Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of 'sporadic' cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS. Overall design: The BAC vectors were retrofitted via the loxP site within the backbone of pCYPAC2 with a vector containing PhiC31 integrase attB sites and a promoterless Neomycin selection cassette. The vectors were then targeted to the Rosa26 (Gt(ROSA26)Sor) locus in embryonic stem cells (ESCs) by PhiC31 integrase-mediated cassette exchange into IDG26.10-3 ESCs, which harbour an PhiC31 attB flanked hygromycin cassette, pre-integrated at the Rosa26 (Gt(ROSA26)Sor) locus (Chen et al., 2011). G418 resistant ESCs were screened by PCR screening and immunoblotting to 1) confirm correct exchange (integration) at the 5' and 3' end using the two primer combination ExPGK3 (5'-CACGCTTCAAAAGCGCACGTCTG- 3') and ExNeo2 (5'-GTTGTGCCCAGTCATAGCCGAATAG-3'); allBac-F1 (5'- TTAGGTCCCTCGACCTGCAGG-3') and Rosa3HR-R (5'- CGGGAGAAATGGATATGAAGTACTGGGC-3'), respectively and 2) to ensure all regions of the human genomic BAC were present. Candidate TDP-43WT and TDP-43M337V ESCs were injected into C57BL/6 blastocysts and the resulting chimeric embryos transferred to recipient pseudopregnant females. Chimeric male founder mice (F1) were subsequently crossed with C57BL/6J female mice to generate two isogenic human TDP-43 transgenic lines, differing only by the presence or absence of the M337V mutation. Mice were again screened by PCR using a battery of primers to confirm correct integration and presence of the BAC vector regions. Retention of the TDP-43M337V mutation in exon 6 was confirmed by sequencing DNA from ESCs and subsequent chimeric founders. Founder mice were backcrossed to C57BL/6J for a minimum of 3 generations and homozygous and heterozygous transgenic lines were established. To minimise the effects of genetic background, all experimental control animals were age-matched littermates. These TDP-43M337V BAC mice are available from The Jackson Laboratory as strain JAX#029266, https://www.jax.org/strain/029266. Five mice with M337 transgene, two mice with WT transgene and three nontransgenic C57BL/6 mice were used for this experiment, all female.