Decreased expression of Beclin2 and LC3 genes in PGRN deficiency: A CRISPR-Cas9 neuronal cell model.

Progranulin (GRN) is genetically associated with frontotemporal dementia (FTD), with mutations present in 23% of patients with familial FTD. The neurobiology of progranulin (PGRN) is still unclear, although the proposed disease mechanism is linked to a deficiency of the protein. Furthermore, patients with PGRN deficiency express primarily ubiquitin-positive TAR DNA-binding protein 43 (TDP-43) aggregates in the brain. It is possible that misfolded proteins may aggregate similarly to prions, converting a native protein into a pathologic misfolded replicate able to initiate template-directed protein misfolding. We enrolled 256 FTD patients and 300 healthy age-, sex- and geographic region-matched controls and sequenced all the coding exons of GRN and MAPT genes in patients with a familial history for dementia. We identified a rare GRN gene exon six deletion, g10325_10331delCTGCTGT (relative to nt 1 in NG_007886.1), alias Cys157LysfsX97, in three autosomal dominant pedigrees in Southern Italy segregating FTD in three generations. Transcriptional and translational analysis of mRNA from WBC and plasma proteins from FTD patients carrying the mutation exhibited deficiency of the protein. We used mouse embryonic stem cells (E14) with the goal of generating a cell model using CRISPR-Cas9, to test regulatory functions and pathological mechanisms of PGRN in vitro. We designed single guide RNA for the GRN gene with high specific, on-target activity and derived pure cellular clones carrying homozygote GRN gene mutations causing protein deficiency. We subjected wild-type and knock-out E14 cells to neuronal differentiation, generating an efficient model system to dissect the molecular mechanisms of PGRN in pathways potentially involved in FTD-TDP43 proteinopathy. To test the hypothesis that PGRN deficiency results in an impairment of the autophagic-lysosomal pathway, we performed mRNA gene expression analysis of autophagy-related genes using quantitative RT-PCR. Preliminary results demonstrated a decrease of Beclin2 and LC3 genes in PGRN-deficient cells compared to wild-type E14. Our findings support the role of the GRN gene in the etiology of FTD and the hypothesis that PGRN insufficiency can be a predisposing factor in neuronal degeneration. We can also speculate that PGRN plays a role in the regulation of the autophagic-lysosomal pathway. Further analyses are in progress to confirm this hypothesis and to identify the connection with the onset of FTD.