Species: Mouse
Genes: Tardbp
Mutations: Tardp Q331K
Modification: Tardbp: Knock-In
Disease Relevance: Frontotemporal Dementia, Amyotrophic Lateral Sclerosis
Strain Name: N/A
Genetic Background: C57Bl/6J
Availability: Available from Jemeen Sreedharan or Robert H. Brown Jr.

Summary

TDP-43 pathology characterizes a neurodegenerative disease spectrum that encompasses ALS and FTD. Transgenic mice have been used to study the pathological effects of mutant TDP-43, but the interpretation of their phenotypes is complicated by potential artifacts due to overexpression, absence of alternative splicing, and aberrant temporal and cell-type-specific expression that occur when transgenes are expressed under the control of heterologous promoters. To study the effects of a disease-linked TARDP mutation in the context of a structurally intact gene with its natural regulatory elements, CRISPR/Cas9 mutagenesis was used to introduce a point mutation equivalent to human Q331K into the mouse Tardp gene (White et al., 2018).

This knock-in approach yielded four founders. The line derived from founder 52 is the best-characterized and is focused on here. Unless otherwise stated, descriptions refer to male mice.

TDP-43^Q331K mice exhibit cognitive dysfunction but no motor impairment. TDP-43 autoregulation is perturbed, although the protein retains its normal nuclear localization. There is a 25 percent decrease in the number of parvalbumin-positive neurons in frontal cortex, but no loss of spinal motor neurons, and neuromuscular junctions appear to be intact. Interestingly, when mice homozygous for the mutation were stratified according to their performance in a marble-burying task, more than 400 genes were found to be differentially expressed in good performers compared with poor performers, suggesting that TDP-43^Q331K mice may be a useful model for the discovery of disease-modifying genes.

Neuropathology

Cortical thickness and cell density in frontal cortex were similar in homozygous TDP-43^Q331K mice and wild-type mice at five months of age; however, there was an approximate 25 percent reduction in the number of parvalbumin-positive neurons in the mutation carriers. The number and morphology of spinal motor neurons appeared to be normal in mutation carriers, and neuromuscular junctions were intact. No evidence of denervation was observed in mice as old as 23 months.

Neither TDP-43 nor tau pathology—in the form of protein aggregation or mislocalization—were observed in mutation carriers at five months of age. However, subcellular fractionation and immunoblotting revealed an approximate 45 percent increase in nuclear TDP-43 in homozygous mutation carriers compared with wild-type mice.

Cognition/behavior

Screening of homozygotes by automated continuous behavioral monitoring revealed reduced walking and hanging and increased rearing and eating by hand in four-month-old male and female mice. Further behavioral testing focused on males.

Both heterozygous and homozygous TDP-43^Q331K mice performed more poorly than wild-type mice on the rotarod. However, this difference was attributed to hyperphagia and increased body weight in the mutation carriers rather than motor deficits—when TDP-43^Q331K mice were placed on calorie-restricted diets, so that their weights matched those of wild-type mice, the three genotypes performed equally on the rotarod, at least to 16 months of age.

TDP-43^Q331K mice exhibited age-dependent deficits in a five-choice serial reaction time task. At four months, homozygous mutation carriers took longer to reach criterion, but eventually performed as well as heterozygous carriers and wild-type mice. All three genotypes performed similarly in a probe test administered at six months of age. At 12 months, heterozygous and homozygous mutation carriers performed as accurately as wild-type mice (i.e., had similar percentages of correct choices). However, the TDP-43^Q331K mice failed to make any choice more often than did the wild-type mice, a difference that might indicate an attention deficit.

Heterozygous and homozygous TDP-43^Q331K mice showed memory deficits in a novel-object-recognition task when tested at nine months of age. TDP-43^Q331K mice also performed more poorly than wild-type mice in a marble-burying task, although there was a wide range of performance among the mutation carriers.

Transcriptome

Not surprisingly, the transcriptomes of mutation carriers and wild-type mice differed, in an age-dependent manner. Additionally, the transcriptomic profiles differed between mutation carriers stratified according to their performance on the marble-burying task.

RNA-sequencing analysis identified 171 genes that were upregulated and 233 genes that were downregulated in the frontal cortices of five-month-old homozygous TDP-43^Q331K mice compared with wild-type. Heterozygous mutation carriers trended in the same direction, suggesting a gene-dose effect. In addition, 138 splicing changes were found in 106 genes, including Tardp, Sort1, and Mapt. Similar splicing changes were found in the hippocampus, in both males and females. Increased expression of Tardp and splicing changes in Tardp, Sort1, and Mapt were not found in spinal motor neurons, but may occur in spinal interneurons.

In 20-month-old mice, more than 1,000 genes showed differential expression between TDP-43^Q331K mice and wild-type mice, a set that partially overlapped with the differentially expressed genes in five-month mice. Genes involved in inhibitory synaptic transmission were among those downregulated in aged mice, as were the ALS-FTD-linked genes Chmp2b, Erbb4, and Epha4a, and the TDP-43 nuclear import factor Kpnb1. Gene ontology and pathway analysis indicated that other differentially expressed genes were found in networks related to protein ubiquitination, autophagy, glutamate receptor activity, and immune processes.

Among TDP-43^Q331K mice, the transcriptome profile differed between good and poor performers in the marble-burying task. In five-month-old mice, 410 gene-expression and 61 splicing differences were found when homozygous mutation carriers were stratified by behavior. For most of these genes (78 percent), the direction of change, relative to wild-type mice, was opposite in good and poor performers. Gene ontology and pathway analysis suggested that genes involved in transcription, DNA methylation, and chromatin modification were downregulated in good performers, while genes involved in protein translation and myelination were upregulated.

Other

The TDP-43^Q331K mutation may be deleterious for male embryos.

Modification Details

CRISPR/Cas9 mutagenesis was used to introduce a point mutation equivalent to human Q331K into the mouse Tardp gene. Sanger sequencing excluded mutagenesis at predicted off-target locations and at other sites in the Tardp gene. Founder 52 was outcrossed four times to remove other potential off-target mutants.

Related Strain

TDP-43^Q331K (line 3). This line was created in parallel with line 52. Similar to line 52, line 3 mice exhibited impairments in the marble-burying task, increased expression and altered splicing of Tardbp, altered splicing of Sort1 and Mapt, and a reduction in the number of parvalbumin-positive neurons.

Phenotype Characterization

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References

Paper Citations

1. White MA, Kim E, Duffy A, Adalbert R, Phillips BU, Peters OM, Stephenson J, Yang S, Massenzio F, Lin Z, Andrews S, Segonds-Pichon A, Metterville J, Saksida LM, Mead R, Ribchester RR, Barhomi Y, Serre T, Coleman MP, Fallon JR, Bussey TJ, Brown RH Jr, Sreedharan J. TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD. Nat Neurosci. 2018 Apr;21(4):552-563. Epub 2018 Mar 19 PubMed.

External Citations

1. Jemeen Sreedharan
2. Robert H. Brown Jr

Further Reading