RESUMO
UNLABELLED: Intraneuronal calcium stimulates the calpain-dependent conversion of p35 to p25, a CDK5 activator. It is widely believed that amyloid ß peptide (Aß) induces this conversion that, in turn, has an essential role in Alzheimer's disease pathogenesis. However, in vivo studies on p25 generation used transgenic mice overexpressing mutant amyloid precursor protein (APP) and presenilin (PS). Here, using single App knock-in mice, we show that p25 generation is an artifact caused by membrane protein overexpression. We show that massive Aß42 accumulation without overexpression of APP or presenilin does not produce p25, whereas p25 generation occurred with APP/PS overexpression and in postmortem mouse brain. We further support this finding using mice deficient for calpastatin, the sole calpain-specific inhibitor protein. Thus, the intracerebral environment of the APP/PS mouse brain and postmortem brain is an unphysiological state. SIGNIFICANCE STATEMENT: We recently estimated using single App knock-in mice that accumulate amyloid ß peptide without transgene overexpression that 60% of the phenotypes observed in Alzheimer's model mice overexpressing mutant amyloid precursor protein (APP) or APP and presenilin are artifacts (Saito et al., 2014). The current study further supports this estimate by invalidating key results from papers that were published in Cell These findings suggest that more than 3000 publications based on APP and APP/PS overexpression must be reevaluated.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Presenilina-1/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genéticaRESUMO
A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins.
Assuntos
Primers do DNA/síntese química , DNA Complementar/síntese química , DNA/química , Fosfodiesterase I/química , RNA Mensageiro/química , Triazóis/química , Artefatos , Química Click , DNA/genética , Primers do DNA/genética , DNA Complementar/análise , DNA Complementar/química , DNA Complementar/genética , Conversão Gênica , Fosfodiesterase I/genética , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Técnicas de Síntese em Fase Sólida/métodosRESUMO
A new triazole-linked analogue of DNA ((TL)DNA) has been designed and synthesized using click chemistry. The chain elongation reaction using copper-catalyzed Huisgen cycloaddition was successful and gave the artificial oligonucleotide that formed a stable double strand with the complementary strand of natural DNA.