Regulation of the Neuron-specific Ras GTPase-activating Protein, synGAP, by Ca2+/Calmodulin-dependent Protein Kinase II

synGAP is a neuron-specific Ras GTPase-activating protein found in high concentration in the postsynaptic density fraction from mammalian forebrain. Proteins in the postsynaptic density, including synGAP, are part of a signaling complex attached to the cytoplasmic tail of the N-methyl-D-aspartate-type glutamate receptor. synGAP can be phosphorylated by a second prominent component of the complex, Ca2+/calmodulin-dependent protein kinase II. Here we show that phosphorylation of synGAP by Ca2+/calmodulin-dependent protein kinase II increases its Ras GTPase-activating activity by 70-95%. We identify four major sites of phosphorylation, serines 1123, 1058, 750/751/756, and 764/765. These sites together with other minor phosphorylation sites in the carboxyl tail of synGAP control stimulation of GTPase-activating activity. When three of these sites and four other serines in the carboxyl tail are mutated, stimulation of GAP activity after phosphorylation is reduced to 21 ± 5% compared with 70-95% for the wild type protein. We used phosphosite-specific antibodies to show that, as predicted, phosphorylation of serines 765 and 1123 is increased in cultured cortical neurons after exposure of the neurons to the agonist N-methyl-D-aspartate

SynGAP Regulates Steady-State and Activity-Dependent Phosphorylation of Cofilin

SynGAP, a prominent Ras/Rap GTPase-activating protein in the postsynaptic density, regulates the timing of spine formation and trafficking of glutamate receptors in cultured neurons. However, the molecular mechanisms by which it does this are unknown. Here, we show that synGAP is a key regulator of spine morphology in adult mice. Heterozygous deletion of synGAP was sufficient to cause an excess of mushroom spines in adult brains, indicating that synGAP is involved in steady-state regulation of actin in mature spines. Both Ras- and Rac-GTP levels were elevated in forebrains from adult synGAP+/- mice. Rac is a well known regulator of actin polymerization and spine morphology. The steady-state level of phosphorylation of cofilin was also elevated in synGAP+/- mice. Cofilin, an F-actin severing protein that is inactivated by phosphorylation, is a downstream target of a pathway regulated by Rac. We show that transient regulation of cofilin by treatment with NMDA is also disrupted in synGAP mutant neurons. Treatment of wild-type neurons with 25 µM NMDA triggered transient dephosphorylation and activation of cofilin within 15 s. In contrast, neurons cultured from mice with a homozygous or heterozygous deletion of synGAP lacked the transient regulation by the NMDA receptor. Depression of EPSPs induced by a similar treatment of hippocampal slices with NMDA was disrupted in slices from synGAP+/- mice. Our data show that synGAP mediates a rate-limiting step in steady-state regulation of spine morphology and in transient NMDA-receptor-dependent regulation of the spine cytoskeleton

Endogenous Zinc in Neurological Diseases

The use of zinc in medicinal skin cream was mentioned in Egyptian papyri from 2000 BC (for example, the Smith Papyrus), and zinc has apparently been used fairly steadily throughout Roman and modern times (for example, as the American lotion named for its zinc ore, 'Calamine'). It is, therefore, somewhat ironic that zinc is a relatively late addition to the pantheon of signal ions in biology and medicine. However, the number of biological functions, health implications and pharmacological targets that are emerging for zinc indicate that it might turn out to be 'the calcium of the twenty-first century'. Here neurobiological roles of endogenous zinc is summarized

Expression of constitutive hsc70 and stress-inducible hsp70 mRNA and protein in the rabbit central nervous system

grantor: University of TorontoThe induction of hsp70 has been established as a useful marker of cellular stress in the mammalian nervous system. In addition, studies have indicated a role for members of the hsp70 gene family in protecting cells from the lethal effects of heat and other stresses. In this thesis the stress response is examined in the rabbit nervous system following a physiologically relevant heat shock using hsc70 and hsp70-specific riboprobes and antibodies. A rapid and transient induction of hsp70 mRNA and protein was observed following hyperthermic stress. Tissue-specific differences in the magnitude of induction of hsp70 protein were observed. Induction was greatest in non-neural tissues compared to regions of the rabbit nervous system. In unstressed animals, abundant levels of hsc70 protein were detected in brain regions whereas lower levels were detected in non-neural tissues. In addition to hsc70, basal levels of inducible hsp70 isoforms were also observed in control animals. At the cellular level, distinct patterns of hsc70 and hsp70 expression were observed in the rabbit brain. In control animals, hsc70 mRNA and protein exhibited a neuronal pattern of expression. In response to hyperthermia, large neuronal cells (Purkinje neurons, deep cerebellar nuclei of the cerebellum and motor neurons of the spinal cord), which expressed high constitutive levels of hsc70, showed a dampened or delayed accumulation of hsp70 mRNA. Hsp70 protein was not detected in these neurons. Glial cells, which show little detectable levels of hsc70, demonstrate a robust induction of hsp70. To further evaluate the neuronal stress response, nuclear translocation of hsc70 and hsp70 protein was used as an additional indicator of cellular stress. Following a 2-3\sp\circC increase in body temperature, non-neuronal cell types (ependymal cells and oligodendrocytes) exhibited features of a stress response. In contrast, several neuronal cell types (Purkinje, cortical, dentate granule and thalamic neurons) required an increased degree of hyperthermia (3.4 $\pm$.2\sp\circC) before eliciting a feature of the stress response, namely nuclear translocation of hsc70 protein. Taken together, these results suggest that the neural heat shock response may occur in stages depending on the level of stress on individual cell populations and existing cellular levels of hsps.Ph.D

Telling tails

The N-methyl-D-aspartate (NMDA)-type glutamate receptor is one of three major classes of receptors for glutamate, the principle excitatory neurotransmitter in the central nervous system. It plays a key role in learning and in the formation of memories by acting as a "coincidence detector" that initiates changes in synaptic strength that lead to the formation of new neural networks (1). It is also an important mediator of several forms of pathological neuronal toxicity. The NMDA receptor responds at a synapse only when the presynaptic terminal releases glutamate at the same time that the postsynaptic neuron is strongly depolarized by the sum of activating influences impinging on it. In effect, it initiates the strengthening of all synapses that depolarize the same postsynaptic neuron at the same time and thus triggers formation of a new, more stable circuit. When the NMDA-receptor channel opens, it allows passage of calcium ions, as well as sodium and potassium, into the cell. The calcium ions trigger a cascade of biochemical signaling reactions catalyzed by enzymes located just underneath the postsynaptic membrane. These reactions modify other membrane channels in the synapse, ultimately leading to a change in the strength of the electrical signal produced when the synapse is activated again

The neuronal stress response: nuclear translocation of heat shock proteins as an indicator of hyperthermic stress

Two characteristic features of the heat shock response, (i) induction of hsp70 protein and (ii) nuclear translocation of constitutive hsc70 and stress-inducible hsp70 protein, were utilized as markers of cellular stress in the rabbit brain. Following a physiologically relevant increase in body temperature of 2.7 ± .3°C, nonneuronal cell types, such as ependymal cells and oligodendrocytes, undergo a stress response as assayed by the above criteria. In contrast, several neuronal cell populations required an increased degree of hyperthermic stress (3.4 ± .2°C) before exhibiting nuclear translocation of constitutive hsc70 protein. Induction of hsp70 protein was not observed in these neuronal cells at either temperature. The present results suggest that certain neurons in the rabbit brain are buffered against induction of the heat shock response, perhaps due to their high constitutive levels of hsc70 protein