Post-translational Modification Of Sulfur Containing Amino Acid Residue:Role In The Oxidative Damage Of Synaptic Plasticity And Neuron And Mechanisms

Part1Role of redox-dependent modification on cysteine residues in the regulation of synaptic plasticity and its underlied mechanismsObjective:Redox-dependent modification on cysteine residues is a key form of protein post-translational modifications. There are a variety of forms of modified cysteine residue, such as-S-S-,-S-OH,-SO3and-SSG. Similar to protein phosphorylation, redox-dependent modification of cysteine residues can also change the structure and activity of key proteins. At present, only a few reports about the role of redox-dependent modification on cysteine residues in the synaptic plasticity have been demonstrated. In this study, we selectively reduced the oxidation of protein cysteine residues via pharmacological approaches and focused on its effects on two key features of synaptic plasticity:NMDA receptor activation and AMPA receptor trafficking.Methods:Slice preparation, electrophysiological recording, western blot analysis, surface receptor cross-linking with BS3and membrane protein extraction.Results:Thiol-specific oxidant, DTNB, inhibited NMDAR-dependent LTP and NMDAR function in the hippocampus of adult rats. Thiol reductant DTT, enhanced NMDAR-dependent LTP and NMDAR function in the hippocampus of adult rats. DTT, β-ME and glutathione can restore aged-associated deficits in the NMDA receptor function and LTP of aged rats. Antioxidant vitamin C and trolox did not affect the NMDA receptor function and LTP of aged rats. Antioxidant prevented, but reductant reversed H2O2-induced impairment of NMDAR-mediated synaptic plasticity in adult rats. Thiol reductant DTT and P-ME can also up-regulated surface expression and function of GluR2-containing AMPAR in the hippocampus. This effect may result from the inhibition of PICK1-regulated endocytosis of GluR2-containing AMPAR. Thiol reductant DTT and β-ME inhibited LTD in the hippocampus. Furthermore, oxidative stress decreased reduced thiol content and triggered an endocytosis of GluR2-containing AMPAR in the hippocampus.Conclusion:Redox-dependent modification on cysteine residues can inhibit NMDA receptor activation and AMPA receptor surface expression, which may further affect plasticity and memory. Thiol reductants can restore NMDA receptor activation and AMPA receptor surface expression and reverse aging-associated deficits in plasticity. Part2Role of S-sulfhydration on cysteine residues in the regulation of synaptic plasticity and its underlied mchanismsObjective:Recent studies demonstrate that cysteine residues can be modified by not only oxidation but also S-sulfhydration, which seems to enhance their reducing activity. S-sulfhydration is mainly mediated by a gas signaling molecule, hydrogen sulfide (H2S). It is reported that H2S signal is closely related to neurodegenerative diseases. Latest research shows that H2S adjusts the structure and activity of key targets through modifying the protein cysteine residues by S-sulfhydration. But the role of S-sulfhydration in synaptic biology has not been understood largely. In this topic, we investigated the role of H2S-mediated S-sulfhydration on cysteine residues in the regulation of LTP.Methods:Slice preparation, electrophysiological recording, western blot analysis, surface receptor cross-linking with BS3and S-sulfhydration assay. Results:H2S-mediated S-sulfhydration of cysteine residues enhanced NMDA receptor function in hippocampal CA2region and increased surface expression of AMPA receptor in hippocampal CA1region. H2S-mediated S-sulfhydration of cysteine residues up-regulated AMPA receptor function in hippocampal CA1region. This S-sulfhydration can activate postsynaptic signaling pathways that control plasticity. Endogenous hydrogen sulfide signal was essential for high-frequency stimulation-induced LTP in the hippocampus. Furthermore, restoring H2S signal using sodium hydrosulfide can reverse LTP deficits in the hippocampus of aged rats.Conclusion:S-sulfhydration of cysteine residues can enhance NMDA receptor activation and surface expression of AMPA receptor in the hippocampal CA1region, thereby regulating the hippocampal synaptic plasticity in young and aged rats. Part3Role of redox-dependent modification on methionine residue in neuroprotection against oxidative stress and its underlied mechanismsObjective:Protein-bound methionine (Met) residues can react with ROS to form methionine sulfoxide (MetO), following by reduction to methionine under certain conditions. This Met-centered redox cycle plays a critical role in scavenging free radical and maintaining protein function. Overexpression of methionine-sulfoxide reductase A (MsrA) in a variety of cells protects them against oxidative stress. The classic theory of Met-centered redox cycle indicates that the anti-oxidation of this cycle was due to the anti-oxidation activity of protein-bound Met residue; meanwhile the MsrA maintains effective concentration of Met under oxidative stress by reducing MetO to Met timely. However, many findings can not be explained by this theory. For instance, methionine oxidation in vitro requires a relatively high oxidant concentration. In aging process, a variety of oxidative products accumulates but the MetO content does not increase, while the MsrA expression declines significantly. Thus, we hypothesize that methionine oxidation in vivo may require MsrA participation. In this study, we further investigated that the role of MsrA in the anti-oxidation of Met-centered redox cycle. In addition, we firstly found two pharmacological regulators of MsrA and investigated their neuroprotection.Methods:Cell culture, Trypan blue staining, PI staining, LC-MS analysis, western blotting, biochemical assays and recombinant protein expression.Results:MsrA significantly facilitated the reaction of H2O2with L-Met and accelerated the oxidation of DTT in vitro. MsrA activity was required for the anti-oxidation of L-Met at low concentrations (1-5mM). Pre-treatment with RSV increased MsrA expression and activity in SH-SY5Y cells. Up-regulation of MsrA by RSV increased the resistant of SH-SY5Y to oxidant and neurotoxin. DMS can enhance the anti-oxidation of MsrA via participating Met-centered redox cycle and exhibit significant neuroprotection against oxidant and neurotoxin.Conclusion:MsrA significantly facilitated the reaction of ROS with L-Met and was largely responsible for the anti-oxdation of L-Met. Pharmacological regulation of MsrA may serve as a new strategy for neuroprotection, which may be due to catalytic anti-oxidation of Met-centered redox cycle.