DETERMINACIÓN DE LOS PARÁMETROS DE CALIDAD DE LAS

The role of exercise in ALS is controversial – on one hand, high levels of physical activity are correlated with the development of ALS in epidemiological studies (Beghi et al. 2010; Huisman et al. 2013), and on the other hand there are numerous case studies and small trials showing that exercise therapy may help to ameliorate disease progression to a small extent in ALS patients (Bohannon 1983; Johnson 1988; Dal Bello-Haas et al. 2007; Sanjak et al. 2010). In the current study, pre-symptomatic treadmill exercise had no significant impact on disease onset and survival time in female SOD1 mice, showing neither beneficial nor detrimental effects. However, exercised mice did show slightly increased stride length compared to sedentary mice at a few select time points in disease progression (Figure 4.6). Although this increase in stride length is not clear evidence of a protective effect, this suggests that moderate treadmill exercise is, at least, not detrimental to SOD1 mice.

______________________________________________________________________ 162 4.4.2.1 Previous exercise studies in ALS mice

Previous studies of exercise in SOD1 mice have shown varying outcomes, depending on the type of exercise, the intensity of the exercise, mouse gender, and whether or not the effects of environmental enrichment were controlled for. Generally, moderate-intensity treadmill exercise has shown marginal to moderate beneficial effects on survival time, disease onset, and functional parameters when started pre-symptomatically in SOD1 mice (Kirkinezos et al. 2003; Veldink et al. 2003; Liebetanz et al. 2004; Sorrells et al. 2009; Carreras et al. 2010).

Wheel-running exercise and swimming exercise have also shown beneficial effects for survival of SOD1 mice (Kaspar et al. 2005; Deforges et al. 2009; Sorrells et al. 2009), with wheel-running mice showing increased survival even when exercise was started around the onset of disease symptoms (Kaspar et al. 2005). In contrast, high-intensity or forced exercise has shown detrimental effects on survival in SOD1 mice (Mahoney et al. 2004). The exercise intensity in the present study was designated as ‘moderate’ – similar to that used in two previous studies which showed beneficially effects of exercise in SOD1 mice (Kirkinezos et al. 2003; Carreras et al. 2010). However, mindful that forced running had a detrimental effect on SOD1 mouse survival (Mahoney et al. 2004), mice in the current study were encouraged but not forced to keep running on the treadmill – thus, the exercise intensity may not have been high enough to elicit protective effects.

Environmental enrichment may also contribute to the apparent effects of exercise seen in previous studies – SOD1 mice exposed to a novel exercise environment, without undergoing exercise, showed longer survival times than mice not exposed to the novel environment (Sorrells et al. 2009; Gerber et al. 2012). In a treadmill exercise study which controlled for environmental enrichment by placing mouse on a stationary treadmill, no effect of exercise was seen on survival of SOD1 mice (Deforges et al. 2009). The current study also controlled for effects of environmental enrichment by placing mice on a stationary treadmill, and saw no significant effects of exercise on survival times. Thus environmental enrichment may play some role in determining survival time, although the biochemical mechanisms behind these effects remain to be elucidated.

______________________________________________________________________ 163 Gender and background strain, and interactions between the two, are known to affect SOD1 mouse survival time (Heiman-Patterson et al. 2005). Most previous studies on exercise in SOD1 mice involved mice with expression of the SOD1 transgene on a hybrid B6SJL genetic background, and most studies looked at the effects of exercise in male mice alone, with a few looking at a mix of male and female mice. Previous studies involving both female and male SOD1 mice have shown differing responses to exercise between the genders: three studies indicate male SOD1 mice respond more strongly to exercise than their female counterparts, regardless of whether exercise has a beneficial effect (Kirkinezos et al. 2003; Kaspar et al. 2005) or a detrimental effect (Mahoney et al. 2004). In contrast, another study showed exercise had a beneficial effect on survival in female but not male SOD1 mice (Veldink et al. 2003). To the best of my knowledge, the current study is the first to examine the effect of exercise in female SOD1 mice on a congenic B6 genetic background. As male mice were not tested in the present study, there may be an unknown effect of gender, or an interaction between gender and genetic background, which results in female SOD1 mice on a congenic B6 background being unresponsive to exercise treatment.

To further examine the effects of exercise in female SOD1 mice on a congenic B6 background, adaptations of the skeletal muscle to exercise should be examined in detail. As mentioned previously, the percentage innervation of neuromuscular junctions in the muscle was not significantly different between treatment groups (Figure 4.14). However, one of the beneficial effects of swimming exercise in SOD1 mice is the conversion of muscle fibre types from fast-twitch fibres to slow-twitch fibres (Deforges et al. 2009); slow-twitch fibres are more resistant to denervation and atrophy in SOD1- mediated ALS than fast-twitch fibres (Hegedus et al. 2008). Analysis of the muscle fibre type profile of exercised and non-exercised female SOD1 mice might help to determine whether moderate-intensity treadmill exercise does induce a protective conversion of muscle fibre types in SOD1 muscle.

4.4.2.2 Exercise and MT-1/2 induction

Part of the original rationale for examining exercise was the apparent upregulation of MT-1/2 in response to exercise in wild-type mouse spinal cord (Hashimoto et al. 2009). In the present study, the number of spinal cord MT-1/2-positive cells appeared slightly higher in the ventral/lateral and dorsal white matter compared to sedentary controls, but these changes were not significant and were not present in the ventral horn (Figure

______________________________________________________________________ 164 4.10). While the quantitative level of MT-1/2 expression was not directly tested in this study (for example, with western blotting or RT-PCR), Hashimoto and colleagues (2009) saw a widespread increase in both the total spinal cord MT-1/2 levels and in the number of cells displaying strong MT-1/2 immunoreactivity following a two week treadmill exercise program in WT mice (Hashimoto et al. 2009). Although it cannot be definitively stated that exercise failed to increase MT-1/2 levels in the current study due to the lack of direct testing by western blot, the fact that the number of MT-1/2-positive cells did not drastically increase with exercise hints that exercise at the intensity used in the current study may not be sufficient to increase the production of MT-1/2 in the spinal cord above that seen with increased duration of disease (Chapter 2, Figure 2.7). The lack of MT-1/2 upregulation with exercise in the current study may correlate with the minimal effects of exercise on survival time; however, MT-1/2 is unlikely to be the sole factor responsible for any exercise-mediated increase in SOD1 survival. Several other factors, indcluding muscle fibre type switching (Deforges et al. 2009), or the production of BDNF and IGF1 (Kaspar et al. 2005; Carreras et al. 2010) are likely responsible for the exercise-mediated increase in survival time seen in previous studies (McCrate & Kaspar 2008).

Interestingly, a possible interaction might exist between MT2 and exercise which does not involve upregulation of MT-1/2 in the spinal cord. The age at disease onset, as calculated from linear mixed modelling, was later in exercised MT2-treated mice than in sedentary MT2-treated mice, but was not affected by exercise in saline-treated mice (Figure 4.4D). Exercise has been reported to enhance LRP2 expression at the choroid plexus, to transport circulating IGF1 into the brain (Carro et al. 2005). As LRP2 is also a receptor for MT2 protein (Klassen et al. 2004), exercise may also facilitate the entry of MT2 into the central nervous system. As mentioned previously, examination of the spinal cord diffuse MT-1/2 immunolabelling, thought to represent extracellular MT-1/2, may determine whether exercise facilitates spinal cord entry of circulating MT2.

4.4.2.3 Exercise in ALS patients

The majority of studies in SOD1 mouse models of ALS show slight beneficial effects of moderate-intensity exercise. These correspond well with several small studies showing slight delays in disease progression in ALS patients treated with exercise-based therapies, with few adverse effects (Drory et al. 2001; Dal Bello-Haas et al. 2007; Lui

______________________________________________________________________ 165 & Byl 2009; Sanjak et al. 2010). Exercise and physical therapy have been implicated as an important component of multidisciplinary care for ALS patients, in order to strengthen remaining muscles, maintain range of motion, and help combat stiffness and cramping in affected muscles (de Almeida et al. 2012; Skalsky & McDonald 2012; Arbesman & Sheard 2014).

The potential beneficial effects of exercise in ALS patients are in contrast to epidemiological studies implicating high levels of physical activity as a risk factor for ALS (Beghi et al. 2010; Lehman et al. 2012; Huisman et al. 2013). However, the absence of a dose-response effect for increasing levels of exercise somewhat attenuates direct causality between physical activity and ALS (Huisman et al. 2013). An alternate theory proposes that the genetic profiles which naturally confer some higher level of body fitness may also predispose to ALS (Chio & Mora 2012; Mattsson et al. 2012). Thus, rather than physical activity directly causing ALS, it may be that those with a genetic risk profile conferring susceptibility to ALS are also more likely to engage in exercise-based leisure-time activities, or lean towards careers involving physical exertion such as professional sports (Lehman et al. 2012) or military service (Weisskopf et al. 2005).

4.4.3 Summary and conclusions

In summary, this proof-of-concept study reports that pre-symptomatic MT2 treatment resulted in a slight delay in disease onset and tended to increase survival time in female SOD1 mice, while exercise failed to show significant effects on disease onset or survival. No consistent synergistic effects were observed between MT2 treatment and exercise in SOD1 mice.

The current study showed no detrimental effects of moderate-intensity treadmill exercise on SOD1 mice, and is consistent with the concept that physical exercise is not causative of ALS; rather, as-yet-unknown genetic factors may predispose to both a lifestyle involving high levels of physical activity, and the development of ALS. In this context, low-intensity to moderate-intensity exercise may be beneficial for ALS patients by providing a minor neuroprotective effect, and for maintaining strength and range of motion around affected muscle groups. However, caution should be used to avoid strenuous exercise which could potentially accelerate disease progression.

______________________________________________________________________ 166 While MT2 has a solid theoretical basis for attenuating multiple pathological pathways present in ALS, the small magnitude of the effects may be due to the timing and delivery method of MT2 administration. Administration of MT2 directly to the central nervous system through intracerebroventricular injection or intrathecal pump, or via viral delivery, to deliver a consistent amount of MT2, may produce more pronounced effects on SOD1 mouse survival. Additionally, treatment beginning after the onset of disease symptoms is needed to confirm the survival-extending properties of MT2 and evaluate the use of MT2 as a potential therapeutic molecule for human ALS. To this end, the effects of a peptide analogue of MT2 will be discussed in Chapter 5.

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