Degenerating myelin inhibits axon regeneration and is rapidly cleared after peripheral (PNS) but not central nervous system (CNS) injury. To better understand mechanisms underlying rapid PNS myelin clearance, we tested the potential role of the humoral immune system. Here, we show that endogenous antibodies are required for rapid and robust PNS myelin clearance and axon regeneration. B-cell knockout JHD mice display a significant delay in macrophage influx, myelin clearance, and axon regeneration. Rapid clearance of myelin debris is restored in mutant JHD mice by passive transfer of antibodies from naïve WT mice or by an anti-PNS myelin antibody, but not by delivery of nonneural antibodies. We demonstrate that degenerating nerve tissue is targeted by preexisting endogenous antibodies that control myelin clearance by promoting macrophage entrance and phagocytic activity. These results demonstrate a role for immunoglobulin (Ig) in clearing damaged self during healing and suggest that the immune-privileged status of the CNS may contribute to failure of CNS myelin clearance and axon regeneration after injury.humoral immunity | nerve regeneration D uring the process of wound healing, rapid and efficient clearance of cellular debris is necessary for tissue regeneration (1). Myelin debris remains in white matter tracks years after an injury to the CNS in humans and primates (2, 3). The prolonged presence of myelin-associated inhibitors of axon regeneration is thought to contribute to the lack of recovery following CNS injury. Although peripheral myelin also contains inhibitors of axon regeneration, PNS myelin is rapidly cleared after injury, thereby permitting rapid axon regeneration (4-6). It is not known why the rates of clearance of PNS and CNS are so different (7).Antibodies, like other opsonins, coat exogenous debris and pathogens, thereby targeting them for clearance by phagocytes. The recognition of self antigens by natural antibodies produced by B1 cells is well documented (8). Although it is hypothesized that these antibodies may have a physiological role other than immune defense, their role in clearing necrotic cellular debris is not known (8). Therefore, we tested whether endogenous antibodies contribute to rapid removal of degenerating myelin after PNS injury, thereby promoting axon regeneration. ResultsAntibodies Accumulate in Sciatic Nerve After Injury. To investigate whether endogenous antibodies aid in removal of myelin debris, we first examined whether antibodies accumulate in peripheral nerves following injury. We compared nerve injury responses between WT and JHD mice, which have a targeted deletion of the JH locus that prevents VDJ recombination and the formation of mature B-cells and antibodies (9). Control and crushed sciatic nerves were obtained from WT and JHD mice and stained with anti-mouse Ig antibodies. Six days after crush injury, we observed a strong increase in immunoreactivity for Ig on degenerating myelin sheathes distal to the site of injury in WT but not in JHD nerves (Fig. S1A). To ...
The channels associated with glutamate receptor (GluR) subtypes, namely N‐methyl‐d‐aspartate receptors (NMDARs), and Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate receptors (AMPARs) and kainate receptors (KARs), are to varying degrees permeable to Ca2+. To compare the mechanism of Ca2+ influx, we measured Ca2+ permeability relative to that of Na+ (PCa/PNa) using fractional Ca2+ currents (Pf) and reversal potential measurements over a wide voltage and Ca2+ concentration range in recombinant NMDAR NR1‐NR2A, AMPAR GluR‐A(Q) and KAR GluR‐6(Q) channels. For NR1‐NR2A channels, PCa/PNa derived from Pf measurements was voltage independent but showed a weak concentration dependence. A stronger concentration dependence was found when PCa/PNa was derived from changes in reversal potentials on going from a Na+ reference solution to a solution with Ca2+ as the only permeant ion (‘biionic’ condition). In contrast, PCa/PNa was concentration independent when derived from changes in reversal potentials on going from a Na+ reference solution to the same solution with added Ca2+ (‘high monovalent’ condition). For GluR‐A(Q) channels, PCa/PNa derived from all three approaches was concentration independent, and for the reversal potential‐based approaches were of comparable magnitude. Their most distinctive property was that PCa/PNa derived from Pf measurements was strongly voltage dependent. For GluR‐6(Q) channels, PCa/PNa derived from Pf measurements was weakly voltage dependent. On the other hand, PCa/PNa derived from all three approaches was the most strongly concentration dependent of any GluR subtype and, except for low Ca2+ concentrations, the values were of comparable magnitude. Thus, the three Ca2+‐permeable GluR subtypes showed unique patterns of Ca2+ permeability, indicating that distinct biophysical and molecular events underlie Ca2+ influx in each subtype.
The high flux rate of Ca2+ through NMDA receptor (NMDAR) channels is critical for their biological function and may depend on a Ca2+ binding site in the extracellular vestibule. We screened substitutions of hydrophilic residues exposed in the vestibule and identified a cluster of charged residues and a proline, the DRPEER motif, positioned C terminal to M3, that is unique to the NR1 subunit. Charge neutralization or conversion of residues in DRPEER altered fractional Ca2+ currents in a manner consistent with its forming a binding site for Ca2+. Similarly, in a mutant channel in which all of the negative charges are neutralized (ARPAAR), the block by extracellular Ca2+ of single-channel current amplitudes is attenuated. In these same channels, the block by extracellular Mg2+ is unaffected. DRPEER is located extracellularly, and its contribution to Ca2+ influx is distinct from that of the narrow constriction. We conclude that key residues in DRPEER, acting as an external binding site for Ca2+, along with a conserved asparagine in the M3 segment proper, contribute to the high fractional Ca2+ currents in these channels under physiological conditions. Therefore, these domains represent critical molecular determinants of NMDAR function in synaptic physiology.
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