Deletion of novel protein TMEM35 alters stress-related functions and impairs long-term memory in mice.

The mounting of appropriate emotional and neuroendocrine responses to environmental stressors critically depends on the hypothalamic-pituitary-adrenal (HPA) axis and associated limbic circuitry. Although its function is currently unknown, the highly evolutionarily conserved transmembrane protein 35 (TMEM35) is prominently expressed in HPA circuitry and limbic areas, including the hippocampus and amygdala. To investigate the possible involvement of this protein in neuroendocrine function, we generated tmem35 knockout (KO) mice to characterize the endocrine, behavioral, electrophysiological, and proteomic alterations caused by deletion of the tmem35 gene. While capable of mounting a normal corticosterone response to restraint stress, KO mice showed elevated basal corticosterone accompanied by increased anxiety-like behavior. The KO mice also displayed impairment of hippocampus-dependent fear and spatial memories. Given the intact memory acquisition but a deficit in memory retention in the KO mice, TMEM35 is likely required for long-term memory consolidation. This conclusion is further supported by a loss of long-term potentiation in the Schaffer collateral-CA1 pathway in the KO mice. To identify putative molecular pathways underlying alterations in plasticity, proteomic analysis of synaptosomal proteins revealed lower levels of postsynaptic molecules important for synaptic plasticity in the KO hippocampus, including PSD95 and N-methyl-d-aspartate receptors. Pathway analysis (Ingenuity Pathway Analysis) of differentially expressed synaptic proteins in tmem35 KO hippocampus implicated molecular networks associated with specific cellular and behavioral functions, including decreased long-term potentiation, and increased startle reactivity and locomotion. Collectively, these data suggest that TMEM35 is a novel factor required for normal activity of the HPA axis and limbic circuitry.

Fetal iron deficiency alters the proteome of adult rat hippocampal synaptosomes.

Fetal and neonatal iron deficiency results in cognitive impairments in adulthood despite prompt postnatal iron replenishment. To systematically determine whether abnormal expression and localization of proteins that regulate adult synaptic efficacy are involved, we used a quantitative proteomic approach (isobaric tags for relative and absolute quantitation, iTRAQ) and pathway analysis to identify dysregulated proteins in hippocampal synapses of fetal iron deficiency model. Rat pups were made iron deficient (ID) from gestational day 2 through postnatal day (P) 7 by providing pregnant and nursing dams an ID diet (4 ppm Fe) after which they were rescued with an iron-sufficient diet (200 ppm Fe). This paradigm resulted in a 40% loss of brain iron at P15 with complete recovery by P56. Synaptosomes were prepared from hippocampi of the formerly iron-deficient (FID) and always iron-sufficient controls rats at P65 using a sucrose gradient method. Six replicates per group that underwent iTRAQ labeling and LC-MS/MS analysis for protein identification and comparison elucidated 331 differentially expressed proteins. Western analysis was used to confirm findings for selected proteins in the glutamate receptor signaling pathway, which regulates hippocampal synaptic plasticity, a cellular process critical for learning and memory. Bioinformatics were performed using knowledge-based Interactive Pathway Analysis. FID synaptosomes show altered expression of synaptic proteins-mediated cellular signalings, supporting persistent impacts of fetal iron deficiency on synaptic efficacy, which likely cause the cognitive dysfunction and neurobehavioral abnormalities. Importantly, the findings uncover previously unsuspected pathways, including neuronal nitric oxide synthase signaling, identifying additional mechanisms that may contribute to the long-term biobehavioral deficits.

The guanylate kinase domain of the MAGUK PSD-95 binds dynamically to a conserved motif in MAP1a.

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinases are scaffolding proteins, whose modular interaction motifs organize protein complexes at cell junctions. The signature guanylate kinase domain (GK) contains elements of the protein's GMP-binding site but does not bind nucleotide. Instead, the GK domain has evolved from an enzyme to a protein-protein interaction motif. Here, we show that this canonical GMP-binding region interacts with microtubule-associated protein-1a (MAP1a) and we present a structural model. We determine the consensus GK-binding sequence in MAP1a and demonstrate that PSD-95 can use a similar interaction mode to bind diverse protein partners. Furthermore, we show that PSD-95 GK has adopted the conformational flexibility of the ancestral enzyme to bind its varied ligands, which suggests a mechanism of regulation.

Interaction of transmembrane AMPA receptor regulatory proteins with multiple membrane associated guanylate kinases.

Surface expression of AMPA type glutamate receptors requires stargazin or a related transmembrane AMPA receptor regulatory protein (TARP). Furthermore, interaction of the cytosolic tail of TARPs with PDZ domains of PSD-95 targets AMPA receptors to postsynaptic densities. Here, we screened for additional proteins that might interact with the cytosolic domain of TARPs. Screening a rat brain cDNA library with the yeast two-hybrid system yielded six PDZ proteins that can bind tail of TARPs. These PDZ proteins include the four neuronal membrane associated guanylate kinases, PSD-95/SAP-90, PSD-93/Chapsyn-110, SAP-97/hDLG and SAP-102; the multi-PDZ protein, MUPP1; and the mitochondrial PDZ protein, OMP-25. Although all of these proteins can bind to TARPs in vitro, only two of these, PSD-95 and PSD-93 associate with TARPs in brain. We also found that all three PDZ domains from PSD-95 associate with the TARP C-termini with similar affinities. This work identifies biochemical promiscuity for interaction of the TARP C-termini with PDZ domains in vitro, but also shows that only specific synaptic PDZ proteins associate with TARPs in brain.

Isolated hypoglossal schwannoma in a 9-year-old child.

The authors report a case of an isolated schwannoma of left hypoglossal nerve in a 9-year-old girl. To the authors' knowledge, this is the first case report of hypoglossal nerve schwannoma in the pediatric population in the absence of neurofibromatosis Type 2. The patient presented with a 2-month history of morning nausea and vomiting with occasional daytime headaches. Magnetic resonance imaging and subsequent CT scanning revealed a dumbbell tumor with a belly in the lower third of the posterior fossa and head underneath the left jugular foramen. Its neck protruded through an expanded hypoglossal canal. Although the lesion bore radiological characteristics of a hypoglossal schwannoma, the absence of hypoglossal palsy and the apparent lack of such tumors in the pediatric population the preoperative diagnosis was not certain. The tumor was approached via a midline suboccipital craniotomy, and gross-total resection was achieved. Pathological examination confirmed the diagnosis of schwannoma. Blood and tumor tests for mutations in the NF2 gene were negative. Postoperative mild hypoglossal palsy recovered by the 3-month follow-up, and an MRI study obtained at 1 year did not show recurrence.

Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions.

Tissue development, differentiation, and physiology require specialized cellular adhesion and signal transduction at sites of cell-cell contact. Scaffolding proteins that tether adhesion molecules, receptors, and intracellular signaling enzymes organize macromolecular protein complexes at cellular junctions to integrate these functions. One family of such scaffolding proteins is the large group of membrane-associated guanylate kinases (MAGUKs). Genetic studies have highlighted critical roles for MAGUK proteins in the development and physiology of numerous tissues from a variety of metazoan organisms. Mutation of Drosophila discs large (dlg) disrupts epithelial septate junctions and causes overgrowth of imaginal discs. Similarly, mutation of lin-2, a related MAGUK in Caenorhabditis elegans, blocks vulval development, and mutation of the postsynaptic density protein PSD-95 impairs synaptic plasticity in mammalian brain. These diverse roles are explained by recent biochemical and structural analyses of MAGUKs, which demonstrate their capacity to assemble well--efined--yet adaptable--protein complexes at cellular junctions.

A revised mechanism for the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA based on unexpected results with the C114A mutant.

The compound (methylenecyclopropyl)formyl-CoA (MCPF-CoA) has been reported earlier as a potent active site-directed inactivator of bovine liver enoyl-CoA hydratase (ECH). It is believed that the mechanism of inactivation involves the attack of Cys114 at C-2' of MCPF-CoA, resulting in ring cleavage and permanent covalent modification of the enzyme. Here, we describe studies with the C114A mutant of bovine liver ECH, which was constructed and purified to determine the role of this residue in the catalytic mechanism of the enzyme. The C114A mutant, which is catalytically competent, shows an unexpected susceptibility to inactivation by MCPF-CoA, indicating that Cys114 is not the primary nucleophile responsible for the inactivation of the enzyme. To determine if catalytic residues Glu115 and Glu135 play a role in the inactivation of the enzyme, the E115Q and E135Q mutants were also constructed and purified. It was determined that these mutants did not react with MCPF-CoA, indicating a possible role for both residues in the inactivation of the wild-type enzyme. Pepsin digestion and subsequent LC-MS/MS analysis of the inactivated wild-type enzyme and C114A mutant revealed that Glu115 was modified in each case, supporting the hypothesis that this residue is the true nucleophile that traps MCPF-CoA and indicating that the covalent modification of Cys114 reported earlier may be a postinactivation artifact. We propose a modified mechanism of inactivation involving Glu115 and Glu135, and suggest that MCPF-CoA may be a mechanism-based inhibitor for bovine liver ECH.

Synaptic strength regulated by palmitate cycling on PSD-95.

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.