The insulin-degrading enzymefrom molecular evolution and subcellular localization to new roles in microglial physiology

  1. Corraliza Gómez, Miriam
Supervised by:
  1. Dolores Ganfornina Álvarez Director
  2. Eduardo Arranz Sanz Co-director
  3. Irene Cozar Castellano Co-director

Defence university: Universidad de Valladolid

Fecha de defensa: 01 December 2021

  1. Mónica García Alloza Chair
  2. Jorge Valero Gómez-Lobo Secretary
  3. Diego Gómez Nicola Committee member
  1. Biochemistry and Molecular Biology and Physiology

Type: Thesis


Alzheimer’s disease (AD) and diabetes mellitus (DM) are two chronic progressive pathologies with an alarming increase in their incidence worldwide. During the recent years, the term “type 3 diabetes” has been proposed to describe the hypothesis that AD is triggered by a type of insulin resistance that occurs specifically in the brain. Insulin-degrading enzyme (IDE) is a metalloprotease markedly expressed in the brain that has been described to cleave not only insulin but also amyloid-beta (Aβ) peptides, which makes this enzyme a good target to study type 3 diabetes. We demonstrated that chronic high D-glucose exposure stimulates metabolic activity and triggers a mild pro-inflammatory state in microglial cells but does not regulate IDE expression. Instead, we found a link between IDE expression and the polarization state of microglia. Furthermore, we observed that Aβ treatment increased IDE exportation to the extracellular media in a time-dependent manner, and this process was reduced under high D-glucose conditions. To address IDE subcellular localization, we constructed a molecular phylogeny and found homologous proteins from Archaea to Eukarya. Bioinformatic analyses revealed that a shift in subcellular localization took place between prokaryotic (with signal peptide) and eukaryotic cells (without it). Our experiments in microglia indicate that IDE is mostly cytoplasmatic, is not found inside any membranous organelle, and partitions between soluble and membrane fractions. IDE association to membranes only occurs at the cytosolic side, and membrane-associated IDE further partitions between raft and non-raft fractions. Moreover, IDE secretion is mediated by extracellular vesicles originating from multivesicular bodies. This mechanism of IDE exportation is modulated by the polarization of microglial cells. To address the role of IDE in vivo we performed a comprehensive analysis of metabolic, behavioral and molecular parameters on a cohort of 12-month-old wild-type, heterozygous and mice with genetic deletion of the Ide gene. The effects of IDE ablation on metabolic parameters were very subtle; however, memory tests revealed sex- and genotype-dependent differences. In the brain, insulin signaling-related proteins and gliosis markers were differentially expressed between males and females but showed no significant differences across genotypes. Nevertheless, a multivariate analysis revealed correlations between IDE and brain-related variables such as gliosis and Aβ managing. In vitro studies in primary microglial cells demonstrated that Ide deletion significantly decreases microglial proliferation and delays its response to M-CSF (macrophage colony-stimulating factor, an important mitogen for microglia). Cytokine profiling assays revealed that IDE knockout (IDE-KO) microglia have impaired polarization under both pro- and anti-inflammatory stimuli, are more sensitive to oxidative stress, and exhibit a sex-specific pro-inflammatory response to Aβ oligomers. Furthermore, lack of IDE alters Aβ internalization dynamics and impairs Aβ oligomer degradation. A transcriptomic profiling of wild-type and IDE-KO microglia revealed that differentially expressed genes were particularly associated with response to stimuli and stress, regulation of immune processes and signaling pathways, confirming prominent roles for IDE in microglial physiology. The results presented in this PhD Thesis reveal previously unknown biological properties and physiological functions of IDE in the nervous system, particularly in microglial cells, where it modulates their multidimensional response to various damaging conditions relevant to the pathogenesis of AD and DM.