O-linked β-N-acetylglucosamine (O-GlcNAc) site thr-87 regulates synapsin I localization to synapses and size of the reserve pool of synaptic vesicles

Abstract

O-GlcNAc is a carbohydrate modification found on cytosolic and nuclear proteins. Our previous findings implicated O-GlcNAc in hippocampal presynaptic plasticity. An important mechanism in presynaptic plasticity is the establishment of the reserve pool of synaptic vesicles (RPSV). Dynamic association of synapsin I with synaptic vesicles (SVs) regulates the size and release of RPSV. Disruption of synapsin I function results in reduced size of the RPSV, increased synaptic depression, memory deficits, and epilepsy. Here, we investigate whether O-GlcNAc directly regulates synapsin I function in presynaptic plasticity. We found that synapsin I is modified by O-GlcNAc during hippocampal synaptogenesis in the rat. We identified three novel O-GlcNAc sites on synapsin I, two of which are known Ca(2+)/calmodulin-dependent protein kinase II phosphorylation sites. All O-GlcNAc sites mapped within the regulatory regions on synapsin I. Expression of synapsin I where a single O-GlcNAc site Thr-87 was mutated to alanine in primary hippocampal neurons dramatically increased localization of synapsin I to synapses, increased density of SV clusters along axons, and the size of the RPSV, suggesting that O-GlcNAcylation of synapsin I at Thr-87 may be a mechanism to modulate presynaptic plasticity. Thr-87 is located within an amphipathic lipid-packing sensor (ALPS) motif, which participates in targeting of synapsin I to synapses by contributing to the binding of synapsin I to SVs. We discuss the possibility that O-GlcNAcylation of Thr-87 interferes with folding of the ALPS motif, providing a means for regulating the association of synapsin I with SVs as a mechanism contributing to synapsin I localization and RPSV generation.

Keywords: Neurobiology; O-GlcNAc; Signal Transduction; Synapses; Synaptic Plasticity.

FIGURE 1.

Characterization of synapsin I O-GlcNAcylation. A, WB analysis of synapsin I O-GlcNAcylation in rat hippocampi during development. Hippocampi dissected from rat pups of indicated ages were lysed, and synapsin I was pulled down from the lysates containing equal amount of protein. The samples were probed with antibodies that recognize total synapsin (tSyn) and O-GlcNAc (110.6). The two bands detected represent synapsin Ia and Ib. In the middle panel, 110.6 antibodies were pretreated with 500 mm GlcNAc to inhibit the recognition of O-GlcNAc by the antibody. Thus, only a nonspecific signal generated by 110.6Ab is detected. p stands for postnatal day. B, MS/MS electron transfer dissociation fragmentation spectra of a tryptic peptide QAS(O-GlcNAc)ISGPAPTK (starting position Gln-566) from bovine synapsin I identifies O-GlcNAc modification site Ser-568. C, amino acid sequence alignment of domains A, B, and D of synapsin I from indicated species with mapped or predicted O-GlcNAc and phosphorylation sites highlighted in gray. P or G above an amino acid symbol indicates whether it is a phosphorylation or O-GlcNAcylation site; numbering of sites is based on rat synapsin I sequence. Residues in bold were found to be O-GlcNAc-modified in this study on mouse and bovine synapsin I or by others on mouse and rat synapsin I. Underlined stretches of amino acids in bovine synapsin I indicate the peptides that were found to be modified by O-GlcNAc, but the site of the modification has not been assigned.

FIGURE 2.

Targeting of O-GlcNAc site-specific mutants of synapsin I to synapses. A, schematic representation of the domain organization of synapsin I. Residues are numbered as in rat synapsin I. Numbers are assigned to O-GlcNAcylated residues that were mutated to alanines to generate various O-GlcNAc site-specific mutants of synapsin I. B and C, numbers that follow Syn1a_ indicate O-GlcNAc sites that are mutated to alanines. B, WB analysis of O-GlcNAc levels on synapsin mutants. Wild type synapsin I and indicated O-GlcNAc site mutants were expressed in HEK293T cells, pulled down by FLAG-agarose, and in vitro modified by OGT. O-GlcNAc levels were analyzed by WB with 110.6 Ab. *, degradation product. IP, immunoprecipitation. C, localization of synapsin I O-GlcNAc site mutants in primary hippocampal neurons. On day 7 in culture, primary rat hippocampal neurons were cotransfected with plasmids expressing wild type or O-GlcNAc site mutants of EGFP-synapsin I and DsRed2 and were imaged on day 15. Scale bar, 10 μm. D, TF values were calculated from images like those in C. Each value represents an average TF of 3–6 biological replicates. For each biological replicate, TFs for 60–300 synapses were calculated. TF are normalized to TF of wild type synapsin. * indicates Bonferroni-corrected p < 0.0001.

FIGURE 3.

Investigation of interplay between O-GlcNAc and ERK phosphorylation sites in domain B in regulation of synapsin I localization to synapses. Numbers that follow Syn1a_ indicate O-GlcNAc sites that are mutated to alanines, numbered as in Fig. 2A. In Syn1a_S62,67E mutant, serines 62 and 67 both were substituted to glutamate to mimic phosphorylation. In Syna1a_1–3_S62A,S67A mutant O-GlcNAc sites 1–3 as well as ERK phosphorylation sites Ser-62 and Ser-67 were mutated to alanines to create synapsin I that cannot be phosphorylated or modified by O-GlcNAc in domain B. A, HEK293T cells were transfected with wild type synapsin I or indicated mutants of synapsin I, lysed 48 h later, and subjected to WB with antibody that recognize total synapsin and synapsin phosphorylated at indicated ERK phosphorylation sites. *, nonspecific bands. B, localization of O-GlcNAc and ERK mutants of synapsin I in primary hippocampal neurons. Upper panel, neurons were cotransfected with plasmids expressing EGFP-tagged wild type or mutants of synapsin I, as indicated on the right side of the panel and DsRed2 on day 7 in culture, and were imaged on day 15. Scale bar, 10 μm. Lower panel, TFs were calculated from images like those on the upper panel. Each value represents an average TF of three biological replicates. For each biological replicate, TFs for at least 300 synapses were calculated. TFs are reported as relative to WT TF arbitrarily set at 1. * indicates Bonferroni-corrected p < 0.05.

FIGURE 4.

SV cluster density in neurons expressing WT or T87A synapsin I. Hippocampal neurons were cotransfected on day 5 with EGFP-Syn1a or EGFP-Syn1a_T87A and a plasmid for the expression of mRFP-synaptophysin. On day 15, live cultures were imaged. Synaptophysin served as a marker of SV clusters. Scale bar, 10 μm. Right panel, frequency of SV clusters along neurite is plotted. Because the vast majority of mRFP puncta colocalized with EGFP puncta, SV clusters were defined as EGFP-mRFP-positive puncta. Averages of three biological replicates were calculated. At least 800 synapses were counted for each biological replica.

FIGURE 5.

Regulation of the RPSV by T87A synapsin I mutant. A–D, analysis of functional pools of SVs. Primary hippocampal neurons were transfected with EGFP-Syn1a or EGFP-Syn1a_T87A on day 5 and stained with FM 4-64 dye and imaged on day 17. A, example of neurons loaded with FM 4-64 before (left) and after HFS (right). Images are inverted. Puncta that disappeared after HFS represent synapses where all SVs of the recycling pool were released. Scale bar, 10 μm. B, FM 4-64 destaining curves. Brief 20 Hz stimulation releases RRPSV (RRP) and two longer 10 Hz trains release RPSV (RP). Median values that were calculated from 147 (WT) and 207 (T87A) synapses from eight (WT) and seven (T87A) destaining experiments are plotted. C, frequency chart, fractions of synapses with RPSV of indicated sizes in neurons expressing WT and T87A synapsin I are plotted. Inset, average size of RPSV (% of total). Values are calculated from the destaining experiments, which are summarized in B. D, ANCOVA analysis of average RPSV size values between neurons expressing WT and T87A synapsin I mutant, with synaptic expression of EGFP-synapsin as a covariate. Average RPSV and EGFP values are from experiments summarized in B. E, synapsin I dispersion upon HFS. Neurons were transfected with EGFP-Syn1a or EGFP-Syn1a_T87A on day 7 and imaged on day 16. Dispersion of synapsin was triggered by 10 Hz stimulation for 90 s. Nine neurons (total 295 synapses) expressing WT synapsin and five neurons (total 387 synapses) expressing T87A synapsin were imaged. Average dispersion curves are shown. Average half-life of synapsin I at a synapse τ ±S.D. is reported. F, helical wheel projection of ALPS motif of synapsin I (on the left) and synapsin II (on the right). Black circles with white letters represent hydrophobic residues. Gray circles with black residues represent hydrophilic residues. Hydrophobic face of ALPS presumably faces SV and interacts with hydrophobic tails of lipid membrane. Hydrophilic face of ALPS faces cytosol and is proposed to interact with polar headgroups of lipid membrane of SVs. Alignment of ALPS motifs from rat synapsin I and II is shown below. Arrows and arrowheads indicate O-GlcNAc sites.