Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions
Postsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.
KeywordsPSD-95 KIF5 Glutamate receptor 1 Dendritic transport
Analysis of variance
Calcium/calmodulin-dependent serine protein kinase
Discs large homology
Green fluorescent protein
Glutamate receptor 1
Glutamate receptor interacting protein 1
Human embryonic kidney
Kinesin superfamily protein
Membrane associated guanylate kinase inverted-2
Member of membrane-associated guanylate kinase
Phosphate buffered saline
Proximity ligation assay
Postsynaptic density protein 95
Synaptic scaffolding molecule
Transmembrane AMPA receptor regulatory protein
Tyrosine receptor kinase B
In excitatory neurons, neuronal excitability changes synaptic function [1, 2, 3, 4, 5] by regulating expression level of synaptic scaffold proteins such as postsynaptic density protein 95 (PSD-95)—a member of the membrane-associated guanylate kinase (MAGUK) class of proteins at synapses [6, 7, 8]. PSD-95 regulates the trafficking and localization of glutamate receptors such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type or N-methyl-D-aspartate (NMDA) type-receptors . Overexpression of PSD-95 enhances the amplitude of the AMPA receptor-mediated synaptic current [1, 9, 10]. For all these reasons, PSD-95 has been implicated in synaptic development, plasticity [11, 12], and defects across several disorders [13, 14]. Despite essential roles of PSD-95 in synaptic functions, its transport and localization to dendrites are partially understood . One study reported that kinesin superfamily protein 1Bα (KIF1Bα)—a member of the kinesin-3 family—associates with MAGUKs such as PSD-95 and synapse-associated protein (SAP)-97, as well as synaptic scaffolding molecule (S-SCAM) and membrane associated guanylate kinase inverted-2 (MAGI-2), suggesting that KIF1Bα functions as a motor protein for synaptic localization of these proteins .
KIF proteins transport a various molecules, including proteins, synaptic vesicles, and mitochondria along the microtubules cytoskeleton of an axon or dendrites to synaptic regions . In particular, KIF5, which belongs to the recently classified kinesin-1 family, consists of three isoforms (A, B and C) . It transports ribonucleoprotein complexes, synaptic vesicles, mitochondria, AMPA receptor vesicles, tyrosine receptor kinase B (TrkB)-containing vesicles , and γ-aminobutyric acid (GABAA) receptor vesicles  in neurons, as well as slowly transported cargo proteins in axons . KIF5 localizes AMPA receptor vesicles to the postsynaptic regions interacting with glutamate receptor interacting protein 1 (GRIP1)—a scaffolding protein similar to a MAGUK and a member of the PSD-95/SAP90/discs large homology (DLG)/zona occludens (ZO)-1 (PDZ)-domain proteins, functioning as a synaptic scaffolding protein [21, 22]. Several studies have reported that MAGUKs are transported to membrane regions by KIF13B [23, 24].
In the present study, we revealed that KIF5 as a motor protein involved in PSD-95 dendritic transport. The C-terminal tail region of KIF5A was associated with the PDZ3 domain of PSD-95. The expression of the ADPDZ3 domain, which includes an NMDA receptor-associated domain (AD), significantly decreased levels of PSD-95 and surface glutamate receptor 1 (GluA1) at the postsynaptic site. Finally, we found that the KIF5A-PSD-95 complex colocalized with GluA1-immuopositive particles in dendritic regions, indicating that KIF5A mediates the transport of both PSD-95 and GluA1-containing vesicles. Thus, we suggest that PSD-95 works as both a scaffolding protein in the excitatory synapses and an adaptor between a cargo and motor proteins.
Expression of a dominant-negative form of KIF5A reduces level of PSD-95 in dendrites
To examine the relevance of kinesin motor protein to dendritic transport of PSD-95, we expressed either green fluorescent protein (GFP), GFP-tagged wild type (WT) KIF5A (which is enriched in neurons) , or a dominant-negative mutant of KIF5A lacking the N-terminal motor domain (ΔMD)  in cultured hippocampal neurons. We then examined PSD-95 particles in dendrites. Consistent with our previous results , ΔMD expression significantly reduced the number and average size of PSD-95 particles by 78.48 and 61.71% (Fig. 1), respectively, indicating that inhibition of KIF5A functions reduces PSD-95 levels in dendrites. Interestingly, expression of the WT did not induce any significant change.
PSD-95 colocalizes with KIF5
Interaction between PSD-95 and KIF5A requires the PDZ3 domain of PSD-95
ADPDZ3 expression reduces PSD-95 level in dendrites
ADPDZ3 expression reduces surface GluA1 level in dendrites
Staufen modulates the association between PSD-95 and KIF5A
Many previous studies have indicated that PSD-95 work as an adaptor between a motor protein and receptor−containing vesicle cargoes. For example, Mint1/X11 (mLin-10)—a PDZ domain−containing protein—interlinks NMDA receptor−containing vesicles and KIF17, which is a member of kinesin-2 family . Glutamate [NMDA] receptor subunit 1 (GluN1) and GluN2 − containing vesicles are transported to dendrites with SAP97 and calcium/calmodulin-dependent serine protein kinase (CASK), while PDZ domain−containing MAGUKs are transported by KIF17 . In addition, by directly interacting with GRIP1, which is another PDZ domain−containing scaffolding protein, KIF5C is reported to transport GluA2 − containing vesicles to dendrites . Thus, GRIP1 interlinks N-cadherin and GluA2-containing vesicles, transporting them into the dendrites . Interestingly, Huntingtin−associated protein 1 (HAP 1) works as an adaptor molecule for the dendritic transport of GABAA receptor-containing vesicles in the inhibitory synapses . Scaffolding and motor proteins carry out dendritic transport and synaptic localization of transmembrane proteins, such as receptors . Our data agree with these previous results, because GluA1 expression on dendritic membranes was dependent on KIF5 − mediated transport of PSD-95 (Figs. 6, 7a).
GRIP 1 directly interacts with KIF5 through PDZ6 and PDZ7 domain, functioning as an adaptor between KIF5A and GluA2-containing vesicles . The protein mLin-10 also directly interacts with KIF17 via the PDZ1 domain . It is likely that PSD-95 directly interacts with KIF5A, although we did not examine this possibility in the study. However, a previous study identified a putative PDZ interaction motif (class I: Ser/Thr − X − Val, S/TXV)  in the tail regions of KIF5A, and this PDZ interaction motif was only found in the KIF5A isoform . The present study corroborated previous data [21, 28], showing that PSD-95 binding to KIF5 may steer KIF5A to dendrites, as occurs when an adaptor binds to a motor protein. Another previous study suggested that PSD-95 is associated with Staufen in synaptic regions . Staufen also works as an adaptor protein for KIF5 cargoes . Consistent with these studies, in the present study, Staufen expression increased the association of PSD-95 with KIF5 (Fig. 7c and d), indicating that Staufen might modulate the PSD-95-KIF5 complex. Further studies should investigate the detailed molecular configuration of scaffolding protein−motor protein transport complexes modulated by Staufen.
Motor protein expression increases the levels of the corresponding cargo protein or associated protein in dendrites . In the present experiment, even though the dominant mutant form of KIF5A (ΔMD) significantly decreased the level of PSD-95 in dendrites, KIF5A (WT) did not increase PSD-95 transport (Fig. 1). Considering for multiple motors to be involved in the transport of PSD-95, the role of single protein might not be considerable. Alternatively, neuronal activity may be required to increase motor activity. Indeed, increases in expression levels of cargo proteins require neuronal activity . The present study has suggested a new mechanism in the dendritic transport of PSD-95 and receptor-containing vesicles in glutamatergic synapses (Fig. 7e).
Cell cultures and transfections
Rat hippocampal neurons were isolated from 1-day-old pups (Sprague Dawely, Samtako, Osan, Republic of Korea). Cultures were maintained in Neurobasal–A (Life Technologies, Carlsbad, CA, USA) supplemented with B27 supplement (Life Technologies) at 37 °C and in 5% CO2, as previously described . The cultures were performed in accordance with the approved animal protocols and the guidelines of the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-1049-17-01). Human embryonic kidney (HEK) 293 T cells were maintained in Dulbecco’s Modified Eagle’s Medium (Life Technologies) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France). With regards to transfection of the neurons, target genes were subcloned to a pSinRep5 vector for Sindbis viral expressions and packaged into Sindbis virion particles according to Invitrogen’s user manual (Invitrogen, Carlsbad, CA, USA). The Sindibis virion was directly added to the cultured neurons and incubated for 6–12 h. Transfection of the HEK cells was carried out by transferring DNA constructs using a calcium phosphate method.
Complete cDNA of mouse PSD-95 (disks large homolog 4, NM_007864) were amplified using PCR (mPSD95-R1-S → mPSD95-Xho-A) and inserted at the EcoRI/XhoI site of the pCS4-3xHA vector. To construct the mutant form of the PSD-95, full length or partial PSD-95 fragments were amplified using PCR and inserted at the EcoRI/XhoI site of the pCS4-3xHA vector (full length: mPSD95-R1-S → mPSD95-Xho-A; PDZ1: mPSD95-R1-S → PZD1-Xho-A; PDZ1–2: mPSD95-R1-S → PZD2-Xho-A; PDZ1–3: mPSD95-R1-S → PZD3-Xho-A; ∆GK: mPSD95-R1-S → SH3-Xho-A; GK: GMPK-R1-S → mPSD95-Xho-A). To allow fine mapping, PSD-95 fragments were amplified using PCR (ADPDZ3: mPSD95-ADPDZ3-R1-S → PDZ3-Xho-A2; PDZ3: mPSD95-PDZ3-R1-S → PDZ3-Xho-A2; AD: mPSD95-ADPDZ3-R1-S2 → AD-Xho-A). In the case of ADPD3 and PDZ3, the fragments were inserted at the EcoRI/XhoI site of the pCS4-3xHA vector. While in the case of AD, they were inserted at the EcoRI/XhoI site of the pCMV-myc vector. To allow Sindbis viral expression, GFP-tagged PSD-95 (PSD-95-GFP) and GFP-tagged ADPDZ3 (GFP-PDZ3) were amplified using PCR (PSD-95-GFP: mPSD95-Mlu-S → GFP-Sph-A; GFP-ADPDZ3: GFP-Mlu-S → PDZ3-Sph-A) and inserted at the MluI/SphI site of the pSinRep5 vector.
The complete cDNA of mouse Kif5a (NM_008447) was amplified using PCR (mKIF5A-Bam-S → mKIF5A-Apa-A) and inserted at the BamHI/ApaI site of the pCMV-tag2B vector. To construct mutants, the KIF5A fragments were amplified using PCR and inserted at the BamHI/ApaI site of the pCMV-tag2B vector (full length: mKIF5A-Bam-S → mKIF5A-Apa-A; 636: mKIF5A-Bam-S → mKIF5A-636-Apa-A; 826: mKIF5A-Bam-S → mKIF5A-826-Apa-A; 906: mKIF5A-Bam-S → mKIF5A-906-Apa-A; ∆MD: mKIF5A-330-Bam-S → mKIF5A-Apa-A). To allow Sindbis viral expression, wild type and ∆MD mutant cDNA was amplified using PCR (mKIF5A-Sph-S → mKIF5A-Apa-A; ∆MD: mKIF5A-330-Sph-S → mKIF5A-Apa-A) and inserted at the SphI/ApaI site of pSinRep5-mRFP, yielding the DNA constructs encoding for mRFP-tagged WT KIF5A or mRFP-tagged ∆MD. The complete cDNA of mouse Kif5b (NM_008448) was amplified using PCR (mKIF5B-Bam-S → mKIF5B-Apa-A) and inserted at the BamHI/ApaI site of the pCMV-tag2B vector. The complete cDNA of human Kif5c (NM_004522) was amplified using PCR (hKIF5C-R1-S → hKIF5C-Sal-A) and inserted at the EcoRI/SalI site of the pCMV-tag2B vector. The cDNA clones of KIF5s were provided by Dr. EY Shin (Chungbuk National University). All PCR primers for PCR were purchased from Bioneer (Daejeon, Republic of Korea). Restriction enzymes used in our experiments were purchased from New England Biolabs (NEB, Ipswich, MS, USA).
mPSD95-R1-S: 5′- ggaattcaatggactgtctctgtatagtg-3′,
mKIF5A-Apa-A: 5′: 5′-tgggcccccttagctggctgctgtctc-3′
For co-immunoprecipitation (co-IP), cell lysates were prepared by adding lysis buffer (150 mM NaCl, 1% IGEPAL® CA-630, 50 mM Tris·Cl; pH 8.0) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The lysate was immunoprecipitated using 2–3 μg of antibody (specificity indicated in the figures), mouse immunoglobulin G (IgG; Sigma-Aldrich, St. Louis, MO, USA), and incubated with 50 μL of Protein G-Sepharose (GE Healthcare, Chicago, IL, USA). The immunoprecipitates were washed three times in 1 mL of ice-cold lysis buffer, followed by additional wash an additional time with 1 mL of 50 mM Tris·Cl (pH 8.0). The precipitated proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8%–12%). For western blot analysis, the blots were incubated using the antibody indicated in the figures. All co-IPs and western blot analyses were performed more than twice to confirm that the data were reproducible. The following antibodies were used in the co-IPs and western blot analyses: monoclonal anti-FLAG antibody (1:2000, Clone M2; Sigma-Aldrich), monoclonal anti-HA antibody (1:2000, Clone HA-7; Sigma-Aldrich), and monoclonal anti-Myc antibody (1:2000, Clone 9E10; Sigma-Aldrich).
Immunocytochemistry and proximity ligation assay
For the immunocytochemistry, cultures were fixed using a fixative (4% paraformaldehyde, 4% sucrose, pH 7.2) and permeabilized using PBT (0.1% TritonX-100, 0.1% BSA in PBS). In the case of surface GluA1 immunocytochemistry, no permeabilization step was performed. The cultures were pretreated using the preblock solution (2% BSA, 0.08 TritonX-100 in PBS) for 1 h and each antibody was directly added to the preblock solution for 2 h. The following antibodies were used for staining, each at a dilution of 1:50; monoclonal anti-PSD-95 antibody (clone 6G6-1C9; Affinity Bioreagents, Golden, CO, USA), polyclonal anti-PSD-95 antibody (Cell Signaling, Danvers, MA, USA), monoclonal anti-kinesin antibody (Clone: H2; Millipore, Temecula, CA, USA), polyclonal anti-synapsin I antibody (Millipore), polyclonal anti-GluA1 antibody (Upstate, Lake Placid, NY), polyclonal anti-GluA1 antibody (Alomone Labs, Jerusalem, Israel) for surface GluA1.The following antibodies were used for secondary staining, each at a dilution of 1:200: Alexa Fluor® 488 anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA), Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), and Alexa Fluor® 647 anti-rabbit IgG antibody (Molecular Probes).
For PLA using Duolink® In Situ-Fluorescence (Sigma-Aldrich), the cultures were infected with Sindbis viruses encoding GFP to visualize whole dendritic structures and then fixed as described above; rabbit polyclonal anti-PSD-95 antibodies (Cell Signaling) and mouse monoclonal anti-KIF5 antibodies (Clone H2, Millipore) were then used. All procedures were performed according to the manufacturers’ instructions. The nucleus of each neuron was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Immunostaining and PLA were visualized using confocal microscopy (Zeiss 710; Carl Zeiss, Oberkochen, Germany).
Secondary or tertiary dendrites with a similar diameter were selected from acquired neuron images and straightened using a plugin of ImageJ program (ver 1.47; National Institute of Health, Bethesda, VA, USA). The images of straightened dendrites were transited to threshold images. The number and size of PSD-95 or GluA1 particles were measured using the particle analysis plugin. Colocalization was measured from the threshold images using colocalization plugins and represented using either Pearson’s correlation coefficient (R − value) or a percentage. All image analyses were performed by blind experiment.
Normality of the data was assessed using either the Kolmogorov-Smirnov test or the D’Agostino and Pearson omnibus normality tests. If the data followed Gaussian distribution, a Student’s t-test was performed to determine statistical significance between two groups, while analysis of variance (ANOVA) was performed among three or more groups, with Newman Keul’s analysis used as a post hoc analysis. If the data did not follow Gaussian distributions, the non-parametric Mann-Whitney test was performed to determine statistical significance between two groups, while the Kruskal-Wallis test combined with Dunn’s multiple comparison test was performed among three or more groups. All statistical analyses were performed using GraphPad prism (ver 5.02; GraphPad Software, San Diego, CA, USA).
We are very grateful to EY Shin for sharing cDNA clones of mouse KIF5A, KIF5B, and human KIF5C.
KSY, JYO, and HL performed the cell biological and biochemical experiments. KL performed the Staufen association experiment. HKK designed the experiments. HP, KSY, YSP and HKK analyzed and interpreted the results. HP and HKK wrote the manuscript. All authors reviewed and approved the manuscript.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01059654).
Ethics approval and consent to participate
Rat hippocampal neuron cultures were performed in accordance with the approved animal protocols and the guidelines of the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-1049-17-01).
The authors declare that they have no competing interests.
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