Network Analysis of the Brain Proteome of Grn Knockout Mice Reveals Pathogenic Mechanisms Shared in Human Frontotemporal dementia caused by GRN mutations.
Authors: Huang Meixiang 
Published: April 2020

Mutations in the GRN gene, which encodes the progranulin (PGRN) protein, are one of the most common causes of Frontotemporal dementia (FTD) and cause disease through haploinsufficiency of PGRN. PGRN is composed of 7.5 repeating domains termed granulins (GRNs 1–7) that are joined by linker regions. We have recently found that PGRN is processed into ~6kDa GRNs within the lysosome, where we hypothesize they are critical for lysosome homeostasis (Holler et al, eNeuro, 2017 Aug 18;4(4)). However, the molecular mechanisms caused by decreased levels of lysosomal PGRN/GRNs that lead to FTD, Alzheimer’s disease, and other neurodegenerative disorders are still unknown. To gain insight into the mechanisms of FTD pathogenesis, we performed an unbiased quantitative proteomic analysis of whole‐brain tissue from wild‐type (WT) and Progranulin knockout (Grn−/−) mice at 3 and 19‐months of age. We utilized a 10‐plex Tandem Mass Tag (TMT) isobaric labeling mass spectrometry approach for peptide labeling and quantification that enabled a deep proteome analysis of mouse brain and detection of 8,695 proteins. Differential expression analysis of the brain proteome of 3‐month old Grn−/− versus WT mice revealed 29 up and 26 down‐regulated proteins, while 119 proteins were up and 20 proteins were down‐regulated in 19‐month old Grn−/− mice. Then we performed weighted correlation network analysis (WGCNA) on the brain proteome of Grn−/− vs WT mice and identified 29 modules of highly co‐expressed proteins. In particular, 3 modules were strongly correlated to Grn deficiency, increased with age, and were enriched with lysosomal proteins (Ctsd, Fuca2, Tpp1) and inflammatory proteins (Gpnmb, Lgals3, GFAP, CD44, S100a C1qa). Subsequent validation of changes identified through proteomics using western blotting and immunohistochemistry revealed age‐dependent increases in multiple lysosomal proteins (i.e. Cathepsin D and Z) and markers of gliosis (GFAP, IBA1) throughout the brain, with a particularly marked increase in the thalamus in old Grn−/− mice versus age‐matched WT. Next, we asked if similar changes occurred in the biofluids or post‐mortem tissue from individuals with GRN mutations associated with FTD. We found that GPNMB and galectin‐3 (LGALS3) were significantly elevated in the lysates of FTD‐GRN brain samples compared to healthy controls. Total levels of GPNMB were also elevated human FTD‐GRN cerebrospinal fluid and plasma samples compared to controls. These data suggest that common pathogenic pathways are dysregulated in human FTD cases with GRN haploinsufficiency and mice completely deficient for Grn. Moreover, our findings support the idea that insufficiency of PGRN and GRNs in humans may cause FTD, AD and related neurodegenerative diseases through lysosomal dysfunction and neuroinflammation and suggest novel therapeutic approaches.

Support or Funding Information

1) National Institute of Health NINDS grants (R01NS105971; R01NS093362)2) the Association for Frontotemporal Degeneration grant3) The Bluefield Project to Cure FTD