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1.
Curr Alzheimer Res ; 11(6): 549-57, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24251389

RESUMO

This study investigated the possible role of the ApoE receptors Lrp1 and Apoer2 in mediating the pathological effects of ApoE4 in ApoE-targeted-replacement mice expressing either the human ApoE3 or ApoE4 allele. In this study we show that activation of the amyloid cascade by inhibition of the Aß-degrading enzyme neprilysin results in upregulation of the ApoE receptor Lrp1 in the CA1 hippocampal neurons of 4-month-old ApoE4 mice, but not in the corresponding ApoE3 or ApoE-deficient (KO) mice. These results are in accordance with the previous findings that activation of the amyloid cascade induces Aß accumulation in the CA1 neurons of ApoE4 mice, but not in ApoE3 or ApoE-KO mice. This suggests that the apoE4-driven elevation of Lrp1 is mediated via a gain of function mechanism and may play a role in mediating the effects of ApoE4 on Aß. In contrast, no changes were observed in the levels of the corresponding Apoer2 receptor following the neprilysin inhibition. The ApoE receptors of naive ApoE4 mice were also affected differentially and isoform specifically by ApoE4. However, under these conditions, the effect was an ApoE4-driven reduction in the levels of Apoer2 in CA1 and CA3 pyramidal neurons, whereas the levels of Lrp1 were not affected. RT-PCR measurements revealed that the levels of Apoer2 and Lrp1 mRNA in the hippocampus of naïve and neprilysin-inhibited mice were not affected by ApoE4, suggesting that the observed effects of ApoE4 on the levels of these receptors is posttranscriptional. In conclusion, this study shows that the levels of hippocampal ApoE receptors Lrp1 and Apoer2 in vivo are affected isoform specifically by ApoE4 and that the type of receptor affected is context dependent.


Assuntos
Apolipoproteína E4/metabolismo , Hipocampo/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Células Piramidais/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Apolipoproteína E3/metabolismo , Hipocampo/efeitos dos fármacos , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Lisossomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neprilisina/antagonistas & inibidores , Neprilisina/metabolismo , Inibidores de Proteases/farmacologia , Células Piramidais/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiorfano/farmacologia
2.
PLoS One ; 7(2): e31977, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22363779

RESUMO

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα(-), PR(-), and Her2(-), named accordingly "Triple-Negative" (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5-12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygro(R)) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50-100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships.


Assuntos
Técnicas de Cultura de Células/métodos , DNA Complementar/genética , Receptor alfa de Estrogênio/genética , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Células Clonais/metabolismo , Genes Reporter/genética , Vetores Genéticos/genética , Humanos , Ligantes , Luciferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção
3.
Int J Alzheimers Dis ; 2011: 792070, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21350674

RESUMO

Activating the amyloid cascade by inhibiting the Aß-degrading enzyme neprilysin in targeted replacement mice, which express either apoE4 or apoE3, results in the specific accumulation of oligomerized Aß42 in hippocampal CA1 neurons of the apoE4 mice. We presently investigated the extent to which the apoE4-driven accumulation of Aß42 and the resulting mitochondrial pathology are due to either gain or loss of function. This revealed that inhibition of neprilysin for one week triggers the accumulation of Aß42 in hippocampal CA1 neurons of the apoE4 mice but not of either the corresponding apoE3 mice or apoE-deficient mice. At 10 days, Aß42 also accumulated in the CA1 neurons of the apoE-deficient mice but not in those of the apoE3 mice. Mitochondrial pathology, which in the apoE4 mice is an early pathological consequence following inhibition of neprilyisn, also occurs in the apoE-deficient but not in the apoE3 mice and the magnitude of this effect correlates with the levels of accumulated Aß42 and oligomerized Aß42 in these mice. These findings suggest that the rate-limiting step in the pathological effects of apoE4 on CA1 neurons is the accumulation of intracellular oligomerized Aß42 which is mediated via a gain of function property of apoE4.

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