A further study of the functional defects involved in Alzheimer diseases (AD)


Project leader


Funding source

Stiftelsen för Gamla Tjänarinnor


Project Details

Start date: 02/05/2011
End date: 31/12/2011
Funding: 30000 SEK


Description

Familial Alzheimer’s disease (FAD)-linked presenilin (PS) mutations are scattered throughout the PS molecule. One third of PS1 residues are highly conserved and 75 % of the FAD mutations are located to highly conserved regions. Defects in the gene PSEN1 are a cause of familial early-onset Alzheimer disease type 3 (AD3). AD3 is the most severe form of the disease with onset as early as 30 years of age.

Polytopic membrane protein biogenesis represents a critical yet poorly understood area of modern biology with important implications for human disease. Inherited mutations in a growing array of membrane proteins frequently lead to improper folding and trafficking. It has been suggested that PS1 is an example in which point mutations lead to a loss of structure and function of the g-secretase biochemically.

Multi-spanning presenilins, PS1 and PS2, are critical components of a large enzyme complex that perform g-secretase cleavage of amyloid-b precursor protein (APP) to generate amyloid b-peptide (Ab. PS1 has a rather complex topology with 10 hydrophobic segments, but only 9 transmembrane (TM) domains, and given its biomedical importance, PS1 is of particularly interest to study membrane insertion of missense mutations in the PSEN1 gene. Many of PS1s gene mutations are located in the TM regions and among them we have a special interest for the non-conservative ones. Many PS mutations change the level of Ab production.

Until now, we have systematically measured membrane integration efficiency of each TM segment, using an assay that allows precise measurement of membrane

insertion of hydrophobic segments engineered into a model protein. Nearly all TM segments of PS1 seem to be hydrophobic enough to integrate into the ER membrane by themselves, except for the hydrophobic segment H7 (0 %) and the TM segment 7 (55 %).

As a second step, we have focused on clinically relevant TM mutations of PS1 and their influence on the integration efficiency into the ER membrane and the level of Ab production. Our preliminary results show that at least four substitutions, TM6(L250S), H7(P284L), TM7(S390I) and TM7(L392P), located in two of the marginally hydrophobic segments, H7 and TM7, and in one of the more efficient ones, TM6, affect the insertion efficiency.

As a continuation, we will investigate PS1´s targeting, translocation and integration with help of SRP, the translocon and the lipid environment. The targeting, translocation and integration processes can be studied in detail using an in vitro transcription/translation system combined with cross-linking and cysteine-labelling.

To further continue the PS study, we will investigate the level of Ab production and the g-secretase activity using mutated PS. Many PS mutations change the level of Ab production and the ratio of Ab42/Ab40 fragments during g-secretase cleavage. We will also investigate the molecular mechanisms whereby mutations in PS1 cause familial AD (FAD). To date, more than 140 mutations in PS1 have been shown co-segregate with the disease. Intriguingly, almost all PS1 FAD mutations increase the Ab42/Ab40 ratio. That the mutations induce a global conformational change of the PS1 protein, thereby subtly altering g-secretase cleavage, has been proposed but remain to be shown.

The plan is to continue the main work to examine the integration of different TM segments containing mutations into the ER membrane and to investigate the ratio of Ab42/Ab40 and AICD formation obtained by the cell based assays during 2011 until the end of 2012. To extend the project during these two years, we will also investigate the proteins that are nearby during assembly and integration of PS1 into the ER membrane.


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Last updated on 2017-24-03 at 12:05