| Part Ⅰ Activity-dependent sulfhydration signal controls long-term potentiation in the rat hippocampusObjective:Hydrogen sulfide (H2S) is an important endogenous gas neurotransmitter /neuromodulator. In recent years, more and more attention has been paid to its closely role in the various neurodegenerative diseases and psychiatric disorders, lonotropic glutamate receptors, especially N-methyl-D-aspartate subtype glutamate receptors (NMDARs) are responsible for the inducement of most synaptic plasticity in the brain, including long-term potentiation (LTP) and long-term depression (LTD). Studies have found that, restoring exogenous H2S could facilitate the induction of LTP in the hippocampus via enhancing the activity of NMDARs, but the mechanism is unknown. Latest research shows that H2S adjusts the structure and activity of key targets through modifying the protein cysteine residues via sulfhydration (SHY, that is, converts cysteine -SH groups to -SSH). But until now, there is much less evidence about the role of this endogenous novel protein post-translational modification in the synaptic plasticity. On this basis, we herein attempt to investigate the role of activity-dependent H2S, a representative molecule of sulfhydration signal, in the rat hippocampal synaptic plasticity.Methods:Hippocampal slices of rats were preparated acutely, after high-frequency stimulation (HFS) or high K+ stimulation, H2S concentrations were measured following the method which is based upon the formation of methylene blue. Treatment of inhibitors of pyridoxal-5’-phosphate (PLP)-dependent H2S-producing enzymes, amino-oxyacetate (AOA) or hydroxylamine (NH2OH), then methylene blue method was used to measure the levels of H2S after HFS, and electrophysiological recording was performed to examine the hippocampal LTP. After using lentiviral-expressed specific short hairpin RNA (shRNA) LV-CBS-RNAi to genetic knockdown cystathionine-β-synthase (CBS), which is the major H2S-producing enzyme in the brain, H2S donor sodium hydrogen sulfide (NaHS) and NMDARs co-agonist D-serine were treated, then electrophysiological recording was performed to examine the hippocampal LTP. After treatment of NaHS, D-amino acid oxidase (DAAO, which was employed to degrade endogenous D-serine) and excess exogenous D-serine, electrophysiological recording was performed to examine the hippocampal LTP. For D-serine concentrations measurement, a method which was based on the DAAO-catalyzed reaction by monitoring the fluorescence of resorufin was established. After incubating NaHS, the D-serine concentration of the hippocampal slices, primary cultures of hppocampal neurons and astrocytes were determined by interpolation from the standard curve of fluorescence.Results:(1) We found that HFS (100 Hz) and high K+ stimulation (50 mM) can both elevate H2S level significantly (Control:100.00% ± 3.26%, HFS:129.74% ± 2.81%; Control:100.00% ± 2.55%, High KC1:114.29% ±3.93%). (2) HFS-induced increase in H2S concentration was abolished by AOA (100 μM) or NH2OH (200 μM; Control:100.00% ±3.26%, HFS:129.74% ± 2.81%, HFS+NH2OH:98.39% ±4.82%, HFS+AOA:101.98% ±2.15%). (3) After treatment of AOA (100 μM) or NH2OH (200 μM), LTP in the Schaffer collateral-CAl pathway was significantly impaired (Control:150.85% ±5.08%, AOA:106.42% ±3.84%, NH2OH:120.59% ±5.17%). (4) The impairment of hippocampal LTP by CBS knockdown (LV-vector:100.00% ± 15.01%, LV-CBS-RNAi:54.23% ±3.39%) could be rescued by NaHS (100 μM) or D-serine (100 μM; LV-vector:133.06% ±3.58%, LV-CBS-RNAi:100.05% ±1.35%, LV-CBS-RNAi+NaHS: 158.50% ±5.71%, LV-CBS-RNAH-D-serine:176.33% ±8.19%). (5) It was observed that NaHS (100 uM) significantly increased hippocampal LTP from 135.58% ± 1.40% to 167.17% ±2.58% of baseline, which could be blocked by DAAO (0.5 U/mL; DAAO:117.00% ±3.03%, DAAO+NaHS:117.36% ±2.86%). (6) But in the presence of exogenous D-serine (100 μM), NaHS (100μM) failed to further increase LTP amplitude (D-serine:169.18% ±4.90%, D-serine+NaHS:164.14% ± 2.17%). (7) We next found that NaHS (50 and 100 μM) significantly increased D-serine level in a concentration-dependent manner (Control:100.00% ±11.53%,50 μM:130.94% ±3.14%,100 μM:162.75% ± 7.50%), which was mainly from astrocytes probably (Astrocyte Control:103.95% ±7.93%, NaHS:133.20% ±6.76%; Neuron Control:100.00% ± 14.42%, NaHS:111.87%±16.83%).Conclusion:Activity-triggered PLP-dependent especially CBS-derived endogenous H2S generation, is essential for the NMDAR-dependent LTP and facilitates hippocampal LTP in the Schaffer collateral-CA1 via an astrocyte-derived D-serine-dependent pathway.Part II The mechanisms underlying the regulation of activity-dependent sulfhydration on long-term potentiation in the rat hippocampusObjective:This topic research proposed the regulation on hippocampal LTP by H2S is closely related to D-serine. Although glutamate-triggered the release of astrocyte-derived D-serine has been recognized, but the mechanism still remains to be unmasked. As the synthase of D-serine, serine racemase (SR) is strongly expressed in astrocytes with CBS, which is a key synthase of H2S. Therefore, we herein attempt to investigate the mechanisms underlying the facilitation of synaptic plasticity by activity-dependent endogenous H2S, a representative molecule of sulfhydration signal, and hypothesize that H2S up-regulates D-serine availability by enhancing SR activity via promoting its sulfhydration, which may lead to glutamate/D-serine cotransmission and facilitates NMDAR-dependent LTP in the Schaffer collateral-CAl pathway.Methods:For SR activity measurement, a method based on the DAAO-catalyzed reaction by monitoring the fluorescence of resorufin was established. After incubating NaHS, the SR activity of the hippocampal slices and recombinant SR were determined by interpolation from the standard curve of fluorescence. After treatment of SR inhibitor L-serine-O-sulfate (LSOS), the D-serine concentrations were determined. We also observed the effect of H2S on sulfh yd rated and nitrosylated SR detected by biotin switch assay, and the effect of dithiothreitol (DTT, which can block the sulfhydration via breaking the disulfide bond) on sulfhydration and activity of SR. After electric stimulation at different frequency or high K+ stimulation, sulfhydration of proteins and SR was detected. After treatment of NaHS or LSOS, electrophysiological recording was performed to examine the hippocampal LTP. Eectrospray ionization mass spectrometry (ESI-MS) was utilized to detected polysulfides (H2Sn) in the degassed solution of NaHS. Hippocampal slices were preparated acutely, after treatment of sodium tetrasulfide (Na2S4, used as a donor of polysulfides), electrophysiobgical recording was performed to examine the hippocampal LTP. A rat model of aging which leads to age-associated memory impairment (A AMI) was employed, to observe the replenishment effect on D-serine lack ing-induced synaptic plasticity impairment after aging by exogenous supplement of H2S.Results:(1) Incubation of hippocampal slices and recombinant SR with NaHS (100μM) markedly elevated SR activity (hippocampal slices:Contro1100.00%±1.49%, NaHS 157.50%± 6.71% and recombinant protein:Control 100.00%±1.38%, NaHS 137.40%±1.05%), which could be prevented by LSOS (4.5 mM; Control:100.00%±11.53%, LSOS:86.51%±2.65%, NaHS:135.82%±5.61%, NaHS+LSOS:91.27%±6.91%). (2) Additonaly, the sulfhydrated SR was from its nitrosylated form probably (Control:100.98%±10.73%, Vitamin C:313.26%± 15.25%, NaHS:321.82%±35.94%, NaHS+Vitamin C:324.37%±30.18%). (3) NaHS (50 and 100μM) significantly increased sulfhydration of proteins in a concentration-dependent manner. (4) Exposure of hippocampal slices and recombinant SR to NaHS (100μM) promoted SR sulfhydration significantly (hippocampal slices:Control 99.88% ± 5.60%, NaHS 413.57%± 48.34% and recombinant protein:Control 95.00%±2.99%, NaHS 439.81%± 33.10%), and SR activity induced by NaHS could be prevented by DTT (50μM; recombinant protein:DTT 100.00% ± 4.66%, NaHS+DTT 108.53% ± 1.13% and hippocampal slices:DTT 100.00% ± 2.99%, NaHS+DTT 96.37% ± 9.31%). (5) We found that the stimulus-coupled SR sulfhydration depended on stimulation frequency (0 Hz:100.73% ± 5.29%,1 Hz:204.64%±11.19%,10 Hz: 322.57% ± 13.89%,100 Hz:506.08% ±17.35%), and the sulfhydration of SR was elevated to 181.32% ±10.86% of control by high K+ stimulation. (6) LSOS (4.5 mM) could block NaHS (100 μM)-increased hippocampal LTP (LSOS:106.51% ±2.06%, NaHS+LSOS:105.69% ± 2.73%). (7) After HFS (100 Hz), the level of D-serine in the hippocampus increased to 140.77% ±8.38% of control, which could be abolished by either NH2OH (200 μM) or AOA (100 μM; NH2OH:94.35% ±6.00%, HFS+NH2OH:94.76%±5.27%, AOA:95.06% ±3.35%, HFS+AOA: 95.82% ± 5.43%). (8) It is observed that the formation of polysulfides in the degassed NaHS solution (100 μM), including [S3]-, [S4]-, [S5]2-, [S6]2-, [S7]2-, [S8]2-. (9) We found that Na2S4 (25 μM) significantly increased hippocampal LTP from 143.04%±3.86% to 168.26%±2.70% of baseline, and rescued the LTP impairment in the CBS knockdown slices (LV-CBS-RNAi:100.05% ± 1.35%, LV-CBS-RNAi+Na2S4:162.78% ±7.17%). (10) We observed declines of H2S level (Adult:100.00% ±5.13%, Aged:66.49% ±1.74%), sulfhydrated proteins including SR (Adult: 106.32% ±3.59%, Aged:55.12% ±5.78%) and LTP in the hippocampus of aged rats, which could be rescued by NaHS (100 μM; SR sulfhydration:Aged 105.80% ±2.13%, Aged+NaHS 283.44% ±38.98%; D-serine level:Aged 100.00% ±5.14%, Aged+NaHS 235.56% ±24.09%; LTP:Aged 113.44% ±3.50%, Aged+NaHS 153.68% ± 3.98%).Conclusion:The representative sulfhydration signal molecule H2S-derived polysulfides, could up-regulates D-serine availability by enhancing SR activity via promoting its sulfhydration, and facilitates hippocampal LTP in the Schaffer collateral-CA1 pathway. Reversal of D-serine signaling by restoring exogenous H2S may have pharmacological significance to against D-serine lacking-induced synaptic plasticity impairment after aging. |
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