Identification of neuroprotective molecules using a C. elegans model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disorder and one of the most common genetic causes of infant mortality. The disease is characterized by a selective degeneration of lower spinal cord motor neurons, which leads to progressive muscle atrophy and death. SMA is caused by mutations of the survival of motor neuron gene, Smn1, which is ubiquitously expressed. Although the genetic bases of SMA have been extensively studied, it is still unknown how the absence of Smn1 induces the selective degeneration of motor neurons, and which are the molecular mechanisms that underlie the disease. This contributes to the lack of an effective treatment being developed. Unbiased chemical screens can be performed in vivo using small animal model organisms, and contribute to identify potential therapeutic compounds as well as elucidate the molecular basis of the disease. In C.elegans two SMA models have been developed by classical genetic approaches: the smn-1(ok355) null mutant (Briese et al., Hum. Mol. Gen. 2009), in which the loss of smn-1 produces pleiotropic phenotypes and lethality, and the smn-1(cb131) hypomorphic mutant (Sleigh et al., Hum. Mol. Gen. 2010), which displays similar but milder defects. However no morphological alteration in the nervous system and no variations in the number of motor neurons have been detected in these genetic mutants. The hypomorphic mutant has been used to screen a chemical library and has led to the identification of some compounds capable of improving at least one of the disease phenotypes of the model. In order to find molecules with a protective role against the neurodegeneration caused by smn-1 loss, we took advantage of a genetic model developed in our laboratory, which is based on neuron-specific RNAi of smn-1 (Esposito et al., Gene 2007). Transgenic strains in which smn-1 is knocked down specifically in the GABAergic motor neurons present an age-dependent neurodegeneration, which results in altered backward movement and in neuronal cell death. Importantly, these animals are viable and fertile allowing us to overcome the lethality problem related to the other C.elegans SMA model carrying smn-1 loss of function. We initially tested on this model a panel of chemical compounds that were selected based on their known function. We have confirmed that valproic acid, a compound successfully used also in mice SMA models (Seo et al., Biochim. Biophys. Acta, 2013), can partially prevent neuronal death in C. elegans. Then, we found that the Ca2+-chelating agent EGTA has a protective function on neurodegeneration, suggesting that Ca2+ plays a role in smn-1-induced GABAergic cell death. Moreover, we have tested the effects of natural compounds and have obtained interesting results with juices of broccoli sprouts, which are currently under characterization. Finally, we are setting up the conditions to carry out an automated screen of a chemical library, consisting of 1280 FDA-approved compounds.