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An RNA interference screen identifies druggable regulators of MeCP2 stability

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Science Translational Medicine  23 Aug 2017:
Vol. 9, Issue 404, eaaf7588
DOI: 10.1126/scitranslmed.aaf7588

An HIP strategy for treating MeCP2 disorders

The role of altered gene dosage is increasingly being recognized in neuropsychiatric disorders and intellectual disability (ID). Even a twofold change in the dose of methyl-CpG–binding protein 2 (MeCP2)—either increased or decreased—results in distinct disorders with overlapping features including ID, autistic behavior, and severe motor dysfunction. In a new work, Lombardi et al. identified four regulators of MeCP2 stability and validated regulation of MeCP2 by PP2A and HIPK2 in vivo. They then demonstrated that pharmacological inhibition of PP2A was sufficient to partially rescue MeCP2 overexpression and certain motor abnormalities in a mouse model of MECP2 duplication syndrome.

Abstract

Alterations in gene dosage due to copy number variation are associated with autism spectrum disorder, intellectual disability (ID), and other psychiatric disorders. The nervous system is so acutely sensitive to the dose of methyl-CpG–binding protein 2 (MeCP2) that even a twofold change in MeCP2 protein—either increased or decreased—results in distinct disorders with overlapping features including ID, autistic behavior, and severe motor dysfunction. Rett syndrome is caused by loss-of-function mutations in MECP2, whereas duplications spanning the MECP2 locus result in MECP2 duplication syndrome (MDS), which accounts for ~1% of X-linked ID. Despite evidence from mouse models that restoring MeCP2 can reverse the course of disease, there are currently no U.S. Food and Drug Administration–approved therapies available to clinically modulate MeCP2 abundance. We used a forward genetic screen against all known human kinases and phosphatases to identify druggable regulators of MeCP2 stability. Two putative modulators of MeCP2, HIPK2 (homeodomain-interacting protein kinase 2) and PP2A (protein phosphatase 2A), were validated as stabilizers of MeCP2 in vivo. Further, pharmacological inhibition of PP2A in vivo reduced MeCP2 in the nervous system and rescued both overexpression and motor abnormalities in a mouse model of MDS. Our findings reveal potential therapeutic targets for treating disorders of altered MECP2 dosage.

INTRODUCTION

The human brain’s requirement for precise gene dosage is clear from the overrepresentation of copy number variants (CNVs) in individuals with neuropsychiatric disorders, such as autism spectrum disorder (ASD), intellectual disability (ID), and schizophrenia (13). A prime example of this dosage sensitivity is embodied by methyl-CpG–binding protein 2 (MECP2) disorders. Devastating neurological disorders characterized by ID, autistic features, and motor dysfunction result from both a twofold increase in MeCP2 protein—as experienced by males with MECP2 duplication syndrome (MDS)—and a decrease or loss of the protein in ~50% of cells occurring in females with Rett syndrome (4). MDS accounts for ~1% of X-linked ID and is further distinguished by epilepsy and premature death (5, 6). Mouse models recapitulate patient symptoms, because male mice expressing twice the normal amount of MeCP2, MECP2TG1, exhibit decreased motor activity, alterations in learning and memory, decreased social interactions, increased anxiety, and seizures reminiscent of patients with MDS (7, 8). Phenotypic severity in mice is further increased with a threefold overexpression of MeCP2, consistent with the devastating nature of observed triplications spanning the MECP2 locus in humans (79). Conversely, classic Rett syndrome is caused in more than 95% of the cases by loss-of-function mutations in MECP2 and occurs in 1 of 10,000 live female births (10, 11). Male mice with even a 50% reduction of MeCP2 exhibit phenotypes reminiscent of Rett syndrome (12). Thus, although it is clinically and experimentally clear that the dose of MeCP2 must be precisely regulated to permit proper neural function, there are currently no U.S. Food and Drug Administration–approved avenues to modulate MeCP2 (11, 13, 14).

MeCP2 binds preferentially to methylated DNA but localizes broadly across the genome (15, 16). In mature neurons, its abundance is similar to that of the histone octamer that makes up nucleosomes (15). Loss of MeCP2 results in various chromatin changes including disruption of chromatin architecture, as observed by mislocalization of the transcriptional regulator ATRX (α-thalassemia/mental retardation, X-linked) (1719) and increased linker histone H1 (15). Loss of MeCP2 also results in misregulation of numerous neuronally important transcripts, such as those encoded by brain-derived neurotrophic factor (Bdnf) (18, 20, 21) and corticotropin-releasing hormone (Crh) (8). Most of these molecular alterations also occur in gain-of-function models but are inversely misregulated. At the cellular level, neurons lacking MeCP2 are hypofunctional, exhibiting decreased soma size (2224) and reduced dendritic branching (2527). On the other hand, neurons from the MDS mouse model display increased synapse density and dendritic arborization (28, 29). Neurological phenotypes are largely reversible in both Rett syndrome and MDS mouse models by normalization of MeCP2 expression (30, 31), consistent with the absence of neurodegeneration and gross anatomical abnormalities. Previous attempts to correct specific molecular abnormalities identified in Mecp2 mutant mice, such as normalization of BDNF or CRH, resulted in only partial phenotypic rescue (8, 32). We posit that given the broad scope of a chromatin protein’s regulon, it is likely that a constellation of misregulation drives the phenotypes in both loss- and gain-of-function MECP2 syndromes. Thus, we propose that the most efficacious treatment of these disorders will involve modulating expression of the MeCP2 protein itself.

For a protein whose expression must be tightly regulated, little is known about the factors that affect MeCP2 turnover or stability. Although regulated posttranscriptionally by various microRNAs (3335), the impact of MeCP2’s numerous posttranslational modifications—including phosphorylation, acetylation, methylation, sumoylation, and ubiquitination—on its stability is largely unknown (36). Given the exquisite sensitivity of brain cells to the amount of MeCP2, we hypothesized that there are multiple endogenous regulators of MeCP2 stability. Thus, the goal of this work was to perform a forward genetic screen to uncover potentially druggable modulators of MeCP2 stability.

RESULTS

Identification of posttranslational regulators of MeCP2 stability

To develop a reporter cell line in which we could monitor MeCP2 expression, we selected Daoy human medulloblastoma cells for screening because of their high small interfering RNA (siRNA) transfection efficiency and their endogenous expression of MeCP2, increasing the probability of regulatory circuits being present for perturbation. Daoy cells were transduced with a lentiviral vector that expresses Discosoma sp. red fluorescent protein (DsRed)–internal ribosomal entry site (IRES)–human MeCP2 (hMeCP2)–enhanced green fluorescent protein (EGFP). This bicistronic transgene allows for unified transcription, but independent translation, of the fluorescent protein DsRed and hMeCP2 with EGFP fused to its C terminus (Fig. 1A). This system robustly controls for variations in transcription of the transgene by normalization to DsRed fluorescence (37, 38), allowing for precise quantification of MeCP2-specific protein regulation. After infection of Daoy cells with lentivirus containing the transgene, cells were analyzed and sorted by fluorescence-activated cell sorting for cells positive for both red and green fluorescence. Multiple stable DsRed-IRES-hMeCP2-EGFP–expressing cell lines were assessed for their suitability for the screen based on their expression of both fluorescent proteins in the experimental range and their sensitivity to short hairpin RNA (shRNA) and siRNA perturbation of MeCP2-EGFP. We chose a clonal cell line with matched dual fluorescence and minimal expression variation for the genetic screen (Fig. 1B).

Fig. 1. Screening for posttranslational regulators of MeCP2.

(A) The bicistronic reporter transgene allowed for monitoring of MeCP2 stability via C-terminal fusion of EGFP while controlling for variation in transgene expression by normalization to the independently translated DsRed. (B) Flow cytometry analysis of a clonal Daoy DsRed-IRES-hMeCP2-EGFP reporter cell line, indicating dual fluorescence and minimal variation in expression. (C) Schematic for arrayed siRNA screen against all known and putative human kinases and phosphatases by flow cytometry analysis of the MeCP2-EGFP/DsRed ratio. (D) One hundred eighty-one siRNAs significantly (P < 0.01) altered the MeCP2-EGFP/DsRed ratio in the primary screen against human phosphatases (711 siRNAs total). (E) Two hundred ninety-nine siRNAs significantly (P < 0.01) altered the MeCP2-EGFP/DsRed ratio in the primary screen against human kinases (1908 siRNAs total). (F) A secondary screen using independent siRNAs confirmed 120 significant siRNAs affecting the MeCP2-EGFP/DsRed ratio. siRNAs that also significantly altered a GFP control cell line were eliminated, revealing 43 MeCP2-specific siRNAs that represent 33 genes.

Using the developed Daoy DsRed-IRES-hMeCP2-EGFP–expressing cell line, we probed the ability of all known human kinases and phosphatases (873 genes per 2619 siRNAs) to modulate MeCP2 protein stability. The reporter cell line was transfected in triplicate for each siRNA (three siRNAs per gene), and the amount of MeCP2-EGFP was assessed using flow cytometry (Fig. 1C). To control for variance in transgene transcription, the MeCP2-EGFP/DsRed ratio was assessed for each siRNA and compared to the effect of three scrambled control siRNAs. We identified 480 siRNAs that significantly (P < 0.01) affected the MeCP2-EGFP/DsRed ratio (Fig. 1, D and E). To enrich for true candidates to follow-up, we required that two of the three siRNAs targeting a candidate gene significantly affected the MeCP2-EGFP/DsRed ratio in the same direction and that the candidate gene be expressed in the brain. This resulted in a list of 81 genes for which we wanted to distinguish MeCP2-specific effects from IRES-EGFP effects. Thus, for the secondary screen, we compared siRNA effects on the EGFP/DsRed ratio in the original DsRed-IRES-hMeCP2-EGFP cell line and a second cell line expressing DsRed-IRES-EGFP alone. This secondary screen was also performed with a set of siRNAs independent of those used in the original screen to reduce the likelihood of any off-target effects and to generate greater confidence in the candidates that continued to show a significant effect on the MeCP2-EGFP/DsRed ratio. This secondary screen validated 33 candidate modulators of MeCP2 (table S1) while eliminating genes whose knockdown altered EGFP levels irrespective of MeCP2 (Fig. 1F).

Validation using endogenous MeCP2 reveals known and unknown regulators

After the validation of siRNAs that specifically affected MeCP2, we wanted to ensure that these candidate genes could modulate endogenous MeCP2, as opposed to MeCP2 overexpressed from the transgene, and that this modulation could be recapitulated in an independent cell type. To this end, we performed a final round of validation in human embryonic kidney (HEK) 293T cells that exhibit robust endogenous MeCP2 expression, using three shRNAs against each candidate gene. Immunoblot analysis was used to determine the effects of candidate knockdown on endogenous MeCP2; RNA analysis enabled evaluation of the efficiency of the knockdown for the candidate modulator. We required the extent of knockdown of the candidate to tightly correspond to effects on MeCP2 protein abundance. Four candidate modulators emerged from this analysis: one phosphatase subunit, protein phosphatase 2 regulatory subunit 1α (PPP2R1A) (Fig. 2A), and three kinases (Fig. 2, B to D). One of these three kinases is a known kinase for MeCP2, homeodomain-interacting protein kinase 2 (HIPK2) (Fig. 2B) (39), intimating the physiological relevance of our screening strategy.

Fig. 2. Validation using endogenous MeCP2 reveals known and unknown regulators.

The effects of candidate knockdown (KD) on endogenous MeCP2 were tested in HEK293T cells along with RNA analysis to determine which shRNAs down-regulated the target. For each candidate, three shRNAs (a to c) were transfected along with vectors expressing scrambled shRNA and shRNA targeting MECP2. Two days after transfection, puromycin was applied to select for cells expressing the silencing plasmid. Selection was maintained for 4 days, and then cells were split for parallel immunoblot analysis (top) and RNA analysis (bottom). (A) The effects of PPP2R1A knockdown on endogenous MeCP2 levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Coupled RNA analysis (bottom) indicated that only the shRNAs "a" and "c," which resulted in significant knockdown of PPP2R1A, decreased MeCP2 protein. (B) As in (A), but for HIPK2. (C) As in (A) and (B), but for HIPK1. (D) As in (A) to (C), but for RIO kinase 1 (RIOK1). n = 3 to 4 cultures per group. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

To determine how these four candidates might regulate MeCP2 protein, we first determined whether the observed decreases in MeCP2 amount occurred posttranscriptionally. For this analysis, RNA from HEK293T cells treated with our candidate shRNAs was assayed for MECP2 transcripts by quantitative real-time polymerase chain reaction. Despite decreases in MeCP2 protein, no significant decreases in MECP2 transcript were detected, except in cells treated with the shRNA targeting MECP2 (fig. S1). Thus, our screening approach using only the coding sequence of MECP2 identified posttranslational regulators of MeCP2.

Posttranslational regulation of MeCP2 by HIPK1 and HIPK2

Next, we sought to assess which of the kinase candidates might directly modify MeCP2. First, we validated our assay by recapitulating previous in vitro results demonstrating the ability of the nuclear kinase HIPK2 to phosphorylate MeCP2 (39). Incubation of recombinant MeCP2 C-terminally tagged with chitin-binding protein (CBP) and HIPK2 with [γ-32P]ATP (adenosine 5′-triphosphate) resulted in both HIPK2 autophosphorylation and MeCP2-CBP phosphorylation (Fig. 3A, lane 1). In contrast, incubating MeCP2-CBP only with [γ-32P]ATP resulted in no detectable radioactivity (Fig. 3A, lane 3). To control for the possibility that the radioactivity associated with MeCP2-CBP was due solely to CBP phosphorylation, chitin-bound MeCP2-CBP from the reaction was washed, such that HIPK2 was depleted (lane 4), and eluted by tag cleavage (lane 5). The radioactivity of tagless MeCP2 demonstrated the specificity of HIPK2’s kinase activity for MeCP2.

Fig. 3. Both HIPK1 and HIPK2 phosphorylate MeCP2 at S216.

(A) Autoradiogram (top) and Coomassie-stained gel (bottom) of kinase reactions with the indicated recombinant proteins in the presence of [γ-32P]ATP. Incubation of HIPK2 and MeCP2-CBP resulted in HIPK2 autophosphorylation and phosphorylation of MeCP2 (lane 1), whereas MeCP2-CBP without HIPK2 resulted in no detectable radioactivity (lane 3). To control for epitope phosphorylation, chitin-bound MeCP2-CBP incubated with HIPK2 was washed (lane 4) and then eluted by tag cleavage (lane 5) (n = 3). GST, glutathione S-transferase. (B) As in (A), but with HIPK1 (n = 3). (C) Tandem mass spectrum of pS216-containing peptides resulting from incubation of HIPK2 with purified MeCP2-CBP. Parental ions, green; b ions, red; y ions, blue. m/z, mass/charge ratio. (D) Percent peptide phosphorylated relative to the sum of modified and unmodified peptides for those peptides containing S80 or S216 for HIPK2 (left) and HIPK1 (right). Area under the peptide curve was used for relative quantitation (n = 5). (E) Incubation of HIPK2 with purified wild-type (WT) MeCP2-CBP or with MeCP2 that could not be phosphorylated at S216, MeCP2 S216A–CBP (n = 3). (F) Immunoprecipitation of MeCP2-EGFP from HEK293T cells demonstrated a HIPK-dependent increase in pS216 comparable to pS80. Cells were harvested 48 hours after cotransfection. *, nonspecific band (n = 3). (G) Stable Daoy cell lines encoding doxycycline-inducible wild-type or S216D MECP2-GFP were used to investigate differential protein dynamics. After inducing transgene expression with doxycycline (Dox) for 48 hours, cells were provided with fresh doxycycline-deficient medium for continued growth (t = 0). −, uninduced (n = 6). (H) MeCP2 protein decreased relative to vinculin in Hipk2−/− mutant mice whole-brain lysates. Mice were 2 to 4 months old (n = 5). *P < 0.05, **P < 0.01, two-tailed t test.

We next tested the ability of HIPK1 to phosphorylate MeCP2 and found that incubation of MeCP2-CBP and HIPK1 with [γ-32P]ATP resulted in both HIPK1 and MeCP2 phosphorylation (Fig. 3B). Given the previous work identifying S80 as the primary phosphorylation site on MeCP2 by HIPK2 (39), we sought to determine whether this was also true for HIPK1. To our surprise, incorporated radioactivity did not appear decreased when the only substrate provided to either HIPK2 or HIPK1 was MeCP2 that could not be phosphorylated at S80, MeCP2 S80A (fig. S2A). This suggested the presence of more HIPK2 and HIPK1 phosphorylation sites on MeCP2. To determine what these additional phosphorylation sites might be, we performed tandem mass spectrometry on MeCP2 alone and MeCP2 incubated with either HIPK2 or HIPK1. Two primary phosphosites emerged from this analysis for both HIPK2 and HIPK1, pS80 and pS216 (Fig. 3C). Both sites are HIPK kinase consensus sites (Ser/Thr-Pro) (39, 40) and are highly conserved in MeCP2 orthologs from zebrafish to human (fig. S2B). Relative quantification of peptide phosphorylation was achieved by comparing the area under the curve for each phosphorylated peptide to the total area for modified and unmodified forms of the peptide (Fig. 3D). The apparent percent phosphorylation of S216 indicated the robustness of this modification by both HIPK2 and HIPK1.

To investigate the biological relevance of pS216, we performed tandem mass spectrometry on MeCP2-EGFP immunoprecipitated from brains of transgenic mice expressing the epitope-tagged human gene at approximately endogenous levels (19). Phosphorylation of S216 was detected in immunoprecipitated MeCP2 on ~10% of the corresponding peptides (fig. S2C). Having demonstrated that pS216 occurs in vivo in mouse whole brain, we developed a phospho-specific antibody for pS216 to assess whether this phosphorylation was a bona fide target of the HIPK kinases. Repeating in vitro kinase assays with recombinant wild-type MeCP2 and MeCP2 that could not be phosphorylated at S216 (S216A) demonstrated the ability of both HIPK2 (Fig. 3E) and HIPK1 (fig. S2D) to specifically mediate this phosphorylation. Further, S216 phosphorylation occurred in cells upon expression of either HIPK2 or HIPK1 at similar levels to pS80 (Fig. 3F). Thus, biochemical analyses uncovered both an additional MeCP2 kinase and a new HIPK-mediated MeCP2 phosphorylation site.

To begin to characterize the impact of pS216 on MeCP2’s stability, we generated stable cell lines expressing doxycycline-inducible wild-type MeCP2 or MeCP2 mutated to mimic constitutive S216 phosphorylation (S216D). After induction of transgenic expression, doxycycline was removed, and MeCP2 protein decay was assessed. Phosphomimetic MeCP2 S216D exhibited increased stability relative to wild type (Fig. 3G), suggesting that pS216 may stabilize MeCP2. Consistent with the model of HIPK2-mediated phosphorylation acting to stabilize MeCP2, MeCP2 was decreased in the brains of Hipk2−/− homozygous mice (Fig. 3H).

Unlike the HIPK kinases, RIOK1 is a member of a family of atypical serine-threonine kinases that function in ribosomal 40S biogenesis. RIO kinases lack canonical kinase activation loops and have no known substrates, suggesting that they act as adenosine triphosphatases in ribosome assembly rather than kinases (41, 42). We did not detect any RIOK1 kinase activity toward MeCP2 (fig. S3). Thus, it is unlikely that RIOK1’s stabilization of MeCP2 resulted from phosphorylation by RIOK1.

Inhibition of PP2A decreases MeCP2 in vivo

Protein phosphatase 2A (PP2A) functions as an obligate dimer of the catalytic subunit and the scaffold, the most abundant isoform of which is encoded by PPP2R1A (43). The scaffold further binds to regulatory proteins that modulate the enzymatic activity and specificity of the catalytic subunit (44). To determine the effect of Ppp2r1a knockdown in the mouse brain, we optimized a viral delivery system in which adeno-associated virus 8 (AAV8)–encoding shRNA is injected into the lateral ventricles of neonates, resulting in widespread cortical and hippocampal infection (45). To perform this experiment, two different AAV vectors were used: one encoding shRNA against Ppp2r1a, and the other encoding scrambled shRNA. Knockdown of Ppp2r1a decreased MeCP2 in the cortex, with the decrease in MeCP2 tightly corresponding to the decrease in PPP2R1A (Fig. 4A). This regulation was not specific to cortical tissue, because MeCP2 was also decreased in hippocampal lysates (fig. S4). We performed a subsequent series of injections of AAV vectors encoding shRNAs to test whether the observed decrease in MeCP2 occurred because of transcriptional or posttranscriptional regulation. Cortical lysates were fractionated for protein and RNA analyses on the same tissue. Consistent with the findings of the cell-based screen, knockdown of Ppp2r1a decreased MeCP2 protein without affecting Mecp2 transcript levels (Fig. 4B). Thus, PPP2R1A appeared to regulate MeCP2 posttranscriptionally.

Fig. 4. Inhibition of PP2A decreases MeCP2 in vivo.

(A) Intraventricular injection into postnatal day 0 wild-type mice of AAV8 encoding shRNA targeting Ppp2r1a resulted in decreased PPP2R1A and MeCP2 protein compared to animals injected with virus expressing a scrambled shRNA (Scr). Brain cortical tissue was harvested at 2 weeks after injection (n = 6). (B) As in (A), but an independent cohort of postnatal day 0 mice in which cortical lysates were generated in TRIzol for protein and RNA analysis of the same tissue (n = 8). (C) Decreased MeCP2 occurred upon treatment of cerebellar granule neuron precursors with the PP2A inhibitors okadaic acid (OKA) [median inhibitory concentration (IC50), 0.1 to 0.5 nM] and fostriecin (FOS) (IC50, 1.5 nM). Neurons were harvested after 9 days of treatment (n = 3). MeOH, methanol. (D) Decreased MeCP2 in cortical tissue upon intraventricular injection of 40 ng of okadaic acid compared to injection with solvent [2% dimethyl sulfoxide (DMSO)] in both wild-type (n = 8) and an MDS mouse model, MECP2TG1 (TG1) (n = 6). Brain cortices were harvested 2 weeks after injection. (E) Decreased MeCP2 in cortical tissue upon intraventricular injection of 240 μM fostriecin compared to injection with solvent (saline) in both wild-type (n = 4) and MECP2TG1 mice (TG1, n = 6). Brain cortices were harvested 2 weeks after injection. (F) Fostriecin treatment of MECP2TG1 mice partially rescued abnormal motor persistence on the accelerating rotating rod as evidenced by decreased latency to fall compared to MECP2TG1 mice treated with saline alone (Sal). Mice were assessed 2 weeks after injection. n = 10 to 16. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test (A to E) and repeated-measures two-way analysis of variance (ANOVA), by day, followed by post hoc t tests (F).

To test whether this regulation by PPP2R1A was occurring through the PP2A holoenzyme (catalytic subunit and scaffold), we determined the effect of two distinct PP2A catalytic inhibitors, okadaic acid and fostriecin (44), on endogenous MeCP2 in cultured cerebellar granule neurons. Treatment with either inhibitor decreased MeCP2 levels (Fig. 4C), suggesting that the observed decrease in MeCP2 upon PPP2R1A/Ppp2r1a knockdown was the result of decreased PP2A catalytic activity. Okadaic acid’s activity was not restricted to murine MeCP2, because it also decreased human MeCP2 in medulloblastoma cells (fig. S5A). This decrease in hMeCP2 occurred at an okadaic acid dose sufficient to result in the hyperphosphorylation of PP2A’s neuronal substrate tau (fig. S5B) (46). Given the resultant feasibility of pharmacological intervention, we next sought to determine whether these findings from cultured neurons could be translated into the mature mouse brain.

Cognizant of the potential limitations of any single drug, we tested both okadaic acid and fostriecin in vivo. Each PP2A inhibitor was intracerebroventricularly injected into 7-week-old wild-type mice, and the effects on MeCP2 relative to the solvent-matched control were detected by immunoblotting. In vivo treatment with either okadaic acid or fostriecin decreased MeCP2 (Fig. 4, D and E, and fig. S6), suggesting that the decrease in MeCP2 occurred via inhibition of PP2A rather than any divergent off-target activities. Next, we sought to determine the effect of PP2A inhibition in a mouse model of MDS, MECP2TG1. The extra copy of MECP2 in MECP2TG1 mice is the human gene, making this a good model for assessing regulation of human MeCP2 in vivo (7). Inhibition of PP2A via okadaic acid or fostriecin markedly decreased MeCP2 in MDS mice [Fig. 4, D (P = 0.02) and E (P = 0.01)], partially rescuing MeCP2 overexpression. Although mice treated with okadaic acid, which is a more potent PP2A inhibitor than fostriecin, maintained brain weights indistinguishable from solvent-treated animals (fig. S7A), we chose to move forward with fostriecin, the more selective PP2A inhibitor (47), to determine whether partial rescue of MeCP2 overexpression might result in behavioral benefit. Given the short time scale of treatment because of the limitations of using fostriecin in the long term (requiring more invasive cannulation), we assayed treated mice for the behavior that is the most sensitive to MeCP2 levels and the first to be rescued upon genetic normalization of MeCP2 in MECP2TG1 mice, the accelerating rotating rod (rotarod) (30). MECP2TG1 mice exhibited abnormal perseverance on the rotarod with the greatest deviation from wild type occurring on the second day of trials (7, 30). A 20 to 25% decrease in MeCP2 in MECP2TG1 mice treated with fostriecin was sufficient to limit their rotarod persistence on day 2 relative to MECP2TG1 mice treated with saline (vehicle). By the final trial of day 2, fostriecin-treated MECP2TG1 mice were not distinguishable from wild-type animals but were significantly different from MECP2TG1 mice treated with saline (Fig. 4F). Unlike MECP2TG1 mice, wild-type mice treated with fostriecin exhibited no decrease in rotarod performance (fig. S7B). Thus, genetic and pharmacological inhibition of PP2A in vivo destabilized MeCP2, partially rescuing both MeCP2 overexpression and motor abnormalities in a mouse model of MDS.

DISCUSSION

The role of altered gene dosage is increasingly being recognized in neuropsychiatric disorders. Examples include a variety of aberrations from single duplications (PMP22) and deletions (SHANK3) to classic aneuploidies, such as Down syndrome (1). The exponential rise in diagnosed cases of autism and ID, coupled with our increasing awareness of de novo CNVs, necessitates innovation in our approaches to developing treatments for these disorders. To evaluate the feasibility of identifying modifiers of dose-sensitive disease drivers, we began with MECP2 due to the validity of available mouse models of disease (48). Even without a direct genetic basis, abnormalities in MeCP2 expression have been observed in ASD, Prader-Willi syndrome (49, 50), and ID patients with NUDT21-spanning CNVs (51), suggesting that modulation of MeCP2 may serve a broad therapeutic purpose. Here, we have identified four regulators of MeCP2 stability and have validated regulation of MeCP2 by PP2A and HIPK2 in vivo. We have demonstrated that pharmacological inhibition of PP2A was sufficient to partially rescue MeCP2 overexpression and certain motor abnormalities in a mouse model of MDS.

The feasibility of pharmacological intervention propelled us to use PP2A as our proof-of-principle candidate in a mouse model of MDS. Both knockdown of Ppp2r1a and pharmacological inhibition of PP2A’s catalytic activity decreased MeCP2 in vivo. Consistent with the screen’s design, inhibition of PP2A also decreased human MeCP2 in the MECP2TG1 mouse model of MDS, in which the extra copy of MECP2 is the human gene. Notably, two distinct inhibitors of PP2A produced similar effects on MeCP2. Further, having more than one inhibitor to choose from enabled us to then move forward to behavioral studies with the most selective PP2A inhibitor. Our results suggest that even partial rescue of MeCP2 overexpression in MECP2TG1 mice may be sufficient to alleviate some behavioral abnormalities.

Given that the prime purpose of this study was to demonstrate a potent cell-based screening strategy for druggable regulators of dose-sensitive genes, it is important to note that our in vivo drug studies are preliminary in nature. Okadaic acid and fostriecin are commercially available tool compounds that were used to validate the regulation of MeCP2 by PP2A in vivo. Future work will require in-depth pharmacological studies of optimized drugs, as opposed to tool compounds. In addition, we believe that it may be necessary to delve further into the mechanism of PP2A’s regulation of MeCP2 to enable more precise pharmacological manipulation. However, the emergence of PPP2R1A loss-of-function mutations as dominant drivers of ID (52) suggests that we have homed in on an important aspect of neuronal physiology.

The identification of a known MeCP2 kinase was unanticipated, given that HIPK2 activity has not been reported to affect the stability of MeCP2. HIPK2 and HIPK1 are nuclear kinases whose phosphorylation of chromatin-associated proteins often modulates their ability to regulate transcription (5356). Here, we recapitulated MeCP2 S80 phosphorylation by HIPK2, as previously shown in cultured cells (39). Further, we demonstrated that this phosphorylation can also be mediated by HIPK1. pS80 appears to be a constitutive MeCP2 phosphorylation site in rodent brain that, when mutated, decreases the amount of chromatin-associated MeCP2 in vivo (57), consistent with potential regulation of both MeCP2’s chromatin function and possibly its stability. We also uncovered a new HIPK-mediated MeCP2 phosphorylation site, pS216. This phosphorylation site has been detected by numerous groups, both in rodents (5759) and humans (6064), but not in brain tissue and thus far has been an orphan MeCP2 phosphorylation site. It is tantalizing to note that this serine-proline motif is conserved from zebrafish to human and falls within MeCP2’s transcriptional repression domain, a domain critical for mediating MeCP2’s interaction with coregulators such as the NCoR/SMRT (nuclear receptor corepressor/silencing mediator for retinoid and thyroid receptors) complex (65, 66). Moving forward, it will be important to determine whether the apparently increased stability of the MeCP2 S216D phosphomimetic is due to a change in MeCP2 interactors.

Consistent with the model of HIPK2-mediated phosphorylation acting to stabilize MeCP2, MeCP2 was found to be decreased in the brains of Hipk2−/− homozygous mice. However, given the genetic redundancy of Hipk1 and Hipk2 (67), the extent of HIPK regulation of MeCP2 will not be clear until the function of both genes is restricted simultaneously. Further experimentation will be required to determine whether MeCP2 S80 and MeCP2 S216 are HIPK1 and HIPK2 phosphorylation sites in vivo and whether these phosphorylation events mediate the apparent stabilization of MeCP2 by HIPK1 and HIPK2.

The fact that our screening approach identified multiple regulators of MeCP2 stability may hold promise beyond uncovering individual druggable targets. Recognizing that inhibiting any one target to effectively normalize MeCP2 might lead to some untoward effects or toxicity, we propose that future studies should focus on partial inhibition of targets that function in two or more independent pathways. In this manner, multiple minor inhibitions would converge on MeCP2 resulting in cumulative MeCP2 normalization without the potential for toxicity from fully inhibiting any one pathway.

Together, we believe we have uncovered four potential biological entry points for therapeutic intervention in patients with MDS and potentially other disorders in which MeCP2 is increased. Further, greater dissection of these nodes of MeCP2 regulation may enable the identification of genes that, when inhibited, increase MeCP2 protein. Such genes could prove valuable therapeutically as certain missense Rett-causing alleles may be amenable to stabilizing therapy or treatment aimed at increasing MeCP2. Specifically, the Mecp2T158A mouse model of the single most abundant Rett-causing MECP2 mutation, T158M, exhibits decreased MeCP2 (68). However, this mutant allele exhibits only a minor decrease in methyl-CpG binding, suggesting that increasing the nuclear concentration of MeCP2 T158A would, in theory, rescue both association with methyl-CpG and any phenotypes resulting from decreased total protein. A recent study showed that increasing expression of MeCP2T158M in mice bearing a T158M knock-in allele rescued multiple RTT-like phenotypes and enhanced the binding of MeCP2T158M to DNA (69). Thus, the ability to discover both positive and negative regulators of MeCP2, as well as the nuances of the underlying biology, may represent an advantage of genetic screening.

MATERIALS AND METHODS

Study design

The primary objective of this study was the identification of genes that modulate MeCP2 protein. The number of biological replicates (n) per experiment is noted in each figure legend. All littermates were randomized before treatment. All mouse experiments were replicated in an independent cohort. An individual blinded to the genotype and treatment performed the behavioral assay and acquired the data. No outlier removal was performed.

Protein analysis

Cultured cells were washed in cold phosphate-buffered saline, lysed in 100 mM tris-HCl (pH 8.0) and 2% SDS supplemented with protease inhibitors (Roche), rotated for 20 min, and then centrifuged at 13,000g. After centrifugation, the supernatant was mixed with 2× Laemmli buffer, heated for 5 min at 95°C, and electrophoresed on a NuPAGE 4 to 12% bis-tris gradient gel. Mouse tissue lysates were similarly prepared but with Dounce homogenization. After electrophoresis, gels were transferred onto polyvinylidene difluoride membranes and probed with rabbit anti-MeCP2 (1:3000; Zoghbi Laboratory, #0535) (70), rabbit anti-GST (1:2000; Sigma-Aldrich, G7781), rabbit anti-HIPK2 pY361 (1:500; Invitrogen), rabbit anti-MeCP2 pS80 (1:500; Active Motif), rabbit anti-PPP2R1A (1:3000; Abcam, ab154551), mouse anti-tau (1:2500; Abcam, ab80579), rabbit anti-tau pS356 (1:1000; Abcam, ab75603), mouse anti-GAPDH 6C5 (1:20,000; Advanced ImmunoChemical, 2-RGM2), and mouse anti-vinculin (1:10,000; Sigma-Aldrich). Secondary antibodies were mouse anti-rabbit horseradish peroxidase (HRP) (1:3000; Jackson ImmunoResearch Laboratories, 211-032-171) and donkey anti-mouse HRP (1:50,000; Jackson ImmunoResearch Laboratories, 715-035-150). Immunoblot images were acquired with the ImageQuant LAS 4000 (GE Healthcare) and quantified with ImageJ.

Statistical analysis

The number of animals (n), statistical tests, and α levels used are indicated for each experiment in the figure legends. Error bars in all figures indicate SEM. ANOVA was calculated using the aov function in R 3.3.1 software.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/9/404/eaaf7588/DC1

Materials and Methods

Fig. S1. Candidate knockdown did not decrease MECP2 transcripts.

Fig. S2. MeCP2 pS216 occurred in vivo.

Fig. S3. No RIOK1 kinase activity for MeCP2 was detected.

Fig. S4. Ppp2r1a knockdown also decreased MeCP2 in mouse hippocampal tissue.

Fig. S5. Okadaic acid treatment was active against human MeCP2.

Fig. S6. In vivo fostriecin titration.

Fig. S7. In vivo PP2A inhibition resulted in no ill effects in wild-type mice at the doses used.

Table S1. MeCP2-specific siRNA gene list.

Table S2. Primers used.

Table S3. shRNAs used.

References (7174)

REFERENCES AND NOTES

Acknowledgments: We thank C. Spencer for motor behavioral analysis training, Y. Shao for stereotactic injection training, and J.-Y. Kim for postnatal day 0 intraventricular injection training. We also thank L. Lavery, T. Westbrook, and M. Rousseaux for their advice. Finally, we thank J. Jankowsky and S. Grunke for their gift of the pAAV-U6-miR-Cag-tdT vector. Funding: This project was funded by the NIH (5R01NS057819 to H.Y.Z.), the Rett Syndrome Research Trust and 401K Project from MDS families, and the Howard Hughes Medical Institute (L.M.L. and H.Y.Z.). This work was also made possible by the following Baylor College of Medicine core facilities: Cell-Based Assay Screening Service (NIH, P30 CA125123), Cytometry and Cell Sorting Core (National Institute of Allergy and Infectious Diseases, P30AI036211; National Cancer Institute P30CA125123; and National Center for Research Resources, S10RR024574), Pathway Discovery Proteomics Core, the DNA Sequencing and Gene Vector Core (Diabetes and Endocrinology Research Center, DK079638), and the mouse behavioral core of the Intellectual and Developmental Disabilities Research Center (NIH, U54 HD083092 from the National Institute of Child Health and Human Development). Author contributions: L.M.L. performed all the molecular experiments. L.M.L., M.Z., Y.S., S.A.B., and H.Y.Z. contributed to the concept and design of the experiments. M.Z. generated the mutant vectors. Y.S. performed the okadaic acid stereotactic injections. S.A.B. assisted with the in vivo immunoblot analysis. T.J.K. generated the doxycycline-inducible stable cell lines. A.A.T. and E.J.H. provided the Hipk2−/− mutant mouse brains. L.M.L., M.Z., S.A.B., and H.Y.Z. collected, analyzed, and interpreted the data. L.M.L. and H.Y.Z. wrote and edited the paper. Competing interests: H.Y.Z. is on the board of Regeneron Pharmaceuticals and is a scientific advisor to the Chan Zuckerberg Initiative, Denali Therapeutics, and AveXis. The other authors declare that they have no competing interests. Data and materials availability: Correspondence and requests for materials should be addressed to H.Y.Z.
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