Abstract
O-Linked N-acetylglucosamine (O-GlcNAc) is a cytosolic and nuclear carbohydrate post-translational modification most abundant in brain. We recently reported uniquely extensive O-GlcNAc modification of proteins that function in synaptic vesicle release and post-synaptic signal transduction. Here we examined potential roles for O-GlcNAc in mouse hippocampal synaptic transmission and plasticity. O-GlcNAc modifications and the enzyme catalyzing their addition (O-GlcNAc transferase) were enriched in hippocampal synaptosomes. Pharmacological elevation or reduction of O-GlcNAc levels had no effect on Schaffer collateral CA1 basal hippocampal synaptic transmission. However, in vivo elevation of O-GlcNAc levels enhanced long term potentiation (LTP), an electrophysiological correlate to some forms of learning/memory. Reciprocally, pharmacological reduction of O-GlcNAc levels blocked LTP. Additionally, elevated O-GlcNAc led to reduced paired-pulse facilitation, a form of short term plasticity attributed to presynaptic mechanisms. Synapsin I and II are presynaptic proteins that increase synaptic vesicle availability for release when phosphorylated, thus contributing to hippocampal synaptic plasticity. Synapsins are among the most extensively O-GlcNAc-modified proteins known. Elevating O-GlcNAc levels increased phosphorylation of Synapsin I/II at serine 9 (cAMP-dependent protein kinase substrate site), serine 62/67 (Erk 1/2 (MAPK 1/2) substrate site), and serine 603 (calmodulin kinase II site). Activation-specific phosphorylation events on Erk 1/2 and calmodulin kinase II, two proteins required for CA1 hippocampal LTP establishment, were increased in response to elevation of O-GlcNAc levels. Thus, O-GlcNAc is a novel regulatory signaling component of excitatory synapses, with specific roles in synaptic plasticity that involve interplay with phosphorylation.
Highlights
Alzheimer’s Association and start-up funds from Drexel University
O-GlcNAc transferase (OGT) and O-GlcNAc are most abundant in hippocampus [16], a brain region implicated in learning and memory through forms of synaptic plasticity such as long term potentiation (LTP)
The enrichment of OGT and O-GlcNAc we observe in synaptosomes indicates unique targeting of O-GlcNAc modifications to neuronal synapses and suggests evolutionarily conserved functions in regulating synaptic transmission/plasticity
Summary
Reagents—Antibodies used in Western blotting were as follows: anti-synaptophysin mouse monoclonal IgG (Chemicon, Temecula, CA); anti-O-GlcNAc transferase (OGT) rabbit polyclonal (kind gift of Gerald Hart, The Johns Hopkins University, Baltimore); anti-O-GlcNAc (110.6) IgM monoclonal [36]; anti-active CaM kinase II (Thr(P)286) (Promega, Madison, WI); rabbit polyclonal IgG anti-synapsin (Cell Signaling, Danvers, MA); rabbit polyclonal IgG anti-phospho-synapsin (Ser9) (Cell Signaling); rabbit polyclonal IgG anti-phospho-Ser62/67 synapsin (PhosphoSolutions, Aurora, CO); rabbit polyclonal IgG anti-ERK 1/2 (Promega, Madison, WI); rabbit polyclonal IgG and anti-ERK 1/2 (pTpY185/187) rabbit polyclonal IgG (BIOSOURCE). Hippocampal slices were placed in the recording chamber, and the slices were completely submerged and continuously superfused with gassed ACSF (31 °C) at a constant rate (2 ml/min) for the remainder of the experiment. The mean initial slopes (between the 0 and 50% points on the rising phase) of two averaged fEPSPs were compared between the slice groups over the 60 min following LTP generation. Tissue Lysis and Western Blotting—Samples for Western blotting of hippocampal slices and fractions from synaptosome preparation were obtained by lysis in 6 M urea buffer containing 200 mM Tris, pH 7.8, and 5 mM EDTA added to two slices and subjected to sonication with a microtip on ice using a Branson Sonifier 250. Samples were sonicated on ice as described for hippocampal slice lysis, left on ice for 10 min, and clarified at 14,000 rpm for 10 min. Immunoprecipitates were washed three times in ice-cold RIPA buffer and eluted by boiling in 2ϫ SDS sample buffer
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