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Title: Role of ApoEr2 isoforms in the cellular processing of the Alzheimer's amyloid precursor protein : insights into the post translational processing of ApoEr2 and identification of a novel mechanism of APP regulation
Author: King, Henry Owain
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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Abstract:
Alzheimer's disease (AD) is a debilitating neurodegenerative disease that affects millions of people worldwide. AD is characterised by the accumulation aggregation and deposition within the brain of amyloid ~ (A~), a 40-42 residue peptide, and hyperphosphorylated Tau. A~ is a cleavage product of the amyloid precursor protein (APP) produced by the sequential cleavage of the ~-secretase, 8ACE1 (~-sjte APP Cleaving enzyme), and the v-secretase. APP can also be proteolytically processed by an alternative pathway where an a-secretase cleaves APP within the A~ region followed by v-cleavage. This precludes the formation of A~. Protein-protein interactions play an important role in regulating protein function; elucidating how the interactions between APP and other proteins affect A~ accumulation in an age-dependent manner is crucial to understanding AD progression. The Apolipoprotein E receptor 2 (ApoEr2) interacts with APP and alters its cleavage, though its exact effect is unclear as it has been shown to both increase and decrease ~-secretase cleavage of APP. To understand the reason for this disparity, 3 different splice variants of ApoEr2 have been expressed in HEK cells and their effects on the levels of APP cleavage products assessed. The isoforms chosen were a full length form (ApoEr2 FL), a form lacking the exon 5 and 15 encoded regions (ApoEr2 1:::.5,15), and a form lacking exon 18 (ApoEr2 1118) which encodes a cytoplasmic proline rich domain. These were chosen as all 3 isoforrns are brain expressed and have been used by different groups in investigating the effects of ApoEr2 on APP processing. It was found that 2 isoforms of ApoEr2 (ApoEr2 FL and 1:::.5,15) caused a reduction in ~- and a-cleavage of APP of -50%, where as ApoEr2 l::.18 increased ~-cleavage -200% whilst having no effect on a- cleavage, To identify the mechanism behind the variation between the isoforms effect on APP Cleavage, the post translational processing of ApoEr2 has been investigated, ApoEr2 was identified as being both N- and O-glycosylated and differences between the isoforms of ApoEr2 in the extent of fully glycosylatyed protein were identified. ApoEr2 l::.5,15 was produced as a fully glycosylated protein. With ApoEr2 FL and 1118 a partially glycosylated protein is also detectable at -25% of total ApoEr2 1:.18 and 40% of total ApoEr2 FL ApoEr2 is also shed from the plasma membrane and there were differences in the shedding of the isoforms of ApoEr2, with ApoEr2 1:.5,15 shed to a lower level than ApoEr2 FL or 1:::.18. The enzyme responsible for the shedding of ApoEr2 has been identified as ADAM10, an enzyme also involved in cleaving APP. The subcellular localisation of ApoEr2 was also investigated and increased detection of ApoEr2 in the early endosome may explain the isoform specific effect of ApoEr2 on APP cleavage. This work has shown for the first time that different splice forms of ApoEr2 have radically different effects on APP proteolytic processing, The fact that splice forms of a protein can have such different effects on APP processing shows that the regulation of APP cleavage is exceedingly complex. This work has also significantly extended what is known about the post translational processing of ApoEr2 with the identification of the enzyme responsible for the shedding of ApoEr2 and has shown that there is substantial variation in post translational processing of the different isoforms of ApoEr2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.574497  DOI: Not available
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