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Förslaget inkom 2010-04-20

Increased APP expression and formation of aggresomes of Alzheimer disease-related protein APP: A response to cellular stress in AD

Aggresome formation is a cellular response to accumulation of misfolded proteins and has been described as cytoplasmic membrane protein aggregations that are induced by proteasome inhibition or over-expression of certain proteins. Aggresomes formation has been reported in many disease-related proteins including the cystic fibrosis. The proteins involved in neurodegenerative diseases are also forming aggresomes. Examples are: the polyglutamine containing huntingtin (Huntington’s disease), ubiquitin carboxyl-terminal esterase L1 (UCH-L1), ?-synuclein (Parkinson’s diseases), superoxide dismutase (ALS) and presenilin (PS) (Alzheimer disease (AD)). Previously, in vitro studies demonstrated that inhibition of proteasome in AD resulted in aggresome-like structure in cytoplasm of the cells.
Amyloid precursor protein (APP) is a transmembrane protein and an important challenge of APP biology has been determined where in the cell APP processing and in particular, the amyloidogenic cleavage occurs.
There are few studies showing why and what causes the induction of APP expression in AD. However, a stress responsive element has been found in the promoter of the APP gene. Johanson et al have also showed altered expression of APP in heat shocked neuronal PC12 cells and they suggest that the amount of APP mRNA correlates inversely with the amount of Hsp70 mRNA. The increased APP expression in AD brain could also form aggresomes as a consequence of stress response.

Various mechanisms have been proposed to explain neuronal death. One of them is formation of aggresomes in neural cells leading to the activation of caspases that in turn cause cell death. Disease-related prion proteins also form aggresomes in neuronal cells and initiate caspase-dependent apoptosis. In vitro studies have demonstrated that compromised proteasome pathway in AD results in aggresome-like structures in the cytoplasm of cells.
Membrane proteins such as APP are co-translationally translocated to the membrane of the ER. Some molecules fold to adopt a maturation-component conformation and others misfold and are translocated from the ER membranes. In eukaryotic cells, mutant, damaged, misfolded or unwanted intracytoplasmic proteins are normally cleared by attachment of an ubiquitin molecule, which signals for protein transport to the proteasome for degradation. Dislocated, ubiqutinated, misfolded proteins can either be rapidly degraded by cytosolic proteasomes or form aggregates. Up till now, there are no studies showing that APP forms aggresomes in response to either cellular stress or as a cause of dysfunction of Omi/HtrA2 protease activity or proteasome inhibition.

Specific aim

•To see if APP forms aggresomes in response to stress by investigating Omi/HtrA2 protease activity and the proteasome pathway.

Methods: Western and Northern blotting, Co-immunoprecipitation (Co-IP), cellular fractionation, Flow Cytometry, immunofluorescent staining (IFS), confocal microscopy. Cell model: Mouse embryonic fibroblasts (MEF) derived from Omi-wt and Omi-KO embryos as well as HEK293 and HEK293/APP over-expressing cells.

About 24 million people worldwide are estimated to suffer from dementia; the most common cause is AD. The degradation capacity of misfolded or unfolded proteins in the nervous system is known to become impaired in neurodegenerative disease as well as during ageing process itself. Brains of patients affected by AD or other neurodegenerative disease exhibit several features that indicate the presence of stress. The aim of this project is to investigate whether aggresome formation of APP is directly related to HS, proteasome activity, stress factors and/or Omi/HtrA2 protease activity. Aggresome formation of, for example, misfolded APP protein can cause crucial damage to cellular functions resulting in increased plaque formation in the brain eventually leading to neuronal cell death. Studying Omi/HtrA2 protease activity and proteasome activity which both participate in a stress-sensing pathway may clear an involvement of aggresomes in the etiology of the majority of AD cases.


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