Article Figures & Data
Figures
- Figure 1
. PR20, but not GR20, is toxic to motor neurons: kinetics of cellular accumulation and disposal. Mixed spinal cord neuron cultures or pure astrocyte cultures were exposed to PR20- or GR20-containing media; survival assays and biochemical interrogations followed. A, The survival of motor neurons was determined 5 d after exposure to various concentrations of PR20. The LD50 of PR20 is ∼ 2 µM. B, The survival of astrocytes was determined using the colorimetric XTT assay 5 d after exposure to various concentrations of PR20 or GR20. No death was seen up to 10 µM DPR. C, The survival of motor neurons was determined 5 d after exposure to various concentrations of GR20. No death was seen up to 35 µM GR20. D, Immunoblotting for HA-tagged PR20 at various time points after addition of 2 µM PR20 to mixed spinal cord cultures. PR20 is first detectable in cell lysates approximately 0.5 h after exposure and rises to a maximum at 1.0-2.0 h. The Western blot signal declines thereafter and is undetectable at the 48-h time point. E, In a 2-h pulse-chase paradigm after 2 µM PR20 application, Western blot signal is detectable in the soluble and insoluble fractions at the 2-h time point, and the signal diminishes thereafter to a barely detectable level by the 6-h time point. F, In a 2-h pulse-chase paradigm after 10 µM GR20 application, Western blot signal is detectable in the soluble fraction only at the 2-h time point, and the signal diminishes thereafter to a barely detectable level by the 4-h time point. G, Mixed spinal cord cultures were pulsed for 2 h with 2 µM PR20 and the chase media contained MG132 (5 µM) or vehicle. Cell lysates were prepared at time intervals thereafter, and quantitative image analysis of the resultant Western blots showed that the decrement in PR20 abundance was the same in the +MG132 condition in comparison with the −MG132 condition. H, Mixed spinal cord cultures were pulsed for 2 h with 2 µM PR20, and the chase media contained bafilomycin A1 (Baf A1) (400 nM). Cell lysates were prepared at time intervals thereafter, and quantitative image analysis of the resultant Western blots showed that the decrement in PR20 abundance was the same in the +Baf A1 condition in comparison with the –Baf A1 condition.
- Figure 2.
PR20 and GR20 accumulate in distinct subcellular locations and distinct cell populations. Mixed spinal cord cultures were exposed to 2 µM PR20 or GR20 for 48 h, fixed, and processed for immunocytochemistry. A, Staining with SMI32 reveals large multipolar motor neurons. The inset shows a single 0.5-µm slice from the confocal data at the level of the nucleus. B, The same field as in A, stained for HA-PR20 reveals nuclear staining as well as scattered puncta. The inset shows a single 0.5-µm slice at the same level as in (A), suggesting nuclear PR20 in the motor neuron. C, A merge image of A and B reveals nuclear PR20 in multiple cells, including the labeled motor neuron. The inset shows the same single 0.5-µm slice from A and B unambiguously demonstrating PR20 immunoreactivity in motor neurons. Calibration bar = 35 µm. D, Staining for GFAP reveals abundant astrocytes in these cultures. E, The same field as in D, stained for HA-PR20 reveals nuclear staining as well as scattered puncta. F, A merge image of D and E reveals nuclear PR20 in astrocytes. G, Mixed spinal cord cultures were exposed to 2 µM GR20 for 48 h, fixed, and processed for immunocytochemistry. Staining with SMI32 reveals large multipolar motor neurons. Inset shows a single 0.5-µm slice from the confocal data at the level of the nucleus. H, The same field as in G, stained for HA-GR20 reveals cytoplasmic staining. Inset show a single 0.5-µm slice at the same level as in G, suggesting cytoplasmic GR20 in the motor neuron. I, A merge image of G and H reveals cytoplasmic GR20 in multiple cells, including the labeled motor neurons. Inset shows the same single 0.5-µm slice from the G and H unambiguously demonstrating GR20 immunoreactivity in motor neurons. Calibration bar = 30 µm. J, Staining for GFAP reveals abundant astrocytes in these cultures. K, The same field as in J stained for HA-GR20 reveals cytoplasmic staining. L, A merge image of J and K reveals cytoplasmic GR20 is not present in astrocytes.
- Figure 3.
PR20 neither influences the activation of TORC or AMPK nor ER stress. Mixed spinal cord cultures were exposed to PR20 for 48 h and then processed for immunoblotting. A, Representative immunoblot images of biochemical markers of TORC activation (e.g., phospho-4EBP1 and phospho-S6K), AMPK activation, or activation of the ER stress response (KDEL) shows no difference between PR20 versus vehicle-treated cells. B, Quantification of the bands with intensity values from samples were averaged and normalized to actin loading controls. Dark grey bars correspond to the presence of PR20, and light grey bars correspond to levels without PR20. Error bars represent SE.
- Figure 4.
Total ubiquitin levels rise over time in the presence of PR20; PR20, but not GR20, inhibits substrate flux through the ubiquitin-proteasomal system. Mixed spinal cord cultures were exposed to PR20 or GR20 and subjected to biochemical interrogations. A, Duplicate or triplicate cell lysate samples probed for ubiquitin in the absence of PR20 (-PR20) or at 2, 4, 8, or 16 h after PR20 application. A progressive build-up in total ubiquitin levels occurs over time. The panel below shows quantification of these data with total ubiquitin levels normalized to actin loading controls. Error bars represent SE. Blotting for HA-tagged PR20 shows that DPR is present in the lysates but does not migrate as a high molecular weight species as would be expected if PR20 was ubiquitylated. Equal amounts of total protein are present in each lane, as reported by the actin blot. B, Cartoon describing two different mechanisms for increased steady state ubiquitin levels: enhanced ubiquitylation versus decreased proteasomal degradation. C, Total ubiquitin levels from cells treated with PR20 or vehicle for 48 h and then MG132 or vehicle for 4 h. The difference between the +/- MG132 lanes represents the flux of ubiquitylated substrates over 4 h. Representative ubiquitin blots demonstrate a smaller difference between the +/- MG132 lanes in PR20-treated cultures versus vehicle-treated cultures. The permutation test was applied to determine whether statistically significant group differences exist, and the results are shown in the lower panel. There is a reduced flux of ubiquitylated substrates in the PR20-treated cells versus vehicle-treated cells. Six independent biological replicates were tested for each condition. *, p < 0.05 by the permutation test. D, Representative ubiquitin blots demonstrate no difference between the +/- MG132 lanes in GR20-treated cultures versus vehicle-treated cultures. The permutation test was applied to determine whether statistically significant group differences exist, and the results are shown in the lower panel. No group differences were found, and at least 10 independent biological replicates were tested for each condition. n.s., not significant.
- Figure 5.
PR20 inhibits flux through the lysosomal-autophagy system. A, Optical pulse labeling of spinal neurons using automated fluorescent microscopy. Following a pulse of 405-nm light, a portion of Dendra2-LC3 is photoconverted such that its peak emission is red-shifted to 573 nm. By measuring the decay in intensity using a TRITC filter, half-life can be determined in individual neurons. The half-life of Dendra2-LC3 is a measurement of autophagosome turnover, with an increased half-life indicating reduced turnover. B, Distribution of Dendra2-LC3 half-lives in cells treated with 2 µM PR20, GR20, and 10 µM MG132 compared with those treated with vehicle. * indicates p < .05 with the two-sided Kolmogorov–Smirnov (KS) test (GR20 p = 0.0038, PR20 p = 2.2 × 10−16, MG132 p = 2.2 × 10−16). C, MG132 significantly extends Dendra2 half-life while 2 µM PR20 and GR20 do not (MG132 p = 3.23 × 10−5, two-sided KS test).
- Figure 6.
PR20, but not GR20 binds to proteasomes; PR20 inhibits the proteasomal degradation of a test substrate. A, By pulling down human proteasomes and then immunoblotting against HA, a direct association of PR20 (but not GR20) peptides to the proteasome is demonstrated. Increasing amounts of proteasome leads to more associated PR20. Proteasomes are identified using an antibody recognizing the α3 subunit. B, Immunoblot images of Ub-Sic1, HA tag, and α3 loading controls. Molar ratios represent the ratio between 26s proteasomes (PTSM) and PR20 peptides. The molar ratios of 1:0, 1:10, and 1:100 were each subject to proteasomal degradation of Ub-Sic1 for 0, 5, and 15 min. Time-dependent loss of Ub-Sic1 is noted. There is no change in the abundance of HA-tagged PR20 or the α3 subunit. C, Quantification of Ub-Sic1 levels normalized to loading controls for each condition. Values represent means (n = 3), and the error bars represent SE. D, Quantification of the difference between starting level of Ub-Sic1 and Ub-Sic1 abundance at the 5- and 15-min time points. *, p < 0.01.
- Figure 7.
MG132 is toxic to motor neurons, inhibits flux through the proteasome, and cell death is rescued by the DUB inhibitor IU1. MG132 was applied one time at varying concentrations to mixed spinal cord cultures and 5 d later processed for immunocytochemical quantification of motor neurons. A, The dose-response curve shows that MG132 kills motor neurons with an LD50 of ∼ 100-150 nM. B, Mixed spinal cord cultures were exposed to 150 nM MG132 or vehicle for 48 h, and the parallel groups of cultures were exposed to 5 µM MG132 or vehicle for 4 h. Cell lysates were prepared and blotted for total ubiquitin levels. The difference of the ubiquitin immunoreactivity in the cultures treated with 5 µM or vehicle reports the flux of substrates through the UPS over 4 h. Representative blots are shown. C, Quantification of the data from the flux assay and statistical analysis by the permutation tests reveals a statistically significant inhibition of flux through the UPS in cultures treated with 150 nM for 48 h (p = 0.027). D, Number of motor neuron in mixed spinal cord cultures after exposure of 150 nM MG132 or vehicle and 5 µM IU1 or vehicle. MG132 leads to a statistically significant reduction in motor neuron number, and this is reversed by IU1 treatment. IU1 alone is not toxic.
- Figure 8.
PR20-evoked motor neuron death is prevented by the proteasome activator, IU1. Mixed spinal cord cultures were exposed to PR20 or vehicle and then dosed with IU1 or vehicle. Motor neuron number was determined 5 d later. In the absence of PR20, IU1 did not affect motor neuron survival. PR20 treatment leads to approximately 50% loss of motor neurons and IU1 application prevents this effect. n.s., not significant; *, p < 0.05.
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