RESUMEN
OBJECTIVE: Children with neurofibromatosis-1 (NF1) are at risk for developing numerous nervous system abnormalities, including cognitive problems and brain tumors (optic pathway glioma). Currently, there are few prognostic factors that predict clinical manifestations or outcomes in patients, even in families with an identical NF1 gene mutation. In this study, we leveraged Nf1 genetically engineered mice (GEM) to define the potential role of sex as a clinically relevant modifier of NF1-associated neuronal dysfunction. METHODS: Deidentified clinical data were analyzed to determine the impact of sex on optic glioma-associated visual decline in children with NF1. In addition, Nf1 GEM were employed as experimental platforms to investigate sexually dimorphic differences in learning/memory, visual acuity, retinal ganglion cell (RGC) death, and Nf1 protein (neurofibromin)-regulated signaling pathway function (Ras activity, cyclic adenosine monophosphate [cAMP], and dopamine levels). RESULTS: Female patients with NF1-associated optic glioma were twice as likely to undergo brain magnetic resonance imaging for visual symptoms and 3× more likely to require treatment for visual decline than their male counterparts. As such, only female Nf1 GEM exhibited a decrement in optic glioma-associated visual acuity, shorter RGC axons, and attenuated cAMP levels. In contrast, only male Nf1 GEM showed spatial learning/memory deficits, increased Ras activity, and reduced dopamine levels. INTERPRETATION: Collectively, these observations establish sex as a major prognostic factor underlying neuronal dysfunction in NF1, and suggest that sex should be considered when interpreting future preclinical and clinical study results.
Asunto(s)
Discapacidades para el Aprendizaje/etiología , Neurofibromatosis 1/complicaciones , Trastornos de la Visión/etiología , Animales , Encéfalo/patología , Niño , Dopamina/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Femenino , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/genética , Hipocampo/metabolismo , Humanos , Discapacidades para el Aprendizaje/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Glioma del Nervio Óptico/genética , Factores Sexuales , Percepción Espacial/fisiologíaRESUMEN
Children with neurofibromatosis type 1 (NF1) are prone to learning and behavioral abnormalities, including problems with spatial learning and attention. The molecular etiology for these deficits is unclear, as previous studies have implicated defective dopamine, cyclic adenosine monophosphate (cAMP), and Ras homeostasis. Using behavioral, electrophysiological, and primary culture, we now demonstrate that reduced dopamine signaling is responsible for cAMP-dependent defects in neuron function and learning. Collectively, these results establish defective dopaminergic function as a contributing factor underlying impaired spatial learning and memory in children and adults with NF1, and support the use of treatments that restore normal dopamine homeostasis for select individuals.
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Dopamina/deficiencia , Discapacidades para el Aprendizaje/metabolismo , Discapacidades para el Aprendizaje/fisiopatología , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/fisiopatología , Animales , Atención/fisiología , AMP Cíclico/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatología , Discapacidades para el Aprendizaje/etiología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurofibromatosis 1/complicaciones , Neurofibromina 1/genética , Transducción de Señal/fisiología , Área Tegmental Ventral/metabolismo , Área Tegmental Ventral/fisiopatologíaRESUMEN
Children with the neurofibromatosis-1 (NF1) cancer predisposition syndrome exhibit numerous clinical problems that reflect defective central nervous system (CNS) neuronal function, including learning disabilities, attention deficit disorder, and seizures. These clinical features result from reduced NF1 protein (neurofibromin) expression in NF1+/- (NF1 heterozygosity) brain neurons. Previous studies have shown that mouse CNS neurons are sensitive to the effects of reduced Nf1 expression and exhibit shorter neurite lengths, smaller growth cone areas, and attenuated survival, reflecting attenuated neurofibromin cAMP regulation. In striking contrast, Nf1+/- peripheral nervous system (PNS) neurons are nearly indistinguishable from their wild-type counterparts, and complete neurofibromin loss leads to increased neurite lengths and survival in a RAS/Akt-dependent fashion. To gain insights into the differential responses of CNS and PNS neurons to reduced neurofibromin function, we designed a series of experiments to define the molecular mechanism(s) underlying the unique CNS neuronal sensitivity to Nf1 heterozygosity. First, Nf1 heterozygosity decreases cAMP levels in CNS, but not in PNS, neurons. Second, CNS neurons exhibit Nf1 gene-dependent increases in RAS pathway signaling, but no further decreases in cAMP levels were observed in Nf1-/- CNS neurons relative to their Nf1+/- counterparts. Third, neurofibromin regulates CNS neurite length and growth cone areas in a cAMP/PKA/Rho/ROCK-dependent manner in vitro and in vivo. Collectively, these findings establish cAMP/PKA/Rho/ROCK signaling as the responsible axis underlying abnormal Nf1+/- CNS neuronal morphology with important implications for future preclinical and clinical studies aimed at improving cognitive and behavioral deficits in mice and children with reduced brain neuronal NF1 gene expression.
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Sistema Nervioso Central/ultraestructura , AMP Cíclico/metabolismo , Heterocigoto , Neurofibromina 1/genética , Neuronas/ultraestructura , Transducción de Señal/fisiología , Animales , Células Cultivadas , Sistema Nervioso Central/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Genes de Neurofibromatosis 1 , Conos de Crecimiento/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/metabolismo , Neuritas/ultraestructura , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Neurofibromina 1/metabolismo , Neuronas/citología , Neuronas/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
The intestinal peptides GLP-1 and GIP potentiate glucose-mediated insulin release. Agents that increase GLP-1 action are effective therapies in type 2 diabetes mellitus (T2DM). However, GIP action is blunted in T2DM, and GIP-based therapies have not been developed. Thus, it is important to increase our understanding of the mechanisms of GIP action. We developed mice lacking GIP-producing K cells. Like humans with T2DM, "GIP/DT" animals exhibited a normal insulin secretory response to exogenous GLP-1 but a blunted response to GIP. Pharmacologic doses of xenin-25, another peptide produced by K cells, restored the GIP-mediated insulin secretory response and reduced hyperglycemia in GIP/DT mice. Xenin-25 alone had no effect. Studies with islets, insulin-producing cell lines, and perfused pancreata indicated xenin-25 does not enhance GIP-mediated insulin release by acting directly on the beta-cell. The in vivo effects of xenin-25 to potentiate insulin release were inhibited by atropine sulfate and atropine methyl bromide but not by hexamethonium. Consistent with this, carbachol potentiated GIP-mediated insulin release from in situ perfused pancreata of GIP/DT mice. In vivo, xenin-25 did not activate c-fos expression in the hind brain or paraventricular nucleus of the hypothalamus indicating that central nervous system activation is not required. These data suggest that xenin-25 potentiates GIP-mediated insulin release by activating non-ganglionic cholinergic neurons that innervate the islets, presumably part of an enteric-neuronal-pancreatic pathway. Xenin-25, or molecules that increase acetylcholine receptor signaling in beta-cells, may represent a novel approach to overcome GIP resistance and therefore treat humans with T2DM.
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Polipéptido Inhibidor Gástrico/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Glucosa/farmacología , Neurotensina/farmacología , Animales , Glucemia/metabolismo , Western Blotting , Carbacol/farmacología , Línea Celular Tumoral , Agonistas Colinérgicos/farmacología , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Ayuno/sangre , Femenino , Polipéptido Inhibidor Gástrico/genética , Polipéptido Inhibidor Gástrico/farmacología , Humanos , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurotensina/sangre , Páncreas/efectos de los fármacos , Páncreas/metabolismoRESUMEN
Diversifying the scientific workforce remains a national priority due to the continued lack of representation from underrepresented individuals in STEM fields. Quality mentoring has been identified as a stimulus to enhance not only research success, but also recruitment and retention of underrepresented groups pursuing STEM careers. Utilizing the Entering Mentoring training curriculum framework, this report provides a brief synopsis and key takeaways from the 2019 NIH-ASCB Accomplishing Career Transition (ACT) workshop, "Introduction to Effective Mentorship for Scientists" for 30 senior postdoctoral and early-career faculty researchers from historically underrepresented racial and ethnicity backgrounds. In addition, effective strategies and best practices to enhance STEM mentoring for early-career researchers are provided, which have practical applications for diverse mentoring relationships across disciplines, career stages, and mentee types.
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Cognitive dysfunction, including significant impairments in learning, behavior, and attention, is found in over 10% of children in the general population. However, in the common inherited cancer predisposition syndrome, neurofibromatosis type 1 (NF1), the prevalence of these cognitive deficits approaches 70%. As a monogenic disorder, NF1 provides a unique genetic tool to identify and dissect mechanistically the molecular and cellular bases underlying cognitive dysfunction. In this review, we discuss Nf1 fly and mouse systems that mimic many of the cognitive abnormalities seen in children with NF1. Further, we describe discoveries from these models that have uncovered defects in the regulation of Ras activity, cAMP generation, and dopamine homeostasis as key mechanisms important for cognitive dysfunction in children with NF1.
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Trastornos del Conocimiento , Modelos Animales de Enfermedad , Neurofibromatosis 1/genética , Animales , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/metabolismo , AMP Cíclico/metabolismo , Humanos , Neurofibromatosis 1/metabolismo , Transducción de SeñalRESUMEN
Children with neurofibromatosis type 1 (NF1) frequently have cognitive and behavioral deficits. Some of these deficits have been successfully modeled in Nf1 genetically-engineered mice that develop optic gliomas (Nf1 OPG mice). In the current study, we show that abnormal motivational influences affect the behavior of Nf1 OPG mice, particularly with regard to their response to novel environmental stimuli. For example, Nf1 OPG mice made fewer spontaneous alternations in a Y-maze and fewer arm entries relative to WT controls. However, analysis of normalized alternation data demonstrated that these differences were not due to a spatial working memory deficit. Other reported behavioral results (e.g., open-field test, below) suggest that differential responses to novelty and/or other motivational influences may be more important determinants of these kinds of behavior than simple differences in locomotor activity/spontaneous movements. Importantly, normal long-term depression was observed in hippocampal slices from Nf1 OPG mice. Results from elevated plus maze testing showed that differences in exploratory activity between Nf1 OPG and WT control mice may be dependent on the environmental context (e.g., threatening or non-threatening) under which exploration is being measured. Nf1 OPG mice also exhibited decreased exploratory hole poking in a novel holeboard and showed abnormal olfactory preferences, although L-dopa (50 mg/kg) administration resolved the abnormal olfactory preference behaviors. Nf1 OPG mice displayed an attenuated response to a novel open field in terms of decreased ambulatory activity and rearing but only during the first 10 min of the session. Importantly, Nf1 OPG mice demonstrated investigative rearing deficits with regard to a novel hanging object suspended on one side of the field which were not rescued by L-dopa administration. Collectively, our results provide new data important for evaluating therapeutic treatments aimed at ameliorating NF1-associated cognitive/behavioral deficits.
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Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Dopaminérgicos/administración & dosificación , Levodopa/administración & dosificación , Motivación/efectos de los fármacos , Neurofibromatosis 1/tratamiento farmacológico , Neurofibromina 1/fisiología , Animales , Depresión/diagnóstico , Depresión/tratamiento farmacológico , Depresión/etiología , Electrofisiología , Femenino , Humanos , Integrasas/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/diagnóstico , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/etiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Neurofibromatosis 1/complicaciones , Neurofibromatosis 1/metabolismoRESUMEN
Attention system abnormalities represent a significant barrier to scholastic achievement in children with neurofibromatosis-1 (NF1). Using a novel mouse model of NF1-associated attention deficit (ADD), we demonstrate a presynaptic defect in striatal dopaminergic homeostasis and leverage this finding to apply [(11)C]-raclopride positron-emission tomography (PET) in the intact animal. While methylphenidate and l-Deprenyl correct both striatal dopamine levels on PET imaging and defective attention system function in Nf1 mutant mice, pharmacologic agents that target de-regulated cyclic AMP and RAS signaling in these mice do not. These studies establish a robust preclinical model to evaluate promising agents for NF1-associated ADD.
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Trastorno por Déficit de Atención con Hiperactividad , Metilfenidato/farmacología , Neurofibromatosis 1 , Tomografía de Emisión de Positrones/métodos , Selegilina/farmacología , Animales , Atención/fisiología , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico por imagen , Trastorno por Déficit de Atención con Hiperactividad/tratamiento farmacológico , Trastorno por Déficit de Atención con Hiperactividad/etiología , Radioisótopos de Carbono , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/fisiología , Antagonistas de Dopamina , Inhibidores de Captación de Dopamina/farmacología , Neuronas Dopaminérgicas/fisiología , Evaluación Preclínica de Medicamentos/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Neurofibromatosis 1/complicaciones , Neurofibromatosis 1/diagnóstico por imagen , Neurofibromatosis 1/genética , Fármacos Neuroprotectores/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , RaclopridaRESUMEN
OBJECTIVE: Evidence suggests that insulin-sensitive glucose transporters (GLUTs) other than GLUT4 may exist. To investigate whether GLUT12 may represent another insulin-sensitive GLUT, transgenic (TG) mice that overexpress GLUT12 were characterized. RESEARCH DESIGN AND METHODS: TG mice that overexpressed GLUT12 under a ß-actin promoter were generated. Glucose metabolism in TG and wild-type control mice was compared using glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. In addition, basal and insulin-stimulated glucose clearance rates into insulin-sensitive peripheral tissues were measured using [(3)H]-2-deoxy-D-glucose. RESULTS: GLUT12 was overexpressed by 40-75% in TG compared with wild-type mice in insulin-sensitive tissues with no change in GLUT4 content. Body weight and fasting blood glucose did not differ between wild-type and TG mice; however, insulin concentrations were reduced in TG mice. Enhanced oral glucose tolerance was noted in TG mice by a reduced blood glucose excursion compared with wild-type mice (P < 0.05). Enhanced insulin sensitivity was noted by a greater decrease in blood glucose in TG mice during insulin tolerance testing. Hyperinsulinemic-euglycemic clamps confirmed enhanced insulin sensitivity in GLUT12-overexpressing mice (P < 0.01). Tissues of TG mice exhibited normal basal glucose clearance rates; however, under insulin-stimulated conditions, glucose clearance was significantly increased (P < 0.01) in tissues of TG mice. CONCLUSIONS: Increased expression of GLUT12 results in improved whole-body insulin sensitivity mediated by an increased glucose clearance rate in insulin-responsive tissues under insulin-stimulated, but not basal, conditions. These findings provide evidence that GLUT12 represents a novel, second insulin-sensitive GLUT.
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Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Insulina/sangre , Actinas/genética , Animales , Glucemia/metabolismo , Western Blotting , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/genéticaRESUMEN
OBJECTIVE: An impaired ability to sense and appropriately respond to insulin-induced hypoglycemia is a common and serious complication faced by insulin-treated diabetic patients. This study tests the hypothesis that insulin acts directly in the brain to regulate critical glucose-sensing neurons in the hypothalamus to mediate the counterregulatory response to hypoglycemia. RESEARCH DESIGN AND METHODS: To delineate insulin actions in the brain, neuron-specific insulin receptor knockout (NIRKO) mice and littermate controls were subjected to graded hypoglycemic (100, 70, 50, and 30 mg/dl) hyperinsulinemic (20 mU/kg/min) clamps and nonhypoglycemic stressors (e.g., restraint, heat). Subsequently, counterregulatory responses, hypothalamic neuronal activation (with transcriptional marker c-fos), and regional brain glucose uptake (via (14)C-2deoxyglucose autoradiography) were measured. Additionally, electrophysiological activity of individual glucose-inhibited neurons and hypothalamic glucose sensing protein expression (GLUTs, glucokinase) were measured. RESULTS: NIRKO mice revealed a glycemia-dependent impairment in the sympathoadrenal response to hypoglycemia and demonstrated markedly reduced (3-fold) hypothalamic c-fos activation in response to hypoglycemia but not other stressors. Glucose-inhibited neurons in the ventromedial hypothalamus of NIRKO mice displayed significantly blunted glucose responsiveness (membrane potential and input resistance responses were blunted 66 and 80%, respectively). Further, hypothalamic expression of the insulin-responsive GLUT 4, but not glucokinase, was reduced by 30% in NIRKO mice while regional brain glucose uptake remained unaltered. CONCLUSIONS: Chronically, insulin acts in the brain to regulate the counterregulatory response to hypoglycemia by directly altering glucose sensing in hypothalamic neurons and shifting the glycemic levels necessary to elicit a normal sympathoadrenal response.