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.
|