Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury

Stroke and neurotrauma mediate neuronal death through a series of events that involve multiple interdependent molecular pathways. It has been suggested that these pathways are triggered following elevations in extracellular excitatory amino acids, primarily glutamate [1]. This report outlines mechanisms involving glutamate-mediated excitotoxicity with specific focus on (i) the role of Ca²⁺ in neurotoxicity, (ii) The concept of source specificity of neurotoxicity, (iii) the role of the ionotropic N-methyl-D-aspartate (NMDA)-subtype glutamate receptor and its associated submembrane molecules that may give rise to signaling specificity in excitotoxicity and (iv) the role of glutamate-mediated free-radical generation and associated cell death pathways. We also highlight a novel, peptide-based approach for uncoupling NMDA receptors from excitotoxicity in the rat central nervous system subjected to focal ischemia, thereby reducing stroke infarct volume and improving neurological functioning.Received 11 August 2003; received after revision 15 September 2003; accepted 17 September 2003     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998     

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Flavocytochrome b ₅₅₈ is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O₂ into the superoxide anion O₂ ^- in phagocytic cells. Flavocytochrome b ₅₅₈ is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox for phagocyte oxidase) (β subunit) and a small protein p22phox (α subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b ₅₅₈ which becomes activated and generates O₂ ^-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O₂ ^- and the derived metabolites H₂O₂ and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b ₅₅₈ . The p22phox subunit serves as a docking site for the cytosolic phox proteins. This review provides an overview of current knowledge on the structural organization of the O₂ ^--generating flavocytochrome b ₅₅₈ , its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O₂ ^--generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed. Received 28 May 2002; received after revision 20 June 2002; accepted 24 June 2002     

Towards an understanding of the role of glutamate in neurodegenerative disorders: energy metabolism and neuropathology

It is thought that impairment, of energy metabolism that results in deterioration of membrane function, leading to loss of the Mg²⁺ block on NMDA receptors, and allowing persistent activation of these receptors by glutamate, might be a cause of neuronal death in neurodegenerative disorders. Studies in rodents using mitochondrial respiratory chain toxins, such as aminooxyacetic acid, 1-methyl-4-phenylpyridinium, malonic acid and 3-nitropropionic acid, suggest that such processes may indeed be involved in neurotoxicity. Striatal and nigral degeneration induced by mitochondrial toxins in rodents resembles the neuropathology seen in humans suffering from Huntington's or Parkinson's disease, and can be prevented either by decortication or by NMDA receptor antagonists. Such experimental observations suggest that glutamate may be involved in neuronal death leading to neurodegenerative disorders in humans. If so, glutamate antagonists may offer a therapeutic approach for retarding the progression of these disabling disorders.     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997     

Research Articles¶Naturally occurring 2′-O-methylpurine nucleosides with hypotensive properties

2′-O-Methylinosine (1) has been isolated for the first time and shown to be an intrinsic hypotensive principle. Its probable in vivo precursor, 2′-O-methyladenosine (3), showed stronger and even orally potent hypotensive activity. Resistance of the methyladenosine (3) against adenosine deaminase is thought to contribute to its long-lasting activity. The effect of both nucleosides (1 and 3) was not accompanied with any significant change in heart rate, which is often observed with adenosine. Received 2 October 1997; accepted 28 October 1997     

Metabolism of the neuromodulator d-serine

Over the past years, accumulating evidence has indicated that d-serine is the endogenous ligand for the glycine-modulatory binding site on the NR1 subunit of N-methyl-d-aspartate receptors in various brain areas. d-Serine is synthesized in glial cells and neurons by the pyridoxal-5′ phosphate-dependent enzyme serine racemase, and it is released upon activation of glutamate receptors. The cellular concentration of this novel messenger is regulated by both serine racemase isomerization and elimination reactions, as well as by its selective degradation catalyzed by the flavin adenine dinucleotide-containing flavoenzyme d-amino acid oxidase. Here, we present an overview of the current knowledge of the metabolism of d-serine in human brain at the molecular and cellular levels, with a specific emphasis on the brain localization and regulatory pathways of d-serine, serine racemase, and d-amino acid oxidase. Furthermore, we discuss how d-serine is involved with specific pathological conditions related to N-methyl-d-aspartate receptors over- or down-regulation.     

Human and yeast ζ-crystallins bind AU-rich elements in RNA

ζ-crystallins constitute a family of proteins with NADPH:quinone reductase activity found initially in mammalian lenses but now known to be present in many other organisms and tissues. Few proteins from this family have been characterized, and their function remains unclear. In the present work, ζ-crystallins from human and yeast (Zta1p) were expressed, purified and characterized. Both enzymes are able to reduce ortho-quinones in the presence of NADPH but are not active with 2-alkenals. Deletion of the ZTA1 gene makes yeast more sensitive to menadione and hydrogen peroxide, suggesting a role in the oxidative stress response. The human and yeast enzymes specifically bind to adenine-uracil rich elements (ARE) in RNA, indicating that both enzymes are ARE-binding proteins and that this property has been conserved in ζ-crystallins throughout evolution. This supports a role for ζ-crystallins as trans-acting factors that could regulate the turnover of certain mRNAs. Received 21 February 2007; received after revision 16 April 2007; accepted 23 April 2007 M. R. Fernández, S. Porté: These authors contributed equally to this work.     

Ligand recognition by the I domain-containing integrins

Seven of the integrin α subunits described to date, α ₁ , α ₂ , α _L , α _X , α _d , α _M and α _E , contain a highly conserved I (or A) domain of approximately 200 amino acid residues inserted near the amino-terminus of the subunit. As the result of a variety of independent experimental approaches, a large body of data has recently accumulated that indicates that the I domains are independent, autonomously folding domains capable of directly binding ligands that play a necessary and important role in ligand binding by the intact integrins. Recent crystallographic studies have elucidated the structures of recombinant α _M and α _L I domains and also delineated a novel divalent cation-binding motif within the I domains (metal ion-dependent adhesion site, MIDAS) that appears to mediate the divalent cation binding of the I domains and the I domain-containing integrins to their ligands.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

AMPA receptors: potential implications in development and disease

α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors are one type of ionotropic glutamate receptor involved in rapid excitatory synaptic transmission. AMPA receptors have been increasingly implicated in long-term potentiation, and recent evidence suggests that they may play a role in disorders affecting the nervous system. The finding that early in postnatal development AMPA receptors are not expressed has lately been the focus of much attention. Resolving the factors involved in AMPA receptor expression suggests that their induction is a developmentally regulated process with the possibility that alterations in receptor expression may be correlated with pathology in neurological disorders. This paper provides an overview of factors involved in AMPA receptor induction as well as of their role in plasticity and neuronal pathologies. Received 5 December 2000; received after revision 12 January 2001; accepted 2 February 2001     

Hes-1 regulates the excitatory fate of neural progenitors through modulation of <Emphasis Type="Italic">Tlx3 (HOX11L2)</Emphasis> expression

Tlx3 (HOX11L2) is regarded as one of the selector genes in excitatory versus inhibitory fate specification of neurons in distinct regions of the nervous system. Expression of Tlx3 in a post-mitotic immature neuron favors a glutamatergic over GABAergic fate. The factors that regulate Tlx3 have immense importance in the fate specification of glutamatergic neurons. Here, we have shown that Notch target gene, Hes-1, negatively regulates Tlx3 expression, resulting in decreased generation of glutamatergic neurons. Down-regulation of Hes-1 removed the inhibition on Tlx3 promoter, thus promoting glutamatergic differentiation. Promoter–protein interaction studies with truncated/mutated Hes-1 protein suggested that the co-repressor recruitment mediated through WRPW domain of Hes-1 has contributed to the repressive effect. Our results clearly demonstrate a new and unique role for canonical Notch signaling through Hes-1, in neurotransmitter/subtype fate specification of neurons in addition to its known functional role in proliferation/maintenance of neural progenitors.     

The distribution of α2β1, α3β1 and α6β4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle

The human hair follicle is composed of different concentric compartments, which reflect different programmes of differentiation. Using monoclonal antibodies against α2β1 and α3β1 integrins we demonstrated a shift in their expression, from a basolateral distribution in the basal cells of the lower outer root sheath, to an apicolateral expression in the upper outer root sheath, as in epidermis. This shift takes place in a transition zone, localized to the midpart of the follicle. The distinct basolateral distribution of α2β1 and α3β1 integrins in the lower portion of the outer root sheath coincides with the presence of basal cell protrusions and is probably linked to the presence of the vitreous membrane which surrounds the bottom part of the anagen human hair follicle. Moreover, we showed that the expression of α6β4 integrin is discontinuous along the hair follicle and coincides with that of laminin 5. Together these results establish that within a given compartment – namely the outer root sheath – several domains can be clearly identified, which probably reflect the onset of successive differentiation pathways along the hair follicle. Received 17 January 1997; received after revision 18 February 1997; accepted 24 February 1997     

Anticonvulsant,antiepileptogenic, and antiictogenic pharmacostrategies

Pharmacological concepts tailored to status epilepticus, to epileptogenesis following acquired brain insults, and to ictogenesis in established epilepsy vary considerably and should ideally be directed at those pathophysiological mechanisms that presumably underly these conditions. Currently known important molecular targets include voltage-gated sodium and calcium channels, the γ-aminobutyric acid (GABA) system and ionotropic glutamate receptors. Metabotropic glutamate receptors, potassium channels, and neurotransmitters such as acetylcholine, glycine, and monoamines are beyond the scope of this review. In status epilepticus, immediate failure of GABAergic inhibition occurs, and administration of benzodiazepines and barbiturates displays the pharmacostrategic mainstay. In epileptogenesis within limbic structures, the most important underlying pathophysiological mechanisms currently discussed are transient loss of inhibition and aberrant mossy fiber sprouting. Both processes may be facilitated by N-methy-d-aspartat (NMDA) receptor regulation. NMDA antagonists may exhibit antiepileptogenic properties in experimental animals, but reliable data in humans are lacking. In established epilepsy, voltage-gated ion channels and impairment of GABAergic functions contribute to mechanisms facilitating ictogenesis. Blockade of sodium and calcium channels and enhancement of GABAergic inhibition are currently the most important tools to prevent the occurrence of seizures. Received 16 January 2007; received after revision 7 March 2007; accepted 17 April 2007     

Cryo-bioorganic chemistry: freezing effect on stereoselection of l- and dl-leucine cooligomerization in aqueous solution

The chirality of l-/dl-leucine (50–50%) cooligomerization was investigated in liquid and frozen aqueous solutions. Cooligomerization was carried out by carbonyldiimidazole activation without initiator at an ambient (+22°C) and frozen (−18°C) temperature, respectively. The separated samples obtained after different time intervals of treatment were completely hydrolyzed (HCl) and the diastereomeric l- and d-leucine derivates of Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-alanine amide) were then traced and evaluated by RP-HPLC analysis. After 9 days of oligomerization, the l-Leu content was slightly enhanced in the liquid (57%) and somewhat more enhanced in the frozen (64%) samples. After 17 days, however, the l-Leu content had decreased in the liquid (53%) and frozen (56%) conditions. These l-enantiomer amplifications indicate that an l-antipode is preferentially incorporated into the α-helical turn of the oligomer in the earlier stage of cooligomerization, while, later, the d-antipode is also incorporated. The role of ice in the improved stereoselection is discussed. This is the first recorded example of the effect of freezing on stereoselection. Received 27 October 2000; revised 11 December 2000; accepted 4 January 2000     

Integrins and cardiovascular disease

Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor α _IIb  β ₃ , has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of α _IIb  β ₃ inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.     

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease

The amyloid β-peptide (Aβ) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer’s disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Aβ and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Aβ can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Aβ might have a stronger (or weaker) inhibitory effect on the proteasome than does native Aβ. We therefore also investigated the proteasome inhibitory action of Aβ _1–40 (a peptide comprising the first 40 residues of Aβ) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H₂O₂ modification of Aβ _1–40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Aβ _1–40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer’s disease Received 26 September 2000; accepted 26 September 2000     

In pre-sterol carrier protein 2 (SCP2) in solution the leader peptide 1 – 20 is flexibly disordered, and residues 21 – 143 adopt the same globular fold as in mature SCP2

The preform of the rabbit sterol carrier protein 2 (pre-rSCP2) was cloned, the uniformly ¹⁵N-labelled protein expressed in Escherichia coli and studied by three-dimensional ¹⁵N-resolved nuclear magnetic resonance spectroscopy. In spite of its low solubility in aqueous solution of only ∼0.3 mM, sequential ¹⁵N and ¹H backbone resonance assignments were obtained for 105 out of the 143 residues. From comparison of the sequential and medium-range nuclear Overhauser effects (NOEs) in the two proteins, all regular secondary structures previously determined in mature human SCP2 (hSCP2) [Szyperski et al. (1993) FEBS Lett. 335: 18–26] were also identified in pre-rSCP2. Near-identity of the backbone ¹⁵N and ¹H chemical shifts and 1 : 1 correspondence of 24 long-range NOEs to backbone amide groups in the two proteins show that the residues 21 – 143 adopt the same globular fold in pre-rSCP2 and mature hSCP2. The N-terminal 20-residue leader peptide of pre-rSCP2 is flexibly disordered in solution and does not observably affect the conformation of the polypeptide segment 21 – 143. Received 11 May 1998; accepted 15 May 1998     

Characterization of the 5-HT1A receptor of the honeybee (Apis mellifera) and involvement of serotonin in phototactic behavior

Serotonin plays a key role in modulating various physiological and behavioral processes in both protostomes and deuterostomes. The vast majority of serotonin receptors belong to the superfamily of G-protein-coupled receptors. We report the cloning of a cDNA from the honeybee (Am5-ht1A) sharing high similarity with members of the 5-HT₁ receptor class. Activation of Am5-HT_1A by serotonin inhibited the production of cAMP in a dose-dependent manner (EC₅₀ = 16.9 nM). Am5-HT_1A was highly expressed in brain regions known to be involved in visual information processing. Using in vivo pharmacology, we could demonstrate that Am5-HT_1A receptor ligands had a strong impact on the phototactic behavior of individual bees. The data presented here mark the first comprehensive study—from gene to behavior—of a 5-HT_1A receptor in the honeybee, paving the way for the eventual elucidation of additional roles of this receptor subtype in the physiology and behavior of this social insect.