Journal of Neurochemistry

Frenzel, Kristen E.; Falls, Douglas L.

doi: 10.1046/j.1471-4159.2001.00132.xpmid: N/A

Neuregulin‐1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin‐1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N‐terminal fragment containing the bioactive EGF‐like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140‐kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X‐100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low‐density fractions enriched in PSD‐95, a known lipid raft protein. In transfected cells expressing the neuregulin I‐β1a or the III‐β1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X‐100, and neuregulin proproteins and C‐terminal fragments were detected in lipid raft fractions. In contrast, the III‐β1a N‐terminal fragment was detected only in the detergent‐soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.

Gasull, Teresa; DeGregorio‐Rocasolano, Nuria; Trullas, Ramon

doi: 10.1046/j.1471-4159.2001.00187.xpmid: N/A

Glutamate receptor overactivation induces excitotoxic neuronal death, but the contribution of glutamate receptor subtypes to this excitotoxicity is unclear. We have previously shown that excitotoxicity by NMDA receptor overactivation is associated with choline release and inhibition of phosphatidylcholine synthesis. We have now investigated whether the ability of non‐NMDA ionotropic glutamate receptor subtypes to induce excitotoxicity is related to the ability to inhibit phosphatidylcholine synthesis. α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)‐induced a concentration‐dependent increase in extracellular choline and inhibited phosphatidylcholine synthesis when receptor desensitization was prevented. Kainate released choline and inhibited phosphatidylcholine synthesis by an action at AMPA receptors, because these effects of kainate were blocked by the AMPA receptor antagonist LY300164. Selective activation of kainate receptors failed to release choline, even when kainate receptor desensitization was prevented. The inhibition of phosphatidylcholine synthesis evoked by activation of non‐desensitizing AMPA receptors was followed by neuronal death. In contrast, specific kainate receptor activation, which did not inhibit phosphatidylcholine synthesis, did not produce neuronal death. Choline release and inhibition of phosphatidylcholine synthesis were induced by AMPA at non‐desensitizing AMPA receptors well before excitotoxicity. Furthermore, choline release by AMPA required the entry of Ca2+ through the receptor channel. Our results show that AMPA, but not kainate, receptor overactivation induces excitotoxic cell death, and that this effect is directly related to the ability to inhibit phosphatidylcholine synthesis. Moreover, these results indicate that inhibition of phosphatidylcholine synthesis is an early event of the excitotoxic process, downstream of glutamate receptor‐mediated Ca2+ overload.

Marsh, Daniel R.; Holmes, Kevin D.; Dekaban, Gregory A.; Weaver, Lynne C.

doi: 10.1046/j.1471-4159.2001.00182.xpmid: N/A

The NMDA receptor plays an important role in mediating sensory input to the spinal cord. Domains within the C‐terminus of the NMDA receptor bind to cytoskeletal proteins and facilitate membrane targeting and synaptic clustering, and may participate in regulation of receptor function. One strategy to manipulate NMDA receptor function is to express C‐terminal constructs in neurons to disrupt synaptic clustering via competition for binding motifs in cytoskeletal proteins and postsynaptic densities. Biolistic particle‐mediated gene transfer was used to deliver plasmid DNA into organotypic cultures of dorsal root ganglia (DRG). Fusion proteins consisting of recombinant (r)NMDA receptor subunit 1‐1 (rNR1‐1) deletion constructs and enhanced green fluorescent protein (GFP) were expressed in sensory neurons and demonstrated unique distribution patterns within the cell. Expression of the full length rNR1‐1:GFP construct was cytosolic and localized to membranous patches similar to endogenous NR1‐1 protein expression in sensory neurons. Expression of a construct containing only the C‐terminus, GFP:C0C1C2, demonstrated nuclear and membranous localization. When the GFP:C0C1C2 construct was co‐expressed with rNR1‐1 in sensory neurons, membranous localization of rNR1‐1 was disrupted. In contrast, co‐expression of a C‐terminal cassette lacking the C1 exon cassette, GFP:C0C2, with rNR1‐1 did not alter the membranous distribution of rNR1‐1. This observation verifies the utility of a gene transfer strategy to diminish membranous NR1‐1 content by expressing a construct containing the C1 exon cassette.

Ogita, Kiyokazu; Hirata, Koji; Bole, David G.; Yoshida, Sumiko; Tamura, Yutaka; Leckenby, Anne Marie; Ueda, Tetsufumi

doi: 10.1046/j.1471-4159.2001.00200.xpmid: N/A

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [3H]l‐glutamate (Glu) content within the synaptosome (a pinched‐off nerve ending preparation) leads to a change in the amount of exocytotically released [3H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane‐permeant inhibitor (Ki = 19 nm) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H+‐pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [3H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [3H]Glu released in a depolarization‐ (induced by 4‐aminopyridine) and Ca2+‐dependent manner. In contrast, fluorescein, the halogen‐free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [3H]Glu uptake, failed to change the amount of exocytotically released [3H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.

Mukherjee, Piali; Pasinetti, Giulio Maria

doi: 10.1046/j.1471-4159.2001.00167.xpmid: 11279260

We previously reported that pretreatment of murine cortico‐hippocampal neuronal cultures with the complement‐derived anaphylatoxin C5a, protects against glutamate neurotoxicity. In this study we explored the potential mechanisms involved in C5a‐mediated neuroprotection. We found that C5a neuroprotects in vitro through inhibition of apoptotic death because pretreatment with human recombinant (hr)C5a prevented nuclear DNA fragmentation coincidental to inhibition of the pro‐apoptotic caspase 3 activity mediated by glutamate treatment. Also, hrC5a‐mediated responses appeared to be receptor‐mediated because pretreatment of cultures with the specific C5a receptor antagonist C177, prevented hrC5a‐mediated neuroprotection. Based on this evidence, we further explored possible signaling pathways involved in hrC5a inhibition of caspase 3 activation and apoptotic neuronal death. We found that treatment of cultures with the mitogen‐activated protein kinase (MAPK) pathway inhibitor PD98059 prevented hrC5a‐mediated inhibition of caspase 3 and apoptotic neuron death. MAPK pathways, whose activation by hrC5a is inhibited by PD98059 and C177, include the extracellular signal‐regulated kinase (ERK)2 and, to a lesser extent, ERK1. The study suggests that C5a may protect against glutamate‐induced apoptosis in neurons through MAPK‐mediated regulation of caspase cascades.

Satoh, Takumi; Furuta, Kyoji; Tomokiyo, Keiichiro; Namura, Shobu; Nakatsuka, Daisaku; Sugie, Yutaka; Ishikawa, Yasuyuki; Hatanaka, Hiroshi; Suzuki, Masaaki; Watanabe, Yasuyoshi

doi: 10.1046/j.1471-4159.2001.00229.xpmid: N/A

Hallak, Hazem; Ramadan, Bassel; Rubin, Raphael

doi: 10.1046/j.1471-4159.2001.00208.xpmid: N/A

Insulin receptor‐substrate‐1 (IRS‐1) is a docking protein for several tyrosine kinase receptors. Upon tyrosine phosphorylation, IRS‐1 binds to signaling molecules that express Src homology 2 (SH‐2) binding domains, including phosphatidylinositol 3‐kinase (PI 3‐kinase), phosphotyrosine phosphatase SHP‐2 (Syp), Nck, Crk and Grb‐2. Hydrogen peroxide (H2O2) induces tyrosine phosphorylation of key signaling mediators presumably by inhibition of tyrosine phosphatases. In many cell types, the activation of extracellular signal‐related kinases (e.g. MAPK) and other protein kinases by H2O2 leads to transcriptional activation. In the current study, we examined the effect of H2O2 on IRS‐1 tyrosine phosphorylation in primary cultured rat cerebellar granule neurons. H2O2 stimulated the rapid tyrosine phosphorylation of IRS‐1 and p42/p44 MAP kinase, and induced its association with PI 3‐kinase. H2O2‐induced IRS‐1 phosphorylation was rapidly reversible (5 min) whereas MAPK phosphorylation persisted for up to 1 h. NMDA reversed H2O2‐mediated tyrosine phosphorylation of IRS‐1 and its association with PI 3‐kinase. The dephosphorylation of IRS‐1 by NMDA was calcium‐dependent and was inhibited by the calcineurin inhibitor cyclosporine. Calmodulin‐dependent tyrosine phosphatase activity of calcineurin was observed in vitro using both immunoprecipitated and recombinant tyrosine‐phosphorylated IRS‐1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS‐1 tyrosine phosphorylation and identify a novel functional property of calcineurin.

Katoh‐Semba, Ritsuko; Takeuchi, Ikuo K.; Inaguma, Yutaka; Ichisaka, Satoshi; Hata, Yoshio; Tsumoto, Tadaharu; Iwai, Miwako; Mikoshiba, Katsuhiko; Kato, Kanefusa

doi: 10.1046/j.1471-4159.2001.00138.xpmid: N/A

A high level of hippocampal brain‐derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/–)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (γ‐hexachlorocyclohexane); and l‐type voltage‐dependent calcium channel agonist, BAY‐K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY‐K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12–24 h, 8–16 h and 6 h following administration of kainic acid, PTZ and BAY‐K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY‐K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY‐K 8644, but not PTZ, effects. ω‐Conotoxin GVIA and DCG‐IV partially blocked kainic acid‐induced enhancement of BDNF, indicating involvement of l‐type and N‐type voltage‐dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in d‐myo‐inositol‐1,4,5‐triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY‐K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant‐dependent stimulation of BDNF production and involvement of region‐specific voltage‐dependent calcium channels.

Gómez‐Villafuertes, Rosa; Gualix, Javier; Miras‐Portugal, M. Teresa

doi: 10.1046/j.1471-4159.2001.00228.xpmid: N/A

GABAergic terminals from rat midbrain characterized by immunolocalization of glutamic acid decarboxylase and/or the vesicular inhibitory amino acid transporter respond to ATP or P1,P5‐di(adenosine‐5′) pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration measured by a microfluorimetric technique in single synaptic terminals. The ATP response is mediated through the activation of P2X receptors with an abundant presence of P2X3 subunits. Ap5A, however, exerts its effects by acting through a different receptor termed the dinucleotide receptor. Both receptors, once activated in the presence of extrasynaptosomal calcium, induce a concentration‐dependent GABA release from synaptosomal populations with EC50 values of 16 and 20 µm for ATP and Ap5A, respectively. Specific inhibition of GABA release is obtained with pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (80 µm) on the ATP effect and with P1,P5‐di(inosine‐5′) pentaphosphate (100 nm) on the dinucleotide receptor.

Cross, Darren A. E.; Culbert, Ainsley A.; Chalmers, Katy A.; Facci, Laura; Skaper, Stephen D.; Reith, Alastair D.

doi: 10.1046/j.1471-4159.2001.00251.xpmid: N/A

The phosphatidylinositol 3‐kinase (PI 3‐kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase‐3 (GSK‐3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK‐3 in response to insulin and growth factors, which inhibits GSK‐3 activity and leads to the modulation of multiple GSK‐3 regulated cellular processes. We show that the novel potent and selective small‐molecule inhibitors of GSK‐3; SB‐415286 and SB‐216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3‐kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK‐3 activity and modulation of GSK‐3 substrates tau and β‐catenin. Thus, in addition to the previously assigned roles of GSK‐3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK‐3 activity in primary neurones is sufficient to prevent death, implicating GSK‐3 as a physiologically relevant principal regulatory target of the PI 3‐kinase/PKB neuronal survival pathway.