Rapamycin decreases presenilin-1 in SK-N-SH cells

Hontiveros, Silahis
Das, Hriday
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Purpose: Presenilin-1 (PS1) protein is the catalytic subunit of the γ-secretase complex, and participates in the processing of β-amyloid precursor protein (APP) to produce Aβ peptide and Notch 1 receptor to release Notch intracellular domain (NICD) in the cytoplasm. NICD subsequently migrates to the nucleus and causes Notch signaling by increasing the expression of the Hes1 gene. The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that exist as two complexes known as mTORC1 and mTORC2. mTORC1 controls cellular homeostasis, and its activity is inhibited by a FDA approved drug rapamycin. mTOR activity has been directly linked to learning and memory. It has been reported that the buildup of Aβ increases the mTOR signaling, whereas decreasing mTOR signaling reduces Aβ levels suggesting an interrelationship between mTOR signaling and Aβ. Administration of rapamycin in 3XTg-AD mouse model of Alzheimer's disease (AD) rescues cognitive deficits and ameliorates Aβ and Tau pathology. But the precise molecular mechanisms by which rapamycin reduces Aβ in AD is not well characterized. The purpose of this research to dissect the mechanisms by which rapamycin inhibits PS1 expression and PS1/γ-secretase activity in human neuroblastoma SK-N-SH cells. Methods: SK-N-SH cell line was maintained in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, 1% penicillin/streptomycin. Cells were treated with DMSO or different concentrations of rapamycin for 24 h. Total RNA and protein were prepared from treated cells. Amount of mRNA and protein expression were determined by real time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and western blot analysis respectively. Expression of proteins were confirmed by immunofluorescence staining (IFS) of DMSO and rapamycin treated cells. All data was analyzed using prism software and presented as means ± SEM. Comparison was made between groups by one-way ANOVA and Student–Newman–Keuls (SNK) test. A probability was considered to be significant with less than 0.05. Results: Rapamycin decreases the expression of p-mTOR, PS1, NICD, and Hes 1 proteins as well as PS1-mRNA in SK-N-SH cells. Conclusions: Rapamycin decreases PS1 protein levels and PS1/γ-secretase-mediated Notch 1 processing by inhibiting PS1 transcription. Hence we conclude that rapamycin may potentially reduce Aβ in AD by decreasing the transcription of the PS1 gene.