RESUMO
Maintenance of calcium homeostasis is important for proper endoplasmic reticulum (ER) function. When cellular stress conditions deplete the high concentration of calcium in the ER, ER-resident proteins are secreted into the extracellular space in a process called exodosis. Monitoring exodosis provides insight into changes in ER homeostasis and proteostasis resulting from cellular stress associated with ER calcium dysregulation. To monitor cell-type specific exodosis in the intact animal, we created a transgenic mouse line with a Gaussia luciferase (GLuc)-based, secreted ER calcium-modulated protein, SERCaMP, preceded by a LoxP-STOP-LoxP (LSL) sequence. The Cre-dependent LSL-SERCaMP mice were crossed with albumin (Alb)-Cre and dopamine transporter (DAT)-Cre mouse lines. GLuc-SERCaMP expression was characterized in mouse organs and extracellular fluids, and the secretion of GLuc-SERCaMP in response to cellular stress was monitored following pharmacological depletion of ER calcium. In LSL-SERCaMP × Alb-Cre mice, robust GLuc activity was observed only in the liver and blood, whereas in LSL-SERCaMP × DAT-Cre mice, GLuc activity was seen in midbrain dopaminergic neurons and tissue samples innervated by dopaminergic projections. After calcium depletion, we saw increased GLuc signal in the plasma and cerebrospinal fluid collected from the Alb-Cre and DAT-Cre crosses, respectively. This mouse model can be used to investigate the secretion of ER-resident proteins from specific cell and tissue types during disease pathogenesis and may aid in the identification of therapeutics and biomarkers of disease.
Assuntos
Cálcio , Proteostase , Camundongos , Animais , Proteostase/genética , Cálcio/metabolismo , Fígado/metabolismo , Luciferases/metabolismo , Retículo Endoplasmático/genética , Camundongos TransgênicosRESUMO
Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced drug seeking in rodent models correlates with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses in the nucleus accumbens core (NAcore). Matrix metalloproteinases (MMPs) are inducible endopeptidases that degrade extracellular matrix (ECM) proteins, and reveal tripeptide Arginine-Glycine-Aspartate (RGD) domains that bind and signal through integrins. Integrins are heterodimeric receptors composed of αß subunits, and a primary signaling kinase is focal adhesion kinase (FAK). We previously showed that MMP activation is necessary for and potentiates cued reinstatement of cocaine seeking, and MMP-induced catalysis stimulates ß3-integrins to induce t-SP. Here, we determined whether ß3-integrin signaling through FAK and cofilin (actin depolymerization factor) is necessary to promote synaptic growth during t-SP. Using a small molecule inhibitor to prevent FAK activation, we blocked cued-induced cocaine reinstatement and increased spine head diameter (dh). Immunohistochemistry on NAcore labeled spines with ChR2-EYFP virus, showed increased immunoreactivity of phosphorylation of FAK (p-FAK) and p-cofilin in dendrites of reinstated animals compared with extinguished and yoked saline, and the p-FAK and cofilin depended on ß3-integrin signaling. Next, male and female transgenic rats were used to selectively label D1 or D2 neurons with ChR2-mCherry. We found that p-FAK was increased during drug seeking in both D1 and D2-medium spiny neurons (MSNs), but increased p-cofilin was observed only in D1-MSNs. These data indicate that ß3-integrin, FAK and cofilin constitute a signaling pathway downstream of MMP activation that is involved in promoting the transient synaptic enlargement in D1-MSNs induced during reinstated cocaine by drug-paired cues.SIGNIFICANCE STATEMENT Drug-associated cues precipitate relapse, which is correlated with transient synaptic enlargement in the accumbens core. We showed that cocaine cue-induced synaptic enlargement depends on matrix metalloprotease signaling in the extracellular matrix (ECM) through ß3-integrin to activate focal adhesion kinase (FAK) and phosphorylate the actin binding protein cofilin. The nucleus accumbens core (NAcore) contains two predominate neuronal subtypes selectively expressing either D1-dopamine or D2-dopamine receptors. We used transgenic rats to study each cell type and found that cue-induced signaling through cofilin phosphorylation occurred only in D1-expressing neurons. Thus, cocaine-paired cues initiate cocaine reinstatement and synaptic enlargement through a signaling cascade selectively in D1-expressing neurons requiring ECM stimulation of ß3-integrin-mediated phosphorylation of FAK (p-FAK) and cofilin.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Neurônios Dopaminérgicos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Integrina beta3/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Transtornos Relacionados ao Uso de Cocaína/psicologia , Sinais (Psicologia) , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/ultraestrutura , Comportamento de Procura de Droga , Ativação Enzimática , Humanos , Masculino , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos Transgênicos , Recidiva , SinapsesRESUMO
Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis specific member of the DEAD-box family of RNA helicases expressed in meiotic and haploid germ cells which plays an essential role in spermatogenesis. There are two species of GRTH the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in azoospermic men that when expressed in COS1-cells lack the phospho-form of GRTH. To investigate the role of the phospho-GRTH species in spermatogenesis, we generated a GRTH knock-in (KI) transgenic mice with the R242H mutation. GRTH-KI mice are sterile with reduced testis size, lack sperm with spermatogenic arrest at round spermatid stage and loss of the cytoplasmic phospho-GRTH species. Electron microscopy studies revealed reduction in the size of chromatoid bodies (CB) of round spermatids (RS) and germ cell apoptosis. We observed absence of phospho-GRTH in the CB of RS. Complete loss of chromatin remodeling and related proteins such as TP2, PRM2, TSSK6 and marked reduction of their respective mRNAs and half-lives were observed in GRTH-KI mice. We showed that phospho-GRTH has a role in TP2 translation and revealed its occurrence in a 3' UTR dependent manner. These findings demonstrate the relevance of phospho-GRTH in the structure of the chromatoid body, spermatid development and completion of spermatogenesis and provide an avenue for the development of a male contraceptive.
Assuntos
RNA Helicases DEAD-box/metabolismo , Infertilidade Masculina/genética , Mutação de Sentido Incorreto , Processamento de Proteína Pós-Traducional , Espermátides/metabolismo , Animais , Aspermia/genética , Aspermia/metabolismo , Aspermia/fisiopatologia , Montagem e Desmontagem da Cromatina , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/fisiologia , Regulação da Expressão Gênica , Infertilidade Masculina/metabolismo , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Protaminas/genética , Proteínas Serina-Treonina Quinases/genética , Espermátides/patologia , Espermátides/fisiologia , EspermatogêneseRESUMO
Outputs from the nucleus accumbens (NAc) include projections to the ventral pallidum and the ventral tegmental area and subtantia nigra in the ventral mesencephalon. The medium spiny neurons (MSN) that give rise to these pathways are GABAergic and consist of two populations of equal number that are segregated by differentially expressed proteins, including D1- and D2-dopamine receptors. Afferents to the ventral pallidum arise from both D1- and D2-MSNs, whereas the ventral mesencephalon is selectively innervated by D1-MSN. To determine the extent of collateralization of D1-MSN to these axon terminal fields we used retrograde labeling in transgenic mice expressing tdTomato selectively in D1-MSN, and found that a large majority of D1-MSN in either the shell or core subcompartments of the accumbens collateralized to both output structures. Approximately 70% of D1-MSNs projecting to the ventral pallidum collateralized to the ventral mesencephalon, whereas >90% of mesencephalic D1-MSN afferents collateralized to the ventral pallidum. In contrast, <10% of dorsal striatal D1-MSNs collateralized to both the globus pallidus and ventral mesencephalon. D1-MSN activation is required for conditioned cues to induce cocaine seeking. To determine which D1-MSN projection mediates cued cocaine seeking, we selectively transfected D1-MSNs in transgenic rats with an inhibitory Gi-coupled DREADD. Activation of the transfected Gi-DREADD with clozapine-N-oxide administered into the ventral pallidum, but not into the ventral mesencephalon, blocked cue-induced cocaine seeking. These data show that, although accumbens D1-MSNs largely collateralize to both the ventral pallidum and ventral mesencephalon, only D1-MSN innervation of the ventral pallidum is necessary for cue-induced cocaine seeking.SIGNIFICANCE STATEMENT Activity in D1 dopamine receptor-expressing neurons in the NAc is required for rodents to respond to cocaine-conditioned cues and relapse to drug seeking behaviors. The D1-expressing neurons project to both the ventral pallidum and ventral mesencephalon, and we found that a majority of the neurons that innervate the ventral pallidum also collateralize to the ventral mesencephalon. However, despite innervating both structures, only D1 innervation of the ventral pallidum mediates cue-induced cocaine seeking.
Assuntos
Prosencéfalo Basal/fisiologia , Cocaína/administração & dosagem , Comportamento de Procura de Droga/fisiologia , Neurônios/fisiologia , Núcleo Accumbens/fisiologia , Receptores de Dopamina D1/fisiologia , Animais , Prosencéfalo Basal/citologia , Condicionamento Clássico , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios/citologia , Núcleo Accumbens/citologia , Ratos Long-Evans , Ratos TransgênicosRESUMO
BACKGROUND: Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease. METHODS: We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1-/-) and Fabry disease (Glay/-) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses. RESULTS: We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1-/- microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1-/- microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer's disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in "disease-associated microglia" pattern among these diseases. CONCLUSIONS: The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01067742, registered on February 12, 2010.
Assuntos
Microglia/metabolismo , Mucolipidoses/genética , Mucolipidoses/patologia , Transcriptoma , Animais , Doença de Fabry/genética , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Humanos , Camundongos , Camundongos Transgênicos , Microglia/patologia , Mucolipidoses/metabolismoRESUMO
The specific actions of insulin-like growth factor-I (IGF-I) and the role of brain-derived IGF-I during hippocampal neurogenesis have not been fully defined. To address the influence of IGF-I on the stages of hippocampal neurogenesis, we studied a postnatal/adult global Igf-I knockout (KO) mice (Igf-I(-/-) ) and a nervous system Igf-I conditional KO (Igf-I(Δ/Δ) ). In both KO mice we found an accumulation of Tbr2(+) -intermediate neuronal progenitors, some of which were displaced in the outer granule cell layer (GCL) and the molecular layer (ML) of the dentate gyrus (DG). Similarly, more ectopic Ki67(+) - cycling cells were detected. Thus, the GCL was disorganized with significant numbers of Prox1(+) -granule neurons outside this layer and altered morphology of radial glial cells (RGCs). Dividing progenitors were also generated in greater numbers in clonal hippocampal stem cell (HPSC) cultures from the KO mice. Indeed, higher levels of Hes5 and Ngn2, transcription factors that maintain the stem and progenitor cell state, were expressed in both HPSCs and the GCL-ML from the Igf-I(Δ/Δ) mice. To determine the impact of Igf-I deletion on neuronal generation in vivo, progenitors in Igf-I(-/-) and Igf-I(+/+) mice were labeled with a GFP-expressing vector. This revealed that in the Igf-I(-/-) mice more GFP(+) -immature neurons were formed and they had less complex dendritic trees. These findings indicate that local IGF-I plays critical roles during postnatal/adult hippocampal neurogenesis, regulating the transition from HPSCs and progenitors to mature granule neurons in a cell stage-dependent manner. Stem Cells 2016;34:2194-2209.
Assuntos
Envelhecimento/metabolismo , Diferenciação Celular , Hipocampo/citologia , Hipocampo/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Células-Tronco Neurais/citologia , Neurogênese , Neurônios/citologia , Animais , Animais Recém-Nascidos , Contagem de Células , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células , Forma Celular , Células Clonais , Giro Denteado/citologia , Proteínas do Domínio Duplacortina , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Overexpression in humans of KCNH2-3.1, which encodes a primate-specific and brain-selective isoform of the human ether-a-go-go-related potassium channel, is associated with impaired cognition, inefficient neural processing and schizophrenia. Here, we describe a new mouse model that incorporates the KCNH2-3.1 molecular phenotype. KCNH2-3.1 transgenic mice are viable and display normal sensorimotor behaviors. However, they show alterations in neuronal structure and microcircuit function in the hippocampus and prefrontal cortex, areas affected in schizophrenia. Specifically, in slice preparations from the CA1 region of the hippocampus, KCNH2-3.1 transgenic mice have fewer mature dendrites and impaired theta burst stimulation long-term potentiation. Abnormal neuronal firing patterns characteristic of the fast deactivation kinetics of the KCNH2-3.1 isoform were also observed in prefrontal cortex. Transgenic mice showed significant deficits in a hippocampal-dependent object location task and a prefrontal cortex-dependent T-maze working memory task. Interestingly, the hippocampal-dependent alterations were not present in juvenile transgenic mice, suggesting a developmental trajectory to the phenotype. Suppressing KCNH2-3.1 expression in adult mice rescues both the behavioral and physiological phenotypes. These data provide insight into the mechanism of association of KCNH2-3.1 with variation in human cognition and neuronal physiology and may explain its role in schizophrenia.
Assuntos
Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Cognição/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Hipocampo/fisiopatologia , Humanos , Potenciação de Longa Duração/fisiologia , Memória de Curto Prazo , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Neurônios/metabolismo , Patologia Molecular/métodos , Córtex Pré-Frontal/fisiopatologia , Esquizofrenia/genética , Esquizofrenia/metabolismoRESUMO
Glucocorticoids are released in response to stressful experiences and serve many beneficial homeostatic functions. However, dysregulation of glucocorticoids is associated with cognitive impairments and depressive illness. In the hippocampus, a brain region densely populated with receptors for stress hormones, stress and glucocorticoids strongly inhibit adult neurogenesis. Decreased neurogenesis has been implicated in the pathogenesis of anxiety and depression, but direct evidence for this role is lacking. Here we show that adult-born hippocampal neurons are required for normal expression of the endocrine and behavioural components of the stress response. Using either transgenic or radiation methods to inhibit adult neurogenesis specifically, we find that glucocorticoid levels are slower to recover after moderate stress and are less suppressed by dexamethasone in neurogenesis-deficient mice than intact mice, consistent with a role for the hippocampus in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Relative to controls, neurogenesis-deficient mice also showed increased food avoidance in a novel environment after acute stress, increased behavioural despair in the forced swim test, and decreased sucrose preference, a measure of anhedonia. These findings identify a small subset of neurons within the dentate gyrus that are critical for hippocampal negative control of the HPA axis and support a direct role for adult neurogenesis in depressive illness.
Assuntos
Depressão/fisiopatologia , Hipocampo/citologia , Hipocampo/fisiologia , Neurogênese/fisiologia , Estresse Fisiológico/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Corticosterona/análise , Corticosterona/metabolismo , Giro Denteado/citologia , Giro Denteado/efeitos dos fármacos , Giro Denteado/fisiologia , Depressão/tratamento farmacológico , Dexametasona/farmacologia , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Hipocampo/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/efeitos dos fármacos , Neurogênese/efeitos da radiação , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/fisiologia , Receptores de Glucocorticoides/análise , Receptores de Glucocorticoides/metabolismo , Restrição Física/fisiologia , Restrição Física/psicologia , Estresse Fisiológico/efeitos dos fármacos , NataçãoRESUMO
The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE) is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE-specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells.
Assuntos
Proteínas do Olho , Proteínas de Homeodomínio , Fator de Transcrição Associado à Microftalmia , Fatores de Transcrição Box Pareados , Proteínas Repressoras , Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Animais , Transdiferenciação Celular , Desenvolvimento Embrionário , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Glutamate is the major excitatory neurotransmitter in the mammalian CNS and mediates fast synaptic transmission upon activation of glutamate-gated ion channels. In addition, glutamate modulates a variety of other synaptic responses and intracellular signaling by activating metabotropic glutamate receptors (mGluRs), which are G protein-coupled receptors. The mGluRs are also expressed in nonneuronal tissues and are implicated in a variety of normal biological functions as well as diseases. To study mGluR-activated calcium signaling in neurons, we generated mGluR5 transgenic animals using a Thy1 promoter to drive expression in the forebrain, and one founder unexpectedly developed melanoma. To directly investigate the role of mGluR5 in melanoma formation, we generated mGluR5 transgenic lines under a melanocyte-specific promoter, tyrosinase-related protein 1. A majority of the founders showed a severe phenotype with early onset. Hyperpigmentation of the pinnae and tail could be detected as early as 3-5 d after birth for most of the mGluR5 transgene-positive mice. There was 100% penetrance in the progeny from the tyrosinase-related protein 1-mGluR5 lines generated from founders that developed melanoma. Expression of mGluR5 was detected in melanoma samples by RT-PCR, immunoblotting, and immunohistochemistry. We evaluated the expression of several cancer-related proteins in tumor samples and observed a dramatic increase in the phosphorylation of ERK, implicating ERK as a downstream effector of mGluR5 signaling in tumors. Our findings show that mGluR5-mediated glutamatergic signaling can trigger melanoma in vivo. The aggressive growth and severe phenotype make these mouse lines unique and a potentially powerful tool for therapeutic studies.
Assuntos
Melanoma/metabolismo , Melanoma/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Receptores de Glutamato Metabotrópico/metabolismo , Envelhecimento/patologia , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Melanoma/enzimologia , Camundongos , Camundongos Transgênicos , Neoplasias Musculares/metabolismo , Neoplasias Musculares/patologia , Invasividade Neoplásica , Pigmentação , Receptor de Glutamato Metabotrópico 5 , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologiaRESUMO
Relapse to maladaptive eating habits during dieting is often provoked by stress and there is evidence for a role of ovarian hormones in stress responses and feeding. We studied the role of these hormones in stress-induced reinstatement of food seeking and medial prefrontal cortex (mPFC) neuronal activation in c-fos-GFP transgenic female rats, which express GFP in strongly activated neurons. Food-restricted ovariectomized or sham-operated c-fos-GFP rats were trained to lever-press for palatable food pellets. Subsequently, lever-pressing was extinguished and reinstatement of food seeking and mPFC neuronal activation was assessed after injections of the pharmacological stressor yohimbine (0.5-2 mg/kg) or pellet priming (1-4 noncontingent pellets). Estrous cycle effects on reinstatement were also assessed in wild-type rats. Yohimbine- and pellet-priming-induced reinstatement was associated with Fos and GFP induction in mPFC; both reinstatement and neuronal activation were minimally affected by ovarian hormones in both c-fos-GFP and wild-type rats. c-fos-GFP transgenic rats were then used to assess glutamatergic synaptic alterations within activated GFP-positive and nonactivated GFP-negative mPFC neurons following yohimbine-induced reinstatement of food seeking. This reinstatement was associated with reduced AMPA receptor/NMDA receptor current ratios and increased paired-pulse facilitation in activated GFP-positive but not GFP-negative neurons. While ovarian hormones do not appear to play a role in stress-induced relapse of food seeking in our rat model, this reinstatement was associated with unique synaptic alterations in strongly activated mPFC neurons. Our paper introduces the c-fos-GFP transgenic rat as a new tool to study unique synaptic changes in activated neurons during behavior.
Assuntos
Comportamento Alimentar/fisiologia , Genes fos/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Estresse Psicológico/psicologia , Sinapses/fisiologia , Animais , Corticosterona/sangue , Fenômenos Eletrofisiológicos , Ciclo Estral/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Genes fos/genética , Proteínas de Fluorescência Verde/genética , Imuno-Histoquímica , Ovariectomia , Técnicas de Patch-Clamp , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Long-Evans , Ratos Transgênicos , Simpatolíticos/farmacologia , Ioimbina/farmacologiaRESUMO
Recent studies emphasize the importance of mRNA splicing in human genetic disease, as 20-30% of all disease-causing mutations are predicted to result in mRNA splicing defects. The plasticity of the mRNA splicing reaction has made these mutations attractive candidates for the development of therapeutics. Familial dysautonomia (FD) is a severe neurodegenerative disorder, and all patients have an intronic IVS20+6T>C splice site mutation in the IKBKAP gene, which results in tissue-specific skipping of exon 20 and a corresponding reduction in ikappaB kinase complex associated protein (IKAP) levels. We created transgenic mouse lines using a human IKBKAP bacterial artificial chromosome (BAC) into which we inserted the IKBKAP splice mutation (FD BAC) and have shown that the transgenic mice exhibit the same tissue-specific aberrant splicing patterns as seen in FD patients. We have previously demonstrated that the plant cytokinin kinetin can significantly improve the production of wild-type IKBKAP transcripts in FD lymphoblast cell lines by improving exon inclusion. In this study, we tested the ability of kinetin to alter IKBKAP splicing in the transgenic mice carrying the FD BAC and show that it corrects IKBKAP splicing in all major tissues assayed, including the brain. The amount of wild-type IKBKAP mRNA and IKAP protein was significantly higher in the kinetin-treated mice. These exciting results prove that treatment of FD, as well as other mechanistically related splicing disorders, with kinetin holds great promise as a potential therapeutic aimed at increasing normal protein levels, which may, in turn, slow disease progression.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Suplementos Nutricionais , Cinetina/farmacologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Dieta , Relação Dose-Resposta a Droga , Peptídeos e Proteínas de Sinalização Intracelular , Cinetina/administração & dosagem , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
BACKGROUND & AIMS: Obesity-related insulin resistance contributes to cardiovascular disease. Cannabinoid receptor-1 (CB(1)) blockade improves insulin sensitivity in obese animals and people, suggesting endocannabinoid involvement. We explored the role of hepatic CB(1) in insulin resistance and inhibition of insulin signaling pathways. METHODS: Wild-type mice and mice with disruption of CB(1) (CB(1)(-/-) mice) or with hepatocyte-specific deletion or transgenic overexpression of CB(1) were maintained on regular chow or a high-fat diet (HFD) to induce obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp analysis was used to analyze the role of the liver and hepatic CB(1) in HFD-induced insulin resistance. The cellular mechanisms of insulin resistance were analyzed in mouse and human isolated hepatocytes using small interfering or short hairpin RNAs and lentiviral knockdown of gene expression. RESULTS: The HFD induced hepatic insulin resistance in wild-type mice, but not in CB(1)(-/-) mice or mice with hepatocyte-specific deletion of CB(1). CB(1)(-/-) mice that overexpressed CB(1) specifically in hepatocytes became hyperinsulinemic as a result of reduced insulin clearance due to down-regulation of the insulin-degrading enzyme. However, they had increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance; this was further increased by the HFD. In mice with hepatocytes that express CB(1), the HFD or CB(1) activation induced the endoplasmic reticulum stress response via activation of the Bip-PERK-eIF2α protein translation pathway. In hepatocytes isolated from human or mouse liver, CB(1) activation caused endoplasmic reticulum stress-dependent suppression of insulin-induced phosphorylation of akt-2 via phosphorylation of IRS1 at serine-307 and by inducing the expression of the serine and threonine phosphatase Phlpp1. Expression of CB(1) was up-regulated in samples from patients with nonalcoholic fatty liver disease. CONCLUSIONS: Endocannabinoids contribute to diet-induced insulin resistance in mice via hepatic CB(1)-mediated inhibition of insulin signaling and clearance.
Assuntos
Resistência à Insulina , Insulina/metabolismo , Fígado/metabolismo , Receptor CB1 de Canabinoide/fisiologia , Transdução de Sinais , Animais , Ácidos Araquidônicos/farmacologia , Dieta Hiperlipídica , Endocanabinoides , Estresse do Retículo Endoplasmático , Fígado Gorduroso/metabolismo , Intolerância à Glucose/etiologia , Humanos , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica , Fosforilação , Alcamidas Poli-Insaturadas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Various specialized structural/functional properties are considered essential for contextual memory encoding by hippocampal mossy fiber (MF) synapses. Although investigated to exquisite detail in model organisms, synapses, including MFs, have undergone minimal functional interrogation in humans. To determine the translational relevance of rodent findings, we evaluated MF properties within human tissue resected to treat epilepsy. Human MFs exhibit remarkably similar hallmark features to rodents, including AMPA receptor-dominated synapses with small contributions from NMDA and kainate receptors, large dynamic range with strong frequency facilitation, NMDA receptor-independent presynaptic long-term potentiation, and strong cyclic AMP (cAMP) sensitivity of release. Array tomography confirmed the evolutionary conservation of MF ultrastructure. The astonishing congruence of rodent and human MF core features argues that the basic MF properties delineated in animal models remain critical to human MF function. Finally, a selective deficit in GABAergic inhibitory tone onto human MF postsynaptic targets suggests that unrestrained detonator excitatory drive contributes to epileptic circuit hyperexcitability.
Assuntos
Fibras Musgosas Hipocampais , Sinapses , Animais , Humanos , Fibras Musgosas Hipocampais/fisiologia , Sinapses/fisiologia , Potenciação de Longa Duração/fisiologia , Transdução de SinaisRESUMO
Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.
RESUMO
Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.
Assuntos
Encéfalo , Callithrix , Humanos , Animais , Recém-Nascido , Chlorocebus aethiops , Macaca mulatta/genética , Callithrix/genética , Encéfalo/fisiologia , Técnicas de Transferência de Genes , Neurônios , Vetores Genéticos/genéticaRESUMO
Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood-brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.
Assuntos
Capsídeo , Dependovirus , Administração Intravenosa , Animais , Encéfalo/metabolismo , Callithrix/genética , Dependovirus/genética , Vetores Genéticos , Fígado , Camundongos , Camundongos Endogâmicos C57BL , Transdução Genética , TransgenesRESUMO
Protein synthesis inhibitor antibiotics are widely used to produce amnesia, and have been recognized to inhibit general or global mRNA translation in the basic translational machinery. For instance, anisomycin interferes with protein synthesis by inhibiting peptidyl transferase or the 80S ribosomal function. Therefore, de novo general or global protein synthesis has been thought to be necessary for long-term memory formation. However, it is unclear which mode of translation-gene-specific translation or general/global translation-is actually crucial for the memory consolidation process in mammalian brains. Here, we generated a conditional transgenic mouse strain in which double-strand RNA-dependent protein kinase (PKR)-mediated phosphorylation of eIF2alpha, a key translation initiation protein, was specifically increased in hippocampal CA1 pyramidal cells by the chemical inducer AP20187. Administration of AP20187 significantly increased activating transcription factor 4 (ATF4) translation and concomitantly suppressed CREB-dependent pathways in CA1 cells; this led to impaired hippocampal late-phase LTP and memory consolidation, with no obvious reduction in general translation. Conversely, inhibition of general translation by low-dose anisomycin failed to block hippocampal-dependent memory consolidation. Together, these results indicated that CA1-restricted genetic manipulation of particular mRNA translations is sufficient to impair the consolidation and that consolidation of memories in CA1 pyramidal cells through eIF2alpha dephosphorylation depends more on transcription/translation of particular genes than on overall levels of general translation. The present study sheds light on the critical importance of gene-specific translations for hippocampal memory consolidation.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Hipocampo/patologia , Transtornos da Memória/patologia , Células Piramidais/metabolismo , Análise de Variância , Animais , Anisomicina/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Biofísica , Proteína de Ligação a CREB/metabolismo , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fator de Iniciação 2 em Eucariotos/genética , Medo/psicologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/genética , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Técnicas de Patch-Clamp , Fosforilação/genética , Proteínas Quinases/genética , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células Piramidais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estatísticas não Paramétricas , Estilbamidinas/metabolismo , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Fator de Transcrição 4RESUMO
The orbitofrontal cortex (OFC) is a brain region involved in higher-order decision-making. Rodent studies show that cocaine self-administration (CSA) reduces OFC contribution to goal-directed behavior and behavioral strategies to avoid drug intake. This change in OFC function persists for many weeks after cocaine withdrawal, suggesting involvement in the process of addiction. The mechanisms underlying impaired OFC function by cocaine are not well-understood. However, studies implicate altered OFC serotonin (5-HT) function in disrupted cognitive processes during addiction and other psychiatric disorders. Thus, it is hypothesized that cocaine impairment of OFC function involves changes in 5-HT signaling, and previous work shows that 5-HT1A and 5-HT2A receptor-mediated effects on OFC pyramidal neurons (PyNs) are impaired weeks after cocaine withdrawal. However, 5-HT effects on other contributors to OFC circuit function have not been fully investigated, including the parvalbumin-containing, fast-spiking interneurons (OFCPV), whose function is essential to normal OFC-mediated behavior. Here, 5-HT function in naive rats and those withdrawn from CSA were evaluated using a novel rat transgenic line in which the rat parvalbumin promoter drives Cre-recombinase expression to permit identification of OFCPV cells by fluorescent reporter protein expression. We find that whereas CSA altered basal synaptic and membrane properties of the OFCPV neurons in a sex-dependent manner, the effects of 5-HT on these cells were unchanged by CSA. These data suggest that the behavioral effects of dysregulated OFC 5-HT function caused by cocaine experience are primarily mediated by changes in 5-HT signaling at PyNs, and not at OFCPV neurons.
Assuntos
Cocaína , Animais , Integrases , Neurônios , Parvalbuminas , Córtex Pré-Frontal , Ratos , SerotoninaRESUMO
Gonadotropin-regulated testicular RNA helicase is a multifunctional enzyme present in both Leydig and germ cells that is essential for the progress of spermatogenesis. GRTH gene expression is transcriptionally upregulated by human chorionic gonadotropin (hCG) via second messenger (cAMP) and androgen in Leydig cells. The regulatory region(s) in the GRTH gene that is/are required for its cell-specific expression in the testis and hCG/androgen dependent expression were investigated in transgenic mice carrying sequential deletions of 5' flanking sequences of the GRTH gene. GFP-reporter gene expression directed by the GRTH 5' flanking sequences extending to -3.6 kb was specifically located in Leydig cells and the 205 bp minimal promoter domain was sufficient for this cell-specific expression. The 1 kb (5' to the ATG codon) transgene-directed expression was markedly increased by in vivo hCG treatment. Administration of the androgen receptor inhibitor Flutamide blocked the basal and hCG stimulated GFP expression in Leydig cells. We conclude that the expression of GRTH in testicular cells is differentially regulated by its 5' flanking sequence and that the 1 kb fragment of GRTH gene contains sequences for androgen regulation of its expression in Leydig cells.