The atypical vesicular glutamate transporter type 3 (VGLUT3) is expressed by sub-populations 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 central nervous system (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 knockout mice. Finally, we used stimulated emission depletion microscopy (STED) 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 non-uniform 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 (approximately 70%) without altering its function. Furthermore, using stimulated emission depletion (STED) 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.
The authors declare no competing financial interests.
This research was supported by funds from the Fondation pour la Recherche médicale (Équipe FRM DEQ20130326486), the Agence Nationale pour la Recherche (ANR), the Fédération pour la Recherche sur le Cerveau, Labex (Bio-Psy Laboratory of Excellence), INSERM, CNRS and UPMC.; LR received a PhD fellowship from the Ministère de l'enseignement supérieur et de la recherche and from the Fondation pour la Recherche médicale (FDT20140930909).; We thank Géraldine Toutirais from the Service de Microscopie Electronique of the Institut de Biologie Paris-Seine (Université Pierre et Marie Curie, Paris). We thank Valérie Nicolas from the Plateforme d'imagerie cellulaire of the Institut Paris Saclay d'Innovation Thérapeutique (UMS IPSIT Université Paris-Sud — US 31 INSERM — UMS 3679 CNRS, Châtenay-Malabry) for assistance with STED microscopy. We thank Christoph Biesemann for the lentivirus constructs, Stéphanie Pons and Martine Soudant as well as l'École des Neurosciences de Paris (ENP) (“Network for Viral Transfer”) for producing the lentiviruses.