RESUMEN
Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Diferenciación Celular , Movimiento Celular , Neuronas/citología , Neuronas/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Masculino , Ratones Endogámicos C57BL , Células PC12 , Ratas , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructuraRESUMEN
The mitochondrial ATPase Inhibitory Factor 1 (hereafter referred to as IF1) blocks the reversal of the F1Fo-ATPsynthase to prevent detrimental consumption of cellular ATP and associated demise. Herein, we infer further its molecular physiology by assessing its protective function in neurons during conditions of challenged homeostatic respiration. By adopting in vitro and in vivo protocols of hypoxia/ischemia and re-oxygenation, we show that a shift in the IF1:F1Fo-ATPsynthase expression ratio occurs in neurons. This increased IF1 level is essential to induce accumulation of the PTEN-induced putative kinase 1 (PINK-1) and recruitment of the mitophagic ubiquitin ligase PARK-2 to promote autophagic "control" of the mitochondrial population. In IF1 overexpressing neurons ATP depletion is reduced during hypoxia/ischemia and the mitochondrial membrane potential (ΔYm) resilient to re-oxygenation as well as resistant to electrogenic, Ca(2+) dependent depolarization. These data suggest that in mammalian neurons mitochondria adapt to respiratory stress by upregulating IF1, which exerts a protective role by coordinating pro-survival cell mitophagy and bioenergetics resilience.
Asunto(s)
Hipoxia/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Proteínas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Autofagia , Línea Celular Tumoral , Células Cultivadas , Corteza Cerebral/citología , Humanos , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Potencial de la Membrana Mitocondrial , Mitocondrias/fisiología , Ratas , Regulación hacia Arriba , Proteína Inhibidora ATPasaRESUMEN
BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis.
Asunto(s)
Autofagia , Proteínas Portadoras/biosíntesis , Regulación de la Expresión Génica , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Línea Celular , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismoRESUMEN
BACKGROUND: Growing evidence shows that, in vivo, the precursor of Nerve Growth Factor (NGF), proNGF, displays biological activities different from those of its mature NGF counterpart, mediated by distinct, and somewhat complementary, receptor binding properties. NGF and proNGF induce distinct transcriptional signatures in target cells, highlighting their different bioactivities. In vivo, proNGF and mature NGF coexist. It was proposed that the relative proNGF/NGF ratio is important for their biological outcomes, especially in pathological conditions, since proNGF, the principal form of NGF in Central Nervous System (CNS), is increased in Alzheimer's disease brains. These observations raise a relevant question: does proNGF, in the presence of NGF, influence the NGF transcriptional response and viceversa? In order to understand the specific proNGF effect on NGF activity, depending on the relative proNGF/NGF concentration, we investigated whether proNGF affects the pattern of well-known NGF-regulated mRNAs. RESULTS: To test any influence of proNGF on pure NGF expression fingerprinting, the expression level of a set of candidate genes was analysed by qReal-Time PCR in rat adrenal pheochromocytoma cell line PC12, treated with a mixture of NGF and proNGF recombinant proteins, in different stoichiometric ratios. These candidates were selected amongst a set of genes well-known as being rapidly induced by NGF treatment. We found that, when PC12 cells are treated with proNGF/NGF mixtures, a unique pattern of gene expression, which does not overlap with that deriving from treatment with either proNGF or NGF alone, is induced. The specific effect is also dependent on the stoichiometric composition of the mixture. The proNGF/NGF equimolar mixture seems to partially neutralize the specific effects of the proNGF or NGF individual treatments, showing a weaker overall response, compared to the individual contributions of NGF and proNGF alone. CONCLUSIONS: Using gene expression as a functional read-out, our data demonstrate that the relative availability of NGF and proNGF in vivo might modulate the biological outcome of these ligands.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Precursores de Proteínas/metabolismo , Animales , Células PC12 , RatasRESUMEN
MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription-independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function. Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46(P)) and (iii) promotes binding between p53Ser46(P) and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription. Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46(P) to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antineoplásicos/metabolismo , Apoptosis , Carcinoma/genética , Carcinoma/metabolismo , Línea Celular Tumoral , Células Cultivadas , Cisplatino/metabolismo , Citocromos c/metabolismo , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Mitocondrias/ultraestructura , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2 , Ubiquitina-Proteína Ligasas/análisisRESUMEN
The pathogenic model of Alzheimer's disease (AD) posits that aggregates of amyloid ß, a product of amyloid precursor protein (APP) processing, cause dementia. However, alterations of normal APP functions could contribute to AD pathogenesis, and it is therefore important to understand the role of APP. APP is a member of a gene family that shows functional redundancy as documented by the evidence that single knock-out mice are viable, whereas mice with combined deletions of APP family genes die shortly after birth. A residue in the APP intracellular region, Y(682), is indispensable for these essential functions of APP. It is therefore important to identify pathways that regulate phosphorylation of Y(682) as well as the role of Y(682) in vivo. TrkA is associated with both phosphorylation of APP-Y(682) and alteration of APP processing, suggesting that tyrosine phosphorylation of APP links APP processing and neurotrophic signaling to intracellular pathways associated with cellular differentiation and survival. Here we have tested whether the NGF/TrkA signaling pathway is a physiological regulator of APP phosphorylation. We find that NGF induces tyrosine phosphorylation of APP, and that APP interacts with TrkA and this interaction requires Y(682). Unpredictably, we also uncover that APP, and specifically Y(682), regulates activation of the NGF/TrkA signaling pathway in vivo, the subcellular distribution of TrkA and the sensitivity of neurons to the trophic action of NGF. This evidence suggests that these two membrane protein's functions are strictly interconnected and that the NGF/TrkA signaling pathway is involved in AD pathogenesis and can be used as a therapeutic target.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/fisiología , Transducción de Señal/fisiología , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/fisiología , Animales , Células Cultivadas , Femenino , Hipocampo/metabolismo , Hipocampo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor de Crecimiento Nervioso/fisiología , Fosforilación/fisiología , Receptor trkA/metabolismo , Tirosina/fisiologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the progressive degeneration of motoneurons (MNs). Altered electrical properties have been described in familial and sporadic ALS patients. Cortical and spinal neurons cultured from the mutant Cu,Zn superoxide dismutase 1 (SOD1G93A) mouse, a murine model of ALS, exhibit a marked increase in the persistent Na+ currents. Here, we investigated the effects of the SOD1G93A mutation on the expression of the voltage-gated Na+ channel alpha subunit SCN8A (Nav1.6) and the beta subunits SCN1B (beta1), SCN2B (beta2), and SCN3B (beta3) in MNs of the spinal cord in presymptomatic (P75) and symptomatic (P120) mice. We observed a significant increase, within lamina IX, of the beta3 transcript and protein expression. On the other hand, the beta1 transcript was significantly decreased, in the same area, at the symptomatic stage, while the beta2 transcript levels were unaltered. The SCN8A transcript was significantly decreased at P120 in the whole spinal cord. These data suggest that the SOD1G93A mutation alters voltage-gated Na+ channel subunit expression. Moreover, the increased expression of the beta3 subunit support the hypothesis that altered persistent Na+ currents contribute to the hyperexcitability observed in the ALS-affected MNs.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Subunidades de Proteína/metabolismo , Canales de Sodio/metabolismo , Médula Espinal/fisiología , Superóxido Dismutasa/metabolismo , Adulto , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Humanos , Masculino , Ratones , Neuronas Motoras/citología , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Subunidades de Proteína/genética , Canales de Sodio/genética , Médula Espinal/citología , Médula Espinal/patología , Superóxido Dismutasa/genética , Subunidad beta-1 de Canal de Sodio Activado por Voltaje , Subunidad beta-2 de Canal de Sodio Activado por Voltaje , Subunidad beta-3 de Canal de Sodio Activado por VoltajeRESUMEN
Axonal transport and neuronal survival depend critically on active transport and axon integrity both for supplying materials and communication to different domains of the cell body. All these actions are executed through cytoskeleton, transport and regulatory elements that appear to be disrupted in neurodegenerative diseases. Motor-driven transport both supplies and clears distal cellular portions with proteins and organelles. This transport is especially relevant in projection and motor neurons, which have long axons to reach the farthest nerve endings. Thus, any disturbance of axonal transport may have severe consequences for neuronal function and survival. A growing body of literature indicates the presence of alterations to the motor molecules machinery, not only in expression levels and phosphorylation, but also in their subcellular distribution within populations of neurons, which are selectively affected in the course of neurodegenerative diseases. The implications of this altered subcellular localization and how this affects axon survival and neuronal death still remain poorly understood, although several hypotheses have been suggested. Furthermore, cytoskeleton and transport element localization can be selectively disrupted in some disorders suggesting that specific loss of the axonal functionality could be a primary hallmark of the disorder. This can lead to axon degeneration and neuronal death either directly, through the functional absence of essential axonal proteins, or indirectly, through failures in communication among different cellular domains. This review compares the localization of cytoskeleton and transport elements in some neurodegenerative disorders to ask what aspects may be essential for axon survival and neuronal death.
Asunto(s)
Transporte Axonal/fisiología , Axones/metabolismo , Neuronas Motoras/patología , Enfermedades Neurodegenerativas/patología , Axones/patología , Lesiones Encefálicas/patología , Supervivencia Celular , Citoesqueleto/metabolismo , Humanos , Neuronas Motoras/citologíaRESUMEN
Endocannabinoids are neuroprotective in vivo and in vitro, but the mechanisms by which they act are largely unknown. The present study addressed the role of cannabinoid receptors during remote cell death of central neurons in a model that is based on cerebellar lesions. A lesion in one cerebellar hemisphere induced remote cell death and type 2 cannabinoid receptor (CB2R) expression in contralateral precerebellar neurons. Of the selective agonists and antagonists that modulated cannabinoid receptor activity, we found that the CB2R agonist JWH-015 reduced neuronal loss and cytochrome-c release, leading to neurological recovery; these effects were reversed by the selective CB2R antagonist SR144528. Analysis of CB2R-triggered signal transduction demonstrated that in axotomized neurons, CB2R regulated Akt and JNK phosphorylation through a PI3K-dependent pathway, whereas other major signaling routes that are dependent on CB2R, such as ERK1/2 and p38, were not involved. This result was corroborated by the observation that the selective PI3K inhibitor LY294002 blocked the CB2R stimulation effects on neuronal survival as well as Akt and JNK phosphorylation levels. Together, these data demonstrate that axonal damage induces CB2R expression in central neurons and that stimulation of this receptor has a neuroprotective effect that is achieved through PI3K/Akt signaling.
Asunto(s)
Apoptosis/fisiología , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptor Cannabinoide CB2/agonistas , Transducción de Señal/fisiología , Animales , Axotomía , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Cerebelo/patología , Indoles/farmacología , Masculino , Neuronas/enzimología , Neuronas/patología , Ratas , Ratas Wistar , Receptor Cannabinoide CB2/fisiologíaRESUMEN
Nitric oxide (NO) and purinergic ionotropic receptors (P2X) mediate cellular events in the central nervous system (CNS) under physiological conditions as well as during pathological events, and they have been recently proposed to interact in mediating CNS response to injury (Viscomi et al. [2004] Neuroscience 123:393-404; Florenzano et al. [2008] Pflugers Arch. 452:622-644). Trimethyltin (TMT) is an organotin compound that generates neurotoxic effects, and it has been used in a model of neurodegenerative disease and memory dysfunction. TMT causes neuronal death and reactive gliosis primarily in the hippocampus and other limbic regions. In the present study, we examined the degenerative events and the expression of nitric oxide synthase (NOS) and P2X receptor subtypes (P2X(1,2,4,7)Rs) that were induced by TMT administration at different time points (3, 7, 14, and 21 days) by conventional and confocal microscopy and Western blotting. Massive glial activation and neuronal death in the CA1 and CA3 regions were observed after TMT treatment. In these areas, astrocytic P2X(2)R and neuronal NOS were temporarily enhanced in association with the progression of neuronal death. In the hippocampus, the physiological expression of P2X(1)R, P2X(4)R, and P2X(7)R was not modified by TMT. The present data demonstrate that, as in other neurodegenerative models, TMT-induced hippocampal degeneration is associated with nitrergic and purinergic activations. Nevertheless, at odds with previous data, in this model the two systems are active in segregated cell populations, namely, P2XR in astrocytes and NOS in neurons. Finally, the temporal relations between P2XR and NOS expression and neuronal degeneration suggest interactions between P2XR/NO signaling and cell survival.
Asunto(s)
Fármacos del Sistema Nervioso Central/toxicidad , Hipocampo/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Receptores Purinérgicos P2/metabolismo , Compuestos de Trimetilestaño/toxicidad , Animales , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Astrocitos/fisiología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/enzimología , Región CA1 Hipocampal/fisiología , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/enzimología , Región CA3 Hipocampal/fisiología , Muerte Celular/efectos de los fármacos , Femenino , Hipocampo/enzimología , Hipocampo/fisiología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/fisiología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas , Ratas Wistar , Receptores Purinérgicos P2X , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Biological rhythms, and especially the sleep/wake cycle, are frequently disrupted during senescence. This draws attention to the study of aging-related changes in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker. The authors here compared the SCN of young and old mice, analyzing presynaptic terminals, including the gamma-aminobutyric acid (GABA)ergic network, and molecules related to the regulation of GABA, the main neurotransmitter of SCN neurons. Transcripts of the alpha3 subunit of the GABAA receptor and the GABA-synthesizing enzyme glutamic acid decarboxylase isoform 67 (GAD67) were analyzed with real-time RT-PCR and GAD67 protein with Western blotting. These parameters did not show significant changes between the 2 age groups. Presynaptic terminals were identified in confocal microscopy with synaptophysin immunofluorescence, and the GABAergic subset of those terminals was revealed by the colocalization of GAD67 and synaptophysin. Quantitative analysis of labeled synaptic endings performed in 2 SCN subregions, where retinal afferents are known to be, respectively, very dense or very sparse, revealed marked aging-related changes. In both subregions, the evaluated parameters (the number of and the area covered by presynaptic terminals and by their GABAergic subset) were significantly decreased in old versus young mice. No significant differences were found between SCN tissue samples from animals sacrificed at different times of day, in either age group. Altogether, the data point out marked reduction in the synaptic network of the aging biological clock, which also affects GABAergic terminals. Such alterations could underlie aging-related SCN dysfunction, including low-amplitude output during senescence.
Asunto(s)
Envejecimiento/metabolismo , Terminales Presinápticos/metabolismo , Núcleo Supraquiasmático/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Receptores de GABA/genética , Receptores de GABA/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The intrinsically disordered tau protein plays a pivotal role in the pathogenesis of Alzheimer's disease (AD) and other human tauopathies. Abnormal post-translational modifications of tau, such as truncation, are causally involved in the onset/development of these neurodegenerative diseases. In this context, the AD-relevant N-terminal fragment mapping between 26 and 44 amino acids of protein (tau26-44) is interesting, being endowed with potent neurotoxic effects in vitro and in vivo. However, the understanding of the mechanism(s) of tau26-44 toxicity is a challenging task because, similarly to the full-length tau, it does not have a unique 3D structure but exists as dynamic ensemble of conformations. Here we use Atomic Force Spectroscopy, Small Angle X-ray Scattering and Molecular Dynamics simulation to gather structural and functional information on the tau26-44. We highlight the presence, the type and the location of its temporary secondary structures and we unveil the occurrence of relevant transient tertiary conformations that could contribute to tau26-44 toxicity. Data are compared with those obtained on the biologically-inactive, reverse-sequence (tau44-26 peptide) which has the same mass, charge, aminoacidic composition as well as the same overall unfolded character of tau26-44.
Asunto(s)
Simulación de Dinámica Molecular , Péptidos/química , Conformación Proteica , Proteínas tau/química , Enfermedad de Alzheimer , Secuencia de Aminoácidos , Animales , Humanos , Microscopía de Fuerza Atómica , Neuronas/metabolismo , Neuronas/patología , Ratas , Relación Estructura-Actividad , Tauopatías , Difracción de Rayos X , Proteínas tau/metabolismoRESUMEN
In the present study, we used electrophysiological, biochemical, and confocal microscopy techniques, to investigate the functional role of transient receptor potential vanilloid type 1 (TRPV1) and cannabinoid type 1 receptors (CB1-R) in the substantia nigra pars compacta (SNpc) and their stimulation by the endocannabinoid N-arachidonoyl-dopamine (NADA). Liquid chromatography-mass spectrometry analyses revealed that a NADA-like compound is produced in substantia nigra slices, in conditions of hyperactivity. Moreover, the functional role of both TRPV1 and CB1-R in modulating synaptic transmission in this area was suggested by confocal microscopy data, showing TRPV1 and CB1-R immunoreactivity in punctate structures, probably representing synaptic contacts on cell bodies of the SNpc. In patch-clamp recordings from dopamine (DA) neurons of the SNpc, we found that NADA increases or reduces glutamatergic transmission onto DA neurons by activating TRPV1 and CB1 receptors, respectively, whereas it decreases GABAergic transmission via CB1 stimulation. Facilitation of glutamate release through TRPV1 was blocked in the presence of a selective blocker of the putative endocannabinoid membrane transporter (EMT), indicating that NADA needs to be taken up by cells to interact with this receptor. In line with these data, biochemical results demonstrated that NADA selectively acted at CB1-R when its re-uptake was blocked. Altogether these data demonstrate a significant role exerted by the endocannabinoid/endovanilloid NADA in the regulation of synaptic transmission to DA neurons of the SNpc. Moreover, they highlight a key function of the EMT transporter in promoting the stimulation of TRPV1 or CB1-R, thus favoring facilitation or inhibition of glutamate synaptic release.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Dopamina/análogos & derivados , Neuronas/metabolismo , Receptores de Cannabinoides/metabolismo , Sustancia Negra/metabolismo , Transmisión Sináptica/fisiología , Canales Catiónicos TRPV/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Dopamina/metabolismo , Dopamina/farmacología , Técnica del Anticuerpo Fluorescente , Ácido Glutámico/metabolismo , Masculino , Microscopía Confocal , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Proteínas de Transporte de Catión Orgánico/efectos de los fármacos , Proteínas de Transporte de Catión Orgánico/metabolismo , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Receptores de Cannabinoides/efectos de los fármacos , Sustancia Negra/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Canales Catiónicos TRPV/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The accumulation of ß-amyloid (Aß) is one of the hallmarks of Alzheimer disease (AD). Beyond the inflammatory reactions promoted by Aß, it has been demonstrated that the prokineticin (PK) system, composed of the chemokine prokineticin 2 (PK2) and its receptors, is involved in Aß toxicity. In this study we have analyzed how the Aß chronic treatment affects the glutamatergic transmission on neurons from primary cortical cultures, clearly demonstrating the PK system involvement on its action mechanism. In fact, we have observed a significant increase of the ionic current through the AMPA receptors in primary cortical neurons and an up-regulation of the PK system in cultures chronically treated with Aß. All effects were nullified by the prokineticin antagonist PC-1. Moreover, we have herein firstly demonstrated that the incubation of primary cortical culture with Bv8, the amphibian homologue of PK2, was able to increase in neurons the AMPA currents at specific doses and exposure times, measured both as evoked and as spontaneous currents. This effect was not due to a modification of the AMPA receptor subunit expression. In contrast, the up-modulation of AMPA currents were blocked by PC-1 and were mediated by the activation of the intracellular protein kinase C (PKC) transduction pathways because Gö6983, the PKC inhibitor added in the medium, nullified the effect. Finally, cellular death induced by kainate was also reduced following treatment with PC1. In conclusion, our results show that the prokineticin system may be a key mediator in the Aß-induced neuronal damage, suggesting PK antagonists as new therapeutic compounds to ameliorate the AD progression.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Proteínas Anfibias/uso terapéutico , Péptidos beta-Amiloides/toxicidad , Ácido Glutámico/metabolismo , Neuropéptidos/uso terapéutico , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/patología , Animales , Anuros , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Hormonas Gastrointestinales/metabolismo , Indoles/farmacología , Maleimidas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas Wistar , Receptores AMPA/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
The muscarinic receptor response to acetylcholine regulates the hippocampal-related learning, memory, neural plasticity and the production and processing of the pro-nerve growth factor (proNGF) by hippocampal cells. The development and progression of diabetes generate a mild cognitive impairment reducing the functions of the septo-hippocampal cholinergic circuitry, depressing neural plasticity and inducing proNGF accumulation in the brain. Here we demonstrate, in a rat model of early type-1 diabetes, that a physical therapy, the electroacupuncture, counteracts the diabetes-induced deleterious effects on hippocampal physiology by ameliorating hippocampal-related memory functions; recovering the impaired long-term potentiation at the dentate gyrus (DG-LTP) and the lowered expression of the vesicular glutamate transporter 1; normalizing the activity-dependent release of proNGF in diabetic rat hippocampus. Electroacupuncture exerted its therapeutic effects by regulating the expression and activity of M1- and M2-acetylcholine muscarinic receptors subtypes in the dentate gyrus of hippocampus. Our results suggest that a physical therapy based on repetitive sensory stimulation could promote hippocampal neural activity, neuronal metabolism and functions, and conceivably improve the diabetes-induced cognitive impairment. Our data can support the setup of therapeutic protocols based on a better integration between physical therapies and pharmacology for the cure of diabetes-associated neurodegeneration and possibly for Alzheimer's disease.
Asunto(s)
Electroacupuntura , Hipocampo/metabolismo , Hipocampo/fisiopatología , Muscarina/metabolismo , Animales , Recuento de Células , Giro Dentado/metabolismo , Giro Dentado/fisiopatología , Diabetes Mellitus Experimental , Potenciación a Largo Plazo , Memoria , Modelos Biológicos , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Plasticidad Neuronal , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Células Piramidales/metabolismo , Células Piramidales/patología , Ratas , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores Muscarínicos/metabolismoRESUMEN
The largest part of tau secreted from AD nerve terminals and released in cerebral spinal fluid (CSF) is C-terminally truncated, soluble and unaggregated supporting potential extracellular role(s) of NH2 -derived fragments of protein on synaptic dysfunction underlying neurodegenerative tauopathies, including Alzheimer's disease (AD). Here we show that sub-toxic doses of extracellular-applied human NH2 tau 26-44 (aka NH 2 htau) -which is the minimal active moiety of neurotoxic 20-22kDa peptide accumulating in vivo at AD synapses and secreted into parenchyma- acutely provokes presynaptic deficit in K+ -evoked glutamate release on hippocampal synaptosomes along with alteration in local Ca2+ dynamics. Neuritic dystrophy, microtubules breakdown, deregulation in presynaptic proteins and loss of mitochondria located at nerve endings are detected in hippocampal cultures only after prolonged exposure to NH 2 htau. The specificity of these biological effects is supported by the lack of any significant change, either on neuronal activity or on cellular integrity, shown by administration of its reverse sequence counterpart which behaves as an inactive control, likely due to a poor conformational flexibility which makes it unable to dynamically perturb biomembrane-like environments. Our results demonstrate that one of the AD-relevant, soluble and secreted N-terminally truncated tau forms can early contribute to pathology outside of neurons causing alterations in synaptic activity at presynaptic level, independently of overt neurodegeneration.
RESUMEN
Neurophysiologic data suggest that orexin neurons are directly excited by ATP through purinergic receptors (P2XR). Anatomical studies, though reporting P2XR in the hypothalamus, did not describe it in the perifornical hypothalamic area, where orexinergic neurons are located. Here we report the presence of the P2X(2)R subunit in the rat perifornical hypothalamus and demonstrate that hypothalamic orexin neurons express the P2X(2)R. Double immunohistochemistry showed that virtually all orexin-immunoreactive neurons are also P2X(2)R immunoreactive, whereas 80% of P2X(2)R-immunoreactive neurons are also orexin positive. Triple-labeling experiments, combining fluorescence in situ hybridization for P2X(2)R mRNA and P2X(2)R/orexin double immunofluorescence, confirmed these findings. In addition, in situ hybridization demonstrated that P2X(2)R mRNA is localized in cellular processes of orexinergic neurons. The present data support neurophysiologic findings on ATP modulation of orexinergic function and provide direct evidence that the entire population of orexin neurons expresses a P2XR subtype, namely, P2X(2)R. Thus, purinergic transmission might intervene in modulating key functions known to be controlled by the orexinergic system, such as feeding behavior and arousal.
Asunto(s)
Adenosina Trifosfato/metabolismo , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Animales , Nivel de Alerta/fisiología , Dendritas/metabolismo , Dendritas/ultraestructura , Conducta Alimentaria/fisiología , Técnica del Anticuerpo Fluorescente/métodos , Hipotálamo/citología , Inmunohistoquímica , Hibridación in Situ , Masculino , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Receptores de Orexina , Orexinas , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Receptores Purinérgicos P2X2RESUMEN
Neuroplasticity allows the brain to encode experience and learn behaviors, and also to re-acquire lost functions after damage. The cerebellum is a suitable structure to address this topic because of its strong involvement in learning processes and compensation of lesion-induced deficits. This study was aimed to characterize the effects of a hemicerebellectomy (HCb) combined or not with the exposition to environmental enrichment (EE) on dendritic spine density and size in Purkinje cell proximal and distal compartments of cerebellar vermian and hemispherical regions. Male Wistar rats were housed in enriched or standard environments from the 21st post-natal day (pnd) onwards. At the 75th pnd, rats were submitted to HCb or sham lesion. Neurological symptoms and spatial performance in the Morris water maze were evaluated. At the end of testing, morphological analyses assessed dendritic spine density, area, length, and head diameter on vermian and hemispherical Purkinje cells. All hemicerebellectomized (HCbed) rats showed motor compensation, but standard-reared HCbed animals exhibited cognitive impairment that was almost completely compensated in enriched HCbed rats. The standard-reared HCbed rats showed decreased density with augmented size of Purkinje cell spines in the vermis, and augmented both density and size in the hemisphere. Enriched HCbed rats almost completely maintained the spine density and size induced by EE. Both lesion-induced and activity-dependent cerebellar plastic changes may be interpreted as "beneficial" brain reactions, aimed to support behavioral performance rescuing.
Asunto(s)
Conducta Animal , Vermis Cerebeloso/citología , Vermis Cerebeloso/fisiología , Espinas Dendríticas/fisiología , Plasticidad Neuronal , Células de Purkinje/citología , Células de Purkinje/fisiología , Animales , Tamaño de la Célula , Vermis Cerebeloso/lesiones , Ambiente , Locomoción , Masculino , Aprendizaje por Laberinto , Ratas , Ratas Wistar , Navegación Espacial/fisiologíaRESUMEN
BACKGROUND: For long time Alzheimer's disease has been attributed to a cholinergic deficit. More recently, it has been considered dependent on the accumulation of the amyloid beta peptide (Aß), which promotes neuronal loss and impairs neuronal function. Results/methodology: In the present study, using biophysical and biochemical experiments we tested the hypothesis that in addition to its role as a neurotransmitter, acetylcholine may exert its action as an anti-Alzheimer agent through a direct interaction with Aß. CONCLUSION: Our data provide evidence that acetylcholine favors the soluble peptide conformation and exerts a neuroprotective effect against the neuroinflammatory and toxic effects of Aß. The present paper paves the way toward the development of new polyfunctional anti-Alzheimer therapeutics capable of intervening on both the cholinergic transmission and the Aß aggregation.
Asunto(s)
Acetilcolina/metabolismo , Acetilcolina/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Colinérgicos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Acetilcolina/química , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Supervivencia Celular/efectos de los fármacos , Colinérgicos/química , Colinérgicos/metabolismo , Dicroismo Circular , Humanos , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/metabolismo , Agregado de Proteínas/efectos de los fármacosRESUMEN
The occurrence of neurogenesis in the hippocampus of the adult rat during trimethyltin (TMT)-induced neurodegeneration was investigated using bromodeoxyuridine (BrdU). Fifteen days after TMT intoxication, BrdU-labeled cells were significantly more numerous in the hippocampus of treated animals, gradually decreasing towards the control value 21 days after intoxication in the dentate gyrus (DG), while in the CA3/hilus region BrdU-labeled cells were still more numerous in TMT-treated rats. In order to investigate the fate of newly-generated cells double labeling experiments using neuronal or glial markers were performed. Colocalization of the neuronal marker NeuN was detected in many BrdU-positive cells in the DG, while in the CA3/hilus region no colocalization of NeuN and BrdU could be observed. No colocalization of BrdU and the astroglial marker GFAP or the microglial marker OX-42 was detected either in the DG and or in the CA3/hilus region. The results indicate an enhancement of endogenous neurogenesis in the hippocampus during TMT-induced neurodegeneration, with the development of a subpopulation of regenerated cells into neurons in the DG, while in the CA3/hilus region the population of newly-generated cells should be regarded as undifferentiated.