Caenorhabditis elegans glutamate transporter deletion induces AMPA-receptor/adenylyl cyclase 9-dependent excitotoxicity

In stroke and several neurodegenerative diseases, malfunction of glutamate (Glu) transporters causes Glu accumulation and triggers excitotoxicity. Many details on the cascade of events in the neurodegenerative process remain unclear. As molecular components of glutamatergic synapses are assembled in Caenorhabditis elegans and as many fundamental cellular processes are conserved from nematodes to humans, we studied Glu-induced necrosis in C. elegans and probed its genetic requirements. We combined Δglt-3 , a Glu transporter-null mutation, with expression of a constitutively active form of the alpha subunit of the G protein Gs. While neither Δglt-3 nor expression of the constitutively active form of the alpha subunit of the G protein Gs is severely toxic to C. elegans head interneurons, their combination induces extensive neurodegeneration. Δglt-3 -dependent neurodegeneration acts through Ca²⁺-permeable Glu receptors of the α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, requires calreticulin function, and is modulated by calcineurin and type-9 adenylyl cyclase (AC9). We further show that mammalian AC9 hyperactivates mammalian AMPA-receptors (AMPA-Rs) in a Xenopus oocyte expression system, supporting that the relationship between AMPA-Rs hyperactivation and AC9 might be conserved between nematodes and mammals. AMPA-Rs–AC9 synergism is thus critical for nematode excitotoxicity and could potentially be involved in some forms of mammalian neurodegeneration.     

A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning

Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Galpha regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP(2) synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Galpha regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP(2). We propose that asymmetric generation of PIP(2) by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement.     

Overexpression of PPK-1, the Caenorhabditis elegans Type I PIP kinase, inhibits growth cone collapse in the developing nervous system and causes axonal degeneration in adults

Growth cones are dynamic membrane structures that migrate to target tissue by rearranging their cytoskeleton in response to environmental cues. The lipid phosphatidylinositol (4,5) bisphosphate (PIP₂) resides on the plasma membrane of all eukaryotic cells and is thought to be required for actin cytoskeleton rearrangements. Thus PIP₂ is likely to play a role during neuron development, but this has never been tested in vivo. In this study, we have characterized the PIP₂ synthesizing enzyme Type I PIP kinase (ppk-1) in Caenorhabditis elegans. PPK-1 is strongly expressed in the nervous system, and can localize to the plasma membrane. We show that PPK-1 purified from C. elegans can generate PIP₂in vitro and that overexpression of the kinase causes an increase in PIP₂ levels in vivo. In developing neurons, PPK-1 overexpression leads to growth cones that become stalled, produce ectopic membrane projections, and branched axons. Once neurons are established, PPK-1 overexpression results in progressive membrane overgrowth and degeneration during adulthood. These data suggest that overexpression of the Type I PIP kinase inhibits growth cone collapse, and that regulation of PIP₂ levels in established neurons may be important to maintain structural integrity and prevent neuronal degeneration.     

Imbalance of neurotrophin receptor isoforms TrkB-FL/TrkB-T1 induces neuronal death in excitotoxicity

A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies.     

Novel osmotin attenuates glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat brain

The glutamate-induced excitotoxicity pathway has been reported in several neurodegenerative diseases. Molecules that inhibit the release of glutamate or cause the overactivation of glutamate receptors can minimize neuronal cell death in these diseases. Osmotin, a homolog of mammalian adiponectin, is a plant protein from Nicotiana tabacum that was examined for the first time in the present study to determine its protective effects against glutamate-induced synaptic dysfunction and neurodegeneration in the rat brain at postnatal day 7. The results indicated that glutamate treatment induced excitotoxicity by overactivating glutamate receptors, causing synaptic dysfunction and neuronal apoptosis after 4 h in the cortex and hippocampus of the postnatal brain. In contrast, post-treatment with osmotin significantly reversed glutamate receptor activation, synaptic deficit and neuronal apoptosis by stimulating the JNK/PI3K/Akt intracellular signaling pathway. Moreover, osmotin treatment abrogated glutamate-induced DNA damage and apoptotic cell death and restored the localization and distribution of p53, p-Akt and caspase-3 in the hippocampus of the postnatal brain. Finally, osmotin inhibited glutamate-induced PI3K-dependent ROS production in vitro and reversed the cell viability decrease, cytotoxicity and caspase-3/7 activation induced by glutamate. Taken together, these results suggest that osmotin might be a novel neuroprotective agent in excitotoxic diseases.     

Asymmetric Neuroblast Divisions Producing Apoptotic Cells Require the Cytohesin GRP-1 in Caenorhabditis elegans

Cytohesins are Arf guanine nucleotide exchange factors (GEFs) that regulate membrane trafficking and actin cytoskeletal dynamics. We report here that GRP-1, the sole Caenorhabditis elegans cytohesin, controls the asymmetric divisions of certain neuroblasts that divide to produce a larger neuronal precursor or neuron and a smaller cell fated to die. In the Q neuroblast lineage, loss of GRP-1 led to the production of daughter cells that are more similar in size and to the transformation of the normally apoptotic daughter into its sister, resulting in the production of extra neurons. Genetic interactions suggest that GRP-1 functions with the previously described Arf GAP CNT-2 and two other Arf GEFs, EFA-6 and BRIS-1, to regulate the activity of Arf GTPases. In agreement with this model, we show that GRP-1’s GEF activity, mediated by its SEC7 domain, is necessary for the posterior Q cell (Q.p) neuroblast division and that both GRP-1 and CNT-2 function in the Q.posterior Q daughter cell (Q.p) to promote its asymmetry. Although functional GFP-tagged GRP-1 proteins localized to the nucleus, the extra cell defects were rescued by targeting the Arf GEF activity of GRP-1 to the plasma membrane, suggesting that GRP-1 acts at the plasma membrane. The detection of endogenous GRP-1 protein at cytokinesis remnants, or midbodies, is consistent with GRP-1 functioning at the plasma membrane and perhaps at the cytokinetic furrow to promote the asymmetry of the divisions that require its function.     

Regulation of PIP5K activity by Arf6 and its physiological significance

The phospholipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the phosphorylation of the membrane phospholipid phosphatidylinositol 4-phosphate to generate the pleiotropic phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2) ]. To date, three mammalian PIP5K isozymes, α, β, and γ, and several splicing variants of the γ isozyme have been identified. These PIP5K isozymes and PIP5Kγ variants play critical roles in various cellular functions through their product PI(4,5)P(2) . The small GTPase Arf6 is one of the key activators of PIP5K. Increasing evidence suggests that PIP5K functions as a downstream effector of Arf6 to regulate a wide variety of cellular functions, such as exocytosis, endocytosis, endosomal recycling, membrane ruffle formation, immune response, and bacterial invasion. In this review, we place our focus on the recent advances in Arf6/PIP5K signaling and its linkage to cellular functions.     

G-protein-coupled receptor activation induces the membrane translocation and activation of phosphatidylinositol-4-phosphate 5-kinase I alpha by a Rac- and Rho-dependent pathway.

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K I alpha activation following the addition of thrombin. Under resting condition expressed PIP5K I alpha was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of G alpha(q), PIP5K I alpha translocated to the plasma membrane. Movement of PIP5K I alpha to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active G alpha(q), 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K I alpha translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K I alpha. In addition, a dominant negative variant of Rho blocked the PIP5K I alpha membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K I alpha membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K I alpha by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K I alpha, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K I alpha correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K I alpha with either constitutively active G alpha(q), Rac, or Rho led to a 5- to 7-fold increase in PIP5K I alpha activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of G alpha(q), Rac, Rho, and PIP5K I alpha. Once activated and translocated to the cell membrane, PIP5K I alpha becomes available to phosphorylate PI4P to generate PI4,5P(2) on the plasma membrane.     

PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila

The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.     

Dynamic interaction between Arf GAP and PH domains of ASAP1 in the regulation of GAP activity

ASAP family Arf GAPs induce the hydrolysis of GTP bound to the Ras superfamily protein Arf1, regulate cell adhesion and migration and have been implicated in carcinogenesis. The ASAP proteins have a core catalytic domain of PH, Arf GAP and Ank repeat domains. The PH domain is necessary for both biological and catalytic functions of ASAP1 and has been proposed to be integrally folded with the Arf GAP domain. Protection studies and analytical ultracentrifugation studies previously reported indicated that the domains are, at least partly, folded together. Here, using NMR spectroscopy and biochemical analysis, we have further tested this hypothesis and characterized the interdomain interaction. A comparison of NMR spectra of three recombinant proteins comprised of either the isolated PH domain of ASAP1, the Arf GAP and ankyrin repeat domain or all three domains indicated that the PH domain did interact with the Arf GAP and Ank repeat domains; however, we found a significant amount of dynamic independence between the PH and Arf GAP domains, consistent with the interactions being transient. In contrast, the Arf GAP and Ank repeat domains form a relatively rigid structure. The PH-Arf GAP domain interaction partially occluded the phosphoinositide binding site in the soluble protein, but binding studies indicated the PIP2 binding site was accessible in ASAP1 bound to a lipid bilayer surface. Phosphoinositide binding altered the conformation of the PH domain, but had little effect on the structure of the Arf GAP domain. Mutations in a loop of the PH domain that contacts the Arf GAP domain affected PIP2 binding and the K(m) and k(cat) for converting Arf1 GTP to Arf1 GDP. Based on these results, we generated a homology model of a composite PH/Arf GAP/Ank repeat domain structure. We propose that the PH domain contributes to Arf GAP activity by either binding to or positioning Arf1 GTP that is simultaneously bound to the Arf GAP domain.     

Wild-type huntingtin protects neurons from excitotoxicity

Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase-mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor-mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over-expressing full-length wild-type huntingtin were dramatically protected from apoptosis and caspase-3 activation compared with cultured striatal neurons from non-transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase-3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full-length huntingtin, and the generation of a specific N-terminal caspase cleavage product of huntingtin. In vivo, over-expression of wild-type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor-mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtin's anti-apoptotic function will contribute to our understanding of the pathogenesis of Huntingdon's disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.     

Modeling the dynamic behaviors of the COPI vesicle formation regulators,the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain–containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain–containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.     

Activation of ERK1/2 and PI3K/Akt by IGF-1 on GAP-43 Expression in DRG Neurons with Excitotoxicity Induced by Glutamate In Vitro

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Whether IGF-1 influences growth-associated protein 43 (GAP-43) expression and activates the extracellular signal-regulated protein kinase (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in DRG neurons with excitotoxicity induced by glutamate (Glu) remains unknown. In this study, embryonic 15-day-old rat DRG explants were cultured for 48 h and then exposed to IGF-1, Glu, Glu + IGF-1, Glu + IGF-1 + PD98059, Glu + IGF-1 + LY294002, Glu + IGF-1 + PD98059 + LY294002 for additional 12 h. The DRG explants were continuously exposed to growth media as control. The levels of GAP-43 mRNA were detected by real time-PCR analysis. The protein levels of GAP-43, phosphorylated ERK1/2, phosphorylated Akt, total ERK1/2, and total Akt were detected by Western blot assay. GAP-43 expression in situ was determined by immunofluorescent labeling. Apoptotic cell death was monitored by Hoechst 33342 staining. IGF-1 alone increased GAP-43 and its mRNA levels in the absence of Glu. The decreased GAP-43 and its mRNA levels caused by Glu could be partially reversed by the presence of IGF-1. IGF-1 rescued neuronal cell death caused by Glu. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective. To validate the impact of GAP-43 expression by IGF-1, GAP-43 induction was blocked by administration of dexamethasone (DEX). IGF-1 partially rescued the decrease of GAP-43 and its mRNA levels induced by DEX. DEX induced an increase of cell apoptosis. IGF-1 may play an important role in neuroprotective effects on DRG neurons through regulating GAP-43 expression with excitotoxicity induced by Glu and the process was involved in both ERK1/2 and PI3K/Akt signaling pathways.     

Kainate-induced excitotoxicity is dependent upon extracellular potassium concentrations that regulate the activity of AMPA/KA type glutamate receptors

In addition to well-known N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, recent studies suggest that non-NMDA type ionotropic glutamate receptors are also important mediators of excitotoxic neuronal death, and that their functional expression can be regulated by the cellular environment. In this study, we used cerebellar granule cells (CGCs) in culture to investigate kainate (KA)-induced excitotoxicity. Although previous reports indicated that KA induces apoptosis of CGCs in culture, no KA-induced excitotoxic cell death was observed in CGCs treated with KA when cells were maintained in high potassium media (24 mm K+). In contrast, when mature CGCs were shifted into low potassium media (3 mm K+), KA produced significant excitotoxicity. In electrophysiological studies, the KA-induced inward current density was significantly elevated in CGCs shifted into low K+ media compared with those maintained in high K+ media. Non-desensitizing aspects of KA currents observed in this study suggest that these responses were mediated by AMPA rather than KA receptors. In immunofluorescence studies, the surface expression of GluR1 subunits increased when mature CGCs were shifted into a low K+ environment. This study suggests that KA-induced excitotoxicity in mature CGCs is dependent upon the extracellular potassium concentration, which modulates functional expression and excitability of AMPA/KA receptors.     

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP‐1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l ‐Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP‐1 activity using overexpression of the dominant negative DRP‐1^K38A slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild‐type DRP‐1. In contrast, responses to l ‐Glu appeared DRP‐1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca²⁺ uptake or the ability of l ‐Glu to stimulate excitotoxic Ca²⁺ overload. Finally, apoptosis‐induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:335–341, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

Manipulating insulin signaling to enhance mosquito reproduction

Backgrond  

In the mosquito Aedes aegypti the insulin/insulin growth factor I signaling (IIS) cascade is a key regulator of many physiological processes, including reproduction. Two important reproductive events, steroidogenesis in the ovary and yolk synthesis in the fat body, are regulated by the IIS cascade in mosquitoes. The signaling molecule phosphatase and tensin homolog (PTEN) is a key inhibitor of the IIS cascade that helps modulate the activity of the IIS cascade. In Ae. aegypti, six unique splice variants of AaegPTEN were previously identified, but the role of these splice variants, particularly AaegPTEN3 and 6, were unknown.