RESUMEN
There is compelling evidence that oligodendrocyte apoptosis, in response to CNS inflammation, contributes significantly to the development of the demyelinating disorder multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Therefore, approaches designed to protect oligodendrocytes would likely have therapeutic value. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum (ER) stress increases cell survival under various cytotoxic conditions. Moreover, there is evidence that PERK signaling is activated in oligodendrocytes within demyelinating lesions in multiple sclerosis and EAE. Our previous study demonstrated that CNS delivery of the inflammatory cytokine interferon-γ before EAE onset protected mice against EAE, and this protection was dependent on PERK signaling. In our current study, we sought to elucidate the role of PERK signaling in oligodendrocytes during EAE. We generated transgenic mice that allow for temporally controlled activation of PERK signaling, in the absence of ER stress, specifically in oligodendrocytes. We demonstrated that persistent activation of PERK signaling was not deleterious to oligodendrocyte viability or the myelin of adult animals. Importantly, we found that enhanced activation of PERK signaling specifically in oligodendrocytes significantly attenuated EAE disease severity, which was associated with reduced oligodendrocyte apoptosis, demyelination, and axonal degeneration. This effect was not the result of an altered degree of the inflammatory response in EAE mice. Our results provide direct evidence that activation of PERK signaling in oligodendrocytes is cytoprotective, protecting mice against EAE.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/terapia , Oligodendroglía/fisiología , Transducción de Señal/fisiología , eIF-2 Quinasa/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/patología , Bromodesoxiuridina/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inmunosupresores/farmacología , Etiquetado Corte-Fin in Situ , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proteína Básica de Mielina/metabolismo , Proteína Proteolipídica de la Mielina/genética , Infiltración Neutrófila/efectos de los fármacos , Infiltración Neutrófila/genética , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/ultraestructura , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Factores de Tiempo , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/genéticaRESUMEN
Tuberous sclerosis complex (TSC) is caused by heterozygous mutations in either the TSC1 (hamartin) or the TSC2 (tuberin) gene. Among the multisystemic manifestations of TSC, the neurodevelopmental features cause the most morbidity and mortality, presenting a considerable clinical challenge. Hamartin and tuberin form a heterodimer that inhibits the mammalian target of rapamycin complex 1 (mTORC1) kinase, a major cellular regulator of protein translation, cell growth and proliferation. Hyperactivated mTORC1 signaling, an important feature of TSC, has prompted a number of preclinical and clinical studies with the mTORC1 inhibitor rapamycin. Equally exciting is the prospect of treating TSC in the perinatal period to block the progression of brain pathologies and allow normal brain development to proceed. We hypothesized that low-dose rapamycin given prenatally and/or postnatally in a well-established neuroglial (Tsc2-hGFAP) model of TSC would rescue brain developmental defects. We developed three treatment regimens with low-dose intraperitoneal rapamycin (0.1 mg/kg): prenatal, postnatal and pre/postnatal (combined). Combined rapamycin treatment resulted in almost complete histologic rescue, with a well-organized cortex and hippocampus almost identical to control animals. Other treatment regimens yielded less complete, but significant improvements in brain histology. To assess how treatment regimens affected cognitive function, we continued rapamycin treatment after weaning and performed behavioral testing. Surprisingly, the animals treated with the combined therapy did not perform as well as postnatally-treated animals in learning and memory tasks. These results have important translational implications in the optimization of the timing and dosage of rapamycin treatment in TSC affected children.
Asunto(s)
Encéfalo/efectos de los fármacos , Cognición/efectos de los fármacos , Modelos Animales de Enfermedad , Neuroglía/efectos de los fármacos , Sirolimus/administración & dosificación , Esclerosis Tuberosa/tratamiento farmacológico , Esclerosis Tuberosa/psicología , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Humanos , Aprendizaje/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Neuroglía/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/embriología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.
RESUMEN
During development, brain regions follow encoded growth trajectories. Compared to classical brain growth charts, high-definition growth charts could quantify regional volumetric growth and constituent cell types, improving our understanding of typical and pathological brain development. Here, we create high-resolution 3D atlases of the early postnatal mouse brain, using Allen CCFv3 anatomical labels, at postnatal days (P) 4, 6, 8, 10, 12, and 14, and determine the volumetric growth of different brain regions. We utilize 11 different cell type-specific transgenic animals to validate and refine anatomical labels. Moreover, we reveal region-specific density changes in γ-aminobutyric acid-producing (GABAergic), cortical layer-specific cell types, and microglia as key players in shaping early postnatal brain development. We find contrasting changes in GABAergic neuronal densities between cortical and striatal areas, stabilizing at P12. Moreover, somatostatin-expressing cortical interneurons undergo regionally distinct density reductions, while vasoactive intestinal peptide-expressing interneurons show no significant changes. Remarkably, microglia transition from high density in white matter tracks to gray matter at P10, and show selective density increases in sensory processing areas that correlate with the emergence of individual sensory modalities. Lastly, we create an open-access web-visualization (https://kimlab.io/brain-map/epDevAtlas) for cell-type growth charts and developmental atlases for all postnatal time points.
RESUMEN
Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.
RESUMEN
Tuberous sclerosis complex (TSC) is a neurogenetic disorder that often causes brain abnormalities leading to epilepsy, developmental delay, and autism. TSC is caused by inactivating mutations in either of the genes encoding the proteins hamartin (TSC1) and tuberin (TSC2). These proteins form a heterodimer that inhibits the mammalian target of rapamycin complex 1 (mTORC1) pathway, controlling translation and cell growth. Loss of either protein results in dysregulated mTORC1 activation, an important aspect of TSC pathogenesis. About thirty percent of TSC patients have cerebellar pathology that is poorly understood. To investigate the effects of TSC on the cerebellum, we created a mouse model in which the Tsc2 gene was selectively deleted from Purkinje cells starting at postnatal day 6 (P6). The loss of Tsc2 caused a progressive increase in Purkinje cell size and subsequent death from apoptosis. Purkinje cell loss was predominantly cell type specific and associated with motor deficits. Immunohistochemical analysis showed that both endoplasmic reticulum (ER) and oxidative stress were increased in Tsc2-null Purkinje cells. The cell death and ER stress phenotypes were rescued by treatment with the mTORC1 inhibitor rapamycin. To assess whether the murine Purkinje cell loss has a correlate to the human TSC, we analyzed postmortem cerebellum samples from TSC patients and detected Purkinje cell loss in half of the samples. Our results establish a critical role for the TSC complex in Purkinje cell survival by regulating ER and oxidative stress and reveal a novel aspect of TSC neuropathology.
Asunto(s)
Células de Purkinje/patología , Esclerosis Tuberosa/patología , Proteínas Supresoras de Tumor/deficiencia , Adulto , Animales , Apoptosis/fisiología , Tamaño de la Célula , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Femenino , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Mutantes , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Células de Purkinje/metabolismo , Esclerosis Tuberosa/tratamiento farmacológico , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Tuberous sclerosis complex (TSC) is an autosomal dominant, tumor predisposition disorder characterized by significant neurodevelopmental brain lesions, such as tubers and subependymal nodules. The neuropathology of TSC is often associated with seizures and intellectual disability. To learn about the developmental perturbations that lead to these brain lesions, we created a mouse model that selectively deletes the Tsc2 gene from radial glial progenitor cells in the developing cerebral cortex and hippocampus. These Tsc2 mutant mice were severely runted, developed post-natal megalencephaly and died between 3 and 4 weeks of age. Analysis of brain pathology demonstrated cortical and hippocampal lamination defects, hippocampal heterotopias, enlarged dysplastic neurons and glia, abnormal myelination and an astrocytosis. These histologic abnormalities were accompanied by activation of the mTORC1 pathway as assessed by increased phosphorylated S6 in brain lysates and tissue sections. Developmental analysis demonstrated that loss of Tsc2 increased the subventricular Tbr2-positive basal cell progenitor pool at the expense of early born Tbr1-positive post-mitotic neurons. These results establish the novel concept that loss of function of Tsc2 in radial glial progenitors is one initiating event in the development of TSC brain lesions as well as underscore the importance of Tsc2 in the regulation of neural progenitor pools. Given the similarities between the mouse and the human TSC lesions, this model will be useful in further understanding TSC brain pathophysiology, testing potential therapies and identifying other genetic pathways that are altered in TSC.
Asunto(s)
Encéfalo/patología , Neuroglía/metabolismo , Neuroglía/patología , Esclerosis Tuberosa/patología , Proteínas Supresoras de Tumor/deficiencia , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Integrasas/metabolismo , Malformaciones del Desarrollo Cortical del Grupo II/metabolismo , Malformaciones del Desarrollo Cortical del Grupo II/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Neuronas/metabolismo , Neuronas/patología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Proteínas , Células Madre/metabolismo , Células Madre/patología , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismo , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.
Asunto(s)
Encéfalo/citología , Neuronas/clasificación , Neuronas/citología , Análisis de la Célula Individual/métodos , Animales , Conjuntos de Datos como Asunto , Dependovirus , Humanos , Ratones , Ratones TransgénicosRESUMEN
Multiple sclerosis is a chronic demyelinating autoimmune disease of the CNS with no known cure. Although 12 immunomodulatory therapies exist, they have only modest effects on disease progression. The field has therefore focused on the development of alternative treatment strategies, such as enhancement of remyelination and CNS repair. Progress has been made on a third, complementary treatment approach that aims to protect oligodendrocytes-and the myelin they generate and maintain-from inflammation-mediated death by enhancing the integrated stress response. Studies in cells and in mouse models of multiple sclerosis have shown that this innate protective pathway, which maintains proteostasis, can be harnessed effectively to protect oligodendrocytes and myelin during inflammation. With one drug already in clinical development for patients with multiple sclerosis, and several potential therapies under investigation, modulation of the integrated stress response might become an important component of strategies to halt the progression of the disease.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Oligodendroglía/metabolismo , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
Oligodendrocyte death contributes to the pathogenesis of the inflammatory demyelinating disease multiple sclerosis (MS). Nevertheless, current MS therapies are mainly immunomodulatory and have demonstrated limited ability to inhibit MS progression. Protection of oligodendrocytes is therefore a desirable strategy for alleviating disease. Here we demonstrate that enhancement of the integrated stress response using the FDA-approved drug guanabenz increases oligodendrocyte survival in culture and prevents hypomyelination in cerebellar explants in the presence of interferon-γ, a pro-inflammatory cytokine implicated in MS pathogenesis. In vivo, guanabenz treatment protects against oligodendrocyte loss caused by CNS-specific expression of interferon-γ. In a mouse model of MS, experimental autoimmune encephalomyelitis, guanabenz alleviates clinical symptoms, which correlates with increased oligodendrocyte survival and diminished CNS CD4+ T cell accumulation. Moreover, guanabenz ameliorates relapse in relapsing-remitting experimental autoimmune encephalomyelitis. Our results provide support for a MS therapy that enhances the integrated stress response to protect oligodendrocytes against the inflammatory CNS environment.
Asunto(s)
Guanabenzo/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Oligodendroglía/citología , Animales , Linfocitos T CD4-Positivos/citología , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Sistema Nervioso Central/metabolismo , Cerebelo/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Inflamación , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Ratas , Ratas Sprague-Dawley , Células Madre/citologíaRESUMEN
Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with variable expressivity. Heterozygous mutations in either of two genes, TSC1 (hamartin) or TSC2 (tuberin), are responsible for most cases. Hamartin and tuberin form a heterodimer that functions as a major cellular inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) kinase. Genotype-phenotype studies suggest that TSC2 mutations are associated with a more severe neurologic phenotype, although the biologic basis for the difference between TSC1- and TSC2-based disease is unclear. Here we performed a study to compare and contrast the brain phenotypes of Tsc1 and Tsc2 single and double mutants. Using Tsc1 and Tsc2 floxed alleles and a radial glial transgenic Cre driver (FVB-Tg(GFAP-cre)25Mes/J), we deleted Tsc1 and/or Tsc2 in radial glial progenitor cells. Single and double mutants had remarkably similar phenotypes: early postnatal mortality, brain overgrowth, laminar disruption, astrogliosis, a paucity of oligodendroglia, and myelination defects. Double Tsc1/Tsc2 mutants died earlier than single mutants, and single mutants showed differences in the location of heterotopias and the organization of the hippocampal stratum pyramidale. The differences were not due to differential mTORC1 activation or feedback inhibition on Akt. These data provide further genetic evidence for individual hamartin and tuberin functions that may explain some of the genotype-phenotype differences seen in the human disease.
Asunto(s)
Encéfalo/citología , Mutación/genética , Neuroglía/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Diferenciación Celular/genética , Línea Celular Transformada , Supervivencia Celular/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 gene. Patients afflicted with TSC develop tumors in various organ systems, but cerebral pathology is particularly severe. Conventional gene disruption of the Tsc1 or Tsc2 gene in mice cause limited central nervous system pathology. Homozygous deletion of either gene causes midgestation lethality. To circumvent the homozygous lethality of the conventional Tsc2 knockout we have generated a conditional allele of the Tsc2 gene by homologous recombination in mouse ES cells. The homozygous Tsc2(flox/flox) mice are identical to wildtype in many organs typically affected by TSC, especially the brain. Using this Tsc2(flox) allele we have generated a null allele using Cre recombination. This allele will be useful in investigating TSC pathology with appropriate cell and organ specific Cre-transgenic mice.