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Pharmacological reversal of synaptic and network pathology in human MECP2 ‐KO neurons and cortical organoids
2020; Springer Nature; Volume: 13; Issue: 1 Linguagem: Inglês
10.15252/emmm.202012523
ISSN1757-4684
AutoresCleber A. Trujillo, Jason W. Adams, Priscilla D. Negraes, Cassiano Carromeu, Leon Tejwani, Allan Acab, Ben Tsuda, Charles A. Thomas, Neha Sodhi, Katherine Fichter, Sarah Romero, Fabian Zanella, Terrence J. Sejnowski, Henning Ulrich, Alysson R. Muotri,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle8 December 2020Open Access Transparent process Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids Cleber A Trujillo Corresponding Author [email protected] orcid.org/0000-0002-0675-6437 Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USAThese authors contributed equally to the work Search for more papers by this author Jason W Adams Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA, USA Center for Academic Research and Training in Anthropogeny, University of California San Diego, La Jolla, CA, USAThese authors contributed equally to the work Search for more papers by this author Priscilla D Negraes Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Cassiano Carromeu Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Leon Tejwani Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Allan Acab Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Ben Tsuda Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA, USA Computational Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Charles A Thomas Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Neha Sodhi StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Katherine M Fichter StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Sarah Romero StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Fabian Zanella StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Terrence J Sejnowski Computational Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA Institute for Neural Computation, University of California San Diego, La Jolla, CA, USA Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Henning Ulrich Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil Search for more papers by this author Alysson R Muotri Corresponding Author [email protected] orcid.org/0000-0003-0867-2875 Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Center for Academic Research and Training in Anthropogeny, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Cleber A Trujillo Corresponding Author [email protected] orcid.org/0000-0002-0675-6437 Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USAThese authors contributed equally to the work Search for more papers by this author Jason W Adams Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA, USA Center for Academic Research and Training in Anthropogeny, University of California San Diego, La Jolla, CA, USAThese authors contributed equally to the work Search for more papers by this author Priscilla D Negraes Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Cassiano Carromeu Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Leon Tejwani Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Allan Acab Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Ben Tsuda Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA, USA Computational Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Charles A Thomas Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Neha Sodhi StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Katherine M Fichter StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Sarah Romero StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Fabian Zanella StemoniX Inc, Maple Grove, MN, USA Search for more papers by this author Terrence J Sejnowski Computational Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA Institute for Neural Computation, University of California San Diego, La Jolla, CA, USA Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Henning Ulrich Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil Search for more papers by this author Alysson R Muotri Corresponding Author [email protected] orcid.org/0000-0003-0867-2875 Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA Center for Academic Research and Training in Anthropogeny, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Author Information Cleber A Trujillo *,1, Jason W Adams1,2,3, Priscilla D Negraes1,4, Cassiano Carromeu1,4, Leon Tejwani1,†, Allan Acab1, Ben Tsuda2,5, Charles A Thomas1, Neha Sodhi4, Katherine M Fichter4, Sarah Romero4, Fabian Zanella4, Terrence J Sejnowski5,6,7, Henning Ulrich8 and Alysson R Muotri *,1,3 1Department of Pediatrics/Rady Children's Hospital, Department of Cellular & Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA 2Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA, USA 3Center for Academic Research and Training in Anthropogeny, University of California San Diego, La Jolla, CA, USA 4StemoniX Inc, Maple Grove, MN, USA 5Computational Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA 6Institute for Neural Computation, University of California San Diego, La Jolla, CA, USA 7Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA 8Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil †Present address: Interdepartmental Neuroscience Program, Yale School of Medicine, New Haven, CT, USA *Corresponding author. Tel: +1 858 534 9320; E-mail: [email protected] *Corresponding author. Tel: +1 858 534 9320; E-mail: [email protected] EMBO Mol Med (2021)13:e12523https://doi.org/10.15252/emmm.202012523 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Duplication or deficiency of the X-linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell-derived technologies, including human neurons, MECP2-mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2-knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology. Synopsis Deficiency of the X-linked MECP2 gene profoundly impairs neurodevelopment. Clinically, mutations in MECP2 most commonly present as the severe and untreatable disease Rett syndrome. Innovative human pluripotent stem cell (PSC) technology enables the investigation of therapeutic candidates. Neurons differentiated from human MECP2-KO PSCs showed altered expression of synapse-relevant genes, synaptic morphology, and decreased calcium and network activities compared to control neurons. An in silico neural network simulation affirmed that synaptic structural parameters link with neural network activity, supporting our approach of rescuing synaptic structure and increasing activity. Strategic screening of select drug candidates with mechanisms of action that counteract MECP2-KO deficiencies isolated two lead compounds, Nefiracetam and PHA 543613, for subsequent validation in 3D human cell models. MECP2-mosaic neurospheres, a novel model of Rett female brain mosaicism, showed altered calcium activity that could be reversed by Nefiracetam and/or PHA 543613. Nefiracetam and/or PHA 543613 increased synaptic gene expression and reversed network pathology in human MECP2-KO cortical organoids, arguing for their clinical trial in patients with MeCP2-deficient neurodevelopmental disorders. The paper explained Problem The X-linked gene MECP2 encodes an epigenetic regulatory protein that is critical for typical human brain development. MECP2 deficiency causes severe neurodevelopmental impairment that clinically presents most commonly as Rett syndrome. However, despite pinpointing the genetic disruption and the eminent treatability of the disease, no clinically approved treatments for Rett syndrome are currently available. Results By establishing an innovative drug-screening pipeline using human pluripotent stem cells differentiated into neurons, MECP2-mosaic neurospheres, and cortical organoids, we isolated two lead compounds that specifically reversed synaptic and network pathology resulting from MECP2 deficiency while leaving controls unaffected. The two compounds, Nefiracetam and PHA 543613, improved synaptic morphology and function in MECP2-KO human neurons, increased calcium activity in MECP2-mosaic neurospheres, and reversed synaptic transcriptomic pathways and neural network function in MECP2-KO cortical organoids. Impact MECP2 deficiency causes profound neurodevelopmental impairment that urgently demands treatment. This study identified two pharmacological lead compounds, one of which has already been approved for human use commercially, that could specifically reverse MECP2-KO neurocytopathology in human cell models. These compounds may offer meaningful clinical impact for some patients with severe neurodevelopmental impairment resulting from MECP2 deficiency, and trial in a clinical setting is warranted following regulatory approval. Introduction MECP2 is an X-linked gene that encodes an epigenetic regulatory protein, methyl CpG binding protein-2 (MeCP2), that is critical for typical human brain development (Chahrour et al, 2008; Gonzales & LaSalle, 2010). In addition to an array of other neurodevelopmental disorders (Watson et al, 2001; Shibayama et al, 2004; Samaco et al, 2005), MECP2 loss-of-function mutations are the most common etiology of RTT (Amir et al, 1999), a severe neurodevelopmental disorder clinically characterized by head growth deceleration, profound cognitive decline, regression of acquired abilities, and stereotypies in early infancy (Hagberg et al, 2002). Critically, these syndromic clinical features appear following a brief period of normal development (Hagberg et al, 2002). Although the precise neurobiological changes linking MECP2 mutations to the RTT phenotype are unclear, human postmortem tissue from RTT females portrays reduced brain size, decreased dendritic arborization and spine formation, and reduced synapse numbers (Johnston et al, 2003; Gonzales & LaSalle, 2010). Moreover, studies in murine models of Mecp2 deficiency implicate an accordant deficit in synaptic function, including decreased synaptic transmission and plasticity (Moretti et al, 2006; Zhou et al, 2006; Dani & Nelson, 2009). These findings suggest that synaptic dysfunction is a central pathology of MeCP2 deficiency and may be a viable treatment target (Johnston et al, 2003; Gonzales & LaSalle, 2010). Therapeutically targeting pathways downstream from MECP2 has been proposed as a RTT treatment strategy (Braat & Kooy, 2015; Pozzo-Miller et al, 2015; Katz et al, 2016; Benke & Möhler, 2018), and the narrow window of typical development suggests intervention during this period to bolster synaptic function may be opportune to preserve clinical function and ameliorate subsequent decline. Development and discovery of therapeutics for RTT and other neurodevelopmental disorders have been challenged by limited opportunity to investigate disease pathogenesis in a human model. Advances in pluripotent stem cell (PSC) technology, including three-dimensional neural differentiation, offer a promising human-based platform to evaluate candidate therapeutics for neurodevelopmental disorders and hasten clinical translatability (Adams et al, 2019). Here, we employed a series of increasingly complex human stem cell-derived models as a screening platform to identify pharmacological compounds capable of specifically improving the neurocytological deficits caused by knockout of MECP2 (MECP2-KO) without affecting control neurons. Using our strategic pipeline, we demonstrated MECP2-KO-attributable synaptic pathology in genetic expression, morphology, and physiology and screened a series of drugs with synapse-relevant mechanisms of action for their ability to improve these alterations. Two currently available lead compounds, Nefiracetam and PHA 543613, exhibited potential to partially rescue the synaptic defects caused by MeCP2 deficiency and are viable candidates for clinical trial. Results MECP2-KO produces synaptic pathology in human neurons To develop our human models of MeCP2 deficiency, human PSCs with MECP2 exonic loss-of-function mutations (Q83X nonsense or K82 frameshift with familial and isogenic PSC controls, respectively) were differentiated into cortical neurons as previously described (Figs 1A and B and EV1A–I, and EV2A–C; Espuny-Camacho et al, 2013; Nageshappa et al, 2016). We first compared the morphologies between control and MECP2-KO neurons. Compared with controls, MECP2-KO neurons transfected with Syn1::GFP lentiviral vectors had decreased soma areas and spine density (P < 0.0001). The spine-like protrusions in MECP2-KO neurons were less stable (P < 0.01), but there was no observable difference in their formation (P = 0.26), length (P = 0.28), or motility (P = 0.17; Fig EV2D and E), supporting previous descriptions of RTT neuronal pathology (Marchetto et al, 2010; Nguyen et al, 2012). Figure 1. Human MECP2-KO neurons exhibit alterations in synapse-relevant genes and pathways Pluripotent stem cells (PSCs) with one of two distinct exonic loss-of-function mutations (Q83X nonsense or K82 frameshift (K82fs)) at the MECP2 locus were generated and differentiated into neurons via a neural progenitor cell (NPC) intermediate. Immunofluorescent staining confirmed the absence of MeCP2 in MECP2-KO PSCs, NPCs, and neurons. Scale bar = 10 µm. RT–qPCR array revealed differential gene expression between control and MECP2-KO 28-day neurons. The P values are calculated based on a Student's t-test of the replicate 2−ΔΔCt values for each gene in the control group and KO groups (WT83/Q83X cell lines were used; N = 3 clones from each genotype). Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function (days 14 and 28 post-differentiation; normalized by control NPC expression). Single-cell RT–qPCR analysis distinguished populations of control and KO 28-day neurons. Principal component analysis (PCA) of 441 cells projected onto the first two components. Overlaid populations of WT83/Q83X and WT82/K82fs neurons are shown. Violin plots of selected genes showing the comparison between control and MECP2-KO neurons from the single-cell analyses (log2(expression) values) that overlapped with the results obtained via RT–PCR array. Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single-cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student's t-test). Left: Schematic of laminar cortical markers. Right: Top: Quantitation of laminar cell markers based on the single-cell RT–qPCR analysis (two-way analysis of variance (ANOVA), N = 3 replicates per genotype, F3,24 = 3.31, *P = 0.04, with difference in the proportion of cortical layers V and VI, 95% confidence interval (CI) [8.31, 42.41]); Bottom: altered proportions of neurotransmitter markers (two-way ANOVA, N = 3 replicates per genotype, F5,24 = 27.33, ***P < 0.0001, with differences in the proportions of glutamatergic, 95% CI [22.33, 33.60], and cholinergic, 95% CI [0.166, 11.43], neurons). Gene ontology (GO) analysis of targeted genes relevant to synaptic function displaying the differences between MECP2-KO and control neurons. For the full list of markers used see Dataset EV1. Data information: Note that asterisks signify statistically significant difference from MECP2-KO. Data are presented as mean ± standard error of the mean (s.e.m.). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation of MECP2-KO pluripotent stem cells A. Mutations resulting in loss of functional MeCP2 protein shown by Sanger sequencing. CRISPR guide RNA is depicted as a blue bar over WT sequence and the cut site as a red triangle. B. CRISPR guide RNA to generate K82 frameshift mutation. C. Identification of off-target mutations by exome sequencing. D–I. MECP2-KO was successfully generated and did not affect pluripotency, as confirmed by immunochemistry (D), teratoma assay (E), Western blot (F), karyotype (G), and RT–qPCR (H, I). Experiments were performed in triplicate; data are presented as mean ± s.e.m. Scale bar = 200 µm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. MECP2-KO neurons exhibit defects in spine-like morphology A. Schematic showing neuronal differentiation from human PSCs. Scale bar = 200 µm. B. Neuronal markers immunostaining of 28-days MECP2-KO and control neurons. Scale bar = 50 µm. C. No difference in the resultant proportions of neurons or glia was observed between 28-days MECP2-KO and control neural differentiations. Similar results were observed for the proportions of glutamatergic and GABAergic neurons. Experiments were performed in triplicates. D. 28-days MECP2-KO neurons had decreased soma area and spine number (WT83/Q83X cell lines were used; N = 13 neurons per genotype, Student's t-test, t24 = 5.03, ***P < 0.0001). Left: representative images of Syn::GFP + neurons; Right: neuronal feature quantification. Scale bar = 20 µm. E. Observation over time showed decreased spine stability (*P < 0.01), but spine motility (P = 0.17), spine formation (P = 0.26), and spine length (P = 0.28) were unchanged in MECP2-KO neurons. Scale bar = 5 µm. WT83/Q83X cell lines were used, N = 10-15 neurons/condition. Analyses were two-sided Student's t-tests. Top: representative images of neuronal spine dynamics at 5-min intervals; arrows denote representative spines. Bottom: quantification of features of neuronal spine dynamics. Data information: Data are presented as mean ± s.e.m. Download figure Download PowerPoint Because MeCP2 is an epigenetic regulator, we sought to identify changes in gene expression resulting from its deficiency. RT–qPCR array (Figs 1C and D, and EV3A and B) and single-cell RT–qPCR (Figs 1E–G and EV3C–K) analyses at timepoints throughout neuronal differentiation from neural progenitor cells (NPCs; day 0) revealed differences in genetic expression between MECP2-KO and control neurons. Concordant with the prominent roles of MeCP2 in synaptic function and neuronal maturation (Gonzales & LaSalle, 2010), many of the implicated genes appeared relevant to synaptic function and showed differences in expression during differentiation (Fig 1C–G). In further agreement with previous findings of RTT pathology (Stuss et al, 2012), our quantitation of cellular markers of neuronal fate and function in cells positive for the neuronal marker MAP2 revealed a decreased proportion of layer V and VI cortical neurons (CTIP2+ and FOXP2+) in MECP2-KO (P = 0.04; Fig 1H, top) and altered neurotransmitter identity. Transcriptional analysis of genes related to neurotransmission in MECP2-KO populations showed changes in glutamatergic, GABAergic, and cholinergic systems (P < 0.0001; Fig 1H, bottom, Fig EV3G–K; the full list of cellular markers is shown in Dataset EV1). A summary gene ontology analysis confirmed these findings, showing that alterations in gene expression due to MECP2-KO primarily concentrated in neurotransmitter synthesis and receptor pathways in overlapping alignment with the results of our quantitative analysis (Fig 1I). Click here to expand this figure. Figure EV3. RT–PCR array and single-cell RT–qPCR analyses A, B. RT–PCR array revealed that differences in genetic expression showed variation between control and MECP2-KO NPCs and neurons, as depicted on volcano plots (A) and heatmap (B). Each heatmap column is one gene. C–K. Single-cell RT–qPCR analysis likewise showed differences in gene expression throughout neuronal differentiation due to MECP2-KO that appear to involve synaptic function. 28-days WT83/Q83X and WT82/K82fs neurons were used. Principal component analysis (C) of control and MECP2-KO NPCs (day 0 of same cell lines) and comparative expression of select genes between genotypes (D). Principal component analysis shows differences in expression between NPCs and neurons within each genotype (E) and differences in neuronal gene expression between genotypes (F). Heatmap shows differences in expression of particular genes (columns; G), and schematic denotes single-cell qPCR of individual markers (H). Number (H) and graphical depiction (J) of differentially expressed genes between genotypes. Table presents differentially expressed genes (K). Download figure Download PowerPoint Artificial neural network modeling supports targeting synaptic dysfunction for treatment Although synaptic pathology is a prominent consequence of MeCP2 deficiency (Johnston et al, 2003; Gonzales & LaSalle, 2010; Nguyen et al, 2012), many compensatory factors may influence network function, and it is unclear that targeted treatment of synaptic dysfunction will yield measurably linked improvement in neuronal population activity. The variation between individuals in network connectivity patterns or the proportions of excitatory and inhibitory neurons, for example, may modulate the link between synaptic phenotype and altered neural activity (Van Vreeswijk & Sompolinsky, 1996; Pena et al, 2018). Artificial neural networks offer a biologically plausible framework to explore how parameterized manipulation affects network activity (Miconi, 2017; Kim et al, 2019). We generated a neural network in silico to predict the change in neuronal spiking activity expected to result from isolated rescue of synaptic structure. We used co-localized synaptic puncta values from a previously published study (Marchetto et al, 2010) as a proxy for synaptic knockdown. Considering the full range of possible excitatory-inhibitory ratios (E–I balance) and connection sparsity, we simulated neural activity in networks using synaptic levels commensurate with untreated and rescued MECP2-KO. Holding all other parameters equivalent, rescuing the synaptic defect in isolation sufficiently increased neuronal network activity across the parameter space (pairwise delta mean = 0.877, range [−0.002, 7.981]; Fig 2A). The model supports the use of synaptic and neurotransmission phenotypes as actionable targets to improve network function. Figure 2. Screening selected drugs with synaptic action in human neurons identifies Nefiracetam and PHA 543613 as top treatment candidates for MeCP2 deficiency A. In silico neural network modeling using our previous synaptic puncta values for untreated (top, knockdown (KD) [6/16] = 0.375*control) and treated (bottom, KD [24/16] = 1.5*control) RTT neurons (Marchetto et al, 2010) suggested that isolated increase of synaptic knockdown sufficiently increases neural activity (untreated, mean = 0.665, range = [0.050,1.002]; treated, mean = 1.542, range = [0.997,8.395]). For excitatory-inhibitory (E–I) balance, "0" is fully excitatory, "1" is fully inhibitory. B. List of compounds for the phenotypic screening. The compounds were selected for mechanisms of action that counteracted the neurotransmitter deficiencies observed in Fig 1. The final concentration was determined based on previous studies. C. Schematic of drug treatment workflow. Briefly, 28-day-old MECP2-KO neurons were treated for 2 more weeks prior to performing phenotypic reversal experiments. D, E. Western blot quantification showed decreased presynaptic Synapsin1 (D) and postsynaptic PSD-95 (E) in untreated 6-week MECP2-KO neurons that can be increased by drug treatment (Kruskal–Wallis test, *P < 0.05, **P < 0.01, ***P < 0.001; full Western blot, Fig EV4A; WT83/Q83X cell lines were used; N = 3 clones from each genotype). F, G. 6-week MECP2-KO neurons (F) showed a pharmacologically rescuable reduction of co-localized synaptic puncta (G; one-way ANOVA, F16,118 = 9.148, *P < 0.05; Dunnet's multiple comparisons test vs. untreated KO (WT83/Q83X cell lines were used; N = 7–8 neurons/condition). *P < 0.05, **P < 0.01, ***P < 0.001. Z scores relative to KO untreated: Control = 3.903; Nefiracetam = 3.560; Carbamoylcholine = 2.705; Pirenzepine = 0.0448; PHA543613 = 2.121; Acamprosate = 3.339; Baclofen = 0.5672; GR73632 = 2.237; Hyperforin = 3.016, and IGF-1 = 3.791. Scale bar = 5 µm. H. Drug treatment increased calcium transient frequency in 6-week neurons. Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest (one-way ANOVA, F4,52 = 20.28, Dunnett's multiple comparisons test vs. untreated KO: Nefiracetam, **P < 0.01 and Z = 2.364; PHA 543613, ***P < 0.001 and Z = 3.391; Acamprosate, ***P < 0.001 and Z = 3.153; WT83/Q83X cell lines were used; N = 10–14 neurons/condition) and the percentage of active neurons (one-way ANOVA, F4,52 = 23.11; Dunnett's multiple comparison test vs. untreated KO: Nefiracetam, ***P < 0.001 and Z = 3.144; PHA 543613, **P < 0.01 and Z = 2.567; **Acamprosate, P < 0.01 and Z = 2.285; N = 10–14 neurons/condition). I. Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2-KO neurons on MEA (two-sided unpaired Student's t-test compared with KO untreated: Nefiracetam, *P = 0.016 and Z = 2.418; PHA 543613, *P = 0.027 and Z = 2.147; Acamprosate, P = 0.39 and Z = 0.477; WT83/Q83X and WT82/K82fs cell lines were used; N = 4–11 MEA wells/condition). Data information: Note that asterisks signify statistically significant difference from MECP2-KO untreated. Data are presented as mean ± s.e.m. Download figure Download PowerPoint Pharmacological screening identifies two lead compounds that specifically reverse MECP2-KO phenotypes We selected 14 pharmacological compounds with mechanisms of action that counteract the synapse and neurotransmitter pathologies that we identified in Fig 1 (Fig 2B). For instanc
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