Our data on this first C9orf72 mouse model argues in favor for a gain-of-function pathological mechanism in C9orf72 associated ALS and FTD

Our data on this first C9orf72 mouse model argues in favor for a gain-of-function pathological mechanism in C9orf72 associated ALS and FTD. == Physique 2. cells. Alternatively, a pathogenic mechanism has been proposed for the production of toxic dipeptide repeat proteins (DPR) by repeat-associated non-AUG translation (RAN) of the repeat [5,6]. Interestingly, to differentiate between Eperisone repeat RNA-only and DPR protein toxicity fruit travel models carrying a range of real and RNA-only repeats have been generated. These studies exhibited that this major toxic species were the DPR proteins [7]. Here we report on an RNA-only gain-of-function mouse model. To study the intrinsic effect of the repeat without assessing its IFRD2 effect on the C9orf72 gene, we created a spatially and temporally inducible transgenic mouse model. This mouse model has a repeat size of 80 GGGGCC-repeats, without human flanking regions, which may affect repeat translation. This repeat was cloned in the 5 UTR of a GFP reporter gene and controlled by a tetracycline responsive element (TRE) promoter (Physique1A). To enable expression of the TRE-construct we created bigenic mice that possess both the TRE-construct and the tetracycline-responsive transcriptional activator (rtTA) under a general heterogeneous nuclear ribonucleoprotein (hnRNP) promoter [8]. Expression of the repeat was turned on after weaning by adding doxycycline (dox) to the drinking water. Expression of the repeat construct can be stopped at any time by withdrawal of dox, allowing for reversibility studies (Physique1A; more information about the creation of the model can be found in Additional file1). After generation of transgenic mice, the repeat size remained stable for multiple generations (data not shown). GFP expression was seen in bigenic mice as soon as 4 days after dox treatment started and remained stable over time (assessed by western blot of Eperisone liver homogenates, data not shown). Next to liver, multiple other tissues showed GFP expression including lung, kidney and brain; with most prominent expression in the striatum (Physique1B) and the cuneate nucleus in the brainstem. == Physique 1. == Expression of 80GGGGCC-repeats leads to the formation of ubiquitin-positive inclusions in mouse brain. A)Schematic of the model used to produce the inducible mice. Simultaneous expression of rtTA and doxycycline treatment are needed to drive expression of the TRE-80GGGGCC-eGFP construct. PCR for determining repeat size in three transgenic mice made up of the repeat construct.B)GFP expression in the striatum of bigenic mice after 12 weeks dox treatment.C)Intranuclear (arrows) and neuropil (arrowheads) ubiquitin-positive inclusionsin the striatum of bigenic mice after 12 weeks dox treatment. Both ubiquitin-positive, TDP-43-positive and TDP-43 unfavorable neuronal and cytoplasmic inclusions are pathological hallmarks in post mortem brain tissue from C9orf72 FTD and ALS patients. We used the presence of inclusions as a readout for the effect of expression of the repeat in our RNA-only gain-of-function ALS/FTD model [9,10]. We found ubiquitin-positive inclusions in those brain regions that express high levels of GFP, including the striatum (Physique1C) and the cuneate nucleus of bigenic mice after twelve weeks of dox treatment (n = 7 mice per group). Inclusions were found in the nuclei and the neuropil in striatum and mainly in the nuclei in the cuneate nucleus. The presence of ubiquitin-positive inclusions in our mouse model is usually a shared phenomenon with non-C9orf72 mouse models of ALS and FTD [11,12]. We did not observe any TDP-43 positive nor p409/410 TDP-43 positive inclusions in our mouse model after twelve weeks of dox treatment, despite the positive staining in a C9orf72 patient hippocampus control (data not shown). TDP-43 inclusions might only appear after prolonged expression of the repeat or additional genetic or environmental factors might be needed to drive TDP-43 dysfunction. Importantly, TDP-43 function could also be affected without the presence of TDP-43 positive aggregates [13]. The absence of DPR pathology was assessed with a Eperisone poly-GA antibody [6]. We could not detect poly-GA aggregation in brain tissue from bigenic mice, but only revealed GA-positive inclusions in C9orf72 patient hippocampus control material illustrating lack of DPR pathology in this mouse model (Physique2). The mice did not develop any obvious behavioral phenotype and showed no cell loss. The neurotoxic effect of the C9orf72 hexarepeat growth has been suggested by both RNA- and protein-mediated pathology [7]. Due to lack of DPR pathology, this mouse is a good model to investigate whether toxicity could be driven from the do it again RNA only. Long term research concentrating on molecular behavior and adjustments deficits inside our mouse model can offer additional understanding in disease.