C9orf72 deficiency promotes motor deficits of a C9ALS/FTD mouse model in a dose-dependent manner

G4C2 hexanucleotide repeat expansions in the first intron of C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (collectively, C9ALS/FTD) [4, 6, 11, 14]. Haploinsufficiency (loss-of-function) of C9ORF72 protein is a key proposed disease mechanism which may act in parallel with gain-of-function mechanisms, including toxic RNAs from repeat transcription and dipeptide repeat proteins (DPRs) from repeat-associated non-AUG (RAN) translation [5, 9, 17]. However, the effect of C9orf72 deficiency in the background of gain-of-function has not been examined in vivo. Neither heterozygous nor homozygous knockout (KO) of C9orf72 in neurons leads to motor deficits in mice [8]. Recently, gain-of-function mouse models were generated using a C9ORF72 bacterial artificial chromosome (BAC) from C9ALS/FTD patient DNA under the control of the endogenous regulatory elements. Interestingly, three out of four of these C9-BAC transgenic mice did not develop motor behavior deficits, even at advanced ages [7, 12, 13]. Since these C9-BAC mouse models contain elevated C9orf72 proteins from the endogenous mouse gene, we hypothesized that C9orf72 provides neuroprotective effects against motor deficits in C9-BAC mice. To test this hypothesis and investigate the in vivo significance of C9orf72 haploinsufficiency, we crossed C9orf72 mice with C9-BAC mice and examined the consequences of C9orf72 protein dose reduction (loss-of-function) in the background of C9-BAC (gain-of-function). We found that C9orf72 loss and haploinsufficiency exacerbate motor behavior deficits in a dose-dependent manner, and this occurs early in the course of pathogenesis (4 months of age). Among the four published C9-BAC mouse models, we selected the one with motor deficits (we refer to this C9orf72 BAC + model as the C9-BAC line here) [10]. To reduce C9orf72 protein levels at different doses, we crossed C9orf72 and C9-BAC mice for two generations. We isolated proteins from brain tissues and confirmed the expected C9orf72 protein dose reduction (Fig. 1a, Additional file 1: Figure S1A). The unchanged protein level of Atg101, which is associated with the C9orf72/Smcr8 complex based on our previous study [16], suggests the specificity of C9orf72 reduction (Fig. 1a, Additional file 1: Figure S1A). To study effects of C9orf72 deficiency on the motor behaviors of C9-BAC mice, we monitored a cohort of mice [20 WT (10 females + 10 males), 18 C9-BAC (11 females + 7 males), 26 C9orf72;C9-BAC (14 females + 12 males), and 19 C9orf72;C9-BAC (10 females + 9 males)]. We excluded C9orf72 and C9orf72 mice for the following reasons: C9orf72 heterozygous and homozygous KO mice exhibited no neurodegeneration and motor deficits based on previous studies [8]; complete deletion of C9orf72, which does not occur in C9ALS/FTD patients, led to autoimmune disorders and reduced survival in mice [1], which may complicate large-scale behavior and survival studies. We found that there were no significant differences among the four tested groups in their survival around 4months, when behaviors were assessed. They also exhibited similar body weights, taking the sex of the mice into account (Additional file 1: Figure S1B-1C). To examine their general anxiety levels, we performed an open field test [3]. C9-BAC mice with different C9orf72 levels behaved similarly in total distance traveled, * Correspondence: jianfu@usc.edu Qiang Shao and Chen Liang contributed equally to this work. Center for Craniofacial Molecular Biology, University of Southern California (USC), Los Angeles, CA 90033, USA