Analysis of Astroglial Secretomic Profile in the Mecp2-Deficient Male Mouse Model of Rett Syndrome

Mutations in the X-linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder. MECP2 is a transcriptional modulator that finely regulates the expression of many genes, specifically in the central nervous system. Several studies have functionally linked the loss of MECP2 in astrocytes to the appearance and progression of the RTT phenotype in a non-cell autonomous manner and mechanisms are still unknown. Here, we used primary astroglial cells from Mecp2-deficient (KO) pups to identify deregulated secreted proteins. Using a differential quantitative proteomic analysis, twenty-nine proteins have been identified and four were confirmed by Western blotting with new samples as significantly deregulated. To further verify the functional relevance of these proteins in RTT, we tested their effects on the dendritic morphology of primary cortical neurons from Mecp2 KO mice that are known to display shorter dendritic processes. Using Sholl analysis, we found that incubation with Lcn2 or Lgals3 for 48 h was able to significantly increase the dendritic arborization of Mecp2 KO neurons. To our knowledge, this study, through secretomic analysis, is the first to identify astroglial secreted proteins involved in the neuronal RTT phenotype in vitro, which could open new therapeutic avenues for the treatment of Rett syndrome.


Introduction
Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the MECP2 (Methyl CpG binding protein 2) gene, located on the X chromosome [1]. After a period of apparently normal development, females with MECP2 mutations undergo a regression of early neurodevelopment, resulting in the deterioration of motor skills, purposeful use of the hands, speech, and the appearance of severe breathing disturbances as the disease progresses [2]. Mecp2 was originally described as a DNA methylation-dependent transcriptional repressor but has also been shown to play various roles in chromatin compaction and global gene expression [3]. Since Mecp2 is essentially expressed in neurons, where its expression correlates with neuronal maturation [4,5], and because it was thought to be absent in glial cells, RTT was considered to be the result of cell-autonomous neuronal defects.
Neurons have long been considered as the main players in the central nervous system (CNS), while glia was relegated to physical and metabolic support. However, over the past decade new research has highlighted the major roles played by glia in neural development, maintenance and functions [6][7][8]. Absence of Mecp2 in glia [9] has since been disproved [10][11][12][13][14] and astrocytes were finally implicated in RTT pathophysiology and described in many non-cell-autonomous mechanisms [10,11,[15][16][17][18]. These studies suggested that abnormal astrocytes impair the normal neuronal function and participate in the progression of the disease. Conversely, the restoration of Mecp2 expression specifically in astrocytes of Mecp2 KO mice delays the progression of RTT symptoms by improving locomotion, anxiety, breathing abnormalities and increasing their lifespan. Furthermore, reexpression of Mecp2 specifically in astrocytes leads to a non-cell-autonomous positive effect on Mecp2 KO neurons in vivo, restoring normal dendritic morphology, which definitively implicates astrocytes in the pathophysiology of RTT [12].
In the last decade, several studies pointed out the key roles of secreted proteins (glial or non-glial) in neuronal functions in physiological and pathological conditions by acting on cell signaling, communication, transduction, angiogenesis and migration [19,20]. In the meantime, several efforts were done in mass spectrometry-based proteomic research (related to the field of secretomics) to identify and quantify these secreted proteins by minimizing the difficulties inherent to the high dilution factor of proteins and deciphering a deeper secretome, i.e., the set of proteins released from cells into the extracellular space [21]. Kim et al. also pointed out the interest of using proteomic studies to establish astroglial secretome profiles and determine the role of deregulated secreted proteins in neuronal disorders [20]. Interestingly, a study in an in vitro coculture system indicated that Mecp2 KO astrocytes (or their conditioned medium alone) or human RTT astrocytes derived from IPSc have a harmful, non-cell-autonomous effect on wild-type (WT) neurons, resulting in a pathological neuronal phenotype and a short dendritic morphology [10,22]. We therefore hypothesized that the secretion or non-secretion of specific factors by Mecp2 KO astroglia may lead to functional and morphological neuronal anomalies. In order to test this hypothesis, we analyzed the astroglial secretome through a quantitative mass spectrometry-based proteomic approach [19,23]. We evidenced several deregulated secreted proteins in the secretome of Mecp2-deficient astroglia compared to wild-type cells, providing new insights into RTT physiopathology.

Proteomic Analysis of Astroglial Secretome
To determine which secreted proteins were deregulated in RTT, our quantitative proteomic analysis was performed on the astroglial secretome obtained from postnatal day (PND) 2 RTT male mice (Mecp2 KO and WT, n = 6 per condition, split in two experimental replicates) coming from 2 independent experiments ( Figure 1A). This led to the quantification of 557 proteins (Supplementary Table S1) by considering only proteins with at least 1 unique peptide with a confidence score higher than 95% and an FDR of 1%. 105 proteins (listed in Supplementary Table S2a) were found as significantly deregulated in both replicates (with at least 2 unique peptides above 95% confidence level, a p-value ratio lower than 0.05) in the absence of Mecp2. The functional analysis performed using David Bioinformatics resources (https://david.ncifcrf.gov/summary.jsp, accessed on 2019) confirmed that the quantified proteins were mostly localized in extracellular compartments (extracellular exosome, extracellular space and extracellular region associated to a p-value of 2.7 × 10-47, 2.9 × 10-15 and 3.1 × 10-13, respectively) (Supplementary Table S2b). In addition to these Gene Ontology (GO) annotations, the Uniprot annotation provides subcellular localization of proteins with both expertly curated data from literature and rule-based automatic annotation. Therefore, we further filtered the list of our deregulated proteins by using the subcellular localisation of Uniprot annotation. Altogether, by con-sidering GO terms and Uniprot annotations, twenty-nine secreted deregulated proteins were retained (Table 1 and Table S3a) to build a network of secreted deregulated proteins upon Mecp2 deficiency in the RTT astroglial secretome ( Figure 1C). Among these 29 secreted proteins, 11 were down-regulated while 18 proteins were up-regulated. The heat map of deregulated secreted proteins ( Figure 1B) confirmed the coherence of the iTRAQ ratio (Mecp2 KO vs WT) obtained from the two independent replicates with the highest fold change for Lcn2 (iTRAQ ratio KO/WT 0.31), Lgals1 (iTRAQ ratio KO/WT FG 0.35), Mfap4 (iTRAQ ratio KO/WT FG 2.83), and Enpp5 (iTRAQ ratio KO/WT FG 2.54) ( Table 1). Although our analysis highlighted a small number of proteins for global profiling (29 deregulated secreted proteins), this study provides the first proteomic insight into astroglial-secreted factors in RTT. The secretomic data were deposited to the Pro-teomeXchange Consortium via the MassIVE Dataset Submission [24]  automatic annotation. Therefore, we further filtered the list of our deregulated proteins by using the subcellular localisation of Uniprot annotation. Altogether, by considering GO terms and Uniprot annotations, twenty-nine secreted deregulated proteins were retained (Table 1 and Table S3a) to build a network of secreted deregulated proteins upon Mecp2 deficiency in the RTT astroglial secretome ( Figure 1C). Among these 29 secreted proteins, 11 were down-regulated while 18 proteins were up-regulated. The heat map of deregulated secreted proteins ( Figure 1B) Table S4).  Table S4).

Functional and Network Analyses of Secreted Proteins Deregulated upon Mecp2 Deficiency
The STRING network of the secreted proteins significantly deregulated in the RTT astroglial secretome is shown in Figure 1C and Supplementary Table S3b. The highest betweenness centrality scores are obtained for Lcn2 and Lgals3, found downregulated in the RTT astroglial secretome. Knowing that betweenness centrality reflects the power proteins exert on the interactions of other proteins (nodes) in the network, this suggests Lcn2 and Lgals3 as the most pivotal proteins of the network. Based on the functional analysis ( Figure 1D and Supplementary Table S4), the up-regulated proteins in the RTT astroglial secretome are mostly involved in inflammatory response, positive regulation of neuron death, complement activation, metabolic process, cellular response to UV-B, and negative regulation of beta-amyloid formation. The down-regulated proteins are mostly involved in the apoptotic process, maintenance of protein location cellular response to interleukin-1, and response to drug.

Validation of Proteomic Data by Western Blot
Among the 29 deregulated proteins validated by mass-spectrometry analysis, we analyzed by Western blotting the four most relevant proteins on a translational level including both proteins (Lcn2 and Lgals3) with the highest betweenness centrality scores in the network of deregulated secreted proteins. We quantified protein level for Lcn2, Lgals3, B2M and Mfap4 (respectively −45%, −37%, −25%, and +55% of expression level by mass spectrometry in Mecp2 KO secretome compared to WT) in three new secretome samples of astroglial culture from WT and Mecp2 KO mice. Significant downregulation of Lcn2 (−42%), Lgals3 (−44%) and a trend of downregulation of B2M (−43.4%) was confirmed in the Mecp2 KO astroglial secretome samples by Western blot (Figure 2 and Supplementary Figure S1). Although secretomic analysis showed an important upregulation of Mfap4, we only found a trend to an upregulation of Mfap4 ( Figure 2). All Western blots were normalized with total protein staining ( Figure 2).  Figure S1). Although secretomic analysis showed an important upregulation of Mfap4, we only found a trend to an upregulation of Mfap4 ( Figure 2). All Western blots were normalized with total protein staining ( Figure 2). We also analyzed protein expression of these factors in astroglia culture. As Lcn2 is a secretory protein, its cellular levels were too low to be analyzed by Western blot (data not shown). Interestingly, no significant differences in Lgals3, B2M, and Mfap4 protein levels were found between WT and Mecp2 KO astroglial cells (Supplementary Figure S2), which suggests that the observed astroglial secretome deregulations could be the consequence of secretion rather than protein synthesis deficit.

Lcn2 and Lgals3 Supplementation Improves Dendritic Arborization of Mecp2 KO Neurons
An important feature of RTT is the reduced dendritic arborization that can be found in both RTT mouse models and RTT patients [25,26] and was also shown in this study ( Figure 3A). Astrocytes are known to participate in neuronal dendritic growth [27]. To correlate the observed deregulations in the astroglial secretome with RTT features, we tested the effect of two deregulated factors on neuronal dendritic morphology. We fo- We also analyzed protein expression of these factors in astroglia culture. As Lcn2 is a secretory protein, its cellular levels were too low to be analyzed by Western blot (data not shown). Interestingly, no significant differences in Lgals3, B2M, and Mfap4 protein levels were found between WT and Mecp2 KO astroglial cells (Supplementary Figure S2), which suggests that the observed astroglial secretome deregulations could be the consequence of secretion rather than protein synthesis deficit.

Lcn2 and Lgals3 Supplementation Improves Dendritic Arborization of Mecp2 KO Neurons
An important feature of RTT is the reduced dendritic arborization that can be found in both RTT mouse models and RTT patients [25,26] and was also shown in this study ( Figure 3A). Astrocytes are known to participate in neuronal dendritic growth [27]. To correlate the observed deregulations in the astroglial secretome with RTT features, we tested the effect of two deregulated factors on neuronal dendritic morphology. We focused on Lcn2 and Lgals3 for several reasons: (1) Lcn2 mRNA has been already found deregulated in Mecp2 KO astrocytes coming from a mouse model of RTT [17], (2) Lcn2 has been shown to modulate spine morphology and to regulate neuronal excitability [28,29] and (3) Lgals3 is strongly expressed in the brain and modulates gliogenesis and memory [30,31]. Therefore, we supplemented Mecp2 KO primary cortical neurons for 48 h with these factors and analyzed their dendritic morphology by using Sholl analysis. We also supplemented cortical neurons with NGF as a positive control of neurite outgrowth [32], because it leads to an increased branching and an increased total dendritic length in both WT and Mecp2 deregulated in Mecp2 KO astrocytes coming from a mouse model of RTT [17], (2) Lcn2 has been shown to modulate spine morphology and to regulate neuronal excitability [28,29] and (3) Lgals3 is strongly expressed in the brain and modulates gliogenesis and memory [30,31]. Therefore, we supplemented Mecp2 KO primary cortical neurons for 48 h with these factors and analyzed their dendritic morphology by using Sholl analysis. We also supplemented cortical neurons with NGF as a positive control of neurite outgrowth [32], because it leads to an increased branching and an increased total dendritic length in both WT and Mecp2 KO neurons (Figure 3 and Supplementary Figure S3). Sholl analysis showed that Lcn2 treatment significantly increased dendrite branching at 15-50 µm from the soma in Mecp2 KO neurons (Figure 3). Similarly, Lgals3 treatment promoted dendrite branching at 20-60 µm from the soma in Mecp2 KO neurons ( Figure 3B). As shown in Supplementary Figure S3, Lcn2 and Lgals3 treatment significantly increased dendrite branching in WT neurons as well.

Discussion
Astrocytes secrete various substances, including neuromodulators, classical neurotransmitters, growth factors, metabolic mediators, inflammatory factors, molecular regulators of synaptogenesis and synaptic connectivity factors [33]. It is well-documented that astrocytes and astrocyte conditioned medium are involved in RTT neuronal phenotype in vitro [10,11]. In the present study, we report significant changes in the RTT astroglial se-

Discussion
Astrocytes secrete various substances, including neuromodulators, classical neurotransmitters, growth factors, metabolic mediators, inflammatory factors, molecular regulators of synaptogenesis and synaptic connectivity factors [33]. It is well-documented that astrocytes and astrocyte conditioned medium are involved in RTT neuronal phenotype in vitro [10,11]. In the present study, we report significant changes in the RTT astroglial secretome and identify actionable targets. We first characterized the astroglial secretome in the absence of Mecp2 and we further identified and selected deregulated factors, then verified their relevance as potential therapeutic factors in an in vitro model.

Limitations
Despite the difficulties inherent to the high dilution factor of secreted proteins, we obtained reliable quantitative data that enabled us to identify and quantify 557 secreted proteins that represent the main biological pathways affected by RTT.
Astroglial cells were isolated from cortical regions and the deregulated secreted proteins, that have been identified and verified, may be specific to this region. Indeed, recent work from several labs reported the existence of morphological and transcriptional differences in astrocyte populations depending on their brain region [34].
Another limitation is the purity of our astrocyte population. Our purification protocol for cortical astrocytes does not yield pure astrocytes but rather a population highly concentrated in astrocytes with a low percentage of microglial cells, which means that microglial dysfunction may have contributed to our results. Precise astrocyte enrichment has not been assayed. However, proteins found deregulated are fully from astroglial origin since cultures were found free from neurons (Supplementary Figure S4).
The impact of microglia cannot be neglected because, for instance, in the CNS, the Lcn2 protein is not only secreted by astrocytes and endothelial cells [35][36][37], but also by microglia [38,39]. Likewise, Lgals3 has also been identified as secreted by microglia [40,41].

GO Analysis Reveals Enriched Biological ProcessesiIn the RTT Astroglial Secretome
Despite the above-mentioned limitations, we identified 557 proteins, including conventionally secreted and non-conventionally secreted proteins as well as cytosolic proteins. Hence, 105 proteins were significantly deregulated in the Mecp2 KO secretome compared to the WT one, in two independent biological replicates. Among these proteins, we focused our analysis on 29 proteins reported as secreted in the UniprotKB/Swissprot database. Although our analysis highlighted a small number of proteins for global profiling (29 deregulated secreted proteins), this study provides the first proteomic insight into astroglialsecreted factors in RTT. Further analysis based on GO terms revealed a neuro-inflammation signature in the RTT astroglial secretome with the up-regulation of proteins involved in the inflammatory response (C3, C4b, Hyal1) and down-regulation of proteins involved in eosinophil chemotaxis and cellular response to interleukin (Ccl2, Lgals3, Chil1, Lcn2). Our results also reveal the activation of the complement cascade through C3 and C4, important for the regulation of immune processes. These data are consistent with the fact that RTT is associated with neuroinflammation mechanisms and microglial activation [42][43][44]. Interestingly, a down-regulation of pro-apoptotic proteins in the secretome of glial cells (Chil1, Cst3, Lgals1, Lcn2) was observed and corroborates the view that Mecp2 deficiency reduces apoptosis-triggered senescence in neurons [45].

Network Analysis Identifies Two Proteins of Interest: Lcn2 and Lgals3
The highest betweenness centrality scores of the STRING secreted network ( Figure 1C and Supplementary Table S3b) upon Mecp2 deficiency were obtained for lipocalin-2 (Lcn2) and galectin-3 (Lgals3). These proteins observed as downregulated in the RTT astroglial secretome are involved in biological processes (eosinophil chemotaxis, apoptosis, maintenance of protein location, tumor necrosis factor, response to interleukin and several responses to drug) that are themselves known to play a key role in the modulation of neuronal functions in physiological and pathological conditions [46][47][48].

A Well-Known Candidate, Lcn2
Lcn2 has been associated with diverse cellular processes in the CNS such as migration, cell death/survival, morphology and phenotypic polarization [39,[50][51][52][53][54]. Our results showed a downregulation of Lcn2 in the RTT astroglial secretome. Interestingly, Lcn2 has already been identified as deregulated in astrocytes from two different RTT mouse models [17,55], in total brain samples from Mecp2-null mice [56] and in postmortem brains from RTT patients [57]. Lcn2 has been shown to modulate spine morphology and to regulate neuronal excitability, two parameters that are largely deregulated in RTT [28,58]. In addition, Lcn2-null mice display synaptic impairment in hippocampal long-term potentiation [28]. Therefore, a lack of Lcn2 secretion by Mecp2 deficent astrocytes may be involved in spine morphology alteration and neuronal excitability defects in neurons. Indeed, we demonstrated in the present study that in vitro recombinant Lcn2 treatment increased neuronal arborization of Mecp2-deficent neurons. Further experiments are warranted to investigate the role of Lcn2 in the RTT neuropathology in vitro and, ultimately, in vivo.

A Multifunction Protein, Lgals3
Lgals3 acts as an endogenous ligand of TLR4 [40]. Lgals3 is known to be involved in the inflammatory response, and its expression is increased in microglia upon various neuroinflammatory stimuli [41,59]. Interestingly, deregulated inflammatory and immune responses have been found in RTT [60]. Moreover, it has recently been reported that Lgals3 regulates memory formation [61], and during development, Lgals3 has a critical role in driving oligodendrocytes differentiation, myelination, and gliogenesis [30,62].

Lcn2, Lgals3 and BDNF
Lcn2 and Lgals3 seem both involved in microglia and astrocyte activation [39,40]. Furthermore, conditioned media from Lcn2-activated astrocytes, and microglia upregulates pre-and postsynaptic markers such as synaptophysin, synaptotagmin and PSD95 in neural cultures. In addition, Lgals1 and Lgals3 have also been implicated in BDNF regulation [63]. Interestingly, our proteomic results suggest a downregulation of both Lgals1 et Lgals3 in the RTT astroglial secretome.
Given the essential role of BDNF in RTT physiopathology [64,65], Lgals1, Lgals3 and Lcn2 astroglial deficiency could be one of the numerous mechanisms leading to BDNF downregulation in this pathology. In agreement with this hypothesis, an in vitro study showed that Lcn2 treatment of rat astrocytes induced BDNF synthesis and release [66], while Lgals1 KO induces a decrease of BDNF levels in the prefrontal cortex [63] and Lgals3 deficient mice showed lower bdnf mRNA and protein levels in the hippocampus [67].
Based on these findings, we cannot exclude that the arborization improvement observed in Mecp2 KO neurons treated with either recombinant Lgals3 or Lcn2 are BDNFdependent.

Animals
The Mecp2-deficient mice (B6,129P2(C)-Mecp2 tm1-1Bird) were obtained from the Jackson Laboratory (Charles River laboratories, Chatillon-sur-Chalaronne, France) and maintained on a C57BL/6 background. Genotyping was performed by PCR-amplification following a previously described protocol (Miralvès et al., 2007). The animals were housed under a 12:12 h light⁄dark cycle (lights on at 07:00) and given free access to food and water. The experimental procedures were carried out in keeping with the European guidelines for the care and use of laboratory animals (EU directive 2010/63/EU), the guide for the care and use of the laboratory animals of the French national institute for science and health

Reagents
All culture media, supplements and material were purchase from Life Technologies (ThermoFisher Scientific, Courtaboeuf CEDEX, France) unless specified otherwise.

Primary Astroglial Culture and Medium Collection
On PND 2, male pups were rapidly decapitated, and the cortices were dissected in ice-cold sterile DPBS without calcium and magnesium under a binocular microscope. A tail biopsy was also taken and processed for genotyping. Cortical samples from each mouse were processed individually. Samples were washed twice in DPBS and transferred to culture media (DMEM-F12 medium with 10% fetal bovine serum (FBS) and 1X penicillin/streptomycin or pen/strep). Tissues were then cut in small pieces with a scalpel and dissociated by passing five times through a 21 g needle. Cell suspensions were then filtered through a 70-µm cell strainer and pelleted by a 10-min centrifugation at 200× g. Cell pellets were subsequently resuspended in culture media and plated in T25 culture flasks (Dutscher, Brumath, France) coated with poly-D-lysine solution (#P7886, 0.0001%, Sigma Aldrich, Saint-Quentin Fallavier, France). The medium was changed 24 h after plating and then thrice a week. Once confluency was reached, cells were trypsinized by incubating 5 min in phenol red free Tryple express solution, washed, centrifugated at 200× g for 10 min and plated on 10 cm culture dish (Falcon, Dutscher). When cells reached confluency, they were thoroughly washed with DPBS and the medium was replaced by serum free DMEM (#A1443001) with glucose (1 g/L), NaPyruvate (1 mM), 1X Pen/Strep, and without phenol red, glutamine or HEPES. Half the medium was then replaced by fresh medium every 4 days for 16 days. The collected medium was centrifuged at 200× g to pellet any cells or cellular debris and stored at −20 • C until needed. At the last medium collection, cells were washed three time in DPBS and stored at −85 • C until needed.

Sample Purification
Once all medium samples were collected, they were thawed and passed through a 0.22 µm PTFE filter (Agilent Technologies, Montpellier, France). The medium was concentrated by centrifugation at 4000× g through Amicon Ultra-15 filters (PLBC, membrane Ultracel-PL, 3 kD, Merck-Millipore, St Quentin en Yvelines Cedex, France). Once all medium was filtered, the columns were rinsed three time with 10 mL of ultrapure water (Gibco), followed by a 20 min centrifugation at 4000× g. All samples from the same culture and from the same genotype were then pooled and concentrated by SpeedVac for 60 min at room temperature.

Protein Digestion and iTRAQ Labelling
Sixty milligrams of secreted proteins per sample were digested and labeled with isobaric tagging reagents according to the manufacture's recommendations (ITRAQ Reagents 4 plex Application kit; Sciex, Framingham, MA, USA). Briefly, samples were dried in a vacuum concentrator, dissolved in 0.5 M TEAB, reduced (50 mM TCEP), alkylated (200 mM TCEP), and digested by adding the trypsin/LysC Mix (Promega, Madison, WI, USA). Next, samples were labeled using iTRAQ reporter ion tags for 2 h, as follows: WT1 for m/z 114 tag, WT2 for m/z 115 tag, KO1 for m/z 116 tag, and KO2 for m/z 117 tag.

Peptide Separation by OFFGEL Isoelectrofocusing and Nano LC-MS/MS Analysis
Samples containing iTRAQ labeled peptides were then pooled and fractionated in two steps as previously described [23,68]. First, peptide fractionation according to their pI by OFFGEL 3100 fractionator system using OFFGEL kit linear pH 3-10 (Agilent technology) in a 24-well configuration was performed. Peptides were dried, suspended in OFFGEL buffer and loaded into each well. Peptides were focused in a constant current of 50 µA until 50 kVh was accumulated. After isoelectrofocusing, the 24 OFFGEL fractions were desalted with C18 Zip-Tips (Millipore, Burlington, MA, USA) prior a fractionation with nano-HPLC using an Ultimate 3000 C18 RP-nanoLC system (Ultimate3000, Dionex/Thermo Scientific) controlled by Chromeleon v.6.8 software (Dionex/Thermo Scientific/LC Packings, Amsterdam, The Netherlands), and coupled to a Probot MALDI spotting device controlled by the µCarrier 2.0 software (Dionex/Thermo Scientific/LC Packings, Amsterdam, The Netherlands). Peptides were loaded on a trapping column (C18, 3 µm, 100 Å pore size; Thermo Scientific) in 2% ACN and 0.05%TFA for 5 min at a flow rate of 20 µL/min before introducing them into the reverse phase nano-column (Acclaim PepMap 100, 75 µm, 15 cm, nano-viper C18.3 µm, 100 Å pore size; Thermo Scientific) and separating them thanks to a binary gradient of buffer A (2% ACN, 0.05% TFA) and buffer B (80% ACN, 0.

iTRAQ Data Analysis
MS and MS/MS spectra were used for identification and relative quantification using the ProteinPilot software v 4.0 (Sciex, Les Ulis France) and the Paragon TM algorithm. The analysis was performed with the Mus musculus UniProtKB/Swiss-Prot database (European Bioinformatics Institute, Hinxton, UK). The Paragon searches were run using thorough ID and a false discovery rate (FDR) of 1%. Only proteins with at least one peptide above 95% confidence level were included for the quantification analysis. As described previously [23,68,69], the statistical analysis was performed by implementing the area of the iTRAQ reporter ions for each of these validated peptides into the R Package Isobar v. 1.14.0. [70]. A protein was considered as significantly deregulated when pValueRatio was obtained lower than 0.05 in both replicates and at least unique 2 peptides were quantified.
In other words, we targeted our study on proteins recorded with the "secreted" keyword in UniProtKB entries that refers to proteins located outside the cell membrane without external protective or external encapsulating structures (i.e., space outside of plasma membrane and vesicles) after a published literature curation.

Gene Ontology Analysis
A gene ontology analysis based on DAVID v.6.8 Gene Functional Annotation Tool available at https://david.ncifcrf.gov/home.jsp (accessed on 2019) [71,72] was performed on (i) identified proteins to check their localization and determine whether or not they were secreted proteins and on (ii) significantly deregulated secreted proteins to determine in which biological processes these proteins were involved. Only the proteins recorded with the "secreted" keyword in UniProtKB entries (https://www.uniprot.org/) (Uniprot, 2019), that refers to proteins located outside the cell membrane without external protective or external encapsulating structures (i.e., space outside of plasma membrane and vesicles) after a published literature curation, were targeted. A protein-protein interaction analysis of the secreted proteins significantly deregulated in Mecp2 KO astroglial secretome was performed by using Cytoscape (version 3.

Primary Neuronal Cultures and Treatments
On PND 7, male pups were rapidly decapitated, and the cortices were dissected in ice-cold sterile DPBS without calcium and magnesium and 1 g/L glucose under a binocular microscope. A tail biopsy was also taken and processed for genotyping. Each cortical sample from a mouse was processed individually. Primary cortical neurons were obtained by enzymatic digestion using the papain dissociation kit (Worthington, # LK003150, Serlabo Technologies, Vedene Cedex, France) according to the manufacturer's recommendations. Briefly, cortices from one animal were minced by scalpel and incubated in the digestion solution (20 units/mL papain and 0.005% DNase in EBSS or Earle's Balanced Salt Solution) at 37 • C in a rotating incubator for 45 min. The samples were dissociated by pipetting up and down 5 times with 1 mL pipet tip twice and then centrifuged at 300× g for 5 min at room temperature. The cell pellets were resuspended and separated by density gradient using the albumin-inhibitor solution provided. After a centrifugation at 70× g for 6 min at room temperature, the supernatant was discarded and the cell pellet immediately resuspended in culture medium (Neurobasal A medium, B27 1X, Glutamax 0.5 mM, Pen/Strep 1X). Cell suspension was filtered through a 70 µm cell strainer, centrifugated for 10 min at 100× g and resuspended in culture medium. After counting, cells were plated at a density of 40,000 cells/well on poly-d-Lysine-coated coverslips (1 µg/mL) in 24 well plate. After 2 h, the medium was replaced by culture medium with NGF (10 ng/mL) for 24 h. Forty-eight hours after plating, half the medium was replaced with fresh medium without growth factor or with either NGF (control), lipocalin 2 (50 ng/mL final concentration, #1857-CC-050, Bio-techne) or galectin 3 (1 µg/mL, final concentration, #9039-GA-050, Bio-techne). After 48 h of incubation, cells were then rinsed in DPBS and fixed by a 10-min incubation in 4% paraformaldehyde-PBS solution.

Sholl Aanalysis
Pictures of isolated neurons were taken at 40X magnification on a Zeiss Apotome microscope (Carl Zeiss Microimaging, Jena, Germany). Exposition times, images processing and merging were done using the same parameters for each experiment.
Using the Fiji software [74], fluorescence images were converted to 8-bit format, then thresholded using the Li filter, background noise was removed and the Sholl analysis tool (default parameters, v3.7.3) was used to count the number of intersections between neuronal processes and 5 µm concentric circles [75].

Statistical Analysis
Statistical analysis on proteomic data was performed by using the R Package Isobar v. 1.14.0 [70] as already reported [23,68,69]. All other statistical analyses were performed using GraphPad Prism for Windows/MacOS (GraphPad Software, La Jolla, CA, USA, http://www.graphpad.com).

Conclusions
To our knowledge, this in vitro study is the first to identify secreted proteins that are deregulated in the Mecp2 KO astroglia and that could be responsible for the neuronal RTT phenotype. The identification of proteins abnormally secreted by RTT astroglia, in addition to contributing to the RTT phenotype, could potentially lead to the development of new therapeutic strategies.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22094316/s1, Figure S1: Raw data of western blot staining of conditioned-medium from WT and Mecp2 KO astroglial culture, Figure S2: Protein levels of Lgals3, B2M and Mfap4 in WT and Mecp2 KO glial cells, Figure S3: Sholl analysis of WT cortical neurons after specific treatments, Figure S4: Absence of neurons in both WT and Mecp2 KO astroglial culture and Figure S5: STRING network of secreted Lcn2, Lgals3 and Mecp2 proteins. Table S1: List of the 557 secreted proteins identified and quantified in glial secretome of mouse Rett syndrome, Table S2a: List of the 105 secreted proteins significantly deregulated (DEP) in glial secretome of mouse Rett syndrome, Table S2b: Functional annotation of the 105 DEP localization, Table S3a: Refined list of 29 secreted proteins significantly deregulated (DEP) in glial secretome of mouse Rett syndrome, Table S3b: STRING network analysis of the 29 secreted proteins significantly dysregulated (DEP)in glial secretome of mouse Rett syndrome (Cytoscape v 3.7.2), Table S4