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.
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.
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.
A major goal of proteomics is the complete description of the protein interaction network underlying cell physiology. A large number of small scale and, more recently, large-scale experiments have ...contributed to expanding our understanding of the nature of the interaction network. However, the necessary data integration across experiments is currently hampered by the fragmentation of publicly available protein interaction data, which exists in different formats in databases, on authors' websites or sometimes only in print publications. Here, we propose a community standard data model for the representation and exchange of protein interaction data. This data model has been jointly developed by members of the Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organization (HUPO), and is supported by major protein interaction data providers, in particular the Biomolecular Interaction Network Database (BIND), Cellzome (Heidelberg, Germany), the Database of Interacting Proteins (DIP), Dana Farber Cancer Institute (Boston, MA, USA), the Human Protein Reference Database (HPRD), Hybrigenics (Paris, France), the European Bioinformatics Institute's (EMBL-EBI, Hinxton, UK) IntAct, the Molecular Interactions (MINT, Rome, Italy) database, the Protein-Protein Interaction Database (PPID, Edinburgh, UK) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, EMBL, Heidelberg, Germany).
To identify the molecular mechanisms underlying psychostimulant-elicited plasticity in the brain reward system, we undertook a phenotype-driven approach using genome-wide microarray profiling of ...striatal transcripts from three genetic and one pharmacological mouse models of psychostimulant or dopamine supersensitivity. A small set of co-affected genes was identified. One of these genes encoding the synaptic scaffolding protein PSD-95 is downregulated in the striatum of all three mutants and in chronically, but not acutely, cocaine-treated mice. At the synaptic level, enhanced long-term potentiation (LTP) of the frontocortico-accumbal glutamatergic synapses correlates with PSD-95 reduction in every case. Finally, targeted deletion of PSD-95 in an independent line of mice enhances LTP, augments the acute locomotor-stimulating effects of cocaine, but leads to no further behavioral plasticity in response to chronic cocaine. Our findings uncover a previously unappreciated role of PSD-95 in psychostimulant action and identify a molecular and cellular mechanism shared between drug-related plasticity and learning.
Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic ...protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5' rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing.
Beyond finding individual genes that are involved in medical disorders, an important challenge is the integration of sets of disease genes with the complexities of basic biological processes. We ...examine this issue by focusing on neuronal multiprotein complexes and their components encoded on the human X chromosome. Multiprotein signaling complexes in the postsynaptic terminal of central nervous system synapses are essential for the induction of neuronal plasticity and cognitive processes in animals. The prototype complex is the
N-methyl-
d-aspartate
receptor
complex/
membrane-associated guanylate kinase–
associated
signaling
complex (NRC/MASC) comprising 185 proteins and embedded within the postsynaptic density (PSD), which is a set of complexes totaling ∼1,100 proteins. It is striking that 86% (6 of 7) of X-linked NRC/MASC genes and 49% (19 of 39) of X-chromosomal PSD genes are already known to be involved in human psychiatric disorders. Moreover, of the 69 known proteins mutated in X-linked mental retardation, 19 (28%) encode postsynaptic proteins. The high incidence of involvement in cognitive disorders is also found in mouse mutants and indicates that the complexes are functioning as integrated entities or molecular machines and that disruption of different components impairs their overall role in cognitive processes. We also noticed that NRC/MASC genes appear to be more strongly associated with mental retardation and autism spectrum disorders. We propose that systematic studies of PSD and NRC/MASC genes in mice and humans will give a high yield of novel genes important for human disease and new mechanistic insights into higher cognitive functions.
Inhibitors of both phosphatidylinositol-3-kinase (PI3-kinase) and MAPK/ERK (mitogen-activate protein kinase/extracellular signal-related kinase) activation inhibit NMDA receptor-dependent long-term ...potentiation (LTP). PI3-kinase inhibitors also block activation of ERK by NMDA receptor stimulation, suggesting that PI3-kinase inhibitors block LTP because PI3-kinase is an essential upstream regulator of ERK activation. To examine this hypothesis, we investigated the effects of PI3-kinase inhibitors on ERK activation and LTP induction in the CA1 region of mouse hippocampal slices. Consistent with the notion that ERK activation by NMDA receptor stimulation is PI3-kinase dependent, the PI3-kinase inhibitor wortmannin partially inhibited ERK2 activation induced by bath application of NMDA and strongly suppressed ERK2 activation by high-frequency synaptic stimulation. PI3-kinase and MEK (MAP kinase kinase) inhibitors had very different effects on LTP, however. Both types of inhibitors suppressed LTP induced by theta-frequency trains of synaptic stimulation, but only PI3-kinase inhibitors suppressed the induction of LTP by high-frequency stimulation or low-frequency stimulation paired with postsynaptic depolarization. Concentrations of PI3-kinase inhibitors that inhibited LTP when present during high-frequency stimulation had no effect on potentiated synapses when applied after high-frequency stimulation, suggesting that PI3-kinase is specifically involved in the induction of LTP. Finally, we found that LTP induced by theta-frequency stimulation was MEK inhibitor insensitive but still PI3-kinase dependent in hippocampal slices from PSD-95 (postsynaptic density-95) mutant mice. Together, our results indicate that the role of PI3-kinase in LTP is not limited to its role as an upstream regulator of MAPK signaling but also includes signaling through ERK-independent pathways that regulate LTP induction.
At excitatory synapses, the postsynaptic scaffolding protein postsynaptic density 95 (PSD-95) couples NMDA receptors (NMDARs) to the Ras GTPase-activating protein SynGAP. The close association of ...SynGAP and NMDARs suggests that SynGAP may have an important role in NMDAR-dependent activation of Ras signaling pathways, such as the MAP kinase pathway, and in synaptic plasticity. To explore this issue, we examined long-term potentiation (LTP), p42 MAPK (ERK2) signaling, and spatial learning in mice with a heterozygous null mutation of the SynGAP gene (SynGAP(-/+)). In SynGAP(-/+) mutant mice, the induction of LTP in the hippocampal CA1 region was strongly reduced in the absence of any detectable alteration in basal synaptic transmission and NMDAR-mediated synaptic currents. Although basal levels of activated ERK2 were elevated in hippocampal extracts from SynGAP(-/+) mice, NMDAR stimulation still induced a robust increase in ERK activation in slices from SynGAP(-/+) mice. Thus, although SynGAP may regulate the ERK pathway, its role in LTP most likely involves additional downstream targets. Consistent with this, the amount of potentiation induced by stimulation protocols that induce an ERK-independent form of LTP were also significantly reduced in slices from SynGAP(-/+) mice. An elevation of basal phospho-ERK2 levels and LTP deficits were also observed in SynGAP(-/+)/H-Ras(-)/- double mutants, suggesting that SynGAP may normally regulate Ras isoforms other than H-Ras. A comparison of SynGAP and PSD-95 mutants suggests that PSD-95 couples NMDARs to multiple downstream signaling pathways with very different roles in LTP and learning.
Specific patterns of neuronal firing induce changes in synaptic strength that may contribute to learning and memory. If the postsynaptic NMDA (N-methyl-D-aspartate) receptors are blocked, long-term ...potentiation (LTP) and long-term depression (LTD) of synaptic transmission and the learning of spatial information are prevented. The NMDA receptor can bind a protein known as postsynaptic density-95 (PSD-95), which may regulate the localization of and/or signalling by the receptor. In mutant mice lacking PSD-95, the frequency function of NMDA-dependent LTP and LTD is shifted to produce strikingly enhanced LTP at different frequencies of synaptic stimulation. In keeping with neural-network models that incorporate bidirectional learning rules, this frequency shift is accompanied by severely impaired spatial learning. Synaptic NMDA-receptor currents, subunit expression, localization and synaptic morphology are all unaffected in the mutant mice. PSD-95 thus appears to be important in coupling the NMDA receptor to pathways that control bidirectional synaptic plasticity and learning.
How individual receptive field properties are formed in the maturing sensory neocortex remains largely unknown. The shortening of N-methyl-D-aspartate (NMDA) receptor currents by 2A subunit (NR2A) ...insertion has been proposed to delimit the critical period for experience-dependent refinement of circuits in visual cortex. In mice engineered to maintain prolonged NMDA responses by targeted deletion of NR2A, the sensitivity to monocular deprivation was surprisingly weakened but restricted to the typical critical period and delayed normally by dark rearing from birth. Orientation preference instead failed to mature, occluding further effects of dark rearing. Interestingly, a full ocular dominance plasticity (but not orientation bias) was selectively restored by enhanced inhibition, reflecting an imbalanced excitation in the absence of NR2A. Many of the downstream pathways involved in NMDA signaling are coupled to the receptor through a variety of protein-protein interactions and adaptor molecules. To further investigate a mechanistic dissociation of receptive field properties in the developing visual system, mice carrying a targeted disruption of the NR2A-associated 95-kDa postsynaptic density (PSD95) scaffolding protein were analyzed. Although the development and plasticity of ocular dominance was unaffected, orientation preference again failed to mature in these mice. Taken together, our results demonstrate that the cellular basis generating individual sensory response properties is separable in the developing neocortex.
The role of the endoplasmic reticulum (ER) localized in dendritic spines has become a subject of intense interest because of its potential functions in local protein synthesis and signal ...transduction. Although it is recognized from electron microscopic studies that not all spines contain ER, little is know of its dynamic regulation or turnover. Here, we report a surprising degree of turnover of ER within spines. Using confocal microscopy imaging we observed continuity of spine‐ER with dendritic ER in hippocampal primary neurons. Over 24 h, less than 50% of spine ER was stable. Despite this high degree of turn over, we identified a significant subset of spines that maintained ER for at least 4 days. These results indicate that within a single neuron, the organelle composition of a spine is unexpectedly dynamic and may explain aspects of the spine‐to‐spine variation in calcium spike magnitude and localized protein synthesis and trafficking.
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