RESUMEN
Epigenetic gene regulation is a dynamic process orchestrated by chromatin-modifying enzymes. Many of these master regulators exert their function through covalent modification of DNA and histone proteins. Aberrant epigenetic processes have been implicated in the pathophysiology of multiple human diseases. Small-molecule inhibitors have been essential to advancing our understanding of the underlying molecular mechanisms of epigenetic processes. However, the resolution offered by small molecules is often insufficient to manipulate epigenetic processes with high spatiotemporal control. Here we present a generalizable approach, referred to as 'chemo-optical modulation of epigenetically regulated transcription' (COMET), enabling high-resolution, optical control of epigenetic mechanisms based on photochromic inhibitors of human histone deacetylases using visible light. COMET probes may be translated into new therapeutic strategies for diseases where conditional and selective epigenome modulation is required.
Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Luz , Optogenética/métodos , Compuestos Azo/química , Epigénesis Genética , Humanos , Células MCF-7 , Modelos Moleculares , Estructura MolecularRESUMEN
Targeting chromatin-mediated epigenetic regulation has emerged as a potential avenue for developing novel therapeutics for a wide range of central nervous system disorders, including cognitive disorders and depression. Histone deacetylase (HDAC) inhibitors have been pursued as cognitive enhancers that impact the regulation of gene expression and other mechanisms integral to neuroplasticity. Through systematic modification of the structure of crebinostat, a previously discovered cognitive enhancer that affects genes critical to memory and enhances synaptogenesis, combined with biochemical and neuronal cell-based screening, we identified a novel hydroxamate-based HDAC inhibitor, here named neurinostat, with increased potency compared to crebinostat in inducing neuronal histone acetylation. In addition, neurinostat was found to have a pharmacokinetic profile in mouse brain modestly improved over that of crebinostat. This discovery of neurinostat and demonstration of its effects on neuronal HDACs adds to the available pharmacological toolkit for dissecting the molecular and cellular mechanisms of neuroepigenetic regulation in health and disease.
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Compuestos de Bifenilo/química , Encéfalo/metabolismo , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Hidrazinas/química , Acetilación , Animales , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/farmacocinética , Células Cultivadas , Semivida , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/farmacocinética , Histona Desacetilasas/química , Histonas/metabolismo , Hidrazinas/síntesis química , Hidrazinas/farmacocinética , Concentración 50 Inhibidora , Ratones , Neuronas/citología , Neuronas/metabolismo , Unión Proteica , Relación Estructura-ActividadRESUMEN
The structure-activity and structure-kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11Å channel leading to the Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.
Asunto(s)
Anilidas/química , Anilidas/farmacología , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Acetilación/efectos de los fármacos , Aminación , Animales , Células Cultivadas , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Histonas/metabolismo , Humanos , Cinética , Ratones , Simulación del Acoplamiento MolecularRESUMEN
Small molecule fluorophores are indispensable tools for modern biomedical imaging techniques. In this report, we present the development of a new class of BODIPY dyes based on an alkoxy-fluoro-boron-dipyrromethene core. These novel fluorescent dyes, which we term MayaFluors, are characterized by good aqueous solubility and favorable in vitro physicochemical properties. MayaFluors are readily accessible in good yields in a one-pot, two-step approach starting from well-established BODIPY dyes, and allow for facile modification with functional groups of relevance to bioconjugate chemistry and bioorthogonal labeling. Biological profiling in living cells demonstrates excellent membrane permeability, low nonspecific binding, and lack of cytotoxicity.
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Compuestos de Boro/análisis , Colorantes Fluorescentes/análisis , Imagen Molecular/métodos , Compuestos de Boro/síntesis química , Compuestos de Boro/química , Permeabilidad de la Membrana Celular , Supervivencia Celular , Femenino , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Células MCF-7 , Estructura Molecular , Solubilidad , Células Tumorales CultivadasRESUMEN
Frontotemporal dementia (FTD) is a debilitating neurodegenerative disorder with currently no disease-modifying treatment options available. Mutations in GRN are one of the most common genetic causes of FTD, near ubiquitously resulting in progranulin (PGRN) haploinsufficiency. Small molecules that can restore PGRN protein to healthy levels in individuals bearing a heterozygous GRN mutation may thus have therapeutic value. Here, we show that epigenetic modulation through bromodomain and extra-terminal domain (BET) inhibitors (BETi) potently enhance PGRN protein levels, both intracellularly and secreted forms, in human central nervous system (CNS)-relevant cell types, including in microglia-like cells. In terms of potential for disease modification, we show BETi treatment effectively restores PGRN levels in neural cells with a GRN mutation known to cause PGRN haploinsufficiency and FTD. We demonstrate that BETi can rapidly and durably enhance PGRN in neural progenitor cells (NPCs) in a manner dependent upon BET protein expression, suggesting a gain-of-function mechanism. We further describe a CNS-optimized BETi chemotype that potently engages endogenous BRD4 and enhances PGRN expression in neuronal cells. Our results reveal a new epigenetic target for treating PGRN-deficient forms of FTD and provide mechanistic insight to aid in translating this discovery into therapeutics.
Asunto(s)
Demencia Frontotemporal , Humanos , Progranulinas/metabolismo , Demencia Frontotemporal/tratamiento farmacológico , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Mutación , Epigénesis Genética , Proteínas que Contienen Bromodominio , Proteínas de Ciclo Celular/metabolismoRESUMEN
The expanded CAG repeat that causes striatal cell vulnerability in Huntington's disease (HD) encodes a polyglutamine tract in full-length huntingtin that is correlated with cellular [ATP] and [ATP/ADP]. Since striatal neurons are vulnerable to energy deficit, we have investigated, in Hdh CAG knock-in mice and striatal cells, the hypothesis that decreased energetics may affect neuronal (N)-cadherin, a candidate energy-sensitive adhesion protein that may contribute to HD striatal cell sensitivity. In vivo, N-cadherin was sensitive to ischemia and to the effects of full-length mutant huntingtin, progressively decreasing in Hdh(Q111) striatum with age. In cultured striatal cells, N-cadherin was decreased by ATP depletion and STHdh(Q111) striatal cells exhibited dramatically decreased N-cadherin, due to decreased Cdh2 mRNA and enhanced N-cadherin turnover, which was partially normalized by adenine supplementation to increase [ATP] and [ATP/ADP]. Consistent with decreased N-cadherin function, STHdh(Q111) striatal cells displayed profound deficits in calcium-dependent N-cadherin-mediated cell clustering and cell-substratum adhesion, and primary Hdh(Q111) striatal neuronal cells exhibited decreased N-cadherin and an abundance of immature neurites, featuring diffuse, rather than clustered, staining for N-cadherin and synaptic vesicle markers, which was partially rescued by adenine treatment. Thus, mutant full-length huntingtin, via energetic deficit, contributes to decreased N-cadherin levels in striatal neurons, with detrimental effects on neurite maturation, strongly suggesting that N-cadherin-mediated signaling merits investigation early in the HD pathogenic disease process.
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Cadherinas/metabolismo , Cuerpo Estriado/citología , Enfermedad de Huntington/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Adenina , Adenosina Trifosfato/metabolismo , Animales , Adhesión Celular/fisiología , Células Cultivadas , Cuerpo Estriado/metabolismo , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Técnicas de Sustitución del Gen , Humanos , Proteína Huntingtina , Immunoblotting , Inmunohistoquímica , Ratones , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
Asunto(s)
Glutamatos/metabolismo , Procesamiento de Imagen Asistido por Computador , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas/patología , Proteómica , Tauopatías/patología , Proteínas tau/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Piridinas/química , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.
Asunto(s)
Aldehídos/química , Descubrimiento de Drogas/métodos , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/farmacología , Animales , Productos Biológicos/síntesis química , Productos Biológicos/química , Productos Biológicos/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores de Histona Desacetilasas/química , Compuestos Macrocíclicos/química , Ratones , Modelos Moleculares , Conformación Molecular , Estereoisomerismo , Especificidad por SustratoRESUMEN
Background: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods: Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results: Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion: MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Esclerosis Tuberosa , Sistemas CRISPR-Cas , Codón sin Sentido , Edición Génica , Mutación de Línea Germinal , Humanos , Células Madre Pluripotentes Inducidas/citología , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Neurogénesis , Fenotipo , RNA-Seq , Transducción de Señal , Sirolimus , Proteína 1 del Complejo de la Esclerosis Tuberosa/genéticaRESUMEN
Alterations in the autophagosomal-lysosomal pathway are a major pathophysiological feature of CLN3 disease, which is the most common form of childhood-onset neurodegeneration. Accumulating autofluorescent lysosomal storage material in CLN3 disease, consisting of dolichols, lipids, biometals, and a protein that normally resides in the mitochondria, subunit c of the mitochondrial ATPase, provides evidence that autophagosomal-lysosomal turnover of cellular components is disrupted upon loss of CLN3 protein function. Using a murine neuronal cell model of the disease, which accurately mimics the major gene defect and the hallmark features of CLN3 disease, we conducted an unbiased search for modifiers of autophagy, extending previous work by further optimizing a GFP-LC3 based assay and performing a high-content screen on a library of ~2000 bioactive compounds. Here we corroborate our earlier screening results and identify expanded, independent sets of autophagy modifiers that increase or decrease the accumulation of autophagosomes in the CLN3 disease cells, highlighting several pathways of interest, including the regulation of calcium signaling, microtubule dynamics, and the mevalonate pathway. Follow-up analysis on fluspirilene, nicardipine, and verapamil, in particular, confirmed activity in reducing GFP-LC3 vesicle burden, while also demonstrating activity in normalizing lysosomal positioning and, for verapamil, in promoting storage material clearance in CLN3 disease neuronal cells. This study demonstrates the potential for cell-based screening studies to identify candidate molecules and pathways for further work to understand CLN3 disease pathogenesis and in drug development efforts.
Asunto(s)
Autofagosomas/efectos de los fármacos , Descubrimiento de Drogas/métodos , Fluspirileno/farmacología , Lipofuscinosis Ceroideas Neuronales/tratamiento farmacológico , Nicardipino/farmacología , Verapamilo/farmacología , Animales , Autofagosomas/metabolismo , Autofagosomas/patología , Autofagia/efectos de los fármacos , Línea Celular , Mutación con Pérdida de Función , Glicoproteínas de Membrana/genética , Ratones , Chaperonas Moleculares/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Lipofuscinosis Ceroideas Neuronales/patologíaRESUMEN
Tau inclusions are a shared feature of many neurodegenerative diseases, among them frontotemporal dementia caused by tau mutations. Treatment approaches for these conditions include targeting posttranslational modifications of tau proteins, maintaining a steady-state amount of tau, and preventing its tendency to aggregate. We discovered a new regulatory pathway for tau degradation that operates through the farnesylated protein, Rhes, a GTPase in the Ras family. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib reduced Rhes and decreased brain atrophy, tau inclusions, tau sumoylation, and tau ubiquitination in the rTg4510 mouse model of tauopathy. In addition, lonafarnib treatment attenuated behavioral abnormalities in rTg4510 mice and reduced microgliosis in mouse brain. Direct reduction of Rhes in the rTg4510 mouse by siRNA reproduced the results observed with lonafarnib treatment. The mechanism of lonafarnib action mediated by Rhes to reduce tau pathology was shown to operate through activation of lysosomes. We finally showed in mouse brain and in human induced pluripotent stem cell-derived neurons a normal developmental increase in Rhes that was initially suppressed by tau mutations. The known safety of lonafarnib revealed in human clinical trials for cancer suggests that this drug could be repurposed for treating tauopathies.
Asunto(s)
Farnesiltransferasa/antagonistas & inhibidores , Tauopatías/tratamiento farmacológico , Tauopatías/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mutación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Piperidinas/farmacología , Proteolisis/efectos de los fármacos , Piridinas/farmacología , ARN Interferente Pequeño/genética , Tauopatías/patología , Investigación Biomédica Traslacional , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3ß-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3ß/GSK-3α) GSK-3ß inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/ß-catenin signaling activation, was observed in cells.
Asunto(s)
Encéfalo/metabolismo , Descubrimiento de Drogas , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Tomografía de Emisión de Positrones/métodos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Dominio Catalítico , Glucógeno Sintasa Quinasa 3 beta/química , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Neuroimagen , Oxazoles/química , Oxazoles/metabolismo , Oxazoles/farmacología , Inhibidores de Proteínas Quinasas/metabolismo , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
The ankyrin 3 gene (ANK3) is a well-established risk gene for psychiatric illness, but the mechanisms underlying its pathophysiology remain elusive. We examined the molecular effects of disrupting brain-specific Ank3 isoforms in mouse and neuronal model systems. RNA sequencing of hippocampus from Ank3+/- and Ank3+/+ mice identified altered expression of 282 genes that were enriched for microtubule-related functions. Results were supported by increased expression of microtubule end-binding protein 3 (EB3), an indicator of microtubule dynamics, in Ank3+/- mouse hippocampus. Live-cell imaging of EB3 movement in primary neurons from Ank3+/- mice revealed impaired elongation of microtubules. Using a CRISPR-dCas9-KRAB transcriptional repressor in mouse neuro-2a cells, we determined that repression of brain-specific Ank3 increased EB3 expression, decreased tubulin acetylation, and increased the soluble:polymerized tubulin ratio, indicating enhanced microtubule dynamics. These changes were rescued by inhibition of glycogen synthase kinase 3 (GSK3) with lithium or CHIR99021, a highly selective GSK3 inhibitor. Brain-specific Ank3 repression in neuro-2a cells increased GSK3 activity (reduced inhibitory phosphorylation) and elevated collapsin response mediator protein 2 (CRMP2) phosphorylation, a known GSK3 substrate and microtubule-binding protein. Pharmacological inhibition of CRMP2 activity attenuated the rescue of EB3 expression and tubulin polymerization in Ank3-repressed cells by lithium or CHIR99021, suggesting microtubule instability induced by Ank3 repression is dependent on CRMP2 activity. Taken together, our data indicate that ANK3 functions in neuronal microtubule dynamics through GSK3 and its downstream substrate CRMP2. These findings reveal cellular and molecular mechanisms underlying brain-specific ANK3 disruption that may be related to its role in psychiatric illness.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Compuestos de Litio/farmacología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Animales , Ancirinas/genética , Femenino , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microtúbulos/metabolismo , Neuronas/metabolismo , Fosforilación , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacosRESUMEN
Frontotemporal dementia (FTD) arises from neurodegeneration in the frontal, insular, and anterior temporal lobes. Autosomal dominant causes of FTD include heterozygous mutations in the GRN gene causing haploinsufficiency of progranulin (PGRN) protein. Recently, histone deacetylase (HDAC) inhibitors have been identified as enhancers of PGRN expression, although the mechanisms through which GRN is epigenetically regulated remain poorly understood. Using a chemogenomic toolkit, including optoepigenetic probes, we show that inhibition of class I HDACs is sufficient to upregulate PGRN in human neurons, and only inhibitors with apparent fast binding to their target HDAC complexes are capable of enhancing PGRN expression. Moreover, we identify regions in the GRN promoter in which elevated H3K27 acetylation and transcription factor EB (TFEB) occupancy correlate with HDAC-inhibitor-mediated upregulation of PGRN. These findings have implications for epigenetic and cis-regulatory mechanisms controlling human GRN expression and may advance translational efforts to develop targeted therapeutics for treating PGRN-deficient FTD.
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Histona Desacetilasas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Acetilación/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Células Cultivadas , Epigénesis Genética , Demencia Frontotemporal/tratamiento farmacológico , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Haploinsuficiencia/genética , Heterocigoto , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/química , Histona Desacetilasas/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Indoles/química , Indoles/farmacología , Indoles/uso terapéutico , Células Madre Pluripotentes Inducidas/citología , Péptidos y Proteínas de Señalización Intercelular/genética , Luz , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Progranulinas , Regiones Promotoras Genéticas , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Interferencia de ARN , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
The role of Arc in synaptic plasticity and memory consolidation has been investigated for many years with recent evidence that defects in the expression or activity of this immediate-early gene may also contribute to the pathophysiology of brain disorders including schizophrenia and fragile X syndrome. These results bring forward the concept that reversing Arc abnormalities could provide an avenue to improve cognitive or neurological impairments in different disease contexts, but how to achieve this therapeutic objective has remained elusive. Here, we present results from a chemogenomic screen that probed a mechanistically diverse library of small molecules for modulators of BDNF-induced Arc expression in primary cortical neurons. This effort identified compounds with a range of influences on Arc, including promoting its acetylation-a previously uncharacterized post-translational modification of this protein. Together, our data provide insights into the control of Arc that could be targeted to harness neuroplasticity for clinical applications.
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Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Lisina/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Acetilación , Secuencias de Aminoácidos , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas del Citoesqueleto/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Neuronas/química , Neuronas/metabolismo , Estabilidad ProteicaRESUMEN
Fragile X syndrome is due to the absence of the fragile X mental retardation protein (FMRP). Patients are mentally retarded and show physical as well as behavioural abnormalities. Loss of protein in the neurons results in changes of dendrite architecture, and impairment of the pruning process has been indicated. Apart from some minor differences, no severe morphological changes have been observed in the brain. Until now, no therapy is available for fragile X patients. Recently it has been reported, that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to the protein in question. Upon production of a TAT-FMRP fusion protein in a baculovirus-expression system, we used immunohistochemistry to verify TAT-mediated uptake of FMRP in fibroblasts. However, uptake efficiency and velocity was lower than expected. Neuronal uptake was highly inefficient and the fusion protein demonstrated toxicity.
Asunto(s)
Fibroblastos/metabolismo , Síndrome del Cromosoma X Frágil/metabolismo , Productos del Gen tat/farmacología , Proteínas del Tejido Nervioso/farmacología , Neuronas/metabolismo , Proteínas de Unión al ARN/farmacología , Proteínas Recombinantes de Fusión/farmacología , Animales , Células COS , Cricetinae , Fibroblastos/patología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/patología , Productos del Gen tat/genética , Productos del Gen tat/toxicidad , Inmunohistoquímica , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/fisiología , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
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. This article is part of a Special Issue entitled 'Cognitive Enhancers'.
Asunto(s)
Compuestos de Bifenilo/farmacología , Descubrimiento de Drogas/métodos , Inhibidores de Histona Desacetilasas/farmacología , Hidrazinas/farmacología , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Nootrópicos/farmacología , Prosencéfalo/efectos de los fármacos , Acetilación/efectos de los fármacos , Animales , Animales Recién Nacidos , Compuestos de Bifenilo/sangre , Compuestos de Bifenilo/metabolismo , Compuestos de Bifenilo/farmacocinética , Células Cultivadas , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Genes Reporteros/efectos de los fármacos , Inhibidores de Histona Desacetilasas/sangre , Inhibidores de Histona Desacetilasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacocinética , Histona Desacetilasas/química , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Hidrazinas/sangre , Hidrazinas/metabolismo , Hidrazinas/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Nootrópicos/sangre , Nootrópicos/metabolismo , Nootrópicos/farmacocinética , Prosencéfalo/citología , Prosencéfalo/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Epigénesis Genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Sistema Nervioso/crecimiento & desarrollo , Adulto , Estudios de Casos y Controles , Diferenciación Celular/genética , Células Cultivadas , Reprogramación Celular/genética , Preescolar , Islas de CpG/genética , Metilación de ADN/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Mosaicismo , Mutación/genética , Sistema Nervioso/metabolismo , Sistema Nervioso/patología , Neuronas/metabolismo , Neuronas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Donantes de Tejidos , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Carboxylic acids with known central nervous system and histone deacetylase (HDAC) inhibitory activities were converted to hydroxamic acids and tested using a suite of in vitro biochemical assays with recombinant HDAC isoforms, cell based assays in human cervical carcinoma Hela cells and primary cultures from mouse forebrain, and a whole animal (Xenopus laevis) developmental assay. Relative to the parent carboxylic acids, two of these analogs exhibited enhanced potency, and one analog showed altered HDAC isoform selectivity and in vivo activity in the Xenopus assay. We discuss potential uses of these novel hydroxamic acids in studies aimed at determining the utility of HDAC inhibitors as memory enhancers and mood stabilizers.
RESUMEN
Impaired local protein translation at postsynaptic sites has been hypothesized to be the cause of several neurological disorders such as fragile X syndrome, neurofibromatosis-1, Rett syndrome, and other syndromic and non-specific forms of mental retardation. Identification of which mRNAs are present in dendrites and the identification of the molecular pathways that they promote will be imperative to the understanding of the neuropathology of these diseases. Since mouse models are the most widely used animal models of human diseases we developed a cell culture based technique to isolate mRNAs from mouse neurites.