RESUMEN
Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1 -/- synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1 -/- cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.
Asunto(s)
Cerebelo , Proteínas de Transferencia de Fosfolípidos , Sinapsis , Transmisión Sináptica , Vesículas Sinápticas , Animales , Vesículas Sinápticas/metabolismo , Transmisión Sináptica/fisiología , Ratones , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Femenino , Masculino , Cerebelo/citología , Sinapsis/metabolismo , Sinapsis/fisiología , Células Cultivadas , Ratones Noqueados , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/fisiología , Endocitosis/fisiologíaRESUMEN
BACKGROUND: Epidemiological emerging evidence shows that human exposure to some nanosized materials present in the environment would contribute to the onset and/or progression of Alzheimer's disease (AD). The cellular and molecular mechanisms whereby nanoparticles would exert some adverse effects towards neurons and take part in AD pathology are nevertheless unknown. RESULTS: Here, we provide the prime evidence that titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) bind the cellular form of the prion protein (PrPC), a plasma membrane protein well known for its implication in prion diseases and prion-like diseases, such as AD. The interaction between TiO2- or CB-NPs and PrPC at the surface of neuronal cells grown in culture corrupts PrPC signaling function. This triggers PrPC-dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrPC interaction, NPs also promote the activation of 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn provokes the internalization of the neuroprotective TACE α-secretase. This diverts TACE cleavage activity away from (i) TNFα receptors (TNFR), whose accumulation at the plasma membrane augments the vulnerability of NP-exposed neuronal cells to TNFα -associated inflammation, and (ii) the amyloid precursor protein APP, leading to overproduction of neurotoxic amyloid Aß40/42 peptides. The silencing of PrPC or the pharmacological inhibition of PDK1 protects neuronal cells from TiO2- and CB-NPs effects regarding ROS production, TNFα hypersensitivity, and Aß rise. Finally, we show that dysregulation of the PrPC-PDK1-TACE pathway likely occurs in the brain of mice injected with TiO2-NPs by the intra-cerebro-ventricular route as we monitor a rise of TNFR at the cell surface of several groups of neurons located in distinct brain areas. CONCLUSION: Our in vitro and in vivo study thus posits for the first time normal cellular prion protein PrPC as being a neuronal receptor of TiO2- and CB-NPs and identifies PrPC-coupled signaling pathways by which those nanoparticles alter redox equilibrium, augment the intrinsic sensitivity of neurons to neuroinflammation, and provoke a rise of Aß peptides. By identifying signaling cascades dysregulated by TiO2- and CB-NPs in neurons, our data shed light on how human exposure to some NPs might be related to AD.
Asunto(s)
Enfermedad de Alzheimer , Nanopartículas , Priones , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/patología , Animales , Homeostasis , Humanos , Ratones , Nanopartículas/toxicidad , Neuronas/patología , Proteínas Priónicas/metabolismo , Priones/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Hollín/toxicidad , Titanio , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.
Asunto(s)
Neoplasias de la Mama/genética , Exosomas/patología , GTP Fosfohidrolasas/metabolismo , Metástasis de la Neoplasia/genética , Animales , Neoplasias de la Mama/secundario , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Cuerpos Multivesiculares/fisiología , Pez CebraRESUMEN
Advances in microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders originating from the immune response arsenal to gold nanoparticles (AuNPs) as contrasting agents is the method of choice to obtain labeling tools. However, conjugation of the minimal sized 15 kDa nanobody (Nb) to AuNPs remains challenging in comparison to the conjugation of 150 kDa IgG to AuNPs. Herein, effective Nb-AuNP assemblies are built using the selective and almost irreversible non-covalent associations between two peptide sequences deriving from a p53 heterotetramer domain variant. The 15 kDa GFP-binding Nb is fused to one dimerizing motif to obtain a recombinant Nb dimer with improved avidity for GFP while the other complementing dimerizing motif is equipped with thiols and grafted to a 2.4 nm substituted thiobenzoate-coordinated AuNP via thiolate exchange. After pegylation, the modified AuNPs are able to non-covalently anchor Nb dimers and the subsequent complexes demonstrate the ability to form immunogold label GFP-protein fusions within various subcellular locations. These tools open an avenue for precise localization of targets at high resolution by electron microscopy.
RESUMEN
Specific forms of fatty acids are well known to have beneficial health effects, but their precise mechanism of action remains elusive. Phosphatidic acid (PA) produced by phospholipase D1 (PLD1) regulates the sequential stages underlying secretory granule exocytosis in neuroendocrine chromaffin cells, as revealed by pharmacological approaches and genetic mouse models. Lipidomic analysis shows that secretory granule and plasma membranes display distinct and specific composition in PA. Secretagogue-evoked stimulation triggers the selective production of several PA species at the plasma membrane near the sites of active exocytosis. Rescue experiments in cells depleted of PLD1 activity reveal that mono-unsaturated PA restores the number of exocytotic events, possibly by contributing to granule docking, whereas poly-unsaturated PA regulates fusion pore stability and expansion. Altogether, this work provides insight into the roles that subspecies of the same phospholipid may play based on their fatty acyl chain composition.
Asunto(s)
Exocitosis/genética , Células Neuroendocrinas/metabolismo , Ácidos Fosfatidicos/metabolismo , Animales , Humanos , RatonesRESUMEN
Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.
Asunto(s)
Cromogranina A/metabolismo , Aparato de Golgi/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/fisiología , Vesículas Secretoras/fisiología , Animales , Células COS , Chlorocebus aethiops , Ratones , Ratones NoqueadosRESUMEN
The presence of amyloid beta (Aß) plaques in the brain of some individuals with Creutzfeldt-Jakob or Gertsmann-Straussler-Scheinker diseases suggests that pathogenic prions (PrPSc) would have stimulated the production and deposition of Aß peptides. We here show in prion-infected neurons and mice that deregulation of the PDK1-TACE α-secretase pathway reduces the Amyloid Precursor Protein (APP) α-cleavage in favor of APP ß-processing, leading to Aß40/42 accumulation. Aß predominates as monomers, but is also found as trimers and tetramers. Prion-induced Aß peptides do not affect prion replication and infectivity, but display seedable properties as they can deposit in the mouse brain only when seeds of Aß trimers are co-transmitted with PrPSc. Importantly, brain Aß deposition accelerates death of prion-infected mice. Our data stress that PrPSc, through deregulation of the PDK1-TACE-APP pathway, provokes the accumulation of Aß, a prerequisite for the onset of an Aß seeds-induced Aß pathology within a prion-infectious context.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Proteína ADAM17/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Conducta Animal , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/líquido cefalorraquídeo , Placa Amiloide/metabolismo , Enfermedades por Prión/líquido cefalorraquídeo , Enfermedades por Prión/patología , Células MadreRESUMEN
In prion diseases, the brain lesion profile is influenced by the prion "strain" properties, the invasion route to the brain, and still unknown host cell-specific parameters. To gain insight into those endogenous factors, we analyzed the histopathological alterations induced by distinct prion strains in the mouse cerebellum. We show that 22L and ME7 scrapie prion proteins (PrP22L , PrPME7 ), but not bovine spongiform encephalopathy PrP6PB1 , accumulate in a reproducible parasagittal banding pattern in the cerebellar cortex of infected mice. Such banding pattern of PrP22L aggregation did not depend on the neuroinvasion route, but coincided with the parasagittal compartmentation of the cerebellum mostly defined by the expression of zebrins, such as aldolase C and the excitatory amino acid transporter 4, in Purkinje cells. We provide evidence that Purkinje cells display a differential, subtype-specific vulnerability to 22L prions with zebrin-expressing Purkinje cells being more resistant to prion toxicity, while in stripes where PrP22L accumulated most zebrin-deficient Purkinje cells are lost and spongiosis accentuated. In addition, in PrP22L stripes, enhanced reactive astrocyte processes associated with microglia activation support interdependent events between the topographic pattern of Purkinje cell death, reactive gliosis and PrP22L accumulation. Finally, we find that in preclinically-ill mice prion infection promotes at the membrane of astrocytes enveloping Purkinje cell excitatory synapses, upregulation of tumor necrosis factor-α receptor type 1 (TNFR1), a key mediator of the neuroinflammation process. These overall data show that Purkinje cell sensitivity to prion insult is locally restricted by the parasagittal compartmentation of the cerebellum, and that perisynaptic astrocytes may contribute to prion pathogenesis through prion-induced TNFR1 upregulation.
Asunto(s)
Cerebelo/metabolismo , Cerebelo/patología , Proteínas Priónicas/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Bovinos , Encefalopatía Espongiforme Bovina/metabolismo , Encefalopatía Espongiforme Bovina/patología , Transportador 4 de Aminoácidos Excitadores/genética , Transportador 4 de Aminoácidos Excitadores/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inflamación/metabolismo , Inflamación/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Neuronas/metabolismo , Neuronas/patología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Scrapie/metabolismo , Scrapie/patología , Sinapsis/metabolismo , Sinapsis/patologíaRESUMEN
In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.
Asunto(s)
Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Quinasas Asociadas a rho/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Dispositivos Laboratorio en un Chip , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Neurogénesis , Proteínas PrPC/metabolismoRESUMEN
Oligophrenin-1 (OPHN1) is a protein with multiple domains including a Rho family GTPase-activating (Rho-GAP) domain, and a Bin-Amphiphysin-Rvs (BAR) domain. Involved in X-linked intellectual disability, OPHN1 has been reported to control several synaptic functions, including synaptic plasticity, synaptic vesicle trafficking, and endocytosis. In neuroendocrine cells, hormones and neuropeptides stored in large dense core vesicles (secretory granules) are released through calcium-regulated exocytosis, a process that is tightly coupled to compensatory endocytosis, allowing secretory granule recycling. We show here that OPHN1 is expressed and mainly localized at the plasma membrane and in the cytosol in chromaffin cells from adrenal medulla. Using carbon fiber amperometry, we found that exocytosis is impaired at the late stage of membrane fusion in Ophn1 knock-out mice and OPHN1-silenced bovine chromaffin cells. Experiments performed with ectopically expressed OPHN1 mutants indicate that OPHN1 requires its Rho-GAP domain to control fusion pore dynamics. On the other hand, compensatory endocytosis assessed by measuring dopamine-ß-hydroxylase (secretory granule membrane) internalization is severely inhibited in Ophn1 knock-out chromaffin cells. This inhibitory effect is mimicked by the expression of a truncated OPHN1 mutant lacking the BAR domain, demonstrating that the BAR domain implicates OPHN1 in granule membrane recapture after exocytosis. These findings reveal for the first time that OPHN1 is a bifunctional protein that is able, through distinct mechanisms, to regulate and most likely link exocytosis to compensatory endocytosis in chromaffin cells.
Asunto(s)
Células Cromafines/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endocitosis/fisiología , Exocitosis/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Fusión de Membrana/fisiología , Proteínas Nucleares/metabolismo , Animales , Bovinos , Membrana Celular/metabolismo , Ratones , Ratones Noqueados , Vesículas Sinápticas/metabolismoRESUMEN
Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1â»/â» mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis.
Asunto(s)
Células Cromafines/metabolismo , Endocitosis/fisiología , Células Neuroendocrinas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Transporte Biológico Activo/fisiología , Bovinos , Membrana Celular/metabolismo , Células Cromafines/enzimología , Exocitosis/fisiología , Femenino , Metabolismo de los Lípidos/fisiología , Masculino , Ratones , Ratones Transgénicos , Células Neuroendocrinas/enzimología , Células PC12 , RatasRESUMEN
Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1-57 of Dpl replaced by amino acids 1-125 of PrP) and Chi2 (amino acids 1-66 of Dpl replaced by amino acids 1-134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23-134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1-24 of Dpl were replaced by amino acids 1-124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5-7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP(c) but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25-57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.
Asunto(s)
Ataxia/genética , Ataxia/patología , Cerebelo/patología , Priones/genética , Animales , Ataxia/metabolismo , Western Blotting , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Priones/química , Priones/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Quimera por TrasplanteRESUMEN
In secretory cells, calcium-regulated exocytosis is rapidly followed by compensatory endocytosis. Neuroendocrine cells secrete hormones and neuropeptides through various modes of exo-endocytosis, including kiss-and-run, cavicapture and full-collapse fusion. During kiss-and-run and cavicapture modes, the granule membrane is maintained in an omega shape, whereas it completely merges with the plasma membrane during full-collapse mode. As the composition of the granule membrane is very different from that of the plasma membrane, a precise sorting process of granular proteins must occur. However, the fate of secretory granule membrane after full fusion exocytosis remains uncertain. Here, we investigated the mechanisms governing endocytosis of collapsed granule membranes by following internalization of antibodies labeling the granule membrane protein, dopamine-ß-hydroxylase (DBH) in cultured chromaffin cells. Using immunofluorescence and electron microscopy, we observed that after full collapse, DBH remains clustered on the plasma membrane with other specific granule markers and is subsequently internalized through vesicular structures composed mainly of granule components. Moreover, the incorporation of this recaptured granule membrane into an early endosomal compartment is dependent on clathrin and actin. Altogether, these results suggest that after full collapse exocytosis, a selective sorting of granule membrane components is facilitated by the physical preservation of the granule membrane entity on the plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Células Cromafines/fisiología , Exocitosis , Células Neuroendocrinas/metabolismo , Vesículas Secretoras , Actinas/metabolismo , Animales , Bovinos , Clatrina/metabolismo , Humanos , Vesículas Secretoras/fisiologíaRESUMEN
In Ngsk prion protein (PrP)-deficient mice (NP(0/0)), ectopic expression of PrP-like protein Doppel (Dpl) in central neurons induces significant Purkinje cell (PC) death resulting in late-onset ataxia. NP(0/0) PC death is partly prevented by either knocking-out the apoptotic factor BAX or overexpressing the anti-apoptotic factor BCL-2 suggesting that apoptosis is involved in Dpl-induced death. In this study, Western blotting and immunohistofluorescence show that both before and during significant PC loss, the scrapie-responsive gene 1 (Scrg1)--potentially associated with autophagy--and the autophagic markers LC3B and p62 increased in the NP(0/0) PCs whereas RT-PCR shows stable mRNA expression, suggesting that the degradation of autophagic products is impaired in NP(0/0) PCs. At the ultrastructural level, autophagic-like profiles accumulated in somatodendritic and axonal compartments of NP(0/0), but not wild-type PCs. The most robust autophagy was observed in NP(0/0) PC axon compartments in the deep cerebellar nuclei suggesting that it is initiated in these axons. Our previous and present data indicate that Dpl triggers autophagy and apoptosis in NP(0/0) PCs. As observed in amyloid neurodegenerative diseases, upregulation of autophagic markers as well as extensive accumulation of autophagosomes in NP(0/0) PCs are likely to reflect a progressive dysfunction of autophagy that could trigger apoptotic cascades.
Asunto(s)
Priones/genética , Células de Purkinje/metabolismo , Células de Purkinje/patología , Animales , Autofagia , Axones/patología , Axones/ultraestructura , Western Blotting , Muerte Celular , Corteza Cerebelosa/patología , Corteza Cerebelosa/ultraestructura , Núcleos Cerebelosos/patología , Núcleos Cerebelosos/ultraestructura , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Dendritas/patología , Dendritas/ultraestructura , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI , Genotipo , Inmunohistoquímica , Proteínas de Membrana de los Lisosomas/biosíntesis , Proteínas de Membrana de los Lisosomas/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Priones/biosíntesis , Células de Purkinje/ultraestructura , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción TFIIH , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.
Asunto(s)
Membrana Celular/fisiología , Gránulos Citoplasmáticos/metabolismo , Exocitosis/fisiología , Ácidos Fosfatidicos/biosíntesis , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Animales , Membrana Celular/ultraestructura , Células Cromafines/fisiología , Gránulos Citoplasmáticos/ultraestructura , Electrofisiología , Hormona del Crecimiento/metabolismo , Humanos , Lípidos de la Membrana/biosíntesis , Potenciales de la Membrana , Microscopía Inmunoelectrónica , Células PC12 , Plásmidos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , TransfecciónRESUMEN
The Scrg1 gene was initially discovered as one of the genes upregulated in transmissible spongiform encephalopathies (TSE). Scrg1 encodes a highly conserved, cysteine-rich protein expressed principally in the central nervous system. The protein is targeted to the Golgi apparatus and large dense-core vesicles/secretory granules in neurons. We have recently shown that the Scrg1 protein is widely induced in neurons of scrapie-infected mice, suggesting that Scrg1 is involved in the host response to stress and/or the death of neurons. At the ultrastructural level, Scrg1 is associated with dictyosomes of the Golgi apparatus and autophagic vacuoles of degenerative neurons. It is well known that apoptosis plays a major role in the events leading to neuronal cell death in TSE. However, autophagy was identified in experimentally induced scrapie a long time ago and was recently reevaluated as a possible cell death program in prion diseases. The consistent association of Scrg1 with autophagic structures typical of scrapie is in agreement with the recruitment of Golgi-specific proteins in this degradation process and we suggest that Scrg1 might be used as a specific probe to identify neuronal autophagy in TSE.
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Autofagia , Proteínas del Tejido Nervioso/análisis , Enfermedades por Prión/patología , Animales , Biomarcadores/análisis , Ratones , Neuronas/química , Neuronas/ultraestructura , Enfermedades por Prión/metabolismoRESUMEN
The Lurcher mutation in the Grid2 gene causes the cell autonomous death of virtually all cerebellar Purkinje cells and the target-related death of 90% of the granule cells and 60-75% of the olivary neurons. Inactivation of Bax, a pro-apoptotic gene of the Bcl-2 family, in heterozygous Lurcher mutants (Grid2Lc/+) rescues approximately 60% of the granule cells, but does not rescue Purkinje or olivary neurons. Given the larger size of the cerebellar molecular layer in Grid2Lc/+;Bax(-/-) double mutants compared to Grid2Lc/+ mutants, we analyzed the survival of the stellate and basket interneurons as well as the synaptic connectivity of parallel fibers originating from the surviving granule cells in the absence of their Purkinje cell targets in the Grid2Lc/+;Bax(-/-) cerebellum. Quantification showed a significantly higher density of interneurons ( approximately 60%) in the molecular layer of the Grid2Lc/+;Bax(-/-) mice compared to Grid2Lc/+, suggesting that interneurons are subject to a BAX-dependent target-related death in the Lurcher mutants. Furthermore, electron microscopy showed the normal ultrastructural aspect of a number of parallel fibers in the molecular layer of the Grid2Lc/+; Bax(-/-) double mutant mice and preserved their numerous synaptic contacts on interneurons, suggesting that interneurons could play a trophic role for axon terminals of surviving granule cells. Finally, parallel fibers varicosities in the double mutant established "pseudo-synapses" on glia as well as displayed autophagic profiles, suggesting that the connections established by the parallel fibers in the absence of their Purkinje cell targets were subject to a high turnover involving autophagy.
Asunto(s)
Corteza Cerebelosa/anomalías , Interneuronas/metabolismo , Células de Purkinje/metabolismo , Receptores de Glutamato/genética , Sinapsis/metabolismo , Proteína X Asociada a bcl-2/genética , Animales , Apoptosis/genética , Autofagia/genética , Comunicación Celular/genética , Recuento de Células , Diferenciación Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Corteza Cerebelosa/citología , Corteza Cerebelosa/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Interneuronas/ultraestructura , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Microscopía Electrónica de Transmisión , Factores de Crecimiento Nervioso/metabolismo , Neuroglía/metabolismo , Neuroglía/ultraestructura , Células de Purkinje/ultraestructura , Sinapsis/ultraestructura , Regulación hacia Arriba/genéticaRESUMEN
Phocein, a widely expressed intracellular protein involved in clathrin- and dynamin-dependent membrane dynamics, has been shown to interact with members of the striatin family of proteins, striatin, SG2NA, and zinedin. Immunogold labeling was performed to assess subcellular localization of phocein in neurons of the rodent cerebellar cortex and hippocampal Ammon's horn. Most of the phocein-bound gold particles were located within dendritic thorns and spines of the cerebellar Purkinje cells and hippocampal pyramidal neurons, as observed previously for striatin in striatal neurons. The postsynaptic profiles containing phocein were engaged in asymmetric synapses with the main types of afferents in the cerebellum and in the hippocampus. In the cerebellum, phocein-bound immunogold particle numbers ranged from 1-20 in approximately 50% of the Purkinje cell spines. In these spines most of the immunogold particles were found in the neuroplasm ( approximately 70%) and on nonsynaptic plasma membrane domains and related structures such as endocytic-like profiles ( approximately 18%). As soon as the first postnatal week, phocein was detected in the Purkinje cell somatic and dendritic thorns making asymmetric synapses with climbing fibers. During the following weeks the protein was located in the dendritic spines, as observed in the adult molecular layer. Finally, double immunogold labeling revealed a distribution of phocein and SG2NA suggesting that the two proteins could interact in the Purkinje cell spines. The early postnatal expression of phocein, a protein involved in membrane dynamics, suggests that it may have functional relevance in dendritic remodeling during development and potentially in spine plasticity during adulthood.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Espinas Dendríticas/ultraestructura , Proteínas de la Membrana/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/ultraestructura , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratones , RatasRESUMEN
We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In this study, Scrg1-immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie-infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non-infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie-infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non-transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp 0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.
Asunto(s)
Autofagia/genética , Encéfalo/metabolismo , Degeneración Nerviosa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Animales , Aurotioglucosa/farmacología , Encéfalo/patología , Encéfalo/ultraestructura , Modelos Animales de Enfermedad , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/metabolismo , Proteínas Ligadas a GPI , Aparato de Golgi/patología , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/fisiopatología , Priones/genética , Vacuolas/patología , Vacuolas/ultraestructuraRESUMEN
Expression of the cellular prion protein (PrP(c)) by host cells is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. As a consequence, identification of the cell types expressing PrP(c) is necessary to determine the target cells involved in the cerebral propagation of prion diseases. To identify the cells expressing PrP(c) in the mouse brain, the immunocytochemical localization of PrP(c) was investigated at the cellular and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of transgenic mice. By using a preembedding immunogold technique, neuronal PrP(c) was observed mainly bound to the cell surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited PrP(c) antigen. In the olfactory bulb, neocortex, putamen, hippocampus, thalamus, and cerebellum, the distribution pattern of both green fluorescent protein and PrP(c) immunoreactivity suggested that the transgenic regulatory sequences of the bovine PrP gene were sufficient to promote expression of the reporter gene in neurons that express immunodetectable endogenous PrP(c). Transgenic mice expressing PrP-GFP may thus provide attractive murine models for analyzing the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases.