Abdulbaki Agbas

Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of Enhanced Glutamate Release, Altered Synaptic Plasticity, and Selective Neuronal Vulnerability

The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes.

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis

Abstract. As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin α chain and the full-length hydra laminin β chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin α and β subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin β chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin β chain, in situ hybridization experiments revealed that hydra laminin α chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin α chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin α chain is required for head regeneration.

Decreased activity and increased aggregation of brain calcineurin during aging.

Age-related decline in strength of synaptic transmission and memory formation has been attributed to age-associated increases in the activity of calcineurin (Cn) in hippocampus neurons. In the present study, we examined how brain Cn activity, Cn subunit levels, and Cn protein oxidation were changing during the aging process. Cn activity decreased with advancing age in three brain subcellular fractions, homogenate, cytosol, and synaptic membranes, obtained from F344/BNF1 rats of 5-6, 22-24, and 34-36 months of age. Cn activity also decreased during aging in homogenate, cytosol, and a nerve ending-enriched fraction from the hippocampus. Cn protein levels in homogenate and cytosol, as determined by the immune reactivity of its subunits A and B, were not altered during aging. But, in synaptic membranes, there was an age-related decrease in CnA levels, but not of CnB. Another important observation was that of an oxidative modification of CnA, not CnB, with increasing age. Such modification caused the formation of large aggregates of CnA. Aggregate formation was due to SH-group oxidation as the monomeric form of CnA was recovered upon disulfide reduction of the proteins with dithiothreitol. The age-related formation of aggregates of the catalytic subunit of Cn was suggestive of a correlation between aggregate formation and diminished enzyme activity. The loss of Cn activity may alter signal transduction at synapses during the aging process.

Transcriptomic responses in mouse brain exposed to chronic excess of the neurotransmitter glutamate

BackgroundIncreases during aging in extracellular levels of glutamate (Glu), the major excitatory neurotransmitter in the brain, may be linked to chronic neurodegenerative diseases. Little is known about the molecular responses of neurons to chronic, moderate increases in Glu levels. Genome-wide gene expression in brain hippocampus was examined in a unique transgenic (Tg) mouse model that exhibits moderate Glu hyperactivity throughout the lifespan, the neuronal Glutamate dehydrogenase (Glud1) mouse, and littermate 9 month-old wild type mice.ResultsIntegrated bioinformatic analyses on transcriptomic data were used to identify bio-functions, pathways and gene networks underlying neuronal responses to increased Glu synaptic release. Bio-functions and pathways up-regulated in Tg mice were those associated with oxidative stress, cell injury, inflammation, nervous system development, neuronal growth, and synaptic transmission. Increased gene expression in these functions and pathways indicated apparent compensatory responses offering protection against stress, promoting growth of neuronal processes (neurites) and re-establishment of synapses. The transcription of a key gene in the neurite growth network, the kinase Ptk2b, was significantly up-regulated in Tg mice as was the activated (phosphorylated) form of the protein. In addition to genes related to neurite growth and synaptic development, those associated with neuronal vesicle trafficking in the Huntingtons disease signalling pathway, were also up-regulated.ConclusionsThis is the first study attempting to define neuronal gene expression patterns in response to chronic, endogenous Glu hyperactivity at brain synapses. The patterns observed were characterized by a combination of responses to stress and stimulation of nerve growth, intracellular transport and recovery.

Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1-Cn protein interactions occurring in vitro and in vivo

Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2*-). The effects of O2*- on Cn are still controversial. We found that O2*-, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1-rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2*- dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase K(m) without a change in V(max). A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1-Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.

A rat brain bicistronic gene with an internal ribosome entry site codes for a phencyclidine-binding protein with cytotoxic activity

The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24–48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.

Superoxide modification and inactivation of a neuronal receptor-like complex.

Excessive superoxide (O(-)(2)) formation is toxic to cells and organisms. O(-)(2) reacts with either iron-sulfur centers or cysteines (Cys) of cytoplasmic proteins. Reactions with membrane proteins, however, have not been fully characterized. In the present studies, the reaction of O(-)(2) with a protein complex that has glutamate/N-methyl-D-aspartate (NMDA) receptor characteristics and with one of the subunits of this complex was examined. Exposure of the complex purified from neuronal membranes and the recombinant glutamate-binding protein (GBP) subunit of this complex to the O(-)(2)-generating system of xanthine (X) plus xanthine oxidase (XO) caused strong inhibition of L-[3H]glutamate binding. Inhibition of glutamate binding to the complex and GBP by O(-)(2) was greater than that produced by H(2)O(2), another product of the X plus XO reaction. Mutation of two cysteine (Cys) residues in recombinant GBP (Cys(190,191)) eliminated the effect of O(-)(2) on L-[3H]glutamate binding. Both S-thiolation reaction of GBP in synaptic membranes with [35S]cystine and reaction of Cys residues in GBP with [3H]NEM were significantly decreased after exposure of membranes to O(-)(2). Inhibition of cysteylation of membrane GBP by O(-)(2) was still observed after iron chelation by desferrioxamine, albeit diminished, and was not altered by the presence of catalase. Overall, the results indicated that GBP exposure to O(-)(2) modified Cys residues in this protein. The modification was not characterized but it was probably that of disulfide formation.