Ubiquitin-proteasome-mediated Protein Degradation In The Synaptic Plasticity Of Hippocampal Ca1 Region

Synaptic plasticity is tightly associated with use-dependent regulation of protein turnover at the level of protein synthesis, degradation, transport and localization. To address the role of ubiquitin-proteasome mediated protein degradation (UPS) in activity-dependent synaptic plasticity a combination of a viral expression system, fluorescent imaging and electrophysiology will be employed. Our major aims are (ⅰ) to determine the spatiotemporal order of plasticity-related degradation of prominent postsynaptic proteins, (ⅱ) to assess the signaling cascades involved in UPS activation, (ⅲ) to evaluate the degree of input-specificity of protein degradation at the level of individual spines, and (ⅳ) to assess the impact of the degradation of prominent postsynaptic proteins on activity-dependent synaptic plasticity. This study will gain precise insights regarding the spatiotemporal dynamics of synaptic protein turnover and its role in synaptic plasticity.My results are summarized as follows:(1) The UPS inhibitor MG132 prevented the induction of L-LTP when the drug was applied during the induction of LTP, application one hour after LTP induction, MG132 didn' t inhibit the maintenance of L-LTP.(2) Proteasome activity was increased about 50% 10minutes after LTP induction using the "strong" tetanization paradigm, however, one hour after LTP induction, the proteasome activity decreased to control level. In the "Strong before Weak" LTP induction paradigm, weak tetanization was strong enough to increase proteasome activity about 20%, although the increment degree was lower than strong tetanization, this increment was significant difference compared to the control slices without tetanization.(3) In either "Strong before Weak" or "Weak before Strong" LTP induction paradigm, inhibiting proteasome activity during weak tetanization which can tag the synapse, extended early LTP to late LTP in S2, it means inhibiting proteasome activity can block the formation of synaptic tag. Application of UPS inhibitor during "strong" tetanization in the "weak before Strong" LTP induction paradigm prevented reinforcement and LTP induction in the "weakly" and "strongly" tetanized pathways, respectively, which indicates that UPS is involved in PRPs synthesis or tag-PRPs interaction mechanism.(4) One of the key findings was that UPS inhibition led to heterosynaptic depotentiation of established late-LTP in S1 input when inhibited proteasome during weak tetanization in the "strong-before-weak" paradigm. Such heterosynaptic destabilizing effect of UPS inhibition was abolished by concomitant inhibition of NMDA receptors or the calcium/calmodulin dependent phosphatase-calcineurin, indicating that heterosynaptic depotentiation is an active process. In addition, concomitant inhibition of protein synthesis also blocks the MG132-mediated heterosynaptic depotentiation. These results indicated that the balance between synthesis and degradation of key proteins like regulator of calcineurin (PP2B)-RCN, plays a role in conteracting heterosynaptic depotentiation.(5) The induction of L-LTP evoked an increase of PP2B and PP1 activity, in the "Strong before Weak" LTP induction paradigm, weak tetanization was strong enough to increase PP2B and PP1 activity; the increment of PP2B activity not PP1 activity was blocked by MG132. PP2B inhibitor abolished the increment of PP1 activity which indicated PP2B was involved in activating PP1. Concomitant inhibition of PP1 couldn' t block the MG132-mediated heterosynaptic depotentiation.(6) The recombinant SPAR-eGFP DNA was subcloned in a viral transfection system (SFV) and used for SPAR protein overexpression in hippocampal CA1 neurons. The SPAR overexpressing CA1 neurons show healthy morphology but with prominent spine labeling. The dendrite spine density of CA1 pyramidal cells in overexpressed eGFP-SPAR neurons was 2.67spines/μm and in overexpressing eGFP neurons is 1.26 spines/μm.(7) The strong tetanization initiated Plks mRNA transcription.(8) The strong tetanization induced the reduction of SPAR fluoresence; the reduction of SPAR fluoresence was blocked by proteasome inhibitor MG132 and cyclin dependent kinase 5 inhibitor-roscovitine, as well as protein synthesis inhibitor anisomycin, so SPAR degradation is mediated by UPS, it' s suggested that strong tetanization can induce the synthesis of Plk2, and CDK5 phosphorylate specificity site of SPAR and assist the effect of Plk2 on SPAR degradation. (9) Exclude the effect of laser bleaching, SPAR fluorescence were still decreased when protein sythesis was inhibited, the fluorescence reduction of SPAR was blocked by concomitant inhibition of NMDA receptors and proteasome activity.(10) LTP was induced in slices overexpressed eGFP-SPAR and wasn' t induced in slices overexpressed eGFP when protein synthesis was inhibited. We proposed that protein synthesis inhibitor blocked the synthesis of Plk2, SPAR can' t be phosphorylated to be degradated, which indicates that SPAR plays a role in LTP induction.