Identification of a human synaptotagmin-1 mutation that perturbs synaptic vesicle cycling
Kate Baker, … , Michael A. Cousin, F. Lucy Raymond
Kate Baker, … , Michael A. Cousin, F. Lucy Raymond
Published February 23, 2015
Citation Information: J Clin Invest. 2015;125(4):1670-1678. https://doi.org/10.1172/JCI79765.
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Research Article Neuroscience

Identification of a human synaptotagmin-1 mutation that perturbs synaptic vesicle cycling

Abstract

Synaptotagmin-1 (SYT1) is a calcium-binding synaptic vesicle protein that is required for both exocytosis and endocytosis. Here, we describe a human condition associated with a rare variant in SYT1. The individual harboring this variant presented with an early onset dyskinetic movement disorder, severe motor delay, and profound cognitive impairment. Structural MRI was normal, but EEG showed extensive neurophysiological disturbances that included the unusual features of low-frequency oscillatory bursts and enhanced paired-pulse depression of visual evoked potentials. Trio analysis of whole-exome sequence identified a de novo SYT1 missense variant (I368T). Expression of rat SYT1 containing the equivalent human variant in WT mouse primary hippocampal cultures revealed that the mutant form of SYT1 correctly localizes to nerve terminals and is expressed at levels that are approximately equal to levels of endogenous WT protein. The presence of the mutant SYT1 slowed synaptic vesicle fusion kinetics, a finding that agrees with the previously demonstrated role for I368 in calcium-dependent membrane penetration. Expression of the I368T variant also altered the kinetics of synaptic vesicle endocytosis. Together, the clinical features, electrophysiological phenotype, and in vitro neuronal phenotype associated with this dominant negative SYT1 mutation highlight presynaptic mechanisms that mediate human motor control and cognitive development.

Authors

Kate Baker, Sarah L. Gordon, Detelina Grozeva, Margriet van Kogelenberg, Nicola Y. Roberts, Michael Pike, Edward Blair, Matthew E. Hurles, W. Kling Chong, Torsten Baldeweg, Manju A. Kurian, Stewart G. Boyd, Michael A. Cousin, F. Lucy Raymond

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Figure 4

Effect of I368T mutation on SYT1 localization and activity-dependent trafficking.

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Effect of I368T mutation on SYT1 localization and activity-dependent tra...
(A and B) Hippocampal neurons were transfected with either SYT1WT-pHluorin or SYT1I368T-pHluorin. Representative images show a similar punctate distribution of SYT1WT-pHluorin (WT, A) and SYT1I368T-pHluorin (I368T, B) along axonal segments after exposure to alkaline imaging buffer. Image is displayed in false color, with warmer colors indicating more fluorescence intensity. Arrows represent the localization of both pHluorin reporters at nerve terminals. Scale bar: 1 μm. (C and D) Transfected neurons were stimulated with a train of 1,200 action potentials at 10 Hz in the presence of 1 μM bafilomycin to prevent vesicle reacidification and subsequently exposed to NH4Cl to reveal total pHluorin fluorescence (n = 8 WT, n = 6 I368T). (C) Time course of mean ΔF/F0 of SYT1-pHluorin ± SEM, normalized to NH4Cl. *P < 0.05, over time indicated (2-way ANOVA). (D) Exocytosis time constant (τ) determined by fitting a single-phase association curve to the data presented in C. Data are presented as mean ± SEM (n = 8 WT and n = 6 I368T). *P < 0.03 (t test). (E) Neurons were stimulated with a train of 300 action potentials at 10 Hz. Graph displays the time course of mean ΔF/F0 of SYT1-pHluorin ± SEM, normalized to the peak of stimulation (n = 7 WT, n = 6 I368T). *P < 0.05, over time indicated (2-way ANOVA). (F) Surface SYT1-pHluorin fluorescence, determined by normalizing SYT1-pHluorin fluorescence to 100% in alkaline buffer and 0% in acidic buffer. Data are presented as mean ± SEM. n = 4. P = 0.86 (t test).