CLÁUSULAS DE INTERÉS PARA EL ASEGURADO

Repetitive stimulation provides a measure of the fidelity of neuromuscular transmission.

In healthy subjects, no decrement in response will be seen across stimuli; in MND a decrement is seen in most patients (Wang et al., 2001, Iwanami et al., 2011, Killian et al., 1994). The exact aetiology behind this observation remains unclear but is usually interpreted as representing the presence of immature neuromuscular junctions (Iwanami et al., 2011).

A previous study using a mouse model of SMA found that repetitive stimulation at 10Hz showed a decremental response (Bogdanik et al., 2015) but no records of repetitive stimulation in a mouse model of MND could be found. In this study there was no obvious change in response to repetitive stimulation with age in any of the control groups, and no obvious decrement across stimuli was evident in the control groups at 3 or 10Hz. It is probable that the decrement seen at 50Hz in the control groups at 6 months was related to a change in confirmation of the muscle due the tetanic stimulus response.

In comparison, the TDP-43^Q331K mice showed a significant decrement in response at all frequencies and ages, as is consistent with findings in MND patients. This is suggestive of a reduction in the safety factor at the neuromuscular junction, potentially a sign of recent reinnervation in which endplates have a lower safety factor (Wang et al., 2001, Iwanami et al., 2011) or a lack of acetylcholine replacement after depletion (Iwanami et al., 2011).

It appears that the decremental response was less severe in the TDP-43^Q331K riluzole

effect. It is possible that this treatment effect could have been clarified by putting more stress on the neuromuscular junction (i.e. a higher frequency of repetitive stimulation).

When this was attempted at 50Hz, it is believed that the frequency was too high, affecting the confirmation of the limb. A compromise of 20 or 30Hz may have been a more appropriate frequency to investigate.

If these findings do demonstrate a treatment effect, it is interesting that no effect was found in any motor function tests such as the catwalk or rotarod. However, it has been found in MND patients that although riluzole extends survival, it does not improve functional outcome measures including motor function, lung function, fasciculations, muscle strength and motor symptoms (as stated in the Summary of Product Characteristics for riluzole). It is possible therefore that any treatment effect at the neuromuscular junction was too subtle to detect using motor function tests.

It is likely that the repetitive stimulation decrement is reduced in the riluzole group because less NMJs are unstable due to a reduction in denervation/reinnervation events, in which the safety factor for action potential conduction is impaired. Unfortunately there is no NMJ immunostaining to support this theory but it is an interesting topic for further research.

There are a number of ways in which riluzole may reduce NMJ instability. Riluzole affects many neural mechanisms and the mechanisms by which it extends life in MND patients remain unclear. The mechanisms of action include: reduced glutamate release from the pre-synaptic terminal, inhibition of voltage-gated calcium currents, potentiation of calcium-dependent potassium currents, inhibition of voltage-gated potassium current, inhibition of sodium currents (both persistent and fast) (Bellingham, 2011) and blocking of muscle ACh receptors in vitro (Deflorio et al., 2012).

MND patients are recorded as having increased levels of persistent sodium and a decreased potassium conductance (Vucic and Kiernan, 2006). Riluzole reduces hyperexcitability, which increases the threshold voltage, requiring further depolarization in order to produce an action potential (Kuo et al., 2006, Del Negro et al., 2002). This reduction in hyperexcitability is as a result of decreased sodium influx and potassium efflux, reducing the occurrence of over-activation of the neurons and in turn, reducing neurodegeneration, preventing NMJ instability (Stys et al., 1992). Hence,

riluzole has a neuroprotective effect, which reduces the number of unstable NMJs, increasing the muscle’s ability to maintain a response to repetitive stimulation.

A second factor is whether the dose of riluzole is high enough. As shown in table 4.1 we have achieved reasonable levels of riluzole in the CNS, but the levels of riluzole may be much higher in the CNS of patients judging by the levels in the systemic circulation in the two species (blood in mice, serum in patients). This raises the question of whether a higher dose of riluzole may show a more profound difference. However, in-house studies have found that high doses of riluzole have a sedative effect in SOD1^G93A mice so higher doses must be approached with caution. It is also possible that an alternative method of dosing may be more effective and should be considered.

4.5.6.3. EMG

Spontaneous EMG activity is not specific to motor neuron loss and may be seen in a range of pathologies such as neuropathies and myopathies (although fasciculation potentials are not seen in myopathic conditions).

Positive sharp waves have been found in the SOD1^G93A (Miana-Mena et al., 2005) and wild type SOD1 (PeledKamar et al., 1997) mouse models. However it must be considered that normal healthy mice may have some nerve injury from normal everyday life, as found in healthy people (Falck and Alaranta, 1983).

Insertional activity occurs due to discharge potentials as the cell membrane is disrupted by the EMG needle. Heightened insertional activity is considered a sign of denervation (Shi et al., 2014) and was not found in the non-transgenic or TDP-43^WT mice, but was identified in the TDP-43^Q331K mice. Heightened insertional activity has also been identified previously in a wild type SOD1 mouse model (PeledKamar et al., 1997) and a knockout SOD1 mouse model (Shi et al., 2014).

Fibrillations and fasciculations are a classic electrophysiological finding in MND and indeed in mouse models of MND (Costa et al., 2012, Azzouz et al., 1997, Miana-Mena et al., 2005). The previous study of TDP-43^Q331K mice reported fibrillation potentials (Arnold et al., 2013) and we also found both fasciculation and fibrillation potentials in TDP-43^Q331K mice in this study. While it is a little surprising to observe spontaneous activity

and fibrillations have been recorded in healthy people (Falck and Alaranta, 1983). It is possible that this activity related to age related changes or minor injuries sustained over time in cages with other mice. It is difficult to quantify the amount of spontaneous activity as it can be subjective and also liable to sampling error, given the small recording area of the EMG needle. As a result we did not attempt quantification of spontaneous activity. However, we did not observe increased insertional activity or fasciculation potentials in the TDP-43^WT mice.

Overall, the spontaneous activity was evident but was not consistently found in all mice tested. This may be due to some reinnervation, as the phenotype does not progress rapidly. Carrying out EMG at earlier time points would have allowed us to investigate this further but the EMG needle is relatively large (30 gauge) and so it was deemed safest to only carry out EMG during terminal procedures (at 6 months of age).

Ultimately, the increased spontaneous activity in the TDP-43^Q331K group is in keeping with the MND phenotype.

4.5.7. Marble Burying

As described previously, marble burying is believed to be a surrogate marker of normal digging behaviour in mice (Deacon, 2006).

As was found in a previous study of non-transgenic mice (Egashira et al., 2008), riluzole had no impact on marble burying behaviour, as demonstrated by no difference in marble burying activity between the TDP-43^Q331K riluzole and vehicle groups.

The TDP-43^Q331K groups buried fewer marbles than the TDP-43^WT and non-transgenic groups, suggesting a decrease in normal digging behaviour. This was evident in the mice during the task, as the TDP-43^Q331K mice would often sit motionless and show no signs of exploration. It is possible that this lack of digging could be due to motor dysfunction, however, the motor phenotype in this model is mild so that appears unlikely, and little attempt at digging was observed in the TDP-43^Q331K mice. The lack of digging behaviour is more likely to signify a cognitive phenotype, namely apathy, or a lack of interest in digging. 10-15% of MND patients will develop a diagnosis of MND-FTD, exhibiting as signs such as inappropriate behaviour, repetitive behaviour, and/or apathy (Chan et al.,

2015). Overall, the lack of marble burying activity in the TDP-43^Q331K mice may be indicative of an FTD phenotype.

4.6. Conclusion

This study has allowed further characterisation of the TDP-43^Q331K mice and highlighted a potential FTD phenotype. The study has also allowed us to define a more reliable neuroscoring system for the TDP-43^Q331K and TDP-43^WT colonies. Riluzole appeared to have no significant effect on gross motor phenotype in the TDP-43^Q331K mice, but did seem to improve the NMJ safety factor, reducing the repetitive stimulation decrement and posing interesting ideas for future studies. Further investigation using immunohistochemistry is needed to determine whether riluzole treatment influences the disease pathology.

5. 31P-MRS Imaging 5.1. Introduction