Contribution of lower motoneurons, muscle fibers, Schwann cells to ALS onset and progression in the G93RSOD zebrafish model

The recently reported transgenic zebrafish expressing the SOD1 ALS-linked mutation G93R (1) might represent a feasible system to investigate the presymptomatic/early symptomatic phases of disease at different developmental stages. The scope of this project was to validate the SOD1G93R zebrafish as a suitable ALS model focusing on the contribution of motoneurons, muscle fibers, Schwann cells to ALS onset and progression. In transgenic zebrafish expressing either SOD1G93R or SOD1wt at different developmental stages and in adults, we tracked: (i) the processes of axonal branching of lower motoneurons and the innervations of muscle fibers, (ii) the organization of the contractile apparatus, and (iii) the behavior of Schwann cells. Confocal fluorescence analyses of SOD1G93R zebrafish embryos and larvae stained with antibodies against acetylated tubulin and SV2A showed that axonal projections of motoneurons were characterized by outbranching defects. SOD1G93R fish showed a significant reduction in motoraxons length before branching already at 28 hours post fertilization (hpf) up to 8 days post fertilization (dpf). Interestingly, a similar phenotype was detected in other ALS zebrafish models suggesting the existence of a common pathway leading to this phenotype (2-3). Double immunofluorescence staining with GFAP and acetylated tubulin, at 48, 72hpf and 8dpf, revealed the presence of Schwann cells associated with axons, along their entire length. No evident changes in the organization of Schwann cells network were detected among zebrafish lines. In young zebrafish (28hpf-8dpf), co-localization studies using SV2A and \u3b1-bungarotoxin demonstrated a complete superimposition of pre- and postsynaptic elements suggesting that no alteration of the neuromuscular connections exist at these early developmental stages. These observations were confirmed by ultrastructural analyses. In SOD1G93R embryos and larvae, no reduction in the muscle mass or differences in the fine structure of the sarcomere have been detected. In contrast, in adult SOD1G93R we observed a significant reduction in the area and in the number of motoneurons in the caudal segments of the spinal cord compared to both non-transgenic and SOD1wt fish, accompanied by a significant reduction of the calibre of the muscle fibers in the corresponding segments of the body wall. Interestingly, the same spinal cords did not show evidences of reactive astrogliosis, as shown by the staining with anti-GFAP antibodies. Our results highlight defects in lower motoneurons axonal growth and formation of a normal neuromuscular junctional apparatus at early development stages and further confirm that SOD1G93R zebrafish might represent a powerful tool in ALS research References: 1) Ramesh T, Lyon AN, Pineda RH et al, Dis Model Mech 2010; 3(9-10):652-62 2) Lemmens R, Van Hoecke A, Hersmus N et al, Hum Mol Genet 2007; 16(19):2359-6 3) Kabashi E, Lin L, Tradewell ML et al, Hum Mol Genet 2010;15;19(4):671-8