The contribution of the PDK1/Akt signaling pathway to Alzheimer disease analyzed by knock- in mutation

Student thesis: Doctoral thesis


Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases. Aging is the most important known nongentic risk factor for AD, which is accompanied by stereotypical structural and neurophysiological changes in the brain and different degrees of cognitive decline. The 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activity is increased in human AD whole brains; PDK1 triggers the phosphorylation and membrane depletion of TNF converting enzyme (TACE), which significantly influence the course of AD. Pharmacological inhibition and genetic depletion of PDK1 not only reduce amyloid-beta (Aβ) levels but also restore TACE-mediated soluble APPα levels and memory deficits in mice models for AD, which suggests that PDK1 may be activated by Aβ in AD. Of note, side-effects caused the dead of the mice after four months of treatment. PDK1 acts downstream of phosphoinositide 3-kinase (PI3K)-mediated signaling, the binding of growth factors with their receptors cause the activation of PI3K, which phosphorylates the PtdIns[4,5]P2 membrane lipid to synthesize the PtdIns[3,4,5]P3 second messenger. Protein kinase B (PKB/Akt) and PDK1 bind to PtdIns[3,4,5]P3 at the plasma membrane through their PH (Plekstrin Homology) domains, where Akt is phosphorylated at its activating residue threonine (Thr) 308 by PDK1. However, activation of the PI3K/Akt signaling pathway in brain by insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) is markedly disturbed In AD. Among the different positively-charged lateral chains conforming the PDK1 PtdIns[3,4,5]P3 binding pocket, mutating Lysine at position 465 to Glutamic acid abolishes the PDK1-phosphoinositide interaction by disrupting the conformation of this lipid-binding pocket. In order to analyse the importance of the PDK1-PtdIns[3,4,5]P3 interaction, homozygous PDK1K465E/K465E knock-in mice were generated, which express the PDK1 K465E mutant protein abrogating phosphoinositide binding resulting in an specific signalling lesion. Consequently, the mutant mice were shown to be smaller than control littermates, hyperinsulinemic and insulin resistant. These PDK1K465E/K465E mutant mice is a genuine model in which activation of Akt is only moderately reduced but not ablated, which has been proved instrumental in dissecting the contribution of Akt to PDK1 signalling. Taken the phenotypes of the PDK1 knock-in models, I postulate that inhibition of Akt might have protected mice against Alzheimer’s disease, whereas inhibition of the docking-site dependent PDK1 substrates might be responsible for the toxicity the treatments. In cortex and hippocampus, the phosphorylation of Akt at Thr308, the PDK1 site, was significantly reduced in the PDK1K465E/K465E mutant mice along the whole adulthood, since increased levels of BDNF synthesis compensate for the defects of the Akt signaling pathway. Importantly, TACE membrane localization, TACE activity and TNFR1 cleavage are increased in the PDK1K465E/K465E mutant mice, which render the mutant neurons resistant against TNFα-induced neurotoxicity, thereby providing strong evidence on the role of the Akt branch of the PDK1 signaling in regulating TACE trafficking and internalization. In the 3xTg-AD mice, the protein kinase RNA-like endoplasmic reticulum kinase (PERK) is hyperactivated and the eukaryotic initiation factor 2 (eIF2α) hyperphosphorylated. By contrast, in the PDK1K465E/K465E mice the age-induced phosphorylation of PERK and eIF2α is attenuated due to the deficient activation of Akt. Furthermore, in cultured primary neurons treated with tunicamycin or Aβ, the phosphorylation of PERK and eIF2α were reduced in the PDK1K465E/K465E cortical neurons compared to PDK1+/+ wild type, which protected cells from both tunycamicin and Aβ-induced neurotoxicity. In summary, this study show that reducing Akt activation can protect neurons against amyloid-beta induced-toxicity by restraining the reduction of TACE activity in the cell membrane and the increase of the unfolding protein response.
Date of Award30 Mar 2017
Original languageEnglish
SupervisorJose Ramon Bayascas Ramirez (Director)

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