Hierarchical organization and genetically separable subfamilies of PSD95 postsynaptic supercomplexes

PSD95 is an abundant postsynaptic scaffold protein in glutamatergic synapses that assembles into supercomplexes composed of over 80 proteins including neurotransmitter receptors, ion channels and adhesion proteins. How these diverse constituents are organized into PSD95 supercomplexes in vivo is poorly understood. Here, we dissected the supercomplexes in mice combining endogenous gene‐tagging, targeted mutations and quantitative biochemical assays. Generating compound heterozygous mice with two different gene‐tags, one on each Psd95 allele, showed that each ~1.5 MDa PSD95‐containing supercomplex contains on average two PSD95 molecules. Gene‐tagging the endogenous GluN1 and PSD95 with identical Flag tags revealed N‐methyl D‐aspartic acid receptors (NMDARs) containing supercomplexes that represent only 3% of the total population of PSD95 supercomplexes, suggesting there are many other subtypes. To determine whether this extended population of different PSD95 supercomplexes use genetically defined mechanisms to specify their assembly, we tested the effect of five targeted mouse mutations on the assembly of known PSD95 interactors, Kir2.3, Arc, IQsec2/BRAG1 and Adam22. Unexpectedly, some mutations were highly selective, whereas others caused widespread disruption, indicating that PSD95 interacting proteins are organized hierarchically into distinct subfamilies of ~1.5 MDa supercomplexes, including a subpopulation of Kir2.3‐NMDAR ion channel‐channel supercomplexes. Kir2.3‐NMDAR ion channel‐channel supercomplexes were found to be anatomically restricted to particular brain regions. These data provide new insight into the mechanisms that govern the constituents of postsynaptic supercomplexes and the diversity of synapse types

SynGAP isoforms exert opposing effects on synaptic strength

Peer reviewedPublisher PD

RNA-seq Analysis Reveals That an ECF σ Factor, AcsS, Regulates Achromobactin Biosynthesis in Pseudomonas syringae pv. syringae B728a

Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a

BOD1 Is Required for Cognitive Function in Humans and <i>Drosophila</i>

Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features

Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties <it>in vitro</it>

<p>Abstract</p> <p>Background</p> <p>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 <it>in vitro</it>. 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.</p> <p>Results</p> <p>Mouse ES cells carrying homozygous null mutations (FAK^-/-) were generated and differentiated <it>in vitro </it>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.</p> <p>Conclusion</p> <p>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.</p

Chronic treatment with the MEK inhibitor PD-0325901 reverses enhanced I-O relationships of excitatory field potentials in Syngap1+/− mice

Background: Synaptic Ras-GTPase-activating protein 1 (SYNGAP1) is an abundant brain-specific protein localized at the postsynaptic density of mammalian excitatory synapses. SYNGAP1 functions as a crucial regulator of downstream intracellular signaling triggered by N-methyl-D-aspartate receptor activation. One of the most important signaling pathways regulated by SYNGAP1 is the Ras-Raf-MEK-ERK pathway. SYNGAP1 deficiency is associated with hyperphosphorylation of MEK and ERK kinases and with altered synaptic function in Syngap1+/− mice. Loss-of-function mutations in the SYNGAP1 gene have been documented in many human cognitive and neurological disorders. However, there are currently no approaches that reverse the phenotypes of SYNGAP1 deficiency. Methods: Using electrophysiological recordings of field responses in hippocampal slices, we examined if disturbances of synaptic physiology in the hippocampus of 7–8-month old Syngap1+/− mice were sensitive to the effect of the MEK inhibitor PD-0325901 given orally for 6 days. Results: We found that in hippocampal slices from vehicle-treated Syngap1+/− mice, basal synaptic responses were higher and their long-term potentiation (LTP) was lower than in slices from wild-type littermates. Chronic administration of PD-0325901 normalized the basal synaptic responses, but did not reverse the LTP deficit. Conclusions: The differential sensitivity of basal synaptic transmission and LTP to MEK inhibition indicates that the effects of SYNGAP1 deficiency on these synaptic parameters are mediated by distinct pathways. Our findings also suggest that at least some physiological phenotypes of the germline Syngap1 mutation can be ameliorated by pharmacological treatment of adult animals

SynGAP isoforms exert opposing effects on synaptic strength.

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

Development of neuroendocrine tumors in the gastro-intestinal tract of transgenic mice: heterogeneity of hormone expression.

AM J PATHO

PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits.

The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity