Zebrafish survival motor neuron mutants exhibit presynaptic neuromuscular junction defects

Abstract

Spinal muscular atrophy (SMA), a recessive genetic disease, affects lower motoneurons leading to denervation, atrophy, paralysis and in severe cases death. Reduced levels of survival motor neuron (SMN) protein cause SMA. As a first step towards generating a genetic model of SMA in zebrafish, we identified three smn mutations. Two of these alleles, smnY262stop and smnL265stop, were stop mutations that resulted in exon 7 truncation, whereas the third, smnG264D, was a missense mutation corresponding to an amino acid altered in human SMA patients. Smn protein levels were low/undetectable in homozygous mutants consistent with unstable protein products. Homozygous mutants from all three alleles were smaller and survived on the basis of maternal Smn dying during the second week of larval development. Analysis of the neuromuscular system in these mutants revealed a decrease in the synaptic vesicle protein, SV2. However, two other synaptic vesicle proteins, synaptotagmin and synaptophysin were unaffected. To address whether the SV2 decrease was due specifically to Smn in motoneurons, we tested whether expressing human SMN protein exclusively in motoneurons in smn mutants could rescue the phenotype. For this, we generated a transgenic zebrafish line with human SMN driven by the motoneuron-specific zebrafish hb9 promoter and then generated smn mutant lines carrying this transgene. We found that introducing human SMN specifically into motoneurons rescued the SV2 decrease observed in smn mutants. Our analysis indicates the requirement for Smn in motoneurons to maintain SV2 in presynaptic terminals indicating that Smn, either directly or indirectly, plays a role in presynaptic integrity.

Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish

“…The antibody is widely used in zebrafish (for example, Jing et al, 2009;Jonz and Nurse, 2003;Neill et al, 2004) and labels, for example, presynaptic terminals of motor axons, as determined by labeling of the postsynapse with bungarotoxin. When synapse formation is compromised in mutant zebrafish, SV2 labeling is absent (Boon et al, 2009), supporting specific labeling of the antibody. In agreement with our own observations, labeling was particular intense in ventral and dorsal horns of the spinal cord of rats using this antibody (Buckley and Kelly, 1985).…”

Spinal Muscular Atrophy: A Clinical and Research Update

“…Patients with spinal muscular atrophy also appear to manifest structural and physiologic abnormalities of the neuromuscular junction [72][73][74][75][76]. Kong et al revealed that in mice severely affected with spinal muscular atrophy, before anterior horn cell death or axonal degeneration, synaptic dysfunction occurred at the neuromuscular junction, with decreased synaptic vesicle density at motor terminals, reduced quantal content, slowed maturation of the acetylcholine receptor, and prolonged retention of fetal characteristics [74].…”

Deficiency in the mRNA export mediator Gle1 impairs Schwann cell development in the zebrafish embryo

“…T h e z e b r a f i s h h a s b e e n e x t e n s i v e l y u s e d a s a m o d e l organism for neurodegenerative disorders (for review see (Kabashi et al, 2010, Xi et al, 2011), including models of motor neuron diseases such as SMA (Boon et al, 2009) and ALS (Ramesh et al, 2010). A zebrafish model of LCCS1 has recently been reported, with gle1 -/-embryos showing multiple defects, including immobility, small eyes, pharyngeal arch defects, CNS cell death, a moderate reduction in motor neuron numbers and motor axon outgrowth defects (Jao et al, 2012).…”