Hapuarachchi, Bimali; (2020) Cell models for the study of the association of EIF2AK3/PERK with progressive supranuclear palsy and neurodegeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A genome-wide association study (GWAS) identified EIF2AK3 as a risk factor for progressive supranuclear palsy (PSP). EIF2AK3 encodes protein kinase R-like endoplasmic reticulum kinase (PERK), which senses unfolded protein accumulation within the endoplasmic reticulum (ER) lumen. PERK kinase activity has been genetically associated with increased PSP risk. The associated single nuclear polymorphism (SNP), rs7571971, is in linkage disequilibrium with coding SNPs of EIF2AK3: rs867529(Ser136Cys), rs13045(Gln166Arg) and rs1805165(Ala704Ser), forming coding haplotypes of three highly conserved residues; Haplotype A (conserved): S136- R166-S704 and Haplotype B (divergent): Cys136-Gln166-Ala704. A previous study showed that the divergent risk Haplotype B (HapB) has increased PERK activity suggesting that this forms the basis of the genetic risk. The polymorphisms could therefore affect either functional domain (or both). Our aim was to investigate if the two major coding haplotypes of PERK impart differences in the activation of PERK either by impaired homodimerization and/or kinase activity. We generated isogenic HEK293 cell lines for Tet-inducible expression of PERK coding haplotypes with a C-terminal myc-tag to discern from endogenous PERK. With Western blot analyses, we demonstrated robust, inducible expression of myc-tagged PERK. Interestingly, with subsequent passages, the freeze-thawed HapB PERK variants alone underwent C-terminal cleavage as evidenced by loss of the myc-tag which resulted not only in increased PERK protein but also reduced levels of activated p-PERK and p-eIF2a. Myc cleaved HapB cells also showed a significant delayed and impaired kinase activity, alongside increased cell death following induction of the UPR. However, we failed to obtain any evidence for a connection between impaired HapB PERK activity and tau aggregation, even with the more aggregation-prone 2N4Rtau (P301L) and additional seeding with brain lysates from the rTg4510 mouse and with inhibition of ubiquitin proteasome system (UPS) with MG132. In summary, we established stable isogenic cell lines for Tet-inducible expression of PERK functional haplotypes. Although the C-terminus myc tag cleavage of the passage 2 HapB cells resulted in reduced PERK activation leading to an impaired UPR, a necessary further control to validate these conclusions would be to include untagged passage 2 HapB cells. This is essential to clarify as to whether the underlying reduced HapB PERK activity following cell passaging is due to an artefact following myc cleavage or due to posttranslational modifications inherent to HapB PERK.

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