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Identifier 000401076
Title Mechanisms of brain neuronal cell death in alzheimer disease
Alternative Title Μηχανισμοί κυτταρικού θανάτου των νευρώνων του εγκεφάλου στη νόσο του Alzheimer
Author Βολουδάκης, Γεώργιος
Thesis advisor Γραβάνης, Αχιλλέας
Reviewer Καρδάσης, Δημήτριος
Ρομπάκης, Νικόλαος
Καραγωγέως, Δόμνα
Μπίτσιος, Παναγιώτης
Τσατσάνης, Χρήστος
Ζαγανάς, Ιωάννης
Abstract AIM OF THE STUDY The study was aimed at elucidating the mechanisms of cortical neuronal cell death in the Alzheimer’s disease. Presenilin 1 (PS1) mutations are responsible for the majority of the identified cases of patients with Familial Alzheimer’s Disease (FAD). That begs the question of the role of PS1 in neuronal cell death and neuroprotection. It has been known in the literature that several growth factors are able to protect neurons from harmful stimuli such as excitotoxicity from excitatory amino acid neurotransmitters, oxidative stress, and nutrient deprivation. Likewise, epidermal growth factors (EGFs) protect neurons from toxic insults by binding epidermal growth factor receptor (EGFR) and stimulating survival signaling. Apart from its role in neuroprotection, EGFR plays pivotal roles in cell proliferation, differentiation, and tissue development, and recent evidence implicates this receptor in neurometabolic disorders like AD and ageing. In this study, I asked the question of whether PS1 is necessary for the neuroprotective capacity of ligands of the EGFR against L-glutamate excitotoxicity, and what is the mode of regulation of this neuroprotective ability. METHODS For this study, we predominantly used the PS1 knockout (PS1-/-) mouse model. Since the PS1-/- pups die shortly after birth, I could not study cortical neuronal cell death in adult mice. For that reason I studied the expression levels of the proteins of interest by Western Blot (WB) and immunohistochemistry (IHC) in embryonic day 15.5 (E15.5) mouse brains. Fortunately, a variety of primary cell cultures can be obtained from the embryos to dissect the molecular pathway of interest. In this study, I prepared primary cortical neuronal cultures (PCNC), primary glial cultures (pGlia; mostly astrocytes) and primary mouse embryonic fibroblasts (pMEF). Survival experiments were performed against glutamate excitotoxicity in PCNCs to evaluate the ability of EGFR ligands (EGF and HB-EGF) to reduce neuronal cell death under excitotoxicity. Neuronal viability was evaluated by MTT assay which measures the reduction potential of the cell and also by the goldstandard nuclear morphology assay by employing a Hoechst dye. The ability of the EGFR ligands to activate key survival pathways was assessed by visualizing the level of phosphorylation of AKT and ERK with WB. Quantification of mRNA levels of EGFR against a housekeeping gene (GAPDH) was performed by Real-time PCR. Finally, I was able to manipulate the levels of expression of PS1 and EGFR in our cell cultures by employing siRNA technology to reduce the levels of PS1 mRNA, and a mammalian expression vector (based on a lentiviral backbone) for expressing either PS1 or EGFR. In addition to the PS1KO mouse model, the PS2KO mouse model was also employed to evaluate the specificity of the findings relative to PS1 and γ-secretase function in PCNCs. RESULTS & CONCLUSIONS We show that absence of PS1 results in a dramatic decrease (>95%) of neuronal EGFR and that PS1-/- brains have reduced amounts (around 60% of WT) of this receptor. PS1−/− cortical neurons contain little EGFR and show no epidermal growth factor–induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. Egfr mRNA is greatly reduced (>95%) in PS1−/− neurons, and PS1−/− brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal Egfr mRNA, while down-regulation of PS1 decreases it. These effects are neuron-specific, as PS1 affects the EGFR of neither glial nor fibroblast cells. In addition, PS1 controls EGFR through novel mechanisms shared with neither γ-secretase nor the paralog PS2. Our data reveal that PS1 functions as a positive transcriptional regulator of neuronal EGFR controlling its expression in a cell-specific manner. Severe downregulation of EGFR may contribute to developmental abnormalities and lethal phenotype found in PS1, but not PS2, null mice. Furthermore, PS1 may affect neuroprotection and Alzheimer disease by controlling survival signaling of neuronal EGFR. In summary, Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via γ-secretase-independent transcriptional mechanisms.
Language English
Subject Epidermal growth factor receptor
Neuroprotection
Presenilin 1
Νευροπροστασία
Πρεσενιλίνη 1
Υποδοχέας επιδερμικού αναπτυξιακού παράγοντα
Issue date 2015-11-13
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/0/f/e/metadata-dlib-1463659981-476158-20941.tkl Bookmark and Share
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