Delineation of the pathogenic presynaptic mechanisms of synaptotagmin-1 variants

Abstract

Missense variants in the presynaptic calcium sensor synaptotagmin-1 (SYT1) are associated with an autosomal dominant neurodevelopmental disorder (NDD). These cause dominant-negative impairment of evoked neurotransmitter release in a mutation-specific manner; however, whether NDD-associated variants also perturb the auxiliary functions of SYT1 remains unknown. We investigated whether the expression of SYT1 variants in cultured hippocampal neurons altered either action potential-independent spontaneous synaptic vesicle exocytosis or synaptic vesicle endocytosis. SYT1 variants did not induce dominant-negative impairment of either process, confirming that defective evoked exocytosis is the major pathogenic mechanism of SYT1-associated NDD. To examine the differential impacts of human variants on evoked exocytosis, both NDD-associated and strategic mutations were used to explore the functional importance of two distinct Ca2+-binding residues in the C2B domain. We show that both the nature of the amino acid change and the specific residues targeted determine the severity of exocytic disturbance. Together, this work informs understanding of SYT1 function and further clarifies potential mechanistic targets for treating SYT1-associated NDD.

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

“…We also explored alternative mechanisms of pathogenicity beyond dominant-negative effects, by examining how SY1 variants impact protein expression and transport. In keeping with observations from C2B variants studied to date (Baker et al, 2015; Baker et al, 2018; Melland et al, 2023), most of the investigated SYT1 variants across the C2A and C2B domains had no impact on nerve terminal expression or trafficking of SYT1, indicating that disruption to protein stability and expression are not common consequences of SYT1 missense variants.…”

“…previously investigated SYT1 C2B variants slow evoked exocytosis without having dominantnegative impacts on the additional functions of SYT1 as a clamp for spontaneous neurotransmitter release and a modulator of endocytosis (Melland et al, 2023). It therefore remains possible that the newly-identified SYT1 variants may additionally impact these other presynaptic processes.…”

“…For example, it would be important to explore whether a recently identified C2A variant (V184A) identified in an individual with a severe, early-lethal neurodevelopmental disorder (Huang et al, 2023), causes severe impairment to evoked neurotransmitter release. Moreover, we have only assessed the impacts of these variants on evoked exocytosis; previously investigated SYT1 C2B variants slow evoked exocytosis without having dominant-negative impacts on the additional functions of SYT1 as a clamp for spontaneous neurotransmitter release and a modulator of endocytosis (Melland et al, 2023). It therefore remains possible that the newly-identified SYT1 variants may additionally impact these other presynaptic processes.…”

“…Secondly, we interrogated whether the observed functional impacts contribute to variable severity of the associated neurodevelopmental condition. The recurrent C2B variant I368T has well-characterised functional impacts, and hence was included as a reference variant (Baker et al, 2015; Baker et al, 2018; Melland et al, 2023). We reveal here a shared mechanism of pathogenicity that correlates with core neurodevelopmental features of SYT1-associated neurodevelopmental disorder.…”