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1.
Figure 5

Figure 5. From: Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis.

AMPAR activity is required for ouabain-induced AMPAR degradation. Cortical neurons were treated with the sodium channel blocker TTX (1 μM), NMDAR antagonist APV (50 μM), AMPAR antagonist CNQX (20 μM) for 70 min alone, or together with ouabain which was added 10 min after the antagonists. Cells were either lysed for western blot (A and B, n=4) or immunolabeled with anti-GluR1 antibodies under permeant conditions (C and D, n=40). Ouabain treatment remained effective in causing GluR1 reduction in the presence of APV and TTX. In contrast, CNQX completely abolished the ouabain effect, indicating the importance of AMPAR-permitted sodium in mediating the ouabain effect. * P<0.05, t test.

Dawei Zhang, et al. J Neurosci. ;29(14):4498-4511.
2.
Figure 7

Figure 7. From: Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis.

Long-lasting suppression of AMPAR-mediated synaptic transmission by NKA inhibition. (A and B) AMPAR-mediated mEPSC was recorded in the presence of TTX (1 μM), APV (50 μM) and bicuculline (20 μM). Cortical neurons were incubated with ouabain in culture medium for 1 hr and transferred to an ACSF-filled chamber for recording. Representative mEPSCs from non-treated (Control) and treated (Ouabain) neurons were shown. Bar graph (B) showed the average mEPSC amplitudes (n=5 cells each). (C) Neurons were treated with ouabain (1 hr) and allowed to recover in culture medium for varied periods of time as indicated before being recorded. Average mEPSC amplitudes were normalized to the non-treated control. Decreases in mEPSC amplitudes remained even 10 hr after ouabain treatment, indicating a long-lasting depression in synaptic activity (n=4-7 cells for each time point). (D) mEPSC amplitudes from control and ouabain-treated neurons were plotted cumulatively. A leftward shift was produced after ouabain treatment. (E) Histogram of mEPSC amplitude.

Dawei Zhang, et al. J Neurosci. ;29(14):4498-4511.
3.
Figure 2

Figure 2. From: Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis.

NKA inhibition induces a reduction in total AMPAR abundance. Cortical neurons were incubated with the NKA inhibitor ouabain (Oua, 50 μM) in culture medium for 1 hr and cell lysates were used for detecting total protein amount. Ouabain treatment drastically reduced the protein amount of GluR1 (A) (n=8) and GluR2/3 (B) (n=3). As an internal control, the protein level of tubulin was not affected in all experiments. A time course of ouabain incubation (C) showed that GluR1 amount was reduced within 30 min, which reached plateau in 2 hr (n=3). In contrast, NMDAR NR1 was not affected by ouabain (D) (n=5). Postsynaptic proteins that associate with AMPARs or NMDARs were differentially regulated. Ouabain treatment reduced the protein level of GRIP (F) (n=5), but not PSD-95 (E) (n=5) or NSF (G) (n=5). Bar graph data present means ± SEM, * P<0.05, t test.

Dawei Zhang, et al. J Neurosci. ;29(14):4498-4511.
4.
Figure 4

Figure 4. From: Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis.

Involvement of sodium and calcium in NKA-dependent AMPAR proteolysis. (A) Cortical neurons were treated for 1 hr with ouabain (50 μM), or KCl (20 mM) to mimic ouabain-induced membrane depolarization. High potassium did not change GluR1 protein levels, whereas ouabain alone or in combination with KCl significantly reduced GluR1 amounts. Tubulin shows equal amount of loading. Bar graphs (right) represent the average from three independent experiments. (B) Extracellular sodium on ouabain effect. Sodium in ACSF was replaced with the membrane-impermeable substitute NMDG or channel permeant substitute lithium (LiCl). When neurons were incubated with ouabain under either sodium-free condition, the ouabain effect on GluR1 abundance was blocked (n=3). (C) Neurons were treated with the sodium channel opener veratridine (Vera, 20 μM) to enhance sodium influx, and KB-R7943 (5 μM) to block the reversed mode of calcium-sodium exchanger, respectively, alone or together with ouabain for 1 hr. Veratridine caused a reduction in GluR1 and markedly enhanced the ouabain effect when used together (Vera+Oua). KB-R7943 showed no effect when used alone and did not affect the ouabain effect when used in combination (n=3). (D) Extracellular calcium on ouabain effect. Neurons were rinsed with and incubated in calcium-free ACSF (ACSF containing 0 calcium plus 1 mM EGTA, Cafree) for 10 min, then supplemented with ouabain for 1 hr. Compared to the ouabain effect in normal solution (Oua), the calcium-free condition largely abolished ouabain-induced GluR1 reduction (n=4). (E) Neurons were incubated in calcium-free solution for 10 min followed by addition of veratridine (Vera, 20 μM) for 1 hr. Under this condition, veratridine still caused reduction in GluR1 amount (n=4). All the bar graph data present means ± SEM, * P<0.05, t test.

Dawei Zhang, et al. J Neurosci. ;29(14):4498-4511.
5.
Figure 1

Figure 1. From: Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis.

NKA is enriched in synapses and associates with AMPARs. (A) Cultured cortical neurons were fixed and immnostained under permeablilized conditions. The upper panel was a representative neuron double-labeled with antibodies against the synaptic marker protein PSD-95 (monoclonal, 1:500) and the NKA α1 subunit (polyclonal, 1:300). PSD-95 showed a typical punctate pattern indicating its synaptic localization. The NKA α1 subunit immunofluorescence was distributed over the whole cell, with stronger intensity at the soma. Note that in the dendrites, NKA (green) forms clusters that were co-localized with PSD-95 (red) as indicated in the merged image (Overlay), indicating synaptic enrichment of NKA. A selected region was enlarged (lower panel) for clarity. Arrows indicate synaptic clusters with strong co-localization (yellow). (B) Double staining of NKA and AMPARs. Under permeant conditions, cortical neurons were incubated with antibodies against NKA α1 subunits (polyclonal, 1:300) and GluR2 subunits (monoclonal, 1:300). A representative neuron was shown in separate channels (GluR2 in red, NKA α1 in green) and overlay (upper panel). A boxed area was enlarged for clarity (lower panel). Arrows indicate sites of co-localization. (C) Quantification of pixel co-localization of NKA α1 with PSD-95 or GluR2. (D) NKA in synaptosome. Synaptosome lysate prepared from rat cortical tissue was separated by SDS-PAGE and probed with different antibodies. Whereas both NKA α and α3 subunits were detected in synaptosome, α showed higher degree of enrichment. (E) Co-immunoprecipitation of NKA and AMPARs. Rat brain cortical tissue was homogenized manually in lysis buffer and further solubilized for 1 h. Equal amounts of supernatant (500 μg/500μl) were incubated with antibodies against the NKA α1, α3 or β subunits (3 μg each), or rabbit IgG (3 μg) as control. Following protein A beads incubation and precipitation, the protein complexes were probed with anti-GluR1and reprobed with anti-GluR2 antibodies. (F) GST pulldown assays. GST fusion proteins (20 μg) of C-terminals of GluR1 (GluR1CT) and GluR2 (GluR2CT), and of GluR2 n-terminal (GluR2NT) were incubated with cortical brain lysate (800 μg). GST alone was used as control. Pellets of GST pulldown were examined by western blotting using anti-NKA α1 antibodies (upper panel). A parallel gel was stained by commassie blue to confirm the loading of fusion proteins (lower panel).

Dawei Zhang, et al. J Neurosci. ;29(14):4498-4511.
6.
Figure 3

Figure 3. From: Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis.

Ouabain reduces AMPAR abundance via proteasome-mediated proteolysis. (A and B) Cortical neurons were first incubated with the protein synthesis inhibitor anisomycin (Aniso, 30 μM) for 1 hr or a specific proteasome inhibitor MG-132 (MG, 10 μM), or mixed protease inhibitors (Pro inhi, 1:300) for 30 min, and then supplemented with ouabain (Oua, 50 μM) for 1 hr. Compared to the control (Con), ouabain treatment significantly reduced GluR1 amount. Suppression of protein synthesis did not affect ouabain-induced GluR1 reduction, indicating the change in AMPAR amount was not due to inhibition of AMPAR synthesis. In contrast, blockage of proteasome activity by MG-132 completely abolished the ouabain effect, whereas a mixture of protease inhibitors had no effect, indicating that the NKA-dependent decrease of AMPARs was caused by proteasome-mediated protein degradation (n=4). * P<0.05, t test (C and D) Cortical neurons were treated with ouabain for 30 min and 60 min and immunostained with antibodies against GluR1 under permeant conditions. Ouabain treatment reduced immunofluorescence intensity at both the soma (n=12-23) and the dendritic puncta (n=400-500 puncta). (E and F) Application of MG-132 30 min before and during 1 hr ouabain treatment largely blocked the ouabain effect on dendritic GluR1 immunointensity (n=23), consistent with the notion of AMPAR degradation via proteasome. (G and H) Live imaging of GluR1 degradation in transfected neurons. A coverslip of neurons transfected with GFP-GluR1 was transferred to a chamber in ACSF under a fluorescence microscope and imaged every 15 min. Control neurons showed stable GFP fluorescence in 1.5 hr. Following addition of ouabain in the bath at time 0, GFP-GluR1 fluorescence intensity dropped to 60% of the control before stabilization at low levels. However, in the presence of the proteasome inhibitor MG-132 (10 μM), no changes in fluorescence intensity were observed during ouabain treatment. Images were representative neurons imaged under control and ouabain conditions, part of which was enlarged for clarity (G). The rainbow bar on the right indicates the relative intensity of the fluorescence signal. Quantitative data were from measurements of the soma (n=7 for each experiment) (H). Bar graph data present means ± SEM, * P<0.05, t test.

Dawei Zhang, et al. J Neurosci. ;29(14):4498-4511.
7.
Figure 6

Figure 6. From: Na, K-ATPase activity regulates AMPA receptor turnover through proteasome-mediated proteolysis.

AMPAR internalization during NKA inhibition. (A) Isolation of surface AMPARs. Live neurons were incubated with antibodies against the extracellular N-terminus of GluR1 (anti-GluR1N, rabbit, 1:100) to label surface AMPARs, or antibodies against the intracellular C-terminus (anti-GluR1C, rabbit, 1:100) or rabbit IgG as a control, for 5 min, then washed to remove the remaining antibodies. Cell lysates were incubated with protein A beads to isolate surface GluR1. Western blot analysis (probed with anti-GluR1C) showed that GluR1 was isolated specifically following anti-GluR1N antibody incubation, compared to anti-GluR1C and IgG incubations that showed a negative detection. Arrow indicates the antibody band. (B) Using surface pulldown assays as in (A), ouabain treatment (1 hr) dramatically reduced surface-localized AMPARs (GluR1-sur). Consistently, the total GluR1 amount from cell lysates (GluR1-lys) also decreased by ouabain (n=3). (C) Surface biotinylation. Neurons were treated with ouabain and then incubated with biotin (1 mg/ml) in ACSF on ice for 30 min. The biotinylated proteins in cell lysates were precipitated with immobilized streptavidin beads and analyzed by westerns. Similar to GluR1N antibody pulldown, surface biotinylation also showed GluR1 reduction by ouabain. Cell lysates were used for probing tubulin as a control. (D) Live imaging of PH-GluR1. Neurons transfected with PH-GluR1 were imaged every 15 min. Fluorescence signal represents the amount of the surface-localized PH-GluR1. Under control conditions the fluorescence intensity remained stable during 75 min recording (n=5). Addition of ouabain into the imaging bath ACSF induced a rapid reduction in PH-GluR1 fluorescence intensity (n=7). (E) Time courses of reduction of surface and total AMPARs. Neurons were incubated with ouabain for varied periods of time as indicated. Surface GluR1 was isolated as described in (A) and total GluR1 was derived from cell lysates. Optical intensity of GluR1 bands was plotted (below) showing an earlier and faster reduction of surface GluR1 compared to the change of total GluR1. (F) Neurons were incubated in hypertonic sucrose (0.45 M) to block receptor endocytosis. No reduction in total GluR1 was observed following 1 hr ouabain treatment (n=3). (G) Neurons were treated with MG-132 30 min prior to and during 1 hr ouabain incubation. Surface GluR1 isolated as described in (A) still showed reduction when proteasome activity was inhibited (n=3). Bar graph data present means ± SEM, * P<0.05, t test.

Dawei Zhang, et al. J Neurosci. ;29(14):4498-4511.

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