RESUMO
To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo.
Assuntos
Colangiocarcinoma/patologia , Embrião de Mamíferos/patologia , Receptores de GABA-A/metabolismo , Taurina/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor , Humanos , Camundongos , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de GABA-A/genética , Transdução de Sinais/efeitos dos fármacos , Técnicas de Hibridização Subtrativa , Taxa de SobrevidaRESUMO
OBJECTIVE: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion-water homeostasis, but its exact role is unknown. We generated Mlc1-null mice for further studies. METHODS: We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. RESULTS: Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. INTERPRETATION: Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC.
Assuntos
Cistos/genética , Cistos/patologia , Cistos/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Adolescente , Adulto , Fatores Etários , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Astrócitos/patologia , Edema Encefálico/etiologia , Cerebelo/patologia , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Criança , Pré-Escolar , Cistos/metabolismo , Modelos Animais de Doenças , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Lactente , Recém-Nascido , Potenciais da Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/genética , Equilíbrio Postural/genética , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Transtornos de Sensação/genética , Substância Branca/metabolismo , Substância Branca/patologia , Substância Branca/ultraestrutura , Adulto JovemRESUMO
Megalencephalic leucoencephalopathy with subcortical cysts is a genetic brain disorder with onset in early childhood. Affected infants develop macrocephaly within the first year of life, after several years followed by slowly progressive, incapacitating cerebellar ataxia and spasticity. From early on, magnetic resonance imaging shows diffuse signal abnormality and swelling of the cerebral white matter, with evidence of highly increased white matter water content. In most patients, the disease is caused by mutations in the gene MLC1, which encodes a plasma membrane protein almost exclusively expressed in brain and at lower levels in leucocytes. Within the brain, MLC1 is mainly located in astrocyte-astrocyte junctions adjacent to the blood-brain and cereborspinal fluid-brain barriers. Thus far, the function of MLC1 has remained unknown. We tested the hypothesis that MLC1 mutations cause a defect in ion currents involved in water and ion homeostasis, resulting in cerebral white matter oedema. Using whole-cell patch clamp studies we demonstrated an association between MLC1 expression and anion channel activity in different cell types, most importantly astrocytes. The currents were absent in chloride-free medium and in cells with disease-causing MLC1 mutations. MLC1-dependent currents were greatly enhanced by hypotonic pretreatment causing cell swelling, while ion channel blockers, including Tamoxifen, abolished the currents. Down regulation of endogenous MLC1 expression in astrocytes by small interfering RNA greatly reduced the activity of this channel, which was rescued by overexpression of normal MLC1. The current-voltage relationship and the pharmacological profiles of the currents indicated that the channel activated by MLC1 expression is a volume-regulated anion channel. Such channels are involved in regulatory volume decrease. We showed that regulatory volume decrease was hampered in lymphoblasts from patients with megalencephalic leucoencephalopathy. A similar trend was observed in astrocytes with decreased MLC1 expression; this effect was rescued by overexpression of normal MLC1. In the present study, we show that absence or mutations of the MLC1 protein negatively impact both volume-regulated anion channel activity and regulatory volume decrease, indicating that megalencephalic leucoencephalopathy is caused by a disturbance of cell volume regulation mediated by chloride transport.
Assuntos
Astrócitos/patologia , Cloretos/metabolismo , Cistos/fisiopatologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Transporte de Íons/fisiologia , Proteínas de Membrana/genética , Astrócitos/metabolismo , Tamanho Celular , Cistos/metabolismo , Cistos/patologia , Células HEK293 , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Proteínas de Membrana/metabolismoRESUMO
We here investigated inhibitory synapse turnover in the adult brain using the hypothalamic supraoptic nucleus where new synapses form during different physiological conditions, in particular on oxytocin neurons largely controlled by GABAergic inputs and locally released oxytocin. Patch clamp recordings and ultrastructural analysis of the nucleus in acute slices from late gestating rats showed that oxytocin and estrogen promoted rapid formation of inhibitory synapses. Thus, after 2-h exposure to a combination of oxytocin and 17-beta estradiol, the frequency of miniature inhibitory postsynaptic currents was significantly enhanced. Since their amplitude and presynaptic GABA release probability were unmodified, this indicated an increased number of synapses. Electron microscopy confirmed increased densities of symmetric, putative GABAergic synapses within 2-h exposure to the peptide or steroid, effects which were reversible and oxytocin receptor mediated. Our observations thus offer direct evidence that hypothalamic GABAergic microcircuitries can undergo rapid and functional remodeling under changing neuroendocrine conditions.
Assuntos
Estrogênios/farmacologia , Neurônios/efeitos dos fármacos , Ocitocina/farmacologia , Núcleo Supraóptico/efeitos dos fármacos , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Estrogênios/metabolismo , Feminino , Técnicas In Vitro , Neurônios/citologia , Neurônios/metabolismo , Ocitocina/metabolismo , Técnicas de Patch-Clamp , Gravidez , Ratos , Ratos Wistar , Núcleo Supraóptico/citologia , Núcleo Supraóptico/metabolismo , Sinapses/química , Sinapses/efeitos dos fármacos , Sinapses/ultraestruturaRESUMO
Repeated exposure to drugs of abuse causes persistent behavioral sensitization and associated adaptations of striatal neurotransmission, which is thought to play an important role in certain aspects of drug addiction. Microdialysis and neurochemical studies suggest that intermittent morphine treatment may lead to a long-term increase in both ACh and dopaminergic neurotransmission in the nucleus accumbens (NAc). This implies that both cholinergic modulation of GABA synapses and their sensitivity to dopaminergic transmission might be changed, ultimately leading to a modified NAc output. Here we investigate to what extent cholinergic modulation and sensitivity to amphetamine, causing endogenous dopamine efflux, of GABAergic transmission in the nucleus accumbens are affected 3 weeks after a period of daily morphine injections in adult rats. To this end, we recorded medium spiny neurons using whole cell voltage clamp and monitored the frequency and amplitude of spontaneous GABAergic synaptic currents. We observed that the effect of nicotine on the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was suppressed in rats pretreated with morphine, whereas the effects of mecamylamine and tetrodotoxin (TTX) were increased. These results indicate that the probability of GABA release was increased and that this effect resulted from an upregulation of the endogenous activation of presynaptic nicotinic receptors. In addition, we observed an increased sensitivity to in vitro application of amphetamine. This suggests that the long-term increase in dopaminergic transmission caused by the morphine treatment affects GABA synapses in the NAc. Hence, there may be two parallel synaptic mechanisms by which drugs of abuse may affect processing and integration of NAc inputs.
Assuntos
Acetilcolina/metabolismo , Morfina/farmacologia , Núcleo Accumbens/citologia , Sinapses/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Anfetamina/farmacologia , Anestésicos Locais/farmacologia , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Interações Medicamentosas , Técnicas In Vitro , Masculino , Mecamilamina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Entorpecentes/farmacologia , Inibição Neural/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Wistar , Sinapses/metabolismo , Tetrodotoxina/farmacologiaRESUMO
A robust increase in dopaminergic transmission in the nucleus accumbens (NAc) shell has been reported to be consistently associated with the long-term expression of behavioural sensitization to drugs of abuse. However, little is known about how this affects the neuronal network of the NAc. We made cellular recordings in NAc slices of saline- and amphetamine-pretreated adult rats and found that expression of behavioural sensitization was associated with long-lasting changes in the basal firing pattern of cholinergic interneurons up to 3 weeks after the last drug injection. Consequently, upon amphetamine sensitization, an inhibiting effect of the nicotinic receptor blocker mecamylamine on the amplitudes of spontaneous GABAergic synaptic currents as well as on the failure rate of electrically evoked GABAergic currents was found that was not present under control conditions. Thus, behavioural sensitization to amphetamine is associated with an up-regulation of the endogenous activation of nicotinic receptors that, in turn, stimulate the GABAergic synaptic transmission within the NAc shell. This is a new mechanism by which drugs of abuse may induce alterations in the processing and integration of NAc inputs involved in psychomotor sensitization.
Assuntos
Anfetamina/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Acetilcolina/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Técnicas In Vitro , Masculino , Mecamilamina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Neurônios/classificação , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Antagonistas Nicotínicos/farmacologia , Núcleo Accumbens/citologia , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The rat nucleus accumbens contains acetylcholine-releasing interneurons, presumed to play a regulatory role in the electrical activity of medium spiny output neurons. In order to examine this issue in detail, we made electrophysiological recordings in rat nucleus accumbens slices. These experiments showed that gamma-aminobutyric acid-mediated inhibition of the output neurons might be facilitated by activation of nicotinic acetylcholine receptors, in addition to being suppressed via activation of muscarinic acetylcholine receptors. In contrast, glutamatergic excitation of output neurons appeared to be inhibited by activation of muscarinic acetylcholine receptors and to be insensitive to activation of nicotinic acetylcholine receptors. The spontaneous firing frequency of cholinergic neurons appeared to be under control of both a muscarinic and a nicotinic pathway in a bi-directional manner. Finally, we made paired recordings in which the functional connection between cholinergic neurons and output neurons was monitored. Driving the cholinergic neurons at physiological firing frequencies stimulated gamma-aminobutyric acid-mediated inhibition of the output neurons, via activation of nicotinic acetylcholine receptors. The onset of this effect was slow and lacked a fixed delay. These data indicate that activation of nicotinic acetylcholine receptors in rat nucleus accumbens may mediate the facilitation of gamma-aminobutyric acid-mediated inhibition of medium spiny output neurons. Possible mechanisms of neurotransmission, mediating this cholinergic modulation are discussed.