Shinshu Med J, 65⑷：211～224, 2017

Protein Components of the Backbone Structure of
Postsynaptic Density of Type I Excitatory Synapses
Weiheng Guo1）, Qian Liu1） and Tatsuo Suzuki1）2）＊
1） Department of Neuroplasticity, Institute of Pathogenesis and Disease Prevention,
Shinshu University Graduate School of Medicine
2） Department of Biological Sciences for Intractable Neurological Diseases, Institute for Biomedical Sciences,
Interdisciplinary Cluster for Cutting Edge Research, Shinshu University

Postsynaptic density (PSD) is a dynamic structure that functions as an essential device for synaptic trans­mission and plasticity, and is critical for normal synaptic and brain activities. Many neuropsychiatric diseases
and symptoms appear to be caused by abnormalities in synapses, including PSD. Therefore, precise and detailed
knowledge of PSD is valuable for understanding neuropsychiatric diseases and developing new therapies. In
this paper, we searched for PSDs with lattice- or mesh-like structures by preparing PSD from rat forebrainsusing different detergents, brain ages, and methods for collecting the brains. We next searched for the protein
components responsible for the mesh-like structure in PSD using sodium dodecyl sulfate polyacrylamide gelelectrophoresis/silver staining coupled with mass spectrometry and quantitative analyses of western blotting.
The results suggest that general cytoskeletal proteins, together with PSD scaffold and adaptor proteins, may
play a role in the core structure of mature PSDs. In the immature PSD core, general cytoskeletal proteins, inparticular actin and α-internexin, and synapse-associated protein 102 may be involved. However, Ca2+/calmod­
ulin-dependent protein kinase II is not a major constituent of the mesh-like PSD basic structure. Shinshu Med J
65 : 211―224, 2017
(Received for publication March 3, 2017 ; accepted in revised form April 10, 2017)
Key words : molecular architecture, postsynaptic density, type I synapse, postsynaptic density lattice,synaptic plasticity
Abbreviations : α-IN, α-internexin ; CaMKII, Ca2+/calmodulin-dependent protein kinase II ; CaMKIIα, αsub­unit of CaMKII ; CaMKIIβ, βsubunit of CaMKII ; NF, neurofilament ; DOC, deoxycholate ; IAA, iodoacetamide ;LN, liquid nitrogen ; OG, n-Octyl-β-D-glucoside ; PSD, postsynaptic density ; SPM, synaptic plasma membrane ;TX-100, Triton X-100

Ⅰ Introduction

calized immediately underneath the postsynaptic
membrane and plays an important role in signal pro­

The synapse is an essential element in neuronalcessing upon receiving neurotransmitters, which is

networks and brain activity, and postsynaptic densi­

related to the generation of synaptic plasticity. It

ty (PSD) is a major structure in the postsynaptic

is well known that abnormalities in synaptic andside of the synapse. PSD is a special cytoskeleton lo­

PSD proteins are related to neuropsychiatric disor­ders and mental retardation1）-3）. Therefore, detailed＊

Corresponding author : Tatsuo Suzuki

knowledge of PSD will provide a valuable clue to un­

Department of Neuroplasticity, Institute of Pathogenesis

derstand and develop new therapies for neuropsy­

and Disease Prevention, Shinshu University Graduate
School of Medicine, 3-1-1 Asahi, Matsumoto Nagano
390-8621, Japan
E-mail : suzukit@shinshu-u.ac.jp

No. 4, 2017

chiatric disorders. Indeed, PSD is a dynamic struc­ture and the shape and size of both PSD and spineschange when long-term potentiation (LTP) or long211

Guo・Liu・Suzuki

term depression (LTD) are induced4）. The PSD, in

Nanoprobes, Inc. (Yaphank, NY, USA). Antibodies

particular the excitatory type I, has been extensive­used in this study are listed in Table 1. All otherly studied and is reported to consist of several hun­chemicals were of reagent grade.

dreds to a thousand kinds of proteins. It is now be­

B

Preparation of synaptic plasma membranes

lieved that PSD comprises various scaffold and(SPMs) and various kinds of PSDs

adaptor proteins-the scaffold/adaptor assembly

Animals were handled according to the Regula­model. In this model, PSD is constituted of the fol­tions for Animal Experimentation of Shinshu Univer­lowing three layers : (1) a surface layer containingsity. The animal protocol, together with animal han­receptors, channels, and cell adhesion molecules ; (2)dling, was approved by the Committee for Animala layer immediately underneath the postsynaptic

Experiments of Shinshu University (Approval Num­membrane containing PSD-95 ; and (3) a layer con­ber 240066). Based on the national regulations andsisting of ProSAP/Shank. The second and third lay­guidelines, all experimental procedures were re­ers are connected mainly by the guanylate kinase-viewed by the Committee for Animal Experiments

associated protein (GKAP)/SAP-associated protein

and finally approved by the president of Shinshu(SAPAP) adaptor protein. Among the scaffold andUniversity.

adaptor proteins, the Shank/ProSAP family is con­

SPMs were prepared from Wistar rats (males, 6

sidered one of the master scaffold and adaptor pro­weeks old, specific pathogen-free ; Japan SLC, Inc.,

teins in PSD, and it nucleates the underlying struc­Hamamatsu, Japan), essentially as described previ­5）

ture of PSD . The assembly of these scaffold andously6）. Forebrains were collected on ice for 15 to 30

adaptor proteins is believed to form the lattice-likemin after dissection and then saved at -80 ℃. In

basement structure for the type I excitatory PSD

some cases, the forebrains (only 6-week-olds) were

(PSD lattice). However, there has been no direct evi­

rapidly frozen with liquid nitrogen (LN) immediately

dence for this concept, and a detailed relationshipfollowing dissection (snap frozen) or treated at 37 ℃

between the scaffold/adaptor assembly model of thefor 5 min to prepare thin or thick PSDs, respec­

PSD architecture and the PSD lattice is not clear.

tively. Preparations using forebrains without snap

In this paper, we investigated major components

freezing or 37 ℃ treatment are defined as “regularof PSDs prepared from rat forebrains under various

preparation” in this paper. Buffers A and B6） wereconditions, in which the mesh-like structure, possi­

supplemented with 2 mM IAA to prevent artificial

bly the PSD backbone structure, was observed inoxidative cross-linking during the preparation ofelectron microscopy. This study is an initial survey

SPMs. Purified SPMs were stored unfrozen in buf­

to elucidate the relationship between the scaffold/fers containing 50% glycerol at -30 ℃. Longer stor­

adaptor assembly model and the PSD lattice, and toage at -80 ℃ did not cause any deterioration of thedetermine the protein components responsible forsamples for this experiment.

the backbone structure of PSD.
Ⅱ
A

Materials and Methods

Materials

PSDs were prepared from stored SPMs by themethod reported previously7）. The flow chart in
Fig. 1 summarizes this method, which is suitable forthe efficient preparation of various kinds of PSDs

Triton X-100 (TX-100), iodoacetamide (IAA), andfrom small amounts of brains and SPMs. In brief,

ImmunoStar LD were purchased from WAKO Pure

PSDs were isolated as pellets after treatment ofChemical Industries Ltd. (Osaka, Japan), n-Octyl-β-

SPMs (500 µg protein/1.75 mL) with TX-100 or OG

D-glucoside (OG) from Dojindo Laboratories (Kuma­

in 20 mM Tris-HCl (pH 7.4) containing 150 mM NaClmoto, Japan), protease inhibitor cocktail (P8340) fromand 1 mM EDTA (TNE buffer), followed by sucrose

Sigma-Aldrich (Saint Louis, MO), pepsin (S3002) fromdensity gradient centrifugation8）9）. TX-100 and OG

DAKO (Carpinteria, CA, USA) and Nano-W fromhave been used for purification of PSD before. We

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PSD lattice proteins
Table 1

List of antibodies used in this study

Antibody

species ofantibody

dilution＊

Product Code Provider

anti-actin

Mouse

1/600

pan Ab-5

Thermo Scientific(Cheshire, UK, USA)anti-α-IN

Rabbit1/100,000

AB5354

Chemicon International(Billerica, MA)

anti-CaMKII

Mouse

1/2,000

611292

Transduction Laboratories(Lexington, KY, USA)anti-chapsyn110

Rabbit1/2,000

PA1-043

Thermo Scientific(Rockford, IL, USA)anti-fodrin (non-erythroidα-spectrin) Mouse

1/500

MAB1685

Chemicon International(Billerica, MA, USA)anti-Homer

Goat

1/400

sc-8921

Santa Cruz Biotechnology Inc.
(Santa Cruz, CA, USA)anti-NF-H/Neurofilament 200

Rabbit1/5,000

N4142

Sigma-Aldrich(Saint Louis, MO, USA)anti-NF-M/Neurofilament 150

Rabbit1/6,000

AB1981

Chemicon International(Billerica, MA, USA)anti-NF-L/Neurofilament 70

Mouse

1/300

MAB1615

Chemicon International(Billerica, MA, USA)anti-PSD-95

Mouse

1/1,000

610496

Transduction Laboratories(Lexington, KY, USA)anti-SAP97

Mouse

1/1,000

VAM-PS005

StressGen Biotechnologies Corp
(Victoria, BC, Canada)anti-SAP102

Mouse

1/1,000

N19/2

UC Davis/NIH NeuroMab Facility(Davis, CA, USA)anti-SAPAP/GKAP

Mouse

1/500

N127/31

UC Davis/NIH NeuroMab Facility(Davis, CA, USA)anti-Shank1

Mouse

1/100

N22/21

UC Davis/NIH NeuroMab Facility(Davis, CA, USA)anti-Shank2

Mouse

1/100

N23B/6

UC Davis/NIH NeuroMab Facility(Davis, CA, USA)anti-tubulin

Rabbit1/100,000

produced in a rabbit using pig
tubulin as antigen＊Membranes were incubated with primary antibodies usually at 4 ℃ overnight. Dilutions are typical ones.

Fig. 1 Flow chart of the preparation of various types of PSDs.
Forebrains of 6-week-old or 7-day-old rats were used. Forebrains were dissected, placed on ice, and subsequent­ly frozen or snap frozen with LN. The preparations using forebrains without snap freezing or 37 ℃ treatment are“regular preparation”. SPM was treated with either TX-100 or OG. Under combinations of different conditions, vari­
ous types of PSDs were prepared. Different types of PSDs used for identification of mesh-like structures in thisstudy are listed. SDG refers to sucrose density gradient centrifugation. PSDs, postsynaptic densities ; LN, liquid ni­
trogen ; SPM, synaptic plasma membrane ; TX, Triton X ; OG, n-Octyl-β-D-glucoside.
No. 4, 2017213

Guo・Liu・Suzuki

prepared two-types of PSDs (TX-100-PSD and OG-

with IAA, and digested in-gel with trypsin. The re­PSD), since we thought that components essential

sulting peptide mixtures were extracted in a buffer

for backbone structures of PSD may be commonly

containing 50% acetonitrile and 5% trifluoroaceticpresent in these different kinds of backbones. All

acid, and analyzed by mass spectrometry using rat

PSD pellets were resuspended in 955 µl of TNE buf­

UniProtKB database and IDENTITYE, which con­fer. Detergent treatments were supplemented withsists of nanoACQUITY, Xevo QTof MS and Protein­protein inhibitor mixtures (P8340 ; Sigma-Aldrich) at

LynxTM Global SERVER (PLGS) 2.5.2. (Nihon Waters

1/200 dilution along with IAA (2 mM).

K.K., Tokyo, Japan).

C

Electron microscopy

Ⅲ

Electron microscopy was carried out as described

Results

previously9）. Briefly, samples were fixed with 1%

The meshwork-like structures of PSD, such as

glutaraldehyde, postfixed with 1% osmiumtetroxide,

PSD-lattice, have been reported since the 1970’s indehydrated, and embedded in Epon. An ultrathin

deoxycholate (DOC)-treated SPM10）-13）. Here, we ob­

section was cut and stained with uranyl acetate andserved the presence of mesh-like structures in thelead citrate. For the negative staining, 5 µl of the

PSDs prepared under various conditions, such as

samples was spotted for a few to 15 min to glow-dis­treatment of SPM with high concentration of TX-

charged formvar/carbon-coated copper grids at 0 ℃,

100, in immature PSDs, and in PSDs obtained fromand then excess liquids were blotted using a pipette.

snap frozen brains. Typical examples of electron mi­

Grids were then negatively stained by placing themcrographs (thin sections) of PSDs prepared underon 5 µl of Nano-W (Methylamine Tungstate) at roomthese different conditions are shown in Fig. 2. Ar­

temperature twice according to the manufacturer’s

rows indicate typical lattice-like structures in PSD.

instructions. Specimens were examined under a

PSDs prepared after treatment of SPM with 5%TX-

JEOL JEM-1400EX (Tokyo, Japan) electron micro­

100 appeared to unfold the tightly organized PSD

scope at 80 kV. Contrast and sharpness (unsharp

structures (Fig. 2B). In immature PSDs (7-day-old),mask) of images were modified using Photoshop if a

the basement backbones with meshwork-like struc­clearer visualization of the image was required.

ture, possibly before heavy association of varyingD

PSD proteins, were visible (Fig. 2C, D). PSDs pre­Analyses of protein and western blotting
For protein profiling, each PSD preparation waspared from snap frozen brains displayed structures

separated via sodium dodecyl sulfate polyacrylamidethat were more scarce than the PSDs prepared fromgel electrophoresis (SDS-PAGE) and the gels werebrains collected without immediate freezing, as westained with silver. Western blotting was carried out

reported previously (Fig. 2E, F)14）. These lattice-likeusing a chemiluminescent substrate and visualized

structures were difficult to detect, in general, in thewith a CCD video camera system (Atto Densito­

1%TX-PSD and 1%OG-PSD prepared under regu­

graph Lumino CCD AE-6930, ATTO Bioscience &lar conditions from adult brains, because of theTechnology, Tokyo). Chemiluminescence of eachdensely packed proteins associated with matureband was measured by ATTO Lane analyzer 10H

PSDs (Fig. 2A).

(Atto Bioscience & Biotechnology, Tokyo). StatisticalIn the negative staining of mature PSDs (6w-

analyses were performed using GraphPad Prism

1%TX-PSD), no mesh-like structure was observed,

version 4.00 (GraphPad Software, San Diego, CA,

which is probably due to the number of mate­USA). Results were considered statistically signifi­

rials packed on them (Fig. 3A). In contrast to thecant at P < 0.05.

1%TX-PSD, it was easier to see the inside structure

Eof PSD and meshwork-like structures in the case of

Mass spectrometry
The protein bands stained with silver were cut,

1%OG-PSDs (Fig. 3B). In the 5%TX-PSD fromde-stained, reduced with dithiothreitol, alkylated

6-week-old rats, some of the PSD structures tended

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PSD lattice proteinsFig. 2 Electron microscopic observation of various PSDs (thin section).
Typical examples of electron micrographs of PSDs purified under various conditions. Conditions are indicatedat the top of the electron micrographs. Typical mesh-like structures are indicated with arrows. 6w (6-week-old)and 7d (7-day-old) indicate the ages of the rats. Scales are 200 nm. PSDs, postsynaptic densities.

to be unfolded and the lattice-like structures were

gested the formation of mesh-like or lattice-likedisclosed (Fig. 3C). In 7d-1%TX-PSD (Fig. 3D), pack­structures.

ing of fine molecules was observed, but not the lat­

Furthermore, we investigated the protein constit­

tice-like structures. In the 7d-1%OG-PSD (Fig. 3E)

uents of PSDs in which lattice-like structures wereand 1%LN-PSD (both 1%TX- and 1%OG-PSDs) (Fig.

observed. Proteins in these PSDs were separated

3F, G), structures inside the PSD were more clearlyand visualized by silver staining (Fig. 4 A, B). Ac­

visible compared with 6w-1%TX-PSD, which sug­cording to the SDS-PAGE profile shown in Fig. 4A,

No. 4, 2017215

Guo・Liu・Suzuki

Fig. 3 Electron microscopic observation of various PSDs (negative staining).
Typical examples of electron micrographs of PSDs purified under various conditions. Conditions areindicated at the top of the micrographs. Arrows indicate typical mesh-like or lattice-like structures.
PSDs in B, C, E, F and G appear to be entirely mesh-like. 6w (6-week-old) and 7d (7-day-old) indicate
the ages of the rats. Scales are 100 nm. PSDs, postsynaptic densities.

concentrations of some proteins were high in 7d-PSD,

profiles visualized with enhanced silver staining (Fig.

in particular the 45-kDa protein in 7d-1%TX-PSDs

4C, D ; see also Table 2). The 45-kDa protein wasand 45-kDa and 31-kDa proteins in 7d-1%OG-PSDs

identified as actin. The 31-kDa protein was a mix­

(open arrowheads in Fig. 4A). In LN-PSDs, pro­ture of at least three proteins, ADP/ATP translo­

tein recoveries were reduced compared withcase (AAT), prohibitin, and 14-3-3, as reported pre­regularly prepared PSDs (lane marked “6w” inviously15）, with AAT being the major one. AbundantFig. 4B). More specifically, CaMKIIα contents

cytoskeletal proteins, although less abundant thanwere reduced in both TX- and OG-PSDs preparedactin, included α-IN, neurofilament-L, and tubulin

from snap frozen forebrains. In contrast, CaMKIIαin the 7d-TX-PSD. Tubulins were also abundant in

was increased in TX- and OG-PSDs prepared fromthe 7d-OG-PSD, but to a lesser extent than actin.

forebrains incubated at 37 ℃ for 5 min immediately

Moreover, α-IN was also detected in 7d-OG-PSDs,

after dissection (Fig. 4B). This result is in good agree­

although to a lesser extent than in 7d-TX-PSDs. In

14）ment with our previous findings . Moreover, the 31-

the 1%TX-LN-PSD, bands including α-IN, actin,

kDa protein (arrow in Fig. 4B) was highly enriched

and tubulin were abundant. Similar to the 7d-OG-in the OG-LN-PSD, as well as in other OG-PSDs.

PSD, actin and 31-kDa protein were also abundant

Major proteins in these PSDs from 7-day-old andin the 1%OG-LN-PSD. Proteins other than the cyto­

snap frozen forebrains were identified by mass spec­

skeletal proteins were also identified, but their in­trometry, and the results are indicated in the protein

volvement in the PSD structure remains to be clari­216

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PSD lattice proteinsFig. 4 Protein components of various PSDs.
A. Protein profiles of PSDs, in both 1%TX(Triton X)-100-PSD and 1%OG(n-Octyl-β-D-glucoside)-PSD, preparedfrom forebrains at 7-day-old and 6-week-old (7d and 6w, respectively). Protein amounts in each lane were adjusted by
the corresponding total protein amount measured by densitometry of silver-stained lanes and analysis using NIH Im­
age. Thus, protein amounts were the same in each lane. B. Protein profiles of PSDs, in both 1%TX-PSD and 1%OGPSD, prepared from snap frozen forebrains with liquid nitrogen (LN), placed on ice for ～30 min or placed at 37 ℃ for
5 min after dissection (LN, 6w or 37, respectively). Equal volumes (20 µl) of PSD samples were loaded in each lane. C, D.
Identification of major protein bands contained in the different PSDs using mass spectrometry. Silver staining was en­
hanced compared with those shown in A and B, and thus, the number of protein bands visible was increased. Num­bers to the left of the gel images indicate molecular weights in kDa. Abbreviation of protein names : A, actin ; α-IN, αinternexin ; AAT, ADP/ATP translocase ; AP, Adaptor related protein complex ; ATS, ATP synthase ; CK, Creatine
kinase ; clasp, CLIP-associating protein ; CLIP, cytoplasmic linker protein ; Dlg, Disks large homolog ; DLAT, Dihydroli­
poyllysine residue acetyltransferase component of pyruvate dehydrogenase complex ; DLST, Dihydrolipoyllysine resi­
due succinyltransferase component of 2 oxoglutarate dehydrogenase complex ; EF, elongation factor Tu or 1α ; FH,
Fumarate hydratase ; Glu Tr, Glutamate transporter, hsc71, heat shock cognate 71 ; hsp70, heat shock protein 70 ; MAP,
microtubule-associated protein ; myosin HC, myosin heavy chain ; NF-M, neurofilament-M ; Ogdh, oxoglutarate dehy­
drogenase ; P-carrier, phosphate carrier protein ; PDH, pyruvate dehydrogenase complex ; PSDs, postsynaptic densi­
ties ; syntaxin-BP, syntaxin-binding protein ; syntaxin-BP, syntaxin-binding protein ; T, tubulin. For details, see Table 1.

fied. Typical scaffold and adaptor proteins in the

TX-PSD was high (data not shown) as well as in 1%

PSD were not identified by mass spectrometry of

TX-PSD, although mesh-like PSD structures werethe major bands, possibly because of the small

observed in the former preparation. Although PSDs

amounts.

were highly unfolded, our observations suggest that

The number of protein bands contained in the 5%
No. 4, 2017

many proteins were not extracted, but remained217

Guo・Liu・Suzuki
Table 2
⑴ List of the major proteins contained in the 7d-1%TX-PSD derived
from forebrain SPM
Protein name
UniprotKB/Swiss-Prot
spectrinβ
Q5D002
myosin HC (myosin heavy chain) Q9JLT0
MAP1b
B0BNK3MAP2P15146, D3ZW41
polyubiquitin
Q63429, P0CG51
oxoglutarate dehydrogenase Q5XI78(Ogdh)
Adaptor related protein complex P 62944 , Q 3 Z B 97 , P 52303 , P 18484 ,(AP1/2)
Q66HM2, D3ZUY8
α-internexin (α-IN)
P23565
P08461
Dihydrolipoyllysine
residue acetyltransferase
component of pyruvate
dehydrogenase complex (DLAT)
neurofilament H (NF-H)
P16884
Fumarate hydratase (FH)
Q5M964
Tubulin (T)P68370, Q6P9V9, Q68FR8,Q6AYZ1, Q5XIF6, Q6AY56,
Q4QRB4, P69897, P85108,
Q3KRE8, Q6P9T8, Q4QQV0,
CaMKIIβ
P08413, Q63094
CaMKIIαP11275Actin (A)P 63259 , P 60711 , P 63269 , P 68035 ,P62738, P68136⑶ List of the major proteins contained in the LN,6w-1%TX-PSD derived
from forebrain
SPM Protein name
UniprotKB/Swiss-Proto x o g l u t a r a t e d e h y d r o g e n a s e D3ZQD3(Ogdh)
Dihydrolipoyllysine residue acetyl­ P08461
transferase component of pyru­
vate dehydrogenase complex(DLAT)
Fumarate hydratase (FH)
P14408syntaxin-binding protein (syntax­ P61765
in-BP)
α-internexin (α-IN)
P23565Glutamate transporter (Glu Tr)
Q8K5B5, Q91ZA9
Dihydrolipoyllysine residue succi­ Q01205, D0VYQ0nyltransferase component of 2 ox­
oglutarate dehydrogenase com­plex (DLST)
ATP synthaseα(ATSα)P15999Tubulin (T)P69897, Q4QRB4, Q3KRE8, P85108
αsubunit of Ca2+/calmodulin-de­ P11275
pendent protein kinase II (CaMKIIα)Actin (A)P 60711 , P 63259 , P 62738 , P 63269 ,P68136, P68035
creatin kinase (CK)Q5BJT9, P25809
pyruvate dehydrogenase complex P26284, Q4FZZ4(PDH)⑵ List of the major proteins contained in the 7d-1%OG-PSD derived
from forebrain SPM
Protein name
UniprotKB/Swiss-Prot
spectrinβ
Q5D002
myosin HC (myosin heavy chain) Q9JLT0
microtubule-associated
B0BNK3protein 1b (MAP1b)
polyubiquitin
Q63429, P0CG51
clasp1
Q5M928clasp2D3ZZU1, D4A289, D3ZG34
CLIP
Q99JD4
Src kinase
Q9QXY2
Leu-rich PPR motif-containing Q5SGE0
proteinneurofilament-M (NF-M)
P12839oxoglutarate dehydrogenase Q5XI78(Ogdh)
Adaptor related protein complex P62944, Q3ZB97, P52303(AP1/2)
trifunctional subunitα
Q6442878 kDa glucose regulated protein P06761
heat shock cognate (hsc71)
P63018heat shock protein (hsp70)
P55063α-internexin (α-IN)
P23565Tubulin (T)P68370, Q6P9V9, Q6AYZ1, Q68FR8,Q5XIF6, Q6AY56, Q4QRB4, P85108,
Q3KRE8, P69897, Q6P9T8, Q4QQV0
CaMKII β
P08413, Q63094
ATP synthase (ATS α )P15999Dihydrolipoyllysine residue suc­ D0VYQ0cinyltransferase component of 2
oxoglutarate dehydrogenase
complex (DLST)EF
P85834, D3ZC88, P62630, D3ZRH9,
D4A0X4, D3ZXS6, P62632
Fumarate hydratase (FH)
P14408, Q5M964
septin5Q9JJM9P-carrierP16036
ADP/ATP translocase (AAT)
Q09073, Q05962
prohibitin
P6777914-3-3 protein
P 63102 , P 61983 , P 68511 , P 68255 ,P35213, P62260⑷ List of the major proteins contained in the LN,6w-1%OG-PSD derived
from forebrain
SPM Protein name
UniprotKB/Swiss-Prot
ATPaseP 06687 , P 06686 , P 06685 , Q 64541 ,Q5PQV3,clathrin H
P11442o x o g l u t a r a t e d e h y d r o g e n a s e Q5XI78(Ogdh)
Disks large homolog2 (Dlg2)
Q63622, D4AA89
Glutamate receptor 2/3 (GLuR2/3) P19491, P19492
Dihydrolipoyllysine residue acetyl­ P08461
transferase component of pyru­
vate dehydrogenase complex(DLAT)
α-internexin (α-IN)
P23565syntaxin-binding protein (syntax­ P61765
in-BP)
ATPase family AAA domain con­ Q3KRE0
taining protein
Glutamate transporter (Glu Tr)
Q8K5B5, Q91ZA9
CaMKIIβ
P08413, Q63094
Tubulin (T)Q6P9V9, P68370, Q6AYZ1, Q5XIF6,Q68FR8, Q6AY56, Q4QRB4, P85108,
Q3KRE8, P69897, Q6P9T8, Q4QQV0
ATP synthaseα(ATSα)P15999CaMKIIαP11275
ATP synthaseβ(ATSβ)P10719Actin (A)P 60711 , P 63259 , P 62738 , P 68035 ,P68136, P63269
creatin kinase (CK)Q5BJT9ADP/ATP translocase (AAT)
Q09073, Q05962
prohibitin
P6777914-3-3 protein
P 63102 , P 61983 , P 68511 , P 68255 ,P62260218

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PSD lattice proteinsattached to the PSD core structures after treatment

most of the scaffold/adaptor proteins and cytoskele­with high concentrations of TX-100.

tal proteins examined in LN-PSDs were at the same

Subsequently, we carried out western blotting tolevel with 6w-PSDs. Chapsyn-110 and SAP102 werecompare the concentrations of typical PSD proteinsconcentrated exceptionally in LN-PSDs for unknownin various PSD preparations. In particular, we com­

reasons. Concentrations of CaMKIIα and CaMKIIβpared between 7d-PSD and 6w-PSD and between

in LN-PSDs were lower than in 6w-PSDs in both

LN- and 6w-PSD, focusing on scaffold/adaptor pro­

TX- and OG-PSDs. The results of the quantitative

teins and cytoskeletal proteins. Figures 5 and 6 illus­

study are summarized in Table 3.

trate representative western blots and their quanti­
tated results. In the comparison between 7d- and 6w-

Ⅳ Discussion

PSDs (Fig. 5), concentrations of scaffold and adaptor

The presence of meshwork-like structures wasproteins, except for SAP102, were very low in bothdemonstrated under various conditions, such as ex­

7d-TX-PSDs and 7d-OG-PSD. The concentration oftreme solubilization of PSDs (e.g., with 5% TX-100

SAP102 was comparable between 7d- and 6w-PSDs.

or DOC) and PSDs prepared from snap frozen brains

The concentration of Shank1 was also comparable

using LN. Snap freezing and 37 ℃-treatment of the

between 7d-TX- and 6w-TX-PSDs, while it decreased

dissected brains result in thinning and thickening ofin 7d-OG-PSDs. These results are in good agreement

PSD, respectively. Thinning or thickening of PSD iswith the developmental changes in the expression of

mainly due to scantiness or abundance of CaMKII14）.

these proteins in the brain and synapses. In particular,

Following brain dissection, CaMKII is activated by

16）
-18）

and

Ca2+ influx into damaged neurons, which causes au­

some type of Shank16）18） supports our results. The

tophosphorylation and clustering of the kinase, andconcentration of cytoskeletal proteins in 7d-PSDs

finally accumulation into PSD20）. This postmortem ef­

was equal to or higher than in 6w-PSDs, except forfect is enhanced by incubation of dissected brains at

NF-H (Fig. 5B). Moreover, concentrations of actin

37 ℃ causing augmented thickening of PSD. In thisand α-IN were significantly high in OG-PSDs pre­paper, we used snap freezing to prepare thin PSD in

pared from 7d-old forebrains, although not signifi­

order to search for its essential components.

early developmental expression of SAP102

cant in TX-PSDs. Concentrations of CaMKIIα andElectron microscopic analyses of 7d-PSDs sug­

CaMKIIβ in 7d-PSDs were lower than those in 6w-

gested that meshwork-like structures are already

PSDs (Fig. 5C). This result is in agreement with theformed in the early synaptogenesis stage10）-13）. Simi­

developmental changes of CaMKII expression in thelar intercalating PSD fiber network structures have19）

brain and synapses .

been clearly shown in a synaptic complex prepared

Fig. 6 illustrates the concentrations of scaffold andusing p-iodonitrotetrazolium and further treatedadaptor proteins in 6w-LN- and 6w-PSDs. Concen­with urea (1 or 8 M) and mild sonication21）. It is like­trations of chapsyn-110 appeared to be high in bothly that p-iodonitrotetrazolium used in order to ex­

TX- and OG-LN-PSDs. The concentration of SAP102

clude mitochondrial contamination unintentionallywas significantly higher in LN-TX-PSDs comparedcross-linked the PSD structure through oxidation.

with 6w-TX-PSDs, whereas there was no difference

Thus, it prevented the disorganization of the PSD

between LN-OG- and 6w-OG-PSDs. The difference

basic core structure when treated with high concen­

between the OG- and TX-PSDs could be caused bytrations of urea, which dissociates most of the pro­the different properties of the two detergents. Other

teins that are attached to the PSD core. PSD proteinsscaffold and adaptor proteins appeared to be theother than those constructing PSD core structures

same between LN- and 6w-PSDs. Concentrations of

may be very densely attached to the core structure.

the cytoskeletal proteins examined were not signifi­

This makes the mesh-like structures underneathcantly different between LN- and 6w-PSDs. Thus,

rarely detectable in regularly prepared PSDs in both

No. 4, 2017219

Guo・Liu・Suzuki

Fig. 5 Western blot analysis of typical PSD proteins contained in the PSDs prepared from 7-day-old rat
forebrains (7d) compared with those in 6w-PSDs (6w).
A. Scaffold and adaptor proteins. B. Cytoskeletal proteins. C. Ca2+/calmodulin-dependent protein kinase II(CaMKII). Same amounts of proteins in the 7d-, LN- and 6w-PSD were run on a single gel. Western blotting of
7d-, LN- and 6w-PSD was carried out in the same blot, and conditions visualizing the immunoreactive bands
are the same in each comparison. Immunoreactivity was measured by densitometry using video CCD camera
and quantified by ATTO lane analyzer, and ratios against the values of standard band were sought. The aver­aged values of the ratios of 6w-1%OG (n-Octyl-β-D-glucoside) and 6w-1%TX (Triton X), respectively, wereset at 100%. Vertical values (means ± standard error of mean) represent percentage of the signals to those of
6w-1%OG and 6w-1%TX. ＊P<0.05, unpaired Student’s test (two-tailed). Arrows indicate 95-kDa GKAP protein
and 110-kDa immunoreactive protein, as commented on by the manufacturer. Only 95-kDa bands were quanti­
tated. Abbreviations of the protein names are the same as those in Fig. 4 legend. PSD, postsynaptic density.

regular thin sections and negatively stained electron

adult PSDs22）, and in purified immature and mature

micrographs (partly shown in Fig. 2, 3). In the 7d-

PSDs by electron cryotomography23）. Therefore, thePSDs, relatively few proteins were associated withmeshwork structure does not appear to be an arti­

the core mesh-like structure, which enabled its

fact produced after treatment of synapses with de­visualization. Mesh-like structures similar to PSD

tergents. Thus, this experiment supports the idea

lattice were also indicated in the platinum-shadowedthat the mesh-like structure is present underneath

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PSD lattice proteinsFig. 6 Western blot analysis of typical PSD proteins contained in the PSDs prepared from LN-treated
forebrains (LN) compared with those in 6w-PSDs.
A. Scaffold and adaptor proteins. B. Cytoskeletal proteins. C. Ca2+/calmodulin-dependent protein kinase II(CaMKII). Experiment was carried out as described in Fig. 5 legend. Vertical values (means ± standard error ofmean) represent percentage of the signals to those of 6w-1%OG (n-Octyl-β-D-glucoside) and 6w-1%TX (Tri­ton X). In the cases indicated by†, where standard values were extremely low, ratios of LN-PSD vs. 6w-PSD
values were processed to avoid high extent of variation of the final ratios. Thus, values in the 6w-PSD columns
are fixed to 100% with no error bars. ＊P<0.05, unpaired Student’s test (two-tailed). PSD, postsynaptic density.

the PSD and that it is a core PSD structure. The

posed mainly of scaffold and adaptor proteins or

meshwork-like structure mentioned above may bewhether other proteins also play a major role. Major

the same as the PSD lattice, which was first report­

or key components and the molecular organization

ed in the 1970’s.

of the PSD lattice have not yet been identified be­

It is believed that different types of scaffold andcause there remain a number of proteins in the DOC-

adaptor proteins are interwoven to make PSD lat­

insoluble PSD lattice preparations10）13）. Therefore, intice-like network structures. However, there has

this work, we surveyed the key components thatbeen no sound evidence supporting this concept. We

construct the core structure of PSD. Firstly, we

also do not know whether the PSD lattice is com­compared the protein profiles between regularly

No. 4, 2017221

Guo・Liu・Suzuki
7d-PSD

LN, 6w-PSD

SAP102～↑chapsyn-110↓↑

other sc/ad proteins↓～

actin & α-IN↑～other CSK proteins～～

CaMKII↓↓

Table 3 Changes in protein amounts in 7d- and LNPSDs compared with regular mature PSDs
(6w-PSDs) sc/ad proteins, scaffold and adaptor pro­teins ; CSK proteins, cytoskeletal proteins ; (～),
same level

prepared PSDs and those enriched with mesh-likeprimarily in the younger age among all the PSD-95-

structures (“regular preparation”).

like subfamily of the membrane-associated gua­

In 7d-1%TX-PSD, actin was the most abundantnylate kinase (MAGuk) proteins (PSD-95, PSD-93,

protein, while 7d-1%OG-PSDs were enriched with

SAP102, and SAP97) 24）. Thus, it is possible that, in­both actin and 31-kDa proteins. AAT, the major

stead of PSD-95, it is SAP102 that plays a role in thecomponent of the 31-kDa proteins, did not appear to

PSD core formation and maintenance in immature

be localized to PSD, because pre-embedding elec­

PSDs. The concentration of Shank1 in 7d-TX-PSD

tron microscopic examination did not detect any sig­

was at the same level as the 6w-TX-PSD, althoughnals of ATT in the PSD (data not shown). In both

the reason remains unknown. In contrast, the con­

TX- and OG-LN-PSDs, CaMKII content was ex­

centrations of general cytoskeletal proteins, except

14）

tremely diminished as reported previously , but nofor NF-H in OG-PSD, were the same or even higher

apparent changes were observed in other proteinsin the immature PSD fractions compared with regu­

as far as silver-stained protein profiles were con­larly prepared adult PSDs. These results suggest an

cerned. Unfortunately, a number of proteins wereinvolvement of general cytoskeletal proteins, except

still observed in these 7d- and LN-PSDs as well as

for NF-H, in the core structure of immature PSDs.

in the regularly prepared adult PSDs in a one-di­

In particular, actin and α-IN may play major roles

mensional gel electrophoresis when the silver stain­in the formation of PSD lattice/meshwork. As foring was enhanced (Fig. 4C, D). Therefore, it was dif­

NF-H, it has been reported, together with NF-M,

ficult to identify key components or acquire infor-

not to be a constituent of PSD25）.

mation on the molecular organization of the PSD core

In the comparison between LN- and 6w-PSDs,

structure even after mass spectrometry of protein

both most scaffold-adaptor proteins and general cy­

bands of both 7d- and LN-PSDs.

toskeletal proteins were comparable with the adult

Subsequently, we focused on the PSD scaffold-

PSD. Chapsyn-110 and SAP102 were concentrated

adaptor proteins and cytoskeletal proteins, and com­in LN-PSDs. The levels of CaMKII, in particular thepared their contents between regularly prepared adultαisoform, were decreased compared with regularly

PSDs and those enriched with mesh-like structures

prepared PSDs, which is in line with our previous

by western blot-based quantitation. The results are

findings14）. These results suggest that both scaf­

summarized in Table 3. In the 7d-PSDs, contents offold-adaptor proteins and general cytoskeletal pro­scaffold and adaptor proteins, except for SAP102, were

teins are part of the core structure proteins in ma­

extremely low. This was also the case for CaMKIIαture PSDs. It appears that brain snap freezing

and CaMKIIβ. These differences may reflect devel­

prevents tight association of CaMKII with PSDs andopental changes in the expression of these pro­

reduces relatively specifically CaMKII in particular,

teins. It is well known that there is a developmental

and possibly CaMKII protein complexes as well, in

switch in the expression level from SAP102 to PSD-

LN-PSD preparations. CaMKII forms clusters and95, which means that SAP102 expression is present

make PSDs highly electron-dense if associated with

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PSD lattice proteins

PSDs, as shown in thickened PSDs14）. In LN-PSDs,

Acknowledgementsinternal structures could be visualized by the ab­

We acknowledge Dr. K. Kametani, Shinshu Uni­sence of most CaMKII and CaMKII clusters.
In summary, our results suggest a role of generalversity, for help with electron microscopy and Dr.

cytoskeletal proteins, in particular actin and α-IN,

Fujii, Shinshu University, for the generous gift ofand SAP102 in the formation of immature and na­

anti-tubulin antibody. This work was funded by two

scent PSDs by constructing the PSD core structure.

grants (to T.S.) from the Institute for Biomedical Sci­

In contrast, scaffold proteins, such as PSD-95, cha­ences, Interdisciplinary Cluster for Cutting Edge Re­

psyn110/PSD-93, SAP97, Shank, GKAP, and Homer

search, Shinshu University, and the Institute of Med­seem to play major roles in mature PSDs, but not in

icine, School of Medicine and Health Sciences, Shin-

immature PSDs. Moreover, CaMKII may also not

shu University Academic Assembly, in addition to aplay a major role in the PSD mesh-like core struc­grant (to W.G.) from Shinshu Public Utility Foundation

ture. Further analyses using different approaches

for Promotion of Medical Sciences. We would like toare expected.

thank Editage (www.editage.jp) for English language
editing.
Declarations of Interest
The authors have no conflicts of interest to de­clare.
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