Presynaptic silencing is an endogenous neuroprotectant during excitotoxic insults

Abstract

Glutamate release is a root cause of acute and delayed neuronal damage in response to hypoxic/ischemic insults. Nevertheless, therapeutics that target the postsynaptic compartment have been disappointing clinically. Here we explored whether presynaptic silencing (muting) of glutamatergic terminals is sufficient to reduce excitotoxic damage resulting from hypoxia and oxygen/glucose deprivation. Our evidence suggests that strong depolarization, previously shown to mute glutamate synapses, protects neurons by a presynaptic mechanism that is sensitive to inhibition of the proteasome. Postsynaptic Ca2+ rises in response to glutamate application and toxicity in response to exogenous glutamate treatment were unaffected by depolarization preconditioning. These features strongly suggest that reduced glutamate release explains preconditioning protection. We addressed whether hypoxic depolarization itself induces presynaptic silencing, thereby participating in the damage threshold for hypoxic insult. Indeed, we found that the hypoxic insult increased the percentage of mute glutamate synapses in a proteasome-dependent manner. Furthermore, proteasome inhibition exacerbated neuronal loss to mild hypoxia and prevented hypoxia-induced muting. In total our results suggest that presynaptic silencing is an endogenous neuroprotective mechanism that could be exploited to reduce damage from insults involving excess synaptic glutamate release.

Figure 1

Depolarization preconditioning protects against hypoxia. A. Schematic of preconditioning/hypoxic exposure paradigm. B. Photomicrographs from a single experiment demonstrating depolarization protection. Upper left: brightfield image from a normoxic control stained with trypan blue 24 hr post-preconditioning with 4 hr of 30 mM NaCl (control preconditioning) and 2.5 hr sham hypoxia. Upper right: A field from a dish preconditioned with 30 mM NaCl and subjected to 2.5 hr hypoxia. Trypan blue positive pyknotic nuclei are apparent. Lower left: a field from a dish preconditioned with 30 mM KCl for 4 hr and then subjected to 2.5 hr hypoxia. Lower right: a protection control in which 1 μM NBQX and 100 μM D-APV, ionotropic glutamate receptor (GluR) antagonists, were included in the hypoxia media. C. Summary of experiments like that depicted in panel B, showing protection afforded by KCl depolarization preconditioning. Cell survival is expressed as the percentage of trypan blue negative cells averaged over ten fields evaluated with a 20x objective (n=7 independent experiments). Asterisks designate p < 0.05 compared with NaCl hypoxia (unpaired, two-tailed t-tests with Bonferroni correction for multiple comparisons). Gray bar emphasizes the major hypothesized result of KCl preconditioning protection.

Figure 2

Preconditioning induces presynaptic muting but no detectable postsynaptic changes. A. Images of vGluT-1 positive puncta from representative fields from cultures preconditioned by 4 hr KCl (30 mM) depolarization or by 30 mM NaCl control preconditioning. Middle panels show uptake of FM1-43fx during brief depolarization in the same field. The merged images reveal more inactive (FM1-43fx negative) vGluT-1 positive puncta after depolarizing preconditioning. Green = FM1-43fx. Red = vGluT-1. Red puncta with no green overlap are mute synapses, while yellow indicates overlap and active synapses. White arrowheads indicate examples of co-labeled, active glutamatergic synapses. B. The percentage of FM1-43fx positive (FM (+)) terminals is summarized from 5 experiments like that depicted in A. C. Representative fluorescence images from somatic Ca²⁺ signals measured in control (n = 18 cells in 6 fields) and depolarization-conditioned (n = 15 cells in 5 fields) cells. Cells were loaded with a 30 min bath application of 2 μM Fluo3- FF AM. Representative regions of interest (labeled with “1”) show typical locations of measurement in the soma, chosen based on a phase contrast image (not shown). Dendrites and axons are below the plane of focus. Images of baseline (prior to glutamate perfusion), peak fluorescence (5 s following 10 μM glutamate onset), and return to baseline (30 s wash) fluorescence are shown. Region 2 indicates a region devoid of cells and typical of regions from which background fluorescence was measured. D. Summary of glutamate-evoked somatic intracellular Ca²⁺ signals measured over the entire experiment (67 images over 40 seconds) with control-conditioned and depolarization-conditioned cells overlaid.

Figure 3

Exogenous glutamate toxicity is unaffected by depolarization preconditioning. A. Brightfield photomicrographs of trypan blue-stained fields from the indicated conditions in a single experiment. Exogenous glutamate (Glu; 10 μM for 5 min) replaced hypoxia in the preconditioning/insult paradigm. Upper left: control cells 24 hr post-preconditioning with 30 mM NaCl and sham glutamate exposure that included media exchange. Upper right: protection control cells preconditioned with 30 mM NaCl, subjected to 10 μM glutamate (5 min) with ionotropic GluR antagonists present (1μM NBQX + 100μM D-APV). Lower left: cells preconditioned with 30 mM NaCl, subjected to 10 μM glutamate for 5 min. Lower right: cells preconditioned with 30 mM KCl, then subjected to 5 min of 10 μM glutamate. B. Summary graph of the conditions shown in A (n=5). Glutamate significantly increased trypan blue staining compared with the NaCl sham condition in this dataset (p < 0.05, Bonferroni corrected t-test). However, depolarization preconditioning did not significantly alter the severity of cell loss. As with hypoxia, GluR block (1 μM NBQX, 100 μM D-APV) effectively protected neurons from glutamate-induced death, consistent with the pivotal role of excitotoxicity in both insults.

Figure 4

Depolarization preconditioning protects against oxygen-glucose deprivation (OGD). A. Brightfield micrographs of trypan blue staining from each condition: (left to right) control dish 24 hr post-preconditioning with 30 mM NaCl and sham OGD; dish preconditioned with 30 mM NaCl, subjected to OGD for 2.5 hr; dish preconditioned with 30 mM KCl, then subjected to OGD for 2.5 hr. B. Summary graph showing protection afforded by KCl depolarization preconditioning (n=4). Asterisks denote p < 0.05 compared with the NaCl OGD condition (Bonferroni corrected t-tests). Gray bar emphasizes the major hypothesized result of KCl preconditioning protection.

Figure 5

Depolarization preconditioning protection from hypoxia does not depend upon GABA_A receptor modulation, adenosine A1 receptor activation, or extracellular Ca²⁺ influx. A. The non-competitive GABA_A antagonist picrotoxin (PTX ;100 μM), applied during hypoxia (2.5 hr), did not prevent depolarization protection. Summary of the indicated experimental conditions (n=6). “N.S.” indicates lack of significant difference in comparisons of the effect of PTX with the corresponding condition in the absence of PTX. B. The A1 receptor antagonists DPCPX (200 nM) did not block depolarization preconditioning protection. Summary of various 4 hr preconditioning conditions. Hypoxic insult was 2.5 hr (n=5). “N.S.” indicates a lack of difference for the indicated comparisons. C. Summary graph showing protection afforded by KCl depolarization preconditioning (n=5) independent of the addition of 1.8 mM extracellular Ca²⁺ to the Ca²⁺ free conditioning media. For all panels, neuronal survival was assessed 24 hr after insult with trypan blue exclusion. Asterisks denote p < 0.05 comparisons (Bonferroni corrected t-tests).

Figure 6

Proteasome inhibition prevents depolarization preconditioning protection through a presynaptic mechanism. A. Summary of neuronal survival after several 4 hr preconditioning conditions and 2.5 hr hypoxic exposure (n=5). MG-132 (3 μM, co-applied for 4 hr with 30 mM KCl) prevented the protective effect of depolarization. Indicated comparisons represent the major hypothesized effects (asterisk denotes p < 0.05 with Bonferroni correction). Other comparisons that showed a significant difference from insult alone (NaCl hypoxia) were the NaCl sham control and the KCl hypoxia conditions. There was no difference between the NaCl hypoxia condition and NaCl hypoxia plus MG-132. B. Summary of cell survival after indicated preconditioning conditions followed by an insult of exogenously applied glutamate (Glu; 10 μM for 5 min). There was no effect of KCl or of MG-132 on cell survival using the glutamate insult (n=5), suggesting a presynaptic mechanism of depolarization preconditioning protection. No comparison of the NaCl/glutamate group with any of the other indicated groups yielded a significant difference. Glutamate treatment caused significant death relative to control (p < 0.05, Bonferroni corrected t-test for multiple comparisons). C. Control experiment testing direct effects of KCl and MG-132 on cell survival during various 4 hr preconditioning paradigms in the absence of hypoxia (n=5). No comparison with the control sham condition exhibited a significant difference (unpaired t-tests vs. control). For all experiments depicted in panels A, B, and C neuronal survival was evaluated with trypan blue 24 hr after the insult.

Figure 7

Hypoxia induces proteasome-dependent presynaptic silencing. A. FM1-43fx/vGluT-1 correspondence assay. Green = FM1-43fx. Red = vGluT-1. Red puncta with no green overlap are mute synapses, while yellow indicates overlap and active synapses. A 2 hr preconditioning period with or without MG-132 (3 μM) was followed by 2 hr hypoxic insult (or sham) with immediately subsequent FM1-43fx assay. B. Summary of experiments depicted in panel A showing the percentage of active synapses after hypoxic insult with and without MG-132 co-incubation (n=25 fields from 5 independent experiments). Asterisk denotes p < 0.05 compared with hypoxia alone (Bonferroni corrected t-tests). Comparisons of control versus MG-132, control versus hypoxia plus MG-132, and MG-132 versus hypoxia plus MG-132 showed no differences.

Figure 8

Proteasome inhibition during hypoxia exacerbates neuronal damage. A. Brightfield trypan blue-stained fields from a representative experiment. Neurons were incubated with or without MG-132 for 2 hr to allow drug penetration, then subsequently co-incubated with MG-132 for additional 2 hr with or without hypoxia. Culture media was then exchanged, and cells were incubated normally for 24 hr after which trypan blue staining was performed. Upper left: control with no MG-132 or hypoxia. Upper right: 4 hr total MG-132 exposure (no hypoxia). Lower left: 2 hr hypoxia alone. Lower right: 2 hr MG-132 followed by hypoxia/MG-132 co-incubation for 2 hr (4 hr total exposure to MG-132). B. Summary of A showing that MG-132 exacerbates hypoxia-induced death (n=7). Asterisk denotes p < 0.05 (Bonferroni corrected t-test). C. Summary of control for postsynaptic effects of MG-132 (n = 5 experiments). 10 μM glutamate (Glu) treatment was used as a surrogate for hypoxic insult. There was no significant exacerbation of glutamate-induced damage by MG-132. As with hypoxia experiments in B, cells were pre-incubated in MG-132 for 2 hr prior to insult.