Durable fear memories require PSD-95.

Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. Although overly persistent fear memories underlie anxiety disorders, such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Postsynaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Using a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95(GK)), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown (KD) approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95(GK) mice to retrieve remote cued fear memory was associated with hypoactivation of the infralimbic (IL) cortex (but not the anterior cingulate cortex (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated virus-mediated PSD-95 KD in the IL, but not the ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories.

De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia.

A small number of rare, recurrent genomic copy number variants (CNVs) are known to substantially increase susceptibility to schizophrenia. As a consequence of the low fecundity in people with schizophrenia and other neurodevelopmental phenotypes to which these CNVs contribute, CNVs with large effects on risk are likely to be rapidly removed from the population by natural selection. Accordingly, such CNVs must frequently occur as recurrent de novo mutations. In a sample of 662 schizophrenia proband-parent trios, we found that rare de novo CNV mutations were significantly more frequent in cases (5.1% all cases, 5.5% family history negative) compared with 2.2% among 2623 controls, confirming the involvement of de novo CNVs in the pathogenesis of schizophrenia. Eight de novo CNVs occurred at four known schizophrenia loci (3q29, 15q11.2, 15q13.3 and 16p11.2). De novo CNVs of known pathogenic significance in other genomic disorders were also observed, including deletion at the TAR (thrombocytopenia absent radius) region on 1q21.1 and duplication at the WBS (Williams-Beuren syndrome) region at 7q11.23. Multiple de novos spanned genes encoding members of the DLG (discs large) family of membrane-associated guanylate kinases (MAGUKs) that are components of the postsynaptic density (PSD). Two de novos also affected EHMT1, a histone methyl transferase known to directly regulate DLG family members. Using a systems biology approach and merging novel CNV and proteomics data sets, systematic analysis of synaptic protein complexes showed that, compared with control CNVs, case de novos were significantly enriched for the PSD proteome (P=1.72 × 10⁻6. This was largely explained by enrichment for members of the N-methyl-D-aspartate receptor (NMDAR) (P=4.24 × 10⁻6) and neuronal activity-regulated cytoskeleton-associated protein (ARC) (P=3.78 × 10⁻8) postsynaptic signalling complexes. In an analysis of 18 492 subjects (7907 cases and 10 585 controls), case CNVs were enriched for members of the NMDAR complex (P=0.0015) but not ARC (P=0.14). Our data indicate that defects in NMDAR postsynaptic signalling and, possibly, ARC complexes, which are known to be important in synaptic plasticity and cognition, play a significant role in the pathogenesis of schizophrenia.

Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice.

Mice with mutations in four nonreceptor tyrosine kinase genes, fyn, src, yes, and abl, were used to study the role of these kinases in long-term potentiation (LTP) and in the relation of LTP to spatial learning and memory. All four kinases were expressed in the hippocampus. Mutations in src, yes, and abl did not interfere with either the induction or the maintenance of LTP. However, in fyn mutants, LTP was blunted even though synaptic transmission and two short-term forms of synaptic plasticity, paired-pulse facilitation and post-tetanic potentiation, were normal. In parallel with the blunting of LTP, fyn mutants showed impaired spatial learning, consistent with a functional link between LTP and learning. Although fyn is expressed at mature synapses, its lack of expression during development resulted in an increased number of granule cells in the dentate gyrus and of pyramidal cells in the CA3 region. Thus, a common tyrosine kinase pathway may regulate the growth of neurons in the developing hippocampus and the strength of synaptic plasticity in the mature hippocampus.

Neonatal hepatitis induced by alpha 1-antitrypsin: a transgenic mouse model.

Transgenic mouse lineages were established that carry the normal (M) or mutant (Z) alleles of the human alpha 1-antitrypsin (alpha 1-Pi) gene. All of the alpha 1-Pi transgenic mice expressed the human protein in the liver, cartilage, gut, kidneys, lymphoid macrophages, and thymus. The human M-allele protein was secreted normally into the serum. However, the human Z-allele protein accumulated in several cell types, but particularly in hepatocytes, and was found in serum in tenfold lower concentrations than the M-allele protein. Mice in one lineage carrying the mutant Z allele expressed high levels of human alpha 1-Pi RNA and displayed significant runting (50% of normal weight) in the neonatal period. This lineage was found to have alpha 1-Pi-induced liver pathology in the neonatal period, concomitant with the accumulation of human Z protein in diastase-resistant cytoplasmic globules that could be revealed in the Periodic acid-Schiff reaction (PAS). The phenotype of mice in the strain expressing high levels of the Z allele is remarkably similar to human neonatal hepatitis, and this strain may prove to be a useful animal model for studying this disease.

Activation of cAMP-responsive genes by stimuli that produce long-term facilitation in Aplysia sensory neurons.

One of the hallmarks of long-term memory in both vertebrates and invertebrates is the requirement for new protein synthesis. In sensitization of the gill-withdrawal reflex in Aplysia, this requirement can be studied on the cellular level. Here, long-term but not short-term facilitation of the monosynaptic connections between the sensory and motor neurons requires new protein synthesis and is reflected in an altered level of expression of specific proteins regulated through the cAMP second-messenger pathway. Using gene transfer into individual sensory neurons of Aplysia, we find that serotonin (5-HT) induces transcriptional activation of a lacZ reporter gene driven by the cAMP response element (CRE) and that this induction requires CRE-binding proteins (CREBs). The induction by 5-HT does not occur following a single pulse, but becomes progressively more effective following two or more pulses. Moreover, expression of GAL4-CREB fusion genes shows that 5-HT induction requires phosphorylation of CREB on Ser119 by protein kinase A. These data provide direct evidence for CREB-modulated transcriptional activation with long-term facilitation.

alpha-Isoform of calcium-calmodulin-dependent protein kinase II and postsynaptic density protein 95 differentially regulate synaptic expression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors in hippocampus.

N-methyl-d-aspartate receptors (NMDARs) are critical determinants of bidirectional synaptic plasticity, however, studies of NMDAR function have been based primarily on pharmacological and electrophysiological manipulations, and it is still debated whether there are subunit-selective forms of long-term potentiation (LTP) and long-term depression (LTD). Here we provide ultrastructural analyses of axospinous synapses in cornu ammonis field 1 of hippocampus (CA1) stratum radiatum of transgenic mice with mutations to two key underlying postsynaptic density (PSD) proteins, postsynaptic density protein 95 (PSD-95) and the alpha-isoform of calcium-calmodulin-dependent protein kinase II (alphaCaMKII). Distribution profiles of synaptic proteins in these mice reveal very different patterns of subunit-specific NMDAR localization, which may be related to the divergent phenotypes of the two mutants. In PSD-95, Dlg, ZO-1/Dlg-homologous region (PDZ) 3-truncated mutant mice in which LTD could not be induced but LTP was found to be enhanced, we found a subtle, yet preferential displacement of synaptic N-methyl-d-aspartate receptor subunit 2B (NR2B) subunits in lateral regions of the synapse without affecting changes in the localization of N-methyl-d-aspartate receptor subunit 2A (NR2A) subunits. In persistent inhibitory alphaCaMKII Thr305 substituted with Asp in alpha-isoform of calcium-calmodulin kinase II (T305D) mutant mice with severely impaired LTP but stable LTD expression, we found a selective reduction of NR2A subunits at both the synapse and throughout the cytoplasm of the spine without any effect on the NR2B subunit. In an experiment of mutual exclusivity, neither PSD-95 nor alphaCaMKII localization was found to be affected by mutations to the corresponding PSD protein suggesting that they are functionally independent of the other in the regulation of NR2A- and NR2B-containing NMDARs preceding synaptic activity. Consequently, there may exist at least two distinct PSD-95 and alphaCaMKII-specific NMDAR complexes involved in mediating LTP and LTD through opposing signal transduction pathways in synapses of the hippocampus. The contrasting phenotypes of the PSD-95 and alphaCaMKII mutant mice further establish the prospect of an independent and, possibly, competing mechanism for the regulation of NMDAR-dependent bidirectional synaptic plasticity.

Supramolecular signalling complexes in the nervous system.

It is now apparent that multiprotein signalling complexes or "signalling machines" are responsible for orchestrating many complex signalling pathways in the cell. The synapse is a sub-cellular specialisation which transmits and converts patterns of electrical activity into cellular memory. This processing of electrical information is mediated by the protein components of the synapse. The organisation of synaptic proteins has been investigated over the last number of years using proteomic methods and with the application ofbioinformatics; a landscape of modular protein complexes at the synapse is emerging. Many share a common organisation centred on a receptor/channel, a protein scaffold, (in which the signalling molecules are localised) and membrane to cytoskeleton interactions. The use of PDZ-domain based protein scaffolds is a particularly common feature in the construction of neuronal protein complexes and the differential presence of these proteins in complexes can have functional consequences. Here we overview current proteomic methodologies for the analysis of multiprotein complexes. In addition, we describe the characterisation of a number of multiprotein complexes associated with ion channels (NMDAR, P2X7 and Kir2) and GPCRs (5-HT2A/5-HT2C, D2 and mGluR5) and discuss common their common components and organisation.

Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

Dlgap1 knockout mice exhibit alterations of the postsynaptic density and selective reductions in sociability.

The scaffold protein DLGAP1 is localized at the post-synaptic density (PSD) of glutamatergic neurons and is a component of supramolecular protein complexes organized by PSD95. Gain-of-function variants of DLGAP1 have been associated with obsessive-compulsive disorder (OCD), while haploinsufficient variants have been linked to autism spectrum disorder (ASD) and schizophrenia in human genetic studies. We tested male and female Dlgap1 wild type (WT), heterozygous (HT), and knockout (KO) mice in a battery of behavioral tests: open field, dig, splash, prepulse inhibition, forced swim, nest building, social approach, and sucrose preference. We also used biochemical approaches to examine the role of DLGAP1 in the organization of PSD protein complexes. Dlgap1 KO mice were most notable for disruption of protein interactions in the PSD, and deficits in sociability. Other behavioral measures were largely unaffected. Our data suggest that Dlgap1 knockout leads to PSD disruption and reduced sociability, consistent with reports of DLGAP1 haploinsufficient variants in schizophrenia and ASD.

Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.

Standardized experiments in mutant mice reveal behavioural similarity on 129S5 and C57BL/6J backgrounds.

Behavioural analysis of mice carrying engineered mutations is widely used to identify roles of specific genes in components of the mammalian behavioural repertoire. The reproducibility and robustness of phenotypic measures has become a concern that undermines the use of mouse genetic models for translational studies. Contributing factors include low individual study power, non-standardized behavioural testing, failure to address confounds and differences in genetic background of mutant mice. We have examined the importance of these factors using a statistically robust approach applied to behavioural data obtained from three mouse mutations on 129S5 and C57BL/6J backgrounds generated in a standardized battery of five behavioural assays. The largest confounding effect was sampling variation, which partially masked the genetic background effect. Our observations suggest that strong interaction of mutation with genetic background in mice in innate and learned behaviours is not necessarily to be expected. We found composite measures of innate and learned behaviour were similarly impacted by mutations across backgrounds. We determined that, for frequently used group sizes, a single retest of a significant result conforming to the commonly used P < 0.05 threshold results in a reproducibility of 60% between identical experiments. Reproducibility was reduced in the presence of strain differences. We also identified a P-value threshold that maximized reproducibility of mutant phenotypes across strains. This study illustrates the value of standardized approaches for quantitative assessment of behavioural phenotypes and highlights approaches that may improve the translational value of mouse behavioural studies.

Proteomics of the nervous system.

The recent success of large-scale industrialized genomic sequencing opens new doors in studies of biological systems. In the current post-genomic era we must ask how to translate this DNA sequence information into an understanding of living cells, tissues and organisms. One of the major goals is to characterize protein function, biochemical pathways and networks. Achieving this aim is greatly advanced by application of new proteomic tools combined with database mining. Neuroscience in particular is poised to benefit from these approaches in light of its high complexity and cross-talk between different neurotransmitter receptors within the same synapse or across the synaptic cleft. Little is known about the global in vivo protein interactions within synapses, and the knowledge of all proteins present in such structures will help in determining sub-complexes and the modular arrangement of proteins within them. This article reviews the impact of and outlines the application of proteomic analysis in the field of neuroscience, illustrating this with the example of NMDA receptor complexes.

Targeting learning.

Novel transgenic approaches provide an exciting opportunity to assess the impact of the loss of specific genes in the biochemistry and electrophysiology of neurons involved in a learned behavior. Recent studies describing mice harboring mutations in five kinase genes expressed in the hippocampus found that two of these kinases, the alpha-Ca(2+)-calmodulin-dependent kinase II and the Fyn tyrosine kinase are necessary for the establishment of long-term potentiation. In addition to providing a new tool for the dissection of the molecular mechanisms of synaptic plasticity, these mutants will be important in determining how changes in synaptic strength affect not only learning and memory, but also a host of other processes thought to be associated with plasticity.

Gene targeting and synaptic plasticity.

Gene targeting is revealing new molecular functions by creating very specific developmental, physiological and behavioral perturbations, and providing new insights into biochemical pathways underlying synaptic plasticity. Recent studies of mice carrying mutations in genes thought to be involved in modulating synaptic transmission have been subject to integrated biochemical, physiological and behavioral analyses.

Multiprotein complex signaling and the plasticity problem.

Synaptic transmission of distinct patterns of spikes, or 'neural code', leads to plastic changes in synapses and other parts of the neuron, as well as learning in animals. Recent findings indicate that specialized multiprotein structures associated with neurotransmitter receptors and cell-adhesion proteins function as molecular devices that both read the neural code and initiate long-term changes in synaptic structure and function.

A novel role for cyclic guanosine 3',5'monophosphate signaling in synaptic plasticity: a selective suppressor of protein kinase A-dependent forms of long-term potentiation.

At excitatory synapses onto hippocampal CA1 pyramidal cells, activation of cyclic AMP-dependent protein kinase and subsequent down-regulation of protein phosphatases has a crucial role in the induction of long-term potentiation by low-frequency patterns of synaptic stimulation. Because the second messenger cyclic guanosine 3',5'monophosphate can regulate the activity of different forms of the cyclic AMP degrading enzyme phosphodiesterase, we examined whether increases in cyclic guanosine 3',5'monophosphate can modulate long-term potentiation induction in the mouse hippocampal CA1 region through effects on cyclic AMP signaling. Using the cyclic guanosine 3',5'monophosphate-specific phosphodiesterase inhibitor zaprinast or the nitric oxide donor S-nitroso-D,L-penicillamine to elevate cyclic guanosine 3',5'monophosphate levels we found that increases in cyclic guanosine 3',5'monophosphate strongly inhibit the induction of long-term potentiation by low-frequency patterns of synaptic stimulation where protein kinase A activation is required for long-term potentiation induction. In contrast, zaprinast and S-nitroso-D,L-penicillamine had no effect on the induction of long-term potentiation by high-frequency patterns of synaptic stimulation that induce long-term potentiation in a protein kinase A-independent manner. Directly activating protein kinase A with the phosphodiesterase-resistant cyclic AMP analog 8-Br-cAMP, blocking all phosphodiesterases with 3-isobutyl-1-methylxanthine, or inhibiting protein phosphatases rescued long-term potentiation induction in zaprinast-treated slices. Together, these results suggest that increases in cyclic guanosine 3',5'monophosphate inhibit long-term potentiation by activating phosphodiesterases that interfere with the protein kinase A-mediated suppression of protein phosphatases needed for long-term potentiation induction. Consistent with the notion that this cyclic guanosine 3',5'monophosphate-mediated inhibitory pathway is recruited by some patterns of synaptic activity, blocking cyclic guanosine 3',5'monophosphate production strongly facilitated the induction of long-term potentiation by long trains of theta-frequency synaptic stimulation. Together, our results indicate that increases in cyclic guanosine 3',5'monophosphate can act as a long-term potentiation suppressor mechanism that selectively constrains the induction of protein kinase A-dependent forms of long-term potentiation induced by low-frequency patterns of synaptic stimulation.

Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro.

BACKGROUND: Genetically manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK), which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. RESULTS: Mouse ES cells carrying homozygous null mutations (FAK-/-) were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. CONCLUSION: FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

Early invasiveness characterizes metastatic carcinoid tumors in transgenic mice.

The conversion of a normal cell into a metastatic tumor is thought to occur in a stepwise progression of genetic changes that affect both growth control and interactions with the extracellular environment. The development of invasiveness allows tumor cells to escape from their primary site. We have investigated transgenic mice that develop both invasive intestinal neuroendocrine tumors and noninvasive tumors of the pancreatic beta-cells. Visual inspection and gene expression studies indicate that the beta-cell tumors rarely metastasize. In contrast, intestinal tumors that first appear in submucosal areas metastasize with high frequency to the lymph nodes and liver. No evidence of preneoplastic mucosal lesions was seen in the intestine, indicating that invasiveness is acquired early in the tumorigenic progression of these cells. Comparison of intestinal and pancreatic neuroendocrine tumors in transgenic mice suggests that an early requirement for invasiveness may contribute to metastatic potential.

Genetic instability and hematologic disease risk in Werner syndrome patients and heterozygotes.

Werner syndrome (WRN) is an uncommon autosomal recessive disease in which progeroid features are associated with genetic instability and an elevated risk of neoplasia. We have used the glycophorin A (GPA) somatic cell mutation assay to analyze genetic instability in vivo in WRN patients and heterozygotes. GPA variant frequencies were determined for 11 WRN patients and for 10 heterozygous family members who collectively carry 10 different WRN mutations. Genetic instability as measured by GPA O/N allele loss variant frequency was significantly increased, and this increase was strongly age-dependent in WRN patients. GPA O/N allele loss variants were also significantly elevated in heterozygous family members, thus providing the first evidence for in vivo genetic instability in heterozygous carriers in an autosomal recessive genetic instability syndrome. Our results and comparable data on other human genetic instability syndromes allow an estimate of the level of genetic instability that increases the risk of human bone marrow dysfunction or neoplasia.