Characterization of a Human Point Mutation of VGLUT3 (p.A211V) in the Rodent Brain Suggests a Nonuniform Distribution of the Transporter in Synaptic Vesicles

Abstract

The atypical vesicular glutamate transporter type 3 (VGLUT3) is expressed by subpopulations of neurons using acetylcholine, GABA, or serotonin as neurotransmitters. In addition, VGLUT3 is expressed in the inner hair cells of the auditory system. A mutation (p.A211V) in the gene that encodes VGLUT3 is responsible for progressive deafness in two unrelated families. In this study, we investigated the consequences of the p.A211V mutation in cell cultures and in the CNS of a mutant mouse. The mutation substantially decreased VGLUT3 expression (-70%). We measured VGLUT3-p.A211V activity by vesicular uptake in BON cells, electrophysiological recording of isolated neurons, and its ability to stimulate serotonergic accumulation in cortical synaptic vesicles. Despite a marked loss of expression, the activity of the mutated isoform was only minimally altered. Furthermore, mutant mice displayed none of the behavioral alterations that have previously been reported in VGLUT3 knock-out mice. Finally, we used stimulated emission depletion microscopy to analyze how the mutation altered VGLUT3 distribution within the terminals of mice expressing the mutated isoform. The mutation appeared to reduce the expression of the VGLUT3 transporter by simultaneously decreasing the number of VGLUT3-positive synaptic vesicles and the amount of VGLUT3 per synapses. These observations suggested that VGLUT3 global activity is not linearly correlated with VGLUT3 expression. Furthermore, our data unraveled a nonuniform distribution of VGLUT3 in synaptic vesicles. Identifying the mechanisms responsible for this complex vesicular sorting will be critical to understand VGLUT's involvement in normal and pathological conditions. VGLUT3 is an atypical member of the vesicular glutamate transporter family. A point mutation of VGLUT3 (VGLUT3-p.A211V) responsible for a progressive loss of hearing has been identified in humans. We observed that this mutation dramatically reduces VGLUT3 expression in terminals (∼70%) without altering its function. Furthermore, using stimulated emission depletion microscopy, we found that reducing the expression levels of VGLUT3 diminished the number of VGLUT3-positive vesicles at synapses. These unexpected findings challenge the vision of a uniform distribution of synaptic vesicles at synapses. Therefore, the overall activity of VGLUT3 is not proportional to the level of VGLUT3 expression. These data will be key in interpreting the role of VGLUTs in human pathologies.

VGLUT3-p.A211V variant fuses stereocilia bundle and elongates synaptic ribbons in the human deafness DFNA25

“…2 C). Therefore, in contrast to what is observed in the central nervous system neurons (Ramet et al, 2017), the p.A22AV variant does not disturb the VGLUT3 expression and targeting within the hair cells cytoplasm. Alteration at the stereociliary bundle in VGLUT3 A224V/A224V mouse.…”

“…A mouse line expressing the p.A224V mutation was generated at Phenomin-Institut Clinique de la Souris Illkirch, France; http://www. phenomin.fr/ and was named VGLUT3 A224V/A224V as previously reported (Ramet et al, 2017). A point mutation was introduced in exon 5 of the mouse Slc17a8 gene: a GCG (coding for an alanine) was exchanged for a GTG (coding for a valine).…”

“…Genotyping. Genotypes of mice was determine by Polymerase Chain Reaction (PCR) analysis of genomic DNA using FastStart PCR Master Mix (Roche Applied Science, Penzberg, Germany) as previously described (Ramet et al, 2017). In brief, tail or toe biopsies were digested overnight at 55°C in 300 µl of lysis buffer (containing in mM: 100 Tris-HCL pH 8.5; 5 EDTA; 0.2% SDS; 200 NaCl; pH : 8.5) with 100µg/ml of proteinase K (Promega, Madison, WI, USA).…”

“…The variant strongly decreases (-70%) the expression of VGLUT3-p.A224V in the brain (Ramet et al, 2017). To determine whether a defect of the VGLUT3 expression might contribute to the threshold shift, we examined the distribution of VGLUT3 in the IHCs from VGLUT3 A224V/A224V mouse.…”

“…The rare variant VGLUT3-p.A211V was identified as the cause of DFNA25 (Ruel et al, 2008). This variation does not impair the glutamate vesicular accumulation, neither the quantal release nor the quantal content in hippocampal autaptic neurons (Ramet et al, 2017). In addition, investigation of a mouse line harboring the p.A221V allele in human (p.A224V in mouse, VGLUT3 A224V/A224V mouse) showed that this variant resulted to a strong decrease of VGLUT3 expression (≈ -70%) in the brain without altering VGLUT3-piloted behavior (Ramet et al, 2017).Thus, it is not so clear whether and how the VGLUT3-p.A211V variant results in the human auditory deficit, DFNA25.…”