Abstract
21 min readArticle Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Inflammation modifies risk and/or severity of a variety of brain diseases through still elusive molecular mechanisms. Here we show that hyperactivation of the interleukin 1 pathway, through either ablation of the interleukin 1 receptor 8 (IL-1R8, also known as SIGIRR or Tir8) or activation of IL-1R, leads to up-regulation of the mTOR pathway and increased levels of the epigenetic regulator MeCP2, bringing to disruption of dendritic spine morphology, synaptic plasticity and plasticity-related gene expression. Genetic correction of MeCP2 levels in IL-1R8 KO neurons rescues the synaptic defects. Pharmacological inhibition of IL-1R activation by Anakinra corrects transcriptional changes, restores MeCP2 levels and spine plasticity and ameliorates cognitive defects in IL-1R8 KO mice. By linking for the first time neuronal MeCP2, a key player in brain development, to immune activation and demonstrating that synaptic defects can be pharmacologically reversed, these data open the possibility for novel treatments of neurological diseases through the immune system modulation. https://doi.org/10.7554/eLife.21735.001 eLife digest Errors that occur while the brain is developing can lead to conditions such as autism and schizophrenia. They can also lead to rare disorders like Rett syndrome and MeCP2 duplication syndromes, which are characterized by severe cognitive and physical disabilities. Many people with these neurodevelopmental disorders have mutations in genes that encode proteins found at synapses, which are the junctions between neurons where the cells exchange information with one another. However, not everyone with these mutations develops a neurodevelopmental disorder, which indicates that other, non-genetic factors also play a part. One of the main non-genetic factors that can influence the risk and severity of neurodevelopmental disorders is inflammation of the brain. Inflammation is a normal part of the body’s immune response to threats such as invading microorganisms or tissue damage. However, abnormal activation of the immune system in early life can trigger excessive inflammation. This increases the risk of a neurodevelopmental disorder, but it is not clear exactly how it does so. Tomasoni et al. set out to test whether the missing link between inflammation and neurodevelopmental disorders might be damage to synapses. The experiments revealed that genetically modified mice with inflammation of the brain have abnormal synapses and are unable to learn properly. These mutant mice also have excessive levels of a protein that influences how synapses function called MeCP2, which is missing in the brains of people with Rett syndrome and abnormally increased in brains of patients affected by MeCP2 Duplication Syndrome. This is thus the first evidence that directly links inflammation of the brain to a synapse protein implicated in a disorder of brain development. Tomasoni et al. also found that a drug called anakinra – which is used to treat an inflammatory disease called rheumatoid arthritis – reduced levels of MeCP2 in the mutant mice and improved their performance in cognitive tasks. Together, these results raise the possibility that anti-inflammatory medications may be beneficial in the treatment of neurodevelopment disorders. https://doi.org/10.7554/eLife.21735.002 Introduction Neurological disorders represent an enormous source of burden to the individual and to society, with many patients failing to respond to available medication. Growing evidence on genetic components of neurological diseases have been collected during recent years; notably, these genes overwhelmingly point to disorders of synaptic transmission, which led to the coinage of the term ‘synaptopathy’ to indicate a brain disease originating from a dysfunction of the synapse (Grant, 2012; Grabrucker, 2014). Disruption of synapse function may also be caused by environmental stimuli, with inflammatory cytokines affecting synaptic transmission and modifying the risk and severity of a variety of brain diseases, including autism spectrum disorders, schizophrenia and cognitive disabilities (Hagberg et al., 2012; Chugh et al., 2013; Steinman, 2013). Among the cytokines known to affect synaptic function, the proinflammatory cytokine IL-1β plays a critical role. IL-1β was found to impair brain-derived neurotrophic factor (BDNF)-induced expression of molecules critical for activity-dependent synaptic plasticity, including cAMP response element binding protein (CREB), Arc, and cofilin, thus reducing actin polymerization and impairing spine morphogenesis (Tong et al., 2012). In addition, IL-1β controls different neuronal functions, including excitability and transmitter release, via multiple biochemical pathways (Weber et al., 2010). However, no evidence has been reported yet that IL-1β may affect synapse development and function acting on molecular pathways known to be at the root of synaptopathies. Genetic mouse models of immune deregulation may serve as reliable and reproducible systems for examination of the effects of inflammation on synapse structure and function and elucidation of the molecular processes involved. IL-1R8, also known as single Ig IL-1-Related Receptor (SIGIRR) or TIR8, belongs to the toll-like receptors (TLRs) and interleukin-1R-like receptors (ILRs), a family of conserved proteins involved in immunity and inflammation (Riva et al., 2012; Garlanda et al., 2013a). TLRs are receptors able to recognize specific pathogen-associated patterns (PAMPs) and necrotic cell-derived danger signals (DAMPs) and act as sensors for microorganisms and tissue damage, whereas the IL-1R subfamily includes components of signalling receptor complexes as well as molecules with regulatory function. IL-1R8 dampens the activation of the TLRs and IL-1R signalling pathways by intracellularly interfering with the association of adaptor molecules to the receptor complexes, including NF-κB and JNK (Riva et al., 2012; Garlanda et al., 2013b). As a consequence, IL-1R8-deficient mice display exaggerated symptoms of inflammatory conditions (Garlanda et al., 2007; Gulen et al., 2010; Drexler et al., 2010) and demonstrate pronounced susceptibility to the inflammatory challenge posed by microbial LPS (Garlanda et al., 2004). IL-1R8 is also present in the brain (Costelloe et al., 2008; Polentarutti et al., 2003). Genetic deficiency for IL-1R8 is associated with inflammatory changes in the brain, including increased levels of LPS-induced tumor necrosis factor α (TNFα) and IL-6 in microglia, higher expression of TLR4, and NF-κB activation (Watson et al., 2010). Reduced IL-1R8 expression has been described in patients affected by psoriatic arthritis (Batliwalla et al., 2005) while SIGIRR variants (characterized by defective SIGIRR function) have been found in humans in association with necrotizing enterocolitis (Sampath et al., 2015) and with systemic lupus erythematosus (SLE; [Zhu et al., 2014]), all pathologies being characterized by cognitive defects and neurodevelopmental impairment (Husted et al., 2013; Rees et al., 2007; Calderón et al., 2014; Muscal et al., 2010). Notably, IL-1R8-deficient mice are impaired in novel object recognition, spatial reference memory and long-term potentiation (LTP), defects that occur in the absence of any external inflammatory stimuli (Costello et al., 2011). However, the molecular mechanisms by which IL-1R8 deficiency results in brain defects are still completely unknown. We used IL-1R8 deficient mice to investigate whether genetic hyperactivation of the IL-1R pathway affects synapse function impinging on molecular players involved in synaptopathies. We demonstrate that the activity of the IL-1R pathway directly affects, in neurons, the levels of expression of the methyl-CpG-binding protein 2 (MeCP2), a synaptopathy protein involved in neurological diseases -Rett syndrome and MeCP2 duplication syndrome- characterized by defective plasticity, impaired cognition and intellectual disability. We also show that pharmacological inhibition of IL-1R activity normalizes MeCP2 expression and cognitive deficits in IL1R8-deficient mice. Results Altered synaptic architecture and function in IL-1R8 KO hippocampal neurons To investigate the impact of IL-1R8 deficiency on synapse structure and function, we examined the morphology and plasticity of dendritic spines in primary cultures established from embryonic IL-1R8 KO or WT mice hippocampi, transfected with GFP at DIV 12. Compared to their WT counterpart, IL-1R8 KO neurons displayed an increased number of immature, thin spines and a decreased number of mature, mushroom spines (Figure 1A–C) along with a significant reduction of spine width (Figure 1D). Also PSD-95 puncta density (Figure 1E) and size (Figure 1F) were significantly lower in IL-1R8 KO neurons. Consistently, the levels of the postsynaptic protein PSD-95, evaluated by western blotting of culture homogenates were significantly reduced (Figure 1G and H). In line with a synaptic defect, patch clamp recording of IL-1R8 deficient cultures revealed that the frequency, but not the amplitude, of miniature excitatory postsynaptic currents (mEPSCs) was significantly reduced (Figure 1I–K). Spine defects (Figure 2A and B) and reduction in synaptic markers (Figure 2C and D) were also detected in CA1 pyramidal neurons of IL1R8 KO mice with respect to age-matched WT controls. Figure 1 Download asset Open asset IL-1R8 silencing affects spine morphology and function. (A) PSD-95 immunocytochemical staining of GFP-transfected, 16 DIV hippocampal cultures from WT or IL-1R8 KO mice. Scale bar 5 mm. (B-F) Quantitative analysis of the following parameters: thin and mushroom spine density (B and C); spine width (D); PSD-95 puncta density (E) and mean size of PSD-95 puncta (F). Number of analyzed neurons: B-D: 32 (WT), 44 (IL-1R8 KO); E-F: 32 (WT), 29 (IL-1R8 KO); Student t test. (G, H) Western blotting analysis of PSD-95 levels in primary hippocampal cultures, 3 independent experiments, Mann Whitney test. (I) Representative mEPSC traces recorded from WT and IL-1R8 KO neurons. (J) mEPSC frequency quantitation (WT: n = 12; IL-1R8 KO: n = 18; Mann Whitney Test). (K) Cumulative distributions and bar graph of mEPSC amplitude (WT: 22,82 ± 2, n = 12; IL-1R8 KO: 22,11 ± 1,5, n = 18; Mann Whitney test. * indicates significance compared to WT, # indicates, significance compared to IL-1R8 KO. https://doi.org/10.7554/eLife.21735.003 Figure 2 Download asset Open asset IL1R8 deficient mice show altered spines and synapses in hippocampal sections. (A) Representative images of secondary branches of apical dendrites of WT and IL-1R8 KO mice (3 months old) stained by the Golgi-Cox method and relative quantitation (B). A significant reduction of spine density in IL-1R8 KO mice was evident with respect to WT mice (number of spines per micron: WT = 1,02 ± 0,03; number of mice analyzed: 3, number of examined dendrites: 75; IL-1R8 KO = 0,80 ± 0,02; number of mice analyzed: 3, number of examined dendrites: 84; Mann-Whitney test). Scale bar, 5 µm. (C) Representative fields of the CA1 hippocampal region (stratum radiatum) of a WT and IL-1R8 KO mouse brain (1 month old mice), stained for the vesicular glutamate transporter, vGLUT1. Scale bar, 15 µm. (D) A significant reduction in vGLUT1 area was found in the stratum radiatum of CA1 field of IL-1R8 KO mice (total area of vGLUT1 positive puncta WT = 0,4644 ± 0,03420; number of examined fields: 35; IL-1R8 KO = 0,2910 ± 0,01984; number of examined fields: 47; Mann-Whitney test). https://doi.org/10.7554/eLife.21735.004 To investigate whether IL-1R8 KO neurons are able to undergo synaptic potentiation, hippocampal cultures were subjected to an established chemical LTP (c-LTP) protocol based on the culture exposure to 100 μM glycine in KRH devoid of Mg, followed by a washout and recovery in neuronal medium for at least 60 min (Menna et al., 2013). Under these conditions, a significant increase in the density of both PSD-95-positive puncta and mushroom spines occurred in WT but not in IL-1R8 KO neurons (Figure 3A–C). Similarly, no increase in mEPSC amplitude and frequency was recorded over time in IL-1R8 KO neurons (Figure 3D–F), univocally indicating that hippocampal IL-1R8 KO neurons are unable to undergo synaptic plasticity. These data show the occurrence of synaptic structural and functional defects in primary cultures from IL-1R8 KO mice. Figure 3 Download asset Open asset IL-1R8 KO neurons do not undergo LTP. (A) PSD-95 immunocytochemical staining of GFP-transfected, DIV 16 hippocampal cultures from WT or IL-1R8 KO mice, subjected or not to the LTP protocol. Scale bar 5 μm. (B and C) Quantitative analysis of PSD-95 and mushroom spine density of neurons treated as above. Number of analyzed neurons, B: 15 (WT, no LTP), 13 (WT, + LTP), 28 (IL-1R8 KO, no LTP), 18 (IL-1R8 KO, + LTP); C: 16 (WT, no LTP), 33 (WT, + LTP), 24 (IL-1R8 KO, no LTP), 34 (IL-1R8 KO, + LTP); one-way ANOVA analysis of variance followed by post hoc Tukey test). (D) Representative traces of mEPSCs recorded from neurons of WT or IL-1R8 KO mice before and 60 min after LTP induction. (E and F) Averaged mEPSC frequency and amplitude of WT and IL-1R8 KO neurons over different recording time points after LTP induction. Normalized mEPSC frequency: WT 0 min: 0,99 ± 0,06, n = 11; 20 min 1,84 ± 0,3, n = 12; 60 min 1,81 ± 0,14, n = 9; IL-1R8 KO 0 min 1,0 ± 0,07, n = 10; 20 min 1,11 ± 0,15, n = 11; 60 min 0,94 ± 0,07, n = 5. Normalized mEPSC amplitude: WT 0 min: 0,99 ± 0,03, n = 11; 20 min 1,29 ± 0,14, n = 12; 60 min 1,36 ± 0,14, n = 9; IL-1R8 KO 0 min 0,96 ± 0,05, n = 10; 20 min 0,91 ± 0,07, n = 11; 60 min 0,99 ± 0,1, n = 5. Mann Whitney test. https://doi.org/10.7554/eLife.21735.005 Defects in the structure and function of IL-1R8 KO neurons are reversed by blocking IL-1 receptor activity To determine whether the synaptic defects of IL-1R8 KO neurons are attributable to IL-1 receptor (IL-1R) or TLR pathway, both negatively regulated by IL-1R8, we analyzed spine density and electrophysiological properties in neurons from mice deficient for both IL-1R8 and IL-1R (IL-1R8 KO IL-1R KO). Double IL-1R8 KO IL-1R KO neurons displayed an increase in mushroom spine density (number of spines/micron, mean ± SEM, WT: 0,1581 ± 0,01508, n = 32; IL-1R8 KO IL-1R KO: 0,2585 ± 0,0154, n = 41, Student t test, p<0,0001), accompanied by enhanced mEPSCs frequency and amplitude (mEPSC frequency, mean ± SEM, WT: 1317 ± 0,1714, n = 12; IL-1R8 KO IL-1R KO: 2432 ± 0,3187, n = 17, Student t test, p<0,05; mEPSC amplitude, mean + SEM, WT: 22,82 ± 2468, n = 12; IL-1R8 KO IL-1R KO: 29,10 ± 2112, n = 17, Student t test, ns, p=0,0644). These data suggest a role for IL-1R signaling in controlling synaptic structure and function. To determine if reducing IL-1R activity could restore synaptic plasticity and spatial learning, hippocampal neurons from IL-1R8 KO mice exposed overnight to IL1Ra (Anakinra), a naturally-occurring IL-1 receptor antagonist (Dinarello, 2009), displayed increased density of both mushroom spines (Figure 4A and B) and PSD-95 puncta (Figure 4A and C). Furthermore, IL1Ra-treated neurons from IL-1R8 KO mice recovered their ability to undergo both structural and functional LTP, as indicated by the increased density of spines and PSD-95 (Figure 4A–C) and mEPSC frequency and amplitude (Figure 4D–F) following the application of the c-LTP protocol. Therefore, the IL-1R8 KO synaptic phenotype results from hyperactivation of the IL-1R pathway as a result of IL-1R8 silencing. Consistently, pharmacological activation of IL-1R pathway in WT neurons through overnight treatment with IL-1β (40 ng/ml for 14 hr) resulted in an increased number of immature thin spines and a decreased number of mature mushroom-type spines (Figure 4G–I) and PSD-95 puncta (Figure 4G and J), accompanied by inability to undergo LTP (Figure 4G–J). Figure 4 Download asset Open asset Inhibition of IL-1R signalling restores LTP in IL-1R8 KO neurons. (A) PSD-95 immunocytochemical staining of 16 DIV hippocampal cultures from GFP transfected WT or IL-1R8 KO mice, treated or not, at DIV 15 with IL1Ra (20 ng/ml) overnight (14 hr). Scale bar 5 μm. (B) Quantitative analysis of mushroom spine density in control or upon LTP protocol application. Similar results were obtained with IL1Ra at 100 ng/ml. Number of analyzed neurons: 14 (WT, no LTP), 16 (WT, + LTP), 10 (WT, IL1Ra, no LTP), 10 (WT, IL1Ra, + LTP), 26 (IL-1R8 KO, no LTP), 29 (IL-1R8 KO, + LTP), 23 (IL-1R8 KO, IL1Ra, no LTP), 32 (IL-1R8 KO, IL1Ra, + LTP); one-way ANOVA analysis of variance followed by post hoc Tukey test). (C) Quantitative analysis of PSD-95 immunoreactivity. Number of analyzed neurons: 9 (WT, no LTP), 26 (WT, + LTP), 27 (WT, IL1Ra, no LTP), 24 (WT, IL1Ra, + LTP), 24 (IL-1R8 KO, no LTP), 62 (IL-1R8 KO, + LTP), 13 (IL-1R8 KO, IL1Ra, no LTP), 12 (IL-1R8 KO, IL1Ra, + LTP). One-way ANOVA analysis of variance followed by post hoc Tukey test. (D) Representative traces of WT and IL-1R8 KO neurons treated with vehicle or IL1Ra (100 ng/ml). (E and F) Quantitation of mEPSC frequency and amplitude recorded 60 min after LTP protocol in WT or IL-1R8 KO neurons, treated as above. Analysis of normalized mEPSC frequency and amplitude reveals that only WT neurons and IL1-Ra-treated IL-1R8 KO neurons undergo LTP. Number of recorded neurons: 6 (WT, no LTP), 6 (WT, + LTP), 8 (WT, IL1Ra, no LTP), 8 (WT, IL1Ra, + LTP), 9 (IL-1R8 KO, no LTP), 6 (IL-1R8 KO, + LTP), 8 (IL-1R8 KO, IL1Ra, no LTP), 12 (IL-1R8 KO, IL1Ra, + LTP), Mann-Whitney test. (G) Immunocytochemical staining for PSD-95 in GFP-transfected WT neurons treated or not with IL-1β (40 ng/ml, overnight) and subjected or not to LTP stimulation. Scale bar 5 μm. (H and I) Quantitative analysis of mushroom and thin spine density. Number of analyzed neurons: 14 (WT, no LTP), 16 (WT, + LTP), 28 (WT, IL-1β, no LTP), 27 (WT, IL-1β, + LTP), one-way ANOVA analysis of variance followed by post hoc Tukey test. (J) Quantitative analysis of PSD-95 density. Number of analyzed neurons: 9 (WT, no LTP), 13 (WT, + LTP), 15 (WT, IL-1β, no LTP), 15 (WT, IL-1β, + LTP), one-way ANOVA analysis of variance followed by post hoc Tukey test. Data indicate that application of IL-1β prevents synaptic potentiation. https://doi.org/10.7554/eLife.21735.006 Of note, exposure of WT neurons to IL-1Ra prevented LTP, as assessed by confocal analysis (Figure 4A–C) or electrophysiological recording (Figure 4D–F), indicating that IL-1R acts positively in supporting LTP, even when IL-1R8 expression is not perturbed. In line with these observations, we found that neurons genetically devoid of IL-1 receptor (IL-1R KO) were unable to undergo plasticity phenomena (mushroom spine density: WT, no LTP: 0,13 ± 0009, n = 19; WT, + LTP: 0,36 ± 0,03, n = 18; Student t test, p<00001. IL-1R KO, no LTP: 0,41 ± 0,03; IL-1R KO, + LTP: 0,49 ± 0,03 n = 18; Student t test, p ns = 0,1024). Therefore, in line with literature data (Costello et al., 2011; Schneider et al., 1998; Coogan and O'Connor, 1999; Avital et al., 2003), either pharmacological or genetic silencing of IL-1R is per se sufficient to alter dendritic spine morphology and plasticity, indicating that physiological levels of IL-1R activation are required for correct long-term potentiation. IL-1β treatment and IL-1R8 deficiency trigger overlapping gene programs related to hippocampal development and synaptic transmission To further dissect changes in WT, IL-1R8 KO and WT mice treated with IL-1β, we conducted transcriptomic analysis on cortical tissues. RNA-seq data revealed that genetic ablation of IL-1R8 and pharmacological activation of IL-1R both lead to altered transcription of a shared subset of genes (Figure 5A and B). Treatment of WT mice with IL-1β led to transcriptional alterations in 1084 genes (60.6% downregulated and IL-1R8 KO mice alterations in expression of genes compared to WT downregulated and the of we a significant of genes expression was altered by either IL-1β treatment and IL-1R8 deficiency = = downregulated and analysis revealed that the genes are in specific to processes such as hippocampal development and synaptic transmission, with results in neuronal cultures, transcriptomic analysis of IL-1R8 KO mice treated with Anakinra revealed that pharmacological treatment reversed the transcriptional alterations in IL-1R8 KO mice including of the shared genes between IL-1R8 KO mice and in mice (Figure and Figure 1 and Notably, the affected synaptic genes downregulated in both IL-1R8 KO and mice, such as (Figure and These data indicate that IL-1β treatment and IL-1R8 deficiency trigger overlapping gene programs which are reversed, in the of IL-1R8 KO mice, by exposure to Figure 5 with 1 all Download asset Open asset analysis of from WT mice treated with IL-1β and IL-1R8 KO mice reveals genes with altered and of altered expression upon treatment of IL-1R8 KO mice with the IL-1β antagonist (A) significant in the number of genes between conditions = test). genes after and IL-1R8 KO show genes in IL-1R8 KO mice. (B) a of the genes based on changes of expression in the in the WT for the is of the WT treatment with IL-1β or IL-1R8 KO The key the of the from to and for (C) IL-1R8 KO, IL-1R8 KO + for genes in IL-1R8 KO mice and also upon to WT mice that were reversed upon treatment of IL-1R8 KO mice with Anakinra are in or downregulated that were not reversed by Anakinra are in (D) graph the number of genes that were either reversed or by treatment of IL-1R8 KO mice with of reversed genes over the of genes in gene is also gene are as IL-1R8 KO IL-1R8 KO IL-1R8 KO IL-1R8 KO genes are as not found to be the indicated significance in in the of IL-1R8 KO + Anakinra WT mice, and that were in but in the found in and IL-1R8 KO IL-1R effects on hippocampal synapses are by the pathway We the signaling pathway through which hyperactivation of the IL-1R pathway in IL-1R8 KO neurons influences spine morphology and function. The signaling pathways of IL-1R8 have been described in cells and found to the inhibition of NF-κB and JNK on or TLRs family activation and the activation of the pathway in et al. on the known of the pathway in the and of LTP in different brain et al., et al., we on the possibility that the of IL-1R8 could result in the hyperactivation of This possibility be in line with the cognitive and plasticity deficits in genetic mouse models characterized by increased mTOR signaling in and treatment of 15 DIV IL-1R8 KO neurons with 20 et al., which both and was sufficient to the defective spine with IL-1R8 KO treated neurons an increase in density of both mushroom spines (Figure and B) and PSD-95 puncta (Figure and C) relative to IL-1R8 KO neurons. Similarly, overnight exposure to μM a specific of or 20 a mushroom spine and PSD-95 in IL-1R8 KO neurons (Figure of the damage to the neurons, at least for the time of These data demonstrate that hyperactivation of IL-1R in IL-1R8 KO neurons spine morphogenesis and plasticity through the WT neurons exposed to the different displayed an increase in spine and postsynaptic density (Figure These data indicate that the activation of pathway, like for IL-1R activation (Figure is required for correct spine Figure 6 Download asset Open asset Inhibition of mTOR signalling restores LTP in IL-1R8 KO neurons. (A) PSD-95 immunocytochemical staining of GFP-transfected 16 DIV hippocampal cultures from WT or IL-1R8 KO mice. DIV 15 neurons were treated with 20 or 20 overnight (14 hr). Scale bar, 5 μm. (B) Quantitative analysis of mushroom spine density. Number of analyzed neurons: (WT), (WT, 10 (WT, 5 (WT, (IL-1R8 (IL-1R8 KO, 28 (IL-1R8 KO, (IL-1R8 KO, one-way ANOVA analysis of variance followed by post hoc Tukey test. (C) Quantitative analysis of PSD-95 puncta density. Number of analyzed neurons: (WT), (WT, 9 (WT, (WT, 9 (IL-1R8 15 (IL-1R8 KO, 15 (IL-1R8 KO, 18 (IL-1R8 KO, one-way ANOVA analysis of variance followed by post hoc Tukey test. Data indicate that of the mTOR pathway restore synaptic potentiation. * indicates significance compared to WT, # indicates significance compared to IL-1R8 KO. The transcriptional regulator MeCP2 the alterations in spine synaptic transmission and synaptic plasticity in IL-1R8 KO hippocampal neurons is known that mTOR is at the of plasticity, memory and disease processes and 2010). Reduced signaling and protein has been described in the brain of the MeCP2 KO Rett syndrome thus indicating that MeCP2 is an regulator of the pathway et al., 2011). Western blotting and analysis of MeCP2 in IL-1R8 KO hippocampal cultures revealed higher levels of the protein with respect to controls (Figure and in the absence of in the levels WT = ± = ± IL-1R8 KO = ± IL-1R8 = ± mean ± SEM, one-way multiple test). However, relative to et al., we found that IL-1R activation and mTOR pathway can be also an regulator of overnight treatment of IL-1R8 KO neurons with either IL1Ra (20 or 100 ng/ml) or (20 resulted in the of MeCP2 as assessed by Western (Figure and B) or (Figure and thus indicating that MeCP2 is increased in IL-1R8 KO neurons as a of the hyperactivation of IL-1R and to the mTOR MeCP2 levels were increased by (Figure and F) and by western blotting (Figure and H) also in WT neurons exposed to IL-1β (40 ng/ml for 14 hr). As a further to a reduction of MeCP2 was detected in WT neurons exposed to either IL-1Ra or and examined by (Figure and Consistently, MeCP2 analyzed by Western were significantly lower in both and of IL-1R KO mice months old) density of MeCP2 levels in WT: 1 ± = IL-1R KO: ± = Student t test, Normalized density of MeCP2 levels in WT: 1 ±
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