RESUMEN
Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.
Asunto(s)
Calcio/metabolismo , Ácido Glutámico/metabolismo , Sinaptotagmina I/metabolismo , Animales , Calcio/farmacología , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , Células Cultivadas , Ratones , Ratones Noqueados , Modelos Moleculares , Mutación/genética , Estructura Terciaria de Proteína , Sinaptotagmina I/química , Sinaptotagmina I/deficiencia , Sinaptotagmina I/genética , Factores de Tiempo , Triptófano/genética , Triptófano/metabolismoRESUMEN
One of the pathophysiological features of Alzheimer's disease is astrocytosis around senile plaques. Reactive astrocytes may produce proinflammatory mediators, nitric oxide, and subsequent reactive oxygen intermediates such as peroxynitrites. In the present study, we investigated the possible role of the C-terminal fragment of amyloid precursor protein (CT-APP), which is another constituent of amyloid senile plaque and an abnormal product of APP metabolism, as an inducer of astrocytosis. We report that 100 nM recombinant C-terminal 105 amino acid fragment (CT105) of APP induced astrocytosis morphologically and immunologically. CT105 exposure resulted in activation of mitogen-activated protein kinase (MAPK) pathways as well as transcription factor NF-kappaB. Pretreatment with PD098059 and/or SB203580 decreased nitric oxide (NO) production and nuclear factor-kappa B (NF-kappaB) activation. But inhibitors of NF-kappaB activation did not affect MAPKs activation whereas they abolished NO production and attenuated astrocytosis. Furthermore, conditioned media derived from CT105-treated astrocytes enhanced neurotoxicity and pretreatment with NO and peroxynitrite scavengers attenuated its toxicity. These suggest that CT-APP may participate in Alzheimer's pathogenesis through MAPKs- and NF-kappaB-dependent astrocytosis and iNOS induction.
Asunto(s)
Precursor de Proteína beta-Amiloide , Gliosis/inducido químicamente , Neurotoxinas , Fragmentos de Péptidos , Precursor de Proteína beta-Amiloide/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Inducción Enzimática , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , FN-kappa B/fisiología , Neurotoxinas/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Fragmentos de Péptidos/farmacología , RatasAsunto(s)
Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/farmacología , Citocinas/biosíntesis , Neuroglía/efectos de los fármacos , Óxido Nítrico/biosíntesis , Fragmentos de Péptidos/farmacología , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Muerte Celular , Células Cultivadas , Corteza Cerebral/citología , Quimiocina CCL2/biosíntesis , Quimiocina CCL4 , Quimiocina CCL5/biosíntesis , Condimentos , Interleucina-1/biosíntesis , Proteínas Inflamatorias de Macrófagos/biosíntesis , Microglía/efectos de los fármacos , Modelos Biológicos , Neuroglía/citología , Neuronas/citología , Ratas , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Several lines of evidence indicate that A beta may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of A beta and the development of the disease. Thus, A beta may not be the sole active fragment of beta-amyloid precursor protein (betaAPP) in the neurotoxicity assiciated with AD. We focused on the amyloidegenic carboxyl terminal fragments of betaAPP containing the full length of A beta (CT105). We synthesized a recombinant carboxyl-terminal 105 amino acid fragment of betaAPP and examined the effects of CT105 and A beta on cultured neurons, Ca++ uptake into rat brain microsomes, Na+-Ca++ exchange activity, ion channel forming activity in lipid bilayers and passive avoidance performance of mice. Our results suggest that the cytotoxic and channel inducing effects of CT105 are much more potent than that of A beta and toxic mechanisms of CT105 are different from those of A beta. Taken together, these lines of evidence postulate that CT is an alternative toxic element important in the generation of the symptoms common to AD.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/fisiología , Precursor de Proteína beta-Amiloide/fisiología , Péptidos beta-Amiloides/envenenamiento , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/envenenamiento , Animales , Reacción de Prevención/efectos de los fármacos , Encéfalo/metabolismo , Calcio/metabolismo , Conductividad Eléctrica , L-Lactato Deshidrogenasa/metabolismo , Membrana Dobles de Lípidos , Microsomas/metabolismo , Fármacos Neuroprotectores/farmacología , Células PC12 , Fragmentos de Péptidos/fisiología , Fragmentos de Péptidos/envenenamiento , Ratas , Intercambiador de Sodio-Calcio/metabolismo , Azul de Tripano , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
The genes defective in familial Alzheimer's disease encode the proteins presenilin 1 and 2 (PS1 and 2). Expression of presenilins (PSs) and their proteolytic processing are regulated during neuronal development. Even though these proteins are detected and regulated mainly in Golgi and endoplasmic reticulum, their subcellular distribution during the development is not known. The present study aimed to investigate the localization of PSs and their role during early developmental stage using mouse embryo model. At preimplantation stage, PSs were detected not only in cytoplasm, but also in the nucleus from oocyte to 2.5 dpc (day postcoitum), then disappeared in the nucleus at blastocyst stage (3.5 dpc). Antisense against PS1 and PS2 decreased the transition to blastocyst stage, whereas each antisense alone had no effect. Treatment with lactacystin (26S proteosome inhibitor), which arrest cell cycle at M phase, redistributed PSs into centrosome-kinetochore microtubule. PS2 overexpression in HEK 293 cell arrested cell cycle at S phase. These data suggest that PSs play key roles in cell division and differentiation during early development.
Asunto(s)
Acetilcisteína/análogos & derivados , Desarrollo Embrionario , Proteínas de la Membrana/análisis , Acetilcisteína/farmacología , Animales , Diferenciación Celular , División Celular , Línea Celular , Núcleo Celular/química , Citoplasma/química , ADN sin Sentido/farmacología , Embrión de Mamíferos/química , Embrión de Mamíferos/efectos de los fármacos , Femenino , Humanos , Inmunohistoquímica , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos ICR , Microtúbulos/química , Mutación , Embarazo , Presenilina-1 , Presenilina-2 , Fase SRESUMEN
Numerous lines of evidence indicate that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (Abeta). However, the possible role of other cleaved products of APP is less clear. We have previously shown that a recombinant carboxy-terminal 105 amino acid fragment (CT 105) of APP induced strong nonselective inward currents in Xenopus oocyte; it also revealed neurotoxicity in PC12 cells and primary cortical neurons, blocked later phase of long-term potentiation in rat hippocampus in vivo, and induced memory deficits and neuropathological changes in mice. We report here that the pretreatment with CT 105 for 24 h at a 10 microM concentration increases intracellular calcium concentration by about twofold in SK-N-SH and PC 12 cells, but not in U251 cells, originated from human glioblastoma. In addition, the calcium increase and toxicity induced by CT 105 were reduced by cholesterol and MK 801 in SK-N-SH and PC 12 cells, whereas the toxicity of Abeta(1-42) was attenuated by nifedipine and verapamil. CT 105 rendered SK-N-SH cells and rat primary cortical neurons more vulnerable to glutamate-induced excitotoxicity. Also, conformational studies using circular dichroism experiments showed that CT 105 has approximately 15% of beta-sheet content in phosphate buffer and aqueous 2,2, 2-trifluoroethanol solutions. However, the content of beta-sheet conformation in dodecyl phosphocholine micelle or in the negatively charged vesicles, is increased to 22%-23%. The results of this study showed that CT 105 disrupts calcium homeostasis and renders neuronal cells more vulnerable to glutamate-induced excitotoxicity, and that some portion of CT 105 has partial beta-sheet conformation in various environments, which may be related to the self-aggregation and toxicity. This may be significantly possibly involved in inducing the neurotoxicity characteristic of AD.
Asunto(s)
Precursor de Proteína beta-Amiloide/toxicidad , Calcio/metabolismo , Homeostasis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fragmentos de Péptidos/toxicidad , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/toxicidad , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Colesterol/farmacología , Dicroismo Circular , Citotoxinas/farmacología , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Ácido Glutámico/farmacología , Humanos , Neuronas/citología , Fármacos Neuroprotectores/farmacología , Nifedipino/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Células Tumorales Cultivadas , Verapamilo/farmacologíaRESUMEN
We examined the effects of systemic administration of monosodium glutamate (MSG) or aspartate (ASP) on the memory retention and neuronal damage in the brains of adult mice. Compared with the control mice, a single intraperitoneal injection of either 4.0 mg/g MSG or 0.5 mg/g ASP after acquisition trial significantly shortened the response latency in the passive avoidance test, accompanying by the transient weight loss. Histopathological analysis of the brains of these mice revealed that neurons in the arcuate nucleus of hypothalamus were damaged markedly by MSG (4.0 mg/g) or ASP (0.5 mg/g). Other brain areas including cerebral cortex and hippocampus did not show any pathological changes. These findings suggest that systemic administration of MSG or ASP could impair memory retention and damage hypothalamic neurons in adult mice.