In this study we demonstrated that our C. elegans and zebrafish ALS models can be used to identify neuroprotective molecules which represents the first in vivo chemical genetic screening platform for ALS. With this platform we discovered that MB is a potent suppressor of mTDP-43 and mFUS motor neuron toxicity in vivo. In both worms and fish MB corrected motor deficits and reduced the level of oxidative stress associated with the expression of mutant proteins.
MB is a pleiotropic molecule with a long and varied history of medical use 
but in the context of neurodegeneration MB has been reported to prevent amyloid-β and tau aggregation in vitro
. A previous study also showed that the treatment of cells with MB inhibited the formation of TDP-43 aggregates 
suggesting this compound might be appropriate for the treatment of ALS and other dementias. The efficacy of MB as a neuroprotective compound has been examined in Alzheimer's disease and ALS models where in some studies it is protective while in others it has no effect 
. We decided to include this compound in our assay from which we identified MB as a potent suppressor of mTDP-43 and mFUS toxicity in both C. elegans
and zebrafish. However, our data do not agree with a recent study examining the effects of MB in a TDP-43 mouse model 
. Mutant TDP-43[G348C] mice treated with MB showed no improvement in motor phenotypes as determined by the rotarod assay. Furthermore no difference in the cytoplasmic localization of TDP-43 was observed in treated mice.
Worms and fish live in aqueous media and a simple explanation for their greater susceptibility may be that they are more permeable to MB. We further hypothesize that the differences in MB efficacy might also be due to variations in timing for delivery of the compound. Specifically, our worms and fish were treated with MB from hatching whereas the TDP-43 mice were treated at 6 months. To confirm this hypothesis we treated mTDP-43 worms with MB at day 5 of adulthood and observed that late administration of the compound was significantly less effective at reducing paralysis. Thus, perhaps earlier (pre-clinical) treatment with MB may have greater effects in mouse models for ALS. Additionally there may be differences between the models since our worm and fish models capture a clinical aspect of ALS, namely progressive paralysis in animals expressing mTDP-43 that is absent from the TDP-43 mouse model.
Aging is a risk factor common to a number of neurodegenerative disorders including ALS, and oxidative stress is suspected to play a key role in the development of the disease by contributing to aging 
. Indeed, interactions between genetic, environmental, and age-dependent risk factors have been hypothesized to trigger disease onset 
. Consequently, we investigated the impact of MB treatment focusing on aging and stress response. Our C. elegans
data are in agreement with the survival data from the mouse studies where we observed no effect on lifespan in MB treated worms even though there was a positive effect on multiple phenotypes associated with mTDP-43 or mFUS. Thus, at least in simple systems lifespan effects can be uncoupled from neuroprotection but it remains to be seen if the same is true for mouse models of neurodegeneration.
In our previous work we showed that our TDP-43 and FUS transgenic C. elegans
models exhibited no difference in lifespan compared to non-transgenic worms 
. Thus, the paralysis phenotypes observed in our models specifically reflect the consequences of the expression of TDP-43 and FUS in motor neurons and are not due to secondary effects from general sickness and reduced lifespan. Therefore, it may be difficult to detect significant improvement on motor function or reflex phenotypes after MB treatment in mice showing generalized defects instead of treating problems resulting from TDP-43 or FUS proteotoxicity alone.
Finally, the TDP-43 mouse study did not examine the effects of MB on synaptic function or oxidative stress where we see clear effects in the worm and zebrafish models. MB can interact with nitric oxide synthase and also has an antioxidant potential by decreasing the generation of reactive oxygen species 
. Using C. elegans
we showed that MB specifically decreased the sensitivity of wild type worms to oxidative stress. We also investigated the impact of MB treatment in the formation of reactive oxygen species in both C. elegans
and D. rerio
and have observed a significant reduction in the generation of reactive oxygen species. Consistent with the literature 
, our data suggest that MB counteracts oxidative stress to provide protection against proteotoxicity in both our in vivo
models. Synaptic function was also restored after treatment with MB in transgenic mTDP-43 worms suggesting that this compound might also have an effect on synaptic transmission.
In summary, we present novel in vivo chemical genetic screening assays that may be useful for ALS drug discovery. Using two genetic models for ALS we report here that MB acts through reduction of oxidative stress and also restoration of normal synaptic function in genetic models of ALS. In addition, an important issue here is that in simple systems like C. elegans, lifespan effects can be uncoupled from neuroprotection. The next step will be to unravel MB's exact target and mechanism of action to develop compounds with more specific activities and also to capitalize on the strength of our assays to screen additional compounds as potential therapeutics in ALS.