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Neuronal ceroid lipofuscinosis (NCL) comprises ∼13 genetically distinct lysosomal disorders primarily affecting the central nervous system. Here we report successful reprograming of patient fibroblasts into induced pluripotent stem cells (iPSCs) for the two most common NCL subtypes: classic late-infantile NCL, caused by TPP1(CLN2) mutation, and juvenile NCL, caused by CLN3 mutation. CLN2/TPP1- and CLN3-iPSCs displayed overlapping but distinct biochemical and morphological abnormalities within the endosomal-lysosomal system. In neuronal derivatives, further abnormalities were observed in mitochondria, Golgi and endoplasmic reticulum. While lysosomal storage was undetectable in iPSCs, progressive disease subtype-specific storage material was evident upon neural differentiation and was rescued by reintroducing the non-mutated NCL proteins. In proof-of-concept studies, we further documented differential effects of potential small molecule TPP1 activity inducers. Fenofibrate and gemfibrozil, previously reported to induce TPP1 activity in control cells, failed to increase TPP1 activity in patient iPSC-derived neural progenitor cells. Conversely, nonsense suppression by PTC124 resulted in both an increase of TPP1 activity and attenuation of neuropathology in patient iPSC-derived neural progenitor cells. This study therefore documents the high value of this powerful new set of tools for improved drug screening and for investigating early mechanisms driving NCL pathogenesis.

Bipolar disorder (BD) is a heritable neuropsychiatric disorder with largely unknown pathogenesis. Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD. Here we show that levels of miR-34a, which is predicted to target multiple genes implicated as genetic risk factors for BD, are increased in postmortem cerebellar tissue from BD patients, as well as in BD patient-derived neuronal cultures generated by reprogramming of human fibroblasts into induced neurons or into induced pluripotent stem cells (iPSCs) subsequently differentiated into neurons. Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets. Using human iPSC-derived neuronal progenitor cells, we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration. Taken together, we propose that miR-34a serves as a critical link between multiple etiological factors for BD and its pathogenesis through the regulation of a molecular network essential for neuronal development and synaptogenesis., Swiss National Science Foundation, Stanley Center for Psychiatric Research, National Institute of Mental Health (U.S.) (R21MH093958), National Institute of Mental Health (U.S.) (R33MH087896), National Institute of Mental Health (U.S.) (R01MH091115), National Institute of Mental Health (U.S.) (R01MH095088)

The focus of this chapter is squarely on bipolar disorder. Bipolar disorder is a severe mood disorder that often has psychotic features. Its most severe forms are more common than thought and significantly more likely to cause disability than originally thought. Studies of high-risk children have found them to be at increased risk for a variety of symptoms and neurobiological abnormalities. In contrast to schizophrenia, there is no formal prodromal syndrome that has been identified and cognitive abnormalities do not precede the onset of the disorder. Abnormal sleep and circadian rhythms are prominent and have led to intriguing biological models. Neurobiological experiments have primarily focused on candidate pathways and include circadian abnormalities, epigenetic processes including histone modification, Wnt/GSK3 signaling, other modulators of neuroplasticity, and mitochondrial dysfunction. Recent data suggest that bipolar disorder is a highly polygenic disease and that integration of prior modeling and data with the wide variety of new genetic risk loci will be productive in the future.

Traumatic events generate some of the most enduring forms of memories. Despite the elevated lifetime prevalence of anxiety disorders, effective strategies to attenuate long-term traumatic memories are scarce. The most efficacious treatments to diminish recent (i.e., day-old) traumata capitalize on memory updating mechanisms during reconsolidation that are initiated upon memory recall. Here, we show that, in mice, successful reconsolidation-updating paradigms for recent memories fail to attenuate remote (i.e., month-old) ones. We find that, whereas recent memory recall induces a limited period of hippocampal neuroplasticity mediated, in part, by S-nitrosylation of HDAC2 and histone acetylation, such plasticity is absent for remote memories. However, by using an HDAC2-targeting inhibitor (HDACi) during reconsolidation, even remote memories can be persistently attenuated. This intervention epigenetically primes the expression of neuroplasticity-related genes, which is accompanied by higher metabolic, synaptic, and structural plasticity. Thus, applying HDACis during memory reconsolidation might constitute a treatment option for remote traumata., National Institute of Neurological Disorders and Stroke (U.S.)/National Institute on Aging (NS078839), Picower Institute for Learning and Memory (Neurological Disorder Fund), Stanley Medical Research Institute, Howard Hughes Medical Institute, Bard Richmond Fellowship

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.

The advent of somatic cell reprogramming technologies, which enables the generation of patient-specific, induced pluripotent stem cell (iPSC) and other trans-differentiated human neuronal cell models, provides new means of gaining insight into the molecular mechanisms and neural substrates of psychiatric disorders. By allowing a more precise understanding of genotype-phenotype relationship in disease-relevant human cell types, the use of reprogramming technologies in tandem with emerging genome engineering approaches provides a previously ‘missing link’ between basic research and translational efforts. In this review, we summarize advances in applying human pluripotent stem cell and reprogramming technologies to generate specific neural subtypes with a focus on the use of these in vitro systems for the discovery of small molecule-probes and novel therapeutics. Examples are given where human cell models of psychiatric disorders have begun to reveal new mechanistic insight into pathophysiology and simultaneously have provided the foundation for developing disease-relevant, phenotypic assays suitable for both functional genomic and chemical screens. A number of areas for future research are discussed, including the need to develop robust methodology for the reproducible, large-scale production of disease-relevant, neural cell types in formats compatible with high-throughput screening modalities, including high-content imaging, multidimensional, signature-based screening, and in vitro network using multielectrode arrays. Limitations, including the challenges in recapitulating neurocircuits and non-cell autonomous phenotypes are discussed. While these technologies are still in active development, we conclude that as our understanding of how to efficiently generate and probe the plasticity of patient-specific stem models improves, their utility is likely to advance rapidly.

Developing novel therapeutics and diagnostic tools based upon an understanding of neuroplasticity is critical in order to improve the treatment and ultimately the prevention of a broad range of nervous system disorders. In the case of mood disorders, such as major depressive disorder (MDD) and bipolar disorder (BPD), where diagnoses are based solely on nosology rather than pathophysiology, there exists a clear unmet medical need to advance our understanding of the underlying molecular mechanisms and to develop fundamentally new mechanism experimental medicines with improved efficacy. In this context, recent preclinical molecular, cellular, and behavioral findings have begun to reveal the importance of epigenetic mechanisms that alter chromatin structure and dynamically regulate patterns of gene expression that may play a critical role in the pathophysiology of mood disorders. Here, we will review recent advances involving the use of animal models in combination with genetic and pharmacological probes to dissect the underlying molecular mechanisms and neurobiological consequence of targeting this chromatin-mediated neuroplasticity. We discuss evidence for the direct and indirect effects of mood stabilizers, antidepressants, and antipsychotics, among their many other effects, on chromatin-modifying enzymes and on the epigenetic state of defined genomic loci, in defined cell types and in specific regions of the brain. These data, as well as findings from patient-derived tissue, have also begun to reveal alterations of epigenetic mechanisms in the pathophysiology and treatment of mood disorders. We summarize growing evidence supporting the notion that selectively targeting chromatin-modifying complexes, including those containing histone deacetylases (HDACs), provides a means to reversibly alter the acetylation state of neuronal chromatin and beneficially impact neuronal activity-regulated gene transcription and mood-related behaviors. Looking beyond current knowledge, we discuss how high-resolution, whole-genome methodologies, such as RNA-sequencing (RNA-Seq) for transcriptome analysis and chromatin immunoprecipitation-sequencing (ChIP-Seq) for analyzing genome-wide occupancy of chromatin-associated factors, are beginning to provide an unprecedented view of both specific genomic loci as well as global properties of chromatin in the nervous system. These methodologies when applied to the characterization of model systems, including those of patient-derived induced pluripotent cell (iPSC) and induced neurons (iNs), will greatly shape our understanding of epigenetic mechanisms and the impact of genetic variation on the regulatory regions of the human genome that can affect neuroplasticity. Finally, we point out critical unanswered questions and areas where additional data are needed in order to better understand the potential to target mechanisms of chromatin-mediated neuroplasticity for novel treatments of mood and other psychiatric disorders.

Population aging is leading to an increase in age-related cognitive disorders, the most prevalent being Alzheimer’s disease. Mechanisms underlying cognition occur through changes in the expression of neuronal activity-dependent genes and neuroplasticity. These changes in gene expressions are regulated by epigenetic post-translational modifications to the chromatin structure. It is now becoming evident that many age-related human cognitive diseases are a consequence of aberrant chromatin-mediated neuroplasticity. Understanding the molecular nature of epigenetic mechanisms in the nervous system has the potential to provide new future therapeutic avenues for cognitive disease. In this chapter, we outline the role of the histone deacetylase (HDAC) family of chromatin modifying enzymes in regards to neurobiology and cognition. We discuss the chemistry of HDAC inhibitors, their biological effects on learning and memory, and their potential relevance towards developing therapeutics to enhance neuroplasticity and memory. Finally, we open suggestions as to where the field of HDAC pharmacology in relation to cognition should be directed.

Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary – albeit often ineffective – treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity., Stanley Medical Research Institute, National Institutes of Health (U.S.) (R01DA028301), National Institutes of Health (U.S.) (R01DA030321)

Long-term memory formation is known to be critically dependent upon de novo gene expression in the brain. As a consequence, pharmacological enhancement of the transcriptional processes mediating long-term memory formation provides a potential therapeutic strategy for cognitive disorders involving aberrant neuroplasticity. Here we focus on the identification and characterization of small molecule inhibitors of histone deacetylases (HDACs) as enhancers of CREB (cAMP response element-binding protein)-regulated transcription and modulators of chromatin-mediated neuroplasticity. Using a CREB reporter gene cell line, we screened a library of small molecules structurally related to known HDAC inhibitors leading to the identification of a probe we termed crebinostat that produced robust activation of CREB-mediated transcription. Further characterization of crebinostat revealed its potent inhibition of the deacetylase activity of recombinant class I HDACs 1, 2, 3, and class IIb HDAC6, with weaker inhibition of the class I HDAC8 and no significant inhibition of the class IIa HDACs 4, 5, 7, and 9. In cultured mouse primary neurons, crebinostat potently induced acetylation of both histone H3 and histone H4 as well as enhanced the expression of the CREB target gene Egr1 (early growth response 1). Using a hippocampus-dependent, contextual fear conditioning paradigm, mice systemically administered crebinostat for a ten day time period exhibited enhanced memory. To gain insight into the molecular mechanisms of memory enhancement by HDAC inhibitors, whole genome transcriptome profiling of cultured mouse primary neurons treated with crebinostat, combined with bioinformatic analyses of CREB-target genes, was performed revealing a highly connected protein–protein interaction network reflecting modules of genes important to synaptic structure and plasticity. Consistent with these findings, crebinostat treatment increased the density of synapsin-1 punctae along dendrites in cultured neurons. Finally, crebinostat treatment of cultured mouse primary neurons was found to upregulate Bdnf (brain-derived neurotrophic factor) and Grn (granulin) and downregulate Mapt (tau) gene expression—genes implicated in aging-related cognitive decline and cognitive disorders. Taken together, these results demonstrate that crebinostat provides a novel probe to modulate chromatin-mediated neuroplasticity and further suggests that pharmacological optimization of selective of HDAC inhibitors may provide an effective therapeutic approach for human cognitive disorders., National Institutes of Health (U.S.) (R01DA028301), National Institutes of Health (U.S.) (R01NS051874), Stanley Medical Research Institute, Howard Hughes Medical Institute

Platelet-derived growth factor receptor-beta (PDGFRβ) is required for the development of mesenchymal cell types, and plays a diverse role in the function of fibroblasts in tissue homeostasis and regeneration. In this study, we characterized the expression of PDGFRβ in fibroblasts derived from human embryonic stem cells and induced pluripotent stem cells, and showed that this expression is important for cellular functions including migration and extracellular matrix production and assembly in 3D self-assembled tissues. To determine potential regulatory regions predictive of expression of PDGFRβ following differentiation from ESC and iPSC, we analyzed the DNA methylation status of a region of the PDGFRβ promoter containing multiple CpG sites before and after differentiation. We demonstrated that this promoter region is extensively demethylated following differentiation, and represents a developmentally-regulated, differentially-methylated region linked to PDGFRβ expression. Understanding the epigenetic regulation of genes such as PDGFRβ, and identifying sites of active DNA demethylation, is essential for future applications of pluripotent stem cell-derived fibroblasts for regenerative medicine.

Cognitive decline is a debilitating feature of most neurodegenerative diseases of the central nervous system, including Alzheimer’s disease [superscript 1]. The causes leading to such impairment are only poorly understood and effective treatments are slow to emerge [superscript 2]. Here we show that cognitive capacities in the neurodegenerating brain are constrained by an epigenetic blockade of gene transcription that is potentially reversible. This blockade is mediated by histone deacetylase 2, which is increased by Alzheimer’s-disease-related neurotoxic insults in vitro, in two mouse models of neurodegeneration and in patients with Alzheimer’s disease. Histone deacetylase 2 associates with and reduces the histone acetylation of genes important for learning and memory, which show a concomitant decrease in expression. Importantly, reversing the build-up of histone deacetylase 2 by short-hairpin-RNA-mediated knockdown unlocks the repression of these genes, reinstates structural and synaptic plasticity, and abolishes neurodegeneration-associated memory impairments. These findings advocate for the development of selective inhibitors of histone deacetylase 2 and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade., Stanley Medical Research Institute, National Institute of Neurological Disorders and Stroke (U.S.) (RO1NS078839), Swiss National Science Foundation, Bard Richmond (Fellowship), Simons Foundation, Theodor und Ida Herzog-Egli Foundation

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5′ untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology., FRAXA Research Foundation, Harvard Stem Cell Institute (seed grant), Stanley Medical Research Institute, National Institute of Mental Health (U.S.) (grant #R33MH087896)

Synaptic function is affected in many brain diseases and disorders. Technologies for large-scale synapse assays can facilitate identification of drug leads. Here we report a “synapse microarray” technology that enables ultra-sensitive, high-throughput, and quantitative screening of synaptogenesis. Our platform enables the induction of synaptic structures in regular arrays by precise positioning of non-neuronal cells expressing synaptic proteins, while allowing neurites to grow freely around these cells. The technology increases by tenfold the sensitivity of the traditional assays, and simultaneously decreases the time required to capture synaptogenic events by an order of magnitude. It is readily incorporated into multiwell formats compatible with industrial high-throughput screening platforms. Using this technology, we screened a chemical library and identified novel histone deacetylase inhibitors that improve neuroligin-1 induced synaptogenesis via modulating class-I histone deacetylases. We also found a structure-activity relationship for designing novel potent histone deacetylase inhibitors, which can be applied towards development of new therapeutics.

Epigenetic mechanisms have emerged as important components of a variety of human diseases, including cancer and central nervous system disorders. Despite recent studies highlighting the role of epigenetic mechanisms in several neurodegenerative and neuropsychiatric disorders, to date, there has been a paucity of studies exploring the role of epigenetic factors in Parkinson’s disease (PD). PD is a progressive neurological disorder with characteristic motor and non-motor symptoms, including a range of neuropsychiatric features, for which neither preventative nor effective long-term treatment strategies are available. It is one of the most common neurodegenerative disorders and the second most prevalent after Alzheimer’s disease. In this review, we present several lines of evidence suggesting that epigenetic factors may play an important role in the pathogenesis of PD and propose on this basis a framework to guide future investigations into epigenetic mechanisms and systems biology of PD. These notions, together with technical advances in the ability to perform genome-wide analysis of epigenomic states, and newly available small-molecule probes targeting chromatin-modifying enzymes, may help design new treatment strategies for PD and other human diseases involving epigenetic dysregulation.

Advancing our understanding of neuroplasticity and the development of novel therapeutics based upon this knowledge is critical in order to improve the treatment and prevention of a myriad of nervous system disorders. Epigenetic mechanisms of neuroplasticity involve the post-translational modification of chromatin and the recruitment or loss of macromolecular complexes that control neuronal activity-dependent gene expression. While over a century after Ramón y Cajal first described nuclear subcompartments and foci that we now know correspond to sites of active transcription with acetylated histones that are under epigenetic control, the rate and extent to which epigenetic processes act in a dynamic and combinatorial fashion to shape experience-dependent phenotypic and behavioral plasticity in response to various types of neuronal stimuli over a range of time scales is only now coming into focus. With growing recognition that a subset of human diseases involving cognitive dysfunction can be classified as ‘chromatinopathies’, in which aberrant chromatin-mediated neuroplasticity plays a causal role in the underlying disease pathophysiology, understanding the molecular nature of epigenetic mechanisms in the nervous system may provide important new avenues for the development of novel therapeutics. In this review, we discuss the chemistry and neurobiology of the histone deacetylase (HDAC) family of chromatin-modifying enzymes, outline the role of HDACs in the epigenetic control of neuronal function, and discuss the potential relevance of these epigenetic mechanisms to the development of therapeutics aiming to enhance memory and neuroplasticity. Finally, open questions, challenges, and critical needs for the field of ‘neuroepigenetics’ in the years to come will be summarized., National Institutes of Health (U.S.) (R01DA028301), National Institutes of Health (U.S.) (RC1AG035711), National Institutes of Health (U.S.) (RO1NS051874), National Institutes of Health (U.S.) (PO1AG027916), Stanley Medical Research Institute, Howard Hughes Medical Institute

Bipolar disorder (BP) is a debilitating psychiatric disorder, affecting ∼2% of the worldwide population, for which the etiological basis, pathogenesis, and neurocircuitry remain poorly understood. Individuals with BP suffer from recurrent episodes of mania and depression, which are commonly treated with the mood stabilizer lithium. However, nearly half of BP patients do not respond adequately to lithium therapy and the clinically relevant mechanisms of lithium for mood stabilization remain elusive. Here, we modeled lithium responsiveness using cellular assays of glycogen synthase kinase 3 (GSK-3) signaling and mood-related behavioral assays in inbred strains of mice that differ in their response to lithium. We found that activating AKT through phosphosrylation of a key regulatory site (Thr308) was associated with lithium response—activation of signaling pathways downstream of GSK-3 in cells and attenuation of mood-related behaviors in mice—and this response was attenuated by selective and direct inhibition of AKT kinase activity. Conversely, the expression of constitutively active AKT1 in both the cellular and behavioral assays conferred lithium sensitivity. In contrast, selective and direct GSK-3 inhibition by the ATP-competitive inhibitor CHIR99021 bypassed the requirement for AKT activation and modulated behavior in both lithium-responsive and non-responsive mouse strains. These results distinguish the mechanism of action of lithium from direct GSK-3 inhibition both in vivo and in vitro, and highlight the therapeutic potential for selective GSK-3 inhibitors in BP treatment.

Discovery of molecular mechanisms and chemical compounds that enhance neuronal regeneration can lead to development of therapeutics to combat nervous system injuries and neurodegenerative diseases. By combining high-throughput microfluidics and femtosecond laser microsurgery, we demonstrate for the first time large-scale in vivo screens for identification of compounds that affect neurite regeneration. We performed thousands of microsurgeries at single-axon precision in the nematode Caenorhabditis elegans at a rate of 20 seconds per animal. Following surgeries, we exposed the animals to a hand-curated library of approximately one hundred small molecules and identified chemicals that significantly alter neurite regeneration. In particular, we found that the PKC kinase inhibitor staurosporine strongly modulates regeneration in a concentration- and neuronal type-specific manner. Two structurally unrelated PKC inhibitors produce similar effects. We further show that regeneration is significantly enhanced by the PKC activator prostratin.

Genetic findings have suggested that neuregulin-1 (Nrg1) and its receptor v-erb-a erythroblastic leukemia viral oncogene homologue 4 (ErbB4) may play a role in neuropsychiatric diseases. However, the downstream signaling events and relevant phenotypic consequences of altered Nrg1 signaling in the nervous system remain poorly understood. To identify small molecules for probing Nrg1−ErbB4 signaling, a PC12-cell model was developed and used to perform a live-cell, image-based screen of the effects of small molecules on Nrg1-induced neuritogenesis. By comparison of the resulting phenotypic data to that of a similar screening performed with nerve growth factor (NGF), this multidimensional screen identified compounds that directly inhibit Nrg1−ErbB4 signaling, such as the 4-anilino-quinazoline Iressa (gefitinib), as well as compounds that potentiate Nrg1−ErbB4 signaling, such as the indolocarbazole K-252a. These findings provide new insights into the regulation of Nrg1−ErbB4 signaling events and demonstrate the feasibility of using such a multidimensional, chemical-genetic approach for discovering probes of pathways implicated in neuropsychiatric diseases.

Behavioral Profiling The complexity of the brain makes it difficult to predict how a drug will affect behavior without direct testing in live animals. Rihel et al. (p. 348 ) developed a high-throughput assay to assess the effects of thousands of drugs on sleep/wake behaviors of zebrafish larvae. The data set reveals a broad conservation of zebrafish and mammalian sleep/wake pharmacology and identifies pathways that regulate sleep. Moreover, the biological targets of poorly characterized small molecules can be predicted by matching their behavioral profiles to those of well-known drugs. Thus, behavioral profiling in zebrafish offers a cost-effective way to characterize neuroactive drugs and to predict biological targets of novel compounds.

Wnts are secreted ligands that activate several receptor-mediated signal transduction cascades. Homeostatic Wnt signaling through beta-catenin is required in adults, because either elevation or attenuation of beta-catenin function has been linked to diverse diseases. To contribute to the identification of both protein and pharmacological regulators of this pathway, we describe a combinatorial screen that merged data from a high-throughput screen of known bioactive compounds with an independent focused small interfering RNA screen. Each screen independently revealed Bruton's tyrosine kinase (BTK) as an inhibitor of Wnt-beta-catenin signaling. Loss of BTK function in human colorectal cancer cells, human B cells, zebrafish embryos, and cells derived from X-linked agammaglobulinemia patients with a mutant BTK gene resulted in elevated Wnt-beta-catenin signaling, confirming that BTK acts as a negative regulator of this pathway. From affinity purification-mass spectrometry and biochemical binding studies, we found that BTK directly interacts with a nuclear component of Wnt-beta-catenin signaling, CDC73. Further, we show that BTK increased the abundance of CDC73 in the absence of stimulation and that CDC73 acted as a repressor of beta-catenin-mediated transcription in human colorectal cancer cells and B cells.

The broad study of histone deacetylases in chemistry, biology and medicine relies on tool compounds to derive mechanistic insights. A phylogenetic analysis of class I and II histone deacetylases (HDACs) as targets of a comprehensive, structurally diverse panel of inhibitors revealed unexpected isoform selectivity even among compounds widely perceived as nonselective. The synthesis and study of a focused library of cinnamic hydroxamates allowed the identification of, to our knowledge, the first nonselective HDAC inhibitor. These data will guide a more informed use of HDAC inhibitors as chemical probes and therapeutic agents.

Chromatin modifications, especially histone-tail acetylation, have been implicated in memory formation. Increased histone-tail acetylation induced by inhibitors of histone deacetylases (HDACis) facilitates learning and memory in wild-type mice as well as in mouse models of neurodegeneration. Harnessing the therapeutic potential of HDACis requires knowledge of the specific HDAC family member(s) linked to cognitive enhancement. Here we show that neuron-specific overexpression of HDAC2, but not that of HDAC1, decreased dendritic spine density, synapse number, synaptic plasticity and memory formation. Conversely, Hdac2 deficiency resulted in increased synapse number and memory facilitation, similar to chronic treatment with HDACis in mice. Notably, reduced synapse number and learning impairment of HDAC2-overexpressing mice were ameliorated by chronic treatment with HDACis. Correspondingly, treatment with HDACis failed to further facilitate memory formation in Hdac2-deficient mice. Furthermore, analysis of promoter occupancy revealed an association of HDAC2 with the promoters of genes implicated in synaptic plasticity and memory formation. Taken together, our results suggest that HDAC2 functions in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory. These observations encourage the development and testing of HDAC2-selective inhibitors for human diseases associated with memory impairment.

Summary Previous work has identified alterations in histone acetylation in animal models of drug addiction and depression. However, the mechanisms which integrate drugs and stress with changes in chromatin structure remain unclear. Here, we identify the activity-dependent class II histone deacetylase, HDAC5, as a central integrator of these stimuli with changes in chromatin structure and gene expression. Chronic, but not acute, exposure to cocaine or stress decreases HDAC5 function in the nucleus accumbens (NAc), a major brain reward region, which allows for increased histone acetylation and transcription of HDAC5 target genes. This regulation is behaviorally important, as loss of HDAC5 causes hypersensitive responses to chronic, not acute, cocaine or stress. These findings suggest that proper balance of histone acetylation is a crucial factor in the saliency of a given stimulus and that disruption of this balance is involved in the transition from an acute adaptive response to a chronic psychiatric illness.

To pursue a systematic approach to the discovery of functional connections among diseases, genetic perturbation, and drug action, we have created the first installment of a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules, together with pattern-matching software to mine these data. We demonstrate that this “Connectivity Map” resource can be used to find connections among small molecules sharing a mechanism of action, chemicals and physiological processes, and diseases and drugs. These results indicate the feasibility of the approach and suggest the value of a large-scale community Connectivity Map project.

BackgroundGraph theory provides a computational framework for modeling a variety of datasets including those emerging from genomics, proteomics, and chemical genetics. Networks of genes, proteins, small molecules, or other objects of study can be represented as graphs of nodes (vertices) and interactions (edges) that can carry different weights. SpectralNET is a flexible application for analyzing and visualizing these biological and chemical networks.ResultsAvailable both as a standalone .NET executable and as an ASP.NET web application, SpectralNET was designed specifically with the analysis of graph-theoretic metrics in mind, a computational task not easily accessible using currently available applications. Users can choose either to upload a network for analysis using a variety of input formats, or to have SpectralNET generate an idealized random network for comparison to a real-world dataset. Whichever graph-generation method is used, SpectralNET displays detailed information about each connected component of the graph, including graphs of degree distribution, clustering coefficient by degree, and average distance by degree. In addition, extensive information about the selected vertex is shown, including degree, clustering coefficient, various distance metrics, and the corresponding components of the adjacency, Laplacian, and normalized Laplacian eigenvectors. SpectralNET also displays several graph visualizations, including a linear dimensionality reduction for uploaded datasets (Principal Components Analysis) and a non-linear dimensionality reduction that provides an elegant view of global graph structure (Laplacian eigenvectors).ConclusionSpectralNET provides an easily accessible means of analyzing graph-theoretic metrics for data modeling and dimensionality reduction. SpectralNET is publicly available as both a .NET application and an ASP.NET web application fromhttp://chembank.broad.harvard.edu/resources/. Source code is available upon request.

SummaryHere we demonstrate that natural variants of the yeast Saccharomyces cerevisiae are a model system for the systematic study of complex traits, specifically the response to small molecules. As a complement to artificial knockout collections of S. cerevisiae widely used to study individual gene function, we used 314- and 1932-member libraries of mutant strains generated by meiotic recombination to study the cumulative, quantitative effects of natural mutations on phenotypes induced by 23 small-molecule perturbagens (SMPs). This approach reveals synthetic lethality between SMPs, and genetic mapping studies confirm the involvement of multiple quantitative trait loci in the response to two SMPs that affect respiratory processes. The systematic combination of natural variants of yeast and small molecules that modulate evolutionarily conserved cellular processes can enable a better understanding of the general features of complex traits.

Chemical genomics aims to systematically explore the interactions between small molecules and biological systems. These efforts aim to annotate genomes using the language of chemistry, and to provide information-rich profiles of chemical and biological systems. Here, I describe recent conceptual and experimental advances toward the goal of mapping multidimensional chemical and biological descriptor spaces. In doing so, I will focus on the complementary nature of these efforts, the importance of recognizing the distinction between computed versus observed descriptors, and highlight recent ‘landmark’ examples of small molecules discovered using phenotypic screens. Future computation and experimental advances will be needed to fully realize the goals of chemical genomics. For those willing to consider both local and global properties of chemical and biological space, and to venture into uncharted territory, there promises to be new vistas and principles to be discovered.

The TOR (target of rapamycin) proteins play important roles in nutrient signaling in eukaryotic cells. Rapamycin treatment induces a state reminiscent of the nutrient starvation response, often resulting in growth inhibition. Using a chemical genetic modifier screen, we identified two classes of small molecules, small-molecule inhibitors of rapamycin (SMIRs) and small-molecule enhancers of rapamycin (SMERs), that suppress and augment, respectively, rapamycin's effect in the yeast Saccharomyces cerevisiae . Probing proteome chips with biotinylated SMIRs revealed putative intracellular target proteins, including Tep1p, a homolog of the mammalian PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor, and Ybr077cp (Nir1p), a protein of previously unknown function that we show to be a component of the TOR signaling network. Both SMIR target proteins are associated with PI(3,4)P 2 , suggesting a mechanism of regulation of the TOR pathway involving phosphatidylinositides. Our results illustrate the combined use of chemical genetics and proteomics in biological discovery and map a path for creating useful therapeutics for treating human diseases involving the TOR pathway, such as diabetes and cancer.

Chemical genomics is concerned with the effects of both genetic variation and chemical perturbation on the cellular effects of small molecules. Chemical genomics relies on selecting biological networks for study, such as those represented by different cell types or disease models, in order to build the desired specificity into the experimental design. The most relevant network property for such experiments is the global connectivity of all cellular proteins comprising the functional ensemble, as illustrated by case studies of the evolution of cyclooxygenase inhibitors and heat-shock protein modulators. Recent examples of chemical genomic profiling, particularly of different cell types, highlight the power of carefully planned experimental approaches in chemical genomics. These new approaches demonstrate the use of the genome to find new targets or new modes of biological interaction.

An objective of chemical genetics is to understand the relationships between the structures of small molecules and their phenotypic effects in intact living systems. We present here the results of a global analysis of a molecular descriptor space constructed using structural descriptors of an aryl 1,3-dioxane-based diversity-oriented synthesis-derived library containing structural biasing elements directed at inhibiting protein deacetylases. Using principal component analysis and three-dimensional visualization, we generated metric space maps with morphological features contributed by different diversity elements within the library. Filtering these maps using phenotypic descriptors derived from measurements of small-molecule activities in an array of cell-based assays revealed different densities of biological activity within specific subspaces. These results provide evidence that certain structural features may be important for conferring potency and selectivity on deacetylase inhibitors with respect to tubulin and histone acetylation. Moreover, these results highlight an example of the importance of using functional measures to assess molecular diversity. Similar analyses of other chemical spaces and activity classes promise to facilitate the development of chemical genetics.

Genome-wide measurements of multiple experimental samples yield rich fingerprints for comparison and interpretation. Here, a two-dimensional matrix of the cellular effects of all possible pairwise combinations of 24 small molecules, each with a different structure and bioactivity, was used to profile otherwise isogenic deletion strains of the yeast Saccharomyces cerevisiae. Using principles from graph theory, we derived a discrete model of the data for each strain by encoding the information in the form of a binary adjacency matrix. This matrix was used to construct a graph composed of nodes representing small molecules and edges connecting combinations that inhibited cell cycle progression. Computation of a set of graph theoretic descriptors for each chemical genetic network provided a topological fingerprint that showed genotype-dependent fluctuations. Because the structure of the genetic network determines the structure of the chemical genetic network, multidimensional chemical genomic profiling can be used for the characterization of perturbations in biological networks or the networks themselves. This application of small molecules could be useful for discerning the molecular basis of highly complex biological phenotypes, including those involved in the susceptibility to or etiology of human disease.

Development of novel treatments and diagnostic tools for psychiatric illness has been hindered by the absence of cellular models of disease. With the advent of cellular reprogramming, it may be possible to recapitulate the disease biology of psychiatric disorders using patient skin cells transdifferentiated to neurons. However, efficiently identifying and characterizing relevant neuronal phenotypes in the absence of well-defined pathophysiology remains a challenge. In this study, we collected fibroblast samples from patients with bipolar 1 disorder, characterized by their lithium response (n=12), and healthy control subjects (n=6). We identified a cellular phenotype in reprogrammed neurons using a label-free imaging assay based on a nanostructured photonic crystal biosensor and found that an optical measure of cell adhesion was associated with clinical response to lithium treatment. This cellular phenotype may represent a useful biomarker to evaluate drug response and screen for novel therapeutics.

An unfortunate error occurred in the legend of Fig. 13b. It should read: (b) compound 12 (70 μM)

Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.

Background Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. Results We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of geneproduct alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a ‘cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. Conclusions We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with smallmolecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

Small-molecule microarrays have been used to discover biologically active small molecules in collections of synthetic compounds. Here we utilize a versatile isocyanate chemistry to immobilize extracts from microorganisms in a microarray format. Specific bioactive small molecules are detected in crude extracts of Streptomyces hygroscopicus, and the amount of specific to nonspecific binding of a protein to an immobilized compound on a microarray can be estimated. These natural product-extract microarrays (NPEMs) provide new tools for characterizing the metabolic products of organisms and for discovering biologically active small molecules from nature.

Autophagy is an evolutionarily conserved process by which cellular structures may be degraded to support ongoing biogenesis. Recent evidence has identified the induction of autophagy following growth factor pathway inhibition, rapid tumor expansion and proteasome inhibition. Thus, autophagy is a highly desirable pathway for the development of targeted therapeutics in cancer, in particular in tumors characterized by misfolded protein stress such as multiple myeloma (MM). Unfortunately, there are only several known classes of autophagy inhibitors and the emerging biology of autophagy has identified few validated targets. We therefore performed a forward chemical genetic study of autophagy using novel techniques in high-content imaging, high-throughput screening and multidimensional data analysis. Using LN229 glioblastoma cells stably transfected with an LC3-EGFP construct, we created a robust screening platform capable of quantifying the number, size and fluorescence intensity of autophagosomes. Assay validation was performed with the known autophagy inhibitor chloroquine, which induces a characteristic shift from diffuse to punctate fluorescence. Initially, we profiled a library of approximately 3,500 structurally diverse bioactive small molecules to leverage possible downstream mechanistic insights. Assay positives and controls were re-tested in the adherent H1299 adenocarcinoma cell line likewise stably expressing LC3-EGFP. Known autophagy inhibitors were enriched with this approach: nocodazole, bafilomycin A1, colchicine and monensin. Structure-activity relationship analysis of the primary screening data and 34 novel autophagy modulators revealed a compelling activity of bis-indolyl maleimides. We confirmed sub-micromolar inhibition of autophagy by three such compounds using a quantitative, radiolabeled protein degradation assay (tritiated tyrosine pulse-chase), LC3-II immunoblotting and quantitative measurement of autophagosomes across three cell lines. From these studies we selected a lead compound, K252a, for further characterization and tested twenty-two closely related analogues. Detailed SAR analysis has identified features of the molecule required for inhibition of autophagy and sites permissive for further functionalization in ongoing target identification studies. Using MM as a translational model system of misfolded protein stress, we found dose-dependent, selective cytotoxicity in MM of K252a compared to analogues lacking autophagy inhibitory activity. We report here the results of a forward chemical genetic study of autophagy which identifies and characterizes novel small molecule inhibitors of autophagy and establishes a rationale for clinical studies of autophagy inhibitors in the treatment of MM.

Target identification remains a challenge for the field of chemical biology. We describe here an integrative chemical genomic and proteomic approach combining the use of differentially active analogs of small-molecule probes with stable isotope labeling by amino acids in cell culture (SILAC)-mediated affinity enrichment, followed by subsequent testing of candidate targets using RNA interference (RNAi)-mediated gene silencing. We applied this approach to characterizing the natural product K252a and its ability to potentiate neuregulin-1 (Nrg1)/ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4)-dependent neurotrophic factor signaling and neuritogenesis. We show that AAK1 (adaptor-associated kinase 1) is a relevant target of K252a, and that the loss of AAK1 alters ErbB4 trafficking and expression levels providing evidence for a previously unrecognized role for AAK1 in Nrg1-mediated neurotrophic factor signaling. Similar strategies should lead to the discovery of novel targets for therapeutic development.

SummaryTo identify new protein and pharmacological regulators of Wnt/β-catenin signaling, we used a cell-based reporter assay to screen a collection of 1857 human-experienced compounds for their ability to enhance activation of the β-catenin reporter by a low concentration of WNT3A. This identified 44 unique compounds, including the FDA-approved drug riluzole, which is presently in clinical trials for treating melanoma. We found that treating melanoma cells with riluzole in vitro enhances the ability of WNT3A to regulate gene expression, to promote pigmentation, and to decrease cell proliferation. Furthermore riluzole, like WNT3A, decreases metastases in a mouse melanoma model. Interestingly, siRNAs targeting the metabotropic glutamate receptor, GRM1, a reported indirect target of riluzole, enhance β-catenin signaling. The unexpected regulation of β-catenin signaling by both riluzole and GRM1 has implications for the future uses of this drug.

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.

After chromosome replication, the intertwined sister chromatids are disentangled by topoisomerases. The integrity of this process is monitored by the chromatid decatenation checkpoint. Here, we describe small molecule modulators of the human chromatid decatenation checkpoint identified using a cell-based, chemical genetic modifier screen. Similar to 1,2,7-trimethylyxanthine (caffeine), these small molecules suppress the G(2)-phase arrest caused by ICRF-193, a small molecule inhibitor of the enzymatic activity of topoisomerase II. Analysis of specific suppressors, here named suptopins for suppressor of Topoisomerase II inhibition, revealed distinct effects on cell cycle progression, microtubule stability, nucleocytoplasmic transport of cyclin B1, and no effect on the chromatin deacetylation checkpoint induced by trichostatin A. The suptopins provide new molecular tools for dissecting the role of topoisomerases in maintaining genomic stability and determining whether inhibiting the chromatid decatenation checkpoint sensitizes tumor cells to chemotherapeutics.

Systematic chemical genetics aims to explore the space representing interactions between small molecules and biological systems. Beyond measuring binding interactions and enzyme inhibition, measuring changes in the activity of proteins in intact signaling networks is necessary. Toward this end, we are partitioning chemical space into regions with different biological activities using a panel of cell-based assays and small molecule “chemical genetic modifiers.” Herein, we report on the use of this methodology for the discovery of 617 small molecule inhibitors of histone deacetylases from a multidimensional screen of an encoded, diversity-oriented synthesis library. Following decoding of chemical tags and resynthesis, we demonstrate the selectivity of one inhibitory molecule (tubacin) toward α-tubulin deacetylation and another (histacin) toward histone deacetylation. These small molecules will facilitate dissecting the role of acetylation in a variety of cell biological processes.

SummaryAlthough human induced pluripotent stem cells (hiPSCs) have enormous potential in regenerative medicine, their epigenetic variability suggests that some lines may not be suitable for human therapy. There are currently few benchmarks for assessing quality. Here we show that X-inactivation markers can be used to separate hiPSC lines into distinct epigenetic classes and that the classes are phenotypically distinct. Loss of XIST expression is strongly correlated with upregulation of X-linked oncogenes, accelerated growth rate in vitro, and poorer differentiation in vivo. Whereas differences in X-inactivation potential result in epigenetic variability of female hiPSC lines, male hiPSC lines generally resemble each other and do not overexpress the oncogenes. Neither physiological oxygen levels nor HDAC inhibitors offer advantages to culturing female hiPSC lines. We conclude that female hiPSCs may be epigenetically less stable in culture and caution that loss of XIST may result in qualitatively less desirable stem cell lines.