ADULT SOD1 G93R ZEBRAFISH MODEL DEVELOPS HALLMARK FEATURES OF ALS AND DISPLAYS NEUROMUSCULAR JUNCTIONS DEFECTS AND SPINAL NEURONS HYPEREXCITABILITY AT EARLY DEVELOPMENTAL STAGES
Creators: name: BENEDETTI, LORENA
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: Amyotrophic Lateral Sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by selective and progressive loss of motor neurons in the spinal cord, brain stem and motor cortex. Approximately 12% of familial and 1% of sporadic cases of ALS are associated to mutations in the gene coding for the antioxidant enzyme Cu-Zn Superoxide Dismutase type 1 (SOD1). Zebrafish is emerging as a powerful experimental model for the study of disorders affecting the nervous system and here we characterized the disease phenotype in adult zebrafish overexpressing the Sod1 ALS-linked mutation G93R (mSod1) and the wild-type Sod1 (wtSod1). Adult zebrafish expressing mutant Sod1 develop most of the main pathological features occurring in ALS: locomotor impairments, motor neurons degeneration, spinal cord and muscle atrophy, neuromuscular junctions loss, astrogliosis and inflammation. Interestingly, as it was reported in mice models of the disease, also in this model the overexpression of the wild-type form of Sod1 is associated to the development of mild alterations. Having completed the characterization of the pathological phenotype in the adult, we searched for precocious ALS hallmarks in zebrafish embryos and larvae. The optical transparency of zebrafish at early developmental stages allowed us to perform whole-mount fluorescence staining from which we studied the effects of the overexpression of wild-type or mutant Sod1 on spinal motor neurons development and neuromuscular junctions maturation. Mutant zebrafish embryos display precocious motor nerves branching defects while larvae present neuromuscular junctions maturation impairments and muscle fibers atrophy. The expression of mutant Sod1 is associated to precocious behavioral alterations in spontaneous tail coilings at 20 hpf, in touched evoked tail coiling responses at 48 hpf and in burst swimming responses at 96 hpf. In Sod1 G93R expressing embryos we detected spinal neurons hyperexcitability associated to the increased activity of the persistent sodium current INaP, and by the administration of riluzole, directly into the embryo water, we managed to pharmacologically modulate the spinal neurons electrical activity, the behavioral phenotype and the motor axons length. These results are extremely relevant since several studies demonstrated that ALS patients exhibit cortical hyperexcitability before any clinical sign of ALS and electrophysiological experiments performed in cultured mice embryonic and neonatal dissociated spinal motor neurons expressing mutant SOD1 G93A demonstrated neuronal hyperexcitability associated to the increased activity of the persistent sodium current INaP. This work demonstrates that the Sod1 G93R zebrafish model not only represents a valuable complement to other animal model for the study of ALS but also that it is a powerful model on its own to investigate ALS mainly thanks to the possibility to study locomotor circuits and potential pathogenetic mechanisms occurring at the very early stages of disease pathogenesis.