Fxr1 regulates sleep and synaptic homeostasis
2020; Springer Nature; Volume: 39; Issue: 21 Linguagem: Inglês
10.15252/embj.2019103864
ISSN1460-2075
AutoresJivan Khlghatyan, Alesya Evstratova, Lusiné Bozoyan, Simon Chamberland, Dipashree Chatterjee, Aleksandra Marakhovskaia, Tiago Soares Silva, Katalin Tóth, Valérie Mongrain, Jean‐Martin Beaulieu,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoArticle7 September 2020Open Access Transparent processSource Data Fxr1 regulates sleep and synaptic homeostasis Jivan Khlghatyan Jivan Khlghatyan Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Québec-City, QC, Canada Search for more papers by this author Alesya Evstratova Alesya Evstratova Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Lusine Bozoyan Lusine Bozoyan Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Simon Chamberland Simon Chamberland Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Québec-City, QC, Canada Search for more papers by this author Dipashree Chatterjee Dipashree Chatterjee Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Aleksandra Marakhovskaia Aleksandra Marakhovskaia Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Tiago Soares Silva Tiago Soares Silva Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Katalin Toth Katalin Toth Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Valerie Mongrain Valerie Mongrain Department of Neuroscience, Université de Montréal and Center for Advanced Research in Sleep Medicine, Hôpital du Sacré-Coeur de Montréal (CIUSSS-NIM), Montreal, QC, Canada Search for more papers by this author Jean-Martin Beaulieu Corresponding Author Jean-Martin Beaulieu [email protected] orcid.org/0000-0002-0446-7447 Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Jivan Khlghatyan Jivan Khlghatyan Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Québec-City, QC, Canada Search for more papers by this author Alesya Evstratova Alesya Evstratova Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Lusine Bozoyan Lusine Bozoyan Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Simon Chamberland Simon Chamberland Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Québec-City, QC, Canada Search for more papers by this author Dipashree Chatterjee Dipashree Chatterjee Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Aleksandra Marakhovskaia Aleksandra Marakhovskaia Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Tiago Soares Silva Tiago Soares Silva Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Katalin Toth Katalin Toth Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada Search for more papers by this author Valerie Mongrain Valerie Mongrain Department of Neuroscience, Université de Montréal and Center for Advanced Research in Sleep Medicine, Hôpital du Sacré-Coeur de Montréal (CIUSSS-NIM), Montreal, QC, Canada Search for more papers by this author Jean-Martin Beaulieu Corresponding Author Jean-Martin Beaulieu [email protected] orcid.org/0000-0002-0446-7447 Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Search for more papers by this author Author Information Jivan Khlghatyan1,2,†, Alesya Evstratova1, Lusine Bozoyan1, Simon Chamberland2,†, Dipashree Chatterjee1, Aleksandra Marakhovskaia1, Tiago Soares Silva1, Katalin Toth3, Valerie Mongrain4 and Jean-Martin Beaulieu *,1 1Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada 2Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Québec-City, QC, Canada 3Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada 4Department of Neuroscience, Université de Montréal and Center for Advanced Research in Sleep Medicine, Hôpital du Sacré-Coeur de Montréal (CIUSSS-NIM), Montreal, QC, Canada †Present address: Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA, USA †Present address: NYU Neuroscience Institute, Langone Medical Center, New York University, New York, NY, USA *Corresponding author. Tel: +1 416 978 1125; E-mail: [email protected] The EMBO Journal (2020)39:e103864https://doi.org/10.15252/embj.2019103864 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The fragile X autosomal homolog 1 (Fxr1) is regulated by lithium and has been GWAS-associated with schizophrenia and insomnia. Homeostatic regulation of synaptic strength is essential for the maintenance of brain functions and involves both cell-autonomous and system-level processes such as sleep. We examined the contribution of Fxr1 to cell-autonomous homeostatic synaptic scaling and neuronal responses to sleep loss, using a combination of gene overexpression and Crispr/Cas9-mediated somatic knockouts to modulate gene expression. Our findings indicate that Fxr1 is downregulated during both scaling and sleep deprivation via a glycogen synthase kinase 3 beta (GSK3β)-dependent mechanism. In both conditions, downregulation of Fxr1 is essential for the homeostatic modulation of surface AMPA receptors and synaptic strength. Preventing the downregulation of Fxr1 during sleep deprivation results in altered EEG signatures. Furthermore, sequencing of neuronal translatomes revealed the contribution of Fxr1 to changes induced by sleep deprivation. These findings uncover a role of Fxr1 as a shared signaling hub between cell-autonomous homeostatic plasticity and system-level responses to sleep loss, with potential implications for neuropsychiatric illnesses and treatments. Synopsis The insomnia-associated RNA-binding protein FXR1 is downregulated in response to sleep deprivation and homeostatic upscaling. This downregulation is necessary and sufficient to increase AMPA receptor surface expression and synaptic strength. Fxr1 downregulation is necessary and sufficient for the induction of upscaling. Fxr1 is involved in modulation of sleep duration and EEG in response to sleep deprivation. Fxr1 blocks increase of synaptic strength with sleep deprivation and upscaling. During sleep deprivation Fxr1 regulates neuronal translatome associated with local translation, synapse organization and homeostatic plasticity. Introduction Regulation of synaptic strength is essential for the maintenance of proper brain functions, and its disruption contributes in circuit-level imbalance of excitatory and inhibitory activity in neuropsychiatric illnesses such as Alzheimer disease, autism, and schizophrenia (Gao & Penzes, 2015; Nelson & Valakh, 2015; Vico Varela et al, 2019). Homeostatic mechanisms, engaged in response to external conditions, are believed to contribute to this regulation both at a cell autonomous and system level (Vyazovskiy et al, 2008; Turrigiano, 2012; Vitureira et al, 2012; Tononi & Cirelli, 2014; Hengen et al, 2016; Diering et al, 2017). However, the relationship between these different spatial scales of regulation is unclear. Synaptic scaling is a form of cell-autonomous homeostatic plasticity used by neurons to maintain net firing rates and is achieved by the modulation of postsynaptic AMPA receptors (Turrigiano, 2008). Prolonged inhibition of neuronal activity induces a multiplicative increase in miniature excitatory postsynaptic currents (mEPSCs) (upscaling) via increase in postsynaptic AMPA receptors. The opposite (downscaling) occurs as a result of prolonged neuronal activation (Turrigiano et al, 1998). However, it should be noted that in addition to global scaling, which affects the whole cell, forms of synapse selective homeostatic scaling have also been reported (Beique et al, 2011). At the system level, homeostatic regulatory mechanisms are engaged during the sleep/wake cycle. Homeostatic sleep pressure accumulates during wake and dissipates during sleep (Mang & Franken, 2015). Moreover, neuronal activity correlates with sleep pressure by increasing during wake and decreasing during sleep (Vyazovskiy et al, 2008; Liu et al, 2010; Tononi & Cirelli, 2014) and neuronal activity increases following prolonged wakefulness a change that can be reversed by a recovery sleep period (Vyazovskiy et al, 2008; Liu et al, 2010; Tononi & Cirelli, 2014). Some lines of evidence suggest that molecular and structural changes involved in scaling also occur during sleep (Diering et al, 2017; de Vivo et al, 2017). This raises the question of whether there are common homeostatic molecular regulators of neuronal activity involved in cell-autonomous synaptic scaling and system-level sleep regulation. The fragile X mental retardation autosomal homolog 1 (Fxr1) might play a role both in sleep and in homeostatic AMPA receptor regulation. Fxr1 is an RNA-binding protein that is expressed in different types of brain cells. In neurons, Fxr1 is localized in the cell bodies and dendrites in association with mRNAs and ribosomes (Cook et al, 2011). Variants of the FXR1 locus are GWAS-identified risk factors for insomnia (Jansen et al, 2019), and a schizophrenia-associated FXR1 variant is linked to sleep duration (Dashti et al, 2019). Interestingly, Fxr1 protein degradation is regulated by glycogen synthase kinase 3 beta (Gsk3β) (Del'Guidice et al, 2015; Qie et al, 2017) and its brain expression is increased in response to lithium, a pharmacological agent used for the treatment of psychiatric disorders, such as bipolar disorder, and of associated sleep disturbances (Billiard, 1987; Freland & Beaulieu, 2012). Fxr1, together with Fxr2, is an autosomal paralog of the fragile X mental retardation protein Fmrp, which is needed for the regulation of synaptic scaling by retinoic acid (Soden & Chen, 2010; Lee et al, 2018). Fxr2 and Fmrp have been shown to regulate synaptic expression of the AMPA receptor GluA1 subunit in different brain regions (Guo et al, 2015; Khlghatyan & Beaulieu, 2018; Khlghatyan et al, 2018). Fxr1 has been shown to regulate de novo synthesis of the AMPA receptor GluA2 subunit during long-lasting synaptic potentiation of hippocampal neurons (Cook et al, 2014). Nevertheless, the roles of Fxr1 in regulating brain functions, and in particular those related to homeostatic mechanisms, mostly remain to be defined. The association of Fxr1 with insomnia led us to test whether it can be a regulator of homeostatic plasticity and responses to sleep loss. Our results indicate that Fxr1 is downregulated at a post-transcriptional level by synaptic upscaling and sleep loss via a Gsk3β–dependent mechanism leading to its degradation. Furthermore, downregulation of Fxr1 is essential and sufficient for the regulation of synaptic AMPA receptors in both cell-autonomous homeostatic plasticity and the network response to sleep loss, thus reveling a shared molecular underpinning between different scales of homeostatic regulation of synaptic strength. Results Fxr1 protein expression is reduced by homeostatic synaptic scaling We evaluated the impact of homeostatic plasticity on Fxr1 in primary cortical cultures. Upscaling was induced by adding tetrodotoxin (TTX) to the cell culture media to block neuronal firing for a period of 48 h. To cause downscaling, the GABA-A receptor antagonist bicuculline (BIC) was used to increase neuronal excitability for the same time period (Turrigiano et al, 1998). Protein levels of all Fxr1 isoforms decreased during upscaling (TTX vs. vehicle) (Fig 1A), but did not change during downscaling (BIC vs. vehicle) (Fig 1B). In contrast, Fxr2 levels were marginally decreased (Fig 1C), while Fmrp levels were not affected by upscaling (Fig 1D). Upscaling did not affect mRNA levels of Fxr1 and Fxr2 (Fig 1E and F) and increased levels of Fmrp-encoding mRNA (Fmr1) (Fig 1G). This indicates that Fxr1 is the fragile X family protein that is the most affected by upscaling and that this regulation occurs at a post-transcriptional level. Figure 1. Fxr1 protein expression is decreased during homeostatic synaptic upscaling A, B. Western blot analysis of Fxr1 during (A), TTX (48 h treatment) induced upscaling (n = 6 in each condition) and (B), BIC (48 h treatment) induced downscaling (n = 8 in each condition) of primary postnatal cortical cultures. Student's t-test *P < 0.05, ***P < 0.001. C, D. Western blot analysis of (C), Fxr2 (Veh n = 8, TTX n = 8) and (D), Fmrp (Veh n = 10, TTX n = 12) during upscaling, Student's t-test *P < 0.05. E–G. RT–qPCR measurement of mRNA for (E), Fxr1, (F), Fxr2, (G), and Fmr1 during upscaling. n = 4 in each condition, Student's t-test *P < 0.05. H. Western blot analysis for Fxr1, pGsk3α/β, Gsk3α/β, and GAPDH in neuronal cultures treated with 1 μM TTX (TTX) or 1 mM LiCI (Li) or 1 mM NaCI (Ctrl) for 48 h. I. Expression of Fxr1 protein in TTX (n = 5) and Li (n = 5) conditions relative to Ctrl (n = 6) condition. One-way ANOVA with Dunnett's multiple comparison test *P < 0.05, **P < 0.01, ***P < 0.001. J. Expression of pGsk3β/Gsk3β or pGsk3α/Gsk3α in TTX (n = 5) and Li (n = 5) conditions relative to Ctrl (n = 6) condition. One-way ANOVA with Dunnett's multiple comparison test *P < 0.05, **P < 0.01, ***P < 0.001. Data information: Error bars are ± SEM. Source data are available online for this figure. Source Data for Figure 1 [embj2019103864-sup-0009-SDataFig1.pdf] Download figure Download PowerPoint We have previously shown that Gsk3β negatively regulates Fxr1 by targeting the protein for degradation (Del'Guidice et al, 2015). Accordingly, together with the decrease in Fxr1 protein, upscaling resulted in an increase in Gsk3α and β activity, resulting from a reduction in the inhibitory phosphorylation of Ser-21/9 (Fig 1H–J). Conversely, the inhibition of Gsk3α and Gsk3β by lithium increased Fxr1 protein levels (Fig 1H–J). Overall, this shows that Fxr1 protein is downregulated specifically during upscaling and that this process involves Gsk3 activity. Fxr1 stimulates GluA1 expression during upscaling To investigate whether the decrease in Fxr1 protein is contributing to upscaling, we verified whether an augmentation of Fxr1 could block upscaling. AMPA receptor expression is known to be modulated during homeostatic scaling (Diering et al, 2014). Thus, we addressed whether Fxr1 can negatively regulate AMPA receptor expression during scaling. Neuronal cultures were infected with high efficiency by AAV SYN GFP-Fxr1 (Fxr1) or AAV SYN GFP (Ctrl) (Figs 2A, and EV1A and B). While GFP was equally expressed in all parts of neurons, GFP-Fxr1 brightly labeled cell bodies and appeared as granules in dendrites (Fig 2G), as previously reported in cultured hippocampal neurons (Cook et al, 2014). Following infection, we performed Western blot measurement of GluA1 and GluA2 protein levels after TTX or BIC treatment to induce either upscaling or downscaling. Total GluA1 protein increased in Ctrl TTX condition as compared to Ctrl Vehicle (Fig 2B). No changes in total GluA1 protein were observed during downscaling (Fig 2C). Total GluA2 protein did not change during upscaling (Fig EV1D) or downscaling (Fig EV1F). Figure 2. Fxr1 suppresses the increase in surface GluA1 during synaptic upscaling Schematic of high-efficiency infection of neuronal cultures by AAV1 viruses followed by detection of AMPA receptor subunits. Western blot analysis of total GluA1 expression in Ctrl or Fxr1 overexpression (Fxr1) condition during upscaling (Ctrl/Veh n = 5, Ctrl/TTX n = 5, Fxr1/Veh n = 6, Fxr1/Veh n = 6). One-way ANOVA with Dunnett's multiple comparison test ***P < 0.001. Western blot analysis of total GluA1 expression in Ctrl or Fxr1 over condition during downscaling (Ctrl/Veh n = 4, Ctrl/TTX n = 4, Fxr1/Veh n = 4, Fxr1/Veh n = 4). RT–qPCR measurement of Gria1 mRNA in Ctrl or Fxr1 over condition during upscaling (Ctrl/Veh n = 3, Ctrl/TTX n = 3, Fxr1/Veh n = 3, Fxr1/Veh n = 3). One-way ANOVA with Bonferroni's multiple comparison test ***P < 0.001. Schematic representation of tagging of luciferase cDNA with 5′UTR, 1–900CDS, 900–1,800CDS, 1,800–2,700CDS, and 3′UTR of GluA1 gene. Measurement of relative luciferase signal after co-transfection of different tagged luciferase constructs along with GFP (Ctrl) or GFP-Fxr1 (Fxr1) plasmids. n = 5 in each condition, Student's t-test *P < 0.05, ***P < 0.001. Immunostaining for surface GluA1 in GFP (Ctrl) or GFP-Fxr1 (Fxr1 over)-infected cultures after treatment with Veh, TTX, or BIC for 48 h. Percentage of surface GluA1 relative to the mean of Ctrl/Veh condition (Ctrl condition: Veh n = 63, TTX n = 34, BIC n = 29, Fxr1 condition: Veh n = 56, TTX n = 30, BIC n = 29). One-way ANOVA with Bonferroni's multiple comparison test *P < 0.05, **P < 0.01, ***P < 0.001. Data information: Error bars are ± SEM. Source data are available online for this figure. Source Data for Figure 2 [embj2019103864-sup-0010-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Expression of GluA2 during synaptic scaling A, B. Quantification of the % of infection by (A) AAV1 Syn GFP (n = 3) or (B) AAV1 Syn GFP-Fxr1 (n = 3). C. Surface expression of GluA2 during upscaling (Veh n = 28, TTX n = 29). D. Expression of total GluA2 during upscaling (Veh n = 7, TTX n = 6). E. Surface expression of GluA2 during downscaling (Veh n = 18, BIC n = 18). F. Expression of total GluA2 during downscaling (Veh n = 4, BIC n = 4). Data information: Error bars are SEM. Download figure Download PowerPoint Surprisingly, the increase in GluA1 protein expression was exacerbated following TTX treatment (Fxr1 TTX vs. Ctrl TTX) in neurons overexpressing Fxr1 (Fig 2B). Consistently with the results at the protein level, GluA1 mRNA increased in Ctrl TTX condition compared with Ctrl Veh and this increase was also further exacerbated in Fxr1 TTX condition (Fxr1 TTX vs. Ctrl TTX) (Fig 2D). This indicates that the increase in GluA1 protein during upscaling can result from a positive regulation by Fxr1 at the mRNA level. To investigate whether Fxr1 can directly bind to GluA1 mRNA, we tagged a luciferase cDNA with 5′UTR, different coding (CDS), or 3′UTR sequences from the GluA1 (Gria1) mRNA (Fig 2E) and performed dual-luciferase assays. We detected an increase in the luciferase signal for CDS 1–900, CDS 900–1,800, and 3′UTR constructs under Fxr1 overexpression in comparison with control (Fig 2F). This suggests that Fxr1 positively regulates GluA1 level during upscaling via direct binding to its mRNA and facilitating its translation. Fxr1 suppresses the increase in surface GluA1 during upscaling Along with expression, surface levels of AMPA receptor are also changed during homeostatic scaling (Diering et al, 2014). Thus, we investigated the effect of Fxr1 on surface levels of AMPA receptors during upscaling and downscaling. Surface expression of the GluA2 subunit did not change during upscaling or downscaling (Fig EV1C and E). Surface expression of GluA1 increased during upscaling in Ctrl (Ctrl TTX vs. Ctrl vehicle), and this increase was abolished by Fxr1 overexpression (Fxr1 TTX vs. Fxr1 overexpression vehicle) (Fig 2G and H). The decrease in surface GluA1 during downscaling was similar in both Ctrl and Fxr1 overexpression conditions (Fig 2G and H). No differences in surface GluA1 expression were observed between Ctrl vehicle and Fxr1 overexpression vehicle conditions (Fig 2G and H). This indicates that an increase in Fxr1 can specifically block the increase in surface GluA1 during upscaling. Overall, this shows that the augmentation of Fxr1 blocks the increase in surface GluA1 during upscaling without directly inhibiting GluA1 expression. Furthermore, this suggests that downregulation of Fxr1 can be required for the increase in surface GluA1 during upscaling. Fxr1 downregulation is necessary and sufficient for the induction of multiplicative upscaling Upscaling is a cell-autonomous process indexed by a multiplicative increase in miniature excitatory postsynaptic currents (mEPSCs) (Turrigiano et al, 1998; Turrigiano, 2008). Fxr1 protein expression is downregulated, likely via a lithium-sensitive Gsk3-dependent mechanism, and controls surface GluA1 during upscaling (Figs 1 and 2). Thus, we tested whether the Gsk3β-Fxr1 signaling module regulates synaptic currents during upscaling. To address the relative contribution of Fxr1 and Gsk3β to upscaling, we used a combination of gene overexpression and Crisps/Cas9-mediated gene inactivation. We designed series of Fxr1 gene targeting guide RNAs (gRNAs) and verified their efficiency at targeting the mouse genome using Neuro2A cells (Fig EV2A–C). The most efficient Fxr1 targeting gRNA and a previously characterized Gsk3β targeting gRNA (Khlghatyan et al, 2018) were used for Crispr/Cas9-mediated gene inactivation in neuronal cultures. All control plasmids contained scrambled gRNAs. An expression vector encoding GFP-Fxr1 and a control vector expressing GFP alone were used for overexpression experiments. Click here to expand this figure. Figure EV2. CRISPR/Cas9-mediated knockout of Gsk3b and Fxr1 in primary neurons A. Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B. Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C. Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t-test, ***P < 0.001). Bars and error bars are mean ± SEM. D. Schematic representation of low-efficiency transfection of primary neuronal cultures with various plasmids. E–G. Evaluation of CRISPR/Cas9 KO of (E) Gsk3b, (F) Fxr1, and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H. Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1. Download figure Download PowerPoint We generated Fxr1 knockout (KO) and Fxr1 Ctrl (both tagged with mCherry), and Gsk3β KO and Gsk3β Ctrl (both tagged with GFP) constructs and performed low-efficiency transfection of neuronal cultures (Fig EV2D). Immunofluorescent staining revealed the presence of single (Fxr1 or Gsk3β) or double (Gsk3β and Fxr1) KO of targeted genes (Fig EV2E–H). Whole-cell patch-clamp recordings showed a multiplicative increase in mEPSC amplitude in control neurons following upscaling (Figs 3A–D and O, and EV3). Fxr1 overexpression or Gsk3β KO prevented the increase in mEPSC amplitude induced by TTX (Figs 3E, F and O, and EV3). Figure 3. The decrease in Fxr1 expression is necessary and sufficient for induction of multiplicative upscaling A. Cumulative probability plots of mEPSC amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical control neurons after 48 h of 1 μM TTX or Veh exposure (Ctrl/Veh n = 16 and Ctrl/TTX n = 17). B. A linear fit of Ctrl/TTX and Ctrl/Veh amplitudes. C. The degrees of overlap between Ctrl/TTX and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant P-value was obtained with 1.47 scaling factor. D. Cumulative probability plots of the mEPSC amplitude of Ctrl/Veh, Ctrl/TTX, and Ctrl/TTX divided by scaling factor 1.47, which yielded the maximum overlap with Ctrl/Veh data. E–G. Cumulative probability plots of mEPSC amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 h of 1 μM TTX or Veh exposure, (E) Fxr1 overexpressing neurons (Fxr1/Veh n = 10 and Fxr1/TTX n = 8), (F) Gsk3 KO neurons (Gsk3KO/Veh n = 10 and Gsk3KO/TTX n = 11), (G) Fxr1 KO neurons (Fxr1KO/Veh n = 16 and Fxr1KO/TTX n = 8). H. A linear fit of Fxr1 KO/Veh and Ctrl/Veh amplitudes. I. The degrees of overlap between Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant P-value was obtained with 1.27 scaling factor. J. Cumulative probability plots of the mEPSC amplitude of Ctrl/Veh, Fxr1 KO/Veh, and Fxr1 KO/Veh divided by scaling factor 1.27, which yielded the maximum overlap with Ctrl/Veh data. K. Cumulative probability plots of mEPSC amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical Gsk3 and Fxr1 KO neurons after 48 h of 1 μM TTX or Veh exposure (Gsk3/Fxr1KO/Veh n = 8 and Gsk3/Fxr1KO/TTX n = 11). M. A linear fit of Gsk3/Fxr1 KO/Veh and Ctrl/Veh amplitudes. N. The degrees of overlap between Gsk3/Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant P-value was obtained with 1.68 scaling factor. O. Cumulative probability plots of the mEPSC amplitude of Ctrl/Veh, Gsk3/Fxr1 KO/Veh, and Gsk3/Fxr1 KO/Veh divided by scaling factor 1.68, which yielded the maximum overlap with Ctrl/Veh data. P. mEPSC mean amplitude of cultured cortical neurons after 48 h of 1 μM TTX or Veh exposure. One-way ANOVA with Bonferroni's multiple comparison test *P < 0.05, **P < 0.01, ***P < 0.001. Q. mEPSC frequency of cultured cortical neurons after 48 h of 1 μM TTX or Veh exposure. Data information: Error bars are ± SEM. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Expanded traces of recordings from primary neuronal cultures- Download figure Download PowerPoint The Fxr1 KO condition resulted in an elevation of mEPSC amplitude in a multiplicative manner (Figs 3G–J and O, and EV3) to a level that was not further increased following activity blockade by TTX (Figs 3G and O, and EV3). Similarly, the double Gsk3β/Fxr1 KO induced a multiplicative elevation of mEPSC amplitude (Figs 3K–N and O, and EV3) that was not further increased by TTX treatment (Figs 3K and O, and EV3). No changes in mEPSC frequency were observed in all conditions (Fig 3P). Overall, these observations confirm that Fxr1 upregulation prevents upscaling and reveal that a decrease in Fxr1 protein is necessary and sufficient for the induction of this form of homeostatic plasticity. Furthermore, genetic inactivation of Gsk3β, which increases Fxr1 levels (Del'Guidice et al, 2015), also inhibits upscaling in an Fxr1-dependent manner. These findings are compatible with a mechanism by which Gsk3β is activated and negatively regulates Fxr1 protein level to contribute to upscaling. Fxr1 modulates sleep duration and the response to sleep deprivation We next investigated the involvement of Fxr1 in the homeostatic regulation of neuronal activity at the system level by verifying whether Fxr1 is involved in the regulation of sleep, which is homeostatically regulated. Forty-eight hours of EEG and electromyographic (EMG) recordings were performed in freely moving mice injected with AAV SYN GFP-Fxr1 (Del'Guidice et al, 2015) or the control AAV SYN GFP into the frontal cortex. Homeostatic sleep pressure accumulates during wake and is thus higher following enforced wakefulness or sleep deprivation (SD). We performed baseline (undisturbed) recordings for 24 h, followed by a 24-h recording including a 6-h SD and 18 h of recovery (termed recovery recording). Measurement of the time spent in wakefulness (WAKE), slow-wave sleep (SWS), and paradoxical sleep (PS) indicated that Fxr1 overexpressing mice have a significantly different distribution of vigilant states over time in baseline, an effect that became more pronounced during the recovery recording (Figs 4A and EV4B). This included an increased time spent in SWS (decreased time spent in WAKE) during the active/dark period that was likely related to longer individual bouts of SWS rather than to more individual bouts (Fig EV4C and D). We computed the power spectra for WAKE, SWS, and PS during baseline and recovery (Fig 4B). When normalized to the Ctrl, we noticed significant differences in the alpha frequency band (8–11.75 Hz) during wakefulness only in the recovery recording (Fig 4C and D), which includes SD. Pronounced effects during recovery can be indicative of involvement of Fxr1 in the response to elevated homeostatic sleep pressure. Figure 4. Fxr1 modulates sleep duration and the response to sleep deprivation A. Hourly distribution of wakefulness (WAKE), slow-wave sleep (SWS), and paradoxical sleep (PS) during a 24-h baseline (BL) recording and a second 24-h starting with 6-h sleep deprivation (SD) (recovery: REC) in Ctrl and Fxr1 overexpressing (Fxr1) mice. Significant group-by-hour interactions were found for wakefulness during BL (F23,230 = 1.67, P = 0.046) and REC (F23,230 = 2.83, P = 0.0029), for SWS during BL (F23,230 = 1.66, P = 0.045) and REC (F23,230 = 2.89, P = 0.0028), and for PS during REC (F16,160 = 2.54, P = 0.0
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