RESUMEN
α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.
Asunto(s)
Cuerpo Estriado/metabolismo , Sustancia Negra/metabolismo , Sinapsis/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Cuerpo Estriado/ultraestructura , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Persona de Mediana Edad , Sustancia Negra/ultraestructura , Sinapsis/ultraestructuraRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease, leading to the impairment of movement execution. PD pathogenesis has been largely investigated, either limited to bulk transcriptomic levels or at certain cell types, which failed to capture the cellular heterogeneity and intrinsic interplays among distinct cell types. Here, we report the application of single-nucleus RNA-seq on midbrain, striatum, and cerebellum of the α-syn-A53T mouse, a well-established PD mouse model, and matched controls, generating the first single cell transcriptomic atlas for the PD model mouse brain composed of 46,174 individual cells. Additionally, we comprehensively depicte the dysfunctions in PD pathology, covering the elevation of NF-κB activity, the alteration of ion channel components, the perturbation of protein homeostasis network, and the dysregulation of glutamatergic signaling. Notably, we identify a variety of cell types closely associated with PD risk genes. Taken together, our study provides valuable resources to systematically dissect the molecular mechanism of PD pathogenesis at the single-cell resolution, which facilitates the development of novel approaches for diagnosis and therapies against PD.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Filamentos Intermediarios/genética , Proteínas Musculares/genética , Enfermedad de Parkinson/genética , Transcriptoma/genética , Animales , Encéfalo/patología , Encéfalo/ultraestructura , Cerebelo/metabolismo , Cerebelo/patología , Cerebelo/ultraestructura , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Cuerpo Estriado/ultraestructura , Modelos Animales de Enfermedad , Humanos , Mesencéfalo/metabolismo , Mesencéfalo/patología , Mesencéfalo/ultraestructura , Ratones , FN-kappa B/genética , Enfermedad de Parkinson/patología , RNA-Seq , Análisis de la Célula Individual/tendenciasRESUMEN
Background: Essential fatty acids (EFAs) and non-essential fatty acids (nEFAs) exert experimental and clinical neuroprotection in neurodegenerative diseases. The main EFAs, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), nEFAs, and oleic acid (OA) contained in olive and fish oils are inserted into the cell membranes, but the exact mechanism through which they exert neuroprotection is still unknown. Objectives and Methods: In this study, we assessed the fatty acids content and membrane fluidity in striatal rat synaptosomes after fatty acid-rich diets (olive- or a fish-oil diet, 15% w/w). Then, we evaluated the effect of enriching striatum synaptosomes with fatty acids on the oxidative damage produced by the prooxidants ferrous sulfate (FeSO4) or quinolinic acid (QUIN). Results and Discussion: Lipid profile analysis in striatal synaptosomes showed that EPA content increased in the fish oil group in comparison with control and olive groups. Furthermore, we found that synaptosomes enriched with fatty acids and incubated with QUIN or FeSO4 showed a significant oxidative damage reduction. Results suggest that EFAs, particularly EPA, improve membrane fluidity and confer antioxidant effect.
Asunto(s)
Membrana Celular/metabolismo , Cuerpo Estriado/metabolismo , Ácidos Grasos/metabolismo , Estrés Oxidativo , Sinaptosomas/metabolismo , Animales , Membrana Celular/ultraestructura , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/ultraestructura , Ácidos Grasos/administración & dosificación , Aceites de Pescado/administración & dosificación , Masculino , Aceites de Plantas/administración & dosificación , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sinaptosomas/ultraestructuraRESUMEN
Huntington's disease (HD) is one of main neurodegenerative diseases, characterized by striatal atrophy, involuntary movements, and motor incoordination. Ginsenoside Rg1 (Rg1), an active ingredient in ginseng, possesses a variety of neuroprotective effects with low toxicity and side effects. In this study, we investigated the potential therapeutic effects of Rg1 in a mouse model of HD and explored the underlying mechanisms. HD was induced in mice by injection of 3-nitropropionic acid (3-NP, i.p.) for 4 days. From the first day of 3-NP injection, the mice were administered Rg1 (10, 20, 40 mg·kg-1, p.o.) for 5 days. We showed that oral pretreatment with Rg1 alleviated 3-NP-induced body weight loss and behavioral defects. Furthermore, pretreatment with Rg1 ameliorated 3-NP-induced neuronal loss and ultrastructural morphological damage in the striatum. Moreover, pretreatment with Rg1 reduced 3-NP-induced apoptosis and inhibited the activation of microglia, inflammatory mediators in the striatum. We revealed that Rg1 exerted neuroprotective effects by suppressing 3-NP-induced activation of the MAPKs and NF-κΒ signaling pathways in the striatum. Thus, our results suggest that Rg1 exerts therapeutic effects on 3-NP-induced HD mouse model via suppressing MAPKs and NF-κΒ signaling pathways. Rg1 may be served as a novel therapeutic option for HD.
Asunto(s)
Cuerpo Estriado/metabolismo , Ginsenósidos/farmacología , Enfermedad de Huntington/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fármacos Neuroprotectores/farmacología , Animales , Apoptosis , Cuerpo Estriado/patología , Cuerpo Estriado/ultraestructura , Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas/efectos de los fármacos , Nitrocompuestos , Propionatos , Transducción de Señal/efectos de los fármacosRESUMEN
B cell leukemia/lymphoma-2 (Bcl-2)-interacting death suppressor (BIS), also identified as Bcl-2-associated athanogene 3 (BAG3), has been reported to be upregulated in reactive astrocytes after brain insults. The present study was designed to further substantiate the involvement of BIS protein in the astroglial reaction in the striatum of rats treated with the mitochondrial toxin, 3-nitropropionic acid. Weak constitutive immunoreactivity for BIS was observed in astrocytes in the control striatum, whereas its expression was upregulated, along with that of nestin, in the lesioned striatum. In the lesion core, where astrocytes are virtually absent, BIS/nestin double-labeled cells were associated with the vasculature and were identified as perivascular adventitial fibroblasts. By contrast, BIS/nestin double-labeled cells in the perilesional area were reactive astrocytes, which were confined to the border zone contributing to the formation of the astroglial scar; this was evident 3 days post-lesion and increased thereafter progressively throughout the 28-day experimental period. At the ultrastructural level, BIS protein was diffusely localized throughout the cytoplasm within the stained cells. Collectively, our results demonstrate the phenotypic and functional heterogeneity of BIS-positive cells in the lesioned striatum, suggesting the involvement of BIS in the formation of astroglial scar and its potential role in the development of fibrotic scar after brain insults.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Astrocitos/patología , Cicatriz/patología , Mitocondrias/patología , Neuronas/patología , Toxinas Biológicas/toxicidad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Cuerpo Estriado/ultraestructura , Fibroblastos/metabolismo , Fibrosis , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Nestina/metabolismo , Neuronas/ultraestructura , Nitrocompuestos , Fenotipo , Propionatos , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Expression of the glutamate transporter GLT-1 in neurons has been shown to be important for synaptic mitochondrial function in the cerebral cortex. Here we determined whether neuronal GLT-1 plays a similar role in the hippocampus and striatum, using conditional GLT-1 knockout mice in which GLT-1 was inactivated in neurons by expression of synapsin-Cre (synGLT-1 KO). Ex vivo 13C-labelling using [1,2-13C]acetate, representing astrocytic metabolism, yielded increased [4,5-13C]glutamate levels, suggesting increased astrocyte-neuron glutamine transfer, in the striatum but not in the hippocampus of the synGLT-1 KO. Moreover, aspartate concentrations were reduced - 38% compared to controls in the hippocampus and the striatum of the synGLT-1 KO. Mitochondria isolated from the hippocampus of synGLT-1 KO mice exhibited a lower oxygen consumption rate in the presence of oligomycin A, indicative of a decreased proton leak across the mitochondrial membrane, whereas the ATP production rate was unchanged. Electron microscopy revealed reduced mitochondrial inter-cristae distance within excitatory synaptic terminals in the hippocampus and striatum of the synGLT-1 KO. Finally, dilution of 13C-labelling originating from [U-13C]glucose, caused by metabolism of unlabelled glutamate, was reduced in hippocampal synGLT-1 KO synaptosomes, suggesting that neuronal GLT-1 provides glutamate for synaptic tricarboxylic acid cycle metabolism. Collectively, these data demonstrate an important role of neuronal expression of GLT-1 in synaptic mitochondrial metabolism in the forebrain.
Asunto(s)
Ácido Aspártico/metabolismo , Cuerpo Estriado/metabolismo , Transportador 2 de Aminoácidos Excitadores/deficiencia , Hipocampo/metabolismo , Mitocondrias/metabolismo , Sinapsis/metabolismo , Animales , Cuerpo Estriado/ultraestructura , Transportador 2 de Aminoácidos Excitadores/genética , Hipocampo/ultraestructura , Homeostasis/fisiología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Sinapsis/ultraestructuraRESUMEN
Despite negative association in cognition and memory, mice harboring Val66Met BDNF SNP (BDNFM/M) exhibit enhanced motor recovery accompanied by elevated excitatory synaptic markers VGLUT1 and VGLUT2 in striatum contralateral to unilateral ischemic stroke. The cortico-striatal pathway is a critical gateway for plasticity of motor/gait function. We hypothesized that enhanced excitability of the cortico-striatal pathway, especially of the contralateral hemisphere, underlies improved motor recovery. To test this hypothesis, we examined the key molecules involving excitatory synaptogenesis: Thrombospondins (TSP1/2) and their neuronal receptor α2δ-1. In WT brains, stroke induced expressions of TSP1/2-mRNA. The contralateral hemisphere of BDNFM/M mice showed heightened TSP2 and α2δ-1 mRNA and protein specifically at 6 months post-stroke. Immunoreactivities of TSPs and α2δ-1 were increased in cortical layers 1/2 of stroked BDNFM/M animals compared with BDNFM/M sham brains at this time. Areal densities of excitatory synapses in cortical layer 1 and striatum were also increased in stroked BDNFM/M brains, relative to stroked WT brains. Notably, the frequency of GABAergic synapses was greatly reduced along distal dendrites in cortical layer 1 in BDNFM/M brains, whether or not stroked, compared with WT brains. There was no effect of genotype or treatment on the density of GABAergic synapses onto striatal medium spiny neurons. The study identified molecular and synaptic substrates in the contralateral hemisphere of BDNFM/M mice, especially in cortical layers 1/2, which indicates selective region-related synaptic plasticity. The study suggests that an increase in excitatory-to-inhibitory synaptic balance along the contralateral cortico-striatal pathway underlies the enhanced functional recovery of BDNFM/M mice.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Accidente Cerebrovascular/metabolismo , Sinapsis/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Corteza Cerebral/ultraestructura , Cuerpo Estriado/ultraestructura , Excitabilidad Cortical , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/ultraestructura , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Polimorfismo de Nucleótido Simple , Trombospondinas/metabolismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of protein inclusions and the loss of dopaminergic neurons. Abnormal mitochondrial homeostasis is thought to be important for the pathogenesis of PD. Transcranial direct current stimulation (tDCS), a noninvasive brain stimulation technique, constitutes a promising approach for promoting recovery of various neurological conditions. However, little is known about its mechanism of action. The present study elucidated the neuroprotective effects of tDCS on the mitochondrial quality control pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We used the MPTP-induced neurotoxicity in vivo model. Mice were stimulated for 5 consecutive days with MPTP treatment. After observation of behavioral alteration using the rotarod test, mice were sacrificed for the measurement of the PD- and mitochondrial quality control-related protein levels in the substantia nigra. tDCS improved the behavioral alterations and changes in tyrosine hydroxylase levels in MPTP-treated mice. Furthermore, tDCS attenuated mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in the MPTP-induced PD mouse model. MPTP significantly increased mitophagy and decreased mitochondrial biogenesis-related proteins. These changes were attenuated by tDCS. Furthermore, MPTP significantly increased fission-related protein dynamin-related protein 1 with no effect on fusion-related protein mitofusin-2, and tDCS attenuated these changes. Our findings demonstrated the neuroprotective effect of anodal tDCS on the MPTP-induced neurotoxic mouse model through suppressing excessive mitophagy and balancing mitochondrial dynamics. The neuroprotective effect of anodal tDCS with modulation of mitochondrial dynamics provides a new therapeutic strategy for the treatment of PD.
Asunto(s)
Intoxicación por MPTP/prevención & control , Dinámicas Mitocondriales/efectos de la radiación , Estimulación Transcraneal de Corriente Directa , Adenosina Trifosfato/análisis , Animales , Cuerpo Estriado/química , Cuerpo Estriado/efectos de la radiación , Cuerpo Estriado/ultraestructura , Electrodos , GTP Fosfohidrolasas/análisis , Glutamato Deshidrogenasa/análisis , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/enzimología , Mitofagia/efectos de la radiación , Proteínas del Tejido Nervioso/análisis , Biogénesis de Organelos , Prueba de Desempeño de Rotación con Aceleración Constante , Sustancia Negra/química , Sustancia Negra/efectos de la radiación , Sustancia Negra/ultraestructura , Tirosina 3-Monooxigenasa/análisisRESUMEN
Striatal medium spiny neurons (MSNs) control motor function. Hyper- or hypo-activity of MSNs coincides with basal ganglia-related movement disorders. Based on the assumption that lasting alterations in neuronal activity lead to structural changes in the brain, understanding these structural alterations may be used to infer MSN functional abnormalities. To infer MSN function from structural data, understanding how long-lasting alterations in MSN activity affect brain morphology is essential. To address this, we utilized a simplified model of functional induction by stimulating MSNs expressing channelrhodopsin 2 (ChR2). Subsequent structural alterations which induced long-term activity changes in these MSNs were investigated in the striatal pathway and its associated regions by diffusion tensor imaging (DTI) and histological assessment with super-resolution microscopy. DTI detected changes in the striatum, substantia nigra, and motor cortex. Histological assessment found a reduction in the diameter of myelinated cortical axons as well as MSN dendrites and axons. The structural changes showed a high correlation between DTI parameters and histological data. These results demonstrated that long-term neural activation in the MSNs alters the diameter of MSN and cortical neurons fibers. This study provides a tool for understanding the causal relationship between functional and structural alterations.
Asunto(s)
Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/ultraestructura , Imagen de Difusión Tensora/métodos , Neuronas/ultraestructura , Optogenética/métodos , Animales , Cuerpo Estriado/química , Masculino , Ratones , Ratones Transgénicos , Microscopía/métodos , Neuronas/químicaRESUMEN
Neuropeptides are essential signaling molecules transported and secreted by dense-core vesicles (DCVs), but the number of DCVs available for secretion, their subcellular distribution, and release probability are unknown. Here, we quantified DCV pool sizes in three types of mammalian CNS neurons in vitro and in vivo Super-resolution and electron microscopy reveal a total pool of 1,400-18,000 DCVs, correlating with neurite length. Excitatory hippocampal and inhibitory striatal neurons in vitro have a similar DCV density, and thalamo-cortical axons in vivo have a slightly higher density. Synapses contain on average two to three DCVs, at the periphery of synaptic vesicle clusters. DCVs distribute equally in axons and dendrites, but the vast majority (80%) of DCV fusion events occur at axons. The release probability of DCVs is 1-6%, depending on the stimulation. Thus, mammalian CNS neurons contain a large pool of DCVs of which only a small fraction can fuse, preferentially at axons.
Asunto(s)
Axones , Cuerpo Estriado , Hipocampo , Neuritas , Vesículas Secretoras , Sinapsis , Animales , Axones/metabolismo , Axones/ultraestructura , Cuerpo Estriado/metabolismo , Cuerpo Estriado/ultraestructura , Hipocampo/metabolismo , Hipocampo/ultraestructura , Ratones , Neuritas/metabolismo , Neuritas/ultraestructura , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Methamphetamine (METH), an amphetamine derivate, may increase the risk of developing Parkinson's disease (PD). Human and animal studies have shown that METH produces persistent dopaminergic neurotoxicity in the nigrostriatal pathway, despite initial partial recovery. To determine the processes leading to early compensation, we studied the detailed morphology and distribution of tyrosine hydroxylase immunoreactive fibers (TH-ir) classified by their thickness (types I-IV) before and after METH. Applying three established neurotoxic regimens of METH: single high dose (1 × 30 mg/kg), multiple lower doses (3 × 5 mg/kg) or (3 × 10 mg/kg), we show that METH primarily damages type I fibers (the thinner ones), and to a much lesser extend types II-IV fibers including sterile axons. The striatal TH terminal partial recovery process, consisting of a progressive regrowth increases in types II, III, and IV fibers, demonstrated by co-localization of GAP-43, a sprouting marker, was observed 3 days post-METH treatment. In addition, we demonstrate the presence of growth-cone-like TH-ir structures, indicative of new terminal generation as well as improvement in motor functions after 3 days. A temporal relationship was observed between decreases in TH-expression and increases in silver staining, a marker of degeneration. Striatal regeneration was associated with an increase in astroglia and decrease in microglia expression, suggesting a possible role for the neuroimmune system in regenerative processes. Identification of regenerative compensatory mechanisms in response to neurotoxic agents could point to novel mechanisms in countering the neurotoxicity and/or enhancing the regenerative processes.
Asunto(s)
Estimulantes del Sistema Nervioso Central/toxicidad , Cuerpo Estriado/fisiopatología , Dopamina/metabolismo , Metanfetamina/toxicidad , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/patología , Animales , Proteínas de Unión al Calcio , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/ultraestructura , Modelos Animales de Enfermedad , Proteína GAP-43/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/patología , Terminales Presinápticos/ultraestructura , Trastornos Psicomotores/etiología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Tinción con Nitrato de Plata , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The heterotrimeric G-protein Go with its splice variants, Go1α and Go2α, seems to be involved in the regulation of motor function but isoform-specific effects are still unclear. We found that Go1α-/- knockouts performed worse on the rota-rod than Go2α-/- and wild-type (WT) mice. In Go1+2α-/- mice motor function was partially recovered. Furthermore, Go1+2α-/- mice showed an increased spontaneous motor activity. Compared to wild types or Go2α-/- mice, Go1+2α-/- mice developed increased behavioural sensitization following repetitive cocaine treatment, but failed to develop conditioned place preference. Analysis of dopamine concentration and expression of D1 and D2 receptors unravelled splice-variant-specific imbalances in the striatal dopaminergic system: In Go1α-/- mice dopamine concentration and vesicular monoamine uptake were increased compared to wild types. The expression of the D2 receptor was higher in Go1α-/- compared to wild type littermates, but unchanged in Go2α-/- mice. Deletion of both Go1α and Go2α re-established both dopamine and D2 receptor levels comparable to those in the wild-type. Cocaine treatment had no effect on the ratio of D1 receptor to D2 receptor in Go1+2α-/- mutants, but decreased this ratio in Go2α-/- mice. Finally, we observed that the deletion of Go1α led to a threefold higher striatal expression of Go2α. Taken together our data suggest that a balance in the expression of Go1α and Go2α sustains normal motor function. Deletion of either splice variant results in divergent behavioural and molecular alterations in the striatal dopaminergic system. Deletion of both splice variants partially restores the behavioural and molecular changes. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Actividad Motora/genética , Animales , Animales Recién Nacidos , Monoaminas Biogénicas/metabolismo , Cocaína/administración & dosificación , Condicionamiento Operante/fisiología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/ultraestructura , Inhibidores de Captación de Dopamina/administración & dosificación , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Transgénicos , Monoaminooxidasa/metabolismo , Actividad Motora/fisiología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Sinapsis/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
The structure and functions of the extracellular matrix (ECM), its spatial distribution and pericellular association of ECM molecules remain poorly understood. Colocalization of ECM molecules with cell phenotypes through immunohistochemistry can provide crucial insights into their juxtacrine signaling role as well as their structural relevance to tissue architecture. As manual quantification of images introduces intra- and inter-user bias and is cumbersome for high-throughput approaches, we implemented an automated high-throughput method to quantify the spatial distribution and cellular association of one ECM molecule, thrombospondin 1 (TSP1) with two major cell phenotypes, neurons, and astrocytes. The distribution of TSP1 was homogeneous throughout the striatum and cortex along the anterior-posterior axis. TSP1 occupied 8.85% of the striatum and 7.40% in the cortex. TSP1 also associated with 94.58% and 88.45% of neurons in the striatum and cortex. The association with astrocytes was significantly lower at 47.55% and 28.09%. These findings highlight the key role that TSP1 plays in neuron physiology in a healthy brain, but also highlights key regional difference in astrocytes secreting ECM molecules. The semiautomated approach implemented here will improve the throughput and reliability of measuring the distribution and cellular colocalization of ECM molecules.
Asunto(s)
Química Encefálica , Matriz Extracelular/química , Trombospondina 1/análisis , Animales , Astrocitos/química , Astrocitos/citología , Astrocitos/ultraestructura , Encéfalo/citología , Encéfalo/ultraestructura , Cuerpo Estriado/química , Cuerpo Estriado/citología , Cuerpo Estriado/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Masculino , Neuronas/química , Neuronas/citología , Neuronas/ultraestructura , Imagen Óptica/métodos , Ratas Sprague-DawleyRESUMEN
Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.
Asunto(s)
Proteína Huntingtina/genética , Enfermedad de Huntington/patología , Animales , Peso Corporal , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/patología , Sistemas CRISPR-Cas/genética , Corteza Cerebral/patología , Corteza Cerebral/ultraestructura , Cuerpo Estriado/patología , Cuerpo Estriado/ultraestructura , Modelos Animales de Enfermedad , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/mortalidad , Imagen por Resonancia Magnética , Neuronas/metabolismo , Neuronas/patología , Técnicas de Transferencia Nuclear , Tasa de Supervivencia , Porcinos , Repeticiones de TrinucleótidosRESUMEN
Several studies have shown that intrastriatal application of 1-methyl-4-phenylpyridinium (MPP+) produces similar biochemical changes in rat to those seen in Parkinson's disease (PD), such as dopaminergic terminal degeneration and consequent appearance of motor deficits, making the MPP+ lesion a widely used model of parkinsonism in rodents. Previous results from our group have shown a neuroprotective effect of the carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) under different types of stress. In the present study, pretreatment with the intraperitoneal injection of Hc-TeTx in rats prevents the decrease of tyrosine hydroxylase immunoreactivity in the striatum due to injury with MPP+, when applied stereotaxically in the striatum. Similarly, striatal catecholamine contents are restored, as well as the levels of two other dopaminergic markers, the dopamine transporter (DAT) and the vesicular monoamine transporter-2 (VMAT-2). Additionally, uptake studies of [3H]-dopamine and [3H]-MPP+ reveal that DAT action is not affected by Hc-TeTx, discarding a protective effect due to a reduced entry of MPP+ into nerve terminals. Behavioral assessments show that Hc-TeTx pretreatment improves the motor skills (amphetamine-induced rotation, forelimb use, and adjusting steps) of MPP+-treated rats. Our results lead us to consider Hc-TeTx as a potential therapeutic tool in pathologies caused by impairment of dopaminergic innervation in the striatum, as is the case of PD.
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Intoxicación por MPTP/prevención & control , Fármacos Neuroprotectores/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Toxina Tetánica/administración & dosificación , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacocinética , Ácido 3,4-Dihidroxifenilacético/metabolismo , Análisis de Varianza , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/ultraestructura , Modelos Animales de Enfermedad , Dopamina/metabolismo , Dopamina/farmacocinética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Esquema de Medicación , Lateralidad Funcional/efectos de los fármacos , Ácido Homovanílico/metabolismo , Intoxicación por MPTP/patología , Masculino , Movimiento/efectos de los fármacos , Fragmentos de Péptidos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Sustancia Negra/patología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Toxina Tetánica/uso terapéutico , Factores de Tiempo , Tritio/farmacocinética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis of the striatum showed increase in long chain sphingomyelins, SM36:1 and SM38:1 after inhibition of nSMase activity. Quantitative proteomic analysis of striatal lipid raft fraction showed many proteins were downregulated by more than 2-fold after inhibition or antisense knockdown of nSMase; consistent with the notion that nSMase2 activity is important for aggregation or clustering of proteins in lipid rafts. Inhibition or antisense knockdown of nSMase2 in the caudate-putamen resulted in motor deficits in the rotarod and narrow beam tests; as well as decreased acoustic startle and improved prepulse inhibition of the startle reflex. Together, results indicate an important function of nSMase2 in the striatum.
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Cuerpo Estriado/enzimología , Microdominios de Membrana/metabolismo , Actividad Motora , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Cuerpo Estriado/citología , Cuerpo Estriado/ultraestructura , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Inhibición Prepulso , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Reflejo Acústico , Reflejo de Sobresalto , Prueba de Desempeño de Rotación con Aceleración Constante , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/genéticaRESUMEN
Earlier, protective role of curcumin in arsenic-induced dopamine (DA)-D2 receptor dysfunctions in corpus striatum has been demonstrated by us. In continuation to that, the present study is focused to decipher the molecular mechanisms associated with alterations in dopaminergic signaling on arsenic exposure in corpus striatum and assess the protective efficacy of curcumin. Exposure to arsenic (20 mg/kg, body weight p.o. for 28 days) in rats resulted to decrease the expression of presynaptic proteins-tyrosine hydroxylase and VMAT2 while no effect was observed on the expression of DAT in comparison to controls. A significant decrease in the expression of DA-D2 receptors associated with alterations in the expression of PKA, pDARPP32 (Thr 34), and PP1 α was clearly evident on arsenic exposure. Expression of BDNF and pGSK3ß in corpus striatum was found decreased in arsenic-exposed rats. Simultaneous treatment with curcumin (100 mg/kg, body weight p.o. for 28 days) resulted to protect arsenic-induced alterations in the expression of DA-D2 receptors, PKA, pDARPP32, pCREB, and pPP1α. Neuroprotective efficacy of curcumin can possibly be attributed to its antioxidant potential which significantly protected arsenic-induced mitochondrial dysfunctions by modulating the ROS generation and apoptosis. Modulation in the expression of BDNF and pGSK3ß in corpus striatum by curcumin exhibits the importance of neuronal survival pathway in arsenic-induced dopaminergic dysfunctions. Interestingly, curcumin was also found to protect arsenic-induced ultrastructural changes in corpus striatum. The results exhibit that curcumin modulates BDNF/DARPP32/CREB in arsenic-induced alterations in dopaminergic signaling in rat corpus striatum.
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Arsénico/toxicidad , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Cuerpo Estriado/patología , Curcumina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Dopamina/metabolismo , Fármacos Neuroprotectores/farmacología , Animales , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cuerpo Estriado/ultraestructura , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptores Dopaminérgicos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Cerium oxide nanoparticles (CeO2NPs) showed promising effects in neurodegenerative diseases including some animal models of Parkinsonism. However, the implication of CeO2NPs in 6-hydroxydopamine (6-OHDA) induced Parkinsonism remains to be investigated. AIM: This study was designed to assess whether CeO2NPs treatment could alleviate neurobehavioral and neurobiochemical deficits in 6-OHDA induced neurotoxicity in rats. MATERIAL AND METHODS: 50 rats received left intrastriatal (IS) injection of either saline (control, n = 10) or 6-OHDA (n = 40). At the third week post-lesion, motor dysfunction was verified using neurobehavioral tests. Then diseased rats received intraperitoneal injection of 0.1, 0.5 or 1 mg/kg of CeO2NPs or vehicle (10 rats each) for 3 weeks. Rats were subjected to behavioral assessments and then sacrificed for biochemical analyses of the striatum. Striatal dopamine levels, oxidative stress markers including total antioxidant capacity (TAC) and malondialdehyde (MDA), and caspase 3 activity as an apoptotic marker were assessed. RESULTS: Different doses of CeO2NPs variably improved motor dysfunctions induced by 6-OHDA injection in open field, Rota Rod and stepping tests. In addition, the neurobiochemical derangements were almost reversed by the 0.5 mg/kg dose of CeO2NPs, while 0.1 mg/kg dose was not sufficient to alter biochemical measurements in the striatum. Administration of 1 mg/kg of CeO2NPs partially ameliorated striatal dopamine and decreased apoptosis without significant effect on oxidative stress. CONCLUSION: The present study showed a putative therapeutic role of CeO2NPs in the treatment of 6-OHDA-induced Parkinsonian rats, and suggested their antioxidant and antiapoptotic effects as possible mechanisms for elevated striatal dopamine level and improved motor performance.
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Cerio/uso terapéutico , Locomoción/efectos de los fármacos , Nanopartículas/uso terapéutico , Oxidopamina/toxicidad , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/tratamiento farmacológico , Animales , Cerio/farmacología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Cuerpo Estriado/ultraestructura , Relación Dosis-Respuesta a Droga , Locomoción/fisiología , Masculino , Trastornos Parkinsonianos/patología , Ratas , Ratas WistarRESUMEN
In Parkinson's disease, striatal dopamine depletion leads to plastic changes at excitatory corticostriatal and thalamostriatal synapses. The functional consequences of these responses on the expression of behavioral deficits are incompletely understood. In addition, most of the information on striatal synaptic plasticity has been obtained in models with severe striatal dopamine depletion, and less is known regarding changes during early stages of striatal denervation. Using a partial model of nigral cell loss based on intranigral injection of the proteasome inhibitor lactacystin, we demonstrate ultrastructural changes at corticostriatal synapses with a 15% increase in the length and 30% increase in the area of the postsynaptic densities at corticostriatal synapses 1 week following toxin administration. This increase was positively correlated with the performance of lactacystin-lesioned mice on the rotarod task, such that mice with a greater increase in the size of the postsynaptic density performed better on the rotarod task. We therefore propose that lengthening of the postsynaptic density at corticostriatal synapses acts as a compensatory mechanism to maintain motor function under conditions of partial dopamine depletion. The ultrastructure of thalamostriatal synapses remained unchanged following lactacystin administration. Our findings provide novel insights into the mechanisms of synaptic plasticity and behavioral compensation following partial loss of substantia nigra pars compacta neurons, such as those occurring during the early stages of Parkinson's disease.
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Corteza Cerebral/fisiopatología , Cuerpo Estriado/fisiopatología , Plasticidad Neuronal , Enfermedad de Parkinson/fisiopatología , Sinapsis/fisiología , Acetilcisteína/administración & dosificación , Acetilcisteína/análogos & derivados , Animales , Conducta Animal , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/ultraestructura , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/ultraestructura , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiopatología , Vías Nerviosas/ultraestructura , Plasticidad Neuronal/efectos de los fármacos , Enfermedad de Parkinson/patología , Trastornos Parkinsonianos/inducido químicamente , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Densidad Postsináptica/efectos de los fármacos , Densidad Postsináptica/ultraestructura , Prueba de Desempeño de Rotación con Aceleración Constante , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructuraRESUMEN
Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions.