Molecular Components of the Hair Cell Synaptic Vesicle Cycle

Hair cells (HCs) of the zebrafish inner ear act as sensory receptors for both auditory and vestibular stimuli, and have glutamatergic ribbon synapses.We have identified and positionally cloned two mutants that interfere with HC synaptic activity from an ENU mutagenesis screen for vestibular dysfunction in zebrafish (Nicolson et al., 1998 and unpublished results). We find that the comet gene encodes the lipid phosphatase synaptojanin 1 (synj1). comet mutant larvae display a balance defect that increases in severity when challenged, suggesting fatigability, perhaps due to insufficient SV recycling.We have sequenced three alleles of comet/synj1, all of which encode severe truncations that presumably lead to functional null phenotypes. We confirm the expression of synj1 in the CNS and, in addition, show expression in HCs of the ear by in situ hybridization. Morpholino-mediated suppression of the short splice variant of synj1, synj1-145 inefficiently generates a balance defect that is dissimilar to that of the comet mutant. Morpholino knockdown of Synj1 produces a phenocopy. At the electron microscopy level, comet/synj1 mutants show a decrease in synaptic ribbon diameter that accompanies a reduced number of ribbon-associated synaptic vesicles. This can be interpreted as evidence for altered release kinetics and ribbon maintenance in comet mutants. We also describe the completely novel phenotype for loss of synj1, basal membrane blebbing. Basal blebbing is stimulation-dependent in comet/synj1 mutants and the absence of synaptic exocytosis in comet/gemini double mutants that lack the synaptic Ca2+ channel cav1.3 (Sidi et al., 2004) abolishes blebbing. In addition, interference with endocytosis by exposing larvae to Latrunculin A phenocopies blebbing in wild-type HCs. Furthermore, we have identified vesicular glutamate transporter 3 (vglut3) as critical for HC function from the asteroid mutant strain. asteroid mutant larvae are deaf and display a profound balance defect, while hair-bundle morphology and FM 1-43 dye uptake appear normal. This phenotype suggests a transmission failure downstream of mechanotransduction. By inducing an early frameshift, the genomic lesion of the asteroid mutant results in a protein null phenotype. We can replicate this phenotype by morpholino-mediated knockdown of vglut3 in the wild-type. Unlike in mammals, vglut3 appears to be exclusively expressed in HCs of the ear and lateral line organ in the zebrafish. Restriction of Vglut3 to HCs and absence in the asteroid mutant were confirmed by immunohistochemistry. asteroid mutants show a 60% decrease in the number of ribbon-associated synaptic vesicles at the ultrastructural level, while ribbon diameters are comparable to the wild-type. This indicates a role for Vglut3 in synaptic vesicle biogenesis and/or trafficking, but Vglut3 is not the sole component required for these processes. Using in situ hybridization, we also detect vglut1 transcript in HCs, but the presence of vglut1 does not compensate for loss of vglut3 under the conditions tested, and qPCR data indicates that neither vglut1 nor vglut3 are significantly upregulated in asteroid/vglut3 mutants. In conclusion, we have identified, cloned and characterized two “novel” genes that are required for proper HC synaptic transmission. Both synj1 and vglut3 are involved in SV generation and recycling and their investigation should aid further elucidation of these mechanisms in future studies