar). RTT is an X chromosome-linked, progressive disorder with a normal prenatal and perinatal period. Rett patients have proper brain development during the first few months of age, achieving normal neurodevelopment, motor function, and communication skills. However, developmental regression appears between 8 and 24 months of age (Trevathan and Naidu, 1988Trevathan E. Naidu S. The clinical recognition and differential diagnosis of Rett syndrome.J. Child Neurol. 1988; 3: S6-S16Crossref PubMed Scopus (18) Google Scholar, Hagberg, 2002Hagberg B. Clinical manifestations and stages of Rett syndrome.Ment. Retard. Dev. Disabil. Res. Rev. 2002; 8: 61-65Crossref PubMed Scopus (258) Google Scholar). RTT patients suffer from growth failure, gastrointestinal problems, seizures, and respiratory and cardiac abnormalities. Epilepsy is also one of the main symptoms, with a frequency between 50% and 90% of all cases (Steffenburg et al., 2001Steffenburg U. Hagberg G. Hagberg B. Epilepsy in a representative series of Rett syndrome.Acta Paediatr. 2001; 90: 34-39Crossref PubMed Google Scholar, Huppke et al., 2007Huppke P. Köhler K. Brockmann K. Stettner G.M. Gärtner J. Treatment of epilepsy in Rett syndrome.Eur. J. Paediatr. Neurol. 2007; 11: 10-16Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), although its severity often tends to decrease after adolescence, with lower seizure frequency and fewer secondary generalized seizures (Krajnc et al., 2011Krajnc N. Župančič N. Oražem J. Epilepsy treatment in Rett syndrome.J. Child Neurol. 2011; 26: 1429-1433Crossref PubMed Scopus (16) Google Scholar). Because glycogen synthase kinase-3B (GSK3B) has crucial roles in neural development, including neurite growth, specification, and synapse (Seira and Del Río, 2014Seira O. Del Río J.A. Glycogen synthase kinase 3 beta (GSK3β) at the tip of neuronal development and regeneration.Mol. Neurobiol. 2014; 49: 931-944Crossref PubMed Scopus (63) Google Scholar), we set out to investigate its role in the pathophysiology of RTT. GSK3 is encoded by two genes, GSK3A and GSK3B, (Frame et al., 2001Frame S. Cohen P. Biondi R.M. A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation.Mol. Cell. 2001; 7: 1321-1327Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, Doble and Woodgett, 2003Doble B.W. Woodgett J.R. GSK-3: tricks of the trade for a multi-tasking kinase.J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1657) Google Scholar). Both kinases are highly expressed in the brain, but enhanced expression of GSK3B above physiological levels results in hyperactivity and mania (Prickaerts et al., 2006Prickaerts J. Moechars D. Cryns K. Lenaerts I. van Craenendonck H. Goris I. Daneels G. Bouwknecht J.A. Steckler T. Transgenic mice overexpressing glycogen synthase kinase 3beta: a putative model of hyperactivity and mania.J. Neurosci. 2006; 26: 9022-9029Crossref PubMed Scopus (270) Google Scholar). GSK3 enzymes also have important functions as regulators of neurotransmitter signaling, as indicated by their control of pathways downstream of dopamine receptors (Beaulieu et al., 2004Beaulieu J.M. Sotnikova T.D. Yao W.D. Kockeritz L. Woodgett J.R. Gainetdinov R.R. Caron M.G. Lithium antagonizes dopamine-dependent behaviors mediated by an AKT/glycogen synthase kinase 3 signaling cascade.Proc. Natl. Acad. Sci. U S A. 2004; 101: 5099-5104Crossref PubMed Scopus (651) Google Scholar). GSK3B is deregulated by tyrosine and serine/threonine phosphorylation; specifically, phosphorylation in Ser9 correlates with the inhibition of its kinase activity (Frame et al., 2001Frame S. Cohen P. Biondi R.M. A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation.Mol. Cell. 2001; 7: 1321-1327Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, Wang et al., 1994Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P.J. Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation.J. Biol. Chem. 1994; 269: 14566-14574Abstract Full Text PDF PubMed Google Scholar, Harwood, 2001Harwood A.J. Regulation of GSK-3: a cellular multiprocessor.Cell. 2001; 105: 821-824Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), while phosphorylation of tyrosine in position 216 is associated with enhanced kinase activity. Several kinases have been reported as targeting the site (Sayas et al., 1999Sayas C.L. Moreno-Flores M.T. Avila J. Wandosell F. The neurite retraction induced by lysophosphatidic acid increases Alzheimer’s disease-like Tau phosphorylation.J. Biol. Chem. 1999; 274: 37046-37052Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, Hartigan et al., 2001Hartigan J.A. Xiong W.C. Johnson G.V. Glycogen synthase kinase 3beta is tyrosine phosphorylated by PYK2.Biochem. Biophys. Res. Commun. 2001; 284: 485-489Crossref PubMed Scopus (84) Google Scholar), and even an intramolecular autophosphorylation event has been described (Cole et al., 2004Cole A. Frame S. Cohen P. Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event.Biochem. J. 2004; 377: 249-255Crossref PubMed Scopus (240) Google Scholar). Overexpression of a constitutively active GSK3B in cultured neurons reduces expression and clustering of the synaptic protein synapsin I (Zhu et al., 2007Zhu L.Q. Wang S.H. Liu D. Yin Y.Y. Tian Q. Wang X.C. Wang Q. Chen J.G. Wang J.Z. Activation of glycogen synthase kinase-3 inhibits long-term potentiation with synapse-associated impairments.J. Neurosci. 2007; 27: 12211-12220Crossref PubMed Scopus (189) Google Scholar), while pharmacological inhibition of GSK3B activity induces synapsin I clustering in developing neurons (Hall et al., 2002Hall A.C. Brennan A. Goold R.G. Cleverley K. Lucas F.R. Gordon-Weeks P.R. Salinas P.C. Valproate regulates GSK-3-mediated axonal remodeling and synapsin I clustering in developing neurons.Mol. Cell. Neurosci. 2002; 20: 257-270Crossref PubMed Scopus (153) Google Scholar). Moreover, activation of GSK3B reduces expression of postsynaptic markers and suppresses release of presynaptic glutamate by inhibiting the synaptic vesicle exocytosis in response to membrane depolarization (Zhu et al., 2010Zhu L.Q. Liu D. Hu J. Cheng J. Wang S.H. Wang Q. Wang F. Chen J.G. Wang J.Z. GSK-3 beta inhibits presynaptic vesicle exocytosis by phosphorylating P/Q-type calcium channel and interrupting SNARE complex formation.J. Neurosci. 2010; 30: 3624-3633Crossref PubMed Scopus (78) Google Scholar). GSK3B negatively regulates synaptic vesicle fusion events through interference with Ca2+-dependent SNARE complex formation. Other studies have also reported functional roles of GSK3B in the regulation of N-methyl-d-aspartic acid (NMDA) receptor-dependent synaptic plasticity (Decker et al., 2010Decker H. Lo K.Y. Unger S.M. Ferreira S.T. Silverman M.A. Amyloid-beta peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons.J. Neurosci. 2010; 30: 9166-9171Crossref PubMed Scopus (168) Google Scholar, Peineau et al., 2008Peineau S. Bradley C. Taghibiglou C. Doherty A. Bortolotto Z.A. Wang Y.T. Collingridge G.L. The role of GSK-3 in synaptic plasticity.Br. J. Pharmacol. 2008; 153: S428-S437Crossref PubMed Scopus (195) Google Scholar, Li et al., 2009Li Y.C. Xi D. Roman J. Huang Y.Q. Gao W.J. Activation of glycogen synthase kinase-3 beta is required for hyperdopamine and D2 receptor-mediated inhibition of synaptic NMDA receptor function in the rat prefrontal cortex.J. Neurosci. 2009; 29: 15551-15563Crossref PubMed Scopus (74) Google Scholar). Activation of GSK3B mediates induction of NMDA-dependent long-term depression (LTD). In contrast, inhibition of GSK3B activity prevents LTD and participates in long-term potentiation (LTP) induction (Hooper et al., 2007Hooper C. Markevich V. Plattner F. Killick R. Schofield E. Engel T. Hernandez F. Anderton B. Rosenblum K. Bliss T. et al.Glycogen synthase kinase-3 inhibition is integral to long-term potentiation.Eur. J. Neurosci. 2007; 25: 81-86Crossref PubMed Scopus (261) Google Scholar). Importantly, an abnormal increase in Gsk3b levels and activity has been associated with neuronal pathologies, and its inhibition has been proposed to be of therapeutic applicability in Alzheimer’s disease (Medina and Avila, 2010Medina M. Avila J. Glycogen synthase kinase-3 (GSK-3) inhibitors for the treatment of Alzheimer’s disease.Curr. Pharm. Des. 2010; 16: 2790-2798Crossref PubMed Scopus (71) Google Scholar) and fragile X syndrome (Mines and Jope, 2011Mines M.A. Jope R.S. Glycogen synthase kinase-3: a promising therapeutic target for fragile x syndrome.Front. Mol. Neurosci. 2011; 4: 35Crossref PubMed Google Scholar). Additionally, GSK3B is involved in inflammation and oxidative damage in the brain, which blocks the production of new neuronal connections in the hippocampus, decreases plasticity and dendritic spine density, and manifests as unsocial behavior. Therefore, drugs that inhibit GSK3B may also prove beneficial in RTT syndrome for controlling cognitive impairments derived from excessive neuroinflammation (Jope et al., 2017Jope R.S. Cheng Y. Lowell J.A. Worthen R.J. Sitbon Y.H. Beurel E. Stressed and Inflamed, Can GSK3 Be Blamed?.Trends Biochem. Sci. 2017; 42: 180-192Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Mecp2-knockout (KO) mice have a range of physiological and neurological abnormalities that imitate the human syndrome. Here we show that Gsk3b activity is increased in Rett Mecp2-KO animals and that by targeting this pathway with a specific inhibitor (SB216763), it is possible to alleviate the symptoms and increase the lifespan of the animals, reduce neuroinflammation markers, and partially restore neuronal connectivity. This makes the Gsk3b signaling pathway an attractive target for therapeutic strategies. Mecp2- KO mice develop a progression of symptoms in comparison with wild-type (WT) animals. KO mice are globally normal until 4 weeks of age; they then begin to suffer cognitive and motor dysfunctions and fully manifest the RTT-like phenotype at about 6–8 weeks of age (Figure S1A). This leads to rapid weight loss and death at approximately 10 weeks of age (Guy et al., 2001Guy J. Hendrich B. Holmes M. Martin J.E. Bird A. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome.Nat. Genet. 2001; 27: 322-326Crossref PubMed Scopus (1137) Google Scholar). To investigate the potential therapeutic role of inhibiting the Gsk3b pathway, three different doses of the specific inhibitor SB216763 were used for the optimization experiment: 0, 0.5, and 1 mg/kg/day. During the whole experiment, none of the selected doses induced a weight loss in the Mecp2-KO mice, indicating that the drug was well tolerated and did not have any negative effect on the animals’ lifespan in comparison with the vehicle-treated Mecp2-KO group. A representative weekly average for a dose of 0.5 mg/kg/day is shown in Figure 1A, which was subsequently used in all experiments and measurements. After dose optimization, symptom onset was evaluated in the SB216763-treated group compared with the vehicle-treated group. In order to test the effect of the Gsk3b inhibitor SB216763 on the symptoms of RTT mice, behavioral tests were used regularly during the whole treatment procedure. We started treatment when the animals were 4 weeks old, before the onset of disease. The mice were treated daily during 6 weeks and scored twice a week for neurological recovery following previous recommendations (Sutherland et al., 1993Sutherland C. Leighton I.A. Cohen P. Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling.Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (703) Google Scholar). To quantify the alleviation of symptoms after drug administration, we measured mobility, tremor, and breathing features and plotted the average of each phenotypic score during each week of the treatment normalized with the vehicle group (Figures 1B–1D). Treatment with the Gsk3b inhibitor improved the general well-being of the mice; the decrease in scores upon inhibitor treatment corresponded to significant improvements in mobility, tremor, and breathing phenotypes from 7 to 10 weeks of age by an average of 50%–60%, with greater differences appearing at earlier ages. Plotting of the average of the six scored symptoms to create a total score showed a significant phenotypic improvement during the whole time of treatment. SB216763 induced slower progression of the disease, and some of the treated mice did not develop any detectable symptoms until after 8 weeks of age (Figure 1E). The vehicle group displayed an average lifespan of 70 days, whereas in the SB216763-treated group, the average lifespan was 91 days, (Figure 1F; Kaplan-Meier log rank test). Considering the whole population of SB216763-treated mice, the survival curve was 30% longer compared with the vehicle group. Thus, the drug administration in Mecp2-KO mice increased their overall well-being by diminishing RTT symptomatology, with no associated intrinsic toxicity, allowing the animals to live longer. To address in an unbiased manner whether SB216763 administration is associated with reduction of the motor impairments in the Mecp2-KO mice, we carried out bar-cross tests. KO mice treated with the drug achieved better scores, suffering fewer slips than vehicle-treated animals (Figures 1G and 1H). Again, the recovery was more noticeable for younger mice. In neophobia or Y-maze tests, we observed not only that the mobility of the KO mice was improved but also that their anxious behavior or susceptibility to stress was reduced (Figures S1B–S1D). We also assessed the effect of starting the treatment at a later time point, from 6 weeks of age, when the symptoms were already present in the animals. As shown in Figures S1E–S1I, drug administration at later times resulted in a slight tendency toward improvement of the phenotype (although not statistically significant). In view of these results, we went on to characterize changes in the Gsk3b pathway following SB216763 administration at the molecular and cellular level. The Gsk3b protein is ubiquitously distributed in mammalian tissues (Plyte et al., 1992Plyte S.E.K. Hughes K. Nikolakaki E. Pulverer B.J. Woodgett J.R. Glycogen synthase kinase-3: functions in oncogenesis and development.Biochim. Biophys. Acta. 1992; 1114: 147-162Crossref PubMed Scopus (326) Google Scholar), and its activity is an important component in the regulation of complex functions (Grimes and Jope, 2001Grimes C.A. Jope R.S. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling.Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1257) Google Scholar). The regulation of Gsk3b can occur through the phosphorylation of specific residues, which may either activate or inactivate the kinase. Inactivation can occur through phosphorylation of serine-9 (Sutherland et al., 1993Sutherland C. Leighton I.A. Cohen P. Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling.Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (703) Google Scholar), serine-389, and threonine-390 (Thornton et al., 2008Thornton T.M. Pedraza-Alva G. Deng B. Wood C.D. Aronshtam A. Clements J.L. Sabio G. Davis R.J. Matthews D.E. Doble B. Rincon M. Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation.Science. 2008; 320: 667-670Crossref PubMed Scopus (342) Google Scholar), or threonine-43 (Ding et al., 2005Ding Q. Xia W. Liu J.C. Yang J.Y. Lee D.F. Xia J. Bartholomeusz G. Li Y. Pan Y. Li Z. et al.Erk associates with and primes GSK-3beta for its inactivation resulting in upregulation of beta-catenin.Mol. Cell. 2005; 19: 159-170Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), while activation can occur through the phosphorylation of tyrosine-216 (Hughes et al., 1993Hughes K. Nikolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (494) Google Scholar). Gsk3b mRNA is broadly distributed in normal mouse brain, as shown by in situ hybridization (ISH) analysis in sagittal brain tissue of P56 male mice. Regions with the highest signal include the isocortex, the hippocampus, and the striatum (Figures 2A and 2B ; mouse Atlas Brain, http://mouse.brain-map.org). Comparison of Gsk3b kinase activity in several brain regions of Mecp2-null mice against WT regions showed an abnormal increase in Gsk3b activity, especially in the cerebellum (Figure 2C). Although no changes in total Gsk3b protein level were observed, we detected a ∼2-fold increase in the phosphorylation levels of Tyr216 (which correlates with enhanced activity of the protein) and a ∼2-fold decrease in the levels of Ser9 phosphorylation (which attenuates activity) (Figure 2D), indicating that Gsk3b activity is dysregulated in the cerebellum of the Mecp2−/y mouse model. Other brain regions of the same mice did not show this pattern of activating phosphorylation (Figures S2A and S2B), pointing to a cerebellum-specific upregulation of the Gsk3b pathway. Male RTT mice models recapitulate most of the human symptoms (Lombardi et al., 2015Lombardi L.M. Baker S.A. Zoghbi H.Y. MECP2 disorders: from the clinic to mice and back.J. Clin. Invest. 2015; 125: 2914-2923Crossref PubMed Scopus (124) Google Scholar) but are arguably a limited source of knowledge for the understanding of a disorder that affects mainly females. We thus analyzed the levels of phosphorylated Gsk3b in the cerebellum of 8-month-old (fully symptomatic) heterozygous Mecp2+/− female mice of the same strain. We also observed an increase in the level of Gsk3b pTyr216 (Figure S2C). Importantly, cerebellar extracts of post-mortem brains from Rett patients displayed the same upregulation (Figure 2E), supporting the idea that aberrant hyperactivation of GSK3 is a hallmark of RTT. Because Akt is one of the main inhibitors of Gsk3b through phosphorylation of Ser9 (Manning and Toker, 2017Manning B.D. Toker A. AKT/PKB signaling: navigating the network.Cell. 2017; 169: 381-405Abstract Full Text Full Text PDF PubMed Scopus (1211) Google Scholar), these results are in accordance with the dysfunction of the Akt/mTOR signaling pathway reported by others in Rett mice models (Ricciardi et al., 2011Ricciardi S. Boggio E.M. Grosso S. Lonetti G. Forlani G. Stefanelli G. Calcagno E. Morello N. Landsberger N. Biffo S. et al.Reduced AKT/mTOR signaling and protein synthesis dysregulation in a Rett syndrome animal model.Hum. Mol. Genet. 2011; 20: 1182-1196Crossref PubMed Scopus (146) Google Scholar). To confirm these observations, we established neuronal primary cultures from either WT or Mecp2−/y newborn mouse brains and carried out immunofluorescence staining to detect Gsk3b phosphorylated residues. As shown in Figures 2F and 2G, Gsk3b pTyr216 signal was increased and pSer9 signal was decreased in neuronal cultures derived from the Mecp2-null brains, pointing to an enhanced activity of Gsk3b also in these primary cultures. Remarkably, treatment of the cultures with the Gsk3b inhibitor SB216763 partially restored both signals to the levels of the WT neurons, with significant differences between the control-treated and the inhibitor-treated Mecp2−/y neurons. Gsk3b activation has been associated with increases in neuroinflammation and microglial activation, partly by enhancing the nuclear factor kappa b subunit 1 (Nfkb1) pathway (Wang et al., 2010Wang M.J. Huang H.Y. Chen W.F. Chang H.F. Kuo J.S. Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades.J. Neuroinflammation. 2010; 7: 99Crossref PubMed Scopus (84) Google Scholar, Hoeflich et al., 2000Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation.Nature. 2000; 406: 86-90Crossref PubMed Scopus (1158) Google Scholar, Ko et al., 2014Ko C.Y. Wang W.L. Wang S.M. Chu Y.Y. Chang W.C. Wang J.M. Glycogen synthase kinase-3β-mediated CCAAT/enhancer-binding protein delta phosphorylation in astrocytes promotes migration and activation of microglia/macrophages.Neurobiol. Aging. 2014; 35: 24-34Crossref PubMed Scopus (25) Google Scholar). To test whether this is the case in the RTT Mecp2-null mice model, we analyzed nuclear Rela/p65 levels (indicative of the functional form as a co-transcriptional regulator) by fluorescence staining in cerebellar neuronal primary cultures. We found that nuclear p65 signaling is upregulated in Mecp2-deficient neurons, whereas treatment with SB216763 restored the WT localization (Figure 3A). Total p65 levels were also increased in KO animals and could be downregulated with the inhibitor, as seen in vivo by western blot analysis of total brain protein extract from WT and Mecp2-KO mice treated with the GSK3b inhibitor (Figure 3B). Mecp2 depletion can trigger the dysregulation of inflammatory responses and the activation of microglia (which is subsequently lost as the disease progresses) in RTT mice models (Cronk et al., 2015Cronk J.C. Derecki N.C. Ji E. Xu Y. Lampano A.E. Smirnov I. Baker W. Norris G.T. Marin I. Coddington N. et al.Methyl-CpG binding protein 2 regulates microglia and macrophage gene expression in response to inflammatory stimuli.Immunity. 2015; 42: 679-691Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Derecki et al., 2013Derecki N.C. Cronk J.C. Kipnis J. The role of microglia in brain maintenance: implications for Rett syndrome.Trends Immunol. 2013; 34: 144-150Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This is in accordance with previous findings describing gliosis and cell loss in the cerebellum of post-mortem RTT brains (Oldfors et al., 1990Oldfors A. Sourander P. Armstrong D.L. Percy A.K. Witt-Engerström I. Hagberg B.A. Rett syndrome: cerebellar pathology.Pediatr. Neurol. 1990; 6: 310-314Abstract Full Text PDF PubMed Scopus (56) Google Scholar). In our study, analysis by immunofluorescence of the microglial marker Cd11b showed high infiltration of microglial cells in the cerebellum of 8-week-old Mecp2-null mice, which was substantially reduced in mice treated with Gsk3b inhibitor (Figure 3C). Similar results were obtained in the analysis of the pons region next to the cerebellum (Figure S3A). This was confirmed by qRT-PCR that measured the levels of Cd11b mRNA (Figure 3D) and of the microglial activator Toll-like receptor 4 (Tlr4) (Figure 3E). To get a broader view of the imbalance in inflammatory cell function and of how SB216763 treatment could restore normal levels, we used a commercial antibody array to interrogate 40 mouse cytokines in the cerebellum of Mecp2-KO control or KO-treated mice (Figures S3B and S3C). As shown in Figure 3F, inhibition of Gsk3b downregulates a number of pro-inflammatory interleukins, including IL-1α, IL-1β, IL-4, IL-12p70, IL-17, and KC/CXCL1. Conversely, Fas ligand increases moderately in SB216763-treated animals, which is indicative of the reaction to an excessive immune response. Similarly, the anti-inflammatory cytokine IL-10 is also upregulated upon Gsk3b inhibition. IL-10 is known to block NFKB1 activity and to be involved in the regulation of the JAK-STAT signaling pathway (Mosser and Zhang, 2008Mosser D.M. Zhang X. Interleukin-10: new perspectives on an old cytokine.Immunol. Rev. 2008; 226: 205-218Crossref PubMed Scopus (704) Google Scholar). We and others have shown that dendrite branching pattern and density is significantly reduced in mice models and post-mortem brain samples from RTT individuals (Szczesna et al., 2014Szczesna K. de la Caridad O. Petazzi P. Soler M. Roa L. Saez M.A. Fourcade S. Pujol A. Artuch-Iriberri R. Molero-Luis M. et al.Improvement of the Rett syndrome phenotype in a MeCP2 mouse model upon treatment with levodopa and a dopa-decarboxylase inhibitor.Neuropsychopharmacology. 2014; 39: 2846-2856Crossref PubMed Scopus (14) Google Scholar, Armstrong et al., 1995Armstrong D. Dunn J.K. Antalffy B. Trivedi R. Selective dendritic alterations in the cortex of Rett syndrome.J. Neuropathol. Exp. Neurol. 1995; 54: 195-201Crossref PubMed Scopus (281) Google Scholar, Bauman et al., 1995Bauman M.L. Kemper T.L. Arin D.M. Pervasive neuroanatomic abnormalities of the brain in three cases of Rett’s syndrome.Neurology. 1995; 45: 1581-1586Crossref PubMed Scopus (128) Google Scholar, Chapleau et al., 2009Chapleau C.A. Calfa G.D. Lane M.C. Albertson A.J. Larimore J.L. Kudo S. Armstrong D.L. Percy A.K. Pozzo-Miller L. Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations.Neurobiol. Dis. 2009; 35: 219-233Crossref PubMed Scopus (161) Google Scholar). We thus wanted to test the effect of SB216763 treatment on dendritic arborization and total spine count in neuronal primary cultures. All neuronal branching was fluorescently labeled when neurons reached 7 days in culture (DIV), using a polyclonal antibody against Map2, a neuron-specific protein localized in dendrites and dendritic spines (Morales and Fifkova, 1989Morales M. Fifkova E. Distribution of MAP2 in dendritic spines and its colocalization with actin. An immunogold electron-microscope study.Cell Tissue Res. 1989; 256: 447-456Crossref PubMed Scopus (82) Google Scholar). As expected, there were significant differences in the branching pattern and density of dendrites, with WT neurons displaying a wider dendritic field than KO neurons. Treatment of Mecp2−/y neurons with SB216763 enabled an important" @default.
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• W2799684901 title "Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice" @default.
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• W2799684901 doi "https://doi.org/10.1016/j.celrep.2018.04.010" @default.
• W2799684901 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29742424" @default.
• W2799684901 hasPublicationYear "2018" @default.
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