Protein fucosylation regulates synapsin Ia/Ib expression and neuronal morphology in primary hippocampal neurons

Abstract

Although fucose-␣(1-2)-galactose [Fuc␣(1-2)Gal] carbohydrates have been implicated in cognitive processes such as long-term memory, the molecular mechanisms by which these sugars influence neuronal communication are not well understood. Here, we present molecular insights into the functions of Fuc␣(1-2)Gal sugars, demonstrating that they play a role in the regulation of synaptic proteins and neuronal morphology. We show that synapsins Ia and Ib, synapse-specific proteins involved in neurotransmitter release and synaptogenesis, are the major Fuc␣(1-2)Gal glycoproteins in mature cultured neurons and the adult rat hippocampus. Fucosylation has profound effects on the expression and turnover of synapsin in cells and protects synapsin from degradation by the calcium-activated protease calpain. Our studies suggest that defucosylation of synapsin has critical consequences for neuronal growth and morphology, leading to stunted neurite outgrowth and delayed synapse formation. We also demonstrate that Fuc␣(1-2)Gal carbohydrates are not limited to synapsin but are found on additional glycoproteins involved in modulating neuronal architecture. Together, our studies identify important roles for Fuc␣(1-2)Gal sugars in the regulation of neuronal proteins and morphological changes that may underlie synaptic plasticity., which exists as a terminal carbohydrate modification to N-and O-linked glycoproteins, has been implicated in cognitive processes such as learning and memory. For instance, preventing formation of Fuc␣(1-2)Gal linkages by incorporation of 2-deoxy-D-galactose (2-dGal) into glycan chains has been shown to cause reversible amnesia in animals (1-3). 2-dGal also interferes with the maintenance of long-term potentiation (LTP), a form of synaptic plasticity that is closely associated with learning and memory (4). Moreover, injection of a monoclonal antibody specific for Fuc␣(1-2)Gal has been found to impair memory formation in animals, presumably by blocking the Fuc␣(1-2)Gal epitope (5, 6). These studies suggest important roles for Fuc␣(1-2)Gal carbohydrates and their associated proteins in modulating neuronal communication in the brain.Interestingly, evidence suggests that protein fucosylation is regulated in response to synaptic activity. Both task-specific learning and LTP have been shown to induce the fucosylation of proteins at the synapse (7,8), and addition of exogenous fucose or 2Јfuco-syllactose was found to enhance LTP in hippocampal slices (9). The activity of fucosyltransferases, enzymes involved in the transfer of fucose to glycoproteins, has also been demonstrated to increase substantially during synaptogenesis (10) and upon passive avoidance training in animals (11). Together, these studies suggest that protein fucosylation may be a highly regulated process in the brain and may contribute to neuronal development and synaptic plasticity. Despite these intriguing observations, little is known about the molecular mechanisms by which Fuc␣(1-2)Gal sugars influence neuronal communication. Surprisingly, no Fuc␣(...

Synapsin I Is an Oligomannose-Carrying Glycoprotein, Acts As an Oligomannose-Binding Lectin, and Promotes Neurite Outgrowth and Neuronal Survival When Released via Glia-Derived Exosomes

“…The mean area intensity of NG2-positive cells was determined to be twofold to threefold higher than that measured for NG2-negative cells. (Murrey et al, 2006). Here, we showed that synapsin I carries Lewis x also on O-glycans and oligomannoses on N-glycans.…”

“…These results show that synapsin carries N-glycans and, thus, is a glycoprotein. Since synapsin has been reported to carry fucose in ␣1,2-linkage to galactose (Murrey et al, 2006) and we showed that it carries oligomannose, we further characterized the glycosylation of purified synapsin using carbohydrate-specific antibodies and lectins. No synapsin staining was observed with antibodies recognizing the HNK-1 epitope or polysialic acid, while L5 antibody, which recognizes Lewis x , bound to synapsin (Fig.…”

The Human Synaptic Vesicle Protein, SV2A, Functions as a Galactose Transporter in Saccharomyces cerevisiae

“…The galactose-containing disaccharide, fucose-␣(1-2)galactose, has been implicated in memory formation, learning, and synaptic plasticity (15)(16)(17)(18)(19)(20)(21). Fucose-␣(1-2)galactose sugars are enriched on glycoproteins in presynaptic nerve terminals (22), and it has been shown that fucose-␣(1-2)-galactose modification has profound effects on the expression and degradation of the synaptic vesicle-associated protein, synapsin (22). Furthermore, the importance of galactose for normal neuronal function is indicated by the fact that B27, the widely used serum replacement for primary neuronal cultures, contains galactose (23).…”

SORLA regulates calpain‐dependent degradation of synapsin

“…Because 14-3-3s might promote interaction of SORLA with synapsin in a phosphorylation-dependent manner, we propose the existence of a tripartite complex that regulates phosphorylation-dependent degradation of synapsins. Based on our data, complex formation identifies phospho-synapsin molecules destined for calpain-mediated degradation, a major pathway for control of synapsin turnover [19]. Importantly, shRNA-mediated knockdown of calpain in SH-SY5Y cells levitated the effect of SORLA overexpression on synapsin levels (Fig.…”

“…Synapsin levels were substantially reduced in SY5Y-S compared to parental SY5Y cells ( It has been shown before that turnover of synapsin is controlled by the calcium-activated protease calpain [19]. To evaluate whether the SORLA-dependent reduction of synapsin levels was mediated by this protease, we generated cell clones from SY5Y and SY5Y-S lines stably overexpressing a small hairpin (sh) RNA directed against calpain 1.…”