RESUMEN
Cytomegalic neurons, characterized by increased size and a hyperactive mechanistic target of rapamycin complex 1 (mTORC1), are pathognomonic for tuberous sclerosis complex (TSC). To model these neurons, we recently generated a murine Tsc1 conditional knockout model in which Tsc1 deletion in late embryonic radial glia results in neuronal hypertrophy of a subset of isocortical pyramidal neurons. In the current study, we compared the cellular pathology of these cytomegalic neurons to those of the enlarged neurons in human cortical tubers. Neurons from the mice showed unique features, such as cytoplasmic vacuoles associated with Golgi complexes and the ectopic formation of perineuronal nets (PNNs), a feature of inhibitory neurons, rarely present in excitatory cortical neurons. The membranes of these vacuoles were enriched for the plasma membrane proteins CD44, KCC2, and Na+/K+ ATPase, suggesting deficits in Golgi membrane trafficking. These aberrant features in the mouse appeared only after the onset of seizures, probably due to the prolonged seizure activity in the context of constitutive mTORC1 activation. Similar PNNs and cytoplasmic vacuoles were present in the cytomegalic neurons of human cortical tubers. Our findings reveal novel pathological features of Golgi complexes and PNNs in the cytomegalic neurons in TSC.
RESUMEN
In the mammalian isocortex, CD44, a cell surface receptor for extracellular matrix molecules, is present in pial-based and fibrous astrocytes of white matter but not in protoplasmic astrocytes. In the hominid isocortex, CD44+ astrocytes comprise the subpial "interlaminar" astrocytes, sending long processes into the cortex. The hippocampus also contains similar astrocytes. We have examined all levels of the human central nervous system and found CD44+ astrocytes in every region. Astrocytes in white matter and astrocytes that interact with large blood vessels but not with capillaries in gray matter are CD44+, the latter extending long processes into the parenchyma. Motor neurons in the brainstem and spinal cord, such as oculomotor, facial, hypoglossal, and in the anterior horn of the spinal cord, are surrounded by CD44+ processes, contrasting with neurons in the cortex, basal ganglia, and thalamus. We found CD44+ processes that intercalate between ependymal cells to reach the ventricle. We also found CD44+ astrocytes in the molecular layer of the cerebellar cortex. Protoplasmic astrocytes, which do not normally contain CD44, acquire it in pathologies like hypoxia and seizures. The pervasive and inducible expression of CD44 in astrocytes is a novel finding that lays the foundations for functional studies into the significance of CD44 in health and disease.
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Receptores de Hialuranos , Hipoxia , Convulsiones , Animales , Humanos , Astrocitos , Receptores de Hialuranos/metabolismo , Hipoxia/metabolismo , Neocórtex , Convulsiones/metabolismo , Sustancia BlancaRESUMEN
Tuberous sclerosis complex (TSC) is a developmental disorder associated with epilepsy, autism, and cognitive impairment. Despite inactivating mutations in the TSC1 or TSC2 genes and hyperactive mechanistic target of rapamycin (mTOR) signaling, the mechanisms underlying TSC-associated neurological symptoms remain incompletely understood. Here we generate a Tsc1 conditional knockout (CKO) mouse model in which Tsc1 inactivation in late embryonic radial glia causes social and cognitive impairment and spontaneous seizures. Tsc1 depletion occurs in a subset of layer 2/3 cortical pyramidal neurons, leading to development of cytomegalic pyramidal neurons (CPNs) that mimic dysplastic neurons in human TSC, featuring abnormal dendritic and axonal overgrowth, enhanced glutamatergic synaptic transmission, and increased susceptibility to seizure-like activities. We provide evidence that enhanced synaptic excitation in CPNs contributes to cortical hyperexcitability and epileptogenesis. In contrast, astrocytic regulation of synapse formation and synaptic transmission remains unchanged after late embryonic radial glial Tsc1 inactivation, and astrogliosis evolves secondary to seizures.
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Esclerosis Tuberosa , Animales , Humanos , Ratones , Células Piramidales , Convulsiones , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
While several studies have attributed the development of tumour-associated seizures to an excitatory-inhibitory imbalance, we have yet to resolve the spatiotemporal interplay between different types of neuron in glioma-infiltrated cortex. Herein, we combined methods for single unit analysis of microelectrode array recordings with wide-field optical mapping of Thy1-GCaMP pyramidal cells in an ex vivo acute slice model of diffusely infiltrating glioma. This enabled simultaneous tracking of individual neurons from both excitatory and inhibitory populations throughout seizure-like events. Moreover, our approach allowed for observation of how the crosstalk between these neurons varied spatially, as we recorded across an extended region of glioma-infiltrated cortex. In tumour-bearing slices, we observed marked alterations in single units classified as putative fast-spiking interneurons, including reduced firing, activity concentrated within excitatory bursts and deficits in local inhibition. These results were correlated with increases in overall excitability. Mechanistic perturbation of this system with the mTOR inhibitor AZD8055 revealed increased firing of putative fast-spiking interneurons and restoration of local inhibition, with concomitant decreases in overall excitability. Altogether, our findings suggest that diffusely infiltrating glioma affect the interplay between excitatory and inhibitory neuronal populations in a reversible manner, highlighting a prominent role for functional mechanisms linked to mTOR activation.
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Glioma , Células Piramidales , Humanos , Potenciales de Acción/fisiología , Células Piramidales/fisiología , Neuronas/fisiología , Convulsiones , Serina-Treonina Quinasas TORRESUMEN
BACKGROUND: Advanced glycation end products (AGEs) are an important risk factor for the development of cognitive decline in aging and late-onset neurodegenerative diseases including Alzheimer's disease. However, whether and how dietary AGEs exacerbate cognitive impairment and brain mitochondrial dysfunction in the aging process remains largely unknown. OBJECTIVE: We investigated the direct effects of dietary AGEs on AGE adducts accumulation, mitochondrial function, and cognitive performance in mice. METHODS: Mice were fed the AGE+ diet or AGE- diet. We examined levels of AGE adducts in serum and cerebral cortexes by immunodetection and immunohistochemistry, determined levels of reactive oxygen species by biochemical analysis, detected enzyme activity associated with mitochondrial respiratory chain complexes I & IV and ATP levels, and assessed learning and memory ability by Morris Water Maze and nesting behavior. RESULTS: Levels of AGE adducts (MG-H1 and CEL) were robustly increased in the serum and brain of AGE+ diet fed mice compared to the AGE- group. Furthermore, greatly elevated levels of reactive oxygen species, decreased activities of mitochondrial respiratory chain complexes I & IV, reduced ATP levels, and impaired learning and memory were evident in AGE+ diet fed mice compared to the AGE- group. CONCLUSION: These results indicate that dietary AGEs are important sources of AGE accumulation in vivo, resulting in mitochondrial dysfunction, impairment of energy metabolism, and subsequent cognitive impairment. Thus, reducing AGEs intake to lower accumulation of AGEs could hold therapeutic potential for the prevention and treatment of AGEs-induced mitochondrial dysfunction linked to cognitive decline.
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Cognición/fisiología , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/metabolismo , Dieta/efectos adversos , Productos Finales de Glicación Avanzada/toxicidad , Mitocondrias/metabolismo , Animales , Cognición/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Femenino , Productos Finales de Glicación Avanzada/administración & dosificación , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Although abnormal mitosis with disarranged metaphase chromosomes or many micronuclei in astrocytes (named "Alzheimer I type astrocytes" and later "Creutzfeldt-Peters cells") have been known for nearly 100 years, the origin and mechanisms of this pathology remain elusive. In experimental brain insults in rats, we show that abnormal mitoses that are not followed by cytokinesis are typical for reactive astrocytes. The pathology originates due to the inability of the cells to form normal mitotic spindles with subsequent metaphase chromosome congression, which, in turn may be due to shape constraints aggravated by cellular enlargement and to the accumulation of large amounts of cytosolic proteins. Many astrocytes escape from arrested mitosis by producing micronuclei. These polyploid astrocytes can survive for long periods of time and enter into new cell cycles.
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Astrocitos/patología , Gliosis/patología , Mitosis/fisiología , Animales , Encefalopatías/patología , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Huntington Disease (HD) is an inherited movement disorder caused by expanded CAG repeats in the Huntingtin gene. We have used single nucleus RNASeq (snRNASeq) to uncover cellular phenotypes that change in the disease, investigating single cell gene expression in cingulate cortex of patients with HD and comparing the gene expression to that of patients with no neurological disease. In this study, we focused on astrocytes, although we found significant gene expression differences in neurons, oligodendrocytes, and microglia as well. In particular, the gene expression profiles of astrocytes in HD showed multiple signatures, varying in phenotype from cells that had markedly upregulated metallothionein and heat shock genes, but had not completely lost the expression of genes associated with normal protoplasmic astrocytes, to astrocytes that had substantially upregulated glial fibrillary acidic protein (GFAP) and had lost expression of many normal protoplasmic astrocyte genes as well as metallothionein genes. When compared to astrocytes in control samples, astrocyte signatures in HD also showed downregulated expression of a number of genes, including several associated with protoplasmic astrocyte function and lipid synthesis. Thus, HD astrocytes appeared in variable transcriptional phenotypes, and could be divided into several different "states", defined by patterns of gene expression. Ultimately, this study begins to fill the knowledge gap of single cell gene expression in HD and provide a more detailed understanding of the variation in changes in gene expression during astrocyte "reactions" to the disease.
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Astrocitos/metabolismo , Expresión Génica , Giro del Cíngulo/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARN , Análisis de la Célula IndividualRESUMEN
The purpose of this study is to develop a platform in which the cellular and molecular underpinnings of chronic focal neocortical lesional epilepsy can be explored and use it to characterize seizure-like events (SLEs) in an ex vivo model of infiltrating high-grade glioma. Microelectrode arrays were used to study electrophysiologic changes in ex vivo acute brain slices from a PTEN/p53 deleted, PDGF-B driven mouse model of high-grade glioma. Electrode locations were co-registered to the underlying histology to ascertain the influence of the varying histologic landscape on the observed electrophysiologic changes. Peritumoral, infiltrated, and tumor sites were sampled in tumor-bearing slices. Following the addition of zero Mg2+ solution, all three histologic regions in tumor-bearing slices showed significantly greater increases in firing rates when compared to the control sites. Tumor-bearing slices demonstrated increased proclivity for SLEs, with 40 events in tumor-bearing slices and 5 events in control slices (p-valueâ¯=â¯.0105). Observed SLEs were characterized by either low voltage fast (LVF) onset patterns or short bursts of repetitive widespread, high amplitude low frequency discharges. Seizure foci comprised areas from all three histologic regions. The onset electrode was found to be at the infiltrated margin in 50% of cases and in the peritumoral region in 36.9% of cases. These findings reveal a landscape of histopathologic and electrophysiologic alterations associated with ictogenesis and spread of tumor-associated seizures.
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Neoplasias Encefálicas/fisiopatología , Encéfalo/fisiopatología , Glioma/fisiopatología , Neuronas/fisiología , Convulsiones/fisiopatología , Potenciales de Acción , Animales , Neoplasias Encefálicas/complicaciones , Modelos Animales de Enfermedad , Glioma/complicaciones , Ratones Transgénicos , Microelectrodos , Convulsiones/complicacionesRESUMEN
Increasing evidence suggests that necroptosis, a form of programmed cell death (PCD), contributes to neurodegeneration in several disorders, including ALS. Supporting this view, investigations in both in vitro and in vivo models of ALS have implicated key molecular determinants of necroptosis in the death of spinal motor neurons (MNs). Consistent with a pathogenic role of necroptosis in ALS, we showed increased mRNA levels for the three main necroptosis effectors Ripk1, Ripk3, and Mlkl in the spinal cord of mutant superoxide dismutase-1 (SOD1G93A) transgenic mice (Tg), an established model of ALS. In addition, protein levels of receptor-interacting protein kinase 1 (RIPK1; but not of RIPK3, MLKL or activated MLKL) were elevated in spinal cord extracts from these Tg SOD1G93A mice. In postmortem motor cortex samples from sporadic and familial ALS patients, no change in protein levels of RIPK1 were detected. Silencing of Ripk3 in cultured MNs protected them from toxicity associated with SOD1G93A astrocytes. However, constitutive deletion of Ripk3 in Tg SOD1G93A mice failed to provide behavioral or neuropathological improvement, demonstrating no similar benefit of Ripk3 silencing in vivo. Lastly, we detected no genotype-specific myelin decompaction, proposed to be a proxy of necroptosis in ALS, in either Tg SOD1G93A or Optineurin knock-out mice, another ALS mouse model. These findings argue against a role for RIPK3 in Tg SOD1G93A-induced neurodegeneration and call for further preclinical investigations to determine if necroptosis plays a critical role in the pathogenesis of ALS.
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Muerte Celular/fisiología , Neuronas Motoras/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Proteínas de Ciclo Celular , Línea Celular , Técnicas de Cocultivo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Femenino , Humanos , Masculino , Proteínas de Transporte de Membrana , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Corteza Motora/metabolismo , Corteza Motora/patología , Neuronas Motoras/patología , Cultivo Primario de Células , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Mitochondrial and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Accumulation of amyloid beta-peptide (Aß) in mitochondria, particularly in synaptic mitochondria, potentiates and amplifies synaptic injury and disruption of synaptic transmission, leading to synaptic dysfunction and ultimately to synaptic failure. Thus, determination of the presence and levels of Aß in synaptic mitochondria associated with amyloid pathology is important for studying mitochondrial amyloid pathology. Here, we present a detailed methodology for the isolation of synaptic mitochondria from brain tissues and the determination of Aß levels in the isolated mitochondria as well as ultrastructural localization of synaptic mitochondrial Aß. These methods have been used successfully for the identification and characterization of Aß accumulation in synaptic mitochondria from mouse brains derived from transgenic AD mouse model. Additionally, we comprehensively discuss the sample preparation, experimental details, our unique procedures, optimization of parameters, and troubleshooting.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/aislamiento & purificación , Encéfalo/citología , Mitocondrias/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Centrifugación por Gradiente de Densidad , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Microscopía Electrónica , Mitocondrias/patología , Mitocondrias/ultraestructura , Sinapsis/metabolismo , Sinapsis/patología , Sinapsis/ultraestructuraRESUMEN
Alexander Disease (AxD) is a degenerative disorder caused by mutations in the GFAP gene, which encodes the major intermediate filament of astrocytes. As other cells in the CNS do not express GFAP, AxD is a primary astrocyte disease. Astrocytes acquire a large number of pathological features, including changes in morphology, the loss or diminution of a number of critical astrocyte functions and the activation of cell stress and inflammatory pathways. AxD is also characterized by white matter degeneration, a pathology that has led it to be included in the "leukodystrophies." Furthermore, variable degrees of neuronal loss take place. Thus, the astrocyte pathology triggers alterations in other cell types. Here, we will review the neuropathology of AxD and discuss how a disease of astrocytes can lead to severe pathologies in non-astrocytic cells. Our knowledge of the pathophysiology of AxD will also lead to a better understanding of how astrocytes interact with other CNS cells and how astrocytes in the gliosis that accompanies many neurological disorders can damage the function and survival of other cells.
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Enfermedad de Alexander/patología , Enfermedad de Alexander/fisiopatología , Astrocitos/patología , Astrocitos/fisiología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones Transgénicos , Neuronas/patología , Oligodendroglía/patologíaRESUMEN
Rosenthal fibers (RFs) are cytoplasmic, proteinaceous aggregates. They are the pathognomonic feature of the astrocyte pathology in Alexander Disease (AxD), a neurodegenerative disorder caused by heterozygous mutations in the GFAP gene, encoding glial fibrillary acidic protein (GFAP). Although RFs have been known for many years their origin and significance remain elusive issues. We have used mouse models of AxD based on the overexpression of human GFAP (transgenic, TG) and a point mutation in mouse GFAP (knock-in, KI) to examine the formation of RFs and to find astrocyte changes that correlate with the appearance of RFs. We found RFs of various sizes and shapes. The smallest ones appear as granular depositions on intermediate filaments. These contain GFAP and the small heat shock protein, alphaB-crystallin. Their aggregation appears to give rise to large RFs. The appearance of new RFs and the growth of previously formed RFs occur over time. We determined that DAPI is a reliable marker of RFs and in parallel with Fluoro-Jade B (FJB) staining defined a high variability in the appearance of RFs, even in neighboring astrocytes. Although many astrocytes in AxD with increased levels of GFAP and with or without RFs change their phenotype, only some cells with large numbers of RFs show a profound reconstruction of cellular processes, with a loss of fine distal processes and the appearance of large, lobulated nuclei, likely due to arrested mitosis. We conclude that 1) RFs appear to originate as small, osmiophilic masses containing both GFAP and alphaB-crystallin deposited on bundles of intermediate filaments. 2) RFs continue to form within AxD astrocytes over time. 3) DAPI is a reliable marker for RFs and can be used with immunolabeling. 4) RFs appear to interfere with the successful completion of astrocyte mitosis and cell division.
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Enfermedad de Alexander/patología , Astrocitos/patología , Citoplasma/patología , Proteína Ácida Fibrilar de la Glía/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alexander/metabolismo , Animales , Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Preescolar , Citoplasma/metabolismo , Femenino , Colorantes Fluorescentes , Proteína Ácida Fibrilar de la Glía/genética , Humanos , Inmunohistoquímica , Indoles , Lactante , Masculino , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitosis/fisiología , Mutación Puntual , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.
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Enfermedad de Alexander , Agregado de Proteínas , Proteoma/análisis , Animales , Astrocitos , Ciclina D2/análisis , ARN Helicasas DEAD-box/análisis , Proteínas de Unión al GTP/análisis , Humanos , Inmunohistoquímica , Ratones , Proteínas de Neoplasias/análisis , Neuropéptidos/análisis , Agregación Patológica de Proteínas , ARN Helicasas/análisis , Receptores de Cinasa C Activada , Receptores de Superficie Celular/análisisRESUMEN
Accumulation of amyloid-ß (Aß) in synaptic mitochondria is associated with mitochondrial and synaptic injury. The underlying mechanisms and strategies to eliminate Aß and rescue mitochondrial and synaptic defects remain elusive. Presequence protease (PreP), a mitochondrial peptidasome, is a novel mitochondrial Aß degrading enzyme. Here, we demonstrate for the first time that increased expression of active human PreP in cortical neurons attenuates Alzheimer disease's (AD)-like mitochondrial amyloid pathology and synaptic mitochondrial dysfunction, and suppresses mitochondrial oxidative stress. Notably, PreP-overexpressed AD mice show significant reduction in the production of proinflammatory mediators. Accordingly, increased neuronal PreP expression improves learning and memory and synaptic function in vivo AD mice, and alleviates Aß-mediated reduction of long-term potentiation (LTP). Our results provide in vivo evidence that PreP may play an important role in maintaining mitochondrial integrity and function by clearance and degradation of mitochondrial Aß along with the improvement in synaptic and behavioral function in AD mouse model. Thus, enhancing PreP activity/expression may be a new therapeutic avenue for treatment of AD.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Serina Endopeptidasas/metabolismo , Sinapsis/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Conducta Animal , Células Cultivadas , Cognición , Modelos Animales de Enfermedad , Expresión Génica , Mediadores de Inflamación/metabolismo , Ratones , Ratones Transgénicos , Estrés Oxidativo , Proteolisis , Serina Endopeptidasas/genéticaRESUMEN
INTRODUCTION: Recent evidence has implicated perituberal, MRI-normal brain tissue as a possible source of seizures in tuberous sclerosis complex (TSC). Data on aberrant structural features in this area that may predispose to the initiation or progression of seizures are very limited. We used immunohistochemistry and confocal microscopy to compare epileptogenic, perituberal, MRI-normal tissue with cortical tubers. RESULTS: In every sample of epileptogenic, perituberal tissue, we found many abnormal cell types, including giant cells and cytomegalic neurons. The majority of giant cells were surrounded by morphologically abnormal astrocytes with long processes typical of interlaminar astrocytes. Perituberal giant cells and astrocytes together formed characteristic "microtubers". A parallel analysis of tubers showed that many contained astrocytes with features of both protoplasmic and gliotic cells. CONCLUSIONS: Microtubers represent a novel pathognomonic finding in TSC and may represent an elementary unit of cortical tubers. Microtubers and cytomegalic neurons in perituberal parenchyma may serve as the source of seizures in TSC and provide potential targets for therapeutic and surgical interventions in TSC.
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Astrocitos/patología , Encéfalo/patología , Gliosis/patología , Neuronas/patología , Convulsiones/patología , Esclerosis Tuberosa/patología , Adolescente , Encéfalo/anomalías , Corteza Cerebral/anomalías , Corteza Cerebral/patología , Niño , Preescolar , Femenino , Gliosis/complicaciones , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Microscopía ConfocalRESUMEN
Diabetes has adverse effects on the brain, especially the hippocampus, which is particularly susceptible to synaptic injury and cognitive dysfunction. The underlying mechanisms and strategies to rescue such injury and dysfunction are not well understood. Using a mouse model of type 2 diabetes (db/db mice) and a human neuronal cell line treated with high concentration of glucose, we demonstrate aberrant mitochondrial morphology, reduced ATP production, and impaired activity of complex I. These mitochondrial abnormalities are induced by imbalanced mitochondrial fusion and fission via a glycogen synthase kinase 3ß (GSK3ß)/dynamin-related protein-1 (Drp1)-dependent mechanism. Modulation of the Drp1 pathway or inhibition of GSK3ß activity restores hippocampal long-term potentiation that is impaired in db/db mice. Our results point to a novel role for mitochondria in diabetes-induced synaptic impairment. Exploration of the mechanisms behind diabetes-induced synaptic deficit may provide a novel treatment for mitochondrial and synaptic injury in patients with diabetes.
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Dinaminas/metabolismo , Mitocondrias/patología , Plasticidad Neuronal/fisiología , Animales , Línea Celular , Dinaminas/genética , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Ratones Endogámicos NOD , Mitocondrias/metabolismo , Neuronas/metabolismoRESUMEN
Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.
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Trastorno Autístico/patología , Autofagia/fisiología , Espinas Dendríticas/genética , Neuronas/patología , Sinapsis/patología , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Factores de Edad , Animales , Trastorno Autístico/genética , Autofagia/efectos de los fármacos , Niño , Preescolar , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Femenino , Humanos , Inmunosupresores/farmacología , Masculino , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Sirolimus/farmacología , Sinapsis/efectos de los fármacos , Lóbulo Temporal/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Adulto JovenRESUMEN
To examine the diversity of astrocytes in the human brain, we immunostained surgical specimens of temporal cortex and hippocampus and autopsy brains for CD44, a plasma membrane protein and extracellular matrix receptor. CD44 antibodies outline the details of astrocyte morphology to a degree not possible with glial fibrillary acidic protein (GFAP) antibodies. CD44+ astrocytes could be subdivided into two groups. First, CD44+ astrocytes with long processes were consistently found in the subpial area ("interlaminar" astrocytes), the deep isocortical layers, and the hippocampus. Many of these processes ended on blood vessels. Some were also found adjacent to large blood vessels, from which they extended long processes. We observed these CD44+, long-process astrocytes in every brain we examined, from fetal to adult. These astrocytes generally displayed high immunostaining for GFAP, S100ß, and CD44, but low immunostaining for glutamine synthetase, excitatory amino-acid transporter 1 (EAAT1), and EAAT2. Aquaporin 4 (AQP4) appeared distributed all over the cell bodies and processes of the CD44+ astrocytes, while, in contrast, AQP4 localized to perivascular end feet in the CD44- protoplasmic astrocytes. Second, there were CD44+ astrocytes without long processes in the cortex. These were not present during gestation or at birth, and in adult brains varied substantially in number, shape, and immunohistochemical phenotype. Many of these displayed a "mixed" morphological and immunocytochemical phenotype between protoplasmic and fibrous astrocytes. We conclude that the diversity of astrocyte populations in the isocortex and archicortex in the human brain reflects both intrinsic and acquired phenotypes, the latter perhaps representing a shift from CD44- "protoplasmic" to CD44+ "fibrous"-like astrocytes.
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Astrocitos/fisiología , Corteza Cerebral/fisiología , Heterogeneidad Genética , Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Fenotipo , Adolescente , Adulto , Anciano , Encéfalo/citología , Encéfalo/fisiología , Corteza Cerebral/citología , Niño , Preescolar , Femenino , Hipocampo/citología , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of Alzheimer's disease (AD). Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in amyloid beta (Aß)-instigated mitochondrial dysfunction. Blockade of CypD prevents Aß-induced mitochondrial malfunction and the consequent cognitive impairments. Here, we showed the elimination of reactive oxygen species (ROS) by antioxidants probucol or superoxide dismutase (SOD)/catalase blocks Aß-mediated inactivation of protein kinase A (PKA)/cAMP regulatory-element-binding (CREB) signal transduction pathway and loss of synapse, suggesting the detrimental effects of oxidative stress on neuronal PKA/CREB activity. Notably, neurons lacking CypD significantly attenuate Aß-induced ROS. Consequently, CypD-deficient neurons are resistant to Aß-disrupted PKA/CREB signaling by increased PKA activity, phosphorylation of PKA catalytic subunit (PKA C), and CREB. In parallel, lack of CypD protects neurons from Aß-induced loss of synapses and synaptic dysfunction. Furthermore, compared to the mAPP mice, CypD-deficient mAPP mice reveal less inactivation of PKA-CREB activity and increased synaptic density, attenuate abnormalities in dendritic spine maturation, and improve spontaneous synaptic activity. These findings provide new insights into a mechanism in the crosstalk between the CypD-dependent mitochondrial oxidative stress and signaling cascade, leading to synaptic injury, functioning through the PKA/CREB signal transduction pathway.
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Péptidos beta-Amiloides/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclofilinas/metabolismo , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Antioxidantes/farmacología , Catalasa/farmacología , Células Cultivadas , Peptidil-Prolil Isomerasa F , Ciclofilinas/genética , Immunoblotting , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Estrés Oxidativo/efectos de los fármacos , Técnicas de Placa-Clamp , Probucol/farmacología , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/farmacologíaRESUMEN
Alexander Disease (AxD) is a primary disorder of astrocytes, caused by heterozygous mutations in GFAP, which encodes the major astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP). Astrocytes in AxD display hypertrophy, massive increases in GFAP, and the accumulation of Rosenthal fibers, cytoplasmic protein inclusions containing GFAP, and small heat shock proteins. To study the effects of GFAP mutations on astrocyte morphology and physiology, we have examined hippocampal astrocytes in three mouse models of AxD, a transgenic line (GFAP(Tg)) in which the normal human GFAP is expressed in several copies, a knock-in line (Gfap(+/R236H)) in which one of the Gfap genes bears an R236H mutation, and a mouse derived from the mating of these two lines (GFAP(Tg); Gfap(+/R236H)). We report changes in astrocyte phenotype in all lines, with the most severe in the GFAP(Tg);Gfap(+/R236H), resulting in the conversion of protoplasmic astrocytes to cells that have lost their bushy-like morphology because of a reduction of distal fine processes, and become multinucleated and hypertrophic. Astrocytes activate the mTOR cascade, acquire CD44, and lose GLT-1. The altered astrocytes display a microheterogeneity in phenotypes, even neighboring cells. Astrocytes also show diminished glutamate transporter current, are significantly depolarized, and not coupled to adjacent astrocytes. Thus, the accumulation of GFAP in the AxD mouse astrocytes initiates a conversion of normal, protoplasmic astrocytes to astrocytes that display severely "reactive" characteristics, many of which may be detrimental to neighboring neurons and oligodendrocytes.