RESUMO
Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid ß-peptide (Aß) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aß(40) and Aß(42) production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aß(42) accompanied by increased intracellular Aß(42) /Aß(40) ratio without changing the Aß secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aß pattern with increased Aß(42) /Aß(40) ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aß(42) /Aß(40) ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-1/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Animais , Apoptose , Caspases/genética , Caspases/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Presenilina-1/genética , Transdução de Sinais , Trissacarídeos/genética , Trissacarídeos/metabolismoRESUMO
Dimebon, a drug currently being evaluated in multiple Phase III Alzheimer's disease trials, has previously been shown to have effects on isolated mitochondria at muM concentrations. Here the effects of nM concentrations of Dimebon on mitochondrial function were investigated both in primary mouse cortical neurons and human neuroblastoma cells (SH-SY5Y). Under non-stress conditions nM concentrations of Dimebon increased succinate dehydrogenase activity (MTT-assay), mitochondrial membrane potential (DeltaPsim), and cellular ATP levels. Dimebon treatment had no effect on mitochondria DNA content, implying that mitochondrial biogenesis was not induced. Under stress conditions, mitochondria in Dimebon-treated neurons showed increased resistance to elevated intracellular calcium concentrations, thus, maintaining their DeltaPsim throughout the experiment, in contrast to control neurons, which rapidly lost their DeltaPsim. Moreover, we show that serum-starved differentiated SH-SY5Y cells treated with Dimebon had an increased survival rate as compared to untreated cells. In conclusion, these data demonstrate that Dimebon enhances mitochondrial function both in the absence and presence of stress and Dimebon-treated cells are partially protected to maintain cell viability.