A de novo missense mutation in synaptotagmin-1 associated with neurodevelopmental disorder desynchronizes neurotransmitter release

Abstract

Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1PL-expressing neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous neurotransmitter release per synapse was increased more than 500% in Syt1PL-expressing neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential induced asynchronous release was increased more than 100%, while synchronous release was not changed. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1PL was expressed in wild type neurons. Our findings show that Syt1PL desynchronizes neurotransmission by reducing the suppression spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increase in release. Syt1PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1’s primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other Syt1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1-associated neurodevelopmental disorder.

Delineation of the pathogenic presynaptic mechanisms of synaptotagmin-1 variants

“…For example, phenotypic heterogeneity within another disorder of synaptic vesicle fusion has been linked to distinct mechanistic aetiologies, where variants in SNAP25 differentially impacted properties of evoked and spontaneous neurotransmitter release (Alten et al, 2021). Together with previous work on Ca 2+ -binding loop variants in SYT1 (Baker et al, 2015;Baker et al, 2018;Bradberry et al, 2020), one recent study of a P401L (human) variant in SYT1 (Cornelisse et al, 2023) supports that such mechanistic heterogeneity may also be present within SYT1-associated neurodevelopmental disorder. Nevertheless, current evidence from four extensively characterised, pathogenic SYT1 variants suggests that impairments in evoked exocytosis is a central presynaptic pathological feature of SYT1associated neurodevelopmental disorder.…”

“…Interestingly, a neurodevelopmental disorder-associated SYT1 variant, P401L (equating to P400L in mouse/rat sequence), was recently demonstrated to increase spontaneous miniature excitatory postsynaptic currents in a dominant-negative manner in autaptic murine neurons (Cornelisse et al, 2023). This variant is notably situated on the opposite side of the C2B domain from the Ca 2+ -binding loop, lying adjacent to the functionally important arginine apex.…”

“…A SYT1 triple mutant ("3DA": D309A/D363A/D365A) showed a dominant-negative increase in spontaneous release when expressed in mammalian cultured neurons (Zhou et al, 2017); however, this highly deleterious triple mutant is expected to abolish Ca 2+ binding to the C2B domain and is not reflective of known pathogenic variants (Shin et al, 2009). Interestingly, a neurodevelopmental disorder-associated SYT1 variant, P401L (equating to P400L in mouse/rat sequence), was recently demonstrated to increase spontaneous miniature excitatory postsynaptic currents in a dominant-negative manner in autaptic murine neurons (Cornelisse et al, 2023). This variant is notably situated on the opposite side of the C2B domain from the Ca 2+ -binding loop, lying adjacent to the functionally important arginine apex.…”

“…Synaptotagmin-1 (SYT1) is an essential synaptic vesicle protein that acts as the major Ca 2+ -sensor mediating fast, synchronous neurotransmitter release in the mammalian cerebrum (Bowers and Reist, 2020; Geppert et al, 1994; Marqueze et al, 1995; Maximov and Sudhof, 2005; Rizo, 2022; Ullrich and Sudhof, 1995; Xu et al, 2007). Heterozygous variants in SYT1 give rise to a neurodevelopmental disorder (MIM 618218; Baker-Gordon Syndrome or SYT1 -associated neurodevelopmental disorder) with 16 published de novo variants linked to the disorder to date (Cornelisse et al, 2023; Melland et al, 2022). Affected individuals present with a spectrum of features including motor delay, intellectual disability, behavioural disturbances, and movement disorders, with abnormal electroencephalograms being an additional hallmark (Baker et al, 2018; Melland et al, 2022).…”

Adaptive functions correlate with evoked neurotransmitter release in SYT1-associated neurodevelopmental disorder

“…Recently, additional disorder-associated SYT1 variants have been identified, both within the C2B domain (including outside of the calcium-binding pocket) and within the C2A domain of the protein (Melland et al, 2022; Huang et al, 2023; Cornelisse et al, 2023). Molecular dynamics simulations demonstrated a lack of convincing evidence of a major structural component to the mechanisms of pathogenicity for most of these variants (Melland et al, 2022), and a pathogenic mechanism for these novel variants is yet to be established.…”