RESUMEN
Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen-glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1ß and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1ß and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Inmunoglobulinas Intravenosas/farmacología , Inflamasomas/metabolismo , Neuronas/metabolismo , Accidente Cerebrovascular/patología , Animales , Isquemia Encefálica/complicaciones , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/patología , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas NLR , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/metabolismo , Resultado del TratamientoRESUMEN
Polysaccharide-based nanoparticles have attracted interest as carriers for imaging and therapeutic agents because of their unique physicochemical properties, including biocompatibility and biodegradability. In addition, the functional groups of the polysaccharide backbone allow facile chemical modification to develop nanoparticles with diverse structures. Some polysaccharides have the intrinsic ability to recognize specific cell types, facilitating the design of targeted-drug delivery systems through receptor-mediated endocytosis. The main objective of this review is to provide an overview of various polysaccharide-based nanoparticles and to highlight the recent efforts that have been made to improve the characteristics of polysaccharide-based nanoparticles for drug delivery and biomedical imaging.
Asunto(s)
Diagnóstico por Imagen/métodos , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/administración & dosificación , Nanopartículas/química , Polisacáridos/administración & dosificación , Polisacáridos/química , Animales , HumanosRESUMEN
Caspase plays an important role in apoptosis. We report here that farnesyltransferase/geranylgeranyltransferase (FTase/GGTase)-alpha, a common subunit of FTase (alpha/beta(FTase)) and GGTase I (alpha/beta(GGTase)), was cleaved by caspase-3 during apoptosis. FTase/GGTase-alpha (49 kDa) was cleaved to 35 kDa (p35) in the Rat-2/H-ras, W4 and Rat-1 cells treated with FTase inhibitor (LB42708), anti-Fas antibody and etoposide, respectively. This cleavage was inhibited by caspase-inhibitors (YVAD-cmk, DEVD-cho). Serial N-terminal deletions and site-directed mutagenesis showed that Asp59 of FTase/GGTase-alpha was cleaved by caspase-3. The common FTase/GGTase-alpha subunit, but not the beta subunits, of the FTase or GGTase I protein complexes purified from baculovirus-infected SF-9 cells was cleaved to be inactivated by purified caspase-3. In contrast, FTase mutant protein complex [(D(59)A)alpha/beta(FTase)] was resistant to caspase-3. Expression of either the cleavage product (60-379) or anti-sense of FTase/GGTase-alpha induced cell death in Rat-2/H-ras cells. Furthermore, expression of (D(59)A)FTase/GGTase-alpha mutant significantly desensitized cells to etoposide-induced death. Taken together, we suggest that cleavage of prenyltransferase by caspase contributes to the progression of apoptosis.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Apoptosis/fisiología , Caspasas/metabolismo , Transferasas Alquil y Aril/efectos de los fármacos , Transferasas Alquil y Aril/genética , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Ácido Aspártico/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Supervivencia Celular/genética , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Farnesiltransferasa , Fibroblastos/metabolismo , Fibroblastos/patología , Linfoma/metabolismo , Mutación , Oligopéptidos/farmacología , Mapeo Peptídico , Prenilación de Proteína , Subunidades de ProteínaRESUMEN
Apoptotic cell death and increased production of amyloid b peptide (Ab) are pathological features of Alzheimer's disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro-apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide-induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+-ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3-induced apoptosis was suppressed by caspase inhibitor Z-VAD and by Bcl-XL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPSW) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C-terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Ab42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis-associated Ab42 production of calsenilin/DREAM/KChIP3.
Asunto(s)
Apoptosis , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Oligonucleótidos Antisentido/metabolismo , Proteínas Represoras/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Western Blotting , Proteínas de Unión al Calcio/genética , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Células Jurkat , Proteínas de Interacción con los Canales Kv , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuroblastoma , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Presenilina-2 , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Proteínas Represoras/genética , Transfección , Proteína bcl-XRESUMEN
Using an in vitro translation assay to screen a human brain cDNA library, we isolated the microtubule-associated protein Tau and determined it to be a caspase-3 substrate whose C-terminal cleavage occurred during neuronal apoptosis. DeltaTau, the 50-kDa cleavage product, was detected by Western blot in apoptotic cortical cells probed with anti-PHF-1 and anti-Tau-5 antibodies, but not anti-T-46 antibody which recognizes the C-terminus. Overexpression of DeltaTau in SK-N-BE2(C) cells significantly increased the incidence of cell death. Staurosporine-induced Tau cleavage was blocked by 20 microM z-Asp-Glu-Val-Asp-chloromethylketone, a caspase-3 inhibitor, and in vitro, Tau was selectively cleaved by caspase-3 or calpain, a calcium-activated protease, but not by caspases-1, -8, or -9. (D421E)-Tau, a mutant in which Asp421 was replaced with a Glu, was resistant to cleavage by caspase-3 and tended to suppress staurosporine-induced cell death more efficiently than did wild-type Tau in both transient and stable expression systems. Finally, the incidence of DeltaTau-induced cell death was augmented by expression of Abeta precursor protein (APP) or Swedish APP mutant. Taken together, these results suggest that the caspase-3 cleavage product of Tau may contribute to the progression of neuronal cell death in Alzheimer's disease.