RESUMEN
The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.
Asunto(s)
Cuerpo Estriado/patología , Cristalinas/genética , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/genética , Animales , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Expresión Génica , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Macaca , Masculino , Ratones , Ratones Transgénicos , Mutación , Ratas , Cristalinas muRESUMEN
Astrocyte reactivity is a hallmark of neurodegenerative diseases (ND), but its effects on disease outcomes remain highly debated. Elucidation of the signaling cascades inducing reactivity in astrocytes during ND would help characterize the function of these cells and identify novel molecular targets to modulate disease progression. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is associated with reactive astrocytes in models of acute injury, but it is unknown whether this pathway is directly responsible for astrocyte reactivity in progressive pathological conditions such as ND. In this study, we examined whether the JAK/STAT3 pathway promotes astrocyte reactivity in several animal models of ND. The JAK/STAT3 pathway was activated in reactive astrocytes in two transgenic mouse models of Alzheimer's disease and in a mouse and a nonhuman primate lentiviral vector-based model of Huntington's disease (HD). To determine whether this cascade was instrumental for astrocyte reactivity, we used a lentiviral vector that specifically targets astrocytes in vivo to overexpress the endogenous inhibitor of the JAK/STAT3 pathway [suppressor of cytokine signaling 3 (SOCS3)]. SOCS3 significantly inhibited this pathway in astrocytes, prevented astrocyte reactivity, and decreased microglial activation in models of both diseases. Inhibition of the JAK/STAT3 pathway within reactive astrocytes also increased the number of huntingtin aggregates, a neuropathological hallmark of HD, but did not influence neuronal death. Our data demonstrate that the JAK/STAT3 pathway is a common mediator of astrocyte reactivity that is highly conserved between disease states, species, and brain regions. This universal signaling cascade represents a potent target to study the role of reactive astrocytes in ND.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Astrocitos , Enfermedad de Huntington/fisiopatología , Quinasas Janus , Factor de Transcripción STAT3 , Transducción de Señal , Enfermedad de Alzheimer/patología , Animales , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Enfermedad de Huntington/patología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat encoding a polyglutamine tract in the huntingtin (Htt) protein. The mutation leads to neuronal death through mechanisms which are still unknown. One hypothesis is that mitochondrial defects may play a key role. In support of this, the activity of mitochondrial complex II (C-II) is preferentially reduced in the striatum of HD patients. Here, we studied C-II expression in different genetic models of HD expressing N-terminal fragments of mutant Htt (mHtt). Western blot analysis showed that the expression of the 30 kDa Iron-Sulfur (Ip) subunit of C-II was significantly reduced in the striatum of the R6/1 transgenic mice, while the levels of the FAD containing catalytic 70 kDa subunit (Fp) were not significantly changed. Blue native gel analysis showed that the assembly of C-II in mitochondria was altered early in N171-82Q transgenic mice. Early loco-regional reduction in C-II activity and Ip protein expression was also demonstrated in a rat model of HD using intrastriatal injection of lentiviral vectors encoding mHtt. Infection of the rat striatum with a lentiviral vector coding the C-II Ip or Fp subunits induced a significant overexpression of these proteins that led to significant neuroprotection of striatal neurons against mHtt neurotoxicity. These results obtained in vivo support the hypothesis that structural and functional alterations of C-II induced by mHtt may play a critical role in the degeneration of striatal neurons in HD and that mitochondrial-targeted therapies may be useful in its treatment.
Asunto(s)
Cuerpo Estriado/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Enfermedad de Huntington/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Células Cultivadas , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Complejo II de Transporte de Electrones/genética , Femenino , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Mitocondrias/genética , Proteínas Mutantes/metabolismo , Mutación , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Due to their pure intracellular compartmentation, the translational diffusion of brain metabolites in vivo depends on the intracellular environment, including viscosity, molecular crowding and subcellular structures. However, as the diffusion time is increased, metabolites have enough time to significantly encounter cell boundaries, so that cell size and geometry are expected to strongly determine metabolite diffusion path. In the present work, diffusion-weighted nuclear magnetic resonance spectroscopy was used to investigate brain metabolite diffusion in vivo, at long and ultra-long diffusion times (from ~80 ms to more than 1 s), in a voxel with equal proportions of white and grey matter in macaque monkeys. No dramatic dependence of the ADC on the diffusion time was observed, suggesting that metabolites' apparent diffusion is largely unrestricted over these time-scales. In an attempt to explain this stability and relate it to plausible cell geometries, data were analyzed with two simple geometrical models describing diffusion either in fibers such as axons, dendrites and astrocytic processes, or in closed cell bodies. Results support the idea that DW-MRS is sensitive to cell shape, and that a vast fraction of brain metabolites is diffusing in long fibers rather than being confined in cell bodies.
Asunto(s)
Encéfalo/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Fibras Nerviosas/metabolismo , Animales , Procesamiento de Imagen Asistido por Computador , Espacio Intracelular/metabolismo , Macaca fascicularis , Imagen por Resonancia MagnéticaRESUMEN
Ciliary neurotrophic factor (CNTF) is neuroprotective against multiple pathologic conditions including metabolic impairment, but the mechanisms are still unclear. To delineate CNTF effects on brain energy homeostasis, we performed a multimodal imaging study, combining in vivo proton magnetic resonance spectroscopy, high-performance liquid chromatography analysis, and in situ glutamate imaging by chemical exchange saturation transfer. Unexpectedly, we found that CNTF expression through lentiviral gene transfer in the rat striatum significantly decreased the levels of neuronal metabolites (N-acetyl-aspartate, N-acetyl-aspartyl-glutamate, and glutamate). This preclinical study shows that CNTF remodels brain metabolism, and suggests that decreased levels of neuronal metabolites may occur in the absence of neuronal dysfunction.
Asunto(s)
Astrocitos/metabolismo , Factor Neurotrófico Ciliar/metabolismo , Cuerpo Estriado/metabolismo , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Ácido Glutámico/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Fármacos Neuroprotectores/metabolismo , Ratas Sprague-DawleyRESUMEN
Astrocytes are key cellular elements in both the tripartite synapse and the neurovascular unit. To fulfill this dual role in synaptic activity and metabolism, they express a panel of receptors and transporters that sense glutamate. Among them, the GLT-1 and GLAST transporters are known to regulate extracellular glutamate concentrations at excitatory synapses and consequently modulate glutamate receptor signaling. These major uptake systems are also involved in energy supply to neurons. However, the functional role of GLAST in concurrent regulation of metabolic and neuronal activity is currently unknown. We took advantage of the attractive structural and functional features of the main olfactory bulb to explore the impact of GLAST on sensory information processing while probing both glutamate uptake and neuronal activity in glomeruli and deeper cellular layers, respectively. Using odor-evoked 2-deoxyglucose imaging and local field potential recordings in GLAST knockout mice, we show in vivo that deletion of GLAST alters both glucose uptake and neuronal oscillations in olfactory bulb networks.
RESUMEN
Tissue-targeted expression is of major interest for studying the contribution of cellular subpopulations to neurodegenerative diseases. However, in vivo methods to investigate this issue are limited. Here, we report an analysis of the cell specificity of expression of fluorescent reporter genes driven by six neuronal promoters, with the ubiquitous phosphoglycerate kinase 1 (PGK) promoter used as a reference. Quantitative analysis of AcGFPnuc expression in the striatum and hippocampus of rodents showed that all lentiviral vectors (LV) exhibited a neuronal tropism; however, there was substantial diversity of transcriptional activity and cell-type specificity of expression. The promoters with the highest activity were those of the 67 kDa glutamic acid decarboxylase (GAD67), homeobox Dlx5/6, glutamate receptor 1 (GluR1), and preprotachykinin 1 (Tac1) genes. Neuron-specific enolase (NSE) and dopaminergic receptor 1 (Drd1a) promoters showed weak activity, but the integration of an amplification system into the LV overcame this limitation. In the striatum, the expression profiles of Tac1 and Drd1a were not limited to the striatonigral pathway, whereas in the hippocampus, Drd1a and Dlx5/6 showed the expected restricted pattern of expression. Regulation of the Dlx5/6 promoter was observed in a disease condition, whereas Tac1 activity was unaffected. These vectors provide safe tools that are more selective than others available, for the administration of therapeutic molecules in the central nervous system (CNS). Nevertheless, additional characterization of regulatory elements in neuronal promoters is still required.
Asunto(s)
Encéfalo/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Encéfalo/patología , Genes Reporteros , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Hipocampo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Lentivirus/genética , Masculino , Ratones , Neuronas/metabolismo , Neuronas/patología , Fosfoglicerato Quinasa/genética , Fosfoglicerato Quinasa/metabolismo , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ratas , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Taquicininas/genética , Taquicininas/metabolismo , Transcripción Genética , Transducción GenéticaRESUMEN
Detection of amyloid plaques in the brain by in vivo neuroimaging is a very promising biomarker approach for early diagnosis of Alzheimer's disease (AD) and evaluation of therapeutic efficacy. Here we describe a new method to detect amyloid plaques by in vivo magnetic resonance imaging (MRI) based on the intracerebroventricular injection of a nontargeted gadolinium (Gd)-based contrast agent, which rapidly diffuses throughout the brain and increases the signal and contrast of magnetic resonance (MR) images by shortening the T1 relaxation time. This gain in image sensitivity after in vitro and in vivo Gd staining significantly improves the detection and resolution of individual amyloid plaques in the cortex and hippocampus of AD transgenic mice. The improved image resolution is sensitive enough to demonstrate an age-dependent increase of amyloid plaque load and a good correlation between the amyloid load measured by µMRI and histology. These results provide the first demonstration that nontargeted Gd staining can enhance the detection of amyloid plaques to follow the progression of AD and to evaluate the activity of amyloid-lowering therapeutic strategies in longitudinal studies.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Encéfalo/patología , Gadolinio , Imagen por Resonancia Magnética/métodos , Placa Amiloide/metabolismo , Animales , Medios de Contraste , Femenino , Gadolinio/farmacocinética , Ratones , Ratones Transgénicos , Imagen Molecular/métodos , Coloración y Etiquetado/métodos , Distribución TisularRESUMEN
Genes selectively expressed in the striatum may be involved in the preferential vulnerability of striatal neurons to Huntington's disease (HD). Here, we investigated whether perturbations of Capucin expression, which is enriched in the striatum and downregulated in Huntington's disease models, could modify the neurotoxicity induced by the injection of a lentiviral vector encoding a short N-terminal fragment of mutant Huntingtin (mHtt) into the mouse striatum. Neither constitutive Capucin deficiency in knockout mice nor lentiviral vector-mediated Capucin overexpression in the striatum of adult wild type mice significantly modified vulnerability to the mHtt fragment in vivo, suggesting that Capucin has no impact on mHtt toxicity.
Asunto(s)
Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Proteína Huntingtina , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , MutaciónRESUMEN
Recent studies have suggested including nonstructural proteins as Tat and Vpr in HIV vaccines. However, little is known about the CD4+ T-cell response that these small proteins induce in humans. We have therefore evaluated these responses by in vitro priming experiments of CD4+ T lymphocytes harvested in healthy donors. In the Tat protein, only one peptide primed CD4+ T cells of eight HLA unrelated healthy donors. T cells induced by this peptide recognized immature DC loaded with the native Tat protein and are restricted by multiple HLA-DR molecules, in agreement with its binding capacity. This peptide was therefore processed in an appropriate manner and was highly immunoprevalent. CD4+ T-cell response to Vpr peptides was more disperse and involved six different peptides depending on the HLA-DR molecules of the donors. Two overlapping peptides were T-cell stimulating in at least half of the donors. T-cell response to Vpr in multiple donors is the result of a combination of several CD4+ T-cell epitopes with good to moderate immunoprevalence. Altogether, our results show that the frequency of responders to HIV Tat or Vpr proteins relies on one or multiple CD4+ T-cell epitopes, respectively.