Mechanisms Underlying The Function Of PKCλ In LTP

Long-term potentiation (LTP) has long been postulated as a physiological substrateof learning and memory. Previous studies illustrate that AMPA receptor incorporationinto postsynaptic membrane is essential for LTP. GluA1and GluA1Ser818phosphorylation by protein kinase C (PKC) are critical for AMPA receptorincorporation. Synaptic activation of NMDAR after LTP induction can activateCaMKII-Ras-PI3K signaling pathway, which can regulate AMPA receptorincorporation into postsynaptic membrane. The product of PI3KPI-3,4,5-trisphosphate (PIP3) functions in many forms of synaptic plasticity. PIP3caninteract and activate atypical PKC (aPKC), however, whether PI3K can regulateGluA1Ser818phsophorylation and promote AMPA receptor incorporation remainsobscure, which is worthy being further studied.PKC is a multigene family consisting of at least10isoforms and can be dividedinto three groups: conventional PKC (cPKC), novel PKC (nPKC) and aPKC. cPKCand nPKC need to be activated by Ca2+and/or diacylglycerol (DAG). There are threetypes of aPKC: PKCλ, PKCζ and PKMζ, but only PKCλ and PKM exist inhippocampus. PKCλ can be activated by the product of PI3K, PIP3. PKMζ isconstitutive active due to lacking regulatory domain. Previous studies have found thatPKMζ is required for maintenance of late LTP and long-term memory. Zeta inhibitorypeptide (ZIP) has been broadly used in previous studies as specific inhibitor of PKMζ,however, it’s worth noting that the sequence of the ZIP (myr-SIYRRGARRWRKL) isalso identical to the autoinhibitory pseudosubstrate domain of PKCλ, therefore, ZIP may also inhibit PKCλ activity. Recent study has shown that the scaffold protein ofaPKC p62can bind AMPA receptor, and the phosphorylation level of AMPA receptorGluA1Ser818site and surface expression of AMPA receptor are reduced afterknock-out (KO) of p62. There is only PKCλ existing in hippocampus that can interactwith p62, because PKMζ can not bind to p62due to lacking of regulatory domain,indicating that P62may form trimeric complex with both AMPAR and PKCλ topromote GluA1Ser818phosphorylation and AMPA receptor trafficking intomembrane. Taken together, these findings give the hint that PKCλ may play a keyrole in LTP.To illustrate the function of PKCλ in LTP, we used a combination of differentmethods. First, we injected rAAV2/1virus containing EGFP-labeled PKCλ-shRNAinto dorsal CA1domain of rat hippocampus in vivo. After2-3weeks, western blotmethod was used to confirm that PKCλ was knocked down by PKCλ-shRNA. Thenwhole-cell voltage-clamped recording was conducted in CA1pyramidal cells in acutehippocampal slices to record EPSCs (excitatory postsynaptic currents). Bothpairing-induced and PI3K activator-induced LTP were disrupted after PKCλknock-down (KD). As a control, the PKCλ-scrambled-shRNA (PKCλ-scr-shRNA)did not effect pairing-induced and PI3K activator-induced LTP. Importantly, LTPdisruption caused by PKCλ KD could be rescued by further infection of mutant PKCλ(mt-PKCλ) with silent mutations resistant to RNA interference by PKCλ-shRNA.Further more, experiments with pharmacological inhibitor Myr-aPKC-PS (ZIP) wasalso carried out to further confirm our results. These results indicate that PKCλ is acritical molecule for LTP expression. Second, we conducted a series of experiments inhippocampal culture neurons, including immunofluorescence, standard surfacebiotinylation assay and mEPSCs (miniature EPSCs) recordings, and found that PKCλcan function downstream of PI3K to promote postsynaptic expression of AMPA receptor. Third, to illustrate molecular mechanisms underlying PKCλ, we usedwestern blotting to detect phosphorylation target of PKCλ and found that PKCλ canpromote AMPA receptor GluA1Ser818but not Ser831phosphorylation. Finally, wedesigned peptides to perturb PKCλ interaction with adaptor protein p62as well asp62interaction with GluA1. These peptides could disrupt AMPA receptor traffickinginto postsynaptic zone and LTP expression, showing that p62may serve as ascaffolding protein to place PKCλ in close proximity to facilitate GluA1phosphorylation and trafficking.In conclusion, we found that PKCλ is a critical signaling molecule for LTPexpression. Besides, we also found that PKCλ can function downstream of PI3K topromote phosphorylation of AMPA receptor GluA1-Ser818and promote synapticincorporation of AMPA receptor into activated synapses. Furthermore, our resultssuggested that p62may serve as a scaffolding protein to place PKCλ in closeproximity to facilitate GluA1phosphorylation and trafficking.