Kibra Modulates Learning And Memory Via Binding To Dendrin

BackgroundKidney and brain expressed protein?Kibra?is encoded by WW domain-containing protein 1?WWC1?and specifically expressed in kidney and in neurons of memory-related brain regions.Kibra is a synaptic scaffold protein that regulates learning and memory.Genome-wide correlation studies suggested that polymorphic loci of Kibra were correlated with memory and cognitive function.Genetic variation of WWC1 is associated with a variety of neurological diseases,such as Alzheimer's disease and Tourette syndrome.In addition,postsynaptic AMPA?alpha-amino-3-hydroxy-5-methyl-4-isoxazopropionic acid?receptors were decreased in Kibra-deficient mice,and impaired synaptic plasticity and learning and memory were observed as well,suggesting that Kibra may participate in learning and memory by regulating AMPA receptors.However,the specific mechanism is still unclear.Kibra has a tandem WW 12 domain,which is a typical class I WW domain binding to PPxY?P represents proline,x represents any amino acid,Y represents tyrosine?.Proteins containing WW domain participate in many signal cascades,such as synaptic signal transduction,Hippo tumor suppressor pathway,slit diaphragm formation and maintenance.Dendrin is mainly distributed in dendrites of pyramidal cells in hippocampal CA1 region.Kibra WW domain can specifically bind to Dendrin PPxY motif and localize Dendrin in cytoplasm and perinucleus.The function of Kibra/Dendrin interaction in the brain has not been studied.Because protein-protein interaction mediated by WW domain is usually weak,it is not known whether Kibra/Dendrin interaction is specific enough to support its cellular function.ObjectiveTo explore the structural characteristics of Kibra/Dendrin interaction and explain the specificity of Kibra/Dendrin interaction.To study the mechanism of Kibra/Dendrin interaction in learning and memory by synthesizing polypeptides to block the interaction between Kibra and Dendrin.To explore the mechanism of learning and memory impairment caused by Kibra mutation in Tourette syndrome and provide theoretical support for the prevention and treatment of Tourette syndrome.MethodsThe binding affinity of Kibra WW12 with Dendrin PY23CT or PY23-derived peptides was analyzed by isothermal titration calorimetry analysis?ITC?.Kibra WW12,Dendrin PY23CT and their complex were stoichiometrically measured by analytical ultracentrifugation?AUC?and size-exclusion chromatography?SEC?.The high-resolution structure of WW12/PY23CT complex was reconstructed by X-ray crystallography to analyze interaction between amino acid residues in the complex.The folding of WW12 and the interaction between PY23CT and WW12 were studied by nuclear magnetic resonance?NMR?.The interaction between Kibra and Dendrin in primary neurons was interrupted by peptide mediated with transmembrane peptides.And adeno-associated virus?AAV-CAG-PB-GFP?was injected in CA1 region of adult mice to block the interaction between Kibra and Dendrin.Immunofluorescence staining was used to analyze number of dendritic spines in CA1 pyramidal neurons or primary neurons after complex formation was blocked.Western Blot was used to detect the expression of synaptic-related proteins after complex formation was blocked.The electrophysiological characteristics of CA1 pyramidal neurons were recorded by whole-cell patch clamp electrophysiological technique after the interaction between Kibra and Dendrin was blocked,and microelectrode array MED64 was used to record the long-term potentiation?LTP?of CA3-CA1 synapses.Open field test,elevated plus mazeand rotarod test were used to test the anxiety,depression and the ability of movement of mice.Morris water maze?MWM?was used to test the spatial learning and memory of mice.ResultsKibra WW12 binds to Dendrin PY23CT with super-high affinity.The association between Kibra WW12 and Dendrin PY123CT,Dendrin PY23CT,Dendrin PY23CT or Dendrin PY1 was detected by ITC.The binding affinity of Dendrin PY123CT with Kibra WW12 was medium.The affinity of PY1 with Kibra WW12 was very weak?K_d=16.5±0.9uM?,while the affinity between PY23CT and Kibra WW12was very high?K_d=4.8±0.3nM?.Dendrin PY23CT promotes the folding of Kibra WW12.The ¹H-¹⁵N heteronuclear single quantum coherence spectra?HSQC?of ¹⁵N-WW12/PY23CT and¹⁵N-WW12 were measured by NMR.¹H-¹⁵N HSQC of Kibra WW12 was very inhomogeneous.The ¹H-¹⁵N HSQC spectrum of the isolated Kibra WW1?residues 5-43?indicated that WW1 is well folded.Upon binding to Dendrin PY23CT,the entire¹H-¹⁵N HSQC spectrum of Kibra WW12 underwent dramatic chemical shift changes and became highly homogenous and well dispersed.The broadening as well as small shifting of the backbone amide peaks in the Kibra WW12 ¹H-¹⁵N HSQC spectrum with respected to those in the isolated WW1 and WW2 spectra suggested that the two WW domains in the tandem transiently interact with each other.Kibra WW12/Dendrin PY23CT complex is a 1:1 hetero-dimer.According to AUC analysis,the molecular weight of WW12/PY23CT was 32.0±1.8kDa and WW12/PY23CT complex was a 1:1 dimer.SEC suggested that the measured molecular weight of WW12 and PY23CT was respectively 13.9 kDa and 18.3 kDa.And the measured molecular weight of WW12/PY23CT was 31.9 kDa,which matched with the theoretical molecular weight of 1:1 complex.However,X-ray crystallographic showed that WW12 and PY23CT formed tetramers in a ratio of 2:2,and the elongated?2 helix at the end of WW12 C?residues 85-127?formed a parallel coile-coil.The ¹H-¹⁵N hetero-nuclear overhauser effect?NOE?analysis showed that the residues from the inter-domain linker and residues from R85 to Q93 at the C-terminal extension of WW2are all well-structured.In solution the?2 of the WW12 in the complex is much shorter,indicating that the elongated coiled-coil dimer of?2 is a crystal-packing artifact.With the above analysis,we built a Kibra WW12/Dendrin PY23CT hetero-dimer structure by converting the swapped WW12 dimer into a monomer at the domain swapping site.Structural factors promoting binding of Kibra WW12/Dendrin PY23CT.According to the high-resolution structure of WW12 and PY23CT composites reconstructed by X-ray crystallography,many hydrophobic interactions,charge interactions and hydrogen bond interactions were found between C-terminal?2 helix and inter-domain linker of WW1 and WW2.Substitution of amino acids or deletion of?2 helix destroyed these effects,and ITC experiments confirmed that the binding affinity was decreased.When V228 was replaced by arginine?Arg?,the charge-charge interaction between Arg and D17/D19 was introduced.Proline?Pro?was also used to replace A229.The mutation of VA-RP further enhanced the binding ability of mutant PY23CT to WW12.Determinants of the Binding Specificity between Kibra and Dendrin.ITC-based experiments showed that the Yes-associated protein?YAP?WW12 tandem binds to Dendrin PY23CT with a K_d of 400 nM,which is⁸0-fold weaker than Kibra WW12does to PY23CT.Both the WW12 and WW34 tandems of itchy E3 ubiquitin protein ligase?ITCH?bind to Dendrin PY23CT with even weaker affinities.A series of Dendrin PY23 derived peptides with one to five Ala as the linker sequences?DN1A to DN5A?.ITC and ¹⁵N-HSQC spectra were used to detect the binding of these peptides to Kibra WW12.It was found that PY motif had the strongest affinity when it was linked by two alanine.The binding affinities of the DN3A,DN4A,and DN5A peptides to Kibra WW12 are similar to that of the wild type PY23CT peptide to WW1.And DN1A peptide has essentially no binding to Kibra WW12.Inhibition of Kibra WW12 Tandem Defects Synaptic Transmission.The binding of Kibra and Dendrin can be blocked by Synthesized PY23CT peptide?P_B peptide?without affecting other functions of Kibra and Dendrin.After the treatment of P_B peptide on primary neurons,the expression of glutamate receptor 1?GluR1?,glutamate receptor 2?GluR2?decreased,and number of dendritic spines decreased.Injection of AAV-CAG-P_B-GFP in CA1 of adult mice inhibited the interaction between Kibra and Dendrin.The number of dendritic spines in CA1 pyramidal neurons was decreased,and the expression of AMPA receptors was also decreased.The amplitude of miniature excitatory postsynaptic currents?mEPSCs?and excitatory postsynaptic currents?EPSCs?in P_B-GFP neurons were significantly decreased,but there was no difference in miniature inhibitory postsynaptic currents?mIPSCs?.And the frequency of mEPSCs and mIPSCs didn't change as well.Blocking the formation of Kibra/Dendrin complex impairs learning and memory.The field excitatory synaptic potentials?fEPSPs?were recorded at the CA1hippocampus from adult mice with the expression of P_B-GFP,P_C-GFP or GFP and LTP of fEPSPs was induced by a brief high frequency stimulation?tetanus?of the afferent fibers.Application of tetanus increased the mean amplitude of fEPSPs at CA1 neurons expressing either P_C-GFP or GFP,and this increase was sustained for more than 60 min.However,the same stimulation caused only a shorter time enhancement of the fEPSPs in neurons expressing P_B-GFP.This enhancement was decayed to the basal level within15 min,showing that inhibition of the Kibra WW12 tandem by P_B-GFP completely blocks the induction of LTP at CA1 synapses.Adult mice with the expression of P_B-GFP,P_C-GFP or GFP was trained in the hidden platform version of the Morris water maze tests.Mice with the expression of P_B-GFP had a significant longer latency and swim path length to reach the platform,as compared to those expressing P_C-GFP or GFP.On probe trials,mice expressing P_B-GFP lost their preference in searching for the target quadrant.Silencing Dendrin Impairs Synaptic Transmission,Learning and Memory.Three weeks after dendrin expression was inhibited by interfering RNA-siDDN in the hippocampus of mice,the expression of GluR1 and GluR2 in the plasma membrane was significantly decreased,while the expression of NR2A,NR2B and PSD95remained unchanged.Silencing Dendrin did not alter the resting membrane potentials?RMP?and input resistance?IR?in the recording cells.The amplitude of mEPSCs in neurons expressing siDDN decreased significantly,while the amplitude and frequency of mIPSC,frequency of mEPSC did not change.Application of tetanus increased the mean amplitude of fEPSPs at CA1 neurons in control,and this increase was sustained for more than 60 min.However,the same stimulation caused only a shorter time enhancement of the fEPSPs in neurons expressing siDDN.This enhancement was decayed to the basal level within 15 min.Mice with the expression of siDDN had a significant longer latency and swim path length to reach the platform.On probe trials,mice expressing siDDN lost their preference in searching for the target quadrant.A W88C Mutation of Kibra Impairs the Interaction of Kibra with Dendrin.¹H-¹⁵N HSQC spectra of Kibra WW12WT?wild type?/PY23CT and Kibra WW12W88C?mutant?/PY23CT were similar in general.The W88C mutation led to disappearances of amide peaks corresponding to the residues from the?2 helix and concomitant appearance of a set of new peaks characteristic of the random coil structure.These newly appeared peaks in the HSQC spectrum of the mutant protein disappeared when pH of the sample was raised to 8.0.WW12WT and WW12W88C purified from column buffer without dithiothreitol?DTT?were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?.WW12WT displayed a small fraction of inter-molecular dimer band,as the protein only contains one Cys?C50?.The WW12W88C only displayed as a monomer band but with obvious migration differences with and without the presence of?-mercaptoethanol in SDS-PAGE.Ellman assay showed that 96%of Cys in WW12W88C was oxidized and only a very small fraction of Cys was oxidized in the WT WW12 when purified under the same conditionConclusionsKibra binds with Dendrin specifically with super affinity.In addition to the direct binding between tandem WW domain and PY motif,the inter-domain linker and?2helix of Kibra WW12CT,two residues connecting two PY motifs,and the subsequent CT terminals of PY motif all participate in the Kibra/Dendrin specific binding.Kibra/Dendrin interaction is involved in synaptic transmission as well as learning and memory by regulating AMPAR targeting and synaptic transmission.Kibra W88C mutation destroys the binding of Kibra and Dendrin.The results of this study provide supports for treatment of learning and memory impairment in Alzheimer's and Tourette's syndrome.