Holly J. Carlisle

Spine architecture and synaptic plasticity.

Many forms of mental retardation and cognitive disability are associated with abnormalities in dendritic spine morphology. Visualization of spines using live-imaging techniques provides convincing evidence that spine morphology is altered in response to certain forms of LTP-inducing stimulation. Thus, information storage at the cellular level appears to involve changes in spine morphology that support changes in synaptic strength produced by certain patterns of synaptic activity. Because the structure of a spine is determined by its underlying actin cytoskeleton, there has been much effort to identify signaling pathways linking synaptic activity to control of actin polymerization. This review, part of the TINS Synaptic Connectivity series, discusses recent studies that implicate EphB and NMDA receptors in the regulation of actin-binding proteins through modulation of Rho family small GTPases.

Integration of biochemical signalling in spines

Short-term and long-term changes in the strength of synapses in neural networks underlie working memory and long-term memory storage in the brain. These changes are regulated by many biochemical signalling pathways in the postsynaptic spines of excitatory synapses. Recent findings about the roles and regulation of the small GTPases Ras, Rap and Rac in spines provide new insights into the coordination and cooperation of different pathways to effect synaptic plasticity. Here, we present an initial working representation of the interactions of five signalling cascades that are usually studied individually. We discuss their integrated function in the regulation of postsynaptic plasticity.

Opposing effects of PSD‐93 and PSD‐95 on long‐term potentiation and spike timing‐dependent plasticity

The membrane‐associated guanylate kinases (MAGUKs) PSD‐95, PSD‐93 and SAP102 are thought to have crucial roles in both AMPA receptor trafficking and formation of NMDA receptor‐associated signalling complexes involved in synaptic plasticity. While PSD‐95, PSD‐93, and SAP102 appear to have similar roles in AMPA receptor trafficking, it is not known whether these MAGUKs also have functionally similar roles in synaptic plasticity. To explore this issue we examined several properties of basal synaptic transmission in the hippocampal CA1 region of PSD‐93 and PSD‐95 mutant mice and compared the ability of a number of different synaptic stimulation protocols to induce long‐term potentiation (LTP) and long‐term depression (LTD) in these mutants. We find that while both AMPA and NMDA receptor‐mediated synaptic transmission are normal in PSD‐93 mutants, PSD‐95 mutant mice exhibit clear deficits in AMPA receptor‐mediated transmission. Moreover, in contrast to the facilitation of LTP induction and disruption of LTD observed in PSD‐95 mutant mice, PSD‐93 mutant mice exhibit deficits in LTP and normal LTD. Our results suggest that PSD‐95 has a unique role in AMPA receptor trafficking at excitatory synapses in the hippocampus of adult mice and indicate that PSD‐93 and PSD‐95 have essentially opposite roles in LTP, perhaps because these MAGUKs form distinct NMDA receptor signalling complexes that differentially regulate the induction of LTP by different patterns of synaptic activity.

SynGAP Regulates Steady-State and Activity-Dependent Phosphorylation of Cofilin

SynGAP, a prominent Ras/Rap GTPase-activating protein in the postsynaptic density, regulates the timing of spine formation and trafficking of glutamate receptors in cultured neurons. However, the molecular mechanisms by which it does this are unknown. Here, we show that synGAP is a key regulator of spine morphology in adult mice. Heterozygous deletion of synGAP was sufficient to cause an excess of mushroom spines in adult brains, indicating that synGAP is involved in steady-state regulation of actin in mature spines. Both Ras- and Rac-GTP levels were elevated in forebrains from adult synGAP+/− mice. Rac is a well known regulator of actin polymerization and spine morphology. The steady-state level of phosphorylation of cofilin was also elevated in synGAP+/− mice. Cofilin, an F-actin severing protein that is inactivated by phosphorylation, is a downstream target of a pathway regulated by Rac. We show that transient regulation of cofilin by treatment with NMDA is also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 μm NMDA triggered transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion of synGAP lacked the transient regulation by the NMDA receptor. Depression of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices from synGAP+/− mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton.

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deletion of Densin-180 Results in Abnormal Behaviors Associated with Mental Illness and Reduces mGluR5 and DISC1 in the Postsynaptic Density Fraction

Densin is an abundant scaffold protein in the postsynaptic density (PSD) that forms a high-affinity complex with αCaMKII and α-actinin. To assess the function of densin, we created a mouse line with a null mutation in the gene encoding it (LRRC7). Homozygous knock-out mice display a wide variety of abnormal behaviors that are often considered endophenotypes of schizophrenia and autism spectrum disorders. At the cellular level, loss of densin results in reduced levels of α-actinin in the brain and selective reduction in the localization of mGluR5 and DISC1 in the PSD fraction, whereas the amounts of ionotropic glutamate receptors and other prominent PSD proteins are unchanged. In addition, deletion of densin results in impairment of mGluR- and NMDA receptor-dependent forms of long-term depression, alters the early dynamics of regulation of CaMKII by NMDA-type glutamate receptors, and produces a change in spine morphology. These results indicate that densin influences the function of mGluRs and CaMKII at synapses and contributes to localization of mGluR5 and DISC1 in the PSD fraction. They are consistent with the hypothesis that mutations that disrupt the organization and/or dynamics of postsynaptic signaling complexes in excitatory synapses can cause behavioral endophenotypes of mental illness.

Phosphorylation of Synaptic GTPase Activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of its GAP Activity Toward Ras and Rap GTPases

Background: synGAP inactivates Ras and Rap at synapses. Results: Phosphorylation of synGAP by CaMKII increases Rap1 GAP activity more than HRas GAP activity; phosphorylation by CDK5 has the opposite effect. Conclusion: Phosphorylation by CaMKII and CDK5 alters the ratio of Rap1 and HRas GAP activities. Significance: Phosphorylation of synGAP by CaMKII and CDK5 can alter the balance of synaptic functions regulated by Ras and Rap. synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAPs HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino‐acid substitution in the polyproline‐binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol‐3‐kinase‐C2α to the SH3‐binding site of PSD95. In wild‐type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.

The Role of the Postsynaptic Density and the SpineCytoskeleton in Synaptic Plasticity

Glutamatergic postsynaptic spines have a specialized cytoskeletal structure consisting of an elaborate submembrane scaffold attached to synaptic receptors, termed the postsynaptic density (PSD), and an actin-based cytoskeleton that maintains their mushroom-like shape. The PSD comprises a layered arrangement of scaffold proteins that organize signaling enzymes to respond to activation of N-methyl-d-aspartate-type and metabotropic glutamate receptors. At every level (receptors, proximal scaffolds, and distal scaffolds), effector proteins, regulatory enzymes, and adaptors are recruited into signal transduction modules that enable specialized adaptive functions. Signaling machinery in the PSD and cytoskeletal regulatory proteins work together to alter the structure of the spine to produce LTP or LTD.

Scaffold Proteins in the Postsynaptic Density

Many intractable neurological and mental diseases, including epilepsy, depression, and schizophrenia, are believed to result, in part, from derangements of regulation of synaptic transmission in the brain. For this reason, much effort has been made to discover how the delicate mechanisms of signal transduction at synapses lead to modification of synaptic strength. One fruitful area of research over the last twenty years has been the postsynaptic signaling apparatus in glutamatergic spines (8, 97, 99, 106). Spines contain clusters of receptors and signaling proteins located in a dense submembranous structure that can be seen in the electron microscope and is called the postsynaptic density or PSD (For review of early work see 98). In the 1970’s, Philip Siekevitz (40) and Carl Cotman (42) worked out subcellular fractionation methods to purify tiny disks from brain that contain many of the proteins that are most tightly bound to the PSD. Their purification methods were later used to obtain material from which several ubiquitous PSD proteins were identified and sequenced, including the prototypical scaffold protein, PSD-95 (37, 109, 113), synGAP (35), the shank/proSAP family (20), and densin (6). At the same time, and independently, some of these proteins, and additional new ones from the PSD fraction, were identified by their interactions in yeast two hybrid screens, including GKAP (104), synGAP (107), shank/proSAP (158), and homer (24). Continuing proteomic analyses of the PSD fraction or related “NMDA-receptor complexes” (89, 127, 177, 235) have produced a catalog of putative PSD-associated proteins that comprises a few hundred individual proteins. More recent studies have begun to address the crucial question of the relative abundance of each protein in the PSD fraction and their stoichiometric ratios (126, 127, 177). However, we are still in the early stages of understanding which core protein complexes are present in most excitatory synapses, and which proteins are present only in a subset of synapses, perhaps conferring specialized properties.