Decreased rotarod performance is evident in both male and female TDP-43Q331K mice by 10 weeks of age and many differences are found in catwalk gait analysis at both 3 and 6 months of age. However, differences in neuroscores did not become apparent until 27 weeks (post the 3 and 6 month time points), suggesting that neuroscoring is not sensitive to onset of disease, as measured by motor function. This is also seen in the SOD1G93A model where a significant rotarod deficit is obvious before the onset of visible signs of disease (Mead et al., 2011), and running wheel performance declines before symptom onset (Bruestle et al., 2009). Indeed, it is often noted by MND patients that they felt different long before onset of their first clinical sign of MND. A classic example of this is Lou Gehrig, the famous American baseball player, whose home-run average declined dramatically before symptom onset and diagnosis of MND (Kasarskis and Winslow, 1989).
Previously published work found significant hindlimb clasping in the TDP-43Q331K mice from 3 months of age (Arnold et al., 2013) and initially hindlimb clasping was measured in this study. However, hindlimb clasping was highly variable within each mouse, so it was deemed unreliable in this model and measurement was stopped.
It must be considered that at the time of neuroscoring in this study, a model-specific neuroscoring protocol had not been developed and therefore, a more specific neuroscoring protocol was subsequently developed for future studies (see chapter 4).
2.5.8. Catwalk Gait Analysis
Gait analysis has been used to detect early signs of disease in the SOD1G93A mouse model, consistently finding increased stand time in the SOD1G93A mice compared to controls (Wooley et al., 2005, Mead et al., 2011), increased base of support (BOS) (Mancuso et al., 2011a, Guillot et al., 2008), and decreased stride length (Mead et al., 2011, Wooley et al., 2005), to the extent of being used as a therapeutic trial readout (Lee et al., 2013, Sun et al., 2014).
2.5.8.1. Differences in Forelimb Gait
Forelimb paw intensity shows a tendency to increase in all groups of males at 10 months of age, possibly due to the increase in weight of all males. This increase is more exaggerated in the TDP-43Q331K females, possibly due to the larger difference in weight between the TDP-43Q331K and other groups. However, weight is significantly increased in TDP-43Q331K males and females by 10 weeks of age, yet no difference in forelimb intensity is found at 3 or 6 months of age. Therefore, although weight may be a contributing factor, it is possible that the forelimb intensity is affected by the unsteadiness of the mice and greater reliance on forepaws.
No differences were found with age, group or gender in forelimb print length, which is to be expected as the size of the paws would not be expected to change post maturity.
Forelimb stride length showed no differences in males with age or group. However, in females, the stride length appeared to increase slightly in the TDP-43WT and non-transgenic groups, whilst reducing slightly in the TDP-43Q331K mice (no findings were significant), as found in SOD1G93A mice (Mead et al., 2011). The forelimbs of SOD1G93A mice tend to be relatively unaffected, whereas there is potentially an effect in the forelimbs of the TDP-43Q331K mice. The reason for an increase in stride length of the TDP-43WT and non-transgenic mice is unknown. It is possible that the decrease in stride length of the TDP-43Q331K mice is due to weakness of the forelimbs, limiting the amount of lift given to each paw, and therefore the amount of distance covered.
An increase in duty cycle may represent a decreased ability to initiate movement of the limb off the ground, as found in TDP-43Q331K males at 10 months of age, and TDP-43Q331K
females at 6 months of age, possibly due to an increased swing time, affectively decreasing the proportion of time spent with the paws on the surface and in turn, decreasing the duty cycle.
Forelimb swing time is increased in the TDP-43Q331K male and female mice at 3, 6 and 10 months. This is suggestive of a generalised slowing of movement, likely due to weakness of the forelimbs. Forelimb swing time shows a slight increase in all groups at 6 months compared to all other time points. The reason for this is unknown.
Forelimb BOS increases in all groups with age in both males and females, possibly due to an increase in size of the mice with age, increasing the distance between the paws.
Forelimb BOS is significantly higher in the female TDP-43Q331K mice at 10 months of age.
In the forepaws, a swimming gait was not observed and cannot therefore explain this finding. It is possible that an increased weight has caused a wider stance. However, this is not evident at 6 months of age, despite a significantly increased weight at this timepoint.
2.5.8.2. Differences in Hindlimb Gait
Hindlimb paw intensity shows a decrease in all groups of both males and females at 6 months of age for an unknown reason. Hindlimb intensity is significantly lower in the TDP-43Q331K mice compared to all other groups at 10 months of age, suggesting that less weight is being placed on the hind paws, despite an increase in the weight of the animal.
This is consistent with a slight increase in forelimb paw intensity at 10 months of age, suggesting that the mice are placing more weight on to the forelimbs, possibly because the hindlimbs are weaker than the forelimbs, increasing reliance on forelimb strength.
However, this finding is not seen in female mice, despite an increase in forelimb intensity.
Hindlimb print length showed no differences with age, group or sex, other than an increase in the TDP-43Q331K females at 10 months of age, the reason for which is unknown as it is not expected that paw size would change post maturity.
Hindlimb stride length showed no differences in males with age or group. However, in females, the stride length appeared to increase slightly in the TDP-43WT and non-transgenic groups, whilst reducing slightly in the TDP-43Q331K mice. As with the forelimb
findings, this is in keeping with findings in SOD1G93A mice (Mead et al., 2011). The reason for an increase in stride length of the TDP-43WT and non-transgenic mice is unknown. It is possible that the decrease in stride length of the TDP-43Q331K mice is due to weakness of the hindlimbs, limiting the amount of lift given to each paw, and therefore the amount of distance covered.
Hindlimb duty cycle expresses the hindlimb stance time as a percentage of the step cycle duration and is as such representative of the amount of time a paw remains on the surface within each stride. A decreased duty cycle was found in TDP-43Q331K males at 3 months of age, the reason for which is unknown. An increase in duty cycle may represent a decreased ability to initiate movement of the limb off the ground, as found in both TDP-43Q331K males at 10 months of age, and females at 6 and 10 months of age.
A decrease in duty cycle of all groups was found in females at 6 months of age, possibly due to an increased swing time, effectively decreasing the percentage of duty cycle.
Hindlimb swing time is increased in the TDP-43Q331K male and female mice at 6 and 10 months of age. This is suggestive of a generalised slowing of movement, likely due to weakness of the muscles, as found in the forelimbs.
Hindlimb BOS increases in all groups with age in both males and females, possibly due to an increase in size of the mice, increasing the distance between the hindpaws. At 6 and 10 months the TDP-43Q331K male and female mice show a larger hindlimb BOS compared to other groups, likely due to their increased weight, which is mainly evident in the lower torso, resulting in an increased distance between the hindlimbs. The waddling gait of the mice will also increase the distance between hindpaw placements, increasing hindlimb BOS further.
2.5.8.3. Differences in Overall Gait Pattern and Duration
Walking duration, indicative of walking speed, remains similar with age in the TDP-43WT and non-transgenic mice. However, in the TDP-43Q331K mice, walking duration increases with age in both males and females, indicating a slowing down of the mice, consistent with the decrease in swing time. This general slowing in walking pace is suggestive of limb weakness and/or a lack of motivation to move.
Ordinarily, mice will spend most of their time walking in a diagonal stepping pattern, meaning they will step with the left forepaw whilst stepping with the right hindpaw;
then step with the right forepaw whilst stepping with the left hindpaw. When a mouse becomes less stable, they may become more reliant on a three paw stepping pattern, spending more time with three paws on the ground. A tendency towards three paw stepping was found in both male and female TDP-43Q331K mice. A decrease in diagonal stepping coincided with an increase in three paw stepping in males at 6 and 10 months, and in females at 10 months of age.
These results indicate that the TDP-43Q331K mice become slower and more unsteady with age.
2.5.9. Litter Statistics
No records could be found for litter size, percentage of transgenic pups and percentage of male pups for the C57BL/6NJ genetic background or for the TDP-43Q331K mouse model. Our own data in SOD1G93A mice indicates a reduction in the percentage of transgenic pups from the expected 50% to approximately 40%.
The TDP-43WT colony have an average litter size of 6 with 50% of pups being transgenic and 48% of pups being male. The TDP-43Q331K colony have an average litter size of 7 with 48% of pups being transgenic and 53% of pups being male. This suggests that there is no particular biological preference specific to gender or genotype. The reason for an increased litter size in the TDP-43Q331K colony is unknown, but may be due to bias in the original breeding trios, which has subsequently been inherited by the following generations.
2.5.10. huTARDBP Gene Expression
Previously published data described the huTARDBP expression levels in TDP-43Q331K mice as being 1-1.5 fold compared to the levels of endogenous expression in non-transgenic mice (Arnold et al., 2013). The non-transgenic mice do not carry any huTDP-43 so ideally, total levels of TDP-43 (both human and mouse) would be compared. However, primer pairs with an equal affinity for both human and mouse TDP-43 could not be
designed (see section 3.5.11. below). Hence, huTDP-43 expression was compared between the TDP-43WT and TDP-43Q331K mice.
The huTDP-43 gene is expressed under a murine prion promotor, which is primarily expressed in the CNS, hence huTDP-43 expression levels were measured in the cortex and spinal cord. Expression levels were also measured in the hindlimb muscle, as this is a potential target tissue for the induction of pathology by the TDP-43Q331K mutation.
In comparison to the TDP-43WT tissue, expression of huTDP-43 in the TDP-43Q331K mice was found to be approximately 2-fold in the spinal cord, 3-fold in the cortex, and 4-fold in the hindlimb muscles. It seems that there is a significantly increased expression of huTDP-43 in the TDP-43Q331K mice as compared to the TDP-43WT mice, which may translate to an increased protein level which could lead to toxicity independent of mutation status.
It is surprising that the highest expression was found in the hindlimb muscle, despite the murine prion promotor being primarily expressed in the CNS (Arnold et al., 2013), and the previously published data suggested that levels of huTDP-43 were relatively low in muscle (Arnold et al., 2013). The reason for the difference in these findings is unclear.