RESUMEN
NMDA receptors (NMDARs) play an essential role in some forms of synaptic plasticity, learning, and memory. Therefore, these receptors are highly regulated with respect to their localization, activation, and abundance both within and on the surface of mammalian neurons. Fundamental questions remain, however, regarding how this complex regulation is achieved. Using cell-based models and F-box Only Protein 2 (Fbxo2) knock-out mice, we found that the ubiquitin ligase substrate adaptor protein Fbxo2, previously reported to facilitate the degradation of the NMDAR subunit GluN1 in vitro, also functions to regulate GluN1 and GluN2A subunit levels in the adult mouse brain. In contrast, GluN2B subunit levels are not affected by the loss of Fbxo2. The loss of Fbxo2 results in greater surface localization of GluN1 and GluN2A, together with increases in the synaptic markers PSD-95 and Vglut1. These synaptic changes do not manifest as neurophysiological differences or alterations in dendritic spine density in Fbxo2 knock-out mice, but result instead in increased axo-dendritic shaft synapses. Together, these findings suggest that Fbxo2 controls the abundance and localization of specific NMDAR subunits in the brain and may influence synapse formation and maintenance.
Asunto(s)
Encéfalo/metabolismo , Proteínas F-Box/metabolismo , Regulación de la Expresión Génica/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Encéfalo/citología , Células Cultivadas , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Proteínas F-Box/genética , Células HEK293 , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Transporte de Proteínas/genética , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructura , Sinaptosomas/metabolismo , Sinaptosomas/ultraestructura , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
For insulin synthesis, the proinsulin precursor is translated at the endoplasmic reticulum (ER), folds to include its three native disulfide bonds, and is exported to secretory granules for processing and secretion. Protein disulfide isomerase (PDI) has long been assumed to assist proinsulin in this process. Herein we have examined the effect of PDI knockdown (PDI-KD) in ß-cells. The data establish that upon PDI-KD, oxidation of proinsulin to form native disulfide bonds is unimpaired and in fact enhanced. This is accompanied by improved proinsulin exit from the ER and increased total insulin secretion, with no evidence of ER stress. We provide evidence for direct physical interaction between PDI and proinsulin in the ER of pancreatic ß-cells, in a manner requiring the catalytic activity of PDI. In ß-cells after PDI-KD, enhanced export is selective for proinsulin over other secretory proteins, but the same effect is observed for recombinant proinsulin trafficking upon PDI-KD in heterologous cells. We hypothesize that PDI exhibits unfoldase activity for proinsulin, increasing retention of proinsulin within the ER of pancreatic ß-cells.
Asunto(s)
Retículo Endoplásmico/metabolismo , Células Secretoras de Insulina/citología , Proinsulina/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Secuencia de Bases , Disulfuros/química , Técnicas de Silenciamiento del Gen , Células HEK293 , Células Hep G2 , Humanos , Proinsulina/química , Proteína Disulfuro Isomerasas/deficiencia , Proteína Disulfuro Isomerasas/genética , Transporte de ProteínasRESUMEN
It has previously been shown that misfolded mutant Akita proinsulin in the endoplasmic reticulum engages directly in protein complexes either with nonmutant proinsulin or with "hProCpepGFP" (human proinsulin bearing emerald-GFP within the C-peptide), impairing the trafficking of these "bystander" proinsulin molecules (Liu, M., Hodish, I., Rhodes, C. J., and Arvan, P. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 15841-15846). Herein, we generated transgenic mice, which, in addition to expressing endogenous proinsulin, exhibit beta-cell-specific expression of hProCpepGFP via the Ins1 promoter. In these mice, hProCpepGFP protein levels are physiologically regulated, and hProCpepGFP is packaged and processed to CpepGFP that is co-stored in beta-secretory granules. Visualization of CpepGFP fluorescence provides a quantifiable measure of pancreatic islet insulin content that can be followed in live animals in states of health and disease. We examined loss of pancreatic insulin in hProCpepGFP transgenic mice mated to Akita mice that develop neonatal diabetes because of the expression of misfolded proinsulin. Loss of bystander insulin in Akita animals is detected initially as a block in CpepGFP/insulin production with intracellular accumulation of the precursor, followed ultimately by loss of pancreatic beta-cells. The data support that misfolded proinsulin perturbs bystander proinsulin in the endoplasmic reticulum, leading to beta-cell failure.
Asunto(s)
Diabetes Mellitus/congénito , Diabetes Mellitus/metabolismo , Proinsulina/química , Proinsulina/metabolismo , Pliegue de Proteína , Animales , Animales Recién Nacidos , Efecto Espectador , Péptido C/metabolismo , Cruzamientos Genéticos , Diabetes Mellitus/patología , Femenino , Fluorescencia , Glucosa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Transgénicos , Especificidad de Órganos , Ratas , Vías Secretoras , Transgenes/genéticaRESUMEN
The most common dominantly inherited ataxia, spinocerebellar ataxia type 3 (SCA3), is an incurable neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene that encodes an abnormally long polyglutamine tract in the disease protein, ATXN3. Mice lacking ATXN3 are phenotypically normal; hence, disease gene suppression offers a compelling approach to slow the neurodegenerative cascade in SCA3. Here we tested antisense oligonucleotides (ASOs) that target human ATXN3 in two complementary mouse models of SCA3: yeast artificial chromosome (YAC) MJD-Q84.2 (Q84) mice expressing the full-length human ATXN3 gene and cytomegalovirus (CMV) MJD-Q135 (Q135) mice expressing a human ATXN3 cDNA. Intracerebroventricular injection of ASOs resulted in widespread delivery to the most vulnerable brain regions in SCA3. In treated Q84 mice, three of five tested ASOs reduced disease protein levels by >50% in the diencephalon, cerebellum, and cervical spinal cord. Two ASOs also significantly reduced mutant ATXN3 in the mouse forebrain and resulted in no signs of astrogliosis or microgliosis. In Q135 mice expressing a single ATXN3 isoform via a cDNA transgene, ASOs did not result in similar robust ATXN3 silencing. Our results indicate that ASOs targeting full-length human ATXN3 would likely be well tolerated and could lead to a preventative therapy for SCA3.
RESUMEN
Hyperglycemia increases insulin flux through the endoplasmic reticulum (ER) of pancreatic ß-cells, and the unfolded protein response pathway is required to enhance insulin processing. Pancreatic and duodenal homeobox 1 (PDX1), a key pancreatic transcription factor, regulates insulin along with targets involved in insulin processing and secretion. Here we find that PDX1 is a direct transcriptional regulator of ER oxidoreductin-1-like ß (Ero1lß), which maintains the oxidative environment of the ER to facilitate disulfide bond formation. PDX1 deficiency reduced Ero1lß transcript levels in mouse islets and mouse insulinoma (MIN6) cells; moreover, PDX1 occupied the Ero1lß promoter in ß-cells. ERO1lß levels were induced by high glucose concentrations and by the reducing agent dithiothreitol, indicating potential roles in adaptation to increased oxidative protein folding load in the ß-cell ER. In MIN6 cells, small interfering RNA-mediated silencing of Ero1lß decreased insulin content and increased susceptibility to ER stress-induced apoptosis. These findings demonstrate roles for the PDX1 target ERO1lß in maintaining insulin content and regulating cell survival during ER stress.
Asunto(s)
Apoptosis , Glicoproteínas/fisiología , Hipoglucemiantes/farmacología , Insulina/farmacología , Islotes Pancreáticos/efectos de los fármacos , Estrés Oxidativo , Animales , Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Línea Celular , Silenciador del Gen , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hiperglucemia/metabolismo , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Insulina/química , Insulina/farmacocinética , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Sustancias Reductoras/toxicidad , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Type 1B diabetes (typically with early onset and without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene Induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed mutant proinsulins exerting dominant-negative (toxic gain of function) behavior in pancreatic beta cells. Here we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that, in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Proinsulina/química , Proinsulina/genética , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Diabetes Mellitus Tipo 1/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/fisiología , Proinsulina/metabolismo , Proinsulina/fisiología , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismoRESUMEN
Single-chain insulins (SCIs) are single polypeptide chains in which the insulin B-chain links contiguously with the insulin A-chain via an uncleaved connecting peptide. Although direct linkage of insulin B- and A-chains produces SCIs with little insulin receptor binding, biologists have been interested in bioengineering linker peptides that form a flexible reverse turn, allowing SCIs to activate insulin receptors. In this report, we have investigated a series of cDNAs intended to explore the significance of linker length, cleavability, and the impact of certain site-dependent residues for the bioactivity of recombinant SCIs on insulin receptors. SCI concentration is readily measured by RIA with a (proinsulin plus insulin)-specific polyclonal antibody. Although dibasic flanking residues may result in potential endoproteolytic susceptibility, a linker with -Gln-Arg- flanking sequences resisted cleavage even in secretory granules, ensuring single-chain behavior. Effective SCIs exhibit favorable and specific binding with insulin receptors. SCIs with linkers bearing an Arg residue immediately preceding the A-chain were most bioactive, although efficient receptor interaction was inhibited as SCI linker length increased, approaching that observed for proinsulin. SCIs activate downstream metabolic signaling, stimulating glucose uptake into adipocytes and suppressing gluconeogenic enzyme biosynthesis in hepatocytes, with only limited cross-reactivity on IGF-I receptors. SCIs might theoretically have utility either in immunotherapy or gene therapy in insulin-deficient diabetes.