R. Scott Duncan

Differential functional interaction of two Vesl/Homer protein isoforms with ryanodine receptor type 1: a novel mechanism for control of intracellular calcium signaling.

Vesl/Homer proteins physically link proteins that mediate cellular signaling [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851] and thereby influence cellular function [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962]. A previous study reported that Vesl-1L/Homer-1c (V-1L) controls the gain of the intracellular calcium activated calcium channel ryanodine receptor type 1 (RyR1) channel [J. Biol Chem. 277 (2002) 44722]. Here, we show that the function of RyR1 is differentially regulated by two isoforms of Vesl-1/Homer-1, V-1L and Vesl-1S/Homer-1a (V-1S). V-1L increases the activity of RyR1 while important regulatory functions and pharmacological characteristics are preserved. V-1S alone had no effect on RyR1, even though, like V-1L, it is directly bound to the channel. However, V-1S dose-dependently decreased the effects of V-1L on RyR1, providing a novel mechanism for the regulation of intracellular calcium channel activity and calcium homeostasis by changing expression levels of Vesl/Homer proteins.

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling.

Cellular signaling proteins such as metabotropic glutamate receptors, Shank, and different types of ion channels are physically linked by Vesl (VASP/Ena-related gene up-regulated during seizure and LTP)/Homer proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851]. Vesl/Homer proteins have also been implicated in differentiation and physiological adaptation processes [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here we provide evidence that a Vesl/Homer subtype, Vesl-1L/Homer-1c (V-1L), reduces the function of the intracellular calcium channel ryanodine receptor type 2 (RyR2). In contrast, Vesl-1S/Homer-1a (V-1S) had no effect on RyR2 function but reversed the effects of V-1L. In live cells, in calcium release studies and in single-channel electrophysiological recordings of RyR2, V-1L reduced RyR2 activity. Important physiological functions and pharmacological properties of RyR2 are preserved in the presence of V-1L. Our findings demonstrate that a protein-protein interaction between V-1L and RyR2 is not only necessary for organizing the structure of intracellular calcium signaling proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23(2000)80; J. Cell Sci. 113 (2000) 1851; Nat Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348; Nature 386 (1997) 284], but that V-1L also directly regulates RyR2 channel activity by changing its biophysical properties. Thereby it may control cellular calcium homeostasis. These observations suggest a novel mechanism for the regulation of RyR2 and calcium-dependent cellular functions.

Effects of Vesl/Homer Proteins on Intracellular Signaling

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 end functional modulation of a plethora of different binding Proteins. Within multlprotein signaling complexes, Vesl/Homer Proteins influence proteins as diverse as metabotropic glutamate receptors; transient receptor potential channels; intracellular 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 Drosoohila to humans.

Progesterone Potentiates IP 3 -Mediated Calcium Signaling Through Akt/PKB

The activity of cells critically depends on the control of their cytosolic free calcium ion (Ca2+) concentration. The objective of the present study was to identify mechanisms of action underlying the control of the gain of intracellular Ca2+ release by circulating gonadal steroid hormones. Acute stimulation of isolated neurons with progesterone led to IP3R-mediated Ca2+ transients that depend on the activation of the PI3 kinase/Akt/PKB signaling pathway. These results were confirmed at the molecular level and phosphorylation of IP3R type 1 by Akt/PKB was identified as the mechanism of action. Hence, it is likely that circulating gonadal steroid hormones control neuronal activity including phosporylation status through receptor- and kinase-mediated signaling. With a direct control of the gain of the Ca2+ second messenger system as a signaling gatekeeper for neuronal activity the present study identifies a novel pathway for interaction of the endocrine and central nervous system.

A novel organotypic culture model of the postnatal mouse retina allows the study of glutamate-mediated excitotoxicity

A novel organotypic culture method of mouse retina explants is being introduced and characterized to evaluate its usefulness in studying glutamate excitotoxicity. Retinal whole-mounts were dissected from eyes of C57BL/6 mice aged P10-14 and transferred to poly-D-lysine/laminin coated round coverslips. After 7 days in vitro, retina explants were treated with varying concentrations of L-glutamate and cell death was accessed with TUNEL histochemistry. Neurofilament-68 kDa immunoreactivity was used to identify retinal ganglion cells (RGC) with immunohistochemistry. Additional cell markers were used to further characterize the cytoarchitecture of the organotypic retina cultures. Retina explants attached very well to the coated coverslips allowing for experimental manipulation and pharmacological access to the tissue. Hematoxylin-Eosin (HE) staining of vertical cryostat sections of retina explants demonstrated well preserved intact cytoarchitecture under organotypic culture conditions and PKCalpha, Calbindin, GABA, Rhodopsin, GFAP and neurofilament immunoreactivities identifying rod bipolar, horizontal, amacrine, photoreceptor, glial, and retinal ganglion cells, respectively, were not different from freshly isolated mouse retina. Dose dependent glutamate toxicity and accompanying RGC apoptotic cell death were determined by TUNEL histochemistry. In contrast to previously published methods using slice or floating whole-mount cultures, the ex vivo culture system presented here combines accessibility to experimental manipulation, and adherence of whole-mount cultures to a substrate with a significant preservation of retinal cell types, numbers and morphology. The described retina explant culture on glass coverslips allows for effective pharmacological manipulation including the study of neuronal cell death and RGC physiology.

The neuroprotective properties of palmitoylethanolamine against oxidative stress in a neuronal cell line

BackgroundN-acylethanolamines (NAEs) are lipids upregulated in response to cell and tissue injury and are involved in cytoprotection. Arachidonylethanolamide (AEA) is a well characterized NAE that is an endogenous ligand at cannabinoid and vanilloid receptors, but it exists in small quantities relative to other NAE types. The abundance of other NAE species, such as palmitoylethanolamine (PEA), together with their largely unknown function and receptors, has prompted us to examine the neuroprotective properties and mechanism of action of PEA. We hypothesized that PEA protects HT22 cells from oxidative stress and activates neuroprotective kinase signaling pathways.ResultsIndeed PEA protected HT22 cells from oxidative stress in part by mediating an increase in phosphorylated Akt (pAkt) and ERK1/2 immunoreactivity as well as pAkt nuclear translocation. These changes take place within a time frame consistent with neuroprotection. Furthermore, we determined that changes in pAkt immunoreactivity elicited by PEA were not mediated by activation of cannabinoid receptor type 2 (CB2), thus indicating a novel mechanism of action. These results establish a role for PEA as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.ConclusionsThe results from this study reveal that PEA protects HT22 cells from oxidative stress and alters the localization and expression levels of kinases known to be involved in neuroprotection by a novel mechanism. Overall, these results identify PEA as a neuroprotectant with potential as a possible therapeutic agent in neurodegenerative diseases involving oxidative stress.

Control of Intracellular Calcium Signaling as a Neuroprotective Strategy

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.

Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons

Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca(2+)) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca(2+) signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP(3)R-mediated intracellular Ca(2+) responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca(2+) signaling and its related functions.

Proteomic Analysis of Mouse Brain Microsomes: Identification and Bioinformatic Characterization of Endoplasmic Reticulum Proteins in the Mammalian Central Nervous System

The endoplasmic reticulum (ER) is the main source for the storage and release of intracellular calcium in neurons and, thus, contributes to the functionality of a diverse set of pathways that control critical aspects of central nervous system function including but not limited to gene expression, neurotransmission, learning, and memory. ER-derived proteins obtained after subcellular fractionation of mouse brain homogenate were digested with trypsin and the corresponding peptides fractionated by strong cation exchange chromatography followed by LC-MS/MS analysis on a hybrid linear ion trap--Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. A comprehensive catalogue representing 1914 proteins was generated from this particular proteomic analysis using identification criteria that corresponded to a false positive identification rate of 0.4%. Various molecular functions and biological processes relevant to the ER were identified upon gene ontology (GO)-based analysis including pathways associated with molecular transport, protein trafficking and localization, and cell signaling. Comparison of the 2D-LC-MS/MS results with those obtained from shotgun LC-MS/MS analyses demonstrated that most molecular functions and biological processes were represented via GO analysis using either methodology. Results from this comparison as well as a focused investigation into components of calcium-mediated signaling in the mouse brain ER are also presented.

Lauroylethanolamide and linoleoylethanolamide improve functional outcome in a rodent model for stroke

Ischemic stroke is a significant health problem affecting over 6 million people in the United States alone. In addition to surgical and thrombolytic therapeutic strategies for stroke, neuroprotective therapies may offer additional benefit. N-acylethanolamines (NAEs) are signaling lipids whose synthesis is upregulated in response to ischemia, suggesting that they may be neuroprotective. To date only three NAEs, arachidonylethanolamide (NAE 20:4), palmitoylethanolamide (NAE 16:0) and oleoylethanolamide (NAE 18:1) have shown to exert neuroprotective effect in animal models for stroke. Here, we describe neuroprotective effects of the hitherto uncharacterized NAEs, lauroylethanolamide (NAE 12:0) and linoleoylethanolamide (NAE 18:2) in a middle cerebral artery occlusion model of stroke. Pretreatment with NAE 18:2 prior to ischemia/reperfusion (I/R) injury resulted in both significantly reduced cortical infarct volume and improved functional outcome as determined using the neurological deficit score. NAE 12:0 improved neurological deficits without a significant reduction lesion size. Our results suggest that NAEs, as a whole, provide neuroprotection during I/R injury and may have therapeutic benefit when used as complementary treatment with other therapies to improve stroke outcome.