1. Synaptotagmin Isoforms and SYT1 Function

Synaptic neurotransmission is the fundamental communication between neurons. At the level of synapse, a transmitter is released from one neuron generating a signal on another neuron or target cell that can excites, inhibits, or modulates its cellular activity ^[1]. The action potential reaching the nerve terminal initiates a rapid influx of Ca²⁺ions into the postsynaptic cell, causing fusion of vesicles and ultimately the release of neurotransmitters from those vesicles (Figure 1). This release is mediated via the assembly and zippering of SNARE (soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) complexes that allow the destined membranes to fuse ^[2]. SNARE complexes in neurons have three components, synaptobrevin or vesicle-associated membrane protein (VAMP), which is the v-SNARE, and two plasma-membrane proteins syntaxin and SNAP-25 (synaptosome-associated protein, 25 kDA) that are the t-SNARES. Once these two membranes combine, they form four-helix bundle aligned in a parallel fashion called the trans-SNARE complex ^[3]. This machinery is essential for vesicle docking and priming at active zones. Previous  studies have indicated the role of synaptotagmin-1 (SYT1)SYT1 as a dual Ca²⁺sensor able to carry out both endocytosis and exocytosis independently of one another ^[4]. These processes are carried out by SYT1’s C2A and C2B domain in a Ca²⁺dependent manner. Synaptotagmins and SNARE proteins play a pivotal role in evoked synchronous neurotransmitter release in a Ca²⁺ dependent and independent manner. For the purposes of these contents, the SYT isoforms of clinical and functional relevance related to SYT1-associated neurodevelopmental disorder will be discussing (Table 1). These isoforms include SYT2, SYT7, and SYT9 ^[5][6]. In the forebrain specifically, SYT2 seems to be concentrated in inhibitory neurons, whereas SYT1 dominates excitatory neurons ^[7]. Interestingly, it has been discovered that KO of SYT2 impairs Ca²⁺ induced vesicle release and produces severe motor deficits in adolescent mice ^[7]. In clinical studies, mutations in SYT2 have been reported in the C2B binding domain, similar to SYT1-associated neurodevelopmental disorder, resulting in motor deficits including muscle weakness, bulbar deficits, and delay in motor developmental that ranges from mild to moderate to severe depending on the mutation ^[8]. SYT9 has the slowest neurotransmitters (NT) release kinetics and lowest affinity for Ca^2+ [5][9]. It is concentrated in dense core vesicles and synaptic vesicles within the limbic system and striatum ^[7][9]. SYT9 is the major isoform responsible for fast-acting synaptic vesicle exocytosis in these neurons ^[7][9]. In addition to striatal neurons, SYT9 has also been found in cortical and hippocampal neurons It has been demonstrated in previous labs that all three of these neuron classes are regulated by SYT1 and SYT9 but have shown that loss of SYT1 inhibited evoked rapid NT release while the loss of SYT9 had no discernable effect ^[5][10]. Interestingly, it has been shown that SYT9 can rescue the loss of SYT1 but only when immensely overexpressed ^[5][7]. Unlike SYT1, C2A is the main Ca²⁺sensing domain in SYT9 ^[5][11] and mutations within this domain disrupts the ability for SYT9 to initiate membrane fusion in the presence of AP-driven Ca²⁺influx ^[5]. It has been found that SYT7 has redundant function with SYT1 and is capable of SV priming and clamping. It is a high affinity Ca²⁺sensor for slow asynchronous neurotransmitter release ^[6][10] and takes part in Ca²⁺dependent short term synaptic facilitation, SV endocytosis and is responsible for maintaining the RRP of vesicles ^[10][12]. Simultaneous loss of function of SYT1 and SYT7 demonstrated a decrease in RRP size in both excitatory and inhibitory synapses, whereas loss of function in only one or the other did not show any effect on RRP size ^[10]. Loss of function of both SYT1 and SYT7 does not seem to alter the rate of vesicle priming into RRP ^[10]. Due to redundancy in function, phenotypic loss of SYT1 can be partially compensated by SYT7, albeit in immature neurons ^[6]. SYT1 is responsible for Ca²⁺dependent and independent fast synchronous NT release ^[13] and plays a role in clamping mini spontaneous release and in the maintenance of readily releasable pools (RRP) in vesicles. SYT1 is the main isoform found to reside within synaptic vesicles and secretory granules in neurons and neuroendocrine cells located mainly in the hippocampus and forebrain ^[9]. SYT1 is a low affinity Ca²⁺sensor that binds to Ca²⁺following an action potential via the C2B domain initiating the fusion of the SNARE complex thereby providing the main mechanism for spontaneous and synchronous release ^[6][13][14]. C2B domain is the main site of mutations associated with SYT1-associated neurodevelopmental disorder. Such mutation has demonstrated decreased evoked synchronous release of NTs due to the inhibition of SV fusion ^[4][13][15]. The precise mechanism will be discussed. In exocytosis, the SYT1 protein senses the rise in intracellular Ca²⁺due to an action potential and in turn interacts with negatively charged phospholipids and SNARE proteins to promote membrane fusion ^[5]. The interaction of SYT1 with negatively charged phospholipid phosphatidylserine is crucial to exocytosis and has been highlighted by previous experiments in C2A mutations that abolished the interaction between SYT1 and phospholipids ^[16]. However, this mutation in C2A has not had the same effects on the interaction between SYT1 and SNARE proteins ^[16]. Upon Ca²⁺binding, SYT1 acts by deforming the plasma membrane or binding to the curved membrane via the C2B domain ^[13]. This interaction is found to be critical in NT release and exocytosis ^[13]. Furthermore, when testing the function of SYT1 during exocytosis it was found that C2B mutations greatly disrupted this process whereas corresponding mutations in the C2A domain did not ^[4]. The efficiency of endocytosis and exocytosis is crucial to establishing efficient neuronal transmission. SYT1 helps to establish this via a bidirectional Ca²⁺ sensing capability that links endocytosis and exocytosis ^[17]. Previous labs have recognized that SYT1 is able to initiate slow and small clathrin mediated endocytosis (CME) and/or block bulk-sized endocytosis during increased neural activity ^[17]. In contrast to the pivotal role of C2B in exocytosis, it has been demonstrated that either C2B or C2A can be involved in SYT1-mediated endocytosis ^[4]. Interestingly, endocytosis can proceed in both a Ca²⁺ dependent and independent manner ^[4]. However, Ca²⁺binding to either the C2A or C2B domain is necessary in the acceleration of endocytosis ^[4]. Experiments have shown that mutations in either C2A or C2B (but not in both simultaneously) rescued knockdown-decrease in transferrin uptake, which represented CME and further established the redundancy of C2AB in endocytosis ^[17]. It was further demonstrated that C2B is needed for bulk endocytosis ^[17].

2. Mutations in C2AB Inhibit Exocytosis

Previous studies indicate that the most consequential clinical manifestations occur with mutations within the amino acid sequence bind domain of the C2B domain ^[4][15][18]. The C2B and C2A domain are structurally similar but the C2B domain has a higher affinity for Ca²⁺-dependent membrane penetration due to the positive cooperativity between C2B and PIP2 ^[19]. This positive cooperativity promotes the oligomerization of SYT1 ^[19]. Interestingly, recent studies have shown that both C2B and C2A work in a cooperative manner to assist SYT1 membrane binding. However, C2B still remains the dominant energetic domain in these transmission interactions ^[16][19][20]. The C2B domain is composed of a series of polylysine residues region at its side that bind to PIP2 in the absence of Ca^2+ [21] and two basic residues region located at its bottom; both of which have been shown to be crucial in synchronous NT release ^[14]. The C2B domain forms a primary and secondary interface with the SNARE complex, which is critical in NT release and exocytosis ^[13]. A primary interface occurs between the C2B domain and SNARE complex via the interaction between arginine amino acids with glutamates and aspartates between SNAP25 and Syyntaxin-1 ^[5]. It has been demonstrated that mutations from arginine to glutamines in C2B show decreases in the rate of fusion between C2B and the SNARE complex, thus disrupting exocytosis ^[13]. Studies have demonstrated that the mutations in C2B domain did not affect the binding rate of Ca²⁺ to C2B, but rather the time bound decreased ^[15] and inhibition of NT release via a dominant-negative manner ^[6][19]. Mutations that neutralized the C2B domain resulted in completely abolishing evoked NT release and disrupt Ca²⁺ independent vesical docking ^[14][20]. However, a mutation in C2A resulted in only a 50% decrease in NT release ^[20]. It is thought that C2B might be the governing domain in evoked NT release due to its increased Ca²⁺ affinity and stronger membrane binding energy ^[20]. The mediating role of C2A is not completely understood on a molecular level and must be studied further.

3. Mutations in SYT1 Result in Synaptic Neuronal Transmission Dysfunction

It has been described by Baker et al. ^[18] that SYT1 mutation causes a dysfunction in synaptic vesicle cycling. In the years since, multiple missense mutations have been implicated in what has been classified as SYT1 -associated neurodevelopmental disorder. Each of the missense mutations causing SYT1associated neurodevelopmental disorder have been implicated in the C2B domain of SYT1 ^[4][15][18] thereby disrupting SYT1 function. Specifically, it has been demonstrated that mutations reduce the binding between C2B and PIP2 and significantly decrease the membrane sensitivity to Ca²⁺influx after an AP ^[21]. On a biomolecular level, this occurs when one of the hydrophobic amino acid residues within the C2B domain are replaced by a neutral or acidic amino acid residue that abolishes Ca²⁺ and phospholipid binding, which ultimately negatively effects NT release ^[15][18]. The consequences of these mutations in SYT1 include disrupted vesicle trafficking, mis-localization of SYT1, deficits in evoked synaptic transmission, dose-dependent dominant negative activity, and diminished rate of exocytosis and SV fusion in mammalian synaptic terminals ^[5][15][18]. However, the severity of the dysfunction depends on the specific mutation. For example, it has been described that while most mutations do impact spontaneous release rates and evoked synchronous NT release, the mutations D303G and I367T in C2B have been witnessed to have a severe impact on evoked synaptic release in comparison to D365E ^[5]. It has also been observed that the three before-mentioned mutations form unstable complexes with Ca²⁺, but, interestingly, have increased rapid release of Ca²⁺, clearly showing that these pathogenic mutations have a reduced Ca²⁺ sensitivity ^[5]. In addition, the first mutation identified in a neurodevelopmental disorder, I368T, was shown to decrease rate of SV NT release and rate of evoked SV fusion due to the isoleucine being replaced by threonine. Isoleucine is of particular interest considering that it is a highly conserved residue in the C2B domain that mediates Ca²⁺ binding ^[15][18]. Interestingly, previous labs have demonstrated that SYT1 variants had roughly the same level of expression at the nerve terminal as wild type SYT1 except for M303K, which showed decreased expression and more diffuse localization ^[15].