The human IDE is a downstream target gene of mitochondrial biogenesis pathway

LEAL M.C; MARINO BUSJLE,, C; MAGNANI, N; EVELSON, P; CASTAÑO E.M; MORELLI L

Buenos Aires

Congreso; 5th Special Conference-ISN; 2012

ISN

Insulin Degrading Enzyme (IDE) is a highly conserved metallopeptidase that contributes in the in vivo metabolism of amyloid b peptide (Ab) of Alzheimer?s disease (AD). IDE mRNA and activity levels are decreased in AD as compared to control brains. In early AD stages, Ab is accumulated in mitochondria impairing cellular respiration. Despite its involvement in extracellular Ab peptide clearance, little is known about the regulation of IDE expression and the relevance of IDE in intracellular Ab metabolism. Our aim was therefore to characterize the human IDE (hIDE) core promoter and define the mechanisms involved in transcription/translation regulation of hIDE using in silico analysis; 5¢RACE; promoter deletions; luciferase (LUC) assays; site directed-mutagenesis; ChiP; EMSA; mitochondria isolation and functionality; ELISA; western blots; qRT-PCR; MALDITOF; cell culture transfection and pharmacological treatments. Our results show that the 5?sequence of hIDE gene is a rich CpG region and lacks a canonical TATA or a similar sequence with promoter function. We detected several TESS from )39 to 28 on hIDE promoter. Transcription produces two types of mRNAs, ??short?? and ??long?? with one or two possible translation start sites (corresponding to M1or M42) and therefore the generation of 2 IDE isoforms depends on the inclusion of the N-terminal region. The longest isoform has a mitochondrial import signal peptide prediction. We found in human brain peptides starting inthe first or second translation start site, showing the in vivo use of the first AUG. The construct promoter )118/59 showed the highest transcriptional activity, suggesting that a core promoter ofIDE is located in this region. In silico analyses of )118/59 region predicted an NRF1 binding site; mutations in this site abolished reporter gene LUC activity. ChiP and EMSA showed that NRF1 binds to the predicted regions in hIDE promoter sequence in vitro and in vivo. Transfections with wild-type or dominant negative versions of NRF1 increased or decreased IDE mRNA expression, respectively. The mitochondrial pathway activation by treatment with PQQ induced NRF1 and long IDE mRNA, decreasing Ab concentration. Our results propose that IDE modulation by mitochondrial cascade may impact on mitochondrial Ab clearance. We demonstrated that NRF1, a well-known regulator of mitochondrial gene expression, regulates the levels of IDE isoforms and determines several subcellular localizations of hIDE with impact on Ab degradation.