Role of chondrolectin in motor axon development in zebrafish

Abstract

Spinal Muscular Atrophy (SMA) is a childhood form of motor neuron disease (MND).

It is monogenic, caused by loss of Smn1. Although Smn is ubiquitously expressed in all cells, and acts in a housekeeping function to correctly assemble the spliceosome, motor neurons are specifically vulnerable to loss of Smn. This leads to degeneration of the motor neurons. Mounting evidence shows that this degeneration starts at the neuromuscular junction and then proceeds to the motor axon. Studies have identified chondrolectin as a downstream gene adversely affected by the Smn deficiency.

Chondrolectin is mis-spliced pre-symptomatically in mouse models of SMA. The protein is required for motor axon outgrowth in zebrafish, and over-expression of chondrolectin partially rescues smn knockdown. In this project, I generated a CRISPR/Cas9 knockout of chondrolectin in the zebrafish.

The mutant is homozygous viable and has no gross morphological defects, but the primary motor axons phenocopy previously published morpholino knockdown. The axons are stalled at a choice point called the horizontal myoseptum, which is known to be rich in ECM proteins such as collagens. Using both acute manipulation and a stable transgenic line, I demonstrate that only full-length chondrolectin is able to rescue the axon length, and loss of any of its protein domains leaves the protein non-functional.

From this, we hypothesise a mechanism of action for this protein where it binds an ECM molecule on the axon surface and transduces a signal via phosphorylation of its intracellular domain. We suggest the binding partner for chondrolectin to be collagenXIXa1.

SMA has been linked to synaptic defects, therefore, I analysed the synaptic compartments of the axons in the chondrolectin mutant at embryonic and larval stages. At embryonic stages, the pre-synaptic compartment is enlarged around the horizontal myoseptum in the mutant compared to wild-types, and fully rescued in a stable chondrolectin-FLAG line. When the secondary motor neurons have also extended axons to innervate the myotome, there are fewer synaptic puncta in the mutant compared to wild-type. This synapse phenotype is partially rescued in the stable transgenic chondrolectin-FLAG line.

Due to the stereotypical phenotype of the chondrolectin mutant, and its relationship with SMA, we developed a drug screening protocol to discover molecules that improve axon growth. We identified two hits after screening a 40-compound library.

Developing this protocol allows refinement of potentially useful compounds from larger libraries to use in other models of SMA.

Overall, this project offers new insight into the mechanism and function of chondrolectin, a gene linked to SMA. Novel findings include the domain analysis demonstrating that all protein domains are required for its function in zebrafish. We also found that loss of chondrolectin leads to synaptic defects, with embryonic synapse puncta enlarged in the mutant, and a loss of synaptic puncta in the larval stage. Finally, we have demonstrated potential translational uses for this mutant by designing a protocol to identify axon growth-enhancing compounds.