Abstract
Glutamate receptors, which play a major role in the physiology and pathology of central nervous system gray matter, are also involved in the pathophysiology of white matter. However, the cellular and molecular mechanisms responsible for excitotoxic damage to white matter elements are not fully understood. We explored the roles of AMPA and GluR5 kainate receptors in axonal Ca(2+) deregulation. Dorsal column axons were loaded with a Ca(2+) indicator and imaged in vitro using confocal microscopy. Both AMPA and a GluR5 kainate receptor agonist increased intraaxonal Ca(2+) in myelinated rat dorsal column fibers. These responses were inhibited by selective antagonists of these receptors. The GluR5-mediated Ca(2+) increase was mediated by both canonical (ie, ionotropic) and noncanonical (metabotropic) signaling, dependent on a pertussis toxin-sensitive G protein/phospholipase C-dependent pathway, promoting Ca(2+) release from inositol triphosphate-dependent stores. In addition, the GluR5 response was reduced by intraaxonal NO scavengers. In contrast, GluR4 AMPA receptors operated via Ca(2+)-induced Ca(2+) release, dependent on ryanodine receptors, and unaffected by NO scavengers. Neither pathway depended on L-type Ca(2+) channels, in contrast with GluR6 kainate receptor action.1 Immunohistochemistry confirmed the presence of GluR4 and GluR5 clustered at the surface of myelinated axons; GluR5 coimmunoprecipitated with nNOS and often colocalized with neuronal nitric oxide synthase clusters on the internodal axon. Central myelinated axons express functional AMPA and GluR5 kainate receptors, and can directly respond to glutamate receptor agonists. These glutamate receptor-dependent signaling pathways promote an increase in intraaxonal Ca(2+) levels potentially contributing to axonal degeneration.
Highlights
GluR5-containing kainate receptors were activated by the selective agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA 10M),[20] whereas the relatively selective agonist amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) (100M) was used to activate AMPA receptors. Both agents induced an increase in axoplasmic Ca2ϩ-dependent fluorescence (FCa.ax), which was largely blocked by the combined AMPA/ kainate receptor antagonist 2,3-dihydroxy-6-nitro-7sulfamoyl-benzoquinoxaline-2,3-dione (NBQX 50M, FCa.ax increase at 30 minutes: 17 Ϯ 14%, n ϭ 32 with ATPA; 18 Ϯ 20%, n ϭ 45 with AMPA; p ϭ 10Ϫ5 vs agonist alone, both groups)
We show that, in addition to a glial effect of glutamate, GluR5-containing kainate receptors and glutamate receptor subunit 4 (GluR4)-containing AMPA receptors are expressed on the internodal axolemma in central myelinated fibers and directly participate in mediating axonal Ca2ϩ increases in responses to agonist
The numerous colocalized and physically associated GluR5 receptors and neuronal nitric oxide synthase (nNOS) clusters distributed along axon cylinders are similar to previously reported Ca2ϩ channel/ryanodine receptor clusters[22] and GluR6/ nNOS “nanocomplexes.”[1] It appears, that the internodal axolemma exhibits an even richer collection of signaling molecules than was previously thought, including GluR4, GluR5, and GluR6, the latter two physically and functionally associated with nNOS
Summary
Glutamate receptors, which play a major role in the physiology and pathology of central nervous system gray matter, are involved in the pathophysiology of white matter. The precise mechanisms of glutamate-mediated toxicity in white matter are not completely established This transmitter likely causes damage to glia given that both astrocytes and oligodendrocytes express a variety of glutamate receptors,[2,3,4,5,6,7,8] with oligodendrocytes being vulnerable to excitotoxic cell death.[9,10,11,12] Whether glutamatergic signaling is directly involved in irreversible axonal injury in disorders such as stroke, multiple sclerosis, and neurotrauma is not known, though a role for glutamate-dependent excitotoxicity is suspected given the protective effects of ␣-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA)/kainate antagonists in models of spinal cord injury, stroke, and experimental autoimmune encephalomyelitis.[2,4,13,14,15,16,17,18] The beneficial effect of glutamate antagonism was hypothesized to be caused by sparing of glia and myelin, but the observed axonal protection remains unexplained. GluR4 AMPA receptors, on the other hand, appear to participate in Ca2ϩinduced Ca2ϩ release (CICR) through ryanodinedependent Ca2ϩ stores
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