Ramet Lauriane, Zimmermann Johannes, Bersot Tiphaine, Poirel Odile, De Gois Stéphanie, Silm Katlin, Sakae Diana Yae, Mansouri-Guilani Nina, Bourque Marie-Josée, Trudeau Louis-Eric, Pietrancosta Nicolas, Daumas Stéphanie, Bernard Véronique, Rosenmund Christian, El Mestikawy Salah
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.
Neurocure NWFZ, Charite Universitaetsmedizin, 10117 Berlin, Germany.
J Neurosci. 2017 Apr 12;37(15):4181-4199. doi: 10.1523/JNEUROSCI.0282-16.2017. Epub 2017 Mar 17.
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.
非典型囊泡谷氨酸转运体3（VGLUT3）由以乙酰胆碱、γ-氨基丁酸或5-羟色胺作为神经递质的神经元亚群表达。此外，VGLUT3在听觉系统的内毛细胞中表达。编码VGLUT3的基因中的一个突变（p.A211V）导致两个不相关家族出现进行性耳聋。在本研究中，我们在细胞培养物和突变小鼠的中枢神经系统中研究了p.A211V突变的后果。该突变显著降低了VGLUT3的表达（-70%）。我们通过BON细胞中的囊泡摄取、分离神经元的电生理记录以及其刺激皮质突触小泡中5-羟色胺积累的能力来测量VGLUT3-p.A211V的活性。尽管表达明显丧失，但突变异构体的活性仅略有改变。此外，突变小鼠没有表现出先前在VGLUT3基因敲除小鼠中报道的任何行为改变。最后，我们使用受激发射损耗显微镜来分析该突变如何改变表达突变异构体的小鼠终末内VGLUT3的分布。该突变似乎通过同时减少VGLUT3阳性突触小泡的数量和每个突触处VGLUT3的量来降低VGLUT3转运体的表达。这些观察结果表明，VGLUT3的整体活性与VGLUT3的表达并非线性相关。此外，我们的数据揭示了VGLUT3在突触小泡中的分布不均匀。确定负责这种复杂囊泡分选的机制对于理解VGLUT在正常和病理状态中的作用至关重要。VGLUT3是囊泡谷氨酸转运体家族的一个非典型成员。在人类中已鉴定出一个导致听力逐渐丧失的VGLUT3点突变（VGLUT3-p.A211V）。我们观察到该突变显著降低了终末中VGLUT3的表达（约70%），而不改变其功能。此外，使用受激发射损耗显微镜，我们发现降低VGLUT3的表达水平会减少突触处VGLUT3阳性小泡的数量。这些意外发现挑战了突触处突触小泡均匀分布的观点。因此，VGLUT3的整体活性与VGLUT3的表达水平不成正比。这些数据对于解释VGLUT在人类病理学中的作用至关重要。
