RESUMEN
During the development of in vivo amyloid imaging agents, an effort was made to use micro-positron emission tomography (PET) imaging in the presenilin-1 (PS1)/amyloid precursor protein (APP) transgenic mouse model of CNS amyloid deposition to screen new compounds and further study Pittsburgh Compound-B (PIB), a PET tracer that has been shown to be retained well in amyloid-containing areas of Alzheimer's disease (AD) brain. Unexpectedly, we saw no significant retention of PIB in this model even at 12 months of age when amyloid deposition in the PS1/APP mouse typically exceeds that seen in AD. This study describes a series of ex vivo and postmortem in vitro studies designed to explain this low retention. Ex vivo brain pharmacokinetic studies confirmed the low in vivo PIB retention observed in micro-PET experiments. In vitro binding studies showed that PS1/APP brain tissue contained less than one high-affinity (K(d) = 1-2 nm) PIB binding site per 1000 molecules of amyloid-beta (Abeta), whereas AD brain contained >500 PIB binding sites per 1000 molecules of Abeta. Synthetic Abeta closely resembled PS1/APP brain in having less than one high-affinity PIB binding site per 1000 molecules of Abeta, although the characteristics of the few high-affinity PIB binding sites found on synthetic Abeta were very similar to those found in AD brain. We hypothesize that differences in the time course of deposition or tissue factors present during deposition lead to differences in secondary structure between Abeta deposited in AD brain and either synthetic Abeta or Abeta deposited in PS1/APP brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Compuestos de Anilina/metabolismo , Encéfalo/metabolismo , Tomografía de Emisión de Positrones/métodos , Tiazoles/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Sitios de Unión/fisiología , Encéfalo/patología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Recent studies indicate that oxysterols, which are ligands for the nuclear hormone liver X receptors (LXR), decrease amyloid beta (Abeta) secretion in vitro. The effect was attributed primarily to the ATP-binding cassette transporter A1 (ABCA1) transcriptionally up-regulated by ligand-activated LXRs. We now examined the effect of the synthetic LXR ligand T0901317, which can be used in vivo, on Abeta production in vitro and in APP23 transgenic mice. T0901317 applied to a variety of in vitro models, including immortalized fibroblasts from Tangier patients, and primary embryonic mouse neurons caused a concentration-dependent decrease in Abeta secretion, and this effect was increased by the addition of apolipoprotein A-I. The inhibition of Abeta production by T0901317 was cell-type specific, being more prominent in primary neurons than in non-neuronal cells. Tangier fibroblasts lacking a functional ABCA1 secreted more Abeta than control fibroblasts, thus demonstrating the role of ABCA1 in amyloid precursor protein (APP) processing and Abeta generation. T0901317 treatment of 11-week-old APP23 mice for 6 days showed a significant increase in ABCA1 expression and a decrease in the ratio of soluble APP (sAPP)beta- to sAPPalpha-cleavage products. Most importantly, the treatment caused a statistically significant reduction in the levels of soluble Abeta40 and of Abeta42 in the brain these mice. Our experiments demonstrate that T0901317 decreases amyloidogenic processing of APP in vitro and in vivo, thus supporting the search for potent and specific LXR ligands with properties allowing therapeutic application.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Anticolesterolemiantes/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Apolipoproteína A-I/química , Western Blotting , Encéfalo/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Humanos , Hidrocarburos Fluorados , Inmunoprecipitación , Ligandos , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Receptores Nucleares Huérfanos , Unión Proteica , Simplexvirus/metabolismo , Sulfonamidas , Factores de Tiempo , Transcripción Genética , Transfección , Regulación hacia ArribaRESUMEN
The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of peripheral cholesterol efflux and plasma high density lipoprotein metabolism. In adult rat brain we found high expression of ABCA1 in neurons in the hypothalamus, thalamus, amygdala, cholinergic basal forebrain, and hippocampus. Large neurons of the cholinergic nucleus basalis together with CA1 and CA3 pyramidal neurons were among the most abundantly immunolabeled neurons. Glia cells were largely negative. Because cholesterol homeostasis may have an essential role in central nervous system function and neurodegeneration, we examined ABCA1 expression and function in different brain cell types using cultures of primary neurons, astrocytes, and microglia isolated from embryonic rat brain. The basal ABCA1 mRNA and protein levels detected in these cell types were increased markedly after exposure to oxysterols and 9-cis-retinoic acid, which are ligands for the nuclear hormone liver X receptors and retinoic X receptors, respectively. Functionally, the increased ABCA1 expression caused by these ligands was followed by elevated apoA-I- and apoE-specific cholesterol efflux in neurons and glia. In non-neuronal and neuronal cells overexpressing a human Swedish variant of amyloid precursor protein, 22R-hydroxycholesterol and 9-cis-retinoic acid induced ABCA1 expression and increased apoA-I-mediated cholesterol efflux consequently decreasing cellular cholesterol content. More importantly, we demonstrated that these ligands alone or in combination with apoA-I caused a substantial reduction in the stability of amyloid precursor protein C-terminal fragments and decreased amyloid beta production. These effects of 22R-hydroxycholesterol may provide a novel strategy to decrease amyloid beta secretion and consequently reduce the amyloid burden in the brain.