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ANRV313-BI76-33 ARI 1 May 2007 19:29

The Postsynaptic Architecture of Excitatory : A More Quantitative View

Morgan Sheng1 and Casper C. Hoogenraad2

1The Picower Institute for Learning and , Howard Hughes Medical Institute, Departments of Brain and Cognitive Sciences, and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; email: [email protected] 2Department of , Erasmus Medical Center, Rotterdam 3015GE, The Netherlands; email: [email protected]

Annu. Rev. Biochem. 2007. 76:823–47 Key Words First published online as a Review in Advance on , , mass spectrometry, December 19, 2006 , PSD-95 The Annual Review of Biochemistry is online at biochem.annualreviews.org Abstract

by SCELC Trial on 03/25/13. For personal use only. This article’s doi: Excitatory (glutamatergic) synapses in the mammalian brain are usu- 10.1146/annurev.biochem.76.060805.160029 ally situated on dendritic spines, a postsynaptic microcompartment Copyright c 2007 by Annual Reviews. that also harbors organelles involved in synthesis, membrane All rights reserved trafficking, and metabolism. The postsynaptic membrane

Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org 0066-4154/07/0707-0823$20.00 contains a high concentration of glutamate receptors, associated sig- naling , and cytoskeletal elements, all assembled by a variety of scaffold proteins into an organized structure called the postsynap- tic density (PSD). A complex machine made of hundreds of distinct proteins, the PSD dynamically changes its structure and composi- tion during development and in response to synaptic activity. The molecular size of the PSD and the stoichiometry of many major con- stituents have been recently measured. The structures of some intact PSD proteins, as well as the spatial arrangement of several proteins within the PSD, have been determined at low resolution by electron microscopy. On the basis of such studies, a more quantitative and geometrically realistic view of PSD architecture is emerging.

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cal to the postsynaptic Contents (excitatory synaptic transmission). The strength of synaptic transmission can INTRODUCTION...... 824 be modified () by various MICROANATOMY OF THE factors, including specific patterns of synaptic EXCITATORY ...... 825 activity (1). By altering synaptic strength in Synaptic Cleft ...... 825 an activity-dependent manner, synaptic plas- The Dendritic Spine...... 827 ticity allows synapses and neural networks to Organelles Within Dendritic store information in response to prior experi- Spines ...... 828 ence, such as occurs during development and THE POSTSYNAPTIC DENSITY . 829 in learning and memory. In parallel with ad- Biochemical Composition of the justing the strength of synaptic transmission, PSD...... 829 can change the morphology of synap- Quantitative View of PSD Protein tic connections and even their physical pat- Composition ...... 831 tern of connectivity (2). Thus information can Three-Dimensional Organization be stored long-term in the brain not only by ofthePSD...... 835 modulation of synaptic strength but also by Three-Dimensional Structures of the formation of new synapses or the elimina- Individual Postsynaptic tion of existing synapses (2, 3). Molecules ...... 837 Synaptic transmission and plasticity are Heterogeneity of PSD crucial for all aspects of func- Composition between Different tion and critical for proper development of Brain Regions and Cell Types. . 838 the . Normal synap- Turnover of Proteins in the PSD . . 839 tic transmission depends on the proper local- Developmental Changes in the ization and arrangement of specific proteins PSD...... 839 on both sides of the synapse. Synaptic plas- EXTRASYNAPTIC MEMBRANE . . 840 ticity is mediated by changes in the molecu- CONCLUDING REMARKS ...... 841 lar composition of synapses and in chemical modification of synaptic proteins. Pathologi- cal synapse development and/or function al- most certainly contribute to many neuropsy- by SCELC Trial on 03/25/13. For personal use only. chiatric disorders (e.g., schizophrenia, autism, INTRODUCTION mental retardation), common neurodegener- The billions of neurons in the mammalian ative diseases (e.g., Alzheimer’s disease), and : the branched projections brain communicate with each other via spe- (4, 5). Therefore, it is of fundamental Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org of a neuron that cialized junctions called synapses. The vast and clinical importance to understand the bio- receive synaptic majority of these synapses occur at contacts chemical and cell biological basis of synaptic inputs conveyed by between presynaptic and postsynaptic function and plasticity. axons from other , and they use glutamate as the exci- We review the molecular and cellular ar- neurons tatory . Glutamate released chitecture of the postsynaptic specialization of AMPA receptor from the presynaptic terminal acts upon post- glutamatergic (glutamate-releasing) synapses, (AMPAR): glutamate receptor synaptic glutamate receptor channels [primar- which make up the vast majority of cen- channel that ily AMPA (α-amino-3-hydroxy-5-methyl-4- tral synapses in mammalian brain. Not cov- mediates most of the isoxazolepropionic acid) receptors (AMPARs) ered are other types of excitatory synapses fast synaptic and NMDA (N-methyl-D-aspartic acid) re- (e.g., those using ) and inhibitory transmission in ceptors (NMDARs)], which open to allow in- synapses, which differ in their molecular or- central excitatory + synapses flux of Na ions (and in the case of NMDARs, ganization (6, 7). The presynaptic special- also Ca2+ ions), thereby propagating electri- ization has been recently reviewed (8). The

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postsynaptic compartment is where most of naptic and extrasynaptic regions. The latter the molecular diversity of synapses lies and two terms are often used interchangeably, al- where the initial signal transduction events though it should be borne in mind that the NMDA receptor take place that lead to long-term synaptic perisynaptic membrane within 100 nm of the (NMDAR): plasticity. On the basis of recent progress in PSD probably differs in molecular content calcium-permeable this area, we aim to give a more quantita- and function from the extrasynaptic mem- glutamate receptor tive description of the postsynaptic special- brane at greater distances from the PSD. channel that ization, focusing on the postsynaptic density Extrasynaptic regions have specialized postsy- contributes to fast synaptic transmission (PSD) and emphasizing three-dimensional naptic functions and are enriched for a distinc- and that regulates (3D) structure, molecular stoichiometry, tive set of proteins, such as metabotropic glu- synaptic plasticity and spatial arrangement of postsynaptic tamate receptors (mGluRs) (14) and proteins Synaptic plasticity: components. involved in endocytosis (15) (Figure 1d ). On the ability of the most principal neurons in the mammalian synapse to alter its brain (e.g., pyramidal neurons of cortex and strength and MICROANATOMY OF THE hippocampus, Purkinje cells of cerebellum, structure medium spiny neurons of ), the post- Excitatory synapse: Ultrastructural features enable unambiguous synaptic specialization is housed on tiny - contact site where synaptic transmission identification of the pre- and postsynaptic rich protrusions called dendritic spines. In occurs by secretion sides of the synapse by electron microscopy contrast, inhibitory synapses are made on the of neurotransmitter (EM). Defining the presynaptic specialization shaft of the dendrite (Figure 1c). In inhibitory (glutamate) from is a cluster of synaptic vesicles (∼40 nm di- local circuit , which usually lack presynaptic neuron ameter), some of which are closely associated spines, the excitatory synapses are also made to postsynaptic neuron (“docked”) with a thickening of the presynap- on the dendritic shaft. The PSD (and hence tic plasma membrane (the ), where the synaptic contact) is typically located on the Postsynaptic density (PSD): an vesicle exocytosis occurs (8) (Figure 1a,b). dilated tip (“head”) of the spine. The dimen- electron-dense Directly apposed to the active zone (and per- sions of the spine head are highly correlated structure at the fectly matched with it in size and shape) is with the size of the PSD and associated active postsynaptic the PSD, an electron-dense thickening of the zone, as well as synaptic strength (16). membrane postsynaptic membrane, where glutamate re- containing many glutamate receptors, ceptor channels and their associated signaling Synaptic Cleft scaffold proteins, and proteins are highly concentrated (Figure 1a- signal transduction by SCELC Trial on 03/25/13. For personal use only. c). The presence of a prominent PSD is char- Separating the PSD and the active zone is molecules acteristic of glutamatergic synapses (hence the synaptic cleft, which measures ∼20 nm EM: electron they are termed asymmetric); in contrast, in- wide in conventional EM and ∼24 nm wide microscopy hibitory (symmetric) synapses lack a prominent in cryo-EM (17, 18) (Figure 1b). Faint lines Active zone: region

Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org postsynaptic thickening (9, 10). bridging the synaptic cleft were noted by early of the presynaptic The presynaptic active zone and the PSD, anatomists (19). Recent cryo-EM and tomog- membrane directly which define the extent of the true synapse raphy studies suggest that the cleft contains apposed to the PSD, where synaptic morphologically, are separated by a gap of 20– more electron-dense material even than the vesicles fuse with the 25 nm (Figure 1a,b). A wide variety of cell ad- cytoplasm; the density peaks midway between presynaptic hesion molecules hold pre- and postsynaptic the pre- and postsynaptic membranes (17, 18). membrane membranes together in register and at the ap- The intracleft material appears to consist of propriate separation (11, 12). Puncta adherens transsynaptic complexes that form extensive junctions and/or cadherin clusters lie adjacent lateral connections within the cleft (17, 18). to the synapse proper or may occur within the The nature of these cleft complexes are un- PSD-active zone apposition (13). known, but cell adhesion molecules such as The postsynaptic membrane can be di- N-cadherin (12) and glutamate receptors may vided into the PSD itself, as well as perisy- contribute (20).

www.annualreviews.org • Excitatory Postsynaptic Structure 825 ANRV313-BI76-33 ARI 1 May 2007 19:29 by SCELC Trial on 03/25/13. For personal use only. Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org

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The Dendritic Spine pecially during development (25, 26). Their number, size, and shape undergo plastic Dendritic spines are small (typically 0.5– changes correlated with long-term modifica- 2 μm in length) membranous protrusions Dendritic spines: tions of synaptic strength and interneuronal that house the essential postsynaptic com- small membranous connectivity (2, 27). Spine shape has been cat- ponents, including the PSD, actin cytoskele- compartments egorized as “mushroom,” “thin,” or “stubby,” protruding from ton, and a variety of “supporting” organelles but EM studies show a continuum between dendrites that (Figure 1d ). Spines occur at a density of 1– these categories. There is growing evidence receive synaptic 10 spines per μm of dendrite length on prin- contacts from that different spine shapes and sizes reflect cipal neurons (21), and they receive most of glutamate-releasing different developmental stages and/or altered the excitatory synapses in the mature mam- axons strength of synapses (2, 16, 27). Sophisticated malian brain. Typical spines have a bulbous imaging experiments indicate that the vol- head (receiving a single synapse) connected ume of spine heads can increase with stim- to the parent dendrite through a thin spine uli that strengthen synapses and can decrease neck (Figure 1c). Because the neck hinders with stimuli that weaken synapses (16, 27). diffusion of molecules to and from the par- The molecular mechanisms that coordinate ent dendritic shaft, spines serve as microcom- synaptic strength with spine morphogenesis partments in which biochemical changes in is a subject of current interest (25). one individual synapse can be isolated from Spines with large heads are generally sta- other synapses on the same neuron (22). The ble, express large numbers of AMPARs, and geometry of the spine neck determines cal- contribute to strong synaptic connections. By cium efflux into the dendrite shaft and hence contrast, spines with small heads are more the degree of calcium elevation in the spine motile, less stable, and contribute to weak head following NMDAR activation (23). Re- synaptic connections (28, 29). In vivo time- cent evidence suggests that the spine neck dif- lapse studies show that spines turn over at fusion barrier can be controlled by neuronal various rates in the mouse brain; a large activity (24). fraction of mushroom spines are persistent, Dendritic spines are highly heterogeneous with lifetimes up to many months (30, 31). structures that show dynamic motility, es-

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

by SCELC Trial on 03/25/13. For personal use only. Figure 1 Microanatomy of the excitatory synapse and dendritic spine. (a) Thin section electron microscopy (EM) image of the CA1 region of the hippocampus. Morphological features are highlighted in color for illustrative purposes: postsynaptic density (PSD); dendrites, including spines; axons, including presynaptic terminals containing synaptic vesicles; processes, which are found frequently at the Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org edge of synapse. The scale bar is 1 μm. (b) EM morphology of an excitatory synapse. The presynaptic terminal contains synaptic vesicles loaded with glutamate, facing the PSD located on the tip of the dendritic spine. The synaptic cleft separating pre- and postsynaptic membranes is 20–25 nm wide. Abbreviation: SV, . (c) Three-dimensional EM reconstruction of a segment of dendrite from the CA1 region of the hippocampus. Red indicates the PSD of an excitatory synapse. Blue indicates inhibitory synapse. Some PSDs are not visible because they face away from the viewer. Note a central perforation in the PSD of the large mushroom spine at bottom right. Examples of thin spines (arrowheads) and mushroom spines (arrows) are indicated. (d) Schematic diagram of mature mushroom-shaped spine, showing the PSD, the perisynaptic membrane, and other organelles. The endocytic zone (EZ) is located lateral of the PSD in extrasynaptic regions of the spine, where it may be associated with clathrin-coated vesicles (CCV) and recycling endosomes (RE). Smooth endoplasmic reticulum (SER), polyribosomes (PR) and mitochondria (M) are found mainly in the dendritic shaft near the base of spines but may extend into the spine. The abundant actin (brown lines) is connected to the PSD and determines spine structure and motility. Other abbreviation: SA, spine apparatus. Images for panels a-c of this figure were kindly provided by Kristen Harris (Medical College of Georgia, USA).

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α-Catenin β-Catenin Actin α δ-Catenin -Actinin α N-Cadherin -Adducin Densin-180 Ankyrins Arp2/3 complex GKAP NCAM-140 Neurofascin Contactin Homer Drebrin Lin-7 HSP40 LIM PSD-95 7% cell Neurabin Shank 6% translation adhesion 2% chaperones α/β-Spectrins 6% scaffolds 12% cytoskeleton actin AMPAR 6% receptors NMDAR and channels MAP1A mGluR1 4% MAP1B cytoskeleton MAP2 others Septin 15% others Tubulin

6% mitochondria Dynein Myosin IIb 4% motor 11% kinases/ Myosin V proteins phosphatases Myosin VI and regulators CaMKIIα/β/δ/ 8% GTPases and Casein kinase 2 7% metabolism regulators 5% membrane PKCγ trafficking ARF3 Protein phosphatase PP1 cAMP-GEFII (RapGEF) AP1 Protein phosphatase PP2A GIT1 (ArfGAP) AP2 Heterotrimeric G proteins Clathrin Kalirin (RhoGEF), NSF PIKE-L (ArfGAP) SNAP-25 IP Ras

by SCELC Trial on 03/25/13. For personal use only. SPAR (RapGAP) Syntaxin BP SynGAP (RasGAP)

Figure 2 The variety of proteins in the PSD fraction. Pie chart showing the wide variety of proteins identified in Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org the PSD fraction of the forebrain, categorized according to cellular function. Only small subsets of identified proteins are shown as examples. PSD proteins are involved in cellular communication and signal transduction (adhesion, GTPases, kinase/phosphates, receptors, and channels), cellular organization (cytoskeleton, membrane traffic, motors, and scaffolds), energy (mitochondria and metabolism), protein synthesis and processing (translation and chaperones), and others. The percentage of PSD proteins per category was obtained from the mass spectrometry data by Peng and colleagues (70).

proteome (63). Current estimates based mitochondrial and glial proteins, abundant on MS analysis range from a few hundred metabolic enzymes, and cytoskeletal ele- (∼100–400) to as many as one thousand ments. Proteomic analysis of the PSD is also proteins in the PSD. However, this number prone to miss bona fide components that includes many potential contaminants of are in low abundance or only transiently the PSD fraction (false positives), such as associated with PSDs (false negatives).

830 Sheng · Hoogenraad ANRV313-BI76-33 ARI 1 May 2007 19:29 by SCELC Trial on 03/25/13. For personal use only. Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org

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Figure 4 Organization of proteins and protein-protein interactions in the postsynaptic density (PSD). Schematic diagram of the network of proteins in the PSD, with edge of PSD depicted at right. Only major families by SCELC Trial on 03/25/13. For personal use only. and certain classes of PSD proteins are shown [in approximate stoichiometric ratio and scaled to molecular size, if known (see text)]. Contacts between proteins indicate an established interaction between them. Domain structure is shown only for PSD-95 (PDZ domain, SH3 domain, GuK domain). Other scaffold proteins are colored yellow; signaling enzymes, green; actin binding proteins, pink. CaMKII (calcium/calmodulin-dependent protein kinase II ) is depicted as dodecamer. Unnamed proteins Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org signify the many other PSD proteins that are not illustrated in this diagram. Abbreviations: AKAP150, A-kinase anchoring protein 150 kDa; CAM, ; Fyn, a Src family kinase; GKAP, guanylate kinase-associated protein; H, Homer; IRSp53, insulin receptor substrate 53 kDa; KCh, K+ channel; mGluR, metabotropic glutamate receptor; nNOS, neuronal nitric oxide synthase; RTK, receptor tyrosine kinases (e.g., ErbB4, TrkB); SPAR, spine-associated RapGAP.

in addition to differential expression of synap- a fraction of Shank and Homer proteins is tic proteins in cultured neurons versus adult lost by Triton X-100 extraction during pu- brain. For instance, the levels of GKAP, as rification of PSDs for MS analysis. Despite well as Shank and Homer family, proteins these quantitative discrepancies, it is clear are influenced by synaptic activity (81), and that the PSD-95/GKAP/Shank/Homer scaf- different levels of activity prevail in culture fold assembly accounts for a substantial pro- versus in vivo. Another possibility is that portion of total protein mass of the PSD (76).

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also be directed to lysosomes for degradation mined. We have obtained a low-resolution (132, 133). picture of the 3D shape of the PSD, but It is generally believed that AMPARs are it was in an unnatural state, divorced from exocytosed at extrasynaptic sites (depending the plasma membrane and actin cytoskeleton on the AMPAR auxiliary subunit stargazin), with which the PSD usually associates. Fur- followed by lateral movement into synapses, ther work combining modern EM and molec- where they become relatively immobile owing ular genetics in more intact preparations is to interactions with, or hindrance by, other essential to reveal the molecular architecture postsynaptic proteins such as PSD-95 (88, of postsynaptic specialization and inform its 134–138) (Figure 5c). Compared with endo- structure-function relationships. cytosis, the precise location on the neuronal Even as we struggle to reach a stoichiomet- surface where AMPAR insertion occurs is less ric and geometric description of the PSD and certain; it could be in extrasynaptic regions of its constituent proteins, it is clear that we are the spine or in the dendritic shaft and . chasing a moving target that changes rapidly A variety of proteins that directly inter- and substantially in response to neural activity act with specific AMPAR subunits play impor- and developmental experience (akin to taking tant roles in AMPAR trafficking (37, 129). For a census of a fluid society). Both the PSD and example, GluR2 binds to N-ethylmaleimide- the dendritic spine, on which it sits, are dy- sensitive factor (NSF) [an ATPase required namic structures made up of parts that move for dissociation of the SNARE protein com- and turn over constantly; components of the plex and recruited to the PSD during brain PSD are in constant flux with the extrasynap- ischemia (105)], and this interaction is crit- tic membrane and cytoplasm, and contents ical for the recycling of GluR2-containing of the spine with the parent dendrite. Reg- AMPARs to the surface and for their incor- ulated modifications of these fluxes (e.g., al- poration into the synapse (129, 139–141). It tered AMPAR trafficking) result in profound is tempting to speculate that NSF functions changes in synaptic strength in response to by dissociating protein interactions that block specific patterns of activity. And yet, despite GluR2 entry into the PSD. being mutable on a timescale of seconds to minutes, and in the face of constant protein turnover, the PSD and dendritic spine remain CONCLUDING REMARKS coordinated with each other and with the by SCELC Trial on 03/25/13. For personal use only. In recent years, a more comprehensive and in- presynaptic specialization, and they are able creasingly quantitative view has been emerg- to maintain over days to months the synaptic ing of the protein composition of the PSD, weights that presumably encode in but numerous details remain to be elucidated, the brain. Perhaps more than anything else,

Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org much of it descriptive in nature. False nega- these remarkable capacities for dynamic plas- tives, which inevitably contaminate the PSD ticity and long-term stability of synapses in- fraction, need to be weeded out, and the bio- trigue neuroscientists. Sorting out the under- chemistry and functional significance of the lying mechanisms will fuel research for years real constituents of the PSD must be deter- to come.

SUMMARY POINTS 1. Excitatory synapses in the mammalian brain mainly use glutamate as the neurotrans- mitter and are usually situated on the tips of specialized postsynaptic compartments called dendritic spines.

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2. Dendritic spines provide synapse-specific biochemical microcompartments; they con- tain not only the synapse proper, but also other organelles involved in protein syn- thesis, membrane trafficking, and ATP and calcium metabolism. 3. The postsynaptic density (PSD) contains the glutamate receptors that detect the release of glutamate from the presynaptic terminal and a host of associated signaling molecules that transduce glutamate binding into postsynaptic biochemical responses. 4. A variety of abundant scaffold proteins (e.g., PSD-95, Shank) assemble the PSD by binding to glutamate receptors, other postsynaptic membrane proteins, cytoplasmic signaling enzymes, and cytoskeletal elements. 5. The molecular size of the PSD and the relative stoichiometry of many major con- stituents have been measured by MS, EM, and other imaging approaches, leading to a more quantitative understanding of protein composition of the PSD. 6. The 3D structures of glutamate receptors and other PSD proteins and their highly organized spatial arrangement within the PSD are emerging through EM and X-ray crystallography. 7. The PSD is a dynamic and heterogeneous structure whose shape, size, and composi- tion changes during development and in response to synaptic activity. 8. The extrasynaptic membrane contains glutamate receptors that are in dynamic flux with those in the PSD, but it is also specialized for different functions, such as endocytosis.

FUTURE ISSUES 1. Hundreds of different proteins have been identified in the PSD, but for most of them, their specific roles in synaptic structure and function are unknown.

by SCELC Trial on 03/25/13. For personal use only. 2. Quantitative stoichiometry, protein-protein interactions, and 3D structures remain to be determined for the vast majority of PSD constituent proteins. 3. The detailed molecular architecture of the PSD, and its relationship with the actin cytoskeleton and other postsynaptic organelles, should be studied in depth with EM

Annu. Rev. Biochem. 2007.76:823-847. Downloaded from www.annualreviews.org tomography combined with molecular manipulations. 4. The dynamics and mechanisms of ontogeny and turnover of PSD proteins are only beginning to be uncovered. The functional significance of the regulated change of PSD proteins needs to be investigated at the cell biology and neural systems levels.

ACKNOWLEDGMENTS We are grateful to Kristen M. Harris for providing EM sections of brain and 3D reconstructions of spines for Figure 1 and to Tom Reese and Xiaobing Chen for providing EM images of purified PSDs for Figure 5. We thank the following for critical comments on the manuscript: Junmin Peng, Yasunori Hayashi, Richard Weinberg, Li-Huei Tsai, Baris Bingol, Honor Hsin, and members of the Hoogenraad lab. M.S. is an Investigator of the Howard Hughes Medical

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