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. 2010 Feb 3;30(5):1798-809.
doi: 10.1523/JNEUROSCI.4965-09.2010.

A Role for the Ubiquitin-Proteasome System in Activity-Dependent Presynaptic Silencing

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Free PMC article

A Role for the Ubiquitin-Proteasome System in Activity-Dependent Presynaptic Silencing

Xiaoping Jiang et al. J Neurosci. .
Free PMC article

Abstract

Chronic changes in electrical excitability profoundly affect synaptic transmission throughout the lifetime of a neuron. We have previously explored persistent presynaptic silencing, a form of synaptic depression at glutamate synapses produced by ongoing neuronal activity and by strong depolarization. Here we investigate the involvement of the ubiquitin-proteasome system (UPS) in the modulation of presynaptic function. We found that proteasome inhibition prevented the induction of persistent presynaptic silencing. Specifically, application of the proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) prevented decreases in the size of the readily releasable pool of vesicles and in the percentage of active synapses. Presynaptic silencing was accompanied by decreases in levels of the priming proteins Munc13-1 and Rim1. Importantly, overexpression of Rim1alpha prevented the induction of persistent presynaptic silencing. Furthermore, strong depolarization itself increased proteasome enzymatic activity measured in cell lysates. These results suggest that modulation of the UPS by electrical activity contributes to persistent presynaptic silencing by promoting the degradation of key presynaptic proteins.

Figures

Figure 1.
Figure 1.
Proteasome inhibition prevents induction of persistent presynaptic silencing. A, Merged images of FM1-43FX stain of active synapses (green) and vGluT-1 immunoreactivity (red) to reveal all glutamatergic terminals. Images are from control (untreated) hippocampal neurons, after a 4 h exposure to 3 μm MG-132, or after depolarization in the presence or absence of MG-132. B, C, Representative traces of autaptic action potential-evoked (B) and sucrose-evoked (C) EPSCs recorded under the same conditions as in A. D–F, Summary of quantitative results from the FM1-43/vGluT-1 correspondence assay (D), from action potential-evoked EPSCs (E), and responses to hypertonic sucrose application (F). n = 15 each; *p < 0.001 compared with the depolarized baseline condition.
Figure 2.
Figure 2.
Effects of MG-132 are specific to the proteasome and unrelated to neurotoxicity. A, Summary of the chymotrypsin-like (LLVY) activity of the proteasome, measured in lysates from control neurons and from neurons treated with MG-132 for 4 h. The effect of 1 μm MG-132 added acutely to control lysate was also measured. n = 4; p < 0.03. B, C, Lack of MG-132 toxicity over 4 h was verified using live cell counts in sibling cultures to those examined in A (B) and using propidium iodide exclusion in separate experiments (C). For B, n = 30; for C, n = 3. D, E, Summary of quantitative results from FM1-43/vGluT-1 correspondence assays performed after 4 h treatment with 3 μm MG-132, 20 μm MDL 28170 (D), or 30 nm epoxomycin (E) in the presence or absence of depolarization. For D and E, n = 10. *p < 0.001 compared with the depolarized baseline condition.
Figure 3.
Figure 3.
Strong depolarization increases proteasome enzymatic activity. A, Representative example (left) and normalized summary (right) of the chymotrypsin-like (LLVY) activity of the proteasome measured in lysates from control neurons, after a 4 h exposure to 3 μm MG-132, or after depolarization in the presence or absence of MG-132. B, Representative example (left) and normalized summary (right) of proteasome activity in control neurons, after a 4 h exposure to 50 μm FSK, or after depolarization in the presence or absence of FSK. For summarized results, n = 4 (A) or 3 (B) experiments. *p < 0.05 compared with the control baseline condition.
Figure 4.
Figure 4.
Strong depolarization decreases Munc13-1 levels, which are rescued by MG-132 or forskolin. A, Representative images of vGluT-1 and Munc13-1 immunostaining in control neurons and after depolarization. B, Representative Western blot image after depolarization, illustrating a decrease in Munc13-1 protein compared with a general protein marker, tubulin, or the synaptic protein SV2. C, E, Summary of the average integrated intensity of Munc13-1 immunostaining at glutamate synapses after depolarization in the presence or absence of 3 μm MG-132 or 50 μm FSK. n = 15 each. For C, **p < 0.001 compared with the control baseline condition and *p < 0.005 compared with the depolarized baseline condition. For E, #p < 0.01 compared with the control baseline condition, **p < 0.03 compared with the control baseline condition, and *p < 0.001 compared with the depolarized baseline condition. D, F, Summary of normalized quantitative results from Western blot analyses in parallel experiments to C and E. Munc13-1 protein was normalized to SV2 protein levels within sample. n = 3 experiments each. For D, **p < 0.05 compared with the control baseline condition and *p < 0.03 compared with the depolarized baseline condition. For F, #p < 0.01 compared with the control baseline condition, **p < 0.01 compared with the control baseline condition, and *p < 0.03 compared with the depolarized baseline condition.
Figure 5.
Figure 5.
Depolarization decreases Rim1 protein levels, which are rescued by MG-132 or forskolin. A, Representative images of vGluT-1 and Rim1α/β immunostaining in control neurons and after depolarization. B, Representative Western blot image after depolarization, illustrating a decrease in Rim1α protein compared with a general protein marker, tubulin, or the synaptic protein SV2. C, E, Summary of the average integrated intensity of Rim1α/β immunostaining at glutamate synapses after depolarization in the presence or absence of 3 μm MG-132 or 50 μm FSK. n = 15–25 each. For C, **p < 0.001 compared with the control baseline condition and *p < 0.01 compared with the depolarized baseline condition. For E, #p < 0.001 compared with the control baseline condition, **p < 0.005 compared with the control baseline condition, and *p < 0.001 compared with the depolarized baseline condition. D, F, Summary of normalized quantitative results from Western blot analyses in parallel experiments to C and E. Rim1α protein was normalized to SV2 protein levels within sample. n = 3 experiments each. For D, **p < 0.03 compared with the control baseline condition and *p < 0.05 compared with the depolarized baseline condition. For F, #p < 0.01 compared with the control baseline condition, **p < 0.02 compared with the control baseline condition, and *p < 0.05 compared with the depolarized baseline condition.
Figure 6.
Figure 6.
Synaptic Munc13-1 and Rim1 levels correlate with presynaptic function. A, Representative images of FM1-43FX uptake and Munc13-1 immunostaining at glutamate synapses under control conditions and after depolarization. Note that Munc13-1 levels are lower at inactive synapses (FM1-43/vGluT-1+; arrowheads) than at active synapses (FM1-43+/vGluT-1+; arrows) in both conditions, although the percentage of inactive synapses in control cultures is much lower overall (see Fig. 1D). B, Summary of average integrated intensity of Munc13-1 immunostaining at active (FM1-43+) and inactive (FM1-43) glutamate synapses. n = 220 (active) or 80 (inactive) for control and 72 (active) or 228 (inactive) for the depolarized condition. *p < 0.001 compared with the active synapses in that condition. C, Representative images of FM1-43FX uptake and Rim1 immunostaining at glutamate synapses under control conditions and after depolarization. Rim1 levels are also lower at inactive synapses (arrowheads) than at active synapses (arrows). Note that synaptic puncta size is slightly smaller in C than in A attributable to differences in cell fixation. D, Summary of average integrated intensity of Rim1 immunostaining at active and inactive glutamate synapses. n = 228 (active) or 122 (inactive) for control and 93 (active) or 257 (inactive) for the depolarized condition. *p < 0.01 compared with the active synapses in that condition. E, F, Measures of FM1-43FX integrated intensity values at individual synapses plotted as a function of the Munc13-1 (E) or the Rim1 (F) integrated intensity values. Data are from the same glutamate synapses as in B and D. For E, r2 = 0.785; p < 0.001. For F, r2 = 0.599; p < 0.001.
Figure 7.
Figure 7.
Several presynaptic proteins are unaffected by depolarization. A, Representative Western blot images from control neurons and after depolarization. As noted in Materials and Methods, some Western blots were performed using tissue from acute hippocampal slices to increase the amount of total protein available for Western blot analysis. We have confirmed previously that persistent presynaptic silencing can be induced in acute hippocampal slices (Moulder et al., 2004). B, Representative images of immunostaining in control neurons and after depolarization. Note that synaptic puncta size can vary attributable to differences in cell fixation and permeabilization. C, Summary of normalized quantitative results from Western blot analyses. The candidate proteins were normalized to SV2 (left) or tubulin (right) protein levels within sample. n = 3 experiments each. Values shown are the percentage of control measured in the depolarized condition. No statistically significant differences were observed after depolarization. vGluT-1 was normalized to SV2 only because of the similarity in size with tubulin. Examples of SV2 Western blot images can be seen in Figures 4 and 5. D, Summary of the average integrated intensity of candidate protein immunostaining at glutamate synapses after depolarization. Analysis was restricted to glutamate synapses through colocalization with vGluT-1 immunostaining. n = 10–15 each. No statistically significant differences were observed after depolarization. A lack of difference in synaptic SV2 and vGluT-1 immunostaining after depolarization has been reported previously (Moulder et al., 2004, 2006).
Figure 8.
Figure 8.
Rim1α overexpression prevents induction of persistent presynaptic silencing. A, B, Representative traces of autaptic action potential-evoked (A) and sucrose-evoked (B) EPSCs recorded under control conditions and after depolarization in neurons transfected with GFP alone or GFP plus Rim1α. C, D, Summary of quantitative results from action potential-evoked EPSCs (C) and responses to hypertonic sucrose application (D) in transfected neurons. n = 13 each; *p < 0.001 compared with the depolarized condition transfected with GFP alone. E, Summary of Munc13-1 integrated intensity at glutamatergic terminals of transfected neurons. n = 214–453 synapses per condition; *p < 0.001 compared with the depolarized condition transfected with synaptophysin–YFP (Syph-YFP) alone.

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