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Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology. Keywords: antisense oligonucleotides, myosin, cardiac hypertrophy, cardiomyopathy, nucleic acid, RNA degradation

Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons.To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice.The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons.This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, increased percentages of Arc- and Egr1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons pointed to an increase in synaptic activity and synaptic strength as compared to control. This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3D context, and could be used to test new therapeutic compounds correcting neuronal development defects in FXS.

Studies supporting a strong association between tau deposition and neuronal loss, neurodegeneration, and cognitive decline have heightened the allure of tau and tau-related mechanisms as therapeutic targets. In February 2020, leading tau experts from around the world convened for the first-ever Tau2020 Global Conference in Washington, DC, co-organized and cosponsored by the Rainwater Charitable Foundation, the Alzheimer's Association, and CurePSP. Representing academia, industry, government, and the philanthropic sector, presenters and attendees discussed recent advances and current directions in tau research. The meeting provided a unique opportunity to move tau research forward by fostering global partnerships among academia, industry, and other stakeholders and by providing support for new drug discovery programs, groundbreaking research, and emerging tau researchers. The meeting also provided an opportunity for experts to present critical research-advancing tools and insights that are now rapidly accelerating the pace of tau research., info:eu-repo/semantics/published

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The cells populated the brain and differentiated into neurons and astrocytes. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons, an increased proportion of Arc-positive FXS neurons and increased dendritic protrusion width of FXS striatal medium spiny neurons. Our data show faster maturation and suggest increased synaptic activity and synaptic strength of FXS transplanted neurons. This model provides new insights into the alterations in FXS neuronal development.

Synaptic dysfunction and loss are core pathological features in Alzheimer disease (AD). In the vicinity of amyloid-β plaques in animal models, synaptic toxicity occurs and is associated with chronic activation of the phosphatase calcineurin (CN). Indeed, pharmacological inhibition of CN blocks amyloid-β synaptotoxicity. We therefore hypothesized that CN-mediated transcriptional changes may contribute to AD neuropathology and tested this by examining the impact of CN over-expression on neuronal gene expression in vivo. We found dramatic transcriptional down-regulation, especially of synaptic mRNAs, in neurons chronically exposed to CN activation. Importantly, the transcriptional profile parallels the changes in human AD tissue. Bioinformatics analyses suggest that both nuclear factor of activated T cells and numerous microRNAs may all be impacted by CN, and parallel findings are observed in AD. These data and analyses support the hypothesis that at least part of the synaptic failure characterizing AD may result from aberrant CN activation leading to down-regulation of synaptic genes, potentially via activation of specific transcription factors and expression of repressive microRNAs. Open practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Read the Editorial Highlight for this article on page 8.

/SummaryPatients with Prader-Willi-Syndrome (PWS) display intellectual impairment, hyperphagia, and various behavioral problems during childhood that converge on a neurologic deficit. The majority of PWS patients have genetic deletions of the paternal 15q11–q13 chromosomal region, with their maternal PWS locus intact but epigenetically silenced by hypermethylation and repressive histone modulation of the PWS imprinting center (PWS-IC). Inhibition of the euchromatin histone methyltransferase G9a by small molecules has been recently reported to reactivate PWS genes in patient fibroblasts and a mouse model. However, it is unknown if inhibition of G9a could have similar effect in human PWS neural cells, the cell types that have direct pathophysiological relevance to PWS. Here, we use neural progenitor cells (NPCs) and cortical excitatory neurons derived from a patient iPSC to model PWS, and quantitatively profile the expression of PWS genes using a NanoString panel. We demonstrated that the methylation of the PWS-IC is stable during neuronal lineage conversion, and that the maternal PWS genes remain silenced in PWS NPCs and neurons. Multiple small molecule inhibitors of G9a activate maternal PWS genes in a dose dependent manner in both NPCs and neurons. In addition, G9a inhibitors induce GNRH1 and HTR2C, two neuronal specific genes that contribute to PWS pathology in neurons. Interestingly, distinct from 5-Azacytidine, G9a inhibition does not induce methylation changes of the maternal PWS-IC, indicating that disruption of the histone repressive complex alone is sufficient to drive an open chromatin state at the PWS-IC that leads to partial reactivation of PWS genes.HighlightsModeling PWS disease in a dish using patient derived NPCs and neuronsG9a inhibition activates maternal PWS genes in patient-derived neural cellsG9a inhibition activates maternal SNORD116 and other PWS genes in patient-derived neuronsInhibition of G9a induces PWS downstream genes GNRH1 and HTR2C in PWS neurons

Duchenne Muscular Dystrophy (DMD) is a fatal disease of skeletal and cardiac muscle characterized by progressive muscle weakness and atrophy. Current average life expectancy is in the mid-20s and mortality is often linked to end stage cardiomyopathy. DMD is a monogenic disease caused by loss-of-function mutations in the dystrophin gene. While dystrophin is critical for muscle function by connecting the cytoskeleton to the sarcolemma and extracellular matrix, its absence can be largely compensated for by up-regulation of utrophin (UTRN), the fetal orthologue of dystrophin. Because of the importance of UTRN expression in the heart in both DMD patients and preclinical models, we utilized DMD patient iPSC-derived cardiomyocytes as a relevant cellular model to perform in vitro pharmacology as well as functional assessment. Human iPSC-derived cardiomyocytes recapitulate key properties of bona fide cardiomyocytes, such as expression of cardiac genes, spontaneous contractility, and response to chronotropic drugs (eg. isoproterenol and hERG channel blockers). We have created an isogenic pair of healthy control and DMD iPSC lines with exon 51 deletion using CRISPR/Cas9 technology. Functional measurements of cardiomyocyte electrophysiology were performed by multielectrode array (MEA) and fluorometric imaging plate reader (FLIPR) platforms. High-throughput assays were developed to measure UTRN expression in cardiomyocyte culture in 384-well plates. Importantly, we have identified small molecules that increased UTRN expression in both patient and isogenic DMD iPSC-derived cardiomyocytes. Our study represents a valuable strategy for small molecule drug discovery for the treatment of DMD cardiomyopathy based on iPSC technology.

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5′ UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS. Rescue of fragile X syndrome neurons by CRISPR-mediated DNA methylation editing of the FMR1 gene., National Institutes of Health (Grant HD045022), National Institutes of Health (Grant MH104610), National Institutes of Health (Grant R01NS088538), National Institutes of Health (Grant R01GM123511)

Small molecule modulators of GPR88 activity (agonists, antagonists, or modulators) are of interest as potential agents for the treatment of a variety of psychiatric disorders including schizophrenia. A series of phenylglycinol and phenylamine analogs have been prepared and evaluated for their GPR88 agonist activity and pharmacokinetic (PK) properties.

Schizophrenia is associated with alterations in working memory that reflect dysfunction of dorsolateral prefrontal cortex (DLPFC) circuitry. Working memory depends on the activity of excitatory pyramidal cells in DLPFC layer 3 and, to a lesser extent, in layer 5. Although many studies have profiled gene expression in DLPFC gray matter in schizophrenia, little is known about cell-type-specific transcript expression in these two populations of pyramidal cells. We hypothesized that interrogating gene expression, specifically in DLPFC layer 3 or 5 pyramidal cells, would reveal new and/or more robust schizophrenia-associated differences that would provide new insights into the nature of pyramidal cell dysfunction in the illness. We also sought to determine the impact of other variables, such as a diagnosis of schizoaffective disorder or medication use at the time of death, on the patterns of gene expression in pyramidal neurons. Individual pyramidal cells in DLPFC layers 3 or 5 were captured by laser microdissection from 36 subjects with schizophrenia or schizoaffective disorder and matched normal comparison subjects. The mRNA from cell collections was subjected to transcriptome profiling by microarray followed by quantitative PCR validation. Expression of genes involved in mitochondrial (MT) or ubiquitin-proteasome system (UPS) functions were markedly downregulated in the patient group (P-values for MT-related and UPS-related pathways were10(-7) and10(-5), respectively). MT-related gene alterations were more prominent in layer 3 pyramidal cells, whereas UPS-related gene alterations were more prominent in layer 5 pyramidal cells. Many of these alterations were not present, or found to a lesser degree, in samples of DLPFC gray matter from the same subjects, suggesting that they are pyramidal cell specific. Furthermore, these findings principally reflected alterations in the schizophrenia subjects were not present or present to a lesser degree in the schizoaffective disorder subjects (diagnosis of schizoaffective disorder was the most significant covariate, P10(-6)) and were not attributable to factors frequently comorbid with schizophrenia. In summary, our findings reveal expression deficits in MT- and UPS-related genes specific to layer 3 and/or layer 5 pyramidal cells in the DLPFC of schizophrenia subjects. These cell type-specific transcriptome signatures are not characteristic of schizoaffective disorder, providing a potential molecular-cellular basis of differences in clinical phenotypes.

Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile.

There is substantial evidence, both pharmacological and genetic, that hypofunction of the N-methyl-d-aspartate receptor (NMDAR) is a core pathophysiological feature of schizophrenia. There are morphological brain changes associated with schizophrenia, including perturbations in the dendritic morphology of cortical pyramidal neurons and reduction in cortical volume. Our experiments investigated whether these changes in dendritic morphology could be recapitulated in a genetic model of NMDAR hypofunction, the serine racemase knockout (SR-/-) mouse. Pyramidal neurons in primary somatosensory cortex (S1) of SR-/- mice had reductions in the complexity, total length, and spine density of apical and basal dendrites. In accordance with reduced cortical neuropil, SR-/- mice also had reduced cortical volume as compared to wild type mice. Analysis of S1 mRNA by DNA microarray and gene expression analysis revealed gene changes in SR-/- that are associated with psychiatric and neurologic disorders, as well as neurodevelopment. The microarray analysis also identified reduced expression of brain derived neurotrophic factor (BDNF) in SR-/- mice. Follow-up analysis by ELISA confirmed a reduction of BDNF protein levels in the S1 of SR-/- mice. Finally, S1 pyramidal neurons in glycine transporter heterozygote (GlyT1+/-) mutants, which display enhanced NMDAR function, had increased dendritic spine density. These results suggest that proper NMDAR function is important for the arborization and spine density of pyramidal neurons in cortex. Moreover, they suggest that NMDAR hypofunction might, in part, be contributing to the dendritic and synaptic changes observed in schizophrenia and highlight this signaling pathway as a potential target for therapeutic intervention.

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein-coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.

The design and implementation of an automated tissue culture flask labeling, delivery, scraping, and retrieval system centered around the CellmateTm cell culture robot is presented. Three new custom subsystems named FlaskMaster, FlaskLabeler, and FlaskScraper were created and integrated into the Cellmate robotic system. Flask-Master is a centralized multi-axis robot with a surrounding enclosure that houses racks of stacked tissue culture flasks (size T-175). The FlaskMaster system interfaces externally with the Cellmate's conveyor system to deliver and retrieve flasks. FlaskLabeler is a print and apply system that labels flasks prior to transfer between FlaskMaster and the Cellmate. FlaskScraper is a scraping rod and blade assembly that was specifically designed for automated scraping of T-175 flasks. The combination of these systems allows for large-scale, unattended cell seeding and harvesting of cells from flasks. The addition of these new capabilities significantly reduces the manpower needed to operate the Cellmate. Moreover, these changes greatly improve the performance and capability of the stand-alone Cellmate system.

High-throughput screening (HTS) can be defined as a process by which hundreds of thousands to millions of compounds are tested for activity against disease targets of interest with the goal of identifying truly active, progressible “hits”. Over the last 20 years high-throughput screening (HTS) has become a successful, reliable component of the drug discovery process. This article focuses on the variety of screening plate formats and the implications of their usage on the supporting technologies and assay designs. Keywords: high-throughput screening (HTS); drug discovery process; automation; quality control

Among the several goals of a high-throughput screening campaign is the identification of as many active chemotypes as possible for further evaluation. Often, however, the number of concentration response curves (e.g., IC(50)s or K(i)s) that can be collected following a primary screen is limited by practical constraints such as protein supply, screening workload, and so forth. One possible approach to this dilemma is to cluster the hits from the primary screen and sample only a few compounds from each cluster. This introduces the question as to how many compounds must be selected from a cluster to ensure that an active compound is identified, if it exists at all. This article seeks to address this question using a Monte Carlo simulation in which the dependence of the success of sampling is directly linked to screening data variability. Furthermore, the authors demonstrate that the use of replicated compounds in the screening collection can easily assess this variability and provide a priori guidance to the screener and chemist as to the extent of sampling required to maximize chemotype identification during the triage process. The individual steps of the Monte Carlo simulation provide insight into the correspondence between the percentage inhibition and eventual IC(50) curves.

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r2 > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

G-protein-coupled receptors (GPCRs) are the most successful target proteins for drug discovery research to date. More than 150 orphan GPCRs of potential therapeutic interest have been identified for which no activating ligands or biological functions are known. One of the greatest challenges in the pharmaceutical industry is to link these orphan GPCRs with human diseases. Highly automated parallel approaches that integrate ultra-high throughput and focused screening can be used to identify small molecule modulators of orphan GPCRs. These small molecules can then be employed as pharmacological tools to explore the function of orphan receptors in models of human disease. In this review, we describe methods that utilize powerful ultra-high-throughput screening technologies to identify surrogate ligands of orphan GPCRs.

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer's disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid.

For decades, researchers have focused primarily on a pathway initiated by amyloid beta aggregation, amyloid deposition, and accumulation in the brain as the key mechanism underlying the disease and the most important treatment target. However, evidence increasingly suggests that amyloid is deposited early during the course of disease, even prior to the onset of clinical symptoms. Thus, targeting amyloid in patients with mild to moderate Alzheimer's disease (AD), as past failed clinical trials have done, may be insufficient to halt further disease progression. Scientists are investigating other molecular and cellular pathways and processes that contribute to AD pathogenesis. Thus, the Alzheimer's Association's Research Roundtable convened a meeting in April 2012 to move beyond amyloid and explore AD as a complex multifactorial disease, with the goal of using a more inclusive perspective to identify novel treatment strategies.