Drosophila studies support a role for a presynaptic synaptotagmin mutation in a human congenital myasthenic syndrome.

During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology

The role of the C2A domain of synaptotagmin 1 in asynchronous neurotransmitter release.

Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo

Data from: Drosophila studies support a role for a presynaptic synaptotagmin mutation in a human congenital myasthenic syndrome

During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology

Data from: Drosophila studies support a role for a presynaptic synaptotagmin mutation in a human congenital myasthenic syndrome

During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology

Transgenic synaptotagmin is expressed at appropriate levels and exhibits appropriate localization.

<p>(A-C) Representative western blots above and average synaptotagmin expression normalized to actin levels below. <i>Syt</i> heterozygotes expressing one copy of the mutant transgene (<i>+/-;P[syt^P-L]/+</i>, n = 6, 5, and 7, respectively) are compared to <i>syt</i> heterozygotes (<i>+/-</i>, n = 5, A), <i>syt</i> homozygotes (<i>+/+</i>, n = 5, B), and <i>syt</i> heterozygotes expressing one copy of the wild type transgene (<i>+/-; P[syt^WT]/+</i>, n = 8, C). The <i>P[syt^P-L]</i> heterozygotes had approximately twice as much synaptotagmin expression as <i>syt</i> heterozygotes (p = 0.0008, student t-test), while there was no significant difference in expression between the other lines compared (Fig 2B, p = 0.69 and Fig 2C, p = 0.25). Error bars depict SEM. (D, E) Third instar larvae stained with antibodies against horseradish peroxidase (HRP) as a general axonal stain (red) and synaptotagmin (green). Scale bars represent 10 μm. (F, G) First instar larvae stained with antibodies against synaptotagmin. Scale bars represent 20 μm.</p

Predicted conformational changes in synaptotagmin’s C₂B Ca²⁺-binding pocket.

<p>(A) Molecular model depicting the crystal structure of the synaptotagmin C₂B Ca²⁺-binding pocket. Flexible loops 1, 2, and 3 are indicated. The proline residue under investigation (P) and the adjacent, Ca²⁺-coordinating aspartate (D2) are shown as stick models. Note that the proline is at one end of β-strand 3 (small arrow) and the aspartate is in loop 1. (B) Predicted structural changes induced by the syt^P-L substitution. The leucine (L) and the adjacent aspartate (D2) residues are shown as stick models and both are now within β-strand 3 (small arrow indicates extension of β-strand 3). The large arrow indicates a newly-formed β-strand. (C) Overlay of wild type (yellow) and syt^P-L mutant (turquoise). The curved arrow indicates the displacement of the D2 Ca²⁺-binding residue. (D) Sequence alignment of the highly conserved SDPYVK amino acid motif of the synaptotagmin C₂B domain from humans, rat, zebra fish, squid, <i>Drosophila</i>, and <i>C</i>. <i>elegans</i>. * locations of the point mutations in the US and UK families, respectively. Note: <i>syt1</i> and <i>syt2</i> in mammals are identical in this region.</p

Synchronous evoked release is impaired in <i>P[syt^P-L]</i> heterozygotes, but quantal content is unchanged.

<p>(A) Representative EJP traces for control and <i>P[syt^P-L]</i> heterozygotes. Scale bars represent 10 mV, 0.2 s. (B) Mean EJP amplitude is significantly less in <i>P[syt^P-L]</i> heterozygotes (n = 19) compared to control (n = 12, *p = 0.002). Error bars depict SEM. (C) Representative consecutive 3 second mEJP traces for control and <i>P[syt^P-L]</i> heterozygotes. Scale bars represent 1 mV, 0.2 s. (D, E) Neither mean mEJP amplitude (p = 0.09, Wilcoxon Rank-Sum Test, D) nor mean mEJP frequency (p = 0.18, student t-test, E) is significantly different between <i>P[syt^P-L]</i> heterozygotes (n = 12 fibers) and control (n = 19 fibers). Error bars depict SEM.</p

The <i>syt^P-L</i> mutation increases the rate of fatigue in <i>Drosophila</i>.

<p>(A, B) Normalized values of distance traveled up a vial wall after repetitive drops over time for 5–7 day old females (A) and males (B). Each point is a binned 1 minute average of 6 drops (1 drop every 10 seconds) normalized to the percentage of the distance traveled after the first drop. Statistical analysis indicates different rates of fatigue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184817#sec011" target="_blank">methods</a>) between the two genotypes for both females (p < 0.0001) and males (p < 0.0001), indicating the <i>P[syt^P-L]</i> heterozygotes (n = 10) fatigued significantly faster than controls (n = 10).</p

<i>P[syt^P-L]</i> heterozygotes exhibit less depression throughout the course of high frequency stimulation, but fail to maintain this relatively increased response upon cessation of stimulation.

<p>(A) Representative traces from control and <i>P[syt^P-L]</i> heterozygotes during a 2 second, 10 Hz stimulation train, and a single stimulus 1 minute following cessation of the train. Dotted line represents the amplitude of the initial response. Scale bar represents 10 mV, 0.2 s. (B) Mean EJP amplitudes of <i>P[syt^P-L]</i> heterozygotes (n = 17) showed less depression compared to control (n = 14, *indicates p < 0.05, and **indicates p < 0.001). However, this relative increase in release is not maintained upon cessation of the stimulus train (p = 0.87). Error bars depict SEM.</p

The <i>syt^P-L</i> mutation affects locomotor activity in <i>Drosophila</i>.

<p>(A, B) Average activity per 30 minutes for an averaged 24-hour day in a 6-day <i>Drosophila</i> Activity Monitoring Assay in 5–7 day old females (A) and males (B). (C, D) Distinct activity periods (AP) were averaged for females (C, n = 48 for each genotype) and males (D, n = 48 for <i>+/-;P[syt^P-L]/+</i>, n = 45 for control). <i>P[syt^P-L]</i> heterozygotes were significantly less active than controls during all activity periods in both males and females (* indicates p < 0.001, student t-tests). Error bars depict SEM.</p