Frontotemporal dementia (FTD) is a neurodegenerative disorder characterized by clinical and neuropathological heterogeneity and high genetic complexity. Granulin gene (GRN) is a major genetic determinant and Progranulin protein (PGRN) haploinsufficiency is the proposed disease mechanism. Patients with PGRN deficiency, show mainly ubiquitin-positive TAR DNA-binding protein 43 (TDP-43) aggregates in the brain, however the neurobiology of this protein remain still unclear. We collected and screened 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 of Southern Italian segregating FTD on three generations. We identified and analyzed the effect of this GRN mutation at transcriptional and translational levels and observed deficiency of the protein. With the goal to provide an efficient model system to dissect the molecular mechanisms of PGRN in pathway potentially involved in FTD-TDP43 proteinopathy, we generated by CRISPR-Cas9 a cellular model of PGRN deficiency. We silenced GRN gene in mouse embryonic stem cells (E14), then subjected wild-type and knock-out E14 cells to neuronal differentiation to have a mature neuronal phenotype lacking PGRN. With the hypothesis of an impairment of autophagic-lysosomal pathway in cases of PGRN deficiency, we performed, by quantitative RT-PCR, mRNA gene expression analysis of autophagy related genes. Preliminary results show a decrease of Beclin2 and LC3 genes in PGRN deficient cells compared to wild-type E14. Our findings support the role of GRN gene in the etiology of FTD and that PGRN insufficiency can predisposes to neurons degeneration. We can moreover speculate a role of PGRN in the regulation of autophagic-lysosomal pathway further analysis are in progress to confirm this hypothesis and to identify the connection with FTD onset.
CRISPR-Cas9 cell model: analysis of neurodegenerative mechanisms in GRN-related Frontotemporal Dementia (FTD).
2017
Abstract
Frontotemporal dementia (FTD) is a neurodegenerative disorder characterized by clinical and neuropathological heterogeneity and high genetic complexity. Granulin gene (GRN) is a major genetic determinant and Progranulin protein (PGRN) haploinsufficiency is the proposed disease mechanism. Patients with PGRN deficiency, show mainly ubiquitin-positive TAR DNA-binding protein 43 (TDP-43) aggregates in the brain, however the neurobiology of this protein remain still unclear. We collected and screened 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 of Southern Italian segregating FTD on three generations. We identified and analyzed the effect of this GRN mutation at transcriptional and translational levels and observed deficiency of the protein. With the goal to provide an efficient model system to dissect the molecular mechanisms of PGRN in pathway potentially involved in FTD-TDP43 proteinopathy, we generated by CRISPR-Cas9 a cellular model of PGRN deficiency. We silenced GRN gene in mouse embryonic stem cells (E14), then subjected wild-type and knock-out E14 cells to neuronal differentiation to have a mature neuronal phenotype lacking PGRN. With the hypothesis of an impairment of autophagic-lysosomal pathway in cases of PGRN deficiency, we performed, by quantitative RT-PCR, mRNA gene expression analysis of autophagy related genes. Preliminary results show a decrease of Beclin2 and LC3 genes in PGRN deficient cells compared to wild-type E14. Our findings support the role of GRN gene in the etiology of FTD and that PGRN insufficiency can predisposes to neurons degeneration. We can moreover speculate a role of PGRN in the regulation of autophagic-lysosomal pathway further analysis are in progress to confirm this hypothesis and to identify the connection with FTD onset.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
