Synapsin II (SYN2) was identified in the late 1970s following studies that began searching for neuronal substrates for endogenous cyclic adenosine 3′:5′ monophosphate (cAMP)-dependent phosphorylation. cAMP had been suggested to mediate neurotransmitter regulation through synaptic membrane proteins and led to the discovery of several proteins including the family of synapsin proteins. Highly specific to nerve terminals, synapsins were among one of the first identified synaptic vesicle-associated protein families (De Camilli et al. 1990).
The first member identified in the synapsin family was synapsin I, initially named protein I, and described by Paul Greengard in 1972 as a primary synaptic membrane phosphorylation target by cAMP-dependent kinases (Johnson et al. 1972). Following the discovery of synapsin I, a second member of the synapsin family – protein III – later renamed synapsin II (SYN2) was identified in the late 1970s and found to constitute approximately 0.2% of total mammalian brain protein and 9% of vesicle proteins (De Camilli et al. 1990). Subsequently, the presence of two distinct SYN2 isoforms was identified and shown to display regional specific expression in the brain of rodents (Walaas et al. 1988). Follow-up research then focused on investigating the exact structure and function of this prominent neuronal phosphoprotein.
Currently, SYN2 is known to act in a phosphorylation-dependent manner, binding to the cytosolic surface of synaptic vesicle membranes as well as actin filaments (De Camilli et al. 1990). Further, studies identified SYN2’s role in the regulation of neurotransmitter release, neurite extension, synapse formation, and its involvement in later steps of the synaptic vesicle cycle, such as delivery, recycling, and exocytosis (De Camilli et al. 1990; Bykhovskaia 2011).
The structure of SYN2 has been evolutionarily conserved across invertebrate and vertebrate species, such as Loligo pealeii (squid), Xenopus laevis (frog), Rattus norvegicus (rat), and Homo sapiens (humans) (Kao et al. 1999). In humans, the SYN2 gene consists of 187,673 bases mapped onto chromosome 3, at region p25. Upon translation, this region is converted into the 582 amino acid SYN2 protein, with a molecular mass of 62,847 Da. Alternative splicing of the SYN2 gene produces two splice variants, named SYN2a and SYN2b. These two isoforms have molecular weights of 74,000 daltons (Da) and 55,000 Da, respectively, and contain many similar structural domains (see https://www.ncbi.nlm.nih.gov/gene/6854 for further details).
Starting at the NH2-terminus, domain A consists of a short region containing a phosphorylation site (Ser10) for protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMK I) (De Camilli et al. 1990). Domain B is a subsequent linker region, rich in prolines, glutamines, alanines, and serines, which connects domains A and C (De Camilli et al. 1990). Within this linker region, two distinct binding sites have been mapped between amino acids 43–74 and 75–121. It is at these sites that SYN2 is able to bind to proteins on the membrane of synaptic vesicles (Thiel et al. 1990). Due to the high degree of homology within the NH2-terminus domains of synapsin I, it is very likely that domain B of SYN2 also contains phosphorylation sites for mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMK II). (Sudhof et al. 1989). Following domain B is the most central and conserved domain of SYN2, domain C. This important domain is approximately 300 amino acids and accounts for more than half of the sequence length (Molinaro et al. 2015). Domain C has been identified as a major binding site for lipid membranes of synaptic vesicles and actin filaments as well as the site for synapsin dimerization (Cheetham et al. 2001). Conserved in this domain are amphipathic and hydrophobic regions essential for high-affinity binding to synaptic vesicles, penetration of their lipid bilayer, and a tyrosine phosphorylation site for tyrosine kinase Src (Cheetham et al. 2001).
Despite the synapsin family similarities in domains A–C, these phosphoprotein structures vary at the COOH-terminus. In SYN2, the major variation lies in the unique presence of domain G, a short proline-rich region that facilitates associations with the cytoskeleton via an actin binding site (De Camilli et al. 1990). Domain G is flanked by domains H and I, present in SYN2a and SYN2b isoforms, respectively. The final domain common to all synapsin “a” isoforms is domain E. This domain is responsible for maintaining the reserve pool by clustering synaptic vesicles and interacting with actin in the cytoskeleton to regulate exocytosis. Domain E is also responsible for mediating synapsin I and II binding, as well as synapsin dimerization (Bykhovskaia 2011). Each domain contains specific binding sites that relate to the multifunctional nature of SYN2.
Localization and Regulation
SYN2 is found in virtually all neuronal presynaptic terminals throughout the central and peripheral nervous systems (Sudhof et al. 1989; De Camilli et al. 1990). Despite being present in all nerve terminals, the relative expression of different synapsin isoforms varies between neuronal cell types. SYN2a and SYN2b are both found in mossy fiber terminals of hippocampal granule cells, while SYN2a is not observed in Purkinje cell axon terminals (Sudhof et al. 1989). The regional distribution of SYN2a and 2b is more consistent and follows a constant 1:2 ratio across various brain regions (Walaas et al. 1988). The exception to this rule is found within the olfactory bulb, where SYN2a concentrations are elevated in comparison to SYN2b, potentially in relation to the ongoing remodeling of synaptic contacts (Walaas et al. 1988).
The final transcription factor responsible for SYN2 regulation is AP-2α, a sequence-specific DNA-binding protein that activates transcription when interacting with enhancer elements (Petersohn et al. 1995). AP-2α binding sites are not found on synapsin I or III, making its regulation specific to SYN2. Two neighboring binding sites for AP-2α are found within the promoter region of SYN2. However, their close proximity likely diminishes the ability of both sites to be occupied simultaneously, with the more distal site being suggested as the more favorable AP-2α binding site (Petersohn et al. 1995). SYN2 levels are influenced by AP-2α and its upstream substrates, such as dopamine (DA). Dopamine indirectly regulates SYN2 expression via AP-2α, through dopamine receptor subtypes (DA-D1 and DA-D2), cAMP and PKA-dependent pathways. DA-D1 receptor stimulation or DA-D2 receptor inhibition increases SYN2 levels (Molinaro et al. 2015) (Fig. 2). Additionally, PKA activates AP-2α which mediates effects, by undergoing posttranslational modifications, such as sumoylation that facilitates changes in protein kinase C (PKC) and cAMP levels, in turn affecting the expression of SYN2 (Petersohn et al. 1995).
In addition to regulation by transcription factors, SYN2 expression levels are also influenced by methylation. The promoter region of SYN2 contains large GC-rich regions, also known as CpG islands, specifically in sequences surrounding transcription start sites. The addition of methyl groups to these CpG islands represses gene expression, and the lack of methylation increases SYN2 expression. CpG methylation mapping of postmortem samples from bipolar disorder and major depressive disorder patients identified DNA hypomethylation and significantly reduced SYN2 levels in patients compared to controls (Cruceanu et al. 2016). Thus, methylation also plays an important role in regulating SYN2 expression levels.
Synapsin II has vital functional roles at multiple stages of the synaptic vesicle cycle, including clustering, reserve pool maintenance, delivery to the active zone, and synchronizing release of neurotransmitters. These functions are largely regulated by the dynamic balance between SYN2 phosphorylation and dephosphorylation occurring at domains C and E (De Camilli et al. 1990; Bykhovskaia 2011). As previously discussed, domains C and E are the major functional domains of SYN2 and the site of homo- and heterodimerization with other synapsins, which will be discussed later. Both of these domains function in an actin-dependent manner, while domain A has an actin-independent functional role in regulating neurotransmitter release (Bykhovskaia 2011). In addition to its performance in the synaptic vesicle cycle, SYN2 appears to influence synaptogenesis, synapse maintenance, synaptic plasticity, and synaptic vesicle membrane stabilization (Brenes et al. 2015).
SYN2 is also implicated in synaptogenesis, synapse maintenance, and plasticity. Knockdown studies of SYN2 demonstrate that a reduction in this phosphoprotein results in impaired cytoskeletal organization in early stages of nerve cell development. Aberrant cytoskeletal activity slows axonal outgrowth and diminishes neurite branching which in turn affects maintenance and functional activity of the synapse (Brenes et al. 2015). If reduced after axonal elongation but prior to synapse formation, then neurons do not form synapses, therefore elucidating the role of SYN2 in formation and maintenance of synapses. SYN2b may be responsible for nerve terminal formation, given that transfection with this isoform increased the number of nerve terminals and synaptic vesicles within each terminal (Brenes et al. 2015).
The final proposed function of SYN2 at presynaptic nerve terminals involves stabilization of synaptic vesicle membranes. When synapsins interact with the phospholipid bilayer, they induce a conformational change that increases alpha helices. This propagates the insertion of the N-terminal end into the hydrophobic core of the membrane. By inserting SYN2 into the membrane, this protein increases stabilization of the lipid bilayer and inhibits vesicle leakage and unregulated vesicle fusion. Additionally, synapsins have been suggested to be obligatory dimers on vesicle membranes, as dimerization promotes vesicle binding and clustering (Hosaka and Sudhof 1999). Dimerization occurs at domain C in all synapsin isoforms and is functionally regulated by activity-dependent Src phosphorylation. Homodimerization occurs in synapsin I, II, and III, and a weak heterodimer is formed between synapsin I and III. On the contrary SYN2 forms strong heterodimers with both synapsin I and synapsin III (Hosaka and Sudhof 1999).
Synapsin II and Disease
Dysfunction of synapsins has been hypothesized in various disease states due to their numerous roles in both development and proper functioning of synapses. Evidence suggests that this dysfunction may manifest in a variety of ways dependent on the isoform affected as well as the temporal span in which it is influenced. Some of the various diseases, which have been implicated with improper functioning of synapsins, include, but are not limited to, schizophrenia (SZ), Huntington’s disease (HD), Alzheimer’s disease (AD), autism spectrum disorder (ASD), epilepsy, Parkinson’s disease (PD), bipolar disorder (BD), and diabetes (Giovedi et al. 2014; Prasad et al. 2014; Molinaro et al. 2015).
Specifically, SYN2 dysfunction has been implicated in several disease states including SZ, AD, ASD, epilepsy, and BD. The role of SYN2 in the diseased state of SZ has been investigated in depth, allowing genetic susceptibility studies to reveal that the SYN2 gene is a region of vulnerability for possible SZ-related mutations (Molinaro et al. 2015). SYN2 has also been found to play an integral role in neuronal nitric oxide synthase (nNOS) localization through its relationship with CAPON. nNOS has functional roles in neurotransmitter release and neuronal process outgrowth and has been implicated in SZ and BD. The CAPON gene has also since been identified as a gene candidate susceptible in the pathogenesis of SZ (Zheng et al. 2005). Accumulating evidence implicates SYN2 in the pathophysiology of SZ including decreased SYN2 levels within the dorsolateral prefrontal cortex of schizophrenic patients (Molinaro et al. 2015). Consolidating this data are SYN2 knockdown experiments producing schizophrenic-like behaviors in rats, reflecting several SZ symptom domains (Molinaro et al. 2015). Knockdown of SYN2 has been established to produce reduced signaling in different neurotransmitter systems including inhibitory GABA neurons as well as excitatory glutamatergic neurons. Modified excitatory and inhibitory signaling has been implicated in SZ symptoms and is the origin of several hypotheses attempting to explain the cause of the disorder (Molinaro et al. 2015). Genetic studies have also revealed a potential role for SYN2 in the etiology of BD (Tan et al. 2014). Both illnesses present with a spectrum of symptoms ranging in severity across individuals, and there remains a great deal of overlap between the clinical manifestation of BD and SZ. These commonalities between BD and SZ suggest potential similarities in their pathophysiology, including SYN2 dysfunction.
The synapsin family of phosphoproteins was first described in the 1970s via research investigating the roles of cAMP. First identified as “protein III,” SYN2 has since been determined to exist in two isoforms (A and B) and constitutes 0.2% of total mammalian brain protein and approximately 9% of total vesicle proteins. Structurally, the synapsin family consists of multiple evolutionarily conserved domains which may vary slightly depending on isoforms. Domain C plays an integral role as it has been identified as a major binding site for lipid membranes, thus showcasing its importance in the synaptic vesicle cycle. Specific to SYN2, domain G is a proline-rich region which may facilitate the binding of synapsin to cytoskeletal elements such as actin.
Regulation of SYN2 expression is achieved by a variety of cellular mechanisms and transcription factors. The promoter region for SYN2 has been found to include binding sites for EGR-1, PEA-3, as well as AP-2α. Individual promoter sites may be indicative of the varying functional roles of SYN2, such as EGR-1 involvement in growth and differentiation. SYN2 is the only phosphoprotein in the synapsin family with known AP-2α binding sites. These binding sites may be responsible for the dopaminergic control of SYN2 as discussed previously. In addition to regulation by transcription factors, SYN2 expression levels can also be influenced by methylation events.
Functionally, SYN2 plays a wide variety of roles in both developing and mature neuronal tissues. Often considered its most important function, SYN2 regulates vesicular release in a phosphorylation-dependent manner. This is accomplished via interaction between SYN2, cytoskeletal elements, and phospholipid membranes. Studies have also determined a developmental role of SYN2 with respect to synaptogenesis, synapse maintenance, as well as plasticity. These developmental functions are often attributed to the SYN2b isoform. In this manner, SYN2 also acts as a phospholipid membrane stabilizer, reducing both vesicular leakage and unintentional vesicle fusion. SYN2 was also found to play roles in the synaptic vesicle cycle (delivery, release, recycling) as well as development (neurite extension and synapse formation).
Various challenges surround research involving SYN2. As discussed above, SYN2 is involved in numerous functions at various points in development and maintenance. Due to the expansive range of functions, determining which are critical to various disease/psychological states can be difficult. In addition, clinically relevant tissues are often difficult to obtain and are often present in the form of postmortem tissues. Unfortunately, these patients have often been treated with various drugs or a combination of drugs, which is an undeniable confounding factor that reduces the significance of these samples.
Future studies should be conducted to specifically determine the full quaternary structure of SYN2. These studies would provide valuable information pertaining to functions and specific details necessary for a full understanding of this particular synapsin. This is important, as much of our current knowledge is inferred from studies of synapsin I due to their structural similarities. With respect to SYN2, future studies should be directed toward the investigation of the temporal importance of expression. Due to the number of varying functions, it is important to determine those most integral to health and disease. Studies which translate this information to a clinical level will provide vital insight to the role of SYN2 in neuropsychological disorders and promote SYN2 as a therapeutic target for these diseases. Furthermore, this research can lead to the discovery of novel treatment options targeting the underlying pathophysiology of these diseases instead of symptom management.
The work described in this review was supported by the Canadian Institute of Health Research (CIHR).
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