RESUMEN
Fluorescence recovery after photobleaching (FRAP) is a laser method of light microscopy to evaluate the rapid movement of fluorescent molecules. To have a more reliable approach to analyze data from FRAP, we designed Fraping, a free access R library to data analysis obtained from FRAP. Unlike other programs, Fraping has a new form of analyzing curves of FRAP using statistical analysis based on the average curve difference. To evaluate our library, we analyzed the differences of actin polymerization in real time between dendrites and secondary neurites of cultured neuron transfected with LifeAct to track F-actin changes of neurites. We found that Fraping provided greater sensitivity than the conventional model using mobile fraction analysis. Likewise, this approach allowed us to normalize the fluorescence to the size area of interest and adjust data curves choosing the best parametric model. In addition, this library was supplemented with data simulation to have a more significant enrichment for the analysis behavior. We concluded that Fraping is a method that reduces bias when analyzing two data groups as compared with the conventional methods. This method also allows the users to choose a more suitable analysis approach according to their requirements. RESEARCH HIGHLIGHTS: Fraping is a new programming tool to analyze FRAP data to normalize fluorescence recovery curves. The conventional method uses one-point analysis, and the new one compares all the points to define the similarity of the fluorescence recovery.
Asunto(s)
Actinas , Recuperación de Fluorescencia tras Fotoblanqueo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Actinas/análisis , Animales , Polimerizacion , Neuritas , Neuronas/metabolismo , Neuronas/química , Células Cultivadas , Dendritas/química , Dendritas/metabolismoRESUMEN
Emerging evidence highlights the relevance of the protein post-translational modification by SUMO (Small Ubiquitin-like Modifier) in the central nervous system for modulating cognition and plasticity in health and disease. In these processes, astrocyte-to-neuron crosstalk mediated by extracellular vesicles (EVs) plays a yet poorly understood role. Small EVs (sEVs), including microvesicles and exosomes, contain a molecular cargo of lipids, proteins, and nucleic acids that define their biological effect on target cells. Here, we investigated whether SUMOylation globally impacts the sEV protein cargo. For this, sEVs were isolated from primary cultures of astrocytes by ultracentrifugation or using a commercial sEV isolation kit. SUMO levels were regulated: 1) via plasmids that over-express SUMO, or 2) via experimental conditions that increase SUMOylation, i.e., by using the stress hormone corticosterone, or 3) via the SUMOylation inhibitor 2-D08 (2',3',4'-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one). Corticosterone and 2-D08 had opposing effects on the number of sEVs and on their protein cargo. Proteomic analysis showed that increased SUMOylation in corticosterone-treated or plasmid-transfected astrocytes increased the presence of proteins related to cell division, transcription, and protein translation in the derived sEVs. When sEVs derived from corticosterone-treated astrocytes were transferred to neurons to assess their impact on protein synthesis using the fluorescence non-canonical amino acid tagging assay (FUNCAT), we detected an increase in protein synthesis, while sEVs from 2-D08-treated astrocytes had no effect. Our results show that SUMO conjugation plays an important role in the modulation of the proteome of astrocyte-derived sEVs with a potential functional impact on neurons.
Asunto(s)
Vesículas Extracelulares , Proteoma , Proteoma/metabolismo , Astrocitos/metabolismo , Sumoilación , Proteómica , Corticosterona/farmacología , Vesículas Extracelulares/metabolismo , Neuronas/metabolismo , Dendritas/metabolismoRESUMEN
Leucine-rich repeat (LRR) transmembrane proteins have been directly linked to neurodevelopmental and cognitive disorders. We have previously shown that the LRR transmembrane protein, leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1), is a physiological regulator of dendrite complexity of hippocampal pyramidal neurons and social behavior. In this study, we performed a battery of behavioral tests to evaluate spatial memory and cognitive capabilities in Lrig1 mutant mice. The cognitive assessment demonstrated deficits in recognition and spatial memory, evaluated by novel object recognition and object location tests. Moreover, we found that Lrig1-deficient mice present specific impairments in the processing of similar but not dissimilar locations in a spatial pattern separation task, which was correlated with an enhanced dendritic growth and branching of Doublecortin-positive immature granule cells of the dentate gyrus. Altogether, these findings indicate that Lrig1 plays an essential role in controlling morphological and functional plasticity in the hippocampus.
Asunto(s)
Cognición , Hipocampo , Animales , Cognición/fisiología , Dendritas/metabolismo , Hipocampo/metabolismo , Dominios de Inmunoglobulinas , Leucina/metabolismo , RatonesRESUMEN
Insulin signaling through the insulin receptor has long been studied in classic target organs, such as adipose tissue and skeletal muscle, where one of its effects is to increase glucose uptake. Insulin and insulin receptor are present in many areas of the brain, but the functions of brain insulin signaling outside feeding circuits are not well defined. It has been proposed that hippocampal insulin signaling is important for memory, that brain insulin signaling is deficient in Alzheimer's disease, and that intranasal insulin treatment improves cognition, but the mechanisms remain unclear and do not seem to involve increased glucose uptake by neurons. The molecular behavior of the insulin receptor itself is not well known in living neurons; therefore, we investigated the spatial dynamics of the insulin receptor on somatodendritic membranes of live rat hippocampal neurons in culture. Using single-molecule tracking of quantum dot-tagged insulin receptors and single-particle tracking photoactivation localization microscopy, we show that the insulin receptor is distributed over both dendritic shafts and spines. Using colocalization with synaptic markers, we also show that in contrast to the glutamate receptor subunit glutamate receptor subunit A1, the dynamics of the insulin receptor are not affected by association with excitatory synapses; however, the insulin receptor is immobilized by components of inhibitory synapses. The mobility of the insulin receptor is reduced both by low concentrations of the pro-inflammatory cytokine tumor necrosis factor α and by cholesterol depletion, suggesting an association with sphingolipid-rich membrane domains. On the other hand, the insulin receptor dynamics in hippocampal neurons are not affected by increased excitatory signaling. Finally, using real-time single-event quantification, we find evidence of strong insulin receptor exocytosis on dendritic shafts. Our results suggest an association of the neuronal insulin receptor with specific elements of the dendritic shaft, rather than excitatory synapses.
Asunto(s)
Dendritas/metabolismo , Hipocampo/metabolismo , Receptor de Insulina/metabolismo , Animales , Células Cultivadas , Femenino , Masculino , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The pro-oxidant compound okadaic acid (OKA) mimics alterations found in Alzheimer's disease (AD) as oxidative stress and tau hyperphosphorylation, leading to neurodegeneration and cognitive decline. Although loss of dendrite complexity occurs in AD, the study of this post-synaptic domain in chemical-induced models remains unexplored. Moreover, there is a growing expectation for therapeutic adjuvants to counteract these brain dysfunctions. Melatonin, a free-radical scavenger, inhibits tau hyperphosphorylation, modulates phosphatases, and strengthens dendritic arbors. Thus, we determined if OKA alters the dendritic arbors of hilar hippocampal neurons and whether melatonin prevents, counteracts, or reverses these damages. Rat organotypic cultures were incubated with vehicle, OKA, melatonin, and combined treatments with melatonin either before, simultaneously, or after OKA. DNA breaks were assessed by TUNEL assay and nuclei were counterstained with DAPI. Additionally, MAP2 was immunostained to assess the dendritic arbor properties by the Sholl method. In hippocampal hilus, OKA increased DNA fragmentation and reduced the number of MAP2(+) cells, whereas melatonin protected against oxidation and apoptosis. Additionally, OKA decreased the dendritic arbor complexity and melatonin not only counteracted, but also prevented and reversed the dendritic arbor retraction, highlighting its role in post-synaptic domain integrity preservation against neurodegenerative events in hippocampal neurons.
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Dendritas/efectos de los fármacos , Dendritas/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Melatonina/farmacología , Ácido Ocadaico/farmacología , Oxidantes/farmacología , Animales , Fragmentación del ADN , Dendritas/patología , Inmunohistoquímica , Fármacos Neuroprotectores/farmacología , Organoides/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Programmed cell death 4 (PDCD4) protein is a tumor suppressor that inhibits translation through the mTOR-dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons. Using loss- and gain-of-function experiments in cortical and dorsal root ganglia primary neurons, we demonstrated the capacity of PDCD4 to negatively control axonal growth. To explore PDCD4 transcriptome and translatome targets, we used Ribo-seq and uncovered a list of potential targets with known functions as axon/neurite outgrowth regulators. In addition, we observed that PDCD4 can be locally synthesized in adult axons in vivo, and its levels decrease at the site of peripheral nerve injury and before nerve regeneration. Overall, our findings demonstrate that PDCD4 can act as a new regulator of axonal growth via the selective control of translation, providing a target mechanism for axon regeneration and neuronal plasticity processes in neurons.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Axones/metabolismo , Dendritas/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Mutación con Ganancia de Función , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Mutación con Pérdida de Función , Masculino , Ratones , Células PC12 , Cultivo Primario de Células , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Ratas , Regulación hacia ArribaRESUMEN
OBJECTIVE: Drebrins are crucial for synaptic function and dendritic spine development, remodeling, and maintenance. In temporal lobe epilepsy (TLE) patients, a significant hippocampal synaptic reorganization occurs, and synaptic reorganization has been associated with hippocampal hyperexcitability. This study aimed to evaluate, in TLE patients, the hippocampal expression of drebrin using immunohistochemistry with DAS2 or M2F6 antibodies that recognize adult (drebrin A) or adult and embryonic (pan-drebrin) isoforms, respectively. METHODS: Hippocampal sections from drug-resistant TLE patients with hippocampal sclerosis (HS; TLE, n = 33), of whom 31 presented with type 1 HS and two with type 2 HS, and autopsy control cases (n = 20) were assayed by immunohistochemistry and evaluated for neuron density, and drebrin A and pan-drebrin expression. Double-labeling immunofluorescences were performed to localize drebrin A-positive spines in dendrites (MAP2), and to evaluate whether drebrin colocalizes with inhibitory (GAD65) and excitatory (VGlut1) presynaptic markers. RESULTS: Compared to controls, TLE patients had increased pan-drebrin in all hippocampal subfields and increased drebrin A-immunopositive area in all hippocampal subfields but CA1. Drebrin-positive spine density followed the same pattern as total drebrin quantification. Confocal microscopy indicated juxtaposition of drebrin-positive spines with VGlut1-positive puncta, but not with GAD65-positive puncta. Drebrin expression in the dentate gyrus of TLE cases was associated negatively with seizure frequency and positively with verbal memory. TLE patients with lower drebrin-immunopositive area in inner molecular layer (IML) than in outer molecular layer (OML) had a lower seizure frequency than those with higher or comparable drebrin-immunopositive area in IML compared with OML. SIGNIFICANCE: Our results suggest that changes in drebrin-positive spines and drebrin expression in the dentate gyrus of TLE patients are associated with lower seizure frequency, more preserved verbal memory, and a better postsurgical outcome.
Asunto(s)
Epilepsia Refractaria/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Hipocampo/metabolismo , Neuropéptidos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Lobectomía Temporal Anterior , Región CA1 Hipocampal/metabolismo , Región CA2 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Estudios de Casos y Controles , Dendritas/metabolismo , Dendritas/patología , Giro Dentado/metabolismo , Epilepsia Refractaria/patología , Epilepsia Refractaria/cirugía , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/cirugía , Femenino , Glutamato Descarboxilasa/metabolismo , Hipocampo/patología , Hipocampo/cirugía , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Plasticidad Neuronal , Esclerosis , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Emerging evidence shows that Rab11 recycling endosomes (REs Rab11) are essential for several neuronal processes, including the proper functioning of growth cones, synapse architecture regulation and neuronal migration. However, several aspects of REs Rab11 remain unclear, such as its sub-cellular distribution across neuronal development, contribution to dendritic tree organization and its consequences in memory formation. In this work we show a spatio-temporal correlation between the endogenous localization of REs Rab11 and developmental stage of neurons. Furthermore, Rab11-suppressed neurons showed an increase on dendritic branching (without altering total dendritic length) and misdistribution of dendritic proteins in cultured neurons. In addition, suppression of Rab11 in adult rat brains in vivo (by expressing shRab11 through lentiviral infection), showed a decrease on both the sensitivity to induce long-term potentiation and hippocampal-dependent memory acquisition. Taken together, our results suggest that REs Rab11 expression is required for a proper dendritic architecture and branching, controlling key aspects of synaptic plasticity and spatial memory formation.
Asunto(s)
Dendritas/metabolismo , Plasticidad Neuronal , Neuronas/fisiología , Memoria Espacial , Proteínas de Unión al GTP rab/genética , Animales , Giro Dentado/fisiología , Fenómenos Electrofisiológicos , Femenino , Hipocampo/fisiología , Potenciación a Largo Plazo , Masculino , Embarazo , Ratas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
In the last few decades, it has been established that astrocytes play key roles in the regulation of neuronal morphology. However, the contribution of astrocyte-derived small extracellular vesicles (sEVs) to morphological differentiation of neurons has only recently been addressed. Here, we showed that cultured astrocytes expressing a GFP-tagged version of the stress-regulated astrocytic enzyme Aldolase C (Aldo C-GFP) release small extracellular vesicles (sEVs) that are transferred into cultured hippocampal neurons. Surprisingly, Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) displayed an exacerbated capacity to reduce the dendritic complexity in developing hippocampal neurons compared to sEVs derived from control (i.e., GFP-expressing) astrocytes. Using bioinformatics and biochemical tools, we found that the total content of overexpressed Aldo C-GFP correlates with an increased content of endogenous miRNA-26a-5p in both total astrocyte homogenates and sEVs. Notably, neurons magnetofected with a nucleotide sequence that mimics endogenous miRNA-26a-5p (mimic 26a-5p) not only decreased the levels of neuronal proteins associated to morphogenesis regulation, but also reproduced morphological changes induced by Aldo-C-GFP sEVs. Furthermore, neurons magnetofected with a sequence targeting miRNA-26a-5p (antago 26a-5p) were largely resistant to Aldo C-GFP sEVs. Our results support a novel and complex level of astrocyte-to-neuron communication mediated by astrocyte-derived sEVs and the activity of their miRNA content.
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Astrocitos/metabolismo , Vesículas Extracelulares/metabolismo , MicroARNs/metabolismo , Animales , Astrocitos/citología , Diferenciación Celular/fisiología , Células Cultivadas , Dendritas/metabolismo , Femenino , Fructosa-Bifosfato Aldolasa/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Adult neurogenesis occurs in the dentate gyrus (DG) of the hippocampus. New neurons help to counteract the effects of stress and several interventions including antidepressant drugs, environmental modifications and internal factors act pro-neurogenic with consequences in the dorsal and ventral DG. Melatonin, the main product synthesized by the pineal gland, induces antidepressant-like effects and modulates several events of the neurogenic process. However, the information related to the capability of melatonin to modulate dendrite maturation and complexity in the dorsal and ventral regions of the DG and their correlation with its antidepressant-like effect is absent. Thus, in this study, we analyzed the impact of melatonin (0, 0.5, 1, 2.5, 5 or 10 mg/kg) administered daily for fourteen days on the number, dendrite complexity and distribution of doublecortin (DCX)-cells in the dorsal-ventral regions of the DG in male Balb/C mice. Doublecortin is a microtubule-associated protein that is expressed during the course of dendritic maturation of newborn neurons. Also, we analyzed the impact of melatonin on despair-like behavior in the forced swim test. We first found a significant increase in the number and higher dendrite complexity, mainly with the doses of 2.5, 5 and 10 mg/kg of melatonin (81%, 122%, 78%). These cells showed more complex dendritic trees in the ventral- and the dorsal- DG. Concomitantly, the doses of 5 and 10 mg/kg of melatonin decreased depressant-like behavior (76%, 82%). Finally, the data corroborate the antidepressant-like effect of melatonin and the increasing number of doublecortin-associated cells. Besides, the data indicate that melatonin favors the number and dendrite complexity of DCX-cells in the dorsal- and ventral- region of the DG, which may explain part of the antidepressant-like effect of melatonin.
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Antidepresivos/uso terapéutico , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Melatonina/uso terapéutico , Animales , Depresión/tratamiento farmacológico , Depresión/metabolismo , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Asociadas a Microtúbulos/metabolismo , Neurogénesis/efectos de los fármacos , Neuropéptidos/metabolismoRESUMEN
The glial cell line-derived neurotrophic factor (GDNF) is required for the survival and differentiation of diverse neuronal populations during nervous system development. Despite the high expression of GDNF and its receptor GFRα1 in the adult hippocampus, the functional role of this system remains unknown. Here, we show that GDNF, acting through its GFRα1 receptor, controls dendritic structure and spine density of adult-born granule cells, which reveals that GFRα1 is required for their integration into preexisting circuits. Moreover, conditional mutant mice for GFRα1 show deficits in behavioral pattern separation, a task in which adult neurogenesis is known to play a critical role. We also find that running increases GDNF in the dentate gyrus and promotes GFRα1-dependent CREB (cAMP response element-binding protein) activation and dendrite maturation. Together, these findings indicate that GDNF/GFRα1 signaling plays an essential role in the plasticity of adult circuits, controlling the integration of newly generated neurons.
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Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Hipocampo/citología , Neurogénesis , Neuronas/metabolismo , Animales , Conducta Animal , Dendritas/metabolismo , Giro Dentado/metabolismo , Ratones , Condicionamiento Físico Animal , Memoria EspacialRESUMEN
N-methyl-D-aspartate receptors (NMDARs) that contain the NR2A and NR2B subunits play a critical role in neuronal plasticity and dendritogenesis. Gain-and-loss-of function studies indicate that NR2B, but not NR2A, promotes dendritic branching. Accumulating evidence indicates that stimulation of NMDARs activates NADPH oxidase (NOX2), thereby generating superoxide. However, the molecular underpinnings of this process are not understood. RasGRF1, a guanine nucleotide exchange factor, is key for several forms of neuronal plasticity and interacts directly with the tail of NR2B. We investigated whether the NR2B-NMDAR/RasGRF1 pathway regulates the activity of NOX2 and whether superoxide production is required for dendritogenesis. We measured superoxide production in developing primary cultures of hippocampal neurons from 3 to 25 days in vitro (DIV) with the probe dihydroethidium (dHE). We found the highest dHE levels at early and intermediate developmental stages (3-15 DIV), when the NR2B-NMDAR expression is abundant. During these early/intermediate developmental stages, but not in mature neurons (>15 DIV), NMDAR activity is required for superoxide production. We also found that disrupting the NR2B-RasGRF1 interaction led to reduced dHE fluorescence intensity and moreover inhibited dendritic branching in hippocampal neurons. Together, our data indicate that superoxide production is induced by the NR2B-NMDARs/RasGRF1/NOX2 pathway and promotes dendritogenesis.
Asunto(s)
NADPH Oxidasa 2/genética , Neurogénesis/genética , Receptores de N-Metil-D-Aspartato/genética , ras-GRF1/genética , Animales , Dendritas/metabolismo , Células Dendríticas/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Plasticidad Neuronal/genética , Neuronas/metabolismo , Ratas , Transducción de Señal/genética , Superóxidos/metabolismoRESUMEN
Microtubules (MTs) are long cylindrical structures of the cytoskeleton that control cell division, intracellular transport, and the shape of cells. MTs also form bundles, which are particularly prominent in neurons, where they help define axons and dendrites. MTs are bio-electrochemical transistors that form nonlinear electrical transmission lines. However, the electrical properties of most MT structures remain largely unknown. Here we show that bundles of brain MTs spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. Under intracellular-like conditions, voltage-clamped MT bundles displayed electrical oscillations with a prominent fundamental frequency at 39 Hz that progressed through various periodic regimes. The electrical oscillations represented, in average, a 258% change in the ionic conductance of the MT structures. Interestingly, voltage-clamped membrane-permeabilized neurites of cultured mouse hippocampal neurons were also capable of both, generating electrical oscillations, and conducting the electrical signals along the length of the structure. Our findings indicate that electrical oscillations are an intrinsic property of brain MT bundles, which may have important implications in the control of various neuronal functions, including the gating and regulation of cytoskeleton-regulated excitable ion channels and electrical activity that may aid and extend to higher brain functions such as memory and consciousness.
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Potenciales de Acción/fisiología , Axones/fisiología , Encéfalo/metabolismo , Dendritas/fisiología , Microtúbulos/fisiología , Neuronas/fisiología , Animales , Axones/metabolismo , Células Cultivadas , Dendritas/metabolismo , Conductividad Eléctrica , Fenómenos Electrofisiológicos , Ratones , Microtúbulos/metabolismo , Neuronas/metabolismo , RatasRESUMEN
The formation of complex dendritic arbors is crucial for the assembly of functional networks as abnormal dendrite formation underlies several neurodevelopmental and psychiatric disorders. Many extracellular factors have been postulated as regulators of dendritic growth. Wnt proteins play a critical role in neuronal development and circuit formation. We previously demonstrated that Wnt7b acts through the scaffold protein dishevelled 1 (Dvl1) to modulate dendrite arborisation by activating a non-canonical Wnt signalling pathway. Here, we identify the seven-transmembrane frizzled-7 (Fz7, also known as FZD7) as the receptor for Wnt7b-mediated dendrite growth and complexity. Importantly, Fz7 is developmentally regulated in the intact hippocampus, and is localised along neurites and at dendritic growth cones, suggesting a role in dendrite formation and maturation. Fz7 loss-of-function studies demonstrated that Wnt7b requires Fz7 to promote dendritic arborisation. Moreover, in vivo Fz7 loss of function results in dendritic defects in the intact mouse hippocampus. Furthermore, our findings reveal that Wnt7b and Fz7 induce the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and JNK proteins, which are required for dendritic development. Here, we demonstrate that Wnt7b-Fz7 signals through two non-canonical Wnt pathways to modulate dendritic growth and complexity.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Dendritas/metabolismo , Hipocampo/crecimiento & desarrollo , MAP Quinasa Quinasa 4/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Dendritas/enzimología , Dendritas/genética , Proteínas Dishevelled/genética , Proteínas Dishevelled/metabolismo , Receptores Frizzled , Hipocampo/metabolismo , MAP Quinasa Quinasa 4/genética , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Proteínas Wnt/genética , Vía de Señalización WntRESUMEN
Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the Huntington disease gene. The symptomatic stage of the disease is defined by the onset of motor symptoms. However, psychiatric disturbances, including depression, are common features of HD and can occur a decade before the manifestation of motor symptoms. We used the YAC128 transgenic mice (which develop motor deficits at a later stage, allowing more time to study depressive behaviors without the confounding effects of motor impairment) to test the effects of intranasal brain-derived neurotrophic factor (BDNF) treatment for 15 days in the occurrence of depressive-like behaviors. Using multiple well-validated behavioral tests, we found that BDNF treatment alleviated anhedonic and depressive-like behaviors in the YAC128 HD mice. Furthermore, we also investigated whether the antidepressant-like effects of BDNF were associated with an increase in adult hippocampal neurogenesis. However, BDNF treatment only increased cell proliferation and neuronal differentiation in the hippocampal dentate gyrus (DG) of wild-type (WT) mice, without altering these parameters in their YAC128 counterparts. Moreover, BDNF treatment did not cause an increase in the number of dendritic branches in the hippocampal DG when compared with animals treated with vehicle. In conclusion, our results suggest that non-invasive administration of BDNF via the intranasal route may have important therapeutic potential for treating mood disturbances in early-symptomatic HD patients.
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Conducta Animal , Factor Neurotrófico Derivado del Encéfalo/uso terapéutico , Depresión/tratamiento farmacológico , Depresión/prevención & control , Enfermedad de Huntington/complicaciones , Enfermedad de Huntington/patología , Administración Intranasal , Animales , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Depresión/complicaciones , Depresión/fisiopatología , Modelos Animales de Enfermedad , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Enfermedad de Huntington/fisiopatología , Masculino , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Neostriado/efectos de los fármacos , Neostriado/patología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
A single stress exposure facilitates memory formation through neuroplastic processes that reshape excitatory synapses in the hippocampus, probably requiring changes in extracellular matrix components. We tested the hypothesis that matrix metalloproteinase 9 (MMP-9), an enzyme that degrades components of extracellular matrix and synaptic proteins such as ß-dystroglycan (ß-DG43), changes their activity and distribution in rat hippocampus during the acute stress response. After 2.5 h of restraint stress, we found (i) increased MMP-9 levels and potential activity in whole hippocampal extracts, accompanied by ß-DG43 cleavage, and (ii) a significant enhancement of MMP-9 immunoreactivity in dendritic fields such as stratum radiatum and the molecular layer of hippocampus. After 24 h of stress, we found that (i) MMP-9 net activity rises at somatic field, i.e., stratum pyramidale and granule cell layers, and also at synaptic field, mainly stratum radiatum and the molecular layer of hippocampus, and (ii) hippocampal synaptoneurosome fractions are enriched with MMP-9, without variation of its potential enzymatic activity, in accordance with the constant level of cleaved ß-DG43. These findings indicate that stress triggers a peculiar timing response in the MMP-9 levels, net activity, and subcellular distribution in the hippocampus, suggesting its involvement in the processing of substrates during the stress response.
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Hipocampo/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Plasticidad Neuronal/fisiología , Sinapsis/metabolismo , Potenciales de Acción/fisiología , Animales , Dendritas/metabolismo , Masculino , Neuronas/metabolismo , Ratas Sprague-Dawley , Estrés Fisiológico/fisiología , Factores de TiempoRESUMEN
The proper formation and morphogenesis of dendrites is essential to the establishment of neuronal connectivity. We report that 2 members of the Pea3 family of transcription factors, Etv4 and Etv5, are expressed in hippocampal neurons during the main period of dendritogenesis, suggesting that they have a function in dendrite development. Here, we show that these transcription factors are physiological regulators of growth and arborization of pyramidal cell dendrites in the developing hippocampus. Gain and loss of function assays indicate that Etv4 and Etv5 are required for proper development of hippocampal dendritic arbors and spines. We have found that in vivo deletion of either Etv4 or Etv5 in hippocampal neurons causes deficits in dendrite size and complexity, which are associated with impaired cognitive function. Additionally, our data support the idea that Etv4 and Etv5 are part of a brain-derived neurotrophic factor-mediated transcriptional program required for proper hippocampal dendrite connectivity and plasticity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Dendritas/metabolismo , Hipocampo/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Cognición/fisiología , Proteínas de Unión al ADN/genética , Hipocampo/crecimiento & desarrollo , Ratones Transgénicos , Proyección Neuronal/fisiología , Plasticidad Neuronal/fisiología , Células PC12 , Proteínas Proto-Oncogénicas c-ets/genética , Ratas , Factores de Transcripción/genéticaRESUMEN
Serotonin acts through its receptors (5-HTRs) to shape brain networks during development and modulates essential functions in mature brain. The 5-HT1AR is mainly located at soma of hippocampal neurons early during brain development and its expression gradually shifts to dendrites during postnatal development. The 5-HT7R expressed early during hippocampus development, shows a progressive reduction in its expression postnatally. Considering these changes during development, we evaluated in cultured hippocampal neurons whether the 5-HT1AR and 5-HT7R change their expression, modulate dendritic growth, and activate signaling pathways such as ERK1/2, AKT/GSK3ß and LIMK/cofilin, which may sustain dendrite outgrowth by controlling cytoskeleton dynamics. We show that mRNA levels of both receptors increase between 2 and 7 DIV; however only protein levels of 5-HT7R increase significantly at 7 DIV. The 5-HT1AR is preferentially distributed in the soma, while 5-HT7R displays a somato-dendritic localization at 7 DIV. Through stimulation with 5-HT at 7 DIV during 24h and using specific antagonists, we determined that 5-HT1AR decreases the number of primary and secondary dendrites and restricts the growth of primary dendrites. The activation of 5-HT1AR and 5-HT7R promotes the growth of short secondary dendrites and triggers ERK1/2 and AKT phosphorylation through MEK and PI3K activation respectively; without changes in the phosphorylation of LIMK and cofilin. We conclude that 5-HT1AR restricts dendritogenesis and outgrowth of primary dendrites, but that both 5-HT1AR and 5-HT7R promote secondary dendrite outgrowth. These data support the role of 5-HT in neuronal outgrowth during development and provide insight into cellular basis of neurodevelopmental disorders.
Asunto(s)
Dendritas/efectos de los fármacos , Dendritas/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Receptores de Serotonina/metabolismo , Serotonina/farmacología , Animales , Células Cultivadas , Hipocampo/metabolismo , Neurogénesis/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Aging increases the vulnerability to stress and risk of developing depression. These changes have been related to a reduction of dehydroepiandrosterone (DHEA) levels, an adrenal steroid with anti-stress effects. Also, adult hippocampal neurogenesis decreases during aging and its alteration or impaired is related to the development of depression. Besides, it has been hypothesized that DHEA increases the formation of new neurons. However, it is unknown whether treatment with DHEA in aging may stimulate the dendrite maturation of newborn neurons and reversing depressive-like signs evoked by chronic stress exposure. Here aged male rats (14 months old) were subjected to a scheme of chronic mild stress (CMS) during six weeks, received a treatment with DHEA from the third week of CMS. Changes in body weight and sucrose preference (SP) were measured once a week. DHEA levels were measured in serum, identification of doublecortin-(DCX)-, BrdU- and BrdU/NeuN-labeled cells was done in the dentate gyrus of the hippocampus. CMS produced a gradual reduction in the body weight, but no changes in the SP were observed. Treatment enhanced levels of DHEA, but lack of recovery on body weight of stressed rats. Aging reduced the number of DCX-, BrdU- and BrdU/NeuN- cells but DHEA just significantly increased the number of DCX-cells in rats under CMS and controls, reaching levels of young non-stressed rats (used here as a reference of an optimal status of health). In rats under CMS, DHEA facilitated dendritic maturation of immature new neurons. Our results reveal that DHEA improves neural plasticity even in conditions of CMS in middle age rats. Thus, this hormone reverted the decrement of DCX-cells caused during normal aging.
Asunto(s)
Envejecimiento/efectos de los fármacos , Deshidroepiandrosterona/farmacología , Dendritas/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Psicotrópicos/farmacología , Estrés Psicológico/tratamiento farmacológico , Envejecimiento/fisiología , Envejecimiento/psicología , Animales , Antígenos Nucleares/metabolismo , Peso Corporal/efectos de los fármacos , Bromodesoxiuridina , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Enfermedad Crónica , Deshidroepiandrosterona/sangre , Dendritas/metabolismo , Dendritas/patología , Giro Dentado/metabolismo , Giro Dentado/patología , Sacarosa en la Dieta , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Neuropéptidos/metabolismo , Psicotrópicos/sangre , Distribución Aleatoria , Ratas Wistar , Estrés Psicológico/metabolismo , Estrés Psicológico/patologíaRESUMEN
Reelin, an extracellular glycoprotein secreted in embryonic and adult brain, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity-dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2-tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippocampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re-insertion of NR2B-NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. J. Cell. Physiol. 232: 1187-1199, 2017. © 2016 Wiley Periodicals, Inc.