Structures of Synapsin Gene Family Members
Synapsins are produced by three different genes in mammals, and the chromosomal locations of these genes have been mapped in human and mouse: SYN1 is on human and mouse chromosome X (Yang-Feng et al. 1986), SYN2 is on human chromosome 3 and mouse chromosome 6 (Li et al. 1995), and SYN3 is on human chromosome 22 and mouse chromosome 10 (Kao et al. 1998). Alternative splicing yields more synapsin isoforms, namely, synapsin Ia and Ib, synapsin IIa and IIb, and synapsin IIIa–f (Sudhof et al. 1989; Kao et al. 1998). Synapsins are mosaics composed of combinations of conserved and non-conserved structural domains (Fig. 1). The N-terminal regions (domains A–C) are highly conserved amongst all major synapsin isoforms, while the C-terminal regions (domains D–J) consist of unique, isoform-specific sequences.
Domain A is known to interact with phospholipids, an interaction that is important for the ability of synapsins to bind to synaptic vesicles. This interaction is inhibited by phosphorylation of a serine residue (serine 9 or site 1, see below) by the cAMP-dependent protein kinase (PKA) or the Ca2+/calmodulin-dependent protein kinase I (CaMKI) (Hosaka and Südhof 1999). Injection of a peptide derived from domain A blocks release of the excitatory neurotransmitter, glutamate, without perturbing synaptic vesicle pool size or neurotransmitter release kinetics (Hilfiker et al. 2005). This effect is regulated by dephosphorylation of serine 9/site 1, suggesting that the peptide may interfere with neurotransmitter release by competing with phosphorylation-sensitive binding of synapsins to phospholipids. Thus, binding to synaptic vesicle phospholipids may be central to the function of synapsins in neurotransmitter release.
Domain B is a small, conserved linker region joining domains A and C. This domain has many amino acids, such as alanine and serine, with compact conformations. The function of domain B is not well known; it contains a site for phosphorylation by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) and presumably confers regulation of synapsin function by this signaling pathway.
Domain C is the region with the highest degree of homology across synapsins. It performs many of the core functions of synapsins, such as interactions with the actin cytoskeleton and SV phospholipids (Cheetham et al. 2001). This domain is amphipathic – containing both hydrophobic and highly charged residues – and is characterized by a compact structure of roughly elliptical shape, composed of several subdomains organized as α-helices or β-sheets, as well as a disordered region at its C-terminus. Domain C is partially surrounded by the hydrophobic phase of phospholipid bilayers, potentially mediating binding of synapsins to SVs (Südhof et al. 1989). Domain C also mediates homo- and heterodimerization of synapsins (Hosaka and Südhof 1999). Perturbation of C domain function by peptide microinjection indicates that interactions mediated by this domain maintain the reserve pool of synaptic vesicles and regulate the kinetics of neurotransmitter release; these actions have been attributed to interfering with binding of synapsins to the actin cytoskeleton (Hilfiker et al. 2005).
The C-terminal region of each synapsin isoform consists of variable domains that define the identity of each synapsin isoform and presumably confer the unique functional properties of each isoform. All of these variable domains are proline rich.
Domain D consist of 27 percent proline and 17 percent glutamine residues, whereas asparagine is totally excluded (Südhof et al. 1989). This domain interacts with SH3 domain-containing proteins, such as c-Src, Grb2, PI3K, PLC-r, and amphiphysin-I and II, as well as CaMKll and Rab3. Domain D contains two CaMKII phosphorylation sites (Fig. 1), and phosphorylation of these sites has been implicated in regulation of glutamate release (Chi et al. 2003). Domain D also serves to inhibit targeting of synapsin Ib to synaptic vesicles (Gitler et al. 2004a).
Domain E is conserved to all “a” synapsin isoforms (Fig. 1). Microinjection of a peptide from domain E decreases the size of the RP and slows release kinetics, indicating that this domain is implicated both in maintaining the RP and in synchronizing exocytosis (Hilfiker et al. 1998, 2005). It has been reported that domain E also regulates synapsin l oligomerization and cross-links synaptic vesicles. For targeting of synapsins to presynaptic terminals, domain E neutralizes the inhibitory effect of the C-terminus of domain D (Gitler et al. 2004a). The molecular mechanism underlying this action of domain E is not yet understood.
Domain G in synapsins IIa and IIb, as well as domain H in synapsin IIb, is proline-rich domains of unknown function. Domains F and I exist only at the C-terminal ends of “b” type isoforms; their functions also are poorly understood.
Domain J is unique to synapsin IIIa and is enriched in proline, glutamine, serine, and alanine residues (Kao et al. 1998). Although the function of this domain is not fully understood, it must be important for the roles of synapsin IIIa in release of the neurotransmitters dopamine and GABA (see below). Serine reside 470 of this domain is phosphorylated by MAPK and is mutated in some schizophrenia patients (see below).
Phosphorylation Control of Synapsin Functions
As mentioned above, synapsins were first identified as substrates for protein kinases and are the most abundant phosphoproteins in the brain. Here we will describe the multitude of proteins kinases that phosphorylate synapsins, using the sequence of rat synapsin I as a reference.
PKA and CaMKI/IV
Site 1 (serine 9) within domain A is phosphorylated by PKA and CaMKI/IV. PKA-induced phosphorylation of this site inhibits binding of domain A to phospholipids, which dissociates synapsins from synaptic vesicles and is involved in synaptic vesicle mobilization (Chi et al. 2003). PKA-induced phosphorylation reportedly has a more pronounced effect on the actin-binding and actin-bundling properties of synapsin II compared to synapsin I.
Sites 2 and 3 in domain D (serines 566 and 600) are phosphorylated by CaMKII (Czernik et al. 1987). Phosphorylation of sites 2 and 3 introduces two negative phosphate groups within the positively charged tail region of synapsin I, causing a major conformational change that reduces the ability of synapsin I to bind to both actin and to synaptic vesicles.
Mitogen-Activated Protein Kinase (MAPK)/Extracellular Signal-Regulated Kinase (Erk)
Sites 4 and 5 (serines 62 and 67) on domain B and site 6 (serine 549) on domain D are phosphorylated by the MAPK/Erk. Phosphorylation at these sites reduces the ability of synapsin I to trigger actin polymerization and bundling. Activation of the MAPK pathway by the brain-derived neurotrophic factor (BDNF) enhances neurotransmitter release by phosphorylating synapsins. A MAPK phosphorylation site in synapsin III, serine 470, is mutated in some schizophrenia patients (see below).
Cyclin-Dependent Kinases 1 and 5 (cdk 1/5)
Site 6 (serine 549) is phosphorylated by both cdk 1 and cdk 5, while site 7 (serine 551) is phosphorylated only by cdk 5. Similar to observations of MAPK of synapsins, phosphorylation of site 6 by cdk 1 reduces the ability of synapsin I polymerize and bundle actin; phosphorylation by cdk 5 affects neither actin polymerization nor bundling. Recently, another cdk 5 phosphorylation site (serine 404) was identified on synapsin III, which is required for early neuronal development such as axon specification and neurite outgrowth as well as regulation of radial migration and orientation of pyramidal neurons during neocortical development.
Site 8 (tyrosine 301) on domain C is phosphorylated by Src. Synapsin I interacts with Src through SH domains and activates Src, which in turn phosphorylates Tyr 301 to increase synapsin binding to synaptic vesicles and actin, as well as enhancing synapsin dimerization.
Synapsin Functions in Synaptic Vesicle Trafficking
Synapsins are key regulators of synaptic vesicle (SV) dynamics in presynaptic terminals. Synaptic vesicles are organelles within presynaptic terminals that store and release neurotransmitters, chemical signals that are secreted during synaptic transmission. Three different pools of SVs can be distinguished based on the kinetics of neurotransmitter release: the readily releasable pool (RRP), which is responsible for fast, synchronous release of neurotransmitters via exocytosis, a reserve pool (RP) that does not immediately participate in neurotransmitter release but instead is clustered some distance away from the site of exocytosis, and a recycling pool that contributes to maintaining neurotransmitter release by providing a supply of recycled SVs. The specific roles that synapsins play in these SV pools apparently differ according to the type of neurotransmitter stored within SVs.
A role for synapsins in maintaining the RP of glutamatergic SVs has been studied through perturbing synapsin function via antibodies, peptides, or gene deletion (Pieribone et al. 1995; Rosahl et al. 1995; Hilfiker et al. 1998; Gitler et al. 2004b). In all cases, the rate of synaptic depression is accelerated, indicating reduced mobilization of SVs from the RP (Gitler et al. 2004b). At glutamatergic hippocampal neurons from synapsin triple knockout (TKO) mice, the number of SVs in the RP is reduced yet the number of SVs in the RRP is unaffected (Gitler et al. 2004b). Together, these results indicate that synapsins regulate the size of the RP by tethering glutamatergic vesicles within the RP. Remarkably, synapsin IIa is the only isoform that significantly rescues the kinetics of synaptic depression and RP size when introduced into TKO neurons, indicating that this synapsin isoform – and its unique H domain – plays a unique role in trafficking of glutamatergic SVs (Gitler et al. 2008).
In contrast, at inhibitory synapses of TKO mice, loss of synapsins reduces the peak amplitude of GABAergic IPSCs evoked by single stimuli, whereas the rate of synaptic depression is unaffected (Gitler et al. 2004b, 2008; Song and Augustine 2016). Detailed analysis indicates that RRP size is identical in TKO and wild-type neurons, while the kinetics of GABA release is slowed in TKO neurons, indicating that synapsins affect the rate of exocytosis of GABAergic SVs within the RRP (Song and Augustine 2016). These phenotypes are rescued by all major synapsin isoforms – synapsins Ia, Ib, IIa, IIb, or IIIa – although synapsin IIIa can not rescue the kinetics of GABA release (Song and Augustine 2016). These results indicate that synapsins play different roles in regulation of GABAergic SVs, and this role depends on the conserved synapsin domains.
Synapses Employing Other Neurotransmitters
Deletion of all synapsin isoforms increases the amount of depolarization-induced catecholamine release from adrenal chromaffin cells (Villanueva et al. 2006). This increase is not caused by changes in the amount of catecholamine released per vesicle or in the speed of discharge of catecholamines from individual vesicles, but instead is due to an increased rate of exocytotic events. This effect is rescued by synapsin IIa, indicating that this isoform serves as a negative regulator of catecholamine release. Synapsins have differential actions in release of two monoamine neurotransmitters, dopamine (DA) and serotonin (5-HT). While loss of synapsins increases DA release from presynaptic terminals, there is no difference in 5-HT release between wild-type and TKO mice (Kile et al. 2010). The negative regulation of DA release apparently is mediated by synapsin IIIa, because a similar enhancement of DA release occurs in synapsin III KO mice. In addition, synapsins appear to provide a RP of DA-containing vesicles that is recruited during cocaine treatment and is reduced in synapsin TKO mice (Venton et al. 2006). Thus, synapsins appear to have two effects in trafficking of DA vesicles: an inhibitory effect on exocytosis of DA vesicles, mediated by synapsin IIIa, and maintenance of a RP of DA vesicles, mediated by a synapsin isoform (or isoforms) that remains to be determined.
In summary, synapsins play different roles in trafficking of SVs containing different neurotransmitters. This yields diverse regulation of the amount or speed of neurotransmitter release from different presynaptic terminals.
Other Functions of Synapsins
Synapsin expression differs during the course of brain development, suggesting different roles for synapsins during development. Expression of synapsins I and II reaches a peak only 1–2 months after birth in rats, while synapsin III is expressed earlier and then decreases in later life. Consistent with this expression pattern, synapsin III is found in nestin-positive neuronal progenitors, later becoming restricted to cells of neuronal lineage. Synapsin III apparently is involved in adult neurogenesis and affects neuronal migration and orientation in neocortex, working downstream of semaphorin 3A and cdk 5. Synapsin III enhances cell survival after neuronal precursors have differentiated into neurons.
Neurite outgrowth and branching are slowed in cultured neurons from synapsin I KO mice, though growth catches up at later developmental stages (Chin et al. 1995; Ferreira et al. 1996). Similarly, neurite growth is slowed in synapsin II KO neurons and synapse formation is delayed (Ferreira et al. 1998), while defective neurite outgrowth is observed in synapsin III KO neurons only at 1 day in vitro and is spontaneously rescued later (Feng et al. 2002). In contrast, cultured neurons from synapsin I and II double KO mice and TKO mice have normal growth (Gitler et al. 2004b).
In summary, the precise roles of synapsins in neural development are not yet clear.
Synapsin Implication in Brain Disorders
Genetic analyses in human populations have implicated abnormalities in synapsin genes in various brain disorders, particularly epilepsy and autism spectrum disorders (ASDs). A nonsense mutation (W356X) in the SYN1 gene that causes mRNA degradation was found in a family predisposed to epilepsy and a Q555X mutation in SYN1 was found in all affected individuals from a large French-Canadian family segregating epilepsy and ASDs. Additional mutations in SYN1 (A51G, A550T, and T567A) were found in 3.5% of French-Canadian individuals with epilepsy. These linkages also extend to 1.0% of ASD patients. Single-nucleotide polymorphisms in SYN2 reportedly contribute significantly to epilepsy and patients with SYN2 gene polymorphisms exhibit epilepsy risk. In addition, nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD. These missense mutations do not rescue the RP of synapsin II KO neurons. Two SYN3 SNPs, S470N and L469L, were identified in schizophrenia patients. S470N appeared more frequently in schizophrenia in a Caucasian population and is a MAPK phosphorylation site, suggesting impairment of the MAPK signaling pathway (Porton et al. 2004).
Synapsins are a well-known family of presynaptic proteins that bind to SVs. Synapsin binding plays different roles in trafficking of different types of SVs and presumably is mediated by different synapsin isoforms. Synapsin function is regulated through phosphorylation by numerous protein kinases and dephosphorylation by protein phosphatases. Defects in synapsins have been implicated in numerous brain disorders, most prominently epilepsy and ASDs. In addition to functioning in the adult nervous system, synapsins may also be important for neural development.
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