RESUMEN
Ischemic lesions stimulate adult neurogenesis in the dentate gyrus, however, this is not associated with better cognitive function. Furthermore, increased neurogenesis is associated with the formation of aberrant neurons. In a previous study, we showed that a running task after a stroke not only increases neurogenesis but also the number of aberrant neurons without improving general performance. Here, we determined whether stimulation in an enriched environment after a lesion could increase neurogenesis and cognitive function without enhancing the number of aberrant neurons. After an ischemic stroke induced by MCAO, animals were transferred to an enriched environment containing a running wheel, tunnels and nest materials. A GFP-retroviral vector was delivered on day 3 post-stroke and a modified water maze test was performed 6 weeks after the lesion. We found that the enriched environment significantly increased the number of new neurons compared with the unstimulated stroke group but not the number of aberrant cells after a lesion. Increased neurogenesis after environmental enrichment was associated with improved cognitive function. Our study showed that early placement in an enriched environment after a stroke lesion markedly increased neurogenesis and flexible learning but not the formation of aberrant neurons, indicating that rehabilitative training, as a combination of running wheel training and enriched environment housing, improved functional and structural outcomes after a stroke.
Asunto(s)
Cognición , Accidente Cerebrovascular , Ratones , Animales , Cognición/fisiología , Accidente Cerebrovascular/patología , Neuronas/fisiología , Neurogénesis/fisiología , Hipocampo/patologíaRESUMEN
In the aftermath of an acute stroke, numerous signaling cascades that reshape the brain both in the perilesional zone as well as in more distal regions are activated. Despite continuous improvement in the acute treatment of stroke and the sustained research efforts into the pathophysiology of stroke, we critically lag in our integrated understanding of the delayed and chronic responses to ischemic injury. As such, the beneficial or maladaptive effect of some stroke-induced cellular responses is unclear, restricting the advancement of therapeutic strategies to target long-term complications. A prominent delayed effect of stroke is the robust increase in adult neurogenesis, which raises hopes for a regenerative strategy to counter neurological deficits in stroke survivors. In the adult brain, two regions are known to generate new neurons from endogenous stem cells: the subventricular zone (SVZ) and the dentate subgranular zone (SGZ) of the hippocampus. While both niches respond with an increase in neurogenesis post-stroke, there are significant regional differences in the ensuing stages of survival, migration, and maturation, which may differently influence functional outcome. External interventions such as rehabilitative training add a further layer of complexity by independently modulating the process of adult neurogenesis. In this review we summarize the current knowledge regarding the effects of ischemic stroke on neurogenesis in the SVZ and in the SGZ, and the influence of exogenous stimuli such as motor activity or enriched environment (EE). In addition, we discuss the contribution of SVZ or SGZ post-stroke neurogenesis to sensory, motor and cognitive recovery.
RESUMEN
Following stroke, neuronal death takes place both in the infarct region and in brain areas distal to the lesion site including the hippocampus. The hippocampus is critically involved in learning and memory processes and continuously generates new neurons. Dysregulation of adult neurogenesis may be associated with cognitive decline after a stroke lesion. In particular, proliferation of precursor cells and the formation of new neurons are increased after lesion. Within the first week, many new precursor cells die during development. How dying precursors are removed from the hippocampus and to what extent phagocytosis takes place after stroke is still not clear. Here, we evaluated the effect of a prefrontal stroke lesion on the phagocytic activity of microglia in the dentate gyrus (DG) of the hippocampus. Three-months-old C57BL/6J mice were injected once with the proliferation marker BrdU (250 mg/kg) 6 hr after a middle cerebral artery occlusion or sham surgery. The number of apoptotic cells and the phagocytic capacity of the microglia were evaluated by means of immunohistochemistry, confocal microscopy, and 3D-reconstructions. We found a transient but significant increase in the number of apoptotic cells in the DG early after stroke, associated with impaired removal by microglia. Interestingly, phagocytosis of newly generated precursor cells was not affected. Our study shows that a prefrontal stroke lesion affects phagocytosis of apoptotic cells in the DG, a region distal to the lesion core. Whether disturbed phagocytosis might contribute to inflammatory- and maladaptive processes including cognitive impairment following stroke needs to be further investigated.
Asunto(s)
Microglía , Accidente Cerebrovascular , Animales , Giro Dentado , Hipocampo/patología , Ratones , Ratones Endogámicos C57BL , Microglía/patología , Neurogénesis/fisiología , Fagocitosis , Accidente Cerebrovascular/patologíaRESUMEN
Sepsis associated encephalopathy (SAE) is a major complication of patients surviving sepsis with a prevalence up to 70%. Although the initial pathophysiological events of SAE are considered to arise during the acute phase of sepsis, there is increasing evidence that SAE leads to persistent brain dysfunction with severe cognitive decline in later life. Previous studies suggest that the hippocampal formation is particularly involved leading to atrophy in later stages. Thereby, the underlying cellular mechanisms are only poorly understood. Here, we hypothesized that endogenous neural stems cells and adult neurogenesis in the hippocampus are impaired following sepsis and that these changes may contribute to persistent cognitive dysfunction when the animals have physically fully recovered. We used the murine sepsis model of peritoneal contamination and infection (PCI) and combined different labeling methods of precursor cells with confocal microscopy studies to assess the neurogenic niche in the dentate gyrus at day 42 postsepsis. We found that following sepsis i) gliogenesis is increased, ii) the absolute number of radial glia-like cells (type 1 cells), which are considered the putative stem cells, is significantly reduced, iii) the generation of new neurons is not significantly altered, while iv) the synaptic spine maturation of new neurons is impaired with a shift to expression of more immature and less mature spines. In conclusion, sepsis mainly leads to depletion of the neural stem cell pool and enhanced gliogenesis in the dentate gyrus which points towards an accelerated aging of the hippocampus due to septic insult.
Asunto(s)
Células Ependimogliales/patología , Hipocampo/patología , Células-Madre Neurales/patología , Neurogénesis/fisiología , Encefalopatía Asociada a la Sepsis/patología , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
The limited proliferative capacity of neuroprogenitor cells (NPCs) within the periventricular germinal niches (PGNs) located caudal of the subventricular zone (SVZ) of the lateral ventricles together with their high proliferation capacity after isolation strongly implicates cell-extrinsic humoral factors restricting NPC proliferation in the hypothalamic and midbrain PGNs. We comparatively examined the effects of norepinephrine (NE) as an endogenous candidate regulator of PGN neurogenesis in the SVZ as well as the periventricular hypothalamus and the periaqueductal midbrain. Histological and neurochemical analyses revealed that the pattern of NE innervation of the adult PGNs is inversely associated with their in vivo NPC proliferation capacity with low NE levels coupled to high NPC proliferation in the SVZ but high NE levels coupled to low NPC proliferation in hypothalamic and midbrain PGNs. Intraventricular infusion of NE decreased NPC proliferation and neurogenesis in the SVZ-olfactory bulb system, while pharmacological NE inhibition increased NPC proliferation and early neurogenesis events in the caudal PGNs. Neurotoxic ablation of NE neurons using the Dsp4-fluoxetine protocol confirmed its inhibitory effects on NPC proliferation. Contrarily, NE depletion largely impairs NPC proliferation within the hippocampus in the same animals. Our data indicate that norepinephrine has opposite effects on the two fundamental neurogenic niches of the adult brain with norepinephrine being a negative regulator of adult periventricular neurogenesis. This knowledge might ultimately lead to new therapeutic approaches to influence neurogenesis in hypothalamus-related metabolic diseases or to stimulate endogenous regenerative potential in neurodegenerative processes such as Parkinson's disease.
Asunto(s)
Células-Madre Neurales/citología , Norepinefrina/farmacología , Nicho de Células Madre , Animales , Proliferación Celular/efectos de los fármacos , Hipocampo/citología , Ventrículos Laterales/citología , Mesencéfalo/citología , Ratones Endogámicos C57BL , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Bulbo Olfatorio/citología , Fenotipo , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Stroke increases neurogenesis in the adult dentate gyrus in the short term, however, long-term effects at the cellular and functional level are poorly understood. Here we evaluated the impact of an early stroke lesion on neurogenesis and cognitive function of the aging brain. We hypothesized that a stroke disturbs dentate neurogenesis during aging correlate with impaired flexible learning. To address this issue a stroke was induced in 3-month-old C57Bl/6 mice by a middle cerebral artery occlusion (MCAO). To verify long-term changes of adult neurogenesis the thymidine analogue BrdU (5-Bromo-2'-deoxyuridine) was administrated at different time points during aging. One and half months after BrdU injections learning and memory performance were assessed with a modified version of the Morris water maze (MWM) that includes the re-learning paradigm, as well as hippocampus-dependent and -independent search strategies. After MWM performance mice were transcardially perfused. To further evaluate in detail the stroke-mediated changes on stem- and progenitor cells as well as endogenous proliferation nestin-green-fluorescent protein (GFP) mice were used. Adult nestin-GFP mice received a retroviral vector injection in the hippocampus to evaluate changes in the neuronal morphology. At an age of 20 month the nestin-GFP mice were transcardially perfused after MWM performance and BrdU application 1.5 months later. The early stroke lesion significantly decreased neurogenesis in 7.5- and 9-month-old animals and also endogenous proliferation in the latter group. Furthermore, immature doublecortin (DCX)-positive neurons were reduced in 20-month-old nestin-GFP mice after lesion. All MCAO groups showed an impaired performance in the MWM and mostly relied on hippocampal-independent search strategies. These findings indicate that an early ischemic insult leads to a dramatical decline of neurogenesis during aging that correlates with a premature development of hippocampal-dependent deficits. Our study supports the notion that an early stroke might lead to long-term cognitive deficits as observed in human patients after lesion.
Asunto(s)
Disfunción Cognitiva/metabolismo , Neurogénesis/fisiología , Accidente Cerebrovascular/metabolismo , Envejecimiento/fisiología , Animales , Encéfalo/patología , Bromodesoxiuridina/farmacología , Cognición/fisiología , Disfunción Cognitiva/fisiopatología , Giro Dentado/patología , Proteína Doblecortina , Hipocampo/patología , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citologíaRESUMEN
Mitochondria are key organelles in regulating the metabolic state of a cell. In the brain, mitochondrial oxidative metabolism is the prevailing mechanism for neurons to generate ATP. While it is firmly established that neuronal function is highly dependent on mitochondrial metabolism, it is less well-understood how astrocytes function rely on mitochondria. In this study, we investigate if astrocytes require a functional mitochondrial electron transport chain (ETC) and oxidative phosphorylation (oxPhos) under physiological and injury conditions. By immunohistochemistry we show that astrocytes expressed components of the ETC and oxPhos complexes in vivo. Genetic inhibition of mitochondrial transcription by conditional deletion of mitochondrial transcription factor A (Tfam) led to dysfunctional ETC and oxPhos activity, as indicated by aberrant mitochondrial swelling in astrocytes. Mitochondrial dysfunction did not impair survival of astrocytes, but caused a reactive gliosis in the cortex under physiological conditions. Photochemically initiated thrombosis induced ischemic stroke led to formation of hyperfused mitochondrial networks in reactive astrocytes of the perilesional area. Importantly, mitochondrial dysfunction significantly reduced the generation of new astrocytes and increased neuronal cell death in the perilesional area. These results indicate that astrocytes require a functional ETC and oxPhos machinery for proliferation and neuroprotection under injury conditions.
RESUMEN
Amyotrophic lateral sclerosis is characterized by progressive death of motor neurons (MNs) with glutamate excitotoxicity and mitochondrial Ca2+ overload as critical mechanisms in disease pathophysiology. We used MNs from G93AhSOD1 and nontransgenic embryonic cultures and adult mice to analyze the expression of the main mitochondrial calcium uniporter (MCU). MCU was overexpressed in cultured embryonic G93AhSOD1 MNs compared to nontransgenic MNs but downregulated in MNs from adult G93AhSOD1 mice. Furthermore, cultured embryonic G93AhSOD1 were rescued from kainate-induced excitotoxicity by the Ca2+/calmodulin-dependent protein kinase type II inhibitor; KN-62, which reduced MCU expression in G93AhSOD1 MNs. MCU activation via kaempferol neither altered MCU expression nor influenced MN survival. However, its acute application served as a fine tool to study spontaneous Ca2+ activity in cultured neurons which was significantly altered by the mutated hSOD1. Pharmacological manipulation of MCU expression might open new possibilities to fight excitotoxic damage in amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Astrocitos/metabolismo , Mitocondrias/metabolismo , Neuronas Motoras/metabolismo , Animales , Canales de Calcio , Modelos Animales de Enfermedad , Ácido Glutámico/metabolismo , Ratones Transgénicos , Superóxido Dismutasa/metabolismoRESUMEN
Stroke robustly stimulates adult neurogenesis in the hippocampal dentate gyrus. It is currently unknown whether this process induces beneficial or maladaptive effects, but morphological and behavioral studies have reported aberrant neurogenesis and impaired hippocampal-dependent memory following stroke. However, the intrinsic function and network incorporation of adult-born granule cells (ABGCs) after ischemia is unclear. Using patch-clamp electrophysiology, we evaluated doublecortin-positive (DCX+) ABGCs as well as DCX- dentate gyrus granule cells 2 weeks after a stroke or sham operation in DCX/DsRed transgenic mice of either sex. The developmental status, intrinsic excitability, and synaptic excitability of ABGCs were accelerated following stroke, while dendritic morphology was not aberrant. Regression analysis revealed uncoupled development of intrinsic and network excitability, resulting in young, intrinsically hyperexcitable ABGCs receiving disproportionately large glutamatergic inputs. This aberrant functional maturation in the subgroup of ABGCs in the hippocampus may contribute to defective hippocampal function and increased seizure susceptibility following stroke.SIGNIFICANCE STATEMENT Stroke increases hippocampal neurogenesis but the functional consequences of the postlesional response is mostly unclear. Our findings provide novel evidence of aberrant functional maturation of newly generated neurons following stroke. We demonstrate that stroke not only causes an accelerated maturation of the intrinsic and synaptic parameters of doublecortin-positive, new granule cells in the hippocampus, but that this accelerated development does not follow physiological dynamics due to uncoupled intrinsic and synaptic maturation. Hyperexcitable immature neurons may contribute to disrupted network integration following stroke.
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Giro Dentado/fisiopatología , Infarto de la Arteria Cerebral Media/fisiopatología , Neurogénesis , Potenciales Sinápticos , Animales , Giro Dentado/patología , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Femenino , Ácido Glutámico/metabolismo , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Neuropéptidos/genética , Neuropéptidos/metabolismoRESUMEN
Motor neurons are markedly vulnerable to excitotoxicity mostly by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) stimulation and are principal targets in the neurodegenerative disease Amyotrophic Lateral Sclerosis. Interferon-gamma (IFN-γ), a pro-inflammatory cytokine, can independently cause neuronal dysfunction by triggering calcium influx through a calcium-permeable complex of IFN-γ receptor 1(IFNGR1) subunit and AMPAR subunit GluR1. This receptor complex is formed via a non-canonical neuron-specific IFN-γ pathway that involves Jak1/Stat1 and Protein Kinase A. In this study, we explore the expression of the pathway's participants for the first time in the hSOD1G93A Amyotrophic Lateral Sclerosis mouse model. Elevated IFNGR1 and GluR1 are detected in motor neurons of hSOD1G93A symptomatic mice ex vivo, unlike the downstream targets - Jak1, Stat1, and Protein Kinase A. We, also, determine effects of IFN-γ alone or in the presence of an excitotoxic agent, kainate, on motor neuron survival in vitro. IFN-γ induces neuronal damage, but does not influence kainate-mediated excitotoxicity. Increased IFNGR1 can most likely sensitize motor neurons to excitotoxic insults involving GluR1 and/or pathways mediated by IFN-γ, thus, serving as a potential direct link between neurodegeneration and inflammation in Amyotrophic Lateral Sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Neuronas Motoras/metabolismo , Receptores AMPA/metabolismo , Receptores de Interferón/metabolismo , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Transducción de Señal , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Regulación hacia Arriba , Receptor de Interferón gammaRESUMEN
Stroke significantly stimulates neurogenesis in the adult dentate gyrus, though the functional role of this postlesional response is mostly unclear. Recent findings suggest that newborn neurons generated in the context of stroke may fail to correctly integrate into pre-existing networks. We hypothesized that increased neurogenesis in the dentate gyrus following stroke is associated with aberrant neurogenesis and impairment of hippocampus-dependent memory. To address these questions we used the middle cerebral artery occlusion model (MCAO) in mice. Animals were housed either under standard conditions or with free access to running wheels. Newborn granule cells were labelled with the thymidine analoque EdU and retroviral vectors. To assess memory performance, we employed a modified version of the Morris water maze (MWM) allowing differentiation between hippocampus dependent and independent learning strategies. Newborn neurons were morphologically analyzed using confocal microscopy and Neurolucida system at 7 weeks. We found that neurogenesis was significantly increased following MCAO. Animals with MCAO needed more time to localize the platform and employed less hippocampus-dependent search strategies in MWM versus controls. Confocal studies revealed an aberrant cell morphology with basal dendrites and an ectopic location (e.g. hilus) of new granule cells born in the ischemic brain. Running increased the number of new neurons but also enhanced aberrant neurogenesis. Running, did not improve the general performance in the MWM but slightly promoted the application of precise spatial search strategies. In conclusion, ischemic insults cause hippocampal-dependent memory deficits which are associated with aberrant neurogenesis in the dentate gyrus indicating ischemia-induced maladaptive plasticity in the hippocampus.
Asunto(s)
Giro Dentado/patología , Neurogénesis , Memoria Espacial , Accidente Cerebrovascular/psicología , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , Aprendizaje por Laberinto , Ratones , Plasticidad Neuronal , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Aberrations in intracellular calcium (Ca2+) have been well established within amyotrophic lateral sclerosis (ALS), a severe motor neuron disease. Intracellular Ca2+ concentration is controlled in part through the endoplasmic reticulum (ER) mitochondria Ca2+ cycle (ERMCC). The ER supplies Ca2+ to the mitochondria at close contacts between the two organelles, i.e. the mitochondria-associated ER membranes (MAMs). The Sigma 1 receptor (Sig1R) is enriched at MAMs, where it acts as an inter-organelle signaling modulator. However, its impact on intracellular Ca2+ at the cellular level remains to be thoroughly investigated. Here, we used cultured embryonic mice spinal neurons to investigate the influence of Sig1R activation on intracellular Ca2+ homeostasis in the presence of G93AhSOD1 (G93A), an established ALS-causing mutation. Sig1R expression was increased in G93A motor neurons relative to non-transgenic (nontg) controls. Furthermore, we demonstrated significantly reduced bradykinin-sensitive intracellular Ca2+ stores in G93A spinal neurons, which were normalized by the Sig1R agonist SA4503. Moreover, SA4503 accelerated cytosolic Ca2+ clearance following a) AMPAR activation by kainate and b) IP3R-mediated ER Ca2+ release following bradykinin stimulation in both genotypes. PRE-084 (another Sig1R agonist) did not exert any significant effects on cytosolic Ca2+. Both Sig1R expression and functionality were altered by the G93A mutation, indicating the centrality of Sig1R in ALS pathology. Here, we showed that intracellular Ca2+ shuttling can be manipulated by Sig1R activation, thus demonstrating the value of using the pharmacological manipulation of Sig1R to understand Ca2+ homeostasis.
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Esclerosis Amiotrófica Lateral/metabolismo , Señalización del Calcio , Neuronas Motoras/metabolismo , Receptores sigma/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Calcio/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Piperazinas/administración & dosificación , Receptores AMPA/metabolismo , Receptores sigma/agonistas , Médula Espinal/metabolismo , Superóxido Dismutasa-1/genética , Receptor Sigma-1RESUMEN
BACKGROUND: Increased motor activity or social interactions through enriched environment are strong stimulators of grey and white matter plasticity in the adult rodent brain. In the present study we evaluated whether specific reaching training of the dominant forelimb (RT) and stimulation of unspecific motor activity through enriched environment (EE) influence the generation of distinct oligodendrocyte subpopulations in the sensorimotor cortex and corpus callosum of the adult rat brain. Animals were placed in three different housing conditions: one group was transferred to an EE, a second group received daily RT, whereas a third group remained in the standard cage. Bromodeoxyuridine (BrdU) was applied at days 2-6 after start of experiments and animals were allowed to survive for 10 and 42 days. RESULTS: Enriched environment and daily reaching training of the dominant forelimb significantly increased the number of newly differentiated GSTπ+ oligodendrocytes at day 10 and newly differentiated CNPase+ oligodendrocytes in the sensorimotor cortex at day 42. The myelin level as measured by CNPase expression was increased in the frontal cortex at day 42. Distribution of newly differentiated NG2+ subpopulations changed between 10 and 42 days with an increase of GSTπ+ subtypes and a decrease of NG2+ cells in the sensorimotor cortex and corpus callosum. Analysis of neuronal marker doublecortin (DCX) showed that more than half of NG2+ cells express DCX in the cortex. The number of new DCX+NG2+ cells was reduced by EE at day 10. CONCLUSIONS: Our results indicate for the first time that specific and unspecific motor training conditions differentially alter the process of differentiation from oligodendrocyte subpopulations, in particular NG2+DCX+ cells, in the sensorimotor cortex and corpus callosum.
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Cuerpo Calloso/fisiología , Vivienda para Animales , Destreza Motora/fisiología , Oligodendroglía/fisiología , Práctica Psicológica , Corteza Sensoriomotora/fisiología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Antígenos/metabolismo , Bromodesoxiuridina , Cuerpo Calloso/citología , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Miembro Anterior/fisiología , Lóbulo Frontal/citología , Lóbulo Frontal/fisiología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Animales , Neurogénesis/fisiología , Neuropéptidos/metabolismo , Oligodendroglía/citología , Proteoglicanos/metabolismo , Distribución Aleatoria , Ratas Wistar , Tiempo de Reacción , Corteza Sensoriomotora/citologíaRESUMEN
Precise regulation of cellular metabolism is hypothesized to constitute a vital component of the developmental sequence underlying the life-long generation of hippocampal neurons from quiescent neural stem cells (NSCs). The identity of stage-specific metabolic programs and their impact on adult neurogenesis are largely unknown. We show that the adult hippocampal neurogenic lineage is critically dependent on the mitochondrial electron transport chain and oxidative phosphorylation machinery at the stage of the fast proliferating intermediate progenitor cell. Perturbation of mitochondrial complex function by ablation of the mitochondrial transcription factor A (Tfam) reproduces multiple hallmarks of aging in hippocampal neurogenesis, whereas pharmacological enhancement of mitochondrial function ameliorates age-associated neurogenesis defects. Together with the finding of age-associated alterations in mitochondrial function and morphology in NSCs, these data link mitochondrial complex function to efficient lineage progression of adult NSCs and identify mitochondrial function as a potential target to ameliorate neurogenesis-defects in the aging hippocampus.
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Células Madre Adultas/metabolismo , Envejecimiento/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Neurogénesis , Neuronas/metabolismo , Células Madre Adultas/citología , Animales , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Hipocampo/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Células-Madre Neurales , Neuronas/citología , Fosforilación OxidativaRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with intracellular Ca(2+) dysregulation. The P2X receptor family is comprised of ligand-gated ion channels that respond to extracellular adenosine triphosphate (ATP) and increases permeability of calcium into the cell. The underlying mechanisms of purinergic signalling on peripheral blood mononuclear cells (PBMCs) in ALS remain unclear. Herein, we studied the expression of P2X4/P2X7 receptors and calcium homeostasis in blood cells of ALS patients. METHODS: We used PBMCs from 42 ALS patients and 19 controls. Purinergic receptors P2X4 (P2X4R) and P2X7 (P2X7R) were examined using western blot analysis. The effect of exogenous ATP on intracellular Ca(2+) homeostasis in monocytes was measured using fluorimetry by Fura-2 on a single-cell level. RESULTS: Western blot analysis revealed stable P2X4R expression in patients and controls. P2X7R expression was significantly reduced (p=0.012) in ALS patients. Repetitive long-term ATP stimulation caused a sustained decrease in Ca(2+) levels in the ALS group as measured by the area under the curve, peak amplitude and peak height. CONCLUSION: These results confirm our hypothesis that Ca(2+) abnormalities in ALS are measurable in immune cells. These findings suggest that the reduction of P2X7 receptor expression on PBMCs leads to intracellular calcium dysregulation. Our study improves the understanding of ALS pathophysiology and proposes PBMCs as a non-invasive source to study ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/metabolismo , Señalización del Calcio , Leucocitos Mononucleares/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Anciano , Regulación hacia Abajo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptores Purinérgicos P2X4/metabolismoRESUMEN
BACKGROUND: Recovery following stroke depends on cellular plasticity in the perilesional zone (PZ). Doublecortin (DCX), a protein mainly labeling immature neurons in neurogenic niches is also highly expressed in the vicinity of focal cortical infarcts. Notably, the number of DCX+ cells positively correlates with the recovery of functional deficits after stroke though the nature and origin of these cells remains unclear. RESULTS: In the present study, we aimed to characterize the population of DCX+ cells in the vicinity of ischemic infarcts in a mouse model in detail. Employing a photothrombosis model, distinct immunohistochemical techniques, stereology and confocal microscopy, we show that: i) DCX+ cells in the perilesional zone do not constitute a homogenous population and two cell types, stellate and polar cells can be distinguished according to their morphology. ii) Stellate cells are mainly located in the lateral and medial vicinity of the insult and express astrocytic markers. iii) Polar cells are found almost exclusively in the corpus callosum region including in the preserved deep cortical layers close to the subventricular zone (SVZ). Further, they do not show any colocalisation of glial markers. Polar morphology and distribution suggest a migration towards the lesion. CONCLUSIONS: In summary, our findings provide evidence that in mice DCX+ cells in the perilesional zone of cortical infarcts comprise a distinct cell population and the majority of cells are of glial nature.
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Proteínas Asociadas a Microtúbulos/metabolismo , Neuroglía/metabolismo , Neuropéptidos/metabolismo , Corteza Sensoriomotora/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , Microscopía Confocal , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Corteza Sensoriomotora/patología , Nicho de Células Madre/fisiología , Accidente Cerebrovascular/patología , Factores de TiempoRESUMEN
Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one-third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4-GFP expression in NSCs, neuronal-committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12-promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2-positive neural stem/early progenitor cells, NeuroD-positive neuronal-committed progenitors, and DCX-positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one-third of the GCL as differentiation area for immature granule neurons.
Asunto(s)
Giro Dentado/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas/fisiología , Receptores CXCR4/metabolismo , Factores de Edad , Animales , Fármacos Anti-VIH/farmacología , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Bencilaminas , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL12/farmacología , Ciclamas , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Regulación del Desarrollo de la Expresión Génica/genética , Compuestos Heterocíclicos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuropéptidos/metabolismo , Receptores CXCR4/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
The distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of neurodegenerative disorders affecting the lower motoneuron. In a family with both autosomal-dominant dHMN and dHMN type V (dHMN/dHMN-V) present in three generations, we excluded mutations in all genes known to be associated with a dHMN phenotype through Sanger sequencing and defined three potential loci through linkage analysis. Whole-exome sequencing of two affected individuals revealed a single candidate variant within the linking regions, i.e., a splice-site alteration in REEP1 (c.304-2A>G). A minigene assay confirmed complete loss of splice-acceptor functionality and skipping of the in-frame exon 5. The resulting mRNA is predicted to be expressed at normal levels and to encode an internally shortened protein (p.102_139del). Loss-of-function REEP1 mutations have previously been identified in dominant hereditary spastic paraplegia (HSP), a disease associated with upper-motoneuron pathology. Consistent with our clinical-genetic data, we show that REEP1 is strongly expressed in the lower motoneurons as well. Upon exogeneous overexpression in cell lines we observe a subcellular localization defect for p.102_139del that differs from that observed for the known HSP-associated missense mutation c.59C>A (p.Ala20Glu). Moreover, we show that p.102_139del, but not p.Ala20Glu, recruits atlastin-1, i.e., one of the REEP1 binding partners, to the altered sites of localization. These data corroborate the loss-of-function nature of REEP1 mutations in HSP and suggest that a different mechanism applies in REEP1-associated dHMN.
Asunto(s)
Proteínas de Transporte de Membrana/genética , Mutación , Enfermedades del Sistema Nervioso Periférico/genética , Línea Celular , Exoma , Femenino , Humanos , Masculino , Linaje , Análisis de Secuencia de ADNRESUMEN
BACKGROUND AND PURPOSE: Adult neurogenesis in the dentate gyrus is a unique form of brain plasticity that is strongly stimulated after stroke. We investigate the morphological properties of new granule cells, which are born and develop after the ischemic insult, and query whether these adult-born neurons properly integrate into the pre-existing hippocampal circuitries. METHODS: Two well-established models were used to induce either small cortical infarcts (photothrombosis model) or large territorial infarcts (transient middle cerebral artery occlusion model). New granule cells were labeled 4 days after the initial insult by intrahippocampal injection of a retroviral vector encoding green fluorescent protein and newborn neurons were morphologically analyzed using a semiautomatic Neurolucida system and confocal laser scanning microscopy at 6 weeks. RESULTS: Approximately 5% to 10% of newborn granule cells displayed significant morphological abnormalities comprising additional basal dendrites and, after middle cerebral artery occlusion, also ectopic cell position. The extent of morphological abnormalities was higher after large territorial infarcts and seems to depend on the severity of ischemic damage. An increased portion of mushroom spines in aberrant neurons suggests stable synaptic integration. However, poststroke generated granule cells with regular appearance also demonstrate alterations in dendritic complexity and spine morphology. CONCLUSIONS: The remarkable stimulation of dentate neurogenesis after stroke coincides with an increased rate of aberrantly integrated neurons, which may contribute to functional impairments and, hypothetically, favor pathogenesis of adjustment disorders, cognitive deficits, or epilepsy often seen in stroke patients.