Abstract
The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Although overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptor-dependent axonal Ca(2+) deregulation. Dorsal column axons were loaded with a Ca(2+) indicator and imaged in vitro using confocal laser-scanning microscopy. Activation of glutamate receptor 6 (GluR6) kainate receptors promoted a substantial increase in axonal [Ca(2+)]. This Ca(2+) accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine-dependent intracellular stores, which, in turn, depended on activation of L-type Ca(2+) channels: ryanodine, nimodipine, or nifedipine blocked the agonist-induced Ca(2+) increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca(2+) response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal Nomega-nitro-L-arginine methyl ester, blocked the Ca(2+) increase. Loading axons with a peptide that mimics the C-terminal PDZ binding sequence of GluR6, thus interfering with the coupling of GluR6 to downstream effectors, greatly reduced the agonist-induced axonal Ca(2+) increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Ca(v)1.2. Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal "nanocomplexes" to release toxic amounts of Ca(2+) may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur.
Highlights
A number of in vitro and in vivo studies have pointed to an important role for non-NMDA glutamate receptors in white matter injury,[6,8,9,16] with glial cells representing an important target given their known expression of AMPA and kainate receptors,[20] and their sensitivity to this excitotoxin.[24,39]
Little is known about functional glutamate receptors on central axons, though experiments indirectly suggest that such receptors may be present.[8,43]
We show that functional kainate receptors are present on myelinated central axons, raising the distinct possibility that loss of axonal function after glutamate exposure may be caused by direct activation of axonal receptors leading to axoplasmic Ca2ϩ deregulation
Summary
The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Interpretation: Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal “nanocomplexes” to release toxic amounts of Ca2ϩ may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur. We found that GluR6containing kainate receptors reside along the internodal axolemma in “nanocomplexes” together with neuronal nitric oxide synthase (nNOS), exerting control over L-type Ca2ϩ channels and causing Ca2ϩ release from intraaxonal Ca2ϩ stores These signaling molecules are organized in a surprisingly intricate arrangement (see Fig 6) reminiscent of what is found at the postsynaptic membrane of conventional glutamatergic synapses
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