RESUMO
Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.
Assuntos
Mitocôndrias/metabolismo , Mitofagia/fisiologia , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Doenças Neurodegenerativas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologiaRESUMO
Sensorimotor information processing underlies normal cognitive and behavioral traits and has classically been evaluated through prepulse inhibition (PPI) of a startle reflex. PPI is a behavioral dimension deregulated in several neurological and psychiatric disorders, yet the mechanisms underlying the cross-diagnostic nature of PPI deficits across these conditions remain to be understood. To identify circuitry mechanisms for PPI, we performed circuitry recording over the prefrontal cortex and striatum, two brain regions previously implicated in PPI, using wild-type (WT) mice compared to Disc1-locus-impairment (LI) mice, a model representing neuropsychiatric conditions. We demonstrated that the corticostriatal projection regulates neurophysiological responses during the PPI testing in WT, whereas these circuitry responses were disrupted in Disc1-LI mice. Because our biochemical analyses revealed attenuated brain-derived neurotrophic factor (Bdnf) transport along the corticostriatal circuit in Disc1-LI mice, we investigated the potential role of Bdnf in this circuitry for regulation of PPI. Virus-mediated delivery of Bdnf into the striatum rescued PPI deficits in Disc1-LI mice. Pharmacologically augmenting Bdnf transport by chronic lithium administration, partly via phosphorylation of Huntingtin (Htt) serine-421 and its integration into the motor machinery, restored striatal Bdnf levels and rescued PPI deficits in Disc1-LI mice. Furthermore, reducing the cortical Bdnf expression negated this rescuing effect of lithium, confirming the key role of Bdnf in lithium-mediated PPI rescuing. Collectively, the data suggest that striatal Bdnf supply, collaboratively regulated by Htt and Disc1 along the corticostriatal circuit, is involved in sensorimotor gating, highlighting the utility of dimensional approach in investigating pathophysiological mechanisms across neuropsychiatric disorders.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Corpo Estriado , Proteínas do Tecido Nervoso , Córtex Pré-Frontal , Inibição Pré-Pulso , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corpo Estriado/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Córtex Pré-Frontal/metabolismo , Inibição Pré-Pulso/fisiologia , Reflexo de Sobressalto/fisiologia , Filtro Sensorial/fisiologiaRESUMO
First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel anti-seizure strategies. It is emerging that impaired K+/Cl- cotransporter 2 (KCC2) activity leads to deficits in GABAergic inhibition and increased seizure vulnerability in patients. In neurons, the with-no-lysine (WNK) kinase-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signalling pathway inhibits KCC2 activity via T1007 phosphorylation. Here, we exploit the selective WNK kinase inhibitor WNK463 to test the effects of pharmacological WNK inhibition on KCC2 function, GABAergic inhibition, and epileptiform activity. Immunoprecipitation and western blotting analysis revealed that WNK463 reduces KCC2-T1007 phosphorylation in vitro and in vivo. Using patch-clamp recordings in primary rat neurons, we further observed that WNK463 hyperpolarized the Cl- reversal potential, and enhanced KCC2-mediated Cl- extrusion. In the 4-aminopyridine slice model of acute seizures, WNK463 administration reduced the frequency and number of seizure-like events. In vivo, C57BL/6 mice that received intrahippocampal WNK463 experienced delayed onset of kainic acid-induced status epilepticus, less epileptiform EEG activity, and did not develop pharmaco-resistance to diazepam. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. In summary, our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant status epilepticus.
Assuntos
Epilepsia , Estado Epiléptico , Simportadores , Animais , Diazepam/metabolismo , Diazepam/farmacologia , Hipocampo/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/metabolismo , Simportadores/metabolismoRESUMO
A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington's disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.
Assuntos
Membrana Celular/metabolismo , Doença de Huntington/genética , Neurônios/enzimologia , Diester Fosfórico Hidrolases/genética , Domínios Proteicos/genética , Animais , Autofagia/genética , Corpo Estriado/citologia , Corpo Estriado/patologia , AMP Cíclico/metabolismo , Embrião de Mamíferos , Células HEK293 , Humanos , Doença de Huntington/patologia , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação , Neurônios/citologia , Técnicas de Patch-Clamp , Diester Fosfórico Hidrolases/metabolismo , Cultura Primária de Células , Proteólise , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The K+/Cl- cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.
Assuntos
Neurônios/metabolismo , Simportadores/metabolismo , Animais , Apoptose , Cloretos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Cultura Primária de Células , Receptores de GABA/metabolismo , Convulsões , Simportadores/fisiologia , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-RESUMO
Activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene product that support neuroplastic changes important for cognitive function and memory formation. As a protein with homology to the retroviral Gag protein, a particular characteristic of Arc is its capacity to self-assemble into virus-like capsids that can package mRNAs and transfer those transcripts to other cells. Although a lot has been uncovered about the contributions of Arc to neuron biology and behavior, very little is known about how different functions of Arc are coordinately regulated both temporally and spatially in neurons. The answer to this question we hypothesized must involve the occurrence of different protein post-translational modifications acting to confer specificity. In this study, we used mass spectrometry and sequence prediction strategies to map novel Arc phosphorylation sites. Our approach led us to recognize serine 67 (S67) and threonine 278 (T278) as residues that can be modified by TNIK, which is a kinase abundantly expressed in neurons that shares many functional overlaps with Arc and has, along with its interacting proteins such as the NMDA receptor, and been implicated as a risk factor for psychiatric disorders. Furthermore, characterization of each residue using site-directed mutagenesis to create S67 and T278 mutant variants revealed that TNIK action at those amino acids can strongly influence Arc's subcellular distribution and self-assembly as capsids. Together, our findings reveal an unsuspected connection between Arc and TNIK. Better understanding of the interplay between these two proteins in neuronal cells could lead to new insights about apparition and progression of psychiatric disorders. Cover Image for this issue: https://doi.org/10.1111/jnc.15077.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Camundongos , Neurônios/metabolismo , Fosforilação/fisiologiaRESUMO
Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.
Assuntos
Esquizofrenia/genética , Nexinas de Classificação/genética , Adulto , Alelos , Autopsia , Encéfalo/metabolismo , Cromatina/metabolismo , Mapeamento Cromossômico/métodos , Metilação de DNA , Éxons/genética , Feminino , Expressão Gênica/genética , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Fatores de Risco , Nexinas de Classificação/metabolismoRESUMO
The type 2 K+/Cl- cotransporter (KCC2) allows neurons to maintain low intracellular levels of Cl-, a prerequisite for efficient synaptic inhibition. Reductions in KCC2 activity are evident in epilepsy; however, whether these deficits directly contribute to the underlying pathophysiology remains controversial. To address this issue, we created knock-in mice in which threonines 906 and 1007 within KCC2 have been mutated to alanines (KCC2-T906A/T1007A), which prevents its phospho-dependent inactivation. The respective mice appeared normal and did not show any overt phenotypes, and basal neuronal excitability was unaffected. KCC2-T906A/T1007A mice exhibited increased basal neuronal Cl- extrusion, without altering total or plasma membrane accumulation of KCC2. Critically, activity-induced deficits in synaptic inhibition were reduced in the mutant mice. Consistent with this, enhanced KCC2 was sufficient to limit chemoconvulsant-induced epileptiform activity. Furthermore, this increase in KCC2 function mitigated induction of aberrant high-frequency activity during seizures, highlighting depolarizing GABA as a key contributor to the pathological neuronal synchronization seen in epilepsy. Thus, our results demonstrate that potentiating KCC2 represents a therapeutic strategy to alleviate seizures.
Assuntos
Epilepsia/metabolismo , Neurônios/metabolismo , Convulsões/metabolismo , Simportadores/metabolismo , Membranas Sinápticas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Substituição de Aminoácidos , Animais , Epilepsia/genética , Epilepsia/patologia , Técnicas de Introdução de Genes , Camundongos , Mutação de Sentido Incorreto , Neurônios/patologia , Convulsões/genética , Convulsões/patologia , Simportadores/genética , Membranas Sinápticas/genética , Membranas Sinápticas/patologia , Ácido gama-Aminobutírico/genética , Cotransportadores de K e Cl-RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease marked by progressive loss of muscle function. It is the most common adult-onset form of motor neuron disease, affecting about 16 000 people in the United States alone. The average survival is about 3 years. Only two interventional drugs, the antiglutamatergic small-molecule riluzole and the more recent antioxidant edaravone, have been approved for the treatment of ALS to date. Therapeutic strategies under investigation in clinical trials cover a range of different modalities and targets, and more than 70 different drugs have been tested in the clinic to date. Here, we summarize and classify interventional therapeutic strategies based on their molecular targets and phenotypic effects. We also discuss possible reasons for the failure of clinical trials in ALS and highlight emerging preclinical strategies that could provide a breakthrough in the battle against this relentless disease.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Ensaios Clínicos como Assunto , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Autofagia/efeitos dos fármacos , Aprovação de Drogas , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêuticoRESUMO
Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.
Assuntos
Estradiol/farmacologia , Inibição Neural/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Animais , Proteínas de Transporte/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Transmissão Sináptica/efeitos dos fármacosRESUMO
BACKGROUND: RNA sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of these biases have focused on adapter ligation bias with limited evaluation of reverse transcription bias or amplification bias. Furthermore, evaluations of the quantification of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited. No study had yet evaluated the quantification of isomiRs of altered length or compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases. RESULTS: All methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency. CONCLUSIONS: Our data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.
Assuntos
Análise de Sequência de RNA/normas , Animais , Viés , Humanos , Camundongos , Isoformas de RNA , RNA Viral , Ratos , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodosRESUMO
Deficits in the basal ganglia pathways modulating cortical motor activity underlie both Parkinson disease (PD) and Huntington disease (HD). Phosphodiesterase 10A (PDE10A) is enriched in the striatum, and animal data suggest that it is a key regulator of this circuitry. Here, we report on germline PDE10A mutations in eight individuals from two families affected by a hyperkinetic movement disorder due to homozygous mutations c.320A>G (p.Tyr107Cys) and c.346G>C (p.Ala116Pro). Both mutations lead to a reduction in PDE10A levels in recombinant cellular systems, and critically, positron-emission-tomography (PET) studies with a specific PDE10A ligand confirmed that the p.Tyr107Cys variant also reduced striatal PDE10A levels in one of the affected individuals. A knock-in mouse model carrying the homologous p.Tyr97Cys variant had decreased striatal PDE10A and also displayed motor abnormalities. Striatal preparations from this animal had an impaired capacity to degrade cyclic adenosine monophosphate (cAMP) and a blunted pharmacological response to PDE10A inhibitors. These observations highlight the critical role of PDE10A in motor control across species.
Assuntos
Corpo Estriado/patologia , Hipercinese/genética , Mutação , Diester Fosfórico Hidrolases/genética , Alelos , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Variação Genética , Células HEK293 , Humanos , Hipercinese/diagnóstico , Hipercinese/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linhagem , Inibidores de Fosfodiesterase/metabolismo , Alinhamento de SequênciaRESUMO
This letter describes progress towards an M4 PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3-c]pyridazine core has been a consistent feature of key M4 PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the ß-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe.
Assuntos
Regulação Alostérica/imunologia , Receptor Muscarínico M4/imunologia , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Esquizofrenia/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Bioensaio , Humanos , Estrutura MolecularRESUMO
This letter describes progress towards an M4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.
Assuntos
Aldeído Oxidase/metabolismo , Miotonia Congênita/metabolismo , Receptor Muscarínico M4/metabolismo , Animais , Descoberta de Drogas , Humanos , Ratos , Relação Estrutura-AtividadeRESUMO
apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/agonistas , Apolipoproteínas E/agonistas , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Animais , Apolipoproteínas E/metabolismo , Aziridinas/química , Aziridinas/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Células Cultivadas , Humanos , Camundongos , Camundongos Knockout , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Antagonistas do Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X7/deficiência , Bibliotecas de Moléculas Pequenas/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.
Assuntos
Etilmaleimida/metabolismo , Simportadores/metabolismo , Animais , Membrana Celular/metabolismo , Embrião de Mamíferos , Humanos , Moduladores de Transporte de Membrana/metabolismo , Neurônios/metabolismo , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Simportadores/fisiologia , Cotransportadores de K e Cl-RESUMO
The behavioral and anatomical deficits seen in fragile X syndrome (FXS) are widely believed to result from imbalances in the relative strengths of excitatory and inhibitory neurotransmission. Although modified neuronal excitability is thought to be of significance, the contribution that alterations in GABAergic inhibition play in the pathophysiology of FXS are ill defined. Slow sustained neuronal inhibition is mediated by γ-aminobutyric acid type B (GABAB) receptors, which are heterodimeric G-protein-coupled receptors constructed from R1a and R2 or R1b and R2 subunits. Via the activation of Gi/o, they limit cAMP accumulation, diminish neurotransmitter release, and induce neuronal hyperpolarization. Here we reveal that selective deficits in R1a subunit expression are seen in Fmr1 knock-out mice (KO) mice, a widely used animal model of FXS, but the levels of the respective mRNAs were unaffected. Similar trends of R1a expression were seen in a subset of FXS patients. GABAB receptors (GABABRs) exert powerful pre- and postsynaptic inhibitory effects on neurotransmission. R1a-containing GABABRs are believed to mediate presynaptic inhibition in principal neurons. In accordance with this result, deficits in the ability of GABABRs to suppress glutamate release were seen in Fmr1-KO mice. In contrast, the ability of GABABRs to suppress GABA release and induce postsynaptic hyperpolarization was unaffected. Significantly, this deficit contributes to the pathophysiology of FXS as the GABABR agonist (R)-baclofen rescued the imbalances between excitatory and inhibitory neurotransmission evident in Fmr1-KO mice. Collectively, our results provided evidence that selective deficits in the activity of presynaptic GABABRs contribute to the pathophysiology of FXS.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Receptores de GABA-B/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Comportamento Animal , Modelos Animais de Doenças , Síndrome do Cromossomo X Frágil/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Receptores de GABA-B/genética , Serina/química , Transdução de Sinais , Transmissão Sináptica , Ácido gama-Aminobutírico/metabolismoRESUMO
Due to increased interest in As(III) S-adenosylmethionine methyltransferase (AS3MT), a search for chemical probes that can help elucidate function was initiated. A homology model was built based on related enzymes, and virtual screening produced 426 potential hits. Evaluation of these compounds in a functional enzymatic assay revealed several modest inhibitors including an O-substituted 2-amino-3-cyano indole scaffold. Two iterations of near neighbor searches revealed compound 5 as a potent inhibitor of AS3MT with good selectivity over representative methyltransferases DOT1L and NSD2 as well as a representative set of diverse receptors. Compound 5 should prove to be a useful tool to investigate the role of AS3MT and a potential starting point for further optimization.
Assuntos
Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , HumanosRESUMO
Alterations in the efficacy of neuronal inhibition mediated by GABAA receptors (GABAARs) containing ß3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABAARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the ß3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in ß3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A). S408/9A homozygotes exhibited increased phasic, but decreased tonic, inhibition, events that correlated with alterations in the membrane stability and synaptic accumulation of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive behavior, and decreased social interaction, hallmarks of ASDs. ASDs are frequently comorbid with epilepsy, and consistent with this comorbidity, S408/9A mice exhibited a marked increase in sensitivity to seizures induced by the convulsant kainic acid. To assess the relevance of our studies using S408/9A mice for the pathophysiology of ASDs, we measured S408/9 phosphorylation in Fmr1 KO mice, a model of fragile X syndrome, the most common monogenetic cause of ASDs. Phosphorylation of S408/9 was selectively and significantly enhanced in Fmr1 KO mice. Collectively, our results suggest that alterations in phosphorylation and/or activity of ß3-containing GABAARs may directly contribute to the pathophysiology of ASDs.