RESUMEN
The post-synaptic density protein 95 (PSD95) is a crucial scaffolding protein participating in the organization and regulation of synapses. PSD95 interacts with numerous molecules, including neurotransmitter receptors and ion channels. The functional dysregulation of PSD95 as well as its abundance and localization has been implicated with several neurological disorders, making it an attractive target for developing strategies able to monitor PSD95 accurately for diagnostics and therapeutics. This study characterizes a novel camelid single-domain antibody (nanobody) that binds strongly and with high specificity to rat, mouse, and human PSD95. This nanobody allows for more precise detection and quantification of PSD95 in various biological samples. We expect that the flexibility and unique performance of this thoroughly characterized affinity tool will help to further understand the role of PSD95 in normal and diseased neuronal synapses.
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Neuronas , Sinapsis , Ratas , Ratones , Humanos , Animales , Homólogo 4 de la Proteína Discs Large/metabolismo , Sinapsis/metabolismo , Neuronas/metabolismo , Densidad Postsináptica/metabolismo , Canales Iónicos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Surfactant protein C (SP-C) modulates cerebrospinal fluid (CSF) rheology. During ageing, its declining levels are accompanied by an increased burden of white matter lesions. Pulmonary SP-C intermediates harbouring the BRICHOS-domain prevent protein misfolding in the lungs. Thus, cerebral SP-C intermediates may counteract cerebral ß-amyloidosis, a hallmark of Alzheimer's disease (AD). However, data on the molecular neuroanatomy of SP-C and its alterations in wildtype and triple transgenic (3xTg) mice, featuring essential elements of AD-neuropathology, are lacking. Therefore, this study investigated SP-C-containing structures in murine forebrains and their spatial relationships with vascular, glial and neuronal components of the neurovascular unit. Fluorescence labelling demonstrated neuronal SP-C in the medial habenula, the indusium griseum and the hippocampus. Glial counterstaining elucidated astrocytes in the corpus callosum co-expressing SP-C and S100ß. Notably, perineuronal nets were associated with SP-C in the nucleus reticularis thalami, the lateral hypothalamus and the retrosplenial cortex. In the hippocampus of aged 3xTg mice, an increased number of dot-like depositions containing SP-C and Reelin, but devoid of BRICHOS-immunoreactivity were observed apart from AD-like lesions. Wildtype and 3xTg mice revealed an age-dependent increase of such deposits markedly pronounced in about 24-month-old 3xTg mice. SP-C levels of the intracellular and extracellular compartments in each group revealed an inverse correlation of SP-C and Reelin, with reduced SP-C and increased Reelin in an age-dependent fashion especially in 3xTg mice. Taken together, extracellular SP-C, as modulator of glymphatic clearance and potential ligand of PNs, declines in 3xTg mice, which show an accumulation of extracellular Reelin depositions during ageing.
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Química Encefálica/fisiología , Hipocampo/metabolismo , Red Nerviosa/metabolismo , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Envejecimiento/metabolismo , Animales , Astrocitos/metabolismo , Espacio Extracelular/metabolismo , Femenino , Sistema Glinfático/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Red Nerviosa/crecimiento & desarrollo , Neuroglía/metabolismo , Acoplamiento Neurovascular/fisiología , Proteína Reelina/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismoRESUMEN
Ischemic stroke causes cellular alterations in the "neurovascular unit" (NVU) comprising neurons, glia, and the vasculature, and affects the blood-brain barrier (BBB) with adjacent extracellular matrix (ECM). Limited data are available for the zone between the NVU and ECM that has not yet considered for neuroprotective approaches. This study describes ischemia-induced alterations for two main components of the neurovascular matrix adhesion zone (NMZ), i.e., collagen IV as basement membrane constituent and fibronectin as crucial part of the ECM, in conjunction with traditional NVU elements. For spatio-temporal characterization of these structures, multiple immunofluorescence labeling was applied to tissues affected by focal cerebral ischemia using a filament-based model in mice (4, 24, and 72 h of ischemia), a thromboembolic model in rats (24 h of ischemia), a coagulation-based model in sheep (2 weeks of ischemia), and human autoptic stroke tissue (3 weeks of ischemia). An increased fibronectin immunofluorescence signal demarcated ischemia-affected areas in mice, along with an increased collagen IV signal and BBB impairment indicated by serum albumin extravasation. Quantifications revealed a region-specific pattern with highest collagen IV and fibronectin intensities in most severely affected neocortical areas, followed by a gradual decline toward the border zone and non-affected regions. Comparing 4 and 24 h of ischemia, the subcortical fibronectin signal increased significantly over time, whereas neocortical areas displayed only a gradual increase. Qualitative analyses confirmed increased fibronectin and collagen IV signals in ischemic areas from all tissues and time points investigated. While the increased collagen IV signal was restricted to vessels, fibronectin appeared diffusely arranged in the parenchyma with focal accumulations associated to the vasculature. Integrin α5 appeared enriched in the vicinity of fibronectin and vascular elements, while most of the non-vascular NVU elements showed complementary staining patterns referring to fibronectin. This spatio-temporal characterization of ischemia-related alterations of collagen IV and fibronectin in various stroke models and human autoptic tissue shows that ischemic consequences are not limited to traditional NVU components and the ECM, but also involve the NMZ. Future research should explore more components and the pathophysiological properties of the NMZ as a possible target for novel neuroprotective approaches.
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Erythropoietin (EPO), named after its role in hematopoiesis, is also expressed in mammalian brain. In clinical settings, recombinant EPO treatment has revealed a remarkable improvement of cognition, but underlying mechanisms have remained obscure. Here, we show with a novel line of reporter mice that cognitive challenge induces local/endogenous hypoxia in hippocampal pyramidal neurons, hence enhancing expression of EPO and EPO receptor (EPOR). High-dose EPO administration, amplifying auto/paracrine EPO/EPOR signaling, prompts the emergence of new CA1 neurons and enhanced dendritic spine densities. Single-cell sequencing reveals rapid increase in newly differentiating neurons. Importantly, improved performance on complex running wheels after EPO is imitated by exposure to mild exogenous/inspiratory hypoxia. All these effects depend on neuronal expression of the Epor gene. This suggests a model of neuroplasticity in form of a fundamental regulatory circle, in which neuronal networks-challenged by cognitive tasks-drift into transient hypoxia, thereby triggering neuronal EPO/EPOR expression.
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Encéfalo/metabolismo , Encéfalo/fisiopatología , Eritropoyetina/metabolismo , Hipoxia/metabolismo , Hipoxia/fisiopatología , Neurogénesis , Plasticidad Neuronal , Animales , Diferenciación Celular/efectos de los fármacos , Cognición/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Eritropoyetina/farmacología , Femenino , Eliminación de Gen , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Neurológicos , Actividad Motora/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Condicionamiento Físico Animal , Resistencia Física/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores de Eritropoyetina/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Reduced expression of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) in humans and mice causes white matter inflammation and catatonic signs. These consequences are experimentally alleviated by microglia ablation via colony-stimulating factor 1 receptor (CSF1R) inhibition using PLX5622. Here we address for the first time preclinical topics crucial for translation, most importantly 1) the comparison of 2 long-term PLX5622 applications (prevention and treatment) vs. 1 treatment alone, 2) the correlation of catatonic signs and executive dysfunction, 3) the phenotype of leftover microglia evading depletion, and 4) the role of intercellular interactions for efficient CSF1R inhibition. Based on our Cnp-/- mouse model and in vitro time-lapse imaging, we report the unexpected discovery that microglia surviving under PLX5622 display a highly inflammatory phenotype including aggressive premortal phagocytosis of oligodendrocyte precursor cells. Interestingly, ablating microglia in vitro requires mixed glial cultures, whereas cultured pure microglia withstand PLX5622 application. Importantly, 2 extended rounds of CSF1R inhibition are not superior to 1 treatment regarding any readout investigated (magnetic resonance imaging and magnetic resonance spectroscopy, behavior, immunohistochemistry). Catatonia-related executive dysfunction and brain atrophy of Cnp-/- mice fail to improve under PLX5622. To conclude, even though microglia depletion is temporarily beneficial and worth pursuing, complementary treatment strategies are needed for full and lasting recovery.-Fernandez Garcia-Agudo, L., Janova, H., Sendler, L. E., Arinrad, S., Steixner, A. A., Hassouna, I., Balmuth, E., Ronnenberg, A., Schopf, N., van der Flier, F. J., Begemann, M., Martens, H., Weber, M. S., Boretius, S., Nave, K.-A., Ehrenreich, H. Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.
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2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/genética , Encéfalo/patología , Encefalitis/genética , Microglía/patología , Eliminación de Secuencia/genética , Adulto , Animales , Encéfalo/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Compuestos Orgánicos/farmacología , Fenotipo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Eliminación de Secuencia/efectos de los fármacosRESUMEN
Self-propagating amyloid-ß (Aß) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aß42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aß42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aß42 peptides and decreases the seeding activity of Aß aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aß plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aß structures with small molecules as a valid therapeutic strategy.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Compuestos Azo/farmacología , Colorantes/farmacología , Inflamación/tratamiento farmacológico , Placa Amiloide/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Compuestos Azo/química , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Colorantes/química , Relación Dosis-Respuesta a Droga , Femenino , Inflamación/genética , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Molecular , Placa Amiloide/genética , Placa Amiloide/metabolismo , Polimerizacion/efectos de los fármacos , Agregado de Proteínas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Autoantibodies of the IgG class against N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1-AB) were considered pathognomonic for anti-NMDAR encephalitis. This view has been challenged by the age-dependent seroprevalence (up to >20%) of functional NMDAR1-AB of all immunoglobulin classes found in >5000 individuals, healthy or affected by different diseases. These findings question a merely encephalitogenic role of NMDAR1-AB. Here, we show that NMDAR1-AB belong to the normal autoimmune repertoire of dogs, cats, rats, mice, baboons, and rhesus macaques, and are functional in the NMDAR1 internalization assay based on human IPSC-derived cortical neurons. The age dependence of seroprevalence is lost in nonhuman primates in captivity and in human migrants, raising the intriguing possibility that chronic life stress may be related to NMDAR1-AB formation, predominantly of the IgA class. Active immunization of ApoE-/- and ApoE+/+ mice against four peptides of the extracellular NMDAR1 domain or ovalbumin (control) leads to high circulating levels of specific AB. After 4 weeks, the endogenously formed NMDAR1-AB (IgG) induce psychosis-like symptoms upon MK-801 challenge in ApoE-/- mice, characterized by an open blood-brain barrier, but not in their ApoE+/+ littermates, which are indistinguishable from ovalbumin controls. Importantly, NMDAR1-AB do not induce any sign of inflammation in the brain. Immunohistochemical staining for microglial activation markers and T lymphocytes in the hippocampus yields comparable results in ApoE-/- and ApoE+/+ mice, irrespective of immunization against NMDAR1 or ovalbumin. These data suggest that NMDAR1-AB of the IgG class shape behavioral phenotypes upon access to the brain but do not cause brain inflammation on their own.
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Encefalitis Antirreceptor N-Metil-D-Aspartato/inmunología , Trastornos Mentales/inmunología , Receptores de N-Metil-D-Aspartato/inmunología , Adulto , Animales , Autoanticuerpos/inmunología , Barrera Hematoencefálica , Encéfalo/inmunología , Gatos , Perros , Femenino , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Masculino , Ratones , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/inmunología , Primates , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Estudios SeroepidemiológicosRESUMEN
Forgetting is important. Without it, the relative importance of acquired memories in a changing environment is lost. We discovered that synaptotagmin-3 (Syt3) localizes to postsynaptic endocytic zones and removes AMPA receptors from synaptic plasma membranes in response to stimulation. AMPA receptor internalization, long-term depression (LTD), and decay of long-term potentiation (LTP) of synaptic strength required calcium-sensing by Syt3 and were abolished through Syt3 knockout. In spatial memory tasks, mice in which Syt3 was knocked out learned normally but exhibited a lack of forgetting. Disrupting Syt3:GluA2 binding in a wild-type background mimicked the lack of LTP decay and lack of forgetting, and these effects were occluded in the Syt3 knockout background. Our findings provide evidence for a molecular mechanism in which Syt3 internalizes AMPA receptors to depress synaptic strength and promote forgetting.
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Endocitosis , Memoria , Receptores AMPA/fisiología , Sinapsis/fisiología , Sinaptotagminas/fisiología , Animales , Calcio/fisiología , Células Cultivadas , Femenino , Células HEK293 , Hipocampo/citología , Hipocampo/fisiología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Potenciación a Largo Plazo , Depresión Sináptica a Largo Plazo , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , Neuronas/fisiología , Transporte de Proteínas , Ratas Wistar , Fracciones Subcelulares , Vesículas Sinápticas , Sinaptosomas , Sinaptotagminas/genética , TransfecciónRESUMEN
Because stroke therapies are still limited and patients are often concerned by long-term sequelae with significant impairment of daily living, elaborated neuroprotective strategies are needed. During the last decades, research substantially improved the knowledge on cellular pathologies responsible for stroke-related tissue damage. In this context, the neurovascular unit (NVU) concept has been established, summarizing the affections of neurons, associated astrocytes and the vasculature. Although oligodendrocytes were already identified to play a major role in other brain pathologies, their role during stroke evolution and long-lasting tissue damage is poorly understood. This study aims to explore oligodendrocyte structures, i.e., oligodendrocytes and their myelin-associated proteins, after experimental focal cerebral ischemia. For translational issues, different ages and genotypes including an Alzheimer-like background were considered to mimic potential co-morbidities. Three- and 12-month-old wild-type and triple-transgenic mice were subjected to unilateral middle cerebral artery occlusion. Immunofluorescence labeling was performed on forebrain tissues affected by 24 h of ischemia to visualize the oligodendrocyte-specific protein (OSP), the myelin basic protein (MBP), and the neuron-glia antigen 2 (NG2) with reference to the ischemic lesion. Subsequent analyses concomitantly detected the vasculature and the 2', 3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) to consider the NVU concept and to explore the functional relevance of histochemical data on applied oligodendrocyte markers. While the immunosignal of NG2 was found to be nearly absent 24 h after ischemia onset, enhanced immunoreactivities for OSP and especially MBP were observed in close regional association to the vasculature. Added quantitative analyses based on inter-hemispheric differences of MBP-immunoreactivity revealed a shell-like pattern with a significant increase directly in the ischemic core, followed by a gradual decline toward the striatum, the ischemic border zone and the lateral neocortex. This observation was consistent in subsequent analyses on the potential impact of age and genetic background. Furthermore, immunoreactivities for CNPase, MBP, and OSP were found to be simultaneously enhanced. In conclusion, this study provides evidence for a critical role of oligodendrocyte structures in the early phase after experimental stroke, strengthening their involvement in the ischemia-affected NVU. Consequently, oligodendrocytes and their myelin-associated proteins may qualify as potential targets for neuroprotective and regenerative approaches in stroke.
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RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca2+ channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1ß are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM-Munc13-RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis.SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca2+ channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.
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Proteínas de Unión al GTP/biosíntesis , Células Fotorreceptoras de Vertebrados/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Femenino , Proteínas de Unión al GTP/análisis , Proteínas de Unión al GTP/genética , Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Células Fotorreceptoras de Vertebrados/química , Sinapsis/química , Sinapsis/genéticaRESUMEN
Current stroke therapy is focused on recanalizing strategies, but neuroprotective co-treatments are still lacking. Modern concepts of the ischemia-affected neurovascular unit (NVU) and surrounding penumbra emphasize the complexity during the transition from initial damaging to regenerative processes. While early treatment with neurotrophic factors was shown to result in lesion size reduction and blood-brain barrier (BBB) stabilization, cellular consequences from these treatments are poorly understood. This study explored delayed cellular responses not only to ischemic stroke, but also to an early treatment with neurotrophic factors. Rats underwent 60 minutes of focal cerebral ischemia. Fluorescence labeling was applied to sections from brains perfused 7 days after ischemia. Analyses focused on NVU constituents including the vasculature, astrocytes and microglia in the ischemic striatum, the border zone and the contralateral hemisphere. In addition to histochemical signs of BBB breakdown, a strong up-regulation of collagen IV and microglia activation occurred within the ischemic core with simultaneous degradation of astrocytes and their endfeet. Activated astroglia were mainly depicted at the border zone in terms of a glial scar formation. Early treatment with pigment epithelium-derived factor (PEDF) resulted in an attenuation of the usually up-regulated collagen IV-immunoreactivity. However, glial activation was not influenced by treatment with PEDF or the epidermal growth factor (EGF). In conclusion, these data on ischemia-induced cellular reactions within the NVU might help to develop treatments addressing the transition from injury towards regeneration. Thereby, the integrity of the vasculature in close relation to neighboring structures like astrocytes appears as a promising target.
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Encéfalo/efectos de los fármacos , Ataque Isquémico Transitorio/patología , Factores de Crecimiento Nervioso/farmacología , Animales , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/patología , Colágeno Tipo IV/metabolismo , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento Epidérmico/uso terapéutico , Proteínas del Ojo/farmacología , Proteínas del Ojo/uso terapéutico , Proteína Ácida Fibrilar de la Glía/metabolismo , Ataque Isquémico Transitorio/diagnóstico por imagen , Ataque Isquémico Transitorio/tratamiento farmacológico , Imagen por Resonancia Magnética , Masculino , Microglía/metabolismo , Microglía/patología , Microscopía Fluorescente , Factores de Crecimiento Nervioso/uso terapéutico , Ratas , Ratas Sprague-Dawley , Serpinas/farmacología , Serpinas/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in ß-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.
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Diabetes Mellitus Tipo 2/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Animales , Diabetes Mellitus Tipo 2/patología , Estrés del Retículo Endoplásmico , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Transgénicos , Oxidación-Reducción , Agregación Patológica de Proteínas , Conformación ProteicaRESUMEN
The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.
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Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Hipotálamo/metabolismo , Neuropéptidos/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Animales , Inmunohistoquímica/métodos , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurotransmisores/fisiología , Núcleo Supraquiasmático/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury.
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Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.
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Autofagia , Vesículas Sinápticas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Cuerpo Celular/metabolismo , Compartimento Celular , Células Cultivadas , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hipocampo/citología , Humanos , Ratones Endogámicos BALB C , Proteínas Mutantes/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Neuronas/citología , Neuronas/metabolismo , Neuronas/ultraestructura , Fagosomas/metabolismo , Ratas , Proteínas de Transporte Vesicular/metabolismoRESUMEN
A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.
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Corticosterona/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Secretagoginas/metabolismo , Estrés Fisiológico/fisiología , Animales , Corticosterona/genética , Hormona Liberadora de Corticotropina/genética , Masculino , Ratones , Neuronas/citología , Núcleo Hipotalámico Paraventricular/citología , Hipófisis/citología , Hipófisis/metabolismo , Interferencia de ARN , Secretagoginas/genética , Transcriptoma/fisiologíaRESUMEN
OBJECTIVE: We previously reported an unexpectedly high seroprevalence (~10%) of N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1) autoantibodies (AB) in healthy and neuropsychiatrically ill subjects (N = 2,817). This finding challenges an unambiguous causal relationship of serum AB with brain disease. To test whether similar results would be obtained for other brain antigen-directed AB previously connected with pathological conditions, we systematically screened serum samples of 4,236 individuals. METHODS: Serum samples of healthy (n = 1,703) versus neuropsychiatrically ill subjects (schizophrenia, affective disorders, stroke, Parkinson disease, amyotrophic lateral sclerosis, personality disorder; total n = 2,533) were tested. For analysis based on indirect immunofluorescence, we used biochip mosaics of frozen brain sections (rat, monkey) and transfected HEK293 cells expressing respective recombinant target antigens. RESULTS: Seroprevalence of all screened AB was comparable in healthy and ill individuals. None of them, however, reached the abundance of NMDAR1 AB (again ~10%; immunoglobulin [Ig] G ~1%). Appreciable frequency was noted for AB against amphiphysin (2.0%), ARHGAP26 (1.3%), CASPR2 (0.9%), MOG (0.8%), GAD65 (0.5%), Ma2 (0.5%), Yo (0.4%), and Ma1 (0.4%), with titers and Ig class distribution similar among groups. All other AB were found in ≤0.1% of individuals (anti-AMPAR-1/2, AQP4, CV2, Tr/DNER, DPPX-IF1, GABAR-B1/B2, GAD67, GLRA1b, GRM1, GRM5, Hu, LGl1, recoverin, Ri, ZIC4). The predominant Ig class depended on antigen location, with intracellular epitopes predisposing to IgG (chi-square = 218.91, p = 2.8 × 10(-48) ). INTERPRETATION: To conclude, the brain antigen-directed AB tested here are comparably detectable in healthy subjects and the disease groups studied here, thus questioning an upfront pathological role of these serum AB.
Asunto(s)
Autoanticuerpos/sangre , Trastornos Mentales/sangre , Trastornos Mentales/epidemiología , Enfermedades del Sistema Nervioso/sangre , Enfermedades del Sistema Nervioso/epidemiología , Adulto , Anciano , Animales , Autoanticuerpos/biosíntesis , Femenino , Alemania/epidemiología , Células HEK293 , Haplorrinos , Humanos , Masculino , Tamizaje Masivo , Trastornos Mentales/inmunología , Persona de Mediana Edad , Enfermedades del Sistema Nervioso/inmunología , Ratas , Receptores de N-Metil-D-Aspartato/inmunología , Valores de Referencia , Estudios SeroepidemiológicosRESUMEN
The pathogenesis of Alzheimer's disease (AD) is believed to be closely dependent on deposits of neurotoxic amyloid-ß peptides (Aß), which become abundantly present throughout the central nervous system in advanced stages of the disease. The different Aß peptides existing are generated by subsequent cleavage of the amyloid-ß protein precursor (AßPP) and may vary in length and differ at their C-terminus. Despite extensive studies on the most prevalent species Aß40 and Aß42, Aß peptides with other C-termini such as Aß38 have not received much attention. In the present study, we used a highly specific and sensitive antibody against Aß38 to analyze the distribution of this Aß species in cases of sporadic and familial AD, as well as in the brains of a series of established transgenic AD mouse models. We found Aß38 to be present as vascular deposits in the brains of the majority of sporadic AD cases, whereas it is largely absent in non-demented control cases. Aß38-positive extracellular plaques were virtually limited to familial cases. Interestingly we observed Aß38-positive plaques not only among familial cases due to AßPP mutations, but also in cases of familial AD caused by presenilin (PSEN) mutations. Furthermore we demonstrate that Aß38 deposits in the form of extracellular plaques are common in several AD transgenic mouse models carrying either only AßPP, or combinations of AßPP, PSEN1, and tau transgenes.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Modelos Animales de Enfermedad , Fragmentos de Péptidos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/análisis , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Fragmentos de Péptidos/análisisRESUMEN
GABAergic interneurons are essential for a functional equilibrium between excitatory and inhibitory impulses throughout the CNS. Disruption of this equilibrium can lead to various neurological or neuropsychiatric disorders such as epilepsy or schizophrenia. Schizophrenia itself is clinically defined by negative (e.g., depression) and positive (e.g., hallucinations) symptoms as well as cognitive dysfunction. GABAergic interneurons are proposed to play a central role in the etiology and progression of schizophrenia; however, the specific mechanisms and the time-line of symptom development as well as the distinct involvement of cortical and hippocampal GABAergic interneurons in the etiology of schizophrenia-related symptoms are still not conclusively resolved. Previous work demonstrated that GABAergic interneurons can be selectively depleted in adult mice by means of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro and in vivo. Given their involvement in schizophrenia-related disease etiology, we ablated GABAergic interneurons in the medial prefrontal cortex (mPFC) and dorsal hippocampus (dHPC) in adult male C57BL/6N mice. Subsequently we assessed alterations in anxiety, sensory processing, hyperactivity and cognition after long-term (>14 days) and short-term (<14 days) GABAergic depletion. Long-term GABAergic depletion in the mPFC resulted in a decrease in sensorimotor-gating and impairments in cognitive flexibility. Notably, the same treatment at the level of the dHPC completely abolished spatial learning capabilities. Short-term GABAergic depletion in the dHPC revealed a transient hyperactive phenotype as well as marked impairments regarding the acquisition of a spatial memory. In contrast, recall of a spatial memory was not affected by the same intervention. These findings emphasize the importance of functional local GABAergic networks for the encoding but not the recall of hippocampus-dependent spatial memories.
RESUMEN
The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function.