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Analysis of the neuroprotective function and the N-glycosylation progranulin (PGRN)

University of British Columbia

Recent studies have identified null mutations in the progranulin (PGRIV) gene as the cause of pathogenesis of Frontotemporal lobe dementia (FTLD). It was found that PGRN protein levels are reduced in patients due to haploinsufficiency. However, the functions of progranulin in the central nervous system remain poorly characterized. We hypothesized that PGRN plays a role in protecting mouse cortical neurons from aggregated amyloid-beta, NMDA and H₂0₂ toxicity. Neuronal cultures were incubated in conditioned medium containing PGRN protein, or infected with lentivirus carrying PGRN fusion gene, then treated with different toxins. The cell viability was evaluated by MTT assay. The results demonstrated that compared to the control groups without toxins added, restoration of PGRN is not protective from three types of insults in vitro. Another interest of our research is to better understand the N-glycosylation significance of PGRN. It has been demonstrated that PGRN is an N-linked glycoprotein, but the glycosylation status and the importance of such modifications to PGRN are still not clear. To investigate this issue, enzymatic digestion with PNGase F and endo H, and treatment with an N-glycosylation blocker, tunicamycin, were applied to demonstrate the existence of the carbohydrate. To further study the specific N glycosylation sites of PGRN, site-directed mutagenesis was used to construct single and multiple N glycosylation site mutants. The results showed that no single site was crucial for PGRN secretion and that multiple PGRN N-glycosylation mutants were retained and accumulated intracellularly. Additionally, the malfunctioning of N-glycosylation of PGRN leads to ER stress and unfolded/misfolded protein response by up-regulating BiP/GRP78 expression level. Taken together, all the N-glycosylation study results suggest that the N-linked oligosaccharide is very important for PGRN, especially the secretion.

Thesis/Dissertation

application/pdf

2009-04-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/7726

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/