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1.
Sci Rep ; 14(1): 18575, 2024 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-39127839

RESUMEN

Triosephosphate isomerase deficiency (TPI Df) is a rare multisystem disorder with severe neuromuscular symptoms which arises exclusively from mutations within the TPI1 gene. Studies of TPI Df have been limited due to the absence of mammalian disease models and difficulties obtaining patient samples. Recently, we developed a novel murine model of TPI Df which models the most common disease-causing mutation in humans, TPI1E105D. Using our model in the present study, the underlying pathogenesis of neuromuscular symptoms has been elucidated. This is the first report detailing studies of neuromuscular pathology within a murine model of TPI Df. We identified several contributors to neuromuscular symptoms, including neurodegeneration in the brain, alterations in neurotransmission at the neuromuscular junction, and reduced muscle fiber size. TPI Df mice also exhibited signs of cardiac pathology and displayed a deficit in vascular smooth muscle functionality. Together, these findings provide insight into pathogenesis of the neuromuscular symptoms in TPI Df and can guide the future development of therapeutics.


Asunto(s)
Modelos Animales de Enfermedad , Unión Neuromuscular , Triosa-Fosfato Isomerasa , Animales , Triosa-Fosfato Isomerasa/deficiencia , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismo , Ratones , Unión Neuromuscular/patología , Unión Neuromuscular/metabolismo , Anemia Hemolítica Congénita no Esferocítica/genética , Anemia Hemolítica Congénita no Esferocítica/patología , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/patología , Enfermedades Neuromusculares/etiología , Errores Innatos del Metabolismo de los Carbohidratos/genética , Mutación , Humanos
2.
Mol Psychiatry ; 2024 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-39210012

RESUMEN

Glycine is an obligatory co-agonist at excitatory NMDA receptors in the brain, especially in the dentate gyrus, which has been postulated to be crucial for the development of psychotic associations and memories with psychotic content. Drugs modulating glycine levels are in clinical development for improving cognition in schizophrenia. However, the functional relevance of the regulation of glycine metabolism by endogenous enzymes is unclear. Using a chromosome-engineered allelic series in mice, we report that a triplication of the gene encoding the glycine-catabolizing enzyme glycine decarboxylase (GLDC) - as found on a small supernumerary marker chromosome in patients with psychosis - reduces extracellular glycine levels as determined by optical fluorescence resonance energy transfer (FRET) in dentate gyrus (DG) and suppresses long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduces the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displays deficits in schizophrenia-like behaviors which are in part known to be dependent on the activity of the dentate gyrus, e.g., prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference. Our results demonstrate that Gldc negatively regulates long-term synaptic plasticity in the dentate gyrus in mice, suggesting that an increase in GLDC copy number possibly contributes to the development of psychosis in humans.

3.
Alcohol ; 120: 99-107, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-38971210

RESUMEN

The white-tufted marmoset is a small, nonhuman primate that is rapidly gaining popularity as a model organism, especially for neuroscience research. To date, little work in the alcohol research field has utilized the marmoset. As a step toward establishing the marmoset as a research model for alcohol experimentation, a series of exploratory studies were undertaken to characterize ethanol drinking behavior. A voluntary drinking paradigm was established whereby the common marmoset would consume pharmacologically relevant amounts of ethanol. To facilitate ethanol consumption, ethanol was mixed with a marshmallow flavored solution (hereafter called marshmallow juice) to mask the presumed adverse taste of ethanol. Using marshmallow juice flavored solutions, marmosets readily consumed ethanol up to 1 g/kg during 10 min binge-like drinking sessions or up to 5 g/kg during ∼4 h drinking sessions. Consumption of 1.0-1.5 g/kg during a 30 min session resulted in blood ethanol concentrations of 49-73 mg/dl, which are predicted to be pharmacologically relevant. In animals that were stably consuming ethanol in marshmallow juice, gradually reducing the concentration of the marshmallow juice flavoring resulted in markedly reduced ethanol consumption. Lastly, when offered a choice between ethanol in marshmallow juice and marshmallow juice alone, marmosets displayed a very strong preference for the marshmallow juice solution without ethanol. From these studies, it is concluded that marmosets will voluntarily consume ethanol if the taste is masked with a sweet solution such as marshmallow juice. These studies represent the first report of alcohol consumption and preference in the white-tufted marmoset.


Asunto(s)
Consumo de Bebidas Alcohólicas , Callithrix , Etanol , Animales , Etanol/sangre , Etanol/administración & dosificación , Masculino , Consumo de Bebidas Alcohólicas/psicología , Femenino , Nivel de Alcohol en Sangre , Consumo Excesivo de Bebidas Alcohólicas
4.
Neuropharmacology ; 257: 110035, 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-38876310

RESUMEN

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.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico , Etanol , Ratones Endogámicos C57BL , Inhibidores de Fosfodiesterasa 4 , Receptores de GABA-A , Talidomida , Animales , Talidomida/farmacología , Talidomida/análogos & derivados , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores de Fosfodiesterasa 4/farmacología , Masculino , Femenino , Etanol/farmacología , Ratones , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-A/efectos de los fármacos , Técnicas de Sustitución del Gen , Fosforilación/efectos de los fármacos , Ataxia/genética , Consumo de Bebidas Alcohólicas/tratamiento farmacológico , Consumo de Bebidas Alcohólicas/genética , Ratones Transgénicos , Diazepam/farmacología
5.
bioRxiv ; 2024 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-38915579

RESUMEN

Prosapip1 is a brain-specific protein localized to the postsynaptic density, where it promotes dendritic spine maturation in primary hippocampal neurons. However, nothing is known about the role of Prosapip1 in vivo . To examine this, we utilized the Cre-loxP system to develop a Prosapip1 neuronal knockout mouse. We found that Prosapip1 controls the synaptic localization of its binding partner SPAR, along with PSD-95 and the GluN2B subunit of the NMDA receptor (NMDAR) in the dorsal hippocampus (dHP). We next sought to identify the potential contribution of Prosapip1 to the activity and function of the NMDAR and found that Prosapip1 plays an important role in NMDAR-mediated transmission and long-term potentiation (LTP) in the CA1 region of the dHP. As LTP is the cellular hallmark of learning and memory, we examined the consequences of neuronal knockout of Prosapip1 on dHP-dependent memory. We found that global or dHP-specific neuronal knockout of Prosapip1 caused a deficit in learning and memory whereas developmental, locomotor, and anxiety phenotypes were normal. Taken together, Prosapip1 in the dHP promotes the proper localization of synaptic proteins which, in turn, facilitates LTP driving recognition, social, and spatial learning and memory.

6.
Alzheimers Dement ; 20(5): 3455-3471, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38574388

RESUMEN

INTRODUCTION: Fundamental questions remain about the key mechanisms that initiate Alzheimer's disease (AD) and the factors that promote its progression. Here we report the successful generation of the first genetically engineered marmosets that carry knock-in (KI) point mutations in the presenilin 1 (PSEN1) gene that can be studied from birth throughout lifespan. METHODS: CRISPR/Cas9 was used to generate marmosets with C410Y or A426P point mutations in PSEN1. Founders and their germline offspring are comprehensively studied longitudinally using non-invasive measures including behavior, biomarkers, neuroimaging, and multiomics signatures. RESULTS: Prior to adulthood, increases in plasma amyloid beta were observed in PSEN1 mutation carriers relative to non-carriers. Analysis of brain revealed alterations in several enzyme-substrate interactions within the gamma secretase complex prior to adulthood. DISCUSSION: Marmosets carrying KI point mutations in PSEN1 provide the opportunity to study the earliest primate-specific mechanisms that contribute to the molecular and cellular root causes of AD onset and progression. HIGHLIGHTS: We report the successful generation of genetically engineered marmosets harboring knock-in point mutations in the PSEN1 gene. PSEN1 marmosets and their germline offspring recapitulate the early emergence of AD-related biomarkers. Studies as early in life as possible in PSEN1 marmosets will enable the identification of primate-specific mechanisms that drive disease progression.


Asunto(s)
Enfermedad de Alzheimer , Callithrix , Presenilina-1 , Animales , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Animales Modificados Genéticamente , Encéfalo/patología , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Mutación/genética , Mutación Puntual/genética , Presenilina-1/genética
7.
Life Sci ; 348: 122673, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38679193

RESUMEN

AIMS: Glycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol. MAIN METHODS: We used electrophysiological recordings to examine ethanol's effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm. KEY FINDINGS: Ethanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice. SIGNIFICANCE: The results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption.


Asunto(s)
Etanol , Técnicas de Sustitución del Gen , Receptores de Glicina , Animales , Etanol/farmacología , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Ratones , Masculino , Núcleo Accumbens/metabolismo , Núcleo Accumbens/efectos de los fármacos , Consumo de Bebidas Alcohólicas/genética , Consumo de Bebidas Alcohólicas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Ratones Transgénicos , Receptores de GABA-A
8.
Brain Behav Immun ; 118: 437-448, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38499210

RESUMEN

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.


Asunto(s)
Etanol , Receptor Toll-Like 3 , Ratones , Masculino , Animales , Receptor Toll-Like 3/metabolismo , Ratones Endogámicos C57BL , Etanol/farmacología , Transducción de Señal , Consumo de Bebidas Alcohólicas/metabolismo , Poli I-C/farmacología
9.
Neurobiol Stress ; 29: 100603, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38234394

RESUMEN

Chronic stress and alcohol (ethanol) use are highly interrelated and can change an individual's behavior through molecular adaptations that do not change the DNA sequence, but instead change gene expression. A recent wealth of research has found that these nongenomic changes can be transmitted across generations, which could partially account for the "missing heritability" observed in genome-wide association studies of alcohol use disorder and other stress-related neuropsychiatric disorders. In this review, we summarize the molecular and behavioral outcomes of nongenomic inheritance of chronic stress and ethanol exposure and the germline mechanisms that could give rise to this heritability. In doing so, we outline the need for further research to: (1) Investigate individual germline mechanisms of paternal, maternal, and biparental nongenomic chronic stress- and ethanol-related inheritance; (2) Synthesize and dissect cross-generational chronic stress and ethanol exposure; (3) Determine cross-generational molecular outcomes of preconception ethanol exposure that contribute to alcohol-related disease risk, using cancer as an example. A detailed understanding of the cross-generational nongenomic effects of stress and/or ethanol will yield novel insight into the impact of ancestral perturbations on disease risk across generations and uncover actionable targets to improve human health.

10.
Mol Psychiatry ; 29(2): 529-542, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-38135755

RESUMEN

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol's direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary ß subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.


Asunto(s)
Consumo de Bebidas Alcohólicas , Etanol , Ratones Endogámicos C57BL , Animales , Etanol/farmacología , Masculino , Ratones , Consumo de Bebidas Alcohólicas/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Femenino
11.
bioRxiv ; 2023 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-37398055

RESUMEN

The biological significance of a small supernumerary marker chromosome that results in dosage alterations to chromosome 9p24.1, including triplication of the GLDC gene encoding glycine decarboxylase, in two patients with psychosis is unclear. In an allelic series of copy number variant mouse models, we identify that triplication of Gldc reduces extracellular glycine levels as determined by optical fluorescence resonance energy transfer (FRET) in dentate gyrus (DG) but not in CA1, suppresses long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduces the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displays deficits in prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference. Our results thus provide a link between a genomic copy number variation, biochemical, cellular and behavioral phenotypes, and further demonstrate that GLDC negatively regulates long-term synaptic plasticity at specific hippocampal synapses, possibly contributing to the development of neuropsychiatric disorders.

12.
Curr Res Neurobiol ; 3: 100062, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36405628

RESUMEN

Triosephosphate isomerase deficiency (TPI Df) is a rare, aggressive genetic disease that typically affects young children and currently has no established treatment. TPI Df is characterized by hemolytic anemia, progressive neuromuscular degeneration, and a markedly reduced lifespan. The disease has predominately been studied using invertebrate and in vitro models, which lack key aspects of the human disease. While other groups have generated mammalian Tpi1 mutant strains, specifically with the mouse mus musculus, these do not recapitulate key characteristic phenotypes of the human disease. Reported here is the generation of a novel murine model of TPI Df. CRISPR-Cas9 was utilized to engineer the most common human disease-causing mutation, Tpi1 E105D , and Tpi1 null mice were also isolated as a frame-shifting deletion. Tpi1 E105D/null mice experience a markedly shortened lifespan, postural abnormalities consistent with extensive neuromuscular dysfunction, hemolytic anemia, pathological changes in spleen, and decreased body weight. There is a ∼95% reduction in TPI protein levels in Tpi1 E105D/null animals compared to wild-type littermates, consistent with decreased TPI protein stability, a known cause of TPI Df. This work illustrates the capability of Tpi1 E105D/null mice to serve as a mammalian model of human TPI Df. This work will allow for advancement in the study of TPI Df within a model with physiology similar to humans. The development of the model reported here will enable mechanistic studies of disease pathogenesis and, importantly, efficacy testing in a mammalian system for emerging TPI Df treatments.

13.
Proc Natl Acad Sci U S A ; 119(40): e2204828119, 2022 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-36161942

RESUMEN

Biased G protein-coupled receptor (GPCR) ligands, which preferentially activate G protein or ß-arrestin signaling pathways, are leading to the development of drugs with superior efficacy and reduced side effects in heart disease, pain management, and neuropsychiatric disorders. Although GPCRs are implicated in the pathophysiology of Alzheimer's disease (AD), biased GPCR signaling is a largely unexplored area of investigation in AD. Our previous work demonstrated that GPR3-mediated ß-arrestin signaling modulates amyloid-ß (Aß) generation in vitro and that Gpr3 deficiency ameliorates Aß pathology in vivo. However, Gpr3-deficient mice display several adverse phenotypes, including elevated anxiety-like behavior, reduced fertility, and memory impairment, which are potentially associated with impaired G protein signaling. Here, we generated a G protein-biased GPR3 mouse model to investigate the physiological and pathophysiological consequences of selective elimination of GPR3-mediated ß-arrestin signaling in vivo. In contrast to Gpr3-deficient mice, G protein-biased GPR3 mice do not display elevated anxiety levels, reduced fertility, or cognitive impairment. We further determined that G protein-biased signaling reduces soluble Aß levels and leads to a decrease in the area and compaction of amyloid plaques in the preclinical AppNL-G-F AD mouse model. The changes in amyloid pathology are accompanied by robust microglial and astrocytic hypertrophy, which suggest a protective glial response that may limit amyloid plaque development in G protein-biased GPR3 AD mice. Collectively, these studies indicate that GPR3-mediated G protein and ß-arrestin signaling produce discrete and separable effects and provide proof of concept for the development of safer GPCR-targeting therapeutics with more directed pharmacological action for AD.


Asunto(s)
Enfermedad de Alzheimer , Amiloidosis , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas de Unión al GTP/metabolismo , Ratones , Ratones Transgénicos , Placa Amiloide/patología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo
14.
Alcohol ; 105: 9-24, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36055466

RESUMEN

Extracellular vesicles (EVs) are important players in normal biological function and disease pathogenesis. Of the many biomolecules packaged into EVs, coding and noncoding RNA transcripts are of particular interest for their ability to significantly alter cellular and molecular processes. Here we investigate how chronic ethanol exposure impacts EV RNA cargo and the functional outcomes of these changes. Following chronic intermittent ethanol (CIE) vapor exposure, EVs were isolated from male and female C57BL/6J mouse brain. Total RNA from EVs was analyzed by lncRNA/mRNA microarray to survey changes in RNA cargo following vapor exposure. Differential expression analysis of microarray data revealed a number of lncRNA and mRNA types differentially expressed in CIE compared to control EVs. Weighted gene co-expression network analysis identified multiple male and female specific modules related to neuroinflammation, cell death, demyelination, and synapse organization. To functionally test these changes, whole-cell voltage-clamp recordings were used to assess synaptic transmission. Incubation of nucleus accumbens brain slices with EVs led to a reduction in spontaneous excitatory postsynaptic current amplitude, although no changes in synaptic transmission were observed between control and CIE EV administration. These results indicate that CIE vapor exposure significantly changes the RNA cargo of brain-derived EVs, which have the ability to impact neuronal function.


Asunto(s)
Encéfalo , Etanol , Vesículas Extracelulares , ARN Largo no Codificante , Animales , Femenino , Masculino , Ratones , Encéfalo/efectos de los fármacos , Etanol/efectos adversos , Ratones Endogámicos C57BL , ARN Mensajero
15.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-34848542

RESUMEN

Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.


Asunto(s)
Corteza Cerebral/citología , Dendritas/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuronas/fisiología , Transducción de Señal/fisiología , Animales , Sistemas CRISPR-Cas , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Genotipo , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones , Ratones Transgénicos , Mutación Missense , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Receptor Nogo 1/genética , Receptor Nogo 1/metabolismo , Maduración Sexual
16.
Genes Brain Behav ; 20(8): e12774, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34677900

RESUMEN

Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. We conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus (LOD = 3.5, 4.2; peak = 21 cM [36 Mb]). For methamphetamine-induced distance traveled following the first and second administration, we identified a genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 coding for the alpha-2 subunit of the GABA-A receptor (LOD = 3.6-5.2; peak = 34-35 cM [66-67 Mb]). Striatal cis-expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies show the power and efficiency of Reduced Complexity Crosses in identifying causal variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for studying psychostimulants in the C57BL/6J strain-the gold standard strain in biomedical research.


Asunto(s)
Trastornos Relacionados con Anfetaminas/genética , Sitios de Carácter Cuantitativo , Receptores de GABA-A/genética , Animales , Estimulantes del Sistema Nervioso Central/toxicidad , Femenino , Predisposición Genética a la Enfermedad , Masculino , Metanfetamina/toxicidad , Ratones , Ratones Endogámicos C57BL , Mutación , Carácter Cuantitativo Heredable
17.
J Neurophysiol ; 126(4): 1090-1100, 2021 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-34406874

RESUMEN

The general anesthetic etomidate, which acts through γ-aminobutyric acid type A (GABAA) receptors, impairs the formation of new memories under anesthesia. This study addresses the molecular and cellular mechanisms by which this occurs. Here, using a new line of genetically engineered mice carrying the GABAA receptor (GABAAR) ß2-N265M mutation, we tested the roles of receptors that incorporate GABAA receptor ß2 versus ß3 subunits to suppression of long-term potentiation (LTP), a cellular model of learning and memory. We found that brain slices from ß2-N265M mice resisted etomidate suppression of LTP, indicating that the ß2-GABAARs are an essential target in this model. As these receptors are most heavily expressed by interneurons in the hippocampus, this finding supports a role for interneuron modulation in etomidate control of synaptic plasticity. Nevertheless, ß2 subunits are also expressed by pyramidal neurons, so they might also contribute. Therefore, using a previously established line of ß3-N265M mice, we also examined the contributions of ß2- versus ß3-GABAARs to GABAA,slow dendritic inhibition, because dendritic inhibition is particularly well suited to controlling synaptic plasticity. We also examined their roles in long-lasting suppression of population activity through feedforward and feedback inhibition. We found that both ß2- and ß3-GABAARs contribute to GABAA,slow inhibition and that both ß2- and ß3-GABAARs contribute to feedback inhibition, whereas only ß3-GABAARs contribute to feedforward inhibition. We conclude that modulation of ß2-GABAARs is essential to etomidate suppression of LTP. Furthermore, to the extent that this occurs through GABAARs on pyramidal neurons, it is through modulation of feedback inhibition.NEW & NOTEWORTHY Etomidate exerts its anesthetic actions through GABAA receptors. However, the mechanism remains unknown. Here, using a hippocampal brain slice model, we show that ß2-GABAARs are essential to this effect. We also show that these receptors contribute to long-lasting dendritic inhibition in feedback but not feedforward inhibition of pyramidal neurons. These findings hold implications for understanding how anesthetics block memory formation and, more generally, how inhibitory circuits control learning and memory.


Asunto(s)
Anestésicos Intravenosos/farmacología , Etomidato/farmacología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos
18.
Mamm Genome ; 32(5): 350-363, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34086081

RESUMEN

Pathogenic variants in epilepsy genes result in a spectrum of clinical severity. One source of phenotypic heterogeneity is modifier genes that affect expressivity of a primary pathogenic variant. Mouse epilepsy models also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion of Scn1a (Scn1a+/-) model Dravet syndrome, a severe epilepsy most often caused by SCN1A haploinsufficiency. Scn1a+/- mice recapitulate features of Dravet syndrome, including spontaneous seizures, sudden death, and cognitive/behavioral deficits. Scn1a+/- mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no spontaneous seizures. In contrast, admixture with C57BL/6J (B6) results in epilepsy and premature lethality. We previously mapped Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival. Gabra2, encoding the GABAA α2 subunit, was nominated as a candidate modifier at Dsm1. Direct measurement of GABAA receptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide deletion within Gabra2 that lowers mRNA and protein by nearly 50%. Repair of this deletion reestablished normal levels of Gabra2 expression. In this study, we used B6 mice with a repaired Gabra2 allele to evaluate Gabra2 as a genetic modifier of severity in Scn1a+/- mice. Gabra2 repair restored transcript and protein expression, increased abundance of α2-containing GABAA receptors in hippocampal synapses, and rescued epilepsy phenotypes of Scn1a+/- mice. These findings validate Gabra2 as a genetic modifier of Dravet syndrome, and support enhancing function of α2-containing GABAA receptors as treatment strategy for Dravet syndrome.


Asunto(s)
Epilepsias Mioclónicas/genética , Receptores de GABA-A/genética , Animales , Epilepsias Mioclónicas/fisiopatología , Ratones , Polimorfismo de Nucleótido Simple
19.
Int Rev Neurobiol ; 156: 217-277, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33461664

RESUMEN

Substance use disorders are highly prevalent and continue to be one of the leading causes of disability in the world. Notably, not all people who use addictive drugs develop a substance use disorder. Although substance use disorders are highly heritable, patterns of inheritance cannot be explained purely by Mendelian genetic mechanisms. Vulnerability to developing drug addiction depends on the interplay between genetics and environment. Additionally, evidence from the past decade has pointed to the role of epigenetic inheritance in drug addiction. This emerging field focuses on how environmental perturbations, including exposure to addictive drugs, induce epigenetic modifications that are transmitted to the embryo at fertilization and modify developmental gene expression programs to ultimately impact subsequent generations. This chapter highlights intergenerational and transgenerational phenotypes in offspring following a history of parental drug exposure. Special attention is paid to parental preconception exposure studies of five drugs of abuse (alcohol, cocaine, nicotine, cannabinoids, and opiates) and associated behavioral and physiological outcomes in offspring. The highlighted studies demonstrate that parental exposure to drugs of abuse has enduring effects that persist into subsequent generations. Understanding the contribution of epigenetic inheritance in drug addiction may provide clues for better treatments and therapies for substance use disorders.


Asunto(s)
Trastornos Relacionados con Sustancias , Epigénesis Genética , Humanos , Trastornos Relacionados con Sustancias/epidemiología , Trastornos Relacionados con Sustancias/genética
20.
Genes Brain Behav ; 20(2): e12698, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32893479

RESUMEN

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The "hub gene" of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of "Response to Ethanol" emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non-coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype.


Asunto(s)
Consumo de Bebidas Alcohólicas/genética , Encéfalo/metabolismo , ARN Largo no Codificante/genética , Consumo de Bebidas Alcohólicas/fisiopatología , Animales , Sitios de Carácter Cuantitativo , ARN Largo no Codificante/metabolismo , Ratas , Ratas Wistar , Transcriptoma
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