Octopamine metabolically reprograms astrocytes to confer neuroprotection against α-synuclein.

Octopamine is a well-established invertebrate neurotransmitter involved in fight or flight responses. In mammals, its function was replaced by epinephrine. Nevertheless, it is present at trace amounts and can modulate the release of monoamine neurotransmitters by a yet unidentified mechanism. Here, through a multidisciplinary approach utilizing in vitro and in vivo models of α-synucleinopathy, we uncovered an unprecedented role for octopamine in driving the conversion from toxic to neuroprotective astrocytes in the cerebral cortex by fostering aerobic glycolysis. Physiological levels of neuron-derived octopamine act on astrocytes via a trace amine-associated receptor 1-Orai1-Ca2+-calcineurin-mediated signaling pathway to stimulate lactate secretion. Lactate uptake in neurons via the monocarboxylase transporter 2-calcineurin-dependent pathway increases ATP and prevents neurodegeneration. Pathological increases of octopamine caused by α-synuclein halt lactate production in astrocytes and short-circuits the metabolic communication to neurons. Our work provides a unique function of octopamine as a modulator of astrocyte metabolism and subsequent neuroprotection with implications to α-synucleinopathies.

A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6.

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

Orai1 Channels Are Essential for Amplification of Glutamate-Evoked Ca2+ Signals in Dendritic Spines to Regulate Working and Associative Memory.

Store-operated Orai1 calcium channels function as highly Ca2+-selective ion channels and are broadly expressed in many tissues including the central nervous system, but their contributions to cognitive processing are largely unknown. Here, we report that many measures of synaptic, cellular, and behavioral models of learning are markedly attenuated in mice lacking Orai1 in forebrain excitatory neurons. Results with focal glutamate uncaging in hippocampal neurons support an essential role of Orai1 channels in amplifying NMDA-receptor-induced dendritic Ca2+ transients that drive activity-dependent spine morphogenesis and long-term potentiation at Schaffer collateral-CA1 synapses. Consistent with these signaling roles, mice lacking Orai1 in pyramidal neurons (but not interneurons) exhibit striking deficits in working and associative memory tasks. These findings identify Orai1 channels as essential regulators of dendritic spine Ca2+ signaling, synaptic plasticity, and cognition.

GAP-43 and BASP1 in Axon Regeneration: Implications for the Treatment of Neurodegenerative Diseases.

Growth-associated protein-43 (GAP-43) and brain acid-soluble protein 1 (BASP1) regulate actin dynamics and presynaptic vesicle cycling at axon terminals, thereby facilitating axonal growth, regeneration, and plasticity. These functions highly depend on changes in GAP-43 and BASP1 expression levels and post-translational modifications such as phosphorylation. Interestingly, examinations of GAP-43 and BASP1 in neurodegenerative diseases reveal alterations in their expression and phosphorylation profiles. This review provides an overview of the structural properties, regulations, and functions of GAP-43 and BASP1, highlighting their involvement in neural injury response and regeneration. By discussing GAP-43 and BASP1 in the context of neurodegenerative diseases, we also explore the therapeutic potential of modulating their activities to compensate for neuron loss in neurodegenerative diseases.

Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice.

BACKGROUND: Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown. PURPOSE: Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal. RESULTS: We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro. CONCLUSIONS: These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.

Discovery and characterization of novel small-molecule CXCR4 receptor agonists and antagonists.

The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV-1 infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. While previous efforts have identified a number of CXCR4 antagonists, there have been no small molecule agonists reported. Herein, we describe the identification of a novel series of CXCR4 modulators, including the first small molecules to display agonist behavior against this receptor, using a combination of structure- and ligand-based virtual screening. These agonists produce robust calcium mobilization in human melanoma cell lines which can be blocked by the CXCR4-selective antagonist AMD3100. We also demonstrate the ability of these new agonists to induce receptor internalization, ERK activation, and chemotaxis, all hallmarks of CXCR4 activation. Our results describe a new series of biologically relevant small molecules that will enable further study of the CXCR4 receptor and may contribute to the development of new therapeutics.

Regulation of neurogenesis by calcium signaling.

Calcium (Ca(2+)) signaling has essential roles in the development of the nervous system from neural induction to the proliferation, migration, and differentiation of neural cells. Ca(2+) signaling pathways are shaped by interactions among metabotropic signaling cascades, intracellular Ca(2+) stores, ion channels, and a multitude of downstream effector proteins that activate specific genetic programs. The temporal and spatial dynamics of Ca(2+) signals are widely presumed to control the highly diverse yet specific genetic programs that establish the complex structures of the adult nervous system. Progress in the last two decades has led to significant advances in our understanding of the functional architecture of Ca(2+) signaling networks involved in neurogenesis. In this review, we assess the literature on the molecular and functional organization of Ca(2+) signaling networks in the developing nervous system and its impact on neural induction, gene expression, proliferation, migration, and differentiation. Particular emphasis is placed on the growing evidence for the involvement of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in these processes.

Stimulation of hERG1 channel activity promotes a calcium-dependent degradation of cyclin E2, but not cyclin E1, in breast cancer cells.

Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment. Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators.

Store-operated CRAC channels regulate gene expression and proliferation in neural progenitor cells.

Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca(2+) signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca(2+) entry (SOCE) mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La(3+), BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca(2+) signaling by Ca(2+) microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca(2+) entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis.

CXCR4 chemokine receptor signaling mediates pain in diabetic neuropathy.

BACKGROUND: Painful Diabetic Neuropathy (PDN) is a debilitating syndrome present in a quarter of diabetic patients that has a substantial impact on their quality of life. Despite this significant prevalence and impact, current therapies for PDN are only partially effective. Moreover, the cellular mechanisms underlying PDN are not well understood. Neuropathic pain is caused by a variety of phenomena including sustained excitability in sensory neurons that reduces the pain threshold so that pain is produced in the absence of appropriate stimuli. Chemokine signaling has been implicated in the pathogenesis of neuropathic pain in a variety of animal models. We therefore tested the hypothesis that chemokine signaling mediates DRG neuronal hyperexcitability in association with PDN. RESULTS: We demonstrated that intraperitoneal administration of the specific CXCR4 antagonist AMD3100 reversed PDN in two animal models of type II diabetes. Furthermore DRG sensory neurons acutely isolated from diabetic mice displayed enhanced SDF-1 induced calcium responses. Moreover, we demonstrated that CXCR4 receptors are expressed by a subset of DRG sensory neurons. Finally, we observed numerous CXCR4 expressing inflammatory cells infiltrating into the DRG of diabetic mice. CONCLUSIONS: These data suggest that CXCR4/SDF-1 signaling mediates enhanced calcium influx and excitability in DRG neurons responsible for PDN. Simultaneously, CXCR4/SDF-1 signaling may coordinate inflammation in diabetic DRG that could contribute to the development of pain in diabetes. Therefore, targeting CXCR4 chemokine receptors may represent a novel intervention for treating PDN.

The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes.

Integrin-mediated adhesion plays an important role in B cell development and activation. Signaling initiated by antigens, chemokines, or phorbol esters can rapidly convert integrins to an activated adhesion-competent state. The binding of integrins to their ligands can then induce actin-dependent cell spreading, which can facilitate cell-cell adhesion or cell migration on extracellular matrices. The signaling pathways involved in integrin activation and post-adhesion events in B cells are not completely understood. We have previously shown that anti-Ig antibodies, the chemokine stromal cell-derived factor-1 (SDF-1; CXCL12), and phorbol esters activate the Rap1 and Rap2 GTPases in B cells and that Rap activation is essential for SDF-1-induced B cell migration (McLeod, S. J., Li, A. H. Y., Lee, R. L., Burgess, A. E., and Gold, M. R. (2002) J. Immunol. 169, 1365-1371; Christian, S. L., Lee, R. L., McLeod, S. J., Burgess, A. E., Li, A. H. Y., Dang-Lawson, M., Lin, K. B. L., and Gold, M. R. (2003) J. Biol. Chem. 278, 41756-41767). We show here that preventing Rap activation by expressing Rap-specific GTPase-activating protein II (RapGAPII) significantly decreased lymphocyte function-associated antigen-1- and alpha(4) integrin-dependent binding of murine B cell lines to purified adhesion molecules and to other cells. Conversely, augmenting Rap activation by expressing a constitutively active form of Rap2 enhanced B cell adhesion, showing for the first time that Rap2 can promote integrin activation. We also show that blocking Rap activation inhibited anti-Ig-induced cell spreading and phorbol ester-induced actin polymerization as well as anti-Ig- and SDF-1-induced phosphorylation of Pyk2, a tyrosine kinase involved in morphological changes and chemokine-induced B cell migration. Thus, the Rap GTPases regulate integrin-mediated B cell adhesion as well as processes that control B cell morphology and migration.

Oxidative neurotoxicity in rat cerebral cortex neurons: synergistic effects of H2O2 and NO on apoptosis involving activation of p38 mitogen-activated protein kinase and caspase-3.

Oxidative stress in the brain has been increasingly associated with the development of numerous human neurological diseases. Microglia, activated upon neuronal injury or inflammatory stimulation, are known to release superoxide anion (*O(2) (-)), hydrogen peroxide (H(2)O(2)), and nitric oxide (NO), thereby further contributing to oxidative neurotoxicity. The reaction of NO and *O(2) (-), forming the toxic peroxynitrite (ONOO(-)), has been proposed to play a pathogenic role in neuronal injury. However, the interactions between H(2)O(2) and NO during oxidative stress, which may promote or diminish cell death, is less clear. In this study, we explored oxidative neurotoxicity induced by H(2)O(2) plus NO in primary cultures of rat cerebral cortex neurons. As the mechanisms may involve reactions between H(2)O(2) and NO, we monitored the production of ONOO(-)and reactive oxygen species (ROS) throughout the experiments. Results indicated that the NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and H(2)O(2) by themselves elicited neuronal death in a concentration- and time-dependent manner. Sublytic concentrations of H(2)O(2) plus SNAP were sufficient to induce neuronal apoptosis as determined by DNA laddering and fluorescent staining of apoptotic nuclei. Transient ONOO(-)increase was accompanied by rapid H(2)O(2) decay and NO production, whereas ROS slowly decreased following treatment. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation and the cleavage of caspase-3 were observed. Conversely, inhibition of p38 MAPK and caspase-3 significantly reduced apoptotic death induced by H(2)O(2) plus SNAP. These data suggest that H(2)O(2) and NO act synergistically to induce neuronal death through apoptosis in which activation of p38 MAPK and caspase-3 is involved.

Piperlactam S suppresses macrophage migration by impeding F-actin polymerization and filopodia extension.

This study was designed to evaluate the anti-inflammatory effect of piperlactam S on chemoattractant-induced migration, functions underlying leukocyte recruitment in vitro. Results showed that RAW264.7 macrophages migrate toward complement 5a, a powerful chemoattractant for macrophages. This phenomenon could be suppressed concentration dependently by piperlactam S (0.3-30 microM). Fluorescence staining demonstrated that piperlactam S and cytochalasin B both effectively reversed complement 5a-induced cell polarization, filopodia extension, as well as the increase in the cell content of F-actin. Functional inhibition by antibodies suggested that Mac-1 (CD11b) integrin plays a central role in complement 5a-induced migration. However, piperlactam S failed to modify Mac-1 expression. Furthermore, complement 5a triggered the activation of Cdc42, a Rho-family protein involved in the regulation of filopodia extension, with a time course that paralleled that of filopodia extension and which was inhibited by piperlactam S. In summary, piperlactam S exerts anti-inflammatory effects possibly by interfering with cell migration, impeding F-actin polymerization, filopodia formation, and/or Cdc42 activation. However, the detailed mechanism by which piperlactam S regulates the above processes needs further study.

Ca2+ channel blocking effect of iso-S-petasin in rat aortic smooth muscle cells.

The purpose of the present study was to examine the mechanisms underlying the putative hypotensive actions of iso-S-petasin, a sesquiterpene extract of Petasites formosanus through both in vivo and in vitro experiments. Intravenous administration of iso-S-petasin elicited dose-dependent (0.1-1.5 mg/kg) hypotensive and bradycardiac responses in anesthetized rats. Isometric tension recording in isolated thoracic aorta revealed that iso-S-petasin (0.01-100 microM) inhibited KCl- or Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2'-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester)-induced vasoconstriction independent of endothelium. Iso-S-Petasin also attenuated Ca(2+)-induced vasoconstriction in a concentration-dependent manner in Ca(2+)-depleted/high K(+)-depolarized ring segments, indicating that iso-S-petasin inhibited Ca(2+) influx into vascular smooth muscle cells. This was confirmed by whole-cell patch-clamp recording in cultured vascular smooth muscle cells where iso-S-petasin (10-100 microM) appeared to directly inhibit the L-type voltage-dependent Ca(2+) channel (VDCC) activity. Intracellular Ca(2+) concentration ([Ca(2+)](i)) measurements using the fluorescent probe fura-2/AM (1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester) showed suppression of the KCl-stimulated increase in [Ca(2+)](i) by iso-S-petasin (10, 100 microM). In conclusion, these results suggest that Ca(2+) antagonism of the L-type VDCC in vascular smooth muscle cells might largely account for the hypotensive action of iso-S-petasin.

Hypoxia/reoxygenation induces cell injury via different mechanisms in cultured rat cortical neurons and glial cells.

Hypoxia/reoxygenation (H/R) causes cell injury/death. We examined the protection by drugs intervening at various stages of the injury cascade in cultured neurons and glia. Primary cultures of rat cortical neurons and mixed glia were subjected to H/R. Measurements of cell death (by lactate dehydrogenase release into the medium) and viability (by MTT reduction) indicated that H/R led to time-dependent injury in both neuronal and mixed glial cultures. The extent of cell injury in neurons was significantly greater than in glia cells. Pretreatment with (+)-MK-801 hydrogen maleate (MK-801) (an N-methyl-D-aspartate antagonist), N(omega)-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of nitric oxide synthase) or free radical scavengers reduced the extent of the H/R-elicited neuronal damage. MK-801, in contrast, was without effect on glial cells while L-NAME was effective. Our results suggest differential mechanism(s) and susceptibility to injury caused by H/R for neurons and mixed glia.