Rett syndrome (RTT; OMIM #312750) is a severe neurodevelopmental disorder characterized by a regression in the neurological development between 6 and 18 months following a normal early development. Patients experience seizures, autistic features, apnea/hyperpnoea and a loss of all the acquired capabilities, speech and non-verbal communication capacity, stereotypes, loss of purposeful use of hands, and organic dysfunctions [1
The complexity of Rett syndrome derives from the MeCP2 protein function, coded by MECP2
(Xq28; MIM* 300005), as most reported cases are associated with its defective activity. MeCP2 is a nuclear protein that acts as an epigenetic regulator, controlling the expression of numerous genes (either as transcription activators or repressors) involved in several biological processes [3
]. Whilst it is a ubiquitous protein, MeCP2 is most highly expressed in the brain [2
], most precisely in post mitotic neurons [5
], and its deficiency results in a global neurodevelopment disturbance [7
]. Neurochemically, Rett syndrome has been associated with an aberrant expression of neurotransmitters, neuromodulators, transporters, and receptors [8
]. Collectively, these alterations may underlie an unbalanced excitatory/inhibitory neurotransmission together with a disturbed synaptic development associated with Rett syndrome [12
]. In particular, an unbalanced excitatory/inhibitory neurotransmission stands out, with a specific γ-aminobutyric acid (GABA)ergic malfunction. GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the brain [14
]. The fast inhibitory actions of GABA are mediated by the GABA(A
) receptors, which are ligand-gated chloride (Cl-) channels consisting on assemblies of five different subunits from eight possible subfamilies [15
], the 2α1 + 2β2 + 1γ2 conformation being the most prevalent, accounting for 43% of the total GABAA
], present in most brain areas. The selective transport of Cl−
when the GABAA
receptors are activated hyperpolarizes the neuron, reducing its likelihood of starting an action potential [17
GABAergic synapses dysfunction has been associated with several Rett features. This altered performance, nevertheless, seems to be region and developmental-stage dependent. In fact, studies in MeCP2-/y mice brain slices show reduced miniature excitatory postsynaptic currents in the somatosensory cortical neurons, together with unaltered miniature inhibitory postsynaptic currents, which result in an overall reduced excitation. Opposite to that, there is a reduced conductance but increased excitatory/inhibitory ratio in the CA1 and CA3 areas of the hippocampus and V1 pyramidal neurons in in vivo visually evoked responses. Many players appear to be participating in this GABAergic neurotransmission alteration, from GABA receptors [18
] to the chloride channels NKCC1 and KCC2 [13
], responsible for the excitatory to inhibitory switch of GABAergic synapses during development, and for which the expression has been found to be altered in Rett patients’ cerebrospinal fluid (CSF).
In agreement with this, the restoration of correct GABAergic neurotransmission partially rescued Rett-like phenotypic abnormalities in mouse models [22
], supporting the GABAergic pathway pivotal role in Rett’s pathophysiology, and opening a window for the treatment-expectancy of the disease [23
Regarding the need for a treatment for the disease, and proving its mentioned potential reversibility, neurotransmission modulation appears to be an attractive therapeutic approach. However, enlightened by very last reports, the question of “when” adds on to the “how” for treating the disease. Given the severity of Rett syndrome and the lack of therapeutic options, there is an urge for the definition of the molecular alterations during development that set the bases for the window travel to address novel therapeutic targets, as intended throughout this work.
Throughout this work, we aimed to define if there was a direct relationship between GABAergic synapses known to alter elements, and MeCP2. Our results show a direct relationship between MeCP2 and GABA ionotropic receptors’ expression, which is not extensible to other GABAergic proteins such as KCC2, altered in the context of MeCP2 dysfunction, but not appearing to be directly regulated by its activity. More important than this, our work points attention towards the importance of the time frame when addressing Rett syndrome, as changes appear to be time-dependent, with greater importance for the pre-symptomatic stages.
The present work arises from the need to deepen into the definition of Rett’s syndrome pathophysiology in order to define new therapeutic strategies. To such end, we have explored the GABAergic neurotransmission system in different evolutionary stages of the disease, setting the focus on the main GABA ionotropic receptor, GABAA-A1R. We have observed a direct relationship between the MeCP2 altered expression and GABAergic receptors disruption, which is strongly dependent on the prodromic stage of the disease, angling the focus towards the time frame, which will be a key factor when looking for therapeutic options.
The triangle conformed by Rett syndrome, MeCP2, and GABAergic synapses has been previously explored by other groups [9
]. An increased MeCP2 expression in GABAergic neurons has been reported, and a reduced GABA release was reported upon MeCP2 knocking out in forebrain GABAergic neurons [27
]. Such a relationship has been shown to be extensive to other neuron types and brain areas, such as CA3 hippocampal neurons or brainstems [12
]. Moreover, in 2016, Dr. Zoghbi’s team showed how the restoration of the MeCP2 expression exclusively in GABAergic neurons was sufficient to rescue some disease features in a mouse model of Rett syndrome [28
]. Even a time-dependent alteration was suggested in male mice by the authors of [26
]. Through our work, we have followed such a line of thought, wondering how the alterations were occurring during development, setting the focus on the pre-symptomatic stages.
We first wondered if there was a direct relationship between MeCP2 and the postsynaptic GABAA
-A1R expression. Previous studies pointing out a relationship between GABA receptors and MeCP2 had been done in the context of whole brain models’ analysis, so it remained unclear if the potential decrease in GABA receptors was strictly related to MeCP2 expression or was a secondary effect to a global dysfunction. To address that question, we performed two complementary experiments. On first term, we overexpressed MeCP2 in a cell system that almost did not express GABA ionotropic receptors (or its expression was mostly below our detection sensitive), namely N2A cells. To increase the assay restricted conditions, we silenced any potential endogenous MeCP2 expression, and overexpressed the human form in either its wild-type or mutated forms. As expected, the GABA ionotropic receptors were only detected upon transfection with the MeCP2 wt
form, not occurring when transfected with the mutated version. These results were complemented with the peer experiment in primary cortical neurons; this time, we imaged a decrease in GABAA
-A1R expression four days after silencing the MeCP2 expression. Our results strongly suggest a direct relationship between MeCP2 and post-synaptic GABA ionotropic receptors’ expression, rather than this being a secondary effect of an overall altered homeostasis. Opposite to that, we found that the KCC2 expression was unaltered in the presented scenario. KCC2 is a chloride channel, essential for GABAergic correct functioning, and described to be down regulated in Rett syndrome [13
]. The fact that its expression was not significantly altered by MeCP2 inhibition suggests that such an altered regulation is more related to the syndrome pathophysiology rather than to the straightforward MeCP2 mutations. In fact, it has been well described how the KCC2 expression and function is, indeed, regulated by the GABA function itself [29
It is largely known that most GABAA
receptor coding genes are clustered in four chromosomal regions in chromosomes 4, 5, 15, and 19 [31
]. These subunits comprising the pentameric GABA receptor formation have a coordinated expression [32
], and, as revealed by human brain transcriptome analysis, this produces a subject and region-specific expression signature of GABAA
receptor subunits [33
]. Enlightened by our results, further studies should be made to elucidate whether MeCP2 acts a transcriptional regulator of these clusters, the mechanisms through which this regulation takes place, and the time during development.
As stated from the beginning of the discussion, our main objective was setting the focus over the evolutionary stages in the disease. As for that, we switched to a model that allowed us to evaluate the different prodromic stages of the disease. We used females Bird’s Rett mice model, as they better recapitulate the pathophysiology [34
]. We focused our analysis on the following three proteins: GABAA
-A1R, KCC2, and GAD67. The first two proteins were selected because of the previous observations, and because they have been proven to be not only crucial in Rett syndrome development, but also potential actionable targets of the disease [21
]. As shown by our results, a markedly reduced expression of GABAA
-A1R and KCC2 was recorded in pre-symptomatic mice, while the GAD67 population (used as a marker for GABAergic neuronal) remained unaltered. These results suggest a reduced GABAergic activity without the affectation of the GABAergic general population, which are aligned with the previously described results. Expanding previous descriptions, our results bring focus to the pre-symptomatic stages of the disease, where the most differences were observed. Furthermore, we did not observe any reliable difference in both proteins’ expressions in fully-symptomatic mice (or even an increase in KCC2 expression), once again, enhancing the importance of the time-frame when addressing Rett syndrome. The variability in KCC2 expression, and its activation through phosphorylation, is a field that further studies should explore, especially under the recent scenario in which KCC2 is being addressed as a therapeutic target [36
]. Preliminary results (data not shown) have pointed towards an over-phosphorylation of KCC2 in symptomatic mice, drawing a scenario in which KCC2 will be under expressed in early pre-symptomatic stages and inactivated in symptomatic phases—again, shaping different therapeutic strategies on different prodromic stages. KCC2 activation and membrane diffusion has been related with GABAergic activity itself, increasing its therapeutic interest [29
During the preparation of this manuscript, Dr. Ben-Ari’s team also pointed out the importance of GABAergic dysfunction during development in Rett syndrome [37
], providing neurophysiological evidence of such a temporal alteration. The detected expression to normal extents of GABAA
-A1R in mature Rett brains reinforces the idea of time-dependence in the MeCP2 control of the GABAergic cluster during the specific developmental stages.
Completing the observed results, we analyzed the aforementioned targets in two Rett patient’s necropsied brains. Backing up our previous description, the results showed an even enhanced expression of GABAA
-A1R and KCC2, without alterations on GAD67. The overall analysis of the GABAergic pathway showed a slightly increased expression of almost all of the implicated genes. These results confirm the previous findings, pointing towards the importance of pre-symptomatic damage. These results are in agreement with the dataset reported by Renthal et al. [38
]. An increasing body of evidence pointing towards the importance of early intervention has been reported in the last few years, as reviewed by Constentino et al. [39
], and has extended from neurotransmission to other therapeutic targets in Rett syndrome, such as energetic dysfunction, as very recently published [40
], or inflammatory processes [41
To summarize, our results show a direct relationship between MeCP2 and GABA ionotropic receptors’ expression, which is not extensible to other GABAergic proteins such as KCC2, which is altered in the context of MeCP2 dysfunction, but does not appear to be directly regulated by its activity. More important than this, our work points attention towards the importance of the time frame when addressing Rett syndrome, as changes appear to be time-dependent, with greater importance in the pre-symptomatic stages.
Therapeutically, early GABAergic modulation in Rett syndrome may represent a promising strategy. While our results suggest that GABA-A1 R can be a potential therapeutic target, the time window of intervention is, according to our findings, critical. Additionally, the development of novel drugs enhancing GABA-A1 R function (for potential use in the initial clinical stages) and devoid of side effects are required, for an early intervention of Rett syndrome.
4. Materials and Methods
4.1. Cell Lines and Samples Utilization
Immortalized Neuro2a cells (also known N2A cells, a fast-growing mouse neuroblastoma cell line) were grown following standard conditions in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% glutamine, 10% fetal bovine serum (FBS), and antibiotics.
For neuronal primary cells cultures, the protocol described in the literature [43
] was followed. All of the experimental procedures were carried out according to European Union guidelines (Directive 2010/63/EU) and following protocols that were approved by the Ethics Committee of the Bellvitge Biomedical Research Institute (IDIBELL, Barcelona, Spain). Briefly, mouse embryos (embryonic day E18) were obtained from pregnant CD1 females, the cortexes were isolated and maintained in cold Hank’s Balanced Salt Solution supplemented with 0.45% glucose (HBSS-Glucose) and digested mildly with trypsin for 17 min at 37 °C, and dissociated. The cells were washed three times in HBSS and resuspended in a Neurobasal medium supplemented with 2 mM Glutamax (Gibco, Waltham, MA, USA) before filtering in 70 mm mesh filters (BD Falcon, San Jose, CA, USA). The cells were then plated onto glass coverslips (5 × 104
) coated with 0.1 mg/mL poly-L-lysine (Sigma, Darmstadt, Germany), and 2 h after seeding, the plating medium was substituted by a complete growth medium, consisting of a Neurobasal medium supplemented with 2% B27 (Invitrogen, Waltham, MA, USA) and 2 mM Glutamax.
In this study, we used post mortem brain samples from two unrelated Rett patients bearing the MECP2
_c.763C>T mutation and an intra-assay control for RNAseq (i.e., an extra sample that due to its pathology could not be used as a bona fide control, but that allowed us to run technical comprobations). This mutation is the second most frequent Rett-causative mutation, present in 10.9% of the cases [44
]. The patients were between 10 and 15 years old at exitus, which is noteworthy, as life expectancy is not highly reduced in Rett syndrome. In all of the cases, RNA was isolated from two brain regions (frontal and occipital cortex) and the samples were treated according to the informed consent of the legal representatives.
The study was approved by the Ethics Committee of Sant Joan de Déu Hospital, project PI15/01159, 01/2016. We are indebted to the “Biobank de Hospital infantil Sant Joan de Déu per la Investigació” integrated in the “Spanish Biobank Network of ISCIII for the sample and the data procurement”.
4.2. Mouse Colony
Cortex samples from one and six-month old Mecp2−/+
female mice [45
] Bird’ model (B6.129P2(C)-Mecp2tm1.1Bird
/J) were obtained after mouse sacrifice and brain dissection. The proteins from cortex were extracted by tissue homogenization with an ice-cold RIPA buffer with protease inhibitors (cOmplete, mini, EDTA-free protease inhibitor cocktail, Merck), for 30 min at 4 °C followed by 15 min of centrifuge at 4 °C. The protein samples were quantified by Bradford assay and stored at −80 °C.
4.3. Plasmids and Mutagenesis
In certain experiments, we attempted to silence the endogenous MECP2 expression and re-express the human gene either in the wild type or mutated form.
For the MECP2 silencing, transient transfection with a vector containing a shRNA targeting mMECP2 was performed. Silencing was done with the MISSION® shRNA technology (Sigma Aldrich, Darmstadt, Germany; Clone TRCN0000304464), and the efficiency was checked at the protein level. To ensure the sole silencing of the endogenous gene, and not the re-expressed form, we used a shRNA directed to the 3′UTR part of the gene, absent in the transfected cDNA. We used, as a control for transfection, a vector with the same backbone but no shRNA.
The MeCP2 c.763C>T genetic variant was introduced by site-directed mutagenesis using the QuickChange II XL Kit (Agilent Technologies, Santa Clara, CA, USA), in the pEGFP-C1-hMeCP2 (wild-type) mammalian expression vector (kindly provided by Dr. Landsberger). The mutation was confirmed by Sanger sequencing. Both vectors, together with the pEGFP-C1 vector (BD Clontech, Palo Alto, CA, USA) and a mock vector with the same backbone, were used for the experiments that required plasmid transfections.
These were carried out using Lipofectamine 2000 (Thermofisher, Waltham, MA, USA) following manufacturer recommendations. For neuronal primary cultures, 0.8 g of total DNA was mixed with Lipofectamine 2000 and incubated with cortical neurons (at DIV11). The transient expression was allowed for 96 h, and the neurons were fixed at DIV14. For the N2A cells, 4 g of DNA was transfected over 300,000 cells grown in 10 cm2 plates, and the cells were collected four days after transfection for RNA analysis.
4.4. RNA Extraction and qRT-PCR
RNA for RNAseq (from post-mortem human brain samples) and for qRT-PCR (from post-mortem human brain samples and N2A cells) was extracted using RNeasy® Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The total RNA was eluted in 40 μL of RNAse-free water and stored at −80 °C. The RNA concentration was measured using the NanoDrop 2000 Spectrophotometer (ThermoScientific, Waltham, MA, USA), and integrity was assessed by running the samples through a 1% agarose gel.
qPCRs were carried out following a two-step protocol. First, cDNA was synthesized from a total of 500 ng of RNA per reaction, following the recommendations provided with SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (InvitrogenTM). After the RT-PCR reaction, the post-mortem brain cDNA from frontal and occipital cortex samples was pooled.
Second, qPCR was performed in a QuantStudioTM 6 Flex Real Time PCR System (Applied BiosystemsTM MA, USA) with PowerUpTM SYBRTM Green Master Mix (Applied BiosystemsTM). The data were analyzed using a comparative method, correlating the initial template concentration with the cycle threshold (Ct) so as to obtain the relative quantity (RQ) of the RNA. The RQ is defined as 2-ΔΔCt, where ΔΔCt is the ΔCt of the patient cell line minus the ΔCt of the control cell line, and ΔCt is the Ct of the target gene minus Ct of the endogenous gene (RPLP0 and GUSB).
The probes design was done through the selection of the exonic areas present in all of the transcript variants of each gene, by the selection of common fragments in the UCSC genome browser, based on GRCh38/hg38 version. Primers for N2A-derived qPCR experiments were, in 5′-3′ sense, as follows: m-Mecp2 (F: ACCATCATCACCACCATCAC; R: GGGCATCTTCTCTTCTTTGC), h-MECP3 (F: AGGAGAGACTGGAAGAAAAGT; R: CTTGAGGGGTTTGTCCTTGA), m-Gabra1 (F: ACCAGTTTCGGACCAGTTTC; R: TACAGCAGAGTGCCATCCTC), m-Gabrb2 (F: TCGCTGGTTAAAGAGACGGT; R: TCTCTCCAGGCTTGCTGAAA) and m-Gabrg2 (F: TGGGCTACTTCACCATCCAG; R: GCCATACTCCACCAAAGCAG). The primers sequence for the brain samples of qPCRs were not included.
4.5. Western Blotting and ICC
Western blot analysis of the cortex protein samples from Mecp2−/+ female mice was performed. The proteins were subjected to SDS-PAGE and transferred to a nitrocellulose membrane using the Pierce® Power Station (Thermo Scientific). The membranes were blocked with milk, as follows: PBST buffer 5% for 1 h at room temperature. Incubation with primary antibodies was directed against GABA-A1 (Neuromab, UCDavis, CA, USA, 75-136) at a concentration of 1:500, MeCP2 (ab2828; Abcam, Madrid, Spain) at a concentration of 1:1000, and vinculin (ab129002, Abcam, Madrid, Spain) was performed o/n. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies (Lfe Technologies, Waltham, MA, USA); these were detected using the Enhanced Chemiluminescence System (GE Healthcare, Berkshire, UK).
Immunochemistry experiments were performed as described in the literature [43
]. Anti-GABA A1R (Neuromab, UCDavis, CA, USA, 75-136) was used at a concentration of 1:100, and MeCP2 (ab2828; Abcam, Spain) at a concentration of 1:250. The conjugated secondary antibodies for the confocal microscopy were used.
Fluorescence was visualized with a Leica TCS-SL spectral confocal microscope (Leica Microsystems, Wetzlar, Germany) using a Plan-Apochromat 63×/1.4 N.A. immersion oil objective (Leica Microsystems). To excite the different fluorophores, the confocal system was equipped with excitation laser beams at 488 and 546 nm. The images were analyzed with ImageJ software. For the intensity quantification of the ICCs, pictures from independent primary cultures were used. Regions Of Interest (ROIs) were defined on the green channel (GFP positive neurons) applying a threshold to only select the desired neuron. The same ROI was exported to the red channel pictures, and the mean gray value was measured.
4.6. RNAseq Data Analysis
Origin of data: A frontal and occipital cortex paired-end RNAseq was performed for two RTT patients bearing the MECP2c.763C>T, p.Arg255* mutation and the mentioned intra-assay control. Technical triplicates (three separate RNA extractions) were run for each of the two brain regions. The RNA samples were sent to the Centre Nacional d’Anàlisi Genòmica (CNAG), where the RNAseq experiment was performed, within the framework of the project FIS PI15/01159 Rett Syndrome (IP: Judith Armstrong, Ph.D.). Both brain areas were sequenced separately, and as no significant differences were observed between them, they were analyzed as a sole data pool. To discard the differences between the brain areas, we performed a principal component analysis (PCA). In such an analysis, we compare the variance between all of the samples (patients and areas). At this point, we also added an “intra-assay control” sample. Principal Component 1 (explaining 75% of the variance), clearly discriminated between the Rett and not-Rett samples, while there was not a principal component separating the brain areas (data not shown).
Because of the lack of true control data, the data from our RNAseq were compared to various public controls’ data. We used data from public controls available on the GTEx (The Genotype-Tissue Expression) Portal. Cortex RNAseq data from five controls were used, two of which were female (GTEX-15ER7 and GTEX-T2IS) and three were male (GTEX-12126, GTEX-T5JC, and GTEX-WHSE), all of them with ages ranging 20–29. We also compared our data to the cortex RNAseq data from two public male controls (ages 24 and 39) available on the Allen Human Brain Atlas, and the RNAseq data from three female control samples (ages 15–25) used in a publication by Lin, et al. in 2016 (3). Two of these samples were a pool of frontal and temporal cortex RNA, and one of them was just temporal cortex RNA.
RNAseq analysis pipeline: As a result of the low performance of the sequencing experiment, the internal control’s occipital cortex data was excluded from the analysis. The RNAseq analysis pipeline was run by the Bioinformatics Unit from the Genetics and Molecular Medicine Service at the Hospital SJD. The FASTQ files passed through a first quality control, after which a trimming was performed and the adapters were removed. Then, low quality bases were eliminated so only reads longer than 70 bp were left to continue the analysis. Here, a second quality control was performed and the reads were mapped with TopHat2 (4). The counting was performed with HTseq (5) and the R package DESeq2 (6) was used for library normalization. The frontal and occipital cortex data from our two RTT patients were averaged.
In order to compare the data obtained from our RNAseq experiment to the public data, an internal normalization over the endogens RPLP0 or GUSB of every patient’s and control’s data was performed.
A validation of the results was carried out at the Hospital Sant Joan de Déu using qRT-PCR, comparing patients with an internal RNAseq control; that is, a sample that could be used for a later comparison of the results through qPCR and therefore RNAseq technical validation, but could not be used to biologically validate the results, as it was not a healthy brain, as previously described. Thus, following the RNAseq analysis, the targeted gene expression of a subset of 21 genes corresponding to different nodes of the GABAergic pathway and differentially expressed between patients and the internal control was validated by qRT-PCR, showing a strong correlation (20 out of 21 transcripts deregulated; Figure S1
), with an overall coincidence between RQ values (qRT-PCR experiments) and fold-change (RNAseq experiment).
4.7. Data Availability Statement
The present study is not a clinical trial of any kind. All of the data, materials, and methods to conduct the research are available in the manuscript. Patient samples are located at the “Biobank de Hospital Infantil Sant Joan de Déu per la Investigació” integrated in the “Spanish Biobank Network of ISCIII for the sample and the data procurement”, to whom we are indebted.