|Home | About | Journals | Submit | Contact Us | Français|
The deleterious effects of glutamate excitoxicity are well described for CNS gray matter. While 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 Ca2+ deregulation.
dorsal column axons were loaded with a Ca2+ indicator and imaged in vitro using confocal laser-scanning microscopy.
Activation of GluR6 kainate receptors promoted a substantial rise in axonal [Ca2+]. This Ca2+ 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 Ca2+ channels: ryanodine, nimodipine or nifedipine blocked the agonist-induced Ca2+ rise. Also, GluR6 stimulation induced intra-axonal production of NO, which greatly enhanced the Ca2+ response: quenching of NO with intra-axonal (but not extracellular) scavengers, or inhibition of nNOS with intra-axonal L-NAME, blocked the Ca2+ rise. 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 Ca2+ increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma co-localized with nNOS and Cav1.2.
Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of post-synaptic membranes of glutamatergic synapses. The ability of such axonal “nanocomplexes” to release toxic amounts of Ca2+ 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.
Glutamate is the main excitatory neurotransmitter in the mammalian CNS, playing a significant role in gray matter injury in many neurodegenerative diseases 1. Prevalent and devastating disorders such as stroke, multiple sclerosis and trauma to the brain and spinal cord invariably affect afferent and efferent white matter tracts, though much less is known about mechanisms of injury to myelinated white matter axons. Voltage-gated Na+ and Ca2+ channels, together with reverse Na+-Ca2+ exchange play important roles 2-4 (for review see Stys 5). Perhaps counterintuitive, given the non-synaptic nature of CNS white matter, are observations of functional protection of this tissue by antagonists of ionotropic glutamate receptors. AMPA/kainate receptor antagonists are protective both in vitro 6-10 and in vivo 11-14, in ischemic, traumatic and autoimmune models of white matter injury. Conversely, activating AMPA/kainate receptors, but not NMDA receptors, or increasing extracellular glutamate levels by blocking glutamate transport, either in vitro 15-17 or in vivo 17-19 is injurious to axons.
The precise mechanisms of injury to white matter elements induced by non-NMDA glutamate receptor activation are unknown. Both astrocytes and oligodendrocytes express AMPA and kainate receptors (for review see Matute et al. 20), and more recently NMDA receptors have been detected on mature oligodendrocytes 21, their processes 22, and even the myelin sheath 23. These receptors are permeable to Ca2+ ions, therefore it is reasonable to conclude that receptor-mediated Ca2+ overload is responsible for excitotoxic glial injury 15,24,25. What is so far unexplained is the observation that central axons per se are damaged by activation of AMPA/kainate receptors 18,19, and in turn protected by blockers of these receptors in various injury models 9,13,26. These latter observations raise the possibility that central myelinated axons themselves express AMPA/kainate receptors, whose overactivation results in damage to the fibers directly. Indeed, antagonists of AMPA/kainate receptors, but not NMDA receptors, were protective against spinal cord dorsal column injury 6-8, and bath application of AMPA, kainate or glutamate, but not NMDA, induced irreversible reduction of compound action potential 6,16. In this report we tested the hypothesis that myelinated axons from rat spinal cord express functional kainate receptors capable of mediating a potentially deleterious axonal Ca2+ rise. We found that GluR6-containing kainate receptors reside along the internodal axolemma in “nanocomplexes” together with nNOS, exerting control over L-type Ca2+ channels and causing Ca2+ release from intra-axonal Ca2+ stores. These signaling molecules are organized in a surprisingly intricate arrangement (see Fig. 6) reminiscent of what is found at the post-synaptic membrane of conventional glutamatergic synapses.
All experiments were performed in accordance with institutional guidelines for the care and use of experimental animals. Additional details can be found in Supplementary Material.
Dorsal columns from deeply anesthetized adult Long Evans male rats were removed from the thoracic region and placed in cold oxygenated zero-Ca2+ solution containing, loaded for 2 hours with Ca2+-insensitive reference dye (red dextran-conjugated Alexa 594, 250 μM) to allow identification of axon profiles (Fig. 1A) together with the dextran-conjugated Ca2+ indicator Oregon Green BAPTA-1 (250 μM), and imaged on a Nikon C1 confocal microscope at 37 °C. All reported axonal [Ca2+] changes (FCa.ax) are ratios of green to red fluorescence after 30 min of drug application.
Two peptides (NH2-Cys-Ahx-Arg-Leu-Pro-Gly-Lys-Glu-Thr-Met-Ala-CONH2 (I), Mol Wt = 1218, and NH2-Cys-Ahx-Cys-Ahx-Cys-Ahx-Cys-Ahx-Arg-Leu-Pro-Gly-Lys-Glu-Thr-Met-Ala-CONH2 (II), Mol Wt = 1864) were designed that contain the C-terminal of GluR6 PDZ1 binding motif, a single or multiple N-terminal Cys residues (for dye conjugation via free SH groups) and one or more Ahx (epsilon amino hexanoic acid) moieties as spacers (for steric reasons). Active and sham dextro-peptides were synthesized using standard methods. The peptides were dissolved to a concentration of 0.1 - 1 mM in the loading pipette yielding ≈1 - 10 μM in the axons.
We measured [Ca2+] changes in live adult rat dorsal column axons in vitro using laser-scanning confocal microscopy (Fig. 1). Activation of kainate receptors (kainate 200 μM), at concentrations that significantly reduced compound action potentials (see below), caused a progressive increase of intra-axonal [Ca2+]. Axoplasmic Ca2+-dependent fluorescence (FCa.ax) showed a robust increase after drug application (mean increase after 30 min, kainate: 110 ± 67%, n = 54 axons) that was strongly reduced by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX 50 μM) (12 ± 15%, n = 35, P ≈ 0). The AMPA receptor antagonists 1-naphtyl acetyl spermine (25 μM) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI52466 100 μM) did not significantly blunt kainate-induced FCa.ax increase (kainate + spermine: 97 ± 64%, n = 54, P = 0.98; kainate + GYKI52466: 79 ± 65%, n = 40, P = 0.24). In contrast, 3-(hydroxyamino)-6-nitro- 6,7,8,9-tetrahydrobenzo[g]indol-2-one (NS-102 10 μM), an antagonist of GluR6-containing kainate receptors 27 , strongly reduced the response induced by kainate (kainate + NS-102: 35 ± 25%, n = 37, P ≈ 0). (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione (UPB-302, 20 μM), a blocker of GluR5-containing kainate receptors 28, was less effective (kainate + UPB-302: 74 ± 40%, n = 36) than CNQX or NS-102 at blocking the kainate-induced Ca2+ responses (P ≤ 0.012: kainate + UPB-302 vs. kainate + CNQX or kainate + NS-102) (Fig. 1C), indicating that kainate mainly (but not exclusively) activated kainate receptors containing GluR6 subunits. (2S,4R)-4-methyl glutamic acid (SYM2081; 100 μM), another kainate receptor agonist 29, induced an increase of FCa.ax (135 ± 67%, n = 79) with a similar pharmacological profile to kainate: the Ca2+ response was reduced by CNQX (SYM2081 + CNQX: 38 ± 24%, n = 29, P = 4×10−10) and NS102 (25 ± 35%, n = 28, P = 4×10−10), and also was modestly reduced by 1-naphtyl acetyl spermine (79 ± 37%, n = 49, P = 4×10−10) or GYKI52466 (83 ± 18%, n = 20, P = 7x10−10), suggesting a partial activation of AMPA receptors by the latter agent at the concentrations used. UPB-302 was also less effective at blocking the SYM 2081 response (94 ± 40%, n = 17).
To further characterize the sources of axonal Ca2+ increase, agonists were applied in the absence of bath Ca2+ (+ 0.5 mM EGTA), which reduced but did not completely prevent FCa.ax increase (kainate + 0Ca2+: 26 ± 20%, n = 33, P ≈ 0 vs. Ca2+-containing perfusate; SYM2081 + 0Ca2+: 42 ± 31%, n = 24, P = 4×10−10). This suggests that a component of the kainate receptor-induced axonal Ca2+ increase originated from intracellular compartments. Previously we reported that ischemic depolarization of spinal axons releases Ca2+ from ryanodine-dependent axonal Ca2+ stores 30. We therefore examined whether kainate receptors might induce Ca2+ release from these stores. Ryanodine (50 μM, in Ca2+-replete perfusate) almost completely blocked the FCa.ax increase (kainate + ryanodine: 2 ± 22%, n = 33, P = 0 vs. kainate alone; SYM2081 + ryanodine: 11 ± 28%, n = 27, P = 4×10−10), indicating that most of the axonal Ca2+ accumulation observed in response to kainate receptor activation originated from axonal ryanodine-sensitive Ca2+ stores (Fig. 2A). More surprisingly, blockade of L-type Ca2+ channels by nimodipine or nifedipine (10 μM) also strongly inhibited axoplasmic Ca2+ rise (kainate + nimodipine: 6 ± 19%, n = 26, P ≈ 0 vs. kainate alone; SYM2081 + nimodipine: 17 ± 22%, n = 43, P = 4×10−10) (Fig. 2B). L-type Ca2+ channels may in turn be modulated by a local membrane depolarization or possibly even by a metabotropic action of kainate receptors 31. Replacing NaCl with impermeant NMDG-Cl to reduce putative agonist-induced axonal depolarization virtually abolished kainate- (kainate + NMDG: -5 ± 13%, n = 37, P ≈ 0 vs. Na+-containing perfusate) and SYM2081-induced Ca2+ rise (SYM2081 + NMDG: 15 ± 17%, n = 32, P = 1.7x10−10). Substitution of NaCl with LiCl, which readily permeates kainate receptors 32, allowed a robust axonal Ca2+ rise after application of kainate (91 ± 50%, n = 52) or SYM2081 (95 ± 24%, n = 34) (Fig. 2C). Taken together, these data suggest that GluR6-containing kainate receptors mediate their actions through a combination of local membrane depolarization and a small influx of Ca2+ triggering a larger release from ryanodine-sensitive Ca2+ stores.
While the results above support the involvement of kainate receptors in the mobilization of Ca2+, they do not prove that these receptors are necessarily axonal; indeed, the protective effects of AMPA/kainate antagonists in white matter injury was suggested to be due to protection of glial elements 33 with indirect sparing of axons (for review see Matute et al.34). The experiments shown in Fig. 3A, relying on selective extracellular vs. intra-axonal application of scavengers, strongly suggest that kainate receptors are expressed directly on axons and stimulate formation of NO within axons, which in turn promotes the above Ca2+ release cascade. Bath-application of the NO scavenger myoglobin35 failed to prevent axoplasmic Ca2+ rise (kainate + myoglobin: 80 ± 66%, n = 27, P = 0.2 vs. kainate alone; SYM2081 + myoglobin: 145 ± 49%, n = 34, P ≈ 1). Hydroxocobalamin, another NO scavenger36 with a much smaller molecular weight (and therefore more readily able to permeate small interstitial spaces between axons, but nevertheless membrane-impermeable), was equally ineffective (kainate + hydroxocobalamin: 90 ± 71%, n = 23, P = 0.94 vs. kainate alone). These experiments indicate that NO synthesized outside the axon did not play a role in kainate receptor-mediated Ca2+ release inside axons. To explore whether intra-axonally generated NO may be important, we selectively loaded myoglobin into axons. In contrast to bath application, intra-axonal scavenger potently blocked kainate- (0 ± 22%, n = 22) and SYM2081-induced (16 ± 33%, n = 25) Ca2+ responses (P ≈ 0). Intra-axonal hydroxocobalamin was also highly effective as was the NOS inhibitor L-NAME (P ≈ 0). Moreover, the effect of intra-axonal NO was synergistic with depolarization, even in the absence of receptor activation (Fig. 3B): neither depolarization alone (45 mM K+ in the perfusate) nor exogenously applied NO (using the NO donor PAPA NONOate (250 μM)) induced an axonal Ca2+ increase. However, applying the NO donor during K+-induced depolarization induced a substantial axonal Ca2+ increase, which was greatly reduced by either nimodipine or ryanodine.
The previous observations suggest a close relationship between axonally-expressed GluR6 kainate receptors and NOS. Immunohistochemistry was performed to further localize these receptors and their associated signaling proteins (Fig. 4). Punctate staining for GluR6/7 (using two different primary antibodies from different species) and nNOS was observed at the periphery of neurofilament-labeled axon cylinders. These clusters were often, but not invariably, co-localized. While we did not attempt to examine the frequency of these complexes along the length of an axon, the representative micrograph in Fig. 4A-C suggests that at least several clusters are present per internode. Immunoelectron microscopy localized GluR6/7 to the axolemma and to clusters beneath the axolemma. Consistent with pharmacological evidence above pointing to a functional interaction between kainate receptors and L-type Ca2+ channels, co-localized GluR6/7 and Cav1.2 clusters were also observed at the surfaces of axons (Fig. 4E-G). Immunoprecipitation of dorsal column lysate with the GluR6/7 antibody yielded a single nNOS-positive band indicating a physical association between this kainate receptor and the enzyme (Fig. 4I). We further hypothesized that a PDZ-binding motif on the C-terminus of GluR6 may mediate an interaction between this receptor and an adaptor protein37, that in turn may scaffold the receptor in proximity to axonal nNOS to support a functional relationship. We constructed a peptide comprising the nine C-terminal residues of GluR6 (RLPGKETMA, see Methods), in order to interfere with such a putative interaction. When this peptide was loaded into axons, both kainate and SYM2081 Ca2+ responses were almost completely blocked (kainate + peptide: 12 ± 28%, n = 77, P = 1.2×10−5 vs. kainate alone; SYM2081 + peptide: 13 ± 27%, n = 78, P = 1.1×10−5). A sham peptide had little effect on the Ca2+ increase induced by kainate (91 ± 28%, n= 45) or SYM2081 (96 ± 30%, n = 42); the responses with the active compared to the sham peptides were highly significantly different (P < 10−9 for both agonists) (Fig. 5A). Further proof of an intra-axonal localization of a GluR6–PDZ domain, that could scaffold this receptor within a signaling nanocomplex containing nNOS, was obtained by loading the synthetic interfering peptide, itself labeled with multiple fluorescent moieties, into axons. As with the fixed immunohistochemical sections, we observed occasional punctate clusters of fluorescent peptide at the periphery of fluorescein-dextran loaded axons (Fig. 5B-D), consistent with the notion that these fibers contain discrete clusters of PDZ domains able to bind and likely cluster kainate receptors.
Having identified such an arrangement of internodal signaling protein clusters capable of significantly increasing axonal Ca2+ levels, we then explored whether such persistent elevations of Ca2+ had any functional implications in otherwise uninjured dorsal columns. Propagated compound action potentials were recorded electrophysiologically and functional integrity of this white matter tract determined by calculating the area under the digitized responses 38. Exposure of dorsal columns to kainate (200 μM) or SYM2081 (100 μM) for 60 min followed by 3 h wash caused an irreversible reduction of mean CAP area to ≈60% of control (data not shown). Addition of the L-type Ca2+ channel blocker nimodipine (10 μM) significantly protected against kainate (CAP area recovery: kainate + nimodipine 93 ± 17%, n = 8 vs. kainate alone 68 ± 10%, n = 8; P = 0.003, Wilcoxon Rank Test) and SYM2081-induced injury (SYM2081 + nimodipine 83 ± 23, n = 9 vs. SYM2081 alone 51 ± 15, n = 7; P = 0.0022).
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. This sensitivity to AMPA/kainate receptor activation also applies to immature oligodendrocyte precursors 40. Glutamate is released from injured myelinated axons via reverse Na+-dependent glutamate transport 7, and via vesicular release from unmyelinated fibers during physiological activation 41,42. In contrast, little is known about functional glutamate receptors on central axons, though experiments indirectly suggest that such receptors may be present 8,43.
Here 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 also be due to direct activation of axonal receptors leading to (possibly focal) axoplasmic Ca2+ deregulation. Curiously, immature premyelinated fibers are reported to suffer ischemic injury independently of glutamate receptors 33. Contrasted with our present findings in mature myelinated axons, this may indicate that myelination induces expression and clustering of axonal glutamate receptors, as it does other nodal and perinodal proteins 44. Immunohistochemistry of dorsal column axons revealed co-localized Glur6/7 and nNOS clusters sparsely distributed along axon cylinders as has been reported previously for Cav and RyR clusters 30. Our results are consistent with the following proposed feed-forward mechanism (Fig. 6): activation of GluR6-containing kainate receptors induces a local depolarization of the internodal axolemma, together with a small amount of Ca2+ influx from a restricted periaxonal space. The local axonal Ca2+ microdomain promotes NO synthesis by nNOS, and the local depolarization activates L-type Ca2+ channels thereby opening ryanodine receptors on subaxolemmal endoplasmic reticulum culminating in a much larger Ca2+ transient than would be possible solely by influx of this ion. This is consistent with previous observations of kainate-receptor mediated depolarization of central axons 43.
Our electrophysiological recordings, which showed that functional injury induced by kainate receptor stimulation was significantly reduced by blocking L-type Ca2+ channels, emphasize two important points: first, given that activation of these receptors in otherwise uninjured dorsal columns results in significant functional impairment, indicates that the observed Ca2+ rise induced by this treatment is pathophysiologically significant, and raises the distinct possibility that exposure of axons to glutamate in inflammatory or ischemic lesions for instance may be directly damaging to axons. Second, the significant reduction in GluR6-mediated electrophysiological injury conferred by an L-type Ca2+ channel blocker further strengthens the functional connection between these receptors and Ca2+ channels, as suggested by the Ca2+ imaging experiments (Fig. 2) and summarized in the proposed model (Fig. 6).
The effect of NO is curious, though this modulator may function to increase the “gain” of the Cav–RyR coupling mechanism, possibly by upregulation of RyR activity 45. This may be necessary to ensure the fidelity of this signaling cascade because unlike neurons and muscle cells which are not ensheathed, voltage-gated proteins such as Cav's which are localized to the internodal axolemma of myelinated fibers, likely experience smaller electric field fluctuations because of the overlying myelin. Given the known promiscuous actions of NO (and its highly reactive derivative peroxynitrite), it is possible that other ion transporters, important for axonal impulse propagation (e.g. voltage-gated Na and K channels, Na-K-ATPase 46) may be modulated as well, in response to kainate receptor/nNOS activation. Thus, central myelinated axons contain functional complexes of several signaling proteins that are arranged in close proximity (eg. GluR6/7, nNOS and Cav1.2, Fig. 4; L-type Ca2+ channels and ryanodine receptors 30), allowing local NO production and depolarization to modulate their function. The purpose of such clusters in mature myelinated fibers is presently unknown; in developing axons however, growth cone dynamics have been shown to be dependent on glutamate receptor activation and release of Ca2+ from intra-axonal Ca2+ stores 47, indicating that ionotropic glutamate receptors and Ca2+ signaling from axonal stores are functionally related from a very early developmental age. Their precise physiological roles in adulthood will require further study. Scaffolding of axonal receptors and effectors such as nNOS in close proximity is reminiscent of the organization of signaling molecules at the post-synaptic density in neurons 48,49, and hints at highly specialized and complex machinery assembled along the internodal axolemma, where little active signaling was thought to take place.
Both glutamate- and NO-dependent toxicity are involved in white matter injury, and particularly in axonal damage, in crippling disorders such as MS 34. The signaling clusters described in this report likely promote and amplify local Ca2+ transients, and may have profound implications for axonal pathophysiology. The local release of potentially high concentrations of Ca2+ through activation of such axonal “nanocomplexes” may play an important role in the genesis of focal swellings and irreversible axonal transactions 50 which render the entire fiber non-functional. The surprisingly complex interaction of glutamate, NO, voltage-gated Ca2+ channels and internal Ca2+ stores in axons may paradoxically present unforeseen opportunities for the development of novel therapeutic strategies.
We thank Drs. B. Barres, E. Peles and M. Rasband for critical reading of the manuscript, and Dr. J. McRory for assistance with co-immunoprecipitations. Supported by: NINDS, CIHR, Heart and Stroke Foundation of Ontario Center for Stroke Recovery, Canadian Stroke Network, HSFO Career Investigator and AHFMR Scientist Awards, and the generosity of private donors to PKS. GWZ is a CIHR Investigator and an AHFMR Scientist. Additionally supported by CIHR (GWZ, JEM), NINDS (BDT) and CCRI (AB).
Abbreviations: please see Supplementary Material