EURON and THEME joint PhD meeting

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EURON and THEME joint meeting 2011
Presenilins regulate induction of autophagy
Nguyen Thanh Tien
Department of Neurology, University of Bonn, Germany
Introduction
Macroautophagy or autophagy, is a lysosome-dependent degradative pathway
for organelles and nutrient recycling. The process starts with formation of
autophagosomes, which then fusing with lysosomes to become autolysosomes.
Autophagy has also been implicated in the Alzheimer’s disease (AD) associated
neurodegeneration. Sequential cleavage of β-amyloid precursor protein by β-
and γ- secretases generates β-amyloid (Aβ), which deposits as amyloid plaques
in the AD brains. Mutations in presenilins (PS1/PS2), the catalytic subunits of
γ-secretase complex, are major cause of early-onset familial AD. Recent studies
show enrichment of PS1 in autophagic vacuoles, and also indicate potential role
of PS1/PS2 and PS1 homolog in regulation of autophagic flux.
Aims
We address the role of presenilins in autophagy in steady-state and adaptive
response to stimuli, and possible involvement of the class III phosphoinositide
3-kinase (PI3K) complex in this process.
Methods
PS knock-out and PS1 expressing mouse embryonice fibroblasts and human
embryonic kidney cells were used as experimental model. Autophagic flux
was induced by starvation or impaired by lysosomal inhibitors. The changes of
autophagy were assessed by biochemical analyses.
Results
LC3 is widely used as a major marker to assess autophagic flux. LC3-I, a
cytosolic form, is lipidated to LC3-II, which is anchored into the membrane of
autophagosomes. At steady state, western blot analysis shows accumulation of
both LC3-I and LC3-II in PS1 wild-type (PS1wt) expressing cells compared to PS
deficient cells. Immunocytochemistry also prove abundance of LC3 puncta per
cells in PS1wt expressing cells in comparison to PS deficient cells. Upon short
starvation, ratio of LC3-II/LC3-I is increased in PS1wt expressing cells as compared
to PS deficient cells.