RESUMO
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
The signalling characteristics of the Zinc-Activated Channel (ZAC), a member of the Cys-loop receptor (CLR) superfamily, are presently poorly elucidated. The ZACN polymorphism c.454G>A encoding for the Thr128Ala variation in ZAC is found in extremely high allele frequencies across ethnicities. In this, the first study of ZAC in Xenopus oocytes by TEVC electrophysiology, ZACThr128 and ZACAla128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn2+- and H+-evoked current amplitudes in ZACAla128-oocytes were dramatically smaller than those in ZACThr128-oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZACThr128 and ZACAla128 in transfected mammalian cells indicated that their distinct functionalities could arise from the latter. In co-expression experiments, wild-type and variant ZAC subunits assembled efficiently into "heteromeric" complexes in HEK293 cells, while the concomitant presence of ZACAla128 in ZACThr128:ZACAla128-oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZACThr128-oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr128 appeared to be subtle, as agonist-evoked current amplitudes in ZACSer128-, ZACVal128- and ZACIle128-oocytes also were substantially lower than those in ZACThr128-oocytes. In conclusion, the functional properties exhibited by ZAC in this work substantiate the notion of it being an atypical CLR. While the impact of the Thr128Ala variation on ZAC functionality in oocytes is striking, it remains to be investigated whether and to which extent this translates into an in vivo setting and thus could constitute a source of inter-individual variation in ZAC physiology.
Assuntos
Canais Iônicos/metabolismo , Zinco/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação , Canais Iônicos/genética , Canais Iônicos/fisiologia , Oócitos/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Xenopus laevisRESUMO
KEY POINTS: Synapses have high energy demands which increase during intense activity. We show that presynaptic terminals can utilise extracellular glucose or lactate to generate energy to maintain synaptic transmission. Reducing energy substrates induces a metabolic stress: presynaptic ATP depletion impaired synaptic transmission through a reduction in the number of functional synaptic vesicle release sites and a slowing of vesicle pool replenishment, without a consistent change in release probability. Metabolic function is compromised in many pathological conditions (e.g. stroke, traumatic brain injury and neurodegeneration). Knowledge of how synaptic transmission is constrained by metabolic stress, especially during intense brain activity, will provide insights to improve cognition following pathological insults. ABSTRACT: The synapse has high energy demands, which increase during intense activity. Presynaptic ATP production depends on substrate availability and usage will increase during activity, which in turn could influence transmitter release and information transmission. We investigated transmitter release at the mouse calyx of Held synapse using glucose or lactate (10, 1 or 0 mm) as the extracellular substrates while inducing metabolic stress. High-frequency stimulation (HFS) and recovery paradigms evoked trains of EPSCs monitored under voltage-clamp. Whilst postsynaptic intracellular ATP was stabilised by diffusion from the patch pipette, depletion of glucose increased EPSC depression during HFS and impaired subsequent recovery. Computational modelling of these data demonstrated a reduction in the number of functional release sites and slowed vesicle pool replenishment during metabolic stress, with little change in release probability. Directly depleting presynaptic terminal ATP impaired transmitter release in an analogous manner to glucose depletion. In the absence of glucose, presynaptic terminal metabolism could utilise lactate from the aCSF and this was blocked by inhibition of monocarboxylate transporters (MCTs). MCT inhibitors significantly suppressed transmission in low glucose, implying that lactate is a presynaptic substrate. Additionally, block of glycogenolysis accelerated synaptic transmission failure in the absence of extracellular glucose, consistent with supplemental supply of lactate by local astrocytes. We conclude that both glucose and lactate support presynaptic metabolism and that limited availability, exacerbated by high-intensity firing, constrains presynaptic ATP, impeding transmission through a reduction in functional presynaptic release sites as vesicle recycling slows when ATP levels are low.
Assuntos
Potenciais de Ação , Tronco Encefálico/fisiologia , Glucose/metabolismo , Ácido Láctico/metabolismo , Terminações Pré-Sinápticas/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Animais , Tronco Encefálico/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos CBARESUMO
The yeast Hsp31 minifamily proteins (Hsp31, Hsp32, Hsp33, Hsp34) belong to the highly conserved DJ-1 superfamily. The human DJ-1 protein is associated with cancer and neurodegenerative disorders, such as Parkinson disease. However, the precise function of human and yeast DJ-1 proteins is unclear. Here we show that the yeast DJ-1 homologs have a role in diauxic-shift (DS), characterized by metabolic reprogramming because of glucose limitation. We find that the Hsp31 genes are strongly induced in DS and in stationary phase (SP), and that deletion of these genes reduces chronological lifespan, impairs transcriptional reprogramming at DS, and impairs the acquisition of several typical characteristics of SP, including autophagy induction. In addition, under carbon starvation, the HSP31 family gene-deletion strains display impaired autophagy, disrupted target of rapamycin complex 1 (TORC1) localization to P-bodies, and caused abnormal TORC1-mediated Atg13 phosphorylation. Repression of TORC1 by rapamycin in the gene-deletion strains completely reversed their sensitivity to heat shock. Taken together, our data indicate that Hsp31 minifamily is required for DS reprogramming and cell survival in SP, and plays a role upstream of TORC1. The enhanced understanding of the cellular function of these genes sheds light into the biological role of other members of the superfamily, including DJ-1, which is an attractive target for therapeutic intervention in cancer and in Parkinson disease.
Assuntos
Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Autofagia/genética , Carbono/metabolismo , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/citologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
We previously showed that the PDE4 inhibitor apremilast reduces ethanol consumption in mice by protein kinase A (PKA) and GABAergic mechanisms. Preventing PKA phosphorylation of GABAA ß3 subunits partially blocked apremilast-mediated decreases in drinking. Here, we produced Gabrb1-S409A mice to render GABAA ß1 subunits resistant to PKA-mediated phosphorylation. Mass spectrometry confirmed the presence of the S409A mutation and lack of changes in ß1 subunit expression or phosphorylation at other residues. ß1-S409A male and female mice did not differ from wild-type C57BL/6J mice in expression of Gabrb1, Gabrb2, or Gabrb3 subunits or in behavioral characteristics. Apremilast prolonged recovery from ethanol ataxia to a greater extent in Gabrb1-S409A mice but prolonged recovery from zolpidem and propofol to a similar extent in both genotypes. Apremilast shortened recovery from diazepam ataxia in wild-type but prolonged recovery in Gabrb1-S409A mice. In wild-type mice, the PKA inhibitor H89 prevented apremilast modulation of ataxia by ethanol and diazepam, but not by zolpidem. In Gabrb1-S409A mice, inhibiting PKA or EPAC2 (exchange protein directly activated by cAMP) partially reversed apremilast potentiation of ethanol, diazepam, and zolpidem ataxia. Apremilast prevented acute tolerance to ethanol ataxia in both genotypes, but there were no genotype differences in ethanol consumption before or after apremilast. In contrast to results in Gabrb3-S408A/S409A mice, PKA phosphorylation of ß1-containing GABAA receptors is not required for apremilast's effects on acute tolerance or on ethanol consumption but is required for its ability to decrease diazepam intoxication. Besides PKA we identified EPAC2 as an additional cAMP-dependent mechanism by which apremilast regulates responses to GABAergic drugs.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Etanol , Camundongos Endogâmicos C57BL , Inibidores da Fosfodiesterase 4 , Receptores de GABA-A , Talidomida , Animais , Talidomida/farmacologia , Talidomida/análogos & derivados , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Masculino , Feminino , Etanol/farmacologia , Camundongos , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-A/efeitos dos fármacos , Técnicas de Introdução de Genes , Fosforilação/efeitos dos fármacos , Ataxia/genética , Consumo de Bebidas Alcoólicas/tratamento farmacológico , Consumo de Bebidas Alcoólicas/genética , Camundongos Transgênicos , Diazepam/farmacologiaRESUMO
LMTK3 is a brain-specific transmembrane serine/threonine protein kinase that acts as a scaffold for protein phosphatase-1 (PP1). Although LMKT3 has been identified as a risk factor for autism and epilepsy, its physiological significance is unknown. Here, we demonstrate that LMTK3 copurifies and binds to KCC2, a neuron-specific K+/Cl- transporter. KCC2 activity is essential for Cl--mediated hyperpolarizing GABAAR receptor currents, the unitary events that underpin fast synaptic inhibition. LMTK3 acts to promote the association of KCC2 with PP1 to promote the dephosphorylation of S940 within its C-terminal cytoplasmic domain, a process the diminishes KCC2 activity. Accordingly, acute inhibition of LMTK3 increases KCC2 activity dependent upon S940 and increases neuronal Cl- extrusion. Consistent with this, LMTK3 inhibition reduced intrinsic neuronal excitability and the severity of seizure-like events in vitro. Thus, LMTK3 may have profound effects on neuronal excitability as an endogenous modulator of KCC2 activity.
RESUMO
Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, ß and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. ß2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while ß4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating ß2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition.
Assuntos
Receptores de GABA-A , Espectrina , Receptores de GABA-A/metabolismo , Espectrina/metabolismo , Sinapses/metabolismo , Proteínas de Membrana/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl- accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury.
Assuntos
Estado Epiléptico , Simportadores , Camundongos , Animais , Benzodiazepinas/farmacologia , Benzodiazepinas/uso terapêutico , Estado Epiléptico/tratamento farmacológico , Convulsões/metabolismo , Ácido gama-Aminobutírico/metabolismo , Simportadores/metabolismoRESUMO
Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABA A Rs), which mediate phasic and tonic inhibition, respectively. These two GABA A R subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABA A Rs contain the α1-3 subunits whereas extrasynaptic GABA A Rs contain the α4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABA A R subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABA A Rs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABA A Rs and interact with distinct sets of binding proteins. We also discovered that the ß3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABA A Rs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABA A Rs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABA A Rs, and thus the efficacy of neuronal inhibition.
RESUMO
Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.
RESUMO
Drug-induced resistance, or tolerance, is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor, the consequence on tumor growth, and therapeutic strategies to address these challenges remain undescribed. Here, we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatiotemporally controlled nanomedicines, which boost therapeutic efficacy and invigorate "disabled" NK cells. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis, NK group 2 member D, and MHC class I polypeptide-related sequence A, B. Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. On the basis of this evidence, we engineered a "chimeric" nanotherapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor, radicicol, which targets the tumor, reduces tolerance, and optimally reprimes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically approved agents. These findings highlight a convergence between drug-induced resistance, the tumor immune contexture, and engineered approaches that consider the tumor and microenvironment to improve the success of combinatorial therapy. SIGNIFICANCE: This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Animais , Antineoplásicos Imunológicos/química , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Colesterol/química , Docetaxel/administração & dosagem , Docetaxel/farmacocinética , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Macrolídeos/química , Macrolídeos/farmacocinética , Macrolídeos/farmacologia , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular/métodos , Nanopartículas/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/cirurgia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Pharmaceutical breakthroughs for anxiety have been lackluster in the last half-century. Converging behavior and limbic molecular heterogeneity has the potential to revolutionize biomarker-driven interventions. However, current in vivo models too often deploy artificial systems including directed evolution, mutations and fear induction, which poorly mirror clinical manifestations. Here, we explore transcriptional heterogeneity of the amygdala in isogenic mice using an unbiased multi-dimensional computational approach that segregates intra-cohort reactions to moderate situational adversity and intersects it with high content molecular profiling. We show that while the computational approach stratifies known features of clinical anxiety including nitric oxide, opioid and corticotropin signaling, previously unrecognized druggable biomarkers emerge, such as calpain11 and scand1. Through ingenuity pathway analyses, we further describe a role for neurosteroid estradiol signaling, heat shock proteins, ubiquitin ligases and lipid metabolism. In addition, we report a remarkable behavioral pattern that maps to molecular features of anxiety in mice through counterphobic social attitudes, which manifest as increased, yet spatially distant socialization. These findings provide an unbiased approach for interrogating anxiolytics, and hint toward biomarkers underpinning behavioral and social patterns that merit further exploration.
Assuntos
Tonsila do Cerebelo , Ansiedade , Comportamento Animal , Modelos Biológicos , Neurociências/métodos , Comportamento Social , Socialização , Transcrição Gênica , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Animais , Ansiedade/genética , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBARESUMO
The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), ß(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments.
Assuntos
Motivos de Aminoácidos/genética , Axônios/metabolismo , Neurônios GABAérgicos/metabolismo , Receptores de GABA-A/metabolismo , Convulsões/metabolismo , Sinapses/metabolismo , Animais , Axônios/fisiologia , Células Cultivadas , Neurônios GABAérgicos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores de GABA-A/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Convulsões/genética , Convulsões/mortalidade , Sinapses/genética , Transmissão Sináptica/fisiologiaRESUMO
Hyperbilirubinemia (jaundice) is caused by raised levels of unconjugated bilirubin in the blood. When severe, susceptible brain regions including the cerebellum and auditory brainstem are damaged causing neurological sequelae such as ataxia, hearing loss and kernicterus. The mechanism(s) by which bilirubin exerts its toxic effect have not been completely understood to date. In this study we investigated the acute mechanisms by which bilirubin causes the neurotoxicity that contributes to hearing loss. We developed a novel mouse model that exhibits the neurological features seen in human Bilirubin-Induced Neurological Dysfunction (BIND) syndrome that we assessed with a behavioural score and auditory brainstem responses (ABR). Guided by initial experiments applying bilirubin to cultured cells in vitro, we performed whole genome gene expression measurements on mouse brain tissue (cerebellum and auditory brainstem) following bilirubin exposure to gain mechanistic insights into biochemical processes affected, and investigated further using immunoblotting. We then compared the gene changes induced by bilirubin to bacterial lipopolysaccharide (LPS), a well characterized inducer of neuroinflammation, to assess the degree of similarity between them. Finally, we examined the extent to which genetic perturbation of inflammation and both known and novel anti-inflammatory drugs could protect hearing from bilirubin-induced toxicity. The in vitro results indicated that bilirubin induces changes in gene expression consistent with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). These gene changes were similar to the gene expression signature of thapsigargin-a known ER stress inducer. It also induced gene expression changes associated with inflammation and NF-κB activation. The in vivo model showed behavioural impairment and a raised auditory threshold. Whole genome gene expression analysis confirmed inflammation as a key mechanism of bilirubin neurotoxicity in the auditory pathway and shared gene expression hallmarks induced by exposure to bacterial lipopolysaccharide (LPS) a well-characterized inducer of neuroinflammation. Interestingly, bilirubin caused more severe damage to the auditory system than LPS in this model, but consistent with our hypothesis of neuroinflammation being a primary part of bilirubin toxicity, the hearing loss was protected by perturbing the inflammatory response. This was carried out genetically using lipocalin-2 (LCN2)-null mice, which is an inflammatory cytokine highly upregulated in response to bilirubin. Finally, we tested known and novel anti-inflammatory compounds (interfering with NF-κB and TNFα signalling), and also demonstrated protection of the auditory system from bilirubin toxicity. We have developed a novel, reversible, model for jaundice that shows movement impairment and auditory loss consistent with human symptoms. We used this model to establish ER-stress and inflammation as major contributors to bilirubin toxicity. Because of the rapid and reversible onset of toxicity in this novel model it represents a system to screen therapeutic compounds. We have demonstrated this by targeting inflammation genetically and with anti-inflammatory small molecules that offered protection against bilirubin toxicity. This also suggests that anti-inflammatory drugs could be of therapeutic use in hyperbilirubinemia.
Assuntos
Bilirrubina/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perda Auditiva/etiologia , Kernicterus/etiologia , Síndromes Neurotóxicas/etiologia , Doença Aguda , Animais , Anti-Inflamatórios/farmacologia , Ataxia/etiologia , Ataxia/metabolismo , Bilirrubina/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Feminino , Perda Auditiva/metabolismo , Perda Auditiva/prevenção & controle , Humanos , Hiperbilirrubinemia/complicações , Hiperbilirrubinemia/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Kernicterus/metabolismo , Lipocalina-2/deficiência , Lipocalina-2/genética , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , NF-kappa B/metabolismo , Síndromes Neurotóxicas/metabolismoRESUMO
Mesial temporal lobe epilepsy (mTLE) is the most common form of epilepsy, believed to arise in part from compromised GABAergic inhibition. The neuronal specific K+/Cl- co-transporter 2 (KCC2) is a critical determinant of the efficacy of GABAergic inhibition and deficits in its activity are observed in mTLE patients and animal models of epilepsy. To test if reductions of KCC2 activity directly contribute to the pathophysiology of mTLE, we locally ablated KCC2 expression in a subset of principal neurons within the adult hippocampus. Deletion of KCC2 resulted in compromised GABAergic inhibition and the development of spontaneous, recurrent generalized seizures. Moreover, local ablation of KCC2 activity resulted in hippocampal sclerosis, a key pathological change seen in mTLE. Collectively, our results demonstrate that local deficits in KCC2 activity within the hippocampus are sufficient to precipitate mTLE.
Assuntos
Epilepsia do Lobo Temporal/genética , Neurônios/metabolismo , Simportadores/genética , Ácido gama-Aminobutírico/genética , Adulto , Animais , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Vetores Genéticos/genética , Genótipo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Neurônios/patologia , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-RESUMO
BACKGROUND: Kynurenine 3-monooxygenase converts kynurenine to 3-hydroxykynurenine, and its inhibition shunts the kynurenine pathway-which is implicated as dysfunctional in various psychiatric disorders-toward enhanced synthesis of kynurenic acid, an antagonist of both α7 nicotinic acetylcholine and N-methyl-D-aspartate receptors. Possibly as a result of reduced kynurenine 3-monooxygenase activity, elevated central nervous system levels of kynurenic acid have been found in patients with psychotic disorders, including schizophrenia. METHODS: In the present study, we investigated adaptive-and possibly regulatory-changes in mice with a targeted deletion of Kmo (Kmo-/-) and characterized the kynurenine 3-monooxygenase-deficient mice using six behavioral assays relevant for the study of schizophrenia. RESULTS: Genome-wide differential gene expression analyses in the cerebral cortex and cerebellum of these mice identified a network of schizophrenia- and psychosis-related genes, with more pronounced alterations in cerebellar tissue. Kynurenic acid levels were also increased in these brain regions in Kmo-/- mice, with significantly higher levels in the cerebellum than in the cerebrum. Kmo-/- mice exhibited impairments in contextual memory and spent less time than did controls interacting with an unfamiliar mouse in a social interaction paradigm. The mutant animals displayed increased anxiety-like behavior in the elevated plus maze and in a light/dark box. After a D-amphetamine challenge (5 mg/kg, intraperitoneal), Kmo-/- mice showed potentiated horizontal activity in the open field paradigm. CONCLUSIONS: Taken together, these results demonstrate that the elimination of Kmo in mice is associated with multiple gene and functional alterations that appear to duplicate aspects of the psychopathology of several neuropsychiatric disorders.
Assuntos
Quinurenina 3-Mono-Oxigenase/deficiência , Quinurenina 3-Mono-Oxigenase/fisiologia , Transtornos Psicóticos/genética , Transtornos Psicóticos/psicologia , Esquizofrenia/genética , Psicologia do Esquizofrênico , Animais , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Dextroanfetamina/farmacologia , Ácido Cinurênico/metabolismo , Quinurenina 3-Mono-Oxigenase/genética , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacosRESUMO
UNLABELLED: Huntington's disease (HD) is a genetic disease caused by a CAG trinucleotide repeat expansion encoding a polyglutamine tract in the huntingtin (HTT) protein, ultimately leading to neuronal loss and consequent cognitive decline and death. As no treatments for HD currently exist, several chemical screens have been performed using cell-based models of mutant HTT toxicity. These screens measured single disease-related endpoints, such as cell death, but had low 'hit rates' and limited dimensionality for therapeutic detection. Here, we have employed gene expression microarray analysis of HD samples--a snapshot of the expression of 25,000 genes--to define a gene expression signature for HD from publically available data. We used this information to mine a database for chemicals positively and negatively correlated to the HD gene expression signature using the Connectivity Map, a tool for comparing large sets of gene expression patterns. Chemicals with negatively correlated expression profiles were highly enriched for protective characteristics against mutant HTT fragment toxicity in in vitro and in vivo models. This study demonstrates the potential of using gene expression to mine chemical activity, guide chemical screening, and detect potential novel therapeutic compounds. KEY MESSAGES: Single-endpoint chemical screens have low therapeutic discovery hit-rates. In the context of HD, we guided a chemical screen using gene expression data. The resulting chemicals were highly enriched for suppressors of mutant HTT fragment toxicity. This study provides a proof of concept for wider usage in all chemical screening.
Assuntos
Conectoma , Doença de Huntington/metabolismo , Animais , Caspases/metabolismo , Linhagem Celular , Análise por Conglomerados , Desferroxamina/farmacologia , Modelos Animais de Doenças , Drosophila , Descoberta de Drogas , Regulação da Expressão Gênica , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Corpos de Inclusão/metabolismo , Camundongos , Modelos Biológicos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oligomicinas/farmacologia , FenótipoRESUMO
Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Homeostase , Membranas Intracelulares/metabolismo , Calmodulina/antagonistas & inibidores , Análise por Conglomerados , Retículo Endoplasmático/efeitos dos fármacos , Gossipol/análogos & derivados , Gossipol/farmacologia , Células HeLa , Homeostase/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Sódio/metabolismo , Molécula 1 de Interação EstromalRESUMO
Connectivity mapping is the process of establishing connections between different biological states using gene-expression profiles or signatures. There are a number of applications but in toxicology the most pertinent is for understanding mechanisms of toxicity. In its essence the process involves comparing a query gene signature generated as a result of exposure of a biological system to a chemical to those in a database that have been previously derived. In the ideal situation the query gene-expression signature is characteristic of the event and will be matched to similar events in the database. Key criteria are therefore the means of choosing the signature to be matched and the means by which the match is made. In this article we explore these concepts with examples applicable to toxicology.