Extensive phenotypic characterisation of a human TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis (ALS)

The majority of preclinical studies in ALS have relied on transgenic models with overexpression of mutant human superoxide dismutase 1 (SOD1), widely regarded to have failed in terms of translation of therapeutic effects. However, there are still no widely accepted models of other genetic subtypes of ALS. The majority of patients show ubiquitinated cytoplasmic inclusions of TAR DNA binding protein of 43 kilodaltons (TDP-43) in spinal motor neurons at the end stage of disease and a small proportion have mutations in TARDBP, the gene encoding TDP-43. TDP-43 transgenic mouse models have been produced, but have not been widely adopted. Here, we characterised one of these models available from the Jackson Laboratory in detail. Compared to TDP-43WT mice, TDP-43Q331K mice had 43% less hindlimb muscle mass at 6 months and a 73% reduction in hindlimb compound muscle action potential at 8 months of age. Rotarod and gait analysis indicated motor system decline with elevated weight gain. At the molecular level, the lack of TDP-43 cellular pathology was confirmed with a surprising increase in nuclear TDP-43 in motor neurons. Power analysis indicated group sizes of 12–14 mice are needed to detect 10–20% changes in measured parameters with a power of 80%, providing valid readouts for preclinical testing. Overall, this model may represent a useful component of multi-model pre-clinical therapeutic studies for ALS.

It is possible that the rotarod findings were influenced by the weight of the mice, as a previous study reported a correlation between increased weight and decreased rotarod performance 23 . Therefore, we corrected for weight by plotting the ratio of latency to fall (seconds)/weight (g) and the significant difference in rotarod performance found in the TDP-43 Q331K mice was still evident (Fig. 1G,H). Gait analysis. Using the CatWalk gait analysis system, it was possible to identify a number of gait abnormalities. Paw prints were wider, with an unsteady gait pattern in the TDP-43 Q331K mice (Fig. 2C), compared to the gait of non-transgenic ( Fig. 2A) and TDP-43 WT transgenic mice (Fig. 2B). Walking duration (Fig. 2D,E) increased significantly in TDP-43 Q331K mice by 6 months of age (p < 0.001) and this slowing of gait is more prominent by 10 months of age (p < 0.0001).
The hind-limb base of support is the distance between left and right hindlimbs perpendicular to the direction of travel and captures the severe waddling or swimming gait of the TDP-43 Q331K mice. This measurement increases from ~ 21 mm to ~ 36 mm between 1 and 10 months of age in both male and female TDP-43 Q331K mice and this increase is statistically greater than that seen in both sets of control mice at 6 and 10 months of age (Fig. 2F,G).
A useful indicator of unsteady gait is the percentage of time spent on diagonal paws, the predominant stepping pattern in healthy mice. The percentage of time spent on diagonal paws is decreased in male mice at 6 and 10 months of age (p < 0.05) and at 10 months of age in female mice indicating an increasingly unsteady gait with age (p < 0.0001) (Fig. 2H,I).

Lumbar spinal motor neurons show increased levels of c-myc-tagged TDP-43. Lumbar spinal
cord sections from TDP-43 WT and TDP-43 Q331K mice showed clear nuclear staining (Fig. 3A) for c-myc-tagged human TDP-43 (green). C-myc-tagged TDP-43 was also seen in the cytoplasm of motor neurons, identified  (Fig. 3B). However, there was no significant difference in cytoplasmic staining intensity of c-myc-tagged TDP-43 between groups (Fig. 3C). Surprisingly, the ratio Female rotarod performance/weight. Error bars represent mean ± SD. All graphs contain data for TDP-43 Q331K (n = 6 per gender-depicted by the blue lines), TDP-43 WT (n = 6 per gender-depicted by the green lines) and non-transgenic (n = 12 per gender-depicted by the red lines) mice compared using a two-way ANOVA with Tukey's post-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 TDP-43 Q331K vs all other groups. Western blotting with an anti-TDP-43 antibody allowed for quantification of both human and mouse TDP-43 protein due to the c-myc tag on the human protein increasing its molecular weight ( Fig. 4D-F). Moderate levels of human TDP-43 were measured in the TDP-43 WT mice at 10 months of age (66.6 ± 21.0% in cortex, 126.1 ± 67.1% in spinal cord compared to endogenous mouse TDP-43), whilst levels in the TDP-43 Q331K mice were significantly higher than endogenous mouse TDP-43 in cortex (151 ± 56.5%, p < 0.001) but not in spinal cord (213.9 ± 154.1%).

TDP-43 Q331K mice have reduced compound muscle action potential (CMAP) amplitude and fasciculations.
In order to confirm that the loss of muscle mass correlated with reduced electrophysiological properties, CMAP amplitude was measured. A significant reduction in CMAP amplitude in the TDP-43 Q331K mice compared to non-transgenic mice was evident in mice aged 9-11 months (7.9 ± 1.7 mV vs 29.8 ± 2.8 mV, p < 0.001, two-way ANOVA). This represents a 73% reduction in CMAP amplitude in the TDP-43 Q331K mice (Fig. 5B). Loss of CMAP amplitude can be a non-specific observation relating, for example, to motor axon loss, or muscle dysfunction. To explore the aetiology of CMAP data we undertook hindlimb electromyography (EMG) in 8 mice. Clear fasciculation potentials were recorded in 2 of the eight mice (Fig. 5C,D).
Optimal study design based on statistical power analysis. One of the key determinants of model utility is the ability to detect therapeutic or other intervention effects with relatively high statistical confidence with reasonable group sizes. For the data generated here we undertook statistical power analysis to identify numbers of animals needed to detect a range of effect sizes (Table 1). We focussed on female mice overall as they showed less variability in the various readouts (e.g. see Fig. 1) meaning that smaller group sizes would be needed for a given effect size and statistical power. We calculated coefficients of variation (CV) for each readout and these are low overall with none above 16% and 6/8 below 11% (Table 1). Power analysis assuming a twotailed Student's t test with α = 0.05 and β 0.8 was performed on 6 and 10 month data with effect sizes between www.nature.com/scientificreports/ 10 and 20%. We have previously used time taken to reach a 20% decline in performance for rotarod data, 26 and so we used this parameter in the present study as it mitigates the relatively high variability seen on individual timepoints. We noted that a 10 month study does not provide significant additional information over a study length of 6 months, with the exception that time to reach a neuroscore of 2 would be missed. A group size of 14 per group is sufficient to capture the lowest effect size (10%) for all measured parameters. Thus, overall we would recommend group sizes of 14 with a 6 month study duration as being able to capture low effect sizes with reasonable confidence in as short a study as possible.

Discussion
For many years, models based on overexpression of mutant SOD1 27 have been the major workhorse for preclinical testing in ALS. This model has provided useful insights, however, the lack of translation of therapeutic effects from this model to clinical ALS indicates a need for new therapeutic testing paradigms 28 . This may be particularly true given that SOD1-related ALS is not a TDP-43 proteinopathy 3 . Previously, serious flaws in experimental design were identified with the majority of studies in SOD1 related models 29 , mandating that new models must be carefully characterised and serious attention paid to study design. There are currently nine TDP-43 mouse models readily available at The Jackson Laboratory (USA) for which the original papers are available, including the model described here. Two models overexpressing wild type TDP-43 12,13 showed a dose-dependent phenotype with some evidence of TDP-43 pathology, indicating that overexpression of TDP-43 alone can cause an ALS-like disease. Mutant TDP-43 models which highly overexpress the TARDBP gene show severe phenotypes 9,30 . It has been reported that a truncated TDP-43 model has a mild motor phenotype, but the TDP-43 protein was unaffected and the truncation is not one found in ALS patients 31 . Aggressive disease progression and early death has been an issue for several models including two with an M337V mutation under the murine prion promoter 14, 30 and a postnatal deletion of TDP-43 which caused a fatal increase in fat metabolism 24 . A mouse model with an A315T mutation expressed using the murine prion promoter 18 was the first published and has been studied by multiple groups. However this mouse suffers from gut immotility, due to increased huTDP-43 expression in the myenteric plexus and degeneration of neurons therein, complicating the phenotype [19][20][21]32 .
The TDP-43 mouse model described in this paper has a minimal overexpression of the human TARDBP gene for both the mutant and wild-type transgenics, where the authors targeted a level of expression similar to endogenous mouse TDP-43 22 . Furthermore, the Q331K mutation has been found in patients 7 , and these mice showed no signs of gastrointestinal problems.
In contrast to the original study describing these mice we found that the level of expression of transcripts encoding human TDP-43 were higher in TDP-43 Q331K transgenic mice compared to TDP-43 WT mice in both Table 1. Numbers of mice/group required for investigation of each parameter. All power analyses are based on detecting a power of 80% in females, using the highest standard deviation (SD) from the relevant time-point. www.nature.com/scientificreports/ cortex and spinal cord and higher at the protein level in cortex. This calls into question whether this is a mutant specific phenotype, one of the initial attractions in characterising this model and should be considered when selecting controls. Despite this, the TDP-43 Q331K mouse on the C57BL/6 N background shows promise as a model of ALS as it demonstrates signs of reduced muscle mass, muscle weakness and motor dysfunction. The most interesting aspect of these findings is that they occur without the presence of canonical TDP-43 proteinopathy. It is conceivable that the model will develop TDP-43 pathology at a later time-point, as shown in a similar strain of TDP-43 Q331K on a C57BL/6 J background at 24 months of age 33 . These mice (line 31) were generated at the same time as the line tested here (line 103) and are described briefly in the Arnold et al. paper 22 although not characterised. Therefore, it is possible that the findings we present here are representative of an early stage of disease, in which a motor phenotype is evident prior to mislocalisation and aggregation of TDP-43. The development of an early phenotype is advantageous as it will allow us to investigate the onset and early stages of disease, as well as providing the potential to investigate disease modifiers such as gene-environment and gene-gene interactions.
Previous investigations of TDP-43 Q331K mice on a C57BL/6 J background found no evidence of nuclear depletion of TDP-43 22 . We found increased cytoplasmic TDP-43 in the TDP-43 Q331K mice compared to the TDP-43 WT mice, at 10 months of age, as described previously in a similar model at 24 months of age 33 . However, we also report increased nuclear TDP-43 in the TDP-43 Q331K mice compared to the TDP-43 WT mice. Overall this may suggest that mislocalisation per se is not required for the onset of motor system degeneration, but the absolute level of TDP-43 in any one compartment (cytoplasmic or nuclear) may be the driving factor. This would fit in with a dominant negative model of TDP-43 dysfunction 34 . This finding has been highlighted also in a knockin mouse model where the Q331K mutation has been introduced into the endogenous mouse tardbp gene 17 . Interestingly these mice also demonstrate increased weight gain and a lack of TDP-43 mislocalisation. This may suggest that mild overexpression of TDP-43 via a transgenic approach is physiologically relevant and we believe these findings have wider implications for TDP-43 biology.
The results we present here consistently demonstrate a detectable decline in motor performance by 6 months of age in the TDP-43 Q331K mice. Progressive gait abnormalities become identifiable on neuroscoring at around 6 months of age, consistent with the significant changes seen in hindlimb base of support and walking duration on quantitative gait analysis, and a 40% loss of gastrocnemius muscle mass.
Rotarod performance is significantly reduced in the TDP-43 Q331K mice by 10 weeks of age, consistent with findings in a previous study of TDP-43 Q331K mice on a C57BL/6 J background, in which the mice were found to have a significantly reduced rotarod performance at 3 months of age 22 .
We observed that 'walking duration' , increases as TDP-43 Q331K mice age when compared to both TDP-43 WT and non-transgenic mice. This suggests a reduction in speed which may be related to motor dysfunction and/or a reduced motivation. In addition, during normal gait, mice predominantly walk with diagonally opposite paws in contact with the ground at any one time in a diagonal stepping pattern. Both male and female TDP-43 Q331K mice showed a reduction in this gait type with aging suggesting increasingly unsteady gait with age.
A link between manipulation of TDP-43 and tbc1d1 expression has previously been described in mouse models where deletion of tardbp decreased tbc1d1 expression and transgene mediated overexpression of tardbp lead to increased tbc1d1 levels 24,25 . The tbc1d1 gene encodes a regulator of the translocation of Glut4, a glucose transporter and so this has been proposed as the mechanism by which TDP-43 regulates fat deposition 24,25 .
We observed increased weight gain in the TDP-43 Q331K model described here with mild overexpression of mutant TDP-43 and investigated whether tbc1d1 gene expression was also elevated as a potential mechanism causing weight gain. Levels of tbc1d1 expression were similar between TDP-43 Q331K mice when compared to TDP-43 WT mice in cortex, spinal cord and hindlimb muscles. In this model at least there is no correlation between levels of huTDP-43, tbc1d1, and the observed weight gain. Further work is needed to identify the mechanism of weight gain. However, we know that TDP-43 pathology defines subtypes of FTD and ALS-FTD patients, in contrast to ALS patients, show significantly elevated BMI 35,36 and a recent TDP-43 Q331K non-overexpressing model also showed weight gain, which was hypothesised as an FTD-related feature as suggested by other FTDlike phenotypes in this model 17 .
CMAP amplitude quantifies the depolarisation of muscle fibres and a reduction in CMAP amplitude may correlate with loss of functional motor axons, as is often seen in ALS patients 37 . CMAP amplitude was significantly lower in TDP-43 Q331K mice compared to non-transgenic mice at 9-11 months of age. This may indicate either fewer functional motor axons, as has been demonstrated previously in the SOD1 G93A mouse model 38,39 , or functional deficits at the level of the neuromuscular junction or muscle. EMG recordings at 6 months revealed fasciculation potentials, evidence of neurogenic (as opposed to myopathic) dysfunction.
A recent study used the same TDP-43 Q331K mice described here to investigate the effect of heterozygous deletion of Sarm 1 on axonal degeneration. Sarm1 is activated during Wallerian degeneration and its deletion was shown to protect motor axons and neuromuscular junctions from degeneration. The mice were also shown to display FTD-like phenotype with enhanced weight gain, brain atrophy and cognitive impairments 40 .
Our findings show consistent disease progression in both male and female TDP-43 Q331K mice. The data from the female mice are less variable, particularly for weight and rotarod performance. This indicates that, in some studies, it may be more efficient to use females alone.
Statistical power analysis for estimation of group sizes needed to detect different effect sizes indicated that differences of 10% can be detected at 6 months of age in neuroscoring, body weight, CatWalk hindlimb base of support and gastrocnemius/soleus muscle weights with group sizes of 14 for a pair-wise analysis (Student's T Test). An extension of 2 weeks in time to reach a 20% decline in rotarod performance would require 11 mice per group. Therefore, we recommend group sizes of 14 females to detect any potential disease-modifying effect in a 6 month therapeutic study with reasonable power (80%). The reliability of multiple readouts of motor system function makes the model attractive for researchers interested in pursuing therapeutic or mechanistic studies. www.nature.com/scientificreports/ The '3Rs' of reduction, replacement and refinement are critical in preclinical research, and we demonstrate that a large body of data can be obtained with 14 animals per group, which compares favourably with the group sizes recommended in the SJLBL6 SOD1 G93A mice of 24 per group 29 . Furthermore, refinement is applied by using a mild disease model in which mice do not reach a distressing 'end-stage' and we suggest that therapeutic studies do not need to continue beyond 6 months. In summary, this study provides a comprehensive analysis of this TDP-43 Q331K transgenic model which should enable other laboratories to include it as a valid system for preclinical screening cascades with a view to clinical translation. The model has a number of advantages over other TDP-43 based systems. Firstly, it is a robust and reproducible model without confounding effects on gut motility. A range of quantitative readouts show low variability enabling well powered studies with reasonable group sizes for statistical analysis. It recapitulates features (increased nuclear TDP-43, weight gain) described in a model with an endogenous TDP-43 Q331K mutation 17 , confirming that it is physiologically relevant. Overall the wider adoption of this model should improve assessment of novel therapeutic strategies for ALS, a key goal of translational ALS research.

Materials and methods
Please note that the material and methods section was adapted from the PhD Thesis of the first author 41  originally generated on a C57BL/6 J background by insertion of c-myc-tagged human TARDBP 22 and were subsequently backcrossed on to a C57BL/6 N background before being deposited at the Jackson Laboratory (stock numbers 017907 and 017933, respectively).
The basic characterisation tests were carried out with 5-7 males and 5-7 females per group (TDP-43 WT and TDP-43 Q331K ), non-transgenic littermates of each mouse were also tested, the data for which were pooled to create the non-transgenic (NTg) group. Mice were recruited at 25 days of age.
The electrophysiology pilot study consisted of 3 female TDP-43 Q331K mice and 3 female NTg mice at ages ranging from 9 to 11 months.
Tissue was collected for histological investigations at 1, 3, 6 and 10 months of age, collecting from 6 mice (3 male, 3 female) from each group (non-transgenic, TDP-43 WT , TDP-43 Q331K ) for each time point 41 . Housing. Mice were bred in a specified pathogen free environment, and transferred to a conventional facility for the studies described here. The facility uses a 12 h light/dark cycle, and room temperature is set to 21 °C. Cages were lined with fine sawdust (Eco-Pure Flakes 6, Datesand, Manchester, UK), which was changed weekly, a plastic house was placed in each cage and paper wool (Datesand, Manchester, UK) was used as bedding material. Mice were fed 2018 rodent diet (Harlan, Bicester, UK) ad libitum. Water was provided ad libitum and changed weekly. Fresh bottles were provided every 2 weeks.
All mice were housed in same-sex littermate pairs or quadruplets. However, some male pairs were separated at around 6 months of age due to fighting, and were subsequently housed singly 41 . Genotyping. Mice were identified by ear clipping and the ear clip was then used for genotyping. DNA was Neuroscoring and weighing. Neuroscores and weight were monitored weekly in the morning, measuring forelimb and hindlimb tremor on a scale of 0-3 (normal-strong). A 'neuroscore' scale was used to delineate motor dysfunction (0-normal, 1-significant hindlimb tremor, 2-abnormal gait, 3-severe waddle, 4-dragging of one hindlimb). These measurements were taken immediately prior to rotarod testing and in the same procedure room as the rotarod 41 .
Accelerating Rotarod test. Mice were place on the accelerating rotarod (Jones & Roberts for mice 7650) for a period of up to 300 s. In this time the rotarod accelerates from 4 to 40 rpm and latency to fall was recorded for each mouse. All rotarod testing was carried out at a similar time of day (am) and all testing consisted of two trials, with the highest score being recorded for analysis.
Mice were initially trained on the rotarod over 3 days (27-29 days of age) and underwent the first testing day at 30 days of age, being tested each week thereafter 41  Netherlands) was used to capture gait parameters at 1, 3, 6 and 10 months of age. Mice were placed on the glass floor of the CatWalk system in complete darkness and left to walk/run freely. Whenever possible, 6 runs were recorded and 3 runs were selected for analysis. CatWalk software 7.1 was used to analyse the gait parameters of the mice. Walking duration is the time taken to cross the length of the recording window (43.8 cm) 41 .
Tissue collection for immunohistochemical staining. Mice were sacrificed by trans-cardiac perfusion following anaesthetic overdose using an intraperitoneal injection of pentobarbitone (2.5 ml/kg). Under deep anaesthesia (checked by absence of pedal reflex) and prior to cessation of cardiac function, a thoracotomy was performed, in which the left ventricle was cannulated with a syringe containing PBS and mild pressure applied to distend the right auricle, which was then cut and the mouse was perfused with phosphate buffered saline (PBS) via the left ventricle of the heart, followed by perfusion with 4% PFA/PBS. Spinal columns were then extracted and placed in 4% PFA/PBS overnight. Next the spinal cord was extracted from the spinal column and cryoprotected by immersion through a gradient of 5, 10, 15 and 20% sucrose/PBS for 5 min in each concentration before overnight incubation in 20% sucrose overnight. The spinal cords were then dissected into cervical, thoracic, lumbar and sacral segments, and frozen in OCT embedding matrix (CellPath, UK) over dry ice, then stored at − 80 °C .
14 μm lumbar sections were sliced using a cryostat and mounted on to uncoated, charged slides (CellPath, Newtown, UK). Sections were mounted serially over a series of 5 slides, with 5 sections per slide, then stored at − 20 °C 41 .
Immunohistochemical staining for c-myc and NeuN. Slides were placed at 37 °C for 30 min and then washed three times in PBS at room temperature with shaking. Sections were then permeabilised in 5% BSA, 0.5% triton X-100/PBS for 90 min at 37 °C and subsequently placed in an antigen access unit (A. Menarini Diagnostics, Winnersh, UK) submerged in Access Revelation (MP-607-× 500, A. Menarini Diagnostics, Winnersh, UK) for a 30 min cycle at 125 °C, with a pressure of 20 psi.
Following washes in PBS, slides were blocked in 5% BSA/PBS for 10 min and then incubated with secondary antibodies at room temperature for 90 min (Alexa Fluor donkey anti-rabbit 488 [Life Technologies A21206], Alexa Fluor donkey anti-mouse 555 [Life Technologies A31570], both at 1:1000 in 1% BSA).
Slides were then washed in PBS and filtered water. Sections were mounted in Vectashield hard set aqueous mountant with 4' ,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK) and left to dry at 4 °C overnight.
Images were captured using the INCell Analyzer 2000 (GE Healthcare, Hatfield, UK) and analysed using INCell analysis software. Image collection and analysis was carried out blinded. Images were produced using ImageJ 41 .
Tissue collection. Mice were sacrificed at 10 months of age by overdose using an intraperitoneal injection of pentobarbitone (2.5 ml/kg) and exsanguination via cardiac puncture using a 25 gauge 0.5 × 16 mm needle (BD microlance, Ireland) and a 1 ml syringe (Terumo Europe, Belgium), after cessation of the pedal reflex. The brain was removed and the blunt edge of a steel back industrial single edge blade (Fisher Scientific, UK) was used to peel off the cortex from the remaining brain. An incision was made into the spine at the level of the hips and PBS was pushed into the spinal cavity using a 20 ml syringe (BD Plastipak, UK) fixed with a pipette tip (Fisherbrand, UK), ejecting the spinal cord from the rostral end of the spinal column.
Soleus/gastrocnemius muscles were removed together by dissection. Cortex, spinal cord and muscles were immediately snap frozen in liquid nitrogen and then stored at − 80 °C. Cortex and spinal cord from non-transgenic mice had been prepared using these methods from a previous cohort. Unfortunately non-transgenic snap frozen muscle tissue was unavailable 41 .
RT-qPCR. RNA was extracted using an RNeasy® lipid tissue mini kit (74804, Qiagen, UK) as per the manufacturer's instructions. Sample RNA concentrations were estimated using the Nanodrop spectrophotometer ND-1000 (Labtech International, UK). cDNA was extracted using a high capacity RNA-to-cDNA kit (4387406, Applied Biosystems, USA). Samples were placed in the PCR machine (G-storm, UK) for 1 h at 37 °C, followed by 5 min at 95 °C.