RESUMEN
Diabetic encephalopathy is one of the risk factors for Alzheimer's disease. Our previous findings indicated that animals with diabetic encephalopathy exhibit learning and memory impairment in addition to hippocampal neurodegeneration, both of which are ameliorated with amyloid precursor protein (APP) 17-mer (APP17) peptide treatment. Although APP17 is neuroprotective, it is susceptible to enzymatic degradation. Derived from the active sequence structure of APP17, we have previously structurally transformed and modified several APP5-mer peptides (APP328-332 [RERMS], APP 5). We have developed seven different derivatives of APP5, including several analogs. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on human neuroblastoma SH-SY5Y cells in the present study showed that P165 was the most neuroprotective APP5 derivative. Furthermore, we tested the effects of APP5 and P165 on the number of cells and the release of lactate dehydrogenase. Western immunoblot analyses were also performed. The digestion rates of P165 and APP5 were determined by the pepsin digestion test. P165 resisted pepsin digestion significantly more than APP5. Therefore, P165 may be optimal for oral administration. Overall, these findings suggest that P165 may be a potential drug for the treatment of diabetic encephalopathy.
Asunto(s)
Precursor de Proteína beta-Amiloide/farmacología , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacología , Administración Oral , Precursor de Proteína beta-Amiloide/farmacocinética , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cromatografía Líquida de Alta Presión , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fármacos Neuroprotectores/farmacocinética , Pepsina A/metabolismo , Fragmentos de Péptidos/farmacocinética , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Accumulating evidence suggests that bone-marrow (BM)-derived mononuclear phagocytes have an important role in the clearance of soluble and aggregated amyloid-beta peptides (Abeta) in Alzheimer's disease (AD) brains. However, the exact kinetics of Abeta clearance in mononuclear phagocytes derived from transgenic animal models of AD expressing beta-amyloid precursor protein (APP) mutants have been poorly characterized. We have examined whether CCL2 and APP expression affects the clearance of Abeta in conjunction with our control, acetylated low-density lipoprotein (AcLDL), using primary cultured BM-derived macrophages derived from adult APP, CCL2, APP/CCL2, and control littermates. Pulse-chase analysis demonstrated three distinct destinations for Abeta40 and AcLDL: intracellular retention, degradation, and secretion. As predicted, 50% of Abeta remained intracellularly contained even 5 days after pulse, while 40% of degraded and 14% of nondegraded Abeta were secreted. APP/CCL2 macrophages show reduced intracellular Abeta retention, along with enhanced secretion of both degraded and nondegraded Abeta. Abeta accumulation in aggresome is also partially reduced in APP/CCL2 macrophages as compared to other APP, CCL2, or control groups, suggesting impaired sorting of aggregated Abeta in aggresomes. The degradation of intracranially injected (125)I-Abeta40 aggregates was also enhanced in adult APP/CCL2 mice as compared to APP littermates in vivo. These data suggest that APP and CCL2 synergistically enhance BM-derived macrophage-mediated clearance of Abeta. In contrast, the clearance of AcLDL by BM-derived macrophages was not significantly enhanced by the presence of either APP or CCL2.
Asunto(s)
Péptidos beta-Amiloides/farmacocinética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/farmacocinética , Quimiocina CCL2/genética , Quimiocina CCL2/farmacocinética , Macrófagos/metabolismo , Péptidos beta-Amiloides/biosíntesis , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/biosíntesis , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Células Cultivadas , Quimiocina CCL2/biosíntesis , Femenino , Humanos , Macrófagos/patología , Masculino , Tasa de Depuración Metabólica/genética , Ratones , Ratones TransgénicosRESUMEN
This study was performed to test the hypothesis that fragments of amyloid precursor protein are able to enter the injured blood-brain barrier (BBB) following brain ischemia. We observed chronic disruption of the BBB after ischemia. The BBB changes did not increase in intensity and frequency with longer periods of recirculation. As an effect of BBB injury, we noted a visible connection of diffuse amyloid plaques with neurovasculature in rats. This pathology appears to have a similar distribution as in Alzheimer's disease, hippocampal and cortical changes being most severe.
Asunto(s)
Precursor de Proteína beta-Amiloide/farmacocinética , Barrera Hematoencefálica , Isquemia Encefálica/fisiopatología , Daño por Reperfusión/fisiopatología , Enfermedad de Alzheimer/fisiopatología , Animales , Isquemia Encefálica/veterinaria , Femenino , Peroxidasa de Rábano Silvestre/farmacocinética , Ratas , Ratas Wistar , Daño por Reperfusión/veterinariaRESUMEN
Recent studies indicate that nicastrin (NCT) and presenilins form functional components of a multimeric gamma-secretase complex required for the regulated intramembraneous proteolysis of Notch and beta-amyloid (Abeta) precursor protein (APP). To determine whether nicastrin is required for proteolytic processing of Notch and APP in mammals and the role of nicastrin in presenilin/gamma-secretase complex assembly, we generated nicastrin-deficient (NCT-/-) mice and derived fibroblasts from NCT-/- embryos. Nicastrin-null embryos died by embryonic day 10.5 and exhibited several patterning defects, including abnormal somite segmentation, phenotypes that are reminiscent of embryos lacking Notch1 or both presenilins. Importantly, secretion of Abeta peptides is abolished in NCT-/- fibroblasts, whereas it is reduced by approximately 50% in NCT+/- cells; the failure to generate Abeta peptides in NCT-/- cells is accompanied by destabilization of the presenilin/gamma-secretase complex and accumulation of APP-C-terminal fragments. Moreover, APP trafficking analysis in NCT-/- fibroblasts revealed a significant delay in the rate of APP reinternalization compared with that of control cells. Together, these results establish that nicastrin is an essential component of the multimeric gamma-secretase complex in mammals required for both gamma-secretase activity and APP trafficking and suggest that nicastrin may be a valuable therapeutic target for Alzheimer's disease.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Anomalías Múltiples/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/farmacocinética , Animales , Ácido Aspártico Endopeptidasas , Células Cultivadas , Cruzamientos Genéticos , Fibroblastos/citología , Fibroblastos/metabolismo , Marcación de Gen , Sustancias Macromoleculares , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Presenilina-1 , Presenilina-2 , Procesamiento Proteico-Postraduccional/fisiología , Transporte de Proteínas/fisiología , Receptores Notch , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
A key commonality of most age-related neurodegenerative diseases is the accumulation of aggregation-prone proteins in the brain. Except for the prionoses, the initiation and propagation of these proteopathies in vivo remains poorly understood. In a previous study, we found that the deposition of the amyloidogenic peptide Abeta can be induced by injection of dilute extracts of Alzheimeric neocortex into the brains of Tg2576 transgenic mice overexpressing the human beta-amyloid precursor protein. The present study was undertaken to assess the pathology after long-term (12 months) incubation, and to clarify the distinctive anatomical distribution of seeded Abeta-immunoreactivity. All mice were injected at 3 months of age; 5 months later, as expected, Abeta deposits were concentrated mostly in the injected hemisphere. After 12 months, abundant, transgene-derived Abeta deposits were present bilaterally in the forebrain, but plaque load was still clearly greater in the extract-injected hemisphere. There was also evidence of tau hyperphosphorylation in axons of the corpus callosum that had been injured by the injection, most prominently in transgenic mice, but also, to a lesser degree, in non-transgenic mice. Five months following injection of AD-extract, an isolated cluster of Abeta-immunoreactive microglia was sometimes evident in the ipsilateral entorhinal cortex; the strong innervation of the hippocampus by entorhinal cortical neurons suggests the possible spread of seeded pathology from the injection site via neuronal transport mechanisms. Finally, using India Ink to map the local dispersion of injectate, we found that Abeta induction is especially potent in places where the injectate is sequestered. The AD-seeding model can illuminate the emergence and spread of cerebral beta-amyloidosis and tau hyperphosphorylation, and thus could enhance our understanding of AD and its pathogenic commonalties with other cerebral proteopathies.
Asunto(s)
Precursor de Proteína beta-Amiloide/farmacocinética , Amiloidosis/inducido químicamente , Corteza Cerebral/patología , Precursor de Proteína beta-Amiloide/administración & dosificación , Precursor de Proteína beta-Amiloide/genética , Amiloidosis/patología , Animales , Axones/metabolismo , Corteza Cerebral/metabolismo , Cuerpo Calloso/citología , Corteza Entorrinal/patología , Humanos , Inyecciones Intraventriculares , Ratones , Ratones Transgénicos , Microglía/patología , Factores de TiempoRESUMEN
Investigations were performed to characterize a recombinant Kunitz protease inhibitory domain of the amyloid beta-protein precursor (rKPI) as anticoagulants. After a single intravenous infusion of wild type rKPI into dogs, its elimination fit a two compartment model with a t1/2alpha and t1/2beta of 5 and 77 min, respectively. Further investigations determined if a variant form of rKPI with 178-fold more potent anti-factor Xa activity (rKPI-DD135, Ki = 0.9 nM) could serve as an anticoagulant in a rabbit model of extracorporeal circulation using a venovenous shunt. A prospective investigation was initiated to compare standard heparin (n = 8) at 400 U/kg with different infusion concentrations of rKPI-DD135. After a single intravenous infusion of 1.89 mg/kg of rKPI-DD135 followed by a constant infusion at 0.003 (n = 3), 0.03 (n = 7), or 0.3 (n = 5) mg/kg/min, the anti-factor Xa activity of the animals' plasma rapidly reaches a steady state for the two lower infusion concentrations of the agent. All infusions of rKPI-DD135 prolong the activated clotting time with less variation than that seen with heparin administration. rKPI-DD135 anticoagulation does not prevent a drop in the platelet counts. Fibrinogen levels decrease only slightly when the circuit is anticoagulated with rKPI-DD135. rKPI-DD135 markedly prolongs the APTT, has little effect on the PT, and reduces plasma prekallikrein and plasminogen activation. The 0.3 mg/kg/min infusion concentration of rKPI-DD135 results in reduced deposition of 111Indium-labeled platelets on the circuit when compared to heparin. Last, after a steady state level is achieved, 60% of the plasma anti-factor Xa activity of rKPI-DD135 is eliminated within 60 min after stopping the infusion. These data show the rKPI-DD135 can provide single agent anticoagulation in a rabbit extracorporeal circuit. Development of short acting factor Xa inhibitors may be useful anticoagulants for cardiopulmonary bypass.
Asunto(s)
Precursor de Proteína beta-Amiloide/farmacología , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Circulación Extracorporea , Inhibidores del Factor Xa , Fragmentos de Péptidos/farmacología , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/farmacocinética , Animales , Anticoagulantes/farmacocinética , Perros , Fibrinógeno/análisis , Hemodinámica , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacocinética , Plasminógeno/análisis , Precalicreína/análisis , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
A central question in Alzheimer's disease (AD) is the role of amyloid in pathogenesis. Recent discoveries implicating the longer A beta 1-42 form of amyloid in pathogenesis led us to characterize the interaction of A beta with cells to elucidate differences that might account for these observations. We characterized the adsorption, internalization and degradation of radiolabeled A beta in NGF-differentiated PC12 cells under conditions that are not acutely toxic. All A beta peptides examined absorb to the surface of PC12 cells and are internalized; however the adsorption and internalization of A beta 1-42 is significantly greater than that of A beta 1-40 and A beta 1-28. The adsorption of A beta 1-42 is decreased by treatment of the cells with neuraminidase, but not heparitinase. The fate of the internalized A beta 1-42 is also very different than shorter A beta peptides; a fraction of the internalized A beta 1-42 accumulates intracellularly and is resistant to degradation for at least 3 days while A beta 1-40 and shorter peptides are eliminated with a half life of about 1 h. A beta 1-42 does not appear to inhibit lysosomal hydrolases, since A beta 1-28 is degraded at the same rate in the presence or absence of A beta 1-42. The intracellular A beta 1-42 is located in a dense organellar compartment and colocalizes with the lysosomal markers Lucifer Yellow and horseradish peroxidase. These data indicate that there are significant differences in the cell surface adsorption, internalization and catabolism of A beta 1-42 compared to A beta 1-40 and A beta 1-28. These differences may be important for the preferential accumulation of the longer A beta 1-42 isoform and its association with AD pathogenesis.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Células PC12/metabolismo , Adsorción , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/farmacocinética , Animales , Compartimento Celular/fisiología , Diferenciación Celular/fisiología , Endosomas/química , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Isomerismo , Isoquinolinas , Lisosomas/química , Microscopía Confocal , Datos de Secuencia Molecular , Degeneración Nerviosa/fisiología , Células PC12/citología , Células PC12/ultraestructura , RatasRESUMEN
Cerebral capillary sequestration and blood-brain barrier (BBB) permeability to apolipoproteins E2 (apoE2), E3 (apoE3), and E4 (apoE4) and to their complexes with sA beta(1-40), a peptide homologous to the major form of soluble Alzheimer's amyloid beta, were studied in perfused guinea pig brain. Cerebrovascular uptake of three apoE isoforms was low, their blood-to-brain transport undetectable, but uptake by the choroid plexus significant. Binding of all three isoforms to sA beta(1-40) in vitro was similar with a K(D) between 11.8 and 12.9 nM. Transport into brain parenchyma and sequestration by BBB and choroid plexus were negligible for sA beta(1-40)-apoE2 and sA beta(1-40)-apoE3, but significant for sA beta(1-40)-apoE4. After 10 min, 85% of sA beta(1-40)-apoE4 taken up at the BBB remained as intact complex, whereas free sA beta(1-40) was 51% degraded. Circulating apoE isoforms have contrasting effects on cerebral capillary uptake of and BBB permeability of sA beta. ApoE2 and apoE3 completely prevent cerebral capillary sequestration and blood-to-brain transport of sA beta(1-40). Conversely, apoE4, by entering brain microvessels and parenchyma as a stable complex with sA beta, reduces peptide degradation and may predispose to cerebrovascular and possibly enhance parenchymal amyloid formation under pathological conditions.
Asunto(s)
Péptidos beta-Amiloides/farmacocinética , Precursor de Proteína beta-Amiloide/farmacocinética , Apolipoproteínas E/farmacocinética , Barrera Hematoencefálica , Permeabilidad Capilar , Circulación Cerebrovascular , Fragmentos de Péptidos/farmacocinética , Secuencia de Aminoácidos , Péptidos beta-Amiloides/sangre , Precursor de Proteína beta-Amiloide/sangre , Animales , Apolipoproteína E2 , Apolipoproteína E3 , Apolipoproteína E4 , Femenino , Cobayas , Humanos , Cinética , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/sangre , Perfusión , Proteínas Recombinantes/farmacocinéticaRESUMEN
We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.