The experimental procedures were performed in accordance with European Community (EU Directive 2010/63/EU for animal experiments) and National Institutes of Health guidelines for the care and use of laboratory animals. The study was approved by the Georg-August-University, Göttingen and the approval ID T12/18 was assigned to this work.

Following with convention, we designated human genes with all capital letters in italics (e.g., MeCP2), murine genes are given in italics with a starting capital letter (e.g., Mecp2) and genotypes are designated by superscript letters with “+” designating the presence of the wild type allele, “-” designating the knock-out of the allele and “y” indicating the male chromosome. Proteins are given in capital letters not differentiating between human and murine origin (e.g., MeCP2). For serotonin receptors, this takes the form of 5-HT_xR, with x designating the subtype. The exception here are the members of serotonin receptor family 5, whose members are given in the form 5-ht_xR.

The Mecp2 knockout mouse (Mecp2^-/y), which is a model for RTT, strain B6.129P2(C)-Mecp2^tm1-1Bird (Guy et al., 2001) maintained on a C57BL/6J background was obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Mecp2 knockout males (Mecp2^-/y) were generated by crossing hemizygous Mecp2^+/- females with C57BL/6J wild type males. Animals were kept in a temperature- und humidity-controlled 12 h light-dark cycle and had free access to water and standardized pellet food.

The genotype of newborn mice was determined using PCR on DNA isolated from mice tail biopsies. Mice tails were incubated in 25 mM NaOH/0.2 mM EDTA for 3 h at 65°C. After neutralization with equal volume of 40 mM Tris/HCl pH 5.5, 1 μl was used as template in a subsequent PCR. Primers for genotyping are given in Table 1.

Gene expression was analyzed by quantitative RT-PCR analysis. The total ribonucleic acid (RNA) of homogenized brain tissue (hippocampus and brainstem) was isolated using the Trizol^® method according to manufacturer’s instructions (GibcoBRL) and its concentration was determined using the nanodrop ND-1000 spectrophotometer followed by its quality and integrity measurement by electrophoresis on RNA 6000 LabChip^® kit (Agilent 2100 Bioanalyzer). The RNA was transcribed into the corresponding deoxyribonucleic acid (cDNA) using the iScript cDNA Synthesis Kit (BioRad). The primer pairs (Table 1) were designed by using the Primer3 program¹.

Gel electrophoresis revealed a single polymerase chain reaction (PCR) product, and the melting curve analysis showed a single peak for all amplification products. The PCR products were sequenced and blasted to confirm the correct identity of each amplicon. Ten-fold serial dilutions generated from cDNA of each sample were used as a reference for the standard curve calculation to determine primer efficiency. Real-time PCR reactions (25 μl) were performed in triplicates containing 1/20 volume of the sample cDNA preparation from 250 ng total RNA, 400 nM of each primers, and 1X iQ-SYBR Green Supermix (BioRad, Laboratories, Ltd). The PCR-reactions were performed as follows: initial denaturation at 98°C for 30 s, 40 cycles of (denaturation 94°C/1 s, annealing 58°C/15 s, extension 72°C/1 s), and a final gradual increase of 0.5°C in temperature from 60°C to 90°C. All real-time quantifications were performed using the iCycler iQ system (BioRad) and were adjusted by using the method according to (Pfaffl, 2001). Hypoxanthine guanine phosphoribosyl transferase (Hprt) served as reference gene (“housekeeping gene”) for normalization in the qPCR.

Murine neuroblastoma cell line N1E-115 was obtained from the American Type Culture collection (ATCC). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37°C under 5% CO₂.

For transient transfection, cells were seeded in cell culture dishes and transfected with indicated plasmids using Lipofectamine2000 Reagent (Invitrogen) according to the manufacturer’s instruction.

Serotonin receptor 5b (5-ht_5b) expression constructs were generated from murine cDNA. Brain tissue was explanted and used for total RNA isolation with the OLS RNA kit (OLS, Germany) according to the manufacturer’s instructions. The total RNA was used in one-step RT-PCR (Invitrogen) and resulting PCR fragment was cloned into pTarget expression vector (Promega). Primer sequences are given in Table 1. To obtain a C-terminal fluorophore fusion construct of 5-ht_5b, the receptor and the fluorophores CFP, GFP, YFP or mCherry were amplified individually, and fusion PCR was used to combine the cDNAs. The resulting PCR fragment was cloned into pTarget expression vector (Promega). To test the hypothesis of a truncated 5-ht_5b receptor, we amplified the unlabeled and the mCherry fusion construct and cloned the fragments into pTarget. Correct sequences of all constructs were determined by sequencing.

The G_i3α-CFP plasmid was kindly provided by Dr. Andrew Tinker (University College, London, UK). The G_sα-GFP plasmid was a kind gift of Dr. Mark Rasenick (U. Illinois College of Medicine, Chicago).

The plasmids to fluorescently label the cell membrane (pYFP-Mem), the endoplasmic reticulum (pYFP-ER), mitochondria (pYFP-mito) and peroxisomes (pEGFP-Pex) were obtained from Clontech. The plasmid to label the endosomes (GFP-Rab5; Addgene plasmid # 31733) was a gift from Richard Pagano (Choudhury et al., 2002). Lysosomes were counterstained with LysoTracker (Thermo Scientific). The golgi apparatus was counterstained using antibodies directed against cis golgi marker GM-130 (sc-55591, Santa Cruz) and trans-golgi marker TGN38 (sc-166594, Santa Cruz).

Co-localization was observed using Zeiss LSM 510 Meta system. Quantitative analysis of co-localization was carried out by calculating Pearson’s correlation coefficients using LSM 510 software.

Membrane preparations of transiently transfected mouse N1E-115 neuroblastoma cells expressing the full-length or truncated 5-ht_5b receptor linked to cherry fluorophore and G-protein α subunits (G_αi3 and G_αs fused to variants of green fluorescent protein) were performed according to the protocol described by Kvachnina et al. (2009). Membrane preparations were diluted in reaction buffer (50 mM Tris/HCl pH 7.4, 2 mM EDTA, 100 mM NaCl, 3 mM MgCl₂, and 1 μM GDP) to a concentration of 1 μg/μl. Each reaction contained 50 μg membranes in a total volume of 100 μl. Reactions were performed in triplicate. After adding [35S]GTPγS (Hartmann Analytic) to a final concentration of 3 nM, samples were incubated for 5 min at 30°C in the presence or absence of 1 μM 5-HT. The reaction was terminated by adding 900 μl of RIPA-buffer (20 mM Tris/HCl pH 7.4, 0.15 M NaCl, 10 mM EDTA, 10 mM Iodacetamide, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) for 30 min on ice. Samples were centrifuged at 13,000 rpm for 10 min. The supernatants were transferred into a new tube and samples were incubated for 2 h after addition of 100 μl protein A-Sepharose (Sigma, 20 mg/ml RIPA) and 0.5 μl of goat anti-GFP antibodies (University of Alberta, TEC Edmonton). Immuno-precipitates were washed three times, boiled in 0.5 ml of 0.5% SDS, and radioactivity was measured by scintillation counting.

Statistical significance of the data was tested by non-parametric Mann–Whiney test using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) on a Microsoft Windows PC. P-value less than 0.05 is considered significant. Data are presented as mean ± standard error of the mean (SEM).

We used quantitative RT-PCR to analyze the complete serotonergic system in hippocampus and brainstem of WT and Mecp2^-/y mice on the transcriptional level. We analyzed a total of 16 genes that included the serotonin receptors Htr1a, Htr1b, Htr1d, Htr1f, Htr2a, Htr2b, Htr2c, Htr3a, Htr4, Htr5a, Htr5b, Htr6, Htr7, the serotonin transporter (Slc6a4), and the synthesizing enzymes dopamine decarboxylase (Ddc) and tryptophan hydroxylase 2 (Tph2). The serotonin receptor Htr1e was not analyzed as it is absent in mice. Also note that Htr1c is not listed above. The gene was, after its initial discovery and characterization, renamed to Htr2c (OMIM 312861). Table 1 lists the accession numbers of the murine genes of the serotonergic system and the sequences of the primers used for RT-qPCR.

Analyses were performed at postnatal day 40 (P40), a stage where Mecp2^-/y mice have already developed a RTT phenotype with hind limb clasping, low body weight and several other of the mentioned symptoms (Stettner et al., 2008). All differentially expressed genes are summarized in Table 2.

The differential expression levels for all 16 components of the murine serotonergic system in the brainstem are given in Figure 1, where the WT was set to 1. In the brainstem, 5 of the 16 genes of the murine serotonergic system were dysregulated between WT and Mecp2^-/y mice at P40. These were the three serotonin receptor genes Htr1d, Htr2c and Htr5b, as well as the non-receptor genes Ddc and Tph2. Also, Htr4 and Htr6 showed strong, although not yet significant dysregulation.

The mRNA level for Htr1d in Mecp2^-/y mice showed a 2-fold up-regulation (Figure 1C; 2.29 ± 0.184, p = 0.0013). Htr1d affects locomotion and anxiety and also induces vascular vasoconstriction in the brain. In addition, 5-HT_1DR has been implicated in controlling activity of raphé neurons and therefore serotonin release. An up-regulation of 5-HT_1DR in the brainstem could therefore inhibit the serotonin release in the raphé nuclei and thus explain the low brain serotonin levels reported previously (Mokler et al., 1998).

The mRNA level of Htr2c was reduced to roughly half compared with WT (Figure 1G; 0.482 ± 0.057; p = 0.005). Htr2c activation inhibits dopamine and norepinephrine release in the striatum, prefrontal cortex, nucleus accumbens, hippocampus, hypothalamus, and amygdala among other areas (Alex et al., 2005). It also regulates mood, anxiety, feeding, and reproductive behavior (Heisler et al., 2007). In the brainstem, Htr2c is associated with respiratory dysfunction (Tecott et al., 1995; Hodges et al., 2009).

The most striking difference was observed for Htr5b with an up-regulation in Mecp2^-/y mice by a factor of 75 at P40 compared to age-matched WT mice (Figure 1K; 76.43 ± 15.86; p = 0.0089). Htr5b showed a tremendous dysregulation with nearly 75-fold upregulation in Mecp2^-/y mice. Unfortunately, little is known about this specific serotonin receptor. Based on molecular studies, Htr5b is grouped with Htr5a forming the fifth class of serotonin receptors. The two 5-ht₅ receptors are the only serotonin receptors that are transcribed from two exons (Matthes et al., 1993). Htr5b is believed to be non-functional in humans because its coding sequence is interrupted by several stop codons and repeated insertions (Grailhe et al., 2001), while rodents carry a fully intact Htr5b gene. Therefore, we performed some basic tests to determine the relevance of Htr5b dysregulation in RTT (see below).

Tryptophan hydroxylase 2 (Tph2) was reduced 2-fold in Mecp2^-/y mice compared to WT (Figure 1P; 0.553 ± 0.379, p = 0.0067). Tph2 is the main synthesizing enzyme for serotonin, hydroxylating the rate-limiting step from L-tryptophan to 5-hydroxy-L-tryptophan (Kuhar et al., 1999). Tph2 is mainly expressed in neurons of the raphé nuclei, which contain the majority of serotonin-containing neurons (Frazer and Hensler, 1999). A defect of Tph2 could explain reduced levels of serotonin in the brain and in the cerebrospinal fluid (Jellinger, 2003; Ramaekers et al., 2003). The dopamine decarboxylase (Ddc) level was increased 3-fold in Mecp2^-/y mice (Figure 1O; 3.48 ± 0.299, p < 0.001).

The differential expression levels for all 16 components of the murine serotonergic system in the hippocampus are given in Figure 2, where the WT was set to 1. In hippocampus, of the 16 genes of the serotonergic system, only two showed differential expression between WT and Mecp2^-/y mice.

Ddc, which was upregulated threefold in the brainstem, was also up-regulated twofold in hippocampus (Figure 2O; 2.459 ± 0.336, p = 0.0015). Ddc catalyzes several different decarboxylation reactions (Siegel et al., 1999), thus mutations in Ddc lead to reduced levels of neurotransmitters dopamine and serotonin (Ramaekers et al., 2003). However, neither for dopamine nor for serotonin synthesis, Ddc is the rate-limiting enzyme (Kuhar et al., 1999), whereas in humans Ddc is the rate-limiting enzyme for the synthesis of trace amine transmitter (Berry, 2004). For the serotonergic system, Ddc catalyzes the decarboxylation of 5-Hydroxy-L-Tryptophan to serotonin, while Ddc also catalyzes the conversion of 3,4-dihydroxyphenylalanine (DOPA) and Levodopa to dopamine. Dopamine in the basal ganglia is important for coordination and smooth movement, and Mecp2^-/y mice show disturbed motor function like hind limb clasping.

The second dysregulated gene in hippocampus was Slc6a4) which was down-regulated in Mecp2^-/y mice (Figure 2N; 0.387 ± 0.611, p < 0.001). Slc6a4 (solute carrier family 6 member 4) encodes the serotonin transporter SERT (Blakely et al., 1991; Hoffman et al., 1991) and removes serotonin from the synaptic cleft, thus terminating its action by transporting 5-HT into the presynaptic neuron in a sodium-dependent manner (Horschia, 2001). Mutations in the Slc6a4 promoter affect the rate of serotonin uptake (Caspi et al., 2003) and are therefore associated with numerous diseases like sudden infant death syndrome (Weese-Mayer et al., 2003), and propensity to post-traumatic stress disorder (Liu et al., 2015) or susceptibility for depression (Lin et al., 2015).

An up-regulation of Ddc in RTT would have little effect on the availability and therefore on the strength of synaptic transmission. However, a down-regulation of Slc6a4 in RTT would result in increased serotonin-mediated synaptic transmission due to longer dwell time of 5-HT in the synaptic cleft. Therefore, and because no other genes were found dysregulated in hippocampus, we believe that upregulation of Ddc and down-regulation of Slc6a4 could be an adaption to low serotonin levels reported previously (Ramaekers et al., 2003).

In our mouse model of RTT syndrome, 6 out of 16 genes constituting the serotonergic system in mouse were differentially regulated. Of these six genes, only two genes were dysregulated in hippocampus and only dopamine decarboxylase (Ddc) was dysregulated in both brain regions. Ddc was up-regulated in Mecp2^-/y mice in hippocampus and brainstem by approximate the same factor, which might suggests a genetic reason for the up-regulation, e.g., the missing of transcriptional inhibitor Mecp2. Nevertheless, in hippocampus, up-regulation of Ddc might compensate for the down-regulation of Slc6a4 or vice versa. There have been reports of reduced serotonin (Jellinger, 2003; Ramaekers et al., 2003) and dopamine (Riederer et al., 1986; Lekman et al., 1989; Wenk et al., 1991) levels in the brain and cerebrospinal fluid of RTT patients which are compensated by increased expression of dopamine receptor (Chiron et al., 1993). The decrease of both neurotransmitters make it like that the dysregulation of both Ddc and Slc6a4 in hippocampus is compensatory.

After identifying Htr5b to be profoundly dysregulated in the brainstem at P40, we profiled the expression of this gene during the development at P7, P14, P21, P40, and P50 (Figure 3A). Up until P21, Htr5b expression increases uniformly in WT and Mecp2^-/y mice. After reaching peak expression around P21, Htr5b expression in WT mice is essentially turned off after P21. However, in Mecp2^-/y mice it remains at the same elevated levels. Thus, the observed dysregulation is not due to an upregulation of Htr5b in Mecp2^-/y mice, but due to a failure to down-regulate Htr5b expression. In hemizygous female mice at P75, we also found a significant dysregulation, albeit not as strong as in male knockout mice (Figure 3B). However, female mice at this age do not display the same advanced disease progression as male mice and they are mosaics, displaying varying gene expression patterns. Considering the identical profiles in WT and Mecp2^-/y mice up to P21, one can speculate that Htr5b expression may serve a physiological purpose in mice. The intracellular localization of 5-ht_5b and the nearly complete down-regulation after P21 indicates that 5-ht_5b might act more like a regulator than a receptor. In addition to Htr5b, we also assayed the other genes found dysregulated in P40 male mice again in P75 female mice (Figures 3C,D). All genes showed a comparable trend in the female mice as found in the male mice, although the differences between WT and Mecp2^+/- animals were much smaller. In brainstem (Figure 3C), Htr2c (0.672 ± 0.124, p = 0.012) and Ddc (1.841 ± 0.198, p = 0.026) were found to be significantly dysregulated. In hippocampus (Figure 3D), only Ddc showed a significant dysregulation (1.452 ± 0.1, p = 0.0286). The lower number of significantly dysregulated genes as well as the smaller differences between WT and Mecp2^+/- females could be explained in two ways. Hemizygous mice are a mosaic regarding Mecp2 activity, thus generating greater variability of gene regulation. Also, females develop symptoms much later, so possible differences may not be detectable at P75.

The tremendous dysregulation of Htr5b and lack of previous research prompted us to analyze this receptor in more detail at the protein level.

If any, only weak 5-ht_5b receptor expression was found in the hippocampus of both WT and Mecp2^-/y mice at P40 (data not shown). The same was true for the brainstem of WT mice (Figures 4A,C), while in MeCP2-deficient mice, 5-ht_5b receptors were found in abundance (Figures 4B,D). Thus, receptor staining was in agreement with our qPCR expression data. Closer inspection of the immune-histochemical data revealed an unusual staining pattern for 5-ht_5b with punctate intracellular pattern instead of uniform labeling of the cytoplasm (Figure 4E). Interestingly, transfection of a 5ht_5b-fluorescent protein fusion construct in neuroblastoma cells produced the same intracellular expression pattern (Figure 4F).

Western blot analyses of both brainstem tissue (Figure 5B, IV) and neuroblastoma cells transfected with Htr5b (Figure 5B, II) verified the presence of a protein of approximately 40 kDa, as expected from the genetic sequence. In addition, western blots of transfected cells and murine tissue revealed a signal at 22.5 kDa (Figure 5B, II, IV). Such a signal is also obtained when a truncated 5-ht_5b is transfected (Figure 5, III). The truncated receptor of 22.5 kDa might be important for Htr5b expression in humans. The human Htr5b gene is considered to be a pseudo-gene, as its first exon is disrupted by several mutations (Grailhe et al., 2001). However, sequence comparison between different species indicated the presence of a highly conserved second start codon surrounded by a classic Kozak sequence (Grailhe et al., 2001) in Htr5b. Transcription starting from this second exon would result in a shortened protein with a predicted molecular mass of 22.5 kDa. An artificially truncated murine 5-ht_5b receptor behaved nearly identical to the full length version (data not shown), leaving the possibility that a truncated 5-ht_5b might also be present in humans and contributes to RTT. Incidentally, two immune-reactive bands were also reported for 5-ht_5a (Dutton et al., 2008). As both class 5 serotonin receptors are transcribed from two exons (Matthes et al., 1993), the presence of two protein variants might be a class-defining feature.

N1E-115 neuroblastoma cells expressing fluorescent 5-ht_5b were either co-transfected with or counter-stained against specific organelle markers to determine 5-ht_5b’s intracellular compartment. As expected, some co-localization was seen with the ER (Figure 6A, correlation coefficient 0.539 ± 0.08) and cis-golgi marker GM130 (Figure 6B, 0.584 ± 0.05) as proteins are produced here and are sorted for trafficking to the membrane. However, no receptor reached the plasma membrane (Figure 6C, 0.086 ± 0.01). Strong co-localization of 5-ht_5b receptors was seen with endosomal compartments, shown exemplarily for early endosome marker rab5 (Figure 6D, 0.873 ± 0.02). No co-localization of 5-ht_5b receptors was detected with markers for trans-golgi marker TGN38 (Figure 6E, 0.094 ± 0.03) or with lysosomes (Figure 6F 0.359 ± 0.04), meaning that the receptor is neither trafficked to the membrane nor sent immediately for degradation. For completeness, we also checked mitochondria (Figure 6G, 0.359 ± 0.04) and peroxisomes (Figure 6H, 0.343 ± 0.06), but found no co-localization with 5-ht_5b.

An immune-gold electron microscopic analysis from mouse brainstem revealed that 5-ht_5b receptors seemed integrated in vesicular organelles (Figures 7A,B), consistent with the light microscopic images (Figures 6A–H). The G-protein binding domain of a receptor integrated into organelles would be accessible from the cytoplasm (Figure 7C). To determine whether 5-ht_5b, which has lost its capability to be expressed at the plasma membrane, can still serve its function as a receptor and interact with G-proteins, we studied the signaling pathway of the full-length 5-ht_5b receptor in neuroblastoma cells by co-expressing G_αi or G_αs protein subunits. A GTPγS-assay revealed that cells co-expressing 5-ht_5b receptors and G_αi proteins exhibited a 220% increase of the baseline signal. Cells co-expressing 5-ht_5b receptor and G_αs as well as the mock-transfected control did not show any significant changes. The expression of G_αi alone resulted in a minor increase of 39% (Figure 7D). Overexpression of fluorescently labeled G_αi protein led to a faint membrane translocation of otherwise strictly intracellular 5-ht_5b, indicating protein–protein interaction (Figure 7D). Although we found no cell surface expression using recombinant fusion constructs (Figures 6A–H), we need to note that early investigations found a reaction of 5-ht_5b to stimulation by serotonin in rodents (Matthes et al., 1993), thus a small amount of receptor may reach the cell surface.