The clustering of signaling molecules at specialized cellular sites allows cells to effectively convert extracellular signals into intracellular signals and to produce a concerted functional output with specific temporal and spatial patterns. A prime example for these molecules and their effects on cellular signaling are the postsynaptic density proteins of the central nervous system. Recently, one group of these proteins, the Vesl/Homer protein family has received increased attention because of its unique molecular properties that allow both the clustering and functional modulation of a plethora of different binding proteins. Within multiprotein signaling complexes, Vesl/Homer proteins influence proteins as diverse as metabotropic glutamate receptors; transient receptor potential channels; intra-cellular calcium channels; the scaffolding protein, Shank; small GTPases; transcription factors; and cytoskeletal proteins. Furthermore, interaction with such functionally relevant proteins also links Vesl/Homer proteins indirectly to an even larger group of cellular effector proteins, putting the Vesl/Homer proteins at the crossroads of several critical intracellular signaling processes. In addition to the initial reports of Vesl/Homer protein expression in the central nervous system, members of this protein family have now been identified in other excitable cells in various muscle types and in a large number of nonexcitable cells. The widespread expression of Vesl/Homer proteins in different organs and their functional importance in cellular protein signaling complexes is further evidenced by their conservation in organisms from Drosophila to humans.

Hyperfiltration subjects podocytes to increased tensile stress and fluid flow shear stress (FFSS). We showed a 1.5- to 2.0-fold increase in FFSS in uninephrectomized animals and altered podocyte actin cytoskeleton and increased synthesis of prostaglandin E2 (PGE2) following in vitro application of FFSS. We hypothesized that increased FFSS mediates cellular changes through specific receptors of PGE2. Presently, we studied the effect of FFSS on cultured podocytes and decapsulated isolated glomeruli in vitro, and on solitary kidney in uninephrectomized sv129 mice. In cultured podocytes, FFSS resulted in increased gene and protein expression of cyclooxygenase (COX)-2 but not COX-1, prostanoid receptor EP2 but not EP4, and increased synthesis and secretion of PGE2, which were effectively blocked by indomethacin. Next, we developed a special flow chamber for applying FFSS to isolated glomeruli to determine its effect on an intact glomerular filtration barrier by measuring change in albumin permeability (Palb) in vitro. FFSS caused an increase in Palb that was blocked by indomethacin (P < 0.001). Finally, we show that unilateral nephrectomy in sv129 mice resulted in glomerular hypertrophy (P = 0.006), increased glomerular expression of COX-2 (P < 0.001) and EP2 (P = 0.039), and increased urinary albumin excretion (P = 0.001). Activation of the COX-2-PGE2-EP2 axis appears to be a specific response to FFSS in podocytes and provides a mechanistic basis for alteration in podocyte structure and the glomerular filtration barrier, leading to albuminuria in hyperfiltration-mediated kidney injury. The COX-2-PGE2-EP2 axis is a potential target for developing specific interventions to ameliorate the effects of hyperfiltration-mediated kidney injury in the progression of chronic kidney disease.

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Minimal change disease (MCD), the most common idiopathic nephrotic syndrome in children, is characterized by proteinuria and loss of glomerular visceral epithelial cell (podocyte) ultrastructure. Lipopolysaccharide (LPS) and puromycin aminonucleoside (PAN) are used to study podocyte injury in models of MCD in vivo and in vitro. We hypothesized that LPS and PAN influence components of the innate immune system in podocytes such as the Toll-Like Receptor (TLRs), TLR adapter molecules, and associated cytokines. Our results show that cultured human podocytes constitutively express TLRs 1-6 and TLR-10, but not TLRs 7-9. LPS (25 μg/ml) or PAN (60 μg/ml) caused comparable derangement of the actin cytoskeleton in podocytes. Quantitative RT-PCR analysis show that LPS differentially up-regulated the expression of genes for TLRs (1 > 4 ≥ 2 > 3 > 6 > 5), the adapter molecule, MyD88, and transcription factor NF-κB within one hour. LPS also caused increased levels of IL-6, IL-8 and MCP1 without exerting any effect on TNF-α, IFN-α or TGF-β1 at 24 h. Immunofluorescence intensity analysis of confocal microscopy images showed that LPS induced a significant increase in nuclear translocation of NF-κB by 6 h. In contrast, PAN-induced only small changes in the expression of TLRs 2-6 that included a persistent increase in TLRs 2 and 5, a transient increase in TLR-4, and a gradual increase in TLRs 3 and 6 between 1 and 6 h. Correspondingly, it did not alter pro-inflammatory cytokine levels in podocytes. However, PAN induced a low but significant increase in NF-κB nuclear translocation within one hour that remained unchanged up to 6 h. In summary, these novel findings show that LPS, a known TLR-4 ligand, induced the gene expression of multiple TLRs with maximum effect on the expression of TLR-1 suggesting a loss of receptor selectivity and induction of receptor interactions in podocytes. A comparable derangement of the podocyte cytoskeleton and significant increase in the nuclear translocation of NF-κB by PAN suggest that disparate but complementary mechanisms may contribute to the development of podocytopathy in MCD.

N-acylethanolamines (NAEs) are endogenous lipids that are synthesized in response to tissue injury, including ischemia and stroke, suggesting they may exhibit neuroprotective properties. We hypothesized that NAE 16:0 (palmitoylethanolemine) is neuroprotective against ischemiareperfusion injury in rats, a widely employed model of stroke, and that neuroprotection is mediated through an intracellular mechanism independent of known NAE receptors. Administration of NAE 16:0 from 30 min. before to 2 hrs after stroke significantly reduced cortical and subcortical infarct volume, and correlated with an improvement of the neurological phenotype, as assessed by the neurological deficit score. We here show that NAE 16:0-mediated neuroprotection was independent of cannabinoid (CB1) and vanilloid (VR1) receptor activation, known NAE receptors on the plasma membrane, as determined by inclusion of specific inhibitors. The inclusion of an NAE uptake inhibitor (AM404), however, completely reversed NAE 16:0-mediated neuroprotection, suggesting that NAE 16:0's effects are through an intracellular mechanism. NAE 16:0 produced a significant reduction in the number of cells undergoing apoptosis and reversed ischemia-induced up-regulation of several proteins, including inducible nitric oxide synthase and transcription factor NFκB. Our findings suggest that NAE 16:0-mediated neuroprotection is due to the reduction of neuronal apoptosis and inflammation in the brain. Keywords neurodegeneration; neuroprotection; receptors; ischemiaStroke is the third leading cause of death in the United States with approximately 5.8 million current cases and approximately 780,000 new cases every year, making it a significant health problem (Lloyd-Jones et al., 2009). Potential neuroprotective treatment and intervention strategies have been tested over the last several years, but with limited success (White et al., 2000;Lo et al., 2003). Studies utilizing animal surgical models of stroke, such as ischemiareperfusion injury induced by middle cerebral artery occlusion (MCAO), have been utilized to identify potential neuroprotective agents (Wen et al., 2004a, b;Hu et al., 2004;Green and Ashwood, 2005). Assessment of the efficacy of neuroprotective compounds against ischemiacCorresponding author: R. Scott Duncan, University of Missouri -Kansas City, School of Medicine, Department of Basic Medical Science, 2411 Holmes Street, Kansas City, MO 64108, Phone: (816) Fax: (816) 235-6594, duncanrs@umkc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manus...

Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed.

Using TLR pathways, primary human cytomegalovirus (HCMV) induces innate responses including the production of inflammatory cytokines. Mounting evidence suggests that LPS recognition by TLR4/MD2/CD14 results in differential utilization of TIRAP-TRAF6 and TRAM-TRIF signaling, thereby leading to transcriptional activation of various cytokine genes. However, relative roles of the TLR4/MD2/CD14 complex and its adaptor proteins TIRAP and TRAM involved in regulating monocyte responses to HCMV are incomplete. Here, we provided evidence supporting the notion that the TLR4/MD2/CD14 complex contributes notably to HCMV-induced signaling and subsequent cytokine production in monocytes. In particular, induction of both IL-6 and IL-8 is associated with elevated TIRAP and reduced TRAM mRNA expression. The latter may serve in a compensatory pathway that yields a robust IFN response when TIRAP signaling is blocked in monocytes incubated with Toledo strain HCMV. Inhibitory studies using antisense oligonucleotides or neutralizing antibodies indicate that IL-6 induction by TLR4/MD2 complex is important for the activation of endogenous CD14 which later acts in concert or synergy with TLR4/MD2 as a factor resulting in IL-8 gene expression. We further show that exogenous recombinant CD14 can potentiate innate immune response via TLR4-dependent and possibly via TLR9-dependent pathways to promote enhanced expression/production of IL-8 and IFN-β, respectively.