Overview indicated the means??SEM of patients (May mutation carriers seed poly\GA aggregates in repeat RNA\expressing cells Next, we asked whether patient\derived DPR aggregates can induce seeding. (DPR) proteins have been suggested as drivers of pathogenesis (Edbauer & Haass, 2016). Animal models expressing the repeat expansion strongly support a gain\of\function mechanism (Mizielinska models (Tran brains, followed by poly\GP and poly\GR, while poly\PA and poly\PR resulting from translation of the antisense transcript are rare. In addition to RNA foci and DPR pathology, patients also develop TDP\43 pathology that correlates well with neurodegeneration like in other forms of FTLD/ALS SCH 23390 HCl (Mackenzie repeat expansion triggers TDP\43 pathology. In contrast, several SCH 23390 HCl neuropathology studies failed to detect a strong correlation of the different DPR species (or RNA foci) with the region\specific neurodegeneration seen in ALS and FTLD patients (Mackenzie brain extracts which further supports the therapeutic potential of our discovery. Results Poly\GA and poly\PR differentially affect repeat RNA expression and translation To allow SCH 23390 HCl better interpretation of DPR seeding experiments, we first analyzed DPR protein co\localization in cell lines co\expressing repeat RNA and synthetic DPR constructs. Thus, we cotransfected ATG\initiated synthetic DPR expression plasmids with GFP tag together with a (G4C2)80 expression vector driven by the strong CMV promoter (Mori mRNA. Data are shown as mean??SD (mRNA. Data are shown as mean??SD (repeat RNA. Taken together, uptake of poly\GA promotes further aggregation of poly\GA, poly\GR, and poly\GP Igf2 in cells expressing the repeat expansion. Dipeptide repeat proteins promote repeat RNA foci formation To corroborate the effect of poly\GA on repeat RNA levels, we analyzed nuclear RNA foci, which are another disease hallmark of FTLD/ALS. We switched from HEK293 to HeLa cells, because they attach better to glass coverslips and can sustain the harsh washing steps for hybridization. As (G4C2)80 expression resulted in many coalescing RNA foci, which made counting their number unreliable, we analyzed the size of RNA foci. Cotransfection of GA175\GFP, PA175\GFP, and GFP\GR149 significantly increased foci size compared to GFP control, while GP47\GFP and PR175\GFP expression had no effect (Fig?4A and B). The effects of DPR proteins on RNA foci in HeLa cells are comparable to their effects on repeat RNA levels in HEK293 cells (compare Figs?4B and ?and11F). Open in a separate window Figure 4 DPR expression promotes formation of repeat RNA foci in HeLa cells and fibroblasts A, B hybridization of RNA foci (red) in HeLa cells cotransfected with (G4C2)80 and GFP or DPR\GFP for 3?days. Representative images (A) and quantification (B) of foci size from three experiments (at least 30 cells per condition per experiment) are shown. DAPI labels nuclei. Scale bar 10?m. Summary indicated the means??SD. GFP vs. GA\GFP hybridization of (G4C2)n RNA foci in fibroblast of patients transduced with GFP or DPR\GFP lentivirus for 8C9?days. Note that we could not analyze poly\GP, because we failed to generate a codon\modified lentivirus. Representative images (C) and quantification of foci number (D) are shown. Brightness and contrast were digitally enhanced for better visibility for the presentation only. Scale bar 40?m. Summary indicated the means??SEM of patients (May mutation carriers seed poly\GA aggregates in repeat RNA\expressing cells Next, we asked whether patient\derived DPR aggregates can induce seeding. Therefore, we homogenized cerebella of FTLD patients with or without mutation, because in this brain region, DPR levels are very high and TDP\43 aggregation is virtually absent (Mackenzie patients increased the number of GA80\flag\positive cells compared to patient compared to a patient also increased the levels of GR80\HA and GP80\myc (Fig?6C and D). Similar to the experiments with cell lysates, this was associated with an upregulation of (G4C2)80 mRNA expression in the cells receiving extracts from different mutant patients (Fig?6E). Thus, uptake of patient\derived DPR proteins SCH 23390 HCl induces DPR aggregation in (G4C2)\repeat\expressing cells by seeding aggregation and increasing repeat RNA levels. Open in a separate window Figure 6 Brain homogenates from patients seed DPR aggregation and promote repeat RNA expressionAnalysis of RAN translation products in HEK293 cells transfected with (G4C2)80 (for 24?h) and incubated with cerebellar extracts of patients.