Characterization of a Human Point Mutation of VGLUT3 (p.A211V) in the Rodent Brain Suggests a Nonuniform Distribution of the Transporter in Synaptic Vesicles.
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.SIGNIFICANCE STATEMENTVGLUT3 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.
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Lauriane
L
Ramet
Neurocure NWFZ, Charite Universitaetsmedizin, 10117 Berlin, Germany.
Johannes
J
Zimmermann
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Tiphaine
T
Bersot
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Odile
O
Poirel
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Stéphanie
S
De Gois
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Katlin
K
Silm
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Diana Yae
DY
Sakae
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Nina
N
Mansouri-Guilani
Departments of Pharmacology and Neurosciences, Faculty of Medicine, Groupe de Recherche sur le Systéme Nerveux Central (GRSNC), Université de Montréal, Montréal, QC H3T 1J4, Quebec, Canada.
Marie-Josée
MJ
Bourque
Departments of Pharmacology and Neurosciences, Faculty of Medicine, Groupe de Recherche sur le Systéme Nerveux Central (GRSNC), Université de Montréal, Montréal, QC H3T 1J4, Quebec, Canada.
Louis-Eric
0000-0003-4684-1377
LE
Trudeau
Université Paris Descartes, Sorbonne Paris Cité, CNRS, UMR 8601, 75006 Paris, France, and.
Nicolas
0000-0002-9934-7818
N
Pietrancosta
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Stéphanie
S
Daumas
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France.
Véronique
V
Bernard
Neurocure NWFZ, Charite Universitaetsmedizin, 10117 Berlin, Germany.
Christian
0000-0002-3905-2444
C
Rosenmund
Centre National de la Recherche Scientifique (CNRS), UMR 8246, Institut National de la Santé et de la Recherche Médicale, UMR-S 1130, Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Institut de Biologie Paris-Seine, UM119 Neuroscience Paris Seine, F-75005 Paris, France, salah.el_mestikawy@upmc.fr.|Douglas Hospital Research Center, Department of Psychiatry, McGill University, Montréal, QC H4H 1R3 Quebec, Canada.
Salah
S
El Mestikawy
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017354:Point Mutation / Q000502:physiology*; D011897:Random Allocation; D013572:Synaptic Vesicles / Q000235:genetics* / Q000378:metabolism*; D050496:Vesicular Glutamate Transport Proteins / Q000235:genetics* / Q000378:metabolism*
C460879:SLC17A8 protein, human; D050496:Vesicular Glutamate Transport Proteins
DFNA25; STED; mutant mice VGLUT3A224V/A224V; point mutation (p.A211V); synaptic vesicles; vesicular glutamate transporter 3 (VGLUT3)