RESUMEN
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
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
BACKGROUND: There is growing evidence that the death receptor CD95 has a wider role in non-apoptotic functions. In the brain, it may contribute to neural death and to the associated inflammatory reaction via a non-apoptotic pathway. Brain injury triggers an inflammatory reaction in which the CD95/CD95L system acts principally through peripheral cells recruited to the lesion. In cases of inflammation within the brain, with no blood-brain barrier leakage, the role of the CD95/CD95L system is thus unclear. We investigated the possible role of CD95 and CD95L in such conditions, by studying the relationships between glial cell activation, neuron death and CD95/CD95L expression in the cerebellum of the Lurcher (Grid2(Lc/+)) mutant mouse, a model of cerebellar neurodegeneration. METHODS: Glial cells in slices of wild-type and Lurcher mouse cerebella were observed by light microscopy at various ages overlapping periods of neuron loss and of pre- and post-neurodegeneration. Subcellular organization was studied by electron microscopy. We assessed CD95 levels by western blotting, RT-PCR and glial cell cultures. The levels of CD95L and IL-6 were studied by ELISA and a biological assay, respectively. RESULTS: In the Grid2(Lc/+)cerebellum, neuron loss triggers a typical, but abnormally persistent, inflammatory reaction. We identified two phases of astrogliosis: an early burst of large glial cell activation, peaking at postnatal days 25 to 26, coinciding with peak cerebellar neuron loss, followed by a long period of slow decline indicating that the strength of the glial reaction is modulated by neuron mortality rates. Comparisons of time-courses of glial cell activation, cytokine production and neuron loss revealed that the number of surviving neurons decreased as CD95 increased. Thus, CD95 cannot be directly involved in neuron death, and its role must be limited to a contribution to the inflammatory reaction. The upregulation of CD95 likely on astrocytes coincides with increases in the levels of IL-6, a cytokine produced principally by astrocytes, and soluble CD95L. CONCLUSIONS: These results suggest that CD95 and soluble CD95L contribute, via non-apoptotic signaling, to the inflammatory reaction initiated early in neuron death within the Grid2(Lc/+) cerebellum.
Asunto(s)
Cerebelo/patología , Proteína Ligando Fas/fisiología , Inmunidad Innata/fisiología , Neuronas/patología , Receptores de Glutamato/fisiología , Degeneraciones Espinocerebelosas/patología , Receptor fas/fisiología , Animales , Astrocitos/fisiología , Western Blotting , Muerte Celular/fisiología , Células Cultivadas , Citocinas/metabolismo , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Microscopía Electrónica , Neuronas/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneraciones Espinocerebelosas/genética , Degeneraciones Espinocerebelosas/inmunologíaRESUMEN
In neuroendocrine cells, annexin-A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)-containing lipid microdomains that are required for calcium-regulated exocytosis. As soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin-A2-induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin-A2 to the plasma membrane, where it colocalizes with SNAP-25 and S100A10. Cross-linking experiments performed in stimulated chromaffin cells indicate that annexin-A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle-associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin-A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin-A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)-bisphosphate (PIP(2)) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin-A2-mediated lipid microdomains and SNAREs during exocytosis.
Asunto(s)
Anexina A2/fisiología , Células Cromafines/metabolismo , Exocitosis/fisiología , Proteínas SNARE/metabolismo , Adrenérgicos/metabolismo , Anexina A2/metabolismo , Anexina A2/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Gránulos Citoplasmáticos/metabolismo , Humanos , Transporte de Proteínas , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/ultraestructura , Proteínas S100/metabolismo , Proteínas S100/ultraestructura , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/ultraestructuraRESUMEN
The cerebellum participates in motor coordination as well as in numerous cerebral processes, including temporal discrimination. Animals can predict daily timing of food availability, as manifested by food-anticipatory activity under restricted feeding. By studying ex vivo clock gene expression by in situ hybridization and recording in vitro Per1-luciferase bioluminescence, we report that the cerebellum contains a circadian oscillator sensitive to feeding cues (i.e., whose clock gene oscillations are shifted in response to restricted feeding). Food-anticipatory activity was markedly reduced in mice injected intracerebroventricularly with an immunotoxin that depletes Purkinje cells (i.e., OX7-saporin). Mice bearing the hotfoot mutation (i.e., Grid2(ho/ho)) have impaired cerebellar circuitry and mild ataxic phenotype. Grid2(ho/ho) mice fed ad libitum showed regular behavioral rhythms and day-night variations of clock gene expression in the hypothalamus and cerebellum. When challenged with restricted feeding, however, Grid2(ho/ho) mice did not show any food-anticipatory rhythms, nor timed feeding-induced changes in cerebellar clock gene expression. In hypothalamic arcuate and dorsomedial nuclei, however, shifts in Per1 expression in response to restricted feeding were similar in cerebellar mutant and wild-type mice. Furthermore, plasma corticosterone and metabolites before mealtime did not differ between cerebellar mutant and wild-type mice. Together, these data define a role for the cerebellum in the circadian timing network and indicate that the cerebellar oscillator is required for anticipation of mealtime.
Asunto(s)
Regulación del Apetito/fisiología , Cerebelo/fisiología , Ritmo Circadiano/genética , Animales , Proteínas CLOCK/metabolismo , Conducta Alimentaria/fisiología , Inmunohistoquímica , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Mutación , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Proteínas Circadianas Period/metabolismo , Células de Purkinje/metabolismo , Ratas , Ratas TransgénicasRESUMEN
The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Exocitosis/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Vesículas Secretoras/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Animales , Fraccionamiento Celular , Membrana Celular/ultraestructura , Medio de Cultivo Libre de Suero , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Inmunohistoquímica , Nucleótidos/metabolismo , Células PC12 , Fosfolipasa D/metabolismo , Potasio/metabolismo , Ratas , Vesículas Secretoras/ultraestructura , TransfecciónRESUMEN
Neuropathological and neuroimaging studies indicate a decrease in Purkinje cell (PC) density in the cerebellum of autistic patients and rodent models of autism. Autism is far more prevalent in males than females, and sex-specific properties of PCs have been reported recently. We investigated the differential sensitivity of PCs in the valproate acid (VPA) mouse model of autism by estimating the linear density of PCs immununolabelled with calbindin in the cerebellum of males and females. Whereas prenatal VPA treatment surprisingly increased PC linear density in both sexes 13 days after birth (P13), it significantly reduced the linear density of PCs in the cerebellum of 40-day-old (P40) males, but not females. In males, PC loss was more pronounced in the posterior part of the cerebellum and was significant in the VIth, VIIth, IXth and paramedian lobules. In females, PC loss was restricted to the paramedian lobule. These results suggest that this sex-specific sensitivity of PCs to VPA may contribute towards the motor disturbances and behavioural abnormalities observed in autism.
Asunto(s)
Trastorno Autístico/inducido químicamente , Trastorno Autístico/patología , Modelos Animales de Enfermedad , Células de Purkinje/patología , Caracteres Sexuales , Ácido Valproico/toxicidad , Animales , Anticonvulsivantes/toxicidad , Recuento de Células , Cerebelo/efectos de los fármacos , Cerebelo/patología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Células de Purkinje/efectos de los fármacosRESUMEN
Information processing by cerebellar molecular layer interneurons (MLIs) plays a crucial role in motor behavior. MLI recruitment is tightly controlled by the profile of short-term plasticity (STP) at granule cell (GC)-MLI synapses. While GCs are the most numerous neurons in the brain, STP diversity at GC-MLI synapses is poorly documented. Here, we studied how single MLIs are recruited by their distinct GC inputs during burst firing. Using slice recordings at individual GC-MLI synapses of mice, we revealed four classes of connections segregated by their STP profile. Each class differentially drives MLI recruitment. We show that GC synaptic diversity is underlain by heterogeneous expression of synapsin II, a key actor of STP and that GC terminals devoid of synapsin II are associated with slow MLI recruitment. Our study reveals that molecular, structural and functional diversity across GC terminals provides a mechanism to expand the coding range of MLIs.
Asunto(s)
Cerebelo/citología , Cerebelo/fisiología , Red Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Animales , Ratones , Sinapsinas/metabolismoRESUMEN
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
The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.
Asunto(s)
Células de Purkinje/patología , Receptores de Glutamato/deficiencia , Animales , Muerte Celular/fisiología , Cerebelo/metabolismo , Cerebelo/patología , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Mutantes Neurológicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Células de Purkinje/metabolismo , Receptores de Glutamato/biosíntesis , Receptores de Glutamato/genéticaRESUMEN
Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at approximately 21 d after birth and control the complex spike waveform.
Asunto(s)
Fibras Nerviosas/fisiología , Células de Purkinje/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Cerebelo/citología , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , N-Metilaspartato/farmacología , Técnicas de Placa-Clamp/métodos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Synaptic partner selection and refinement of projections are important in the development of precise and functional neuronal connections. We investigated the formation of new synaptic connections in a relatively mature system to test whether developmental events can be recapitulated at later stages (i.e., after the mature synaptic organization has been established), using a model of postlesional reinnervation in the olivo-cerebellar pathway. During the development of this pathway, synaptic connections between climbing fibers (CFs) and Purkinje cells (PCs) are diffuse and redundant before synapse elimination refines the pattern. The regression of CFs during the first 2 postnatal weeks in the rat leads to mono-innervation of each PC. After unilateral transection of the rat olivo-cerebellar pathway and intracerebellar injection of BDNF 24 h after lesion, axons from the remaining inferior olive can sprout into the deafferented hemicerebellum and establish new contacts with denervated PCs at later developmental stages. We found that these contacts are first established on somatic thorns before the CFs translocate to the PC dendrites, recapitulating the morphological steps of normal CF-PC synaptogenesis, but on a relatively mature PC. However, electrophysiology of PC reinnervation by transcommissural CFs in these animals showed that each PC is reinnervated by only one CF. This mono-innervation contrasts with the reinnervation of grafted immature PCs in the same cerebellum. Our results provide evidence that relatively mature PCs do not receive several olivary afferents during late reinnervation, suggesting a critical role of the target cell state in the control of CF-PC synaptogenesis. Thus, synapse exuberance and subsequent elimination are not a prerequisite to reach a mature relationship between synaptic partners.
Asunto(s)
Células de Purkinje/citología , Células de Purkinje/fisiología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Femenino , Masculino , Células de Purkinje/ultraestructura , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Different afferent synapse populations interact to control the specificity of connections during neuronal circuit maturation. The elimination of all but one climbing-fiber onto each Purkinje cell during the development of the cerebellar cortex is a particularly well studied example of synaptic refinement. The suppression of granule cell precursors by X irradiation during postnatal days 4 to 7 prevents this synaptic refinement, indicating a critical role for granule cells. Several studies of cerebellar development have suggested that synapse elimination has a first phase which is granule cell-independent and a second phase which is granule cell-dependent. In this study, we show that sufficiently-strong irradiation restricted to postnatal days 5 or 6 completely abolishes climbing fiber synaptic refinement, leaving the olivo-cerebellar circuit in its immature configuration in the adult, with up to 5 climbing fibers innervating the Purkinje cell in some cases. This implies that the putative early phase of climbing fiber synapse elimination can be blocked by irradiation-induced granule cell loss if this loss is sufficiently large, and thus indicates that the entire process of climbing fiber synapse elimination requires the presence of an adequate number of granule cells. The specific critical period for this effect appears to be directly related to the timing of Purkinje cell and granule cell development in different cerebellar lobules, indicating a close, spatiotemporal synchrony between granule-cell development and olivo-cerebellar synaptic maturation.
Asunto(s)
Células de Purkinje/fisiología , Células de Purkinje/efectos de la radiación , Sinapsis/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Axones/fisiología , Cerebelo/crecimiento & desarrollo , Fenómenos Electrofisiológicos , Femenino , Embarazo , Ratas , Ratas WistarRESUMEN
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
Phocein is an intracellular protein highly expressed in neurons. It is the major partner of the striatin family members which are scaffolding proteins involved in signaling and trafficking. Due to its association with dynamin via direct interactions with nucleotide diphosphate kinase (NDPK) and EPS15, phocein has been implicated in vesicular trafficking, acting in particular in the endocytic process. This review focuses on immuno-cytochemical studies showing the strict localization of phocein in Purkinje cell dendritic spines involved in excitatory transmission in the cerebellum of postnatal and adult rodents. Immunogold labeling sometimes detects phocein in close vicinity with endocytic-like membrane profiles suggesting that phocein plays a role in endocytosis. Furthermore, co-localization of phocein and SG2NA within spines suggests that their interactions have a functional significance in the molecular cascades that underly membrane trafficking in post-synaptic structures. As the striatin family members are highly concentrated in dendritic spines, their interactions with phocein might be involved in mediating synaptic plasticity through spine remodeling by endocytosis.
Asunto(s)
Espinas Dendríticas/fisiología , Endocitosis , Proteínas de la Membrana/metabolismo , Células de Purkinje/fisiología , AnimalesRESUMEN
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.