Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

Ca(2+)-permeable AMPA receptors induce phosphorylation of cAMP response element-binding protein through a phosphatidylinositol 3-kinase-dependent stimulation of the mitogen-activated protein kinase signaling cascade in neurons.

Ca(2+)-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neuronal loss in a number of neuropathologies. However, the intracellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen-activated protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved in gene expression, synaptic plasticity, and neuronal stress. Using primary cultures of mouse striatal neurons and a phosphospecific MAPK antibody we addressed whether AMPA receptors can activate the MAPK cascade. We found that in the presence of cyclothiazide, AMPA caused a robust and direct (no involvement of NMDA receptors or L-type voltage-sensitive Ca(2+) channels) Ca(2+)-dependent activation of MAPK through MAPK kinase (MEK). This activation was blocked by GYKI 53655, a noncompetitive selective antagonist of AMPA receptors. Probing the mechanism of this activation revealed an essential role for phosphatidylinositol 3-kinase (PI 3-kinase) and the involvement of a pertussis toxin (PTX)-sensitive G-protein, a Src family protein tyrosine kinase, and Ca(2+)/calmodulin-dependent kinase II. Similarly, kainate activated MAPK in a PI 3-kinase-dependent manner. AMPA receptor-evoked neuronal death and arachidonic acid mobilization did not appear to involve signaling through the MAPK pathway. However, AMPA receptor stimulation led to a Ca(2+)-dependent phosphorylation of the nuclear transcription factor CREB, which could be prevented by inhibitors of MEK or PI 3-kinase. Our results indicate that Ca(2+)-permeable AMPA receptors transduce signals from the cell surface to the nucleus of neurons through a PI 3-kinase-dependent activation of MAPK. This novel pathway may play a pivotal role in regulating synaptic plasticity in the striatum.

A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes).

Ionotropic glutamate receptor agonists, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate, all facilitated 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes). The non-selective, non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked kainate facilitation of glutamate release. AMPA responses were non-desensitizing and insensitive to the AMPA receptor desensitization inhibitor, cyclothiazide. The AMPA receptor antagonist GYKI 52466 failed to block ionotropic glutamate receptor-mediated facilitation, but the ionotropic glutamate receptor 6 kainate receptor subunit antagonist NS-102 was a potent blocker. Furthermore, kainate and AMPA responses were not additive. Taken together, our results indicate that, in the cerebral cortex, both kainate and AMPA may be facilitating glutamate release through the activation of a high-affinity kainate receptor containing glutamate receptor 6 kainate subunits. Kainate enhanced 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential, indicating that a ligand-gated ion channel that conducts cations may underlie the mechanism by which kainate mediates facilitation of glutamate release. While the facilitatory effect of kainate on glutamate release is consistent with a classical ionotropic action of ionotropic glutamate receptors, our observation that kainate inhibits GABA release suggests that alternative presynaptic mechanisms may operate in cerebrocortical nerve terminals to mediate the ionotropic glutamate receptor modulation of glutamate and GABA release. We conclude that high-affinity kainate-type glutamate autoreceptors represent a positive feed-forward system for potentiating the release of glutamate from cerebrocortical nerve terminals.

Hydrogen peroxide enhances signal-responsive arachidonic acid release from neurons: role of mitogen-activated protein kinase.

Hydrogen peroxide (H2O2) is a potent stimulator of signal-responsive phospholipase A2 (PLA2) in vascular smooth muscle and cultured endothelial cells. We investigated whether H2O2 plays a similar regulatory role in neurons. H2O2 did not stimulate a release of arachidonic acid from cultured neurons when applied alone but strongly enhanced the liberation of arachidonic acid evoked by maximally effective concentrations of either glutamate, the glutamate receptor agonist N-methyl-D-aspartate (NMDA), the muscarinic receptor agonist carbachol, the Na+-channel opener veratridine, or the Ca2+-ionophore ionomycin. The potentiating effects of H2O2 were strongly inhibited in the presence of the PLA2 inhibitor mepacrine, suggesting that the site of action was within the signal responsive arachidonic acid cascade. The enhancing effect of H2O2 was not reversed by protein kinase C inhibitors (chelerythrine chloride or GF 109203X) nor was it mimicked by phorbol ester treatment. H2O2 alone strongly enhanced the levels of immunodetectable activated mitogen-activated protein kinase (activated MAP kinases ERK1 and ERK2) in a Ca2+-dependent manner and this effect was additive with increases in the levels of activated MAP kinase evoked by glutamate. The enhanced release of arachidonic acid, however, was not clearly reversed by the MAP kinase kinase (MEK) inhibitor PD 98059, although this treatment effectively abolished H2O2 activation of MAP kinase. Thus, MAP kinase activation and Ca2+-dependent arachidonic acid release are regulated by oxidative stress in cultured striatal neurons.

Presynaptic GABA(B) receptor modulation of glutamate exocytosis from rat cerebrocortical nerve terminals: receptor decoupling by protein kinase C.

GABA and the GABA(B) receptor agonist (-)-baclofen inhibited 4-aminopyridine (4AP)- and KCl-evoked, Ca2+-dependent glutamate release from rat cerebrocortical synaptosomes. The GABA(B) receptor antagonist CGP 35348, prevented this inhibition of glutamate release, but phaclofen had no effect. (-)-Baclofen-mediated inhibition of glutamate release was insensitive to 2 microg/ml pertussis toxin. As determined by examining the mechanism of GABA(B) receptor modulation of glutamate release, (-)-baclofen caused a significant reduction in 4AP-evoked Ca2+ influx into synaptosomes. The agonist did not alter the resting synaptosomal membrane potential or 4AP-mediated depolarization; thus, the inhibition of Ca2+ influx could not be attributed to GABA(B) receptor activation causing a decrease in synaptosomal excitability. Ionomycin-mediated glutamate release was not affected by (-)-baclofen, indicating that GABA(B) receptors in this preparation are not coupled directly to the exocytotic machinery. Instead, the data invoke a direct coupling of GABA(B) receptors to voltage-dependent Ca2+ channels linked to glutamate release. This coupling was subject to regulation by protein kinase C (PKC), because (-)-baclofen-mediated inhibition of 4AP-evoked glutamate release was reversed when PKC was stimulated with phorbol ester. This may therefore represent a mechanism by which inhibitory and facilitatory presynaptic receptor inputs interplay to fine-tune transmitter release.