RESUMEN
The similarities and differences between nervous systems of various species result from developmental constraints and specific adaptations1-4. Comparative analyses of the prefrontal cortex (PFC), a cerebral cortex region involved in higher-order cognition and complex social behaviours, have identified true and potential human-specific structural and molecular specializations4-8, such as an exaggerated PFC-enriched anterior-posterior dendritic spine density gradient5. These changes are probably mediated by divergence in spatiotemporal gene regulation9-17, which is particularly prominent in the midfetal human cortex15,18-20. Here we analysed human and macaque transcriptomic data15,20 and identified a transient PFC-enriched and laminar-specific upregulation of cerebellin 2 (CBLN2), a neurexin (NRXN) and glutamate receptor-δ GRID/GluD-associated synaptic organizer21-27, during midfetal development that coincided with the initiation of synaptogenesis. Moreover, we found that species differences in level of expression and laminar distribution of CBLN2 are, at least in part, due to Hominini-specific deletions containing SOX5-binding sites within a retinoic acid-responsive CBLN2 enhancer. In situ genetic humanization of the mouse Cbln2 enhancer drives increased and ectopic laminar Cbln2 expression and promotes PFC dendritic spine formation. These findings suggest a genetic and molecular basis for the anterior-posterior cortical gradient and disproportionate increase in the Hominini PFC of dendritic spines and a developmental mechanism that may link dysfunction of the NRXN-GRID-CBLN2 complex to the pathogenesis of neuropsychiatric disorders.
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Dendritas/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Corteza Prefrontal/citología , Animales , Proteínas de Unión al Calcio/metabolismo , Elementos de Facilitación Genéticos/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Macaca , Trastornos Mentales/patología , Ratones , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/patología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Filogenia , Regiones Promotoras Genéticas/genética , Factores de Transcripción SOXD/metabolismo , Transcriptoma , Regulación hacia ArribaRESUMEN
The brains of humans and other mammals are highly vulnerable to interruptions in blood flow and decreases in oxygen levels. Here we describe the restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex vivo normothermic conditions up to four hours post-mortem. We have developed an extracorporeal pulsatile-perfusion system and a haemoglobin-based, acellular, non-coagulative, echogenic, and cytoprotective perfusate that promotes recovery from anoxia, reduces reperfusion injury, prevents oedema, and metabolically supports the energy requirements of the brain. With this system, we observed preservation of cytoarchitecture; attenuation of cell death; and restoration of vascular dilatory and glial inflammatory responses, spontaneous synaptic activity, and active cerebral metabolism in the absence of global electrocorticographic activity. These findings demonstrate that under appropriate conditions the isolated, intact large mammalian brain possesses an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.
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Autopsia , Encéfalo/irrigación sanguínea , Encéfalo/citología , Circulación Cerebrovascular , Microcirculación , Porcinos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Caspasa 3/metabolismo , Supervivencia Celular , Arterias Cerebrales/fisiología , Modelos Animales de Enfermedad , Hipoxia Encefálica/metabolismo , Hipoxia Encefálica/patología , Inflamación/metabolismo , Inflamación/patología , Neuroglía/citología , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Perfusión , Daño por Reperfusión/prevención & control , Porcinos/sangre , Sinapsis/metabolismo , Sinapsis/patología , Factores de Tiempo , VasodilataciónRESUMEN
The centrosome is the main microtubule organizing center in stem cells, and its mother centriole, anchored to the cell membrane, serves as the basal body of the primary cilium. Prolonged anchorage of centrosomes and primary cilia to the apical segment of the membrane of apical neural progenitor cells is considered vital for interkinetic nuclear translocation and repetitive cycling in the ventricular zone. In contrast, the basolateral anchorage of primary cilia has been regarded as the first step in delamination and conversion of apical to basal neural progenitor cells or neurons. Using electron microscopy analysis of serial sections, we show that centrosomes, in a fraction of cells, anchor to the basolateral cell membrane immediately after cell division and before development of cilia. In other cells, centrosomes situate freely in the cytoplasm, increasing their probability of subsequent apical anchorage. In mice, anchored centrosomes in the cells shortly after mitosis predominate during the entire cerebral neurogenesis, whereas in macaque monkeys, cytoplasmic centrosomes are more numerous. Species-specific differences in the ratio of anchored and free cytoplasmic centrosomes appear to be related to prolonged neurogenesis in the ventricular zone that is essential for lateral expansion of the cerebral cortex in primates.
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Centrosoma , Corteza Cerebral , Células-Madre Neurales , Neurogénesis , Animales , Centrosoma/metabolismo , Corteza Cerebral/citología , Células-Madre Neurales/fisiología , Ratones , Neurogénesis/fisiologíaRESUMEN
Alzheimer's disease cortical tau pathology initiates in the layer II cell clusters of entorhinal cortex, but it is not known why these specific neurons are so vulnerable. Aging macaques exhibit the same qualitative pattern of tau pathology as humans, including initial pathology in layer II entorhinal cortex clusters, and thus can inform etiological factors driving selective vulnerability. Macaque data have already shown that susceptible neurons in dorsolateral prefrontal cortex express a "signature of flexibility" near glutamate synapses on spines, where cAMP-PKA magnification of calcium signaling opens nearby potassium and hyperpolarization-activated cyclic nucleotide-gated channels to dynamically alter synapse strength. This process is regulated by PDE4A/D, mGluR3, and calbindin, to prevent toxic calcium actions; regulatory actions that are lost with age/inflammation, leading to tau phosphorylation. The current study examined whether a similar "signature of flexibility" expresses in layer II entorhinal cortex, investigating the localization of PDE4D, mGluR3, and HCN1 channels. Results showed a similar pattern to dorsolateral prefrontal cortex, with PDE4D and mGluR3 positioned to regulate internal calcium release near glutamate synapses, and HCN1 channels concentrated on spines. As layer II entorhinal cortex stellate cells do not express calbindin, even when young, they may be particularly vulnerable to magnified calcium actions and ensuing tau pathology.
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Enfermedad de Alzheimer , Animales , Humanos , Enfermedad de Alzheimer/patología , Corteza Entorrinal/patología , Macaca mulatta/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Calcio , Calbindinas , Glutamatos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismoRESUMEN
INTRODUCTION: Tau phosphorylated at threonine-217 (pT217-tau) is a novel fluid-based biomarker that predicts onset of Alzheimer's disease (AD) symptoms, but little is known about how pT217-tau arises in the brain, as soluble pT217-tau is dephosphorylated post mortem in humans. METHODS: We used multilabel immunofluorescence and immunoelectron microscopy to examine the subcellular localization of early-stage pT217-tau in entorhinal and prefrontal cortices of aged macaques with naturally occurring tau pathology and assayed pT217-tau levels in plasma. RESULTS: pT217-tau was aggregated on microtubules within dendrites exhibiting early signs of degeneration, including autophagic vacuoles. It was also seen trafficking between excitatory neurons within synapses on spines, where it was exposed to the extracellular space, and thus accessible to cerebrospinal fluid (CSF)/blood. Plasma pT217-tau levels increased across the age span and thus can serve as a biomarker in macaques. DISCUSSION: These data help to explain why pT217-tau predicts degeneration in AD and how it gains access to CSF and plasma to serve as a fluid biomarker.
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Enfermedad de Alzheimer , Proteínas tau , Animales , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Corteza Prefontal Dorsolateral , Macaca mulatta/metabolismo , Proteínas tau/líquido cefalorraquídeoRESUMEN
Glutamate carboxypeptidase-II (GCPII) expression in brain is increased by inflammation, e.g. by COVID19 infection, where it reduces NAAG stimulation of metabotropic glutamate receptor type 3 (mGluR3). GCPII-mGluR3 signaling is increasingly linked to higher cognition, as genetic alterations that weaken mGluR3 or increase GCPII signaling are associated with impaired cognition in humans. Recent evidence from macaque dorsolateral prefrontal cortex (dlPFC) shows that mGluR3 are expressed on dendritic spines, where they regulate cAMP-PKA opening of potassium (K+) channels to enhance neuronal firing during working memory. However, little is known about GCPII expression and function in the primate dlPFC, despite its relevance to inflammatory disorders. The present study used multiple label immunofluorescence and immunoelectron microscopy to localize GCPII in aging macaque dlPFC, and examined the effects of GCPII inhibition on dlPFC neuronal physiology and working memory function. GCPII was observed in astrocytes as expected, but also on neurons, including extensive expression in dendritic spines. Recordings in dlPFC from aged monkeys performing a working memory task found that iontophoresis of the GCPII inhibitors 2-MPPA or 2-PMPA markedly increased working memory-related neuronal firing and spatial tuning, enhancing neural representations. These beneficial effects were reversed by an mGluR2/3 antagonist, or by a cAMP-PKA activator, consistent with mGluR3 inhibition of cAMP-PKA-K+ channel signaling. Systemic administration of the brain penetrant inhibitor, 2-MPPA, significantly improved working memory performance without apparent side effects, with largest effects in the oldest monkeys. Taken together, these data endorse GCPII inhibition as a potential strategy for treating cognitive disorders associated with aging and/or neuroinflammation.
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COVID-19 , Corteza Prefontal Dorsolateral , Humanos , Animales , Haplorrinos , Macaca , Cognición , GlutamatosRESUMEN
Mitochondrial malfunction and morphologic disorganization have been observed in brain cells as part of complex pathological changes. However, it is unclear what may be the role of mitochondria in the initiation of pathologic processes or if mitochondrial disorders are consequences of earlier events. We analyzed the morphologic reorganization of organelles in an embryonic mouse brain during acute anoxia using an immunohistochemical identification of the disordered mitochondria, followed by electron microscopic three-dimensional (3D) reconstruction. We found swelling of the mitochondrial matrix after 3 h anoxia and probable dissociation of mitochondrial stomatin-like protein 2 (SLP2)-containing complexes after 4.5 h anoxia in the neocortex, hippocampus, and lateral ganglionic eminence. Surprisingly, deformation of the Golgi apparatus (GA) was detected already after 1 h of anoxia, when the mitochondria and other organelles still had a normal ultrastructure. The disordered GA showed concentrical swirling of the cisternae and formed spherical onion-like structures with the trans-cisterna in the center of the sphere. Such disturbance of the Golgi architecture likely interferes with its function for post-translational protein modification and secretory trafficking. Thus, the GA in embryonic mouse brain cells may be more vulnerable to anoxic conditions than the other organelles, including mitochondria.
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Aparato de Golgi , Mitocondrias , Ratones , Animales , Aparato de Golgi/ultraestructura , Orgánulos , Encéfalo , HipoxiaRESUMEN
Galanin, one of the most inducible neuropeptides, is widely present in developing brains, and its expression is altered by pathologic events (e.g., epilepsy, ischemia, and axotomy). The roles of galanin in brain development under both normal and pathologic conditions have been hypothesized, but the question of how galanin is involved in fetal and early postnatal brain development remains largely unanswered. In this study, using granule cell migration in the cerebellum of early postnatal mice (both sexes) as a model system, we examined the role of galanin in neuronal cell migration during normal development and after brain injury. Here we show that, during normal development, endogenous galanin participates in accelerating granule cell migration via altering the Ca2+ and cAMP signaling pathways. Upon brain injury induced by the application of cold insults, galanin levels decrease at the lesion sites, but increase in the surroundings of lesion sites. Granule cells exhibit the following corresponding changes in migration: (1) slowing down migration at the lesion sites; and (2) accelerating migration in the surroundings of lesion sites. Experimental manipulations of galanin signaling reduce the lesion site-specific changes in granule cell migration, indicating that galanin plays a role in such deficits in neuronal cell migration. The present study suggests that manipulating galanin signaling may be a potential therapeutic target for acutely injured brains during development.SIGNIFICANCE STATEMENT Deficits in neuronal cell migration caused by brain injury result in abnormal development of cortical layers, but the underlying mechanisms remain to be determined. Here, we report that on brain injury, endogenous levels of galanin, a neuropeptide, are altered in a lesion site-specific manner, decreasing at the lesion sites but increasing in the surroundings of lesion sites. The changes in galanin levels positively correlate with the migration rate of immature neurons. Manipulations of galanin signaling ameliorate the effects of injury on neuronal migration and cortical layer development. These results shed a light on galanin as a potential therapeutic target for acutely injured brains during development.
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Lesiones Encefálicas/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Movimiento Celular/fisiología , Cerebelo/metabolismo , Galanina/metabolismo , Animales , Animales Recién Nacidos , Lesiones Encefálicas/patología , Células Cultivadas , Cerebelo/lesiones , Cerebelo/patología , Relación Dosis-Respuesta a Droga , Femenino , Masculino , RatonesRESUMEN
The primate cerebrum is characterized by a large expansion of cortical surface area, the formation of convolutions, and extraordinarily voluminous subcortical white matter. It was recently proposed that this expansion is primarily driven by increased production of superficial neurons in the dramatically enlarged outer subventricular zone (oSVZ). Here, we examined the development of the parietal cerebrum in macaque monkey and found that, indeed, the oSVZ initially adds neurons to the superficial layers II and III, increasing their thickness. However, as the oSVZ grows in size, its output changes to production of astrocytes and oligodendrocytes, which in primates outnumber cerebral neurons by a factor of three. After the completion of neurogenesis around embryonic day (E) 90, when the cerebrum is still lissencephalic, the oSVZ enlarges and contains Pax6+/Hopx+ outer (basal) radial glial cells producing astrocytes and oligodendrocytes until after E125. Our data indicate that oSVZ gliogenesis, rather than neurogenesis, correlates with rapid enlargement of the cerebrum and development of convolutions, which occur concomitantly with the formation of cortical connections via the underlying white matter, in addition to neuronal growth, elaboration of dendrites, and amplification of neuropil in the cortex, which are primary factors in the formation of cerebral convolutions in primates.
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Cerebro/crecimiento & desarrollo , Cerebro/metabolismo , Ventrículos Laterales/crecimiento & desarrollo , Ventrículos Laterales/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Animales , Astrocitos/metabolismo , Cerebro/citología , Cerebro/embriología , Embrión de Mamíferos , Proteínas de Homeodominio/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/embriología , Macaca , Oligodendroglía/citología , Oligodendroglía/metabolismo , Factor de Transcripción PAX6/metabolismo , Primates , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Radial glial cells (RGC) are at the center of brain development in vertebrates, acting as progenitors for neurons and macroglia (oligodendrocytes and astrocytes) and as guides for migration of neurons from the ventricular surface to their final positions in the brain. These cells originate from neuroepithelial cells (NEC) from which they inherit their epithelial features and polarized morphology, with processes extending from the ventricular to the pial surface of the embryonic cerebrum. We have learnt a great deal since the first descriptions of these cells at the end of the nineteenth century. However, there are still questions regarding how and when NEC transform into RGC or about the function of intermediate filaments such as glial fibrillary acidic protein (GFAP) in RGCs and their dynamics during neurogenesis. For example, it is not clear why RGCs in primates, including humans, express GFAP at the onset of cortical neurogenesis while in rodents it is expressed when it is essentially complete. Based on an ultrastructural analysis of GFAP expression and cell morphology of dividing progenitors in the developing neocortex of the macaque monkey, we show that RGCs become the main progenitor in the developing cerebrum by the start of neurogenesis, as all dividing cells show glial features such as GFAP expression and lack of tight junctions. Also, our data suggest that RGCs retract their apical process during mitosis. We discuss our findings in the context of the role and molecular characteristics of RGCs in the vertebrate brain, their differences with NECs and their dynamic behavior during the process of neurogenesis.
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Células Ependimogliales/metabolismo , Neurogénesis/fisiología , Animales , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Extensiones de la Superficie Celular/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Macaca , Proteínas del Tejido Nervioso/metabolismo , Células Neuroepiteliales/metabolismoRESUMEN
Hypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide ß-endorphin. CB1R activation selectively increases ß-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.
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Cannabinoides/farmacología , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Hipotálamo/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proopiomelanocortina/metabolismo , Animales , Metabolismo Energético/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiología , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Naloxona/farmacología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , Respuesta de Saciedad/efectos de los fármacos , Respuesta de Saciedad/fisiología , Proteína Desacopladora 2 , alfa-MSH/metabolismo , betaendorfina/metabolismoRESUMEN
The primary stem cells of the cerebral cortex are the radial glial cells (RGCs), and disturbances in their operation lead to myriad brain disorders in all mammals from mice to humans. Here, we found in mice that maternal gestational obesity and hyperglycemia can impair the maturation of RGC fibers and delay cortical neurogenesis. To investigate potential mechanisms, we used optogenetic live-imaging approaches in embryonic cortical slices. We found that Ca2+ signaling regulates mitochondrial transport and is crucial for metabolic support in RGC fibers. Cyclic intracellular Ca2+ discharge from localized RGC fiber segments detains passing mitochondria and ensures their proper distribution and enrichment at specific sites such as endfeet. Impairment of mitochondrial function caused an acute loss of Ca2+ signaling, while hyperglycemia decreased Ca2+ activity and impaired mitochondrial transport, leading to degradation of the RGC scaffold. Our findings uncover a physiological mechanism indicating pathways by which gestational metabolic disturbances can interfere with brain development.
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Señalización del Calcio , Corteza Cerebral/embriología , Diabetes Gestacional/metabolismo , Glucosa/metabolismo , Hiperglucemia/embriología , Neurogénesis , Neuroglía/metabolismo , Animales , Corteza Cerebral/patología , Diabetes Gestacional/genética , Diabetes Gestacional/patología , Femenino , Hiperglucemia/genética , Hiperglucemia/patología , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Neuroglía/patología , EmbarazoRESUMEN
INTRODUCTION: The etiology of sporadic Alzheimer's disease (AD) requires non-genetically modified animal models. METHODS: The relationship of tau phosphorylation to calcium-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) dysregulation was analyzed in aging rhesus macaque dorsolateral prefrontal cortex (dlPFC) and rat primary cortical neurons using biochemistry and immuno-electron microscopy. The influence of calcium leak from ryanodine receptors (RyRs) on neuronal firing and cognitive performance was examined in aged macaques. RESULTS: Aged monkeys naturally develop hyperphosphorylated tau, including AD biomarkers (AT8 (pS202/pT205) and pT217) and early tau pathology markers (pS214 and pS356) that correlated with evidence of increased calcium leak (pS2808-RyR2). Calcium also regulated early tau phosphorylation in vitro. Age-related reductions in the calcium-binding protein, calbindin, and in phosphodiesterase PDE4D were seen within dlPFC pyramidal cell dendrites. Blocking RyRs with S107 improved neuronal firing and cognitive performance in aged macaques. DISCUSSION: Dysregulated calcium signaling confers risk for tau pathology and provides a potential therapeutic target.
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Calcio/metabolismo , Disfunción Cognitiva/patología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Macaca mulatta , Proteínas tau/metabolismo , Envejecimiento/patología , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Humanos , Masculino , Neuronas/metabolismo , Fosforilación , Corteza Prefrontal/patología , Ratas , Canal Liberador de Calcio Receptor de RianodinaRESUMEN
Noradrenergic (NE) α1-adrenoceptors (α1-ARs) contribute to arousal mechanisms and play an important role in therapeutic medications such as those for the treatment of posttraumatic stress disorder (PTSD). However, little is known about how α1-AR stimulation influences neuronal firing in the dorsolateral prefrontal cortex (dlPFC), a newly evolved region that is dysfunctional in PTSD and other mental illnesses. The current study examined the effects of α1-AR manipulation on neuronal firing in dlPFC of rhesus monkeys performing a visuospatial working memory task, focusing on the "delay cells" that maintain spatially tuned information across the delay period. Iontophoresis of the α1-AR antagonist HEAT (2-{[ß-(4-hydroxyphenyl)ethyl]aminomethyl}-1-tetralone) had mixed effects, reducing firing in a majority of neurons but having nonsignificant excitatory effects or no effect in remaining delay cells. These data suggest that endogenous NE has excitatory effects in some delay cells under basal conditions. In contrast, the α1-AR agonists phenylephrine and cirazoline suppressed delay cell firing and this was blocked by coadministration of HEAT. These results indicate an inverted-U dose response for α1-AR actions, with mixed excitatory actions under basal conditions and suppressed firing with high levels of α1-AR stimulation such as with stress exposure. Immunoelectron microscopy revealed α1-AR expression presynaptically in axons and axon terminals and postsynaptically in spines, dendrites, and astrocytes. It is possible that α1-AR excitatory effects arise from presynaptic excitation of glutamate release, whereas postsynaptic actions suppress firing through calcium-protein kinase C opening of potassium channels on spines. The latter may predominate under stressful conditions, leading to loss of dlPFC regulation during uncontrollable stress.SIGNIFICANCE STATEMENT Noradrenergic stimulation of α1-adrenoceptors (α1-ARs) is implicated in posttraumatic stress disorder (PTSD) and other mental disorders that involve dysfunction of the prefrontal cortex, a brain region that provides top-down control. However, the location and contribution of α1-ARs to prefrontal cortical physiology in primates has received little attention. This study found that α1-ARs are located near prefrontal synapses and that α1-AR stimulation has mixed effects under basal conditions. However, high levels of α1-AR stimulation, as occur with stress, suppress neuronal firing. These findings help to explain why we lose top-down control under conditions of uncontrollable stress when there are high levels of noradrenergic release in brain and why blocking α1-AR, such as with prazosin, may be helpful in the treatment of PTSD.
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Corteza Prefrontal/diagnóstico por imagen , Corteza Prefrontal/metabolismo , Desempeño Psicomotor/fisiología , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Antagonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Macaca mulatta , Imagen por Resonancia Magnética/métodos , Masculino , Norepinefrina/farmacología , Estimulación Luminosa/métodos , Desempeño Psicomotor/efectos de los fármacosRESUMEN
BACKGROUND: Cognitive impairment in schizophrenia, aging, and Alzheimer's disease is associated with spine and synapse loss from the dorsolateral prefrontal cortex (dlPFC) layer III. Complement cascade signaling is critical in driving spine loss and disease pathogenesis. Complement signaling is initiated by C1q, which tags synapses for elimination. C1q is thought to be expressed predominately by microglia, but its expression in primate dlPFC has never been examined. The current study assayed C1q levels in aging primate dlPFC and rat medial PFC (mPFC) and used immunoelectron microscopy (immunoEM), immunoblotting, and co-immunoprecipitation (co-IP) to reveal the precise anatomical distribution and interactions of C1q. METHODS: Age-related changes in C1q levels in rhesus macaque dlPFC and rat mPFC were examined using immunoblotting. High-spatial resolution immunoEM was used to interrogate the subcellular localization of C1q in aged macaque layer III dlPFC and aged rat layer III mPFC. co-IP techniques quantified protein-protein interactions for C1q and proteins associated with excitatory and inhibitory synapses in macaque dlPFC. RESULTS: C1q levels were markedly increased in the aged macaque dlPFC. Ultrastructural localization found the expected C1q localization in glia, including those ensheathing synapses, but also revealed extensive localization within neurons. C1q was found near synapses, within terminals and in spines, but was also observed in dendrites, often near abnormal mitochondria. Similar analyses in aging rat mPFC corroborated the findings in rhesus macaques. C1q protein increasingly associated with PSD95 with age in macaque, consistent with its synaptic localization as evidenced by EM. CONCLUSIONS: These findings reveal novel, intra-neuronal distribution patterns for C1q in the aging primate cortex, including evidence of C1q in dendrites. They suggest that age-related changes in the dlPFC may increase C1q expression and synaptic tagging for glial phagocytosis, a possible mechanism for age-related degeneration.
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Envejecimiento/metabolismo , Complemento C1q/análisis , Complemento C1q/metabolismo , Neuronas/metabolismo , Corteza Prefrontal/química , Corteza Prefrontal/metabolismo , Animales , Macaca mulatta , Neuronas/ultraestructura , Corteza Prefrontal/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
The TCF4 gene is the subject of numerous and varied investigations of it's role in the genesis of neuropsychiatric disease. The gene has been identified as the cause of Pitt-Hopkins syndrome (PTHS) and it has been implicated in various other neuropsychiatric diseases, including schizophrenia, depression, and autism. However, the precise molecular mechanisms of the gene's involvement in neurogenesis, particularly, corticogenesis, are not well understood. Here, we present data showing that TCF4 is expressed in a region-specific manner in the radial glia and stem cells of transient embryonic zones at early gestational ages in both humans and mice. TCF4 haploinsufficiency mice exhibit a delay in neuronal migration, and a significant increase in the number of upper-layer cortical neurons, as well as abnormal dendrite and synapse formation. Our research also reveals that TCF3 up-regulates Tcf4 by binding to the specific "E-box" and its flank sequence in intron 2 of the Tcf4 gene. Additionally, our transcriptome study substantiates that Tcf4 transcriptional function is essential for locomotion, cognition, and learning. By activating expression of TCF4 in the regulation of neuronal proliferation and migration to the overlaying neocortex and subsequent differentiation leading to laminar formation TCF4 fulfills its normal function, but if not, abnormalities such as those reported here result. These findings provide new insight into the specific roles of Tcf4 molecular pathway in neocortical development and their relevance in the pathogenesis of neuropsychiatric diseases.
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Malformaciones del Desarrollo Cortical/genética , Trastornos Mentales/genética , Factor de Transcripción 4/genética , Animales , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Femenino , Redes Reguladoras de Genes/genética , Haploinsuficiencia , Humanos , Masculino , Ratones , Ratones Noqueados , Neurogénesis/fisiología , Neuronas/metabolismo , Fenotipo , Esquizofrenia/genética , Factor de Transcripción 4/metabolismo , Factores de Transcripción/genéticaRESUMEN
Cannabinoid type 1 receptor (CB1R) is expressed and participates in several aspects of cerebral cortex embryonic development as demonstrated with whole-transcriptome mRNA sequencing and other contemporary methods. However, the cellular location of CB1R, which helps to specify molecular mechanisms, remains to be documented. Using three-dimensional (3D) electron microscopic reconstruction, we examined CB1R immunolabeling in proliferating neural stem cells (NSCs) and migrating neurons in the embryonic mouse (Mus musculus) and rhesus macaque (Macaca mulatta) cerebral cortex. We found that the mitotic and postmitotic ventricular and subventricular zone (VZ and SVZ) cells are immunonegative in both species while radially migrating neurons in the intermediate zone (IZ) and cortical plate (CP) contain CB1R-positive intracellular vesicles. CB1R immunolabeling was more numerous and more extensive in monkeys compared to mice. In CB1R-knock out mice, projection neurons in the IZ show migration abnormalities such as an increased number of lateral processes. Thus, in radially migrating neurons CB1R provides a molecular substrate for the regulation of cell movement. Undetectable level of CB1R in VZ/SVZ cells indicates that previously suggested direct CB1R-transmitted regulation of cellular proliferation and fate determination demands rigorous re-examination. More abundant CB1R expression in monkey compared to mouse suggests that therapeutic or recreational cannabis use may be more distressing for immature primate neurons than inferred from experiments with rodents.
Asunto(s)
Movimiento Celular , Proliferación Celular , Células-Madre Neurales/metabolismo , Neurogénesis , Neuronas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Macaca mulatta , Ratones , Ratones Noqueados , Células-Madre Neurales/citología , Neuronas/citología , Receptor Cannabinoide CB1/genéticaRESUMEN
Mitochondria play a critical role in various pathways of regulated cell death. Here we propose a novel method for detection of initial derangement of mitochondria in degenerating and dying neuronal cells. The method is based on our recent finding that antibodies directed against the cannabinoid type 1 receptor (CB1) also bind the mitochondrial stomatin-like protein 2 (SLP2) that belongs to an inner mitochondrial membrane protein complex. It is well established that SLP2 regulates mitochondrial biogenesis and respiratory functions. We now show that anti-CB1 antibodies recognize conformational epitopes but not the linear amino acid sequence of SLP2. In addition we found that anti-CB1 serum mostly labels swollen mitochondria with early or advanced stages of pathology in mouse brain while other proteins of the complex may mask epitopes of SLP2 in the normal mitochondria. Although neurons and endothelial cells in healthy brains contain occasional immunopositive mitochondria detectable with anti-CB1 serum, their numbers increase significantly after hypoxic insults in parallel with signs of cellular damage. Moreover, use of electron microscopy suggests relocation of SLP2 from its normal functional position in the inner mitochondrial membrane into the mitochondrial matrix in pathological cells. Thus, SLP2-like immunolabeling serves as an in situ histochemical target detecting early derangement of mitochondria. Anti-CB1 serum is crucial for this purpose because available anti-SLP2 antibodies do not provide selective labeling of mitochondria in the fixed tissue. This new method of detecting mitochondrial dysfunction can benefit the in vitro research of human diseases and developmental disorders by enabling analysis in live animal models.
Asunto(s)
Encéfalo/inmunología , Encéfalo/ultraestructura , Inmunohistoquímica/métodos , Proteínas de la Membrana/inmunología , Mitocondrias/ultraestructura , Proteínas Mitocondriales/inmunología , Proteínas del Tejido Nervioso/inmunología , Neuronas/inmunología , Neuronas/ultraestructura , Receptor Cannabinoide CB1/inmunología , Animales , Anticuerpos , Muerte Celular , Hipoxia de la Célula , Epítopos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Importance: The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. Objective: To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. Design, Setting, and Participants: The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Main Outcomes and Measures: Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Results: Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by ß1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or ß1-adrenoceptor antagonist protected working memory from stress. Conclusions and Relevance: The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.
Asunto(s)
Canales de Calcio Tipo L , Células Piramidales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Humanos , Animales , Células Piramidales/fisiología , Células Piramidales/metabolismo , Masculino , Trastornos del Conocimiento/fisiopatología , Trastornos del Conocimiento/genética , Corteza Prefrontal/metabolismo , Corteza Prefrontal/fisiopatología , Corteza Prefontal Dorsolateral/fisiología , Macaca mulatta , Memoria a Corto Plazo/fisiología , Femenino , TranscriptomaRESUMEN
Anti-cannabinoid type 1 receptor (CB1 ) polyclonal antibodies are widely used to detect the presence of CB1 in a variety of brain cells and their organelles, including neuronal mitochondria. Surprisingly, we found that anti-CB1 sera, in parallel with CB1 , also recognize the mitochondrial protein stomatin-like protein 2. In addition, we show that the previously reported effect of synthetic cannabinoid WIN 55,212-2 on mitochondrial complex III respiration is not detectable in purified mitochondrial preparations. Thus, our study indicates that a direct relationship between endocannabinoid signaling and mitochondrial functions in the cerebral cortex seems unlikely, and that caution should be taken interpreting findings obtained using anti-CB1 antibodies.