mGluR stimulation increases the translation of STEP in the hippocampus
We first examined whether activation of group I mGluRs regulates STEP protein expression. Hippocampal slices were treated with DHPG (10, 50, and 100 μM for 5 min). A dose-dependent increase in STEP61 protein expression is shown in . STEP46 is not expressed in the hippocampus, and no increase was detected for this isoform (not shown). We next tested whether the DHPG-induced increase in STEP (STEP61 isoform) expression was due to translational or transcriptional mechanisms. Anisomycin (40 μM) or actinomycin D (25 μM) were applied 15 min prior to DHPG (50 μM) stimulation. Anisomycin blocked the DHPG-induced increase in STEP protein expression without affecting its basal expression, while actinomycin D showed no effect (). We confirmed these findings by using a second translation inhibitor, cycloheximide (60 nM), which also blocked increased STEP expression (DHPG, 179 ± 19%, p < 0.01 versus control; DHPG and cycloheximide, 104 ± 21%, p < 0.05 versus DHPG; n = 4).
DHPG stimulation increases STEP61 levels in hippocampal slices
DHPG-induced increase in STEP protein expression requires activation of both ERK and PI3K
Both the MAPK and PI3K signaling pathways are required for the translation-dependent form of DHPG-LTD (Gallagher et al., 2004
; Hou and Klann, 2004
). We therefore explored whether these pathways might underlie the DHPG-induced STEP translation. Pre-incubation of the MEK inhibitor SL327 (50 μM) and the PI3K inhibitor LY294002 (50 μM) for 20 min abolished the DHPG-induced increase in STEP translation without affecting STEP basal expression (). We also confirmed that DHPG increased the phosphorylation of members of MAPK and PI3K pathways, including ERK, PDK, Akt, mTOR, and 4E-BP1 as previously described (Banko et al., 2006
; Gallagher et al., 2004
; Hou and Klann, 2004
) (data not shown). SL327 and LY294002 blocked the activation of these downstream effectors. We further showed that 20 min pre-incubation with mTOR inhibitor, rapamycin (200 nM), blocked the increase in STEP expression, confirming the necessary involvement of mTOR in DHPG-induced STEP translation (DHPG, 171 ± 6%, p < 0.01; rapamycin + DHPG, 106 ± 9%, p = 0.57; comparing to the control, n=4).
DHPG-induced increase in STEP translation requires activation of mGluR5
DHPG activates group I mGluRs, mGluR1 and mGluR5, through similar signaling pathways. While most evidence favors a role for mGluR5 in DHPG-stimulated AMPAR internalization and DHPG-LTD (Banko et al., 2006
; Huber et al., 2001
; Moult et al., 2006
), mGluR1 also plays a role (Volk et al., 2006
). To clarify which of these receptors might be involved in the DHPG-induced translation of STEP, we applied specific mGluR5 or mGluR1 inhibitors (MPEP and LY367385, respectively) to hippocampal slices prior to the addition of DHPG. MPEP (10 μM) significantly blocked DHPG-induced increases in STEP translation, but the mGluR1 inhibitor, LY367385 (100 μM), had no significant effect (). STEP translation was completely blocked by mGluR1 and mGluR5 antagonists. The results suggest that the DHPG-induced increase in STEP translation occurs primarily through mGluR5 activation.
DHPG-induced increase in STEP translation occurs in synaptoneurosomes
Some proteins involved in synaptic plasticity are translated locally in dendrites rather than the cell body, so we tested whether the DHPG-induced translation of STEP occurs, at least in part, within synaptoneurosomes. Synaptoneurosomal preparations were enriched for the synaptic proteins, PSD-95 and synaptophysin compared to homogenates, whereas the nuclear marker histone H3 was not detected (). Synaptoneurosomes were stimulated by DHPG and a dose-dependent increase in STEP expression was observed (). The significant increase was detected as early as 2 min after 50 μM DHPG (). STEP synthesis in synaptoneurosomes was blocked by two functionally distinct translational inhibitors, anisomycin and cycloheximide, and was not affected by actinomycin D (). These results suggest that the DHPG-induced increase in STEP expression occurs in synaptoneurosomes.
DHPG stimulation increases STEP61 levels in synaptoneurosomes
DHPG-induced AMPAR endocytosis requires translation of STEP
Group I mGluRs activation triggers protein translation dependent endocytosis of both GluR1 and GluR2 receptors (Snyder et al., 2001
). Moreover, DHPG-induced redistribution of GluR2 requires an unknown PTP (Huang and Hsu, 2006
; Moult et al., 2006
). We therefore tested the hypothesis that DHPG-induced STEP translation may play a role in the regulation of AMPAR endocytosis. Glutamate receptor trafficking has been studied with subcellular fractionation (Dunah and Standaert, 2001
), and we used a similar approach to look at receptor expression in synaptosomal membrane fractions (LP1). Hippocampal slices were pre-incubated with anisomycin (40 μM, 15 min) and processed 30 min after DHPG treatment to LP1 fractions. STEP protein expression increased significantly in the absence of anisomycin (). DHPG treatment significantly decreased levels of GluR1 and GluR2, consistent with previous findings (Snyder et al., 2001
). Anisomycin blocked both STEP translation and the endocytosis of GluR1 and GluR2. These results indicate that increased STEP synthesis is correlated with increased AMPAR internalization.
GluR1 and GluR2 internalization is blocked by STEP substrate-trapping construct
STEP induces AMPAR endocytosis
If STEP is involved in endocytosis of GluR1/GluR2-containing AMPARs, we reasoned that the addition of wild-type STEP (TAT-STEP WT), even in the absence of DHPG stimulation, might increase AMPAR internalization. The addition of TAT-STEP WT (2 μM, 30 min) to hippocampal slices decreased expression of both GluR1 and GluR2 in the synaptic membrane fraction LP1 (47 ± 10 % and 58 ± 8 % comparing to the TAT-Myc treated control, p < 0.01) (). In contrast, the expression of GABAAβ 2/3 in the LP1 fraction was not changed.
TAT-STEP [C/S] blocks DHPG-induced AMPA receptor endocytosis
To test the specificity of the effects of STEP on GluR1 and GluR2 containing AMPARs, we next treated hippocampal slices with TAT-STEP [C/S]. This construct contains a point mutation in the catalytic domain that renders it inactive. It functions as a substrate-trapping protein that binds to substrates but does not release them, as release requires dephosphorylation (Paul et al., 2007
; Snyder et al., 2005
). Hippocampal slices were preincubated with TAT-Myc or TAT-STEP [C/S] (2 μM for 30 min) and then treated with or without DHPG (50 μM for 5 min). Immunofluorescent staining showed nearly 100% transduction of TAT proteins into the cells after 10 min (data not shown), confirming previous results (Paul et al., 2007
). DHPG significantly decreased both GluR1 and GluR2 expression on synaptic membranes (LP1) in the TAT-Myc control group. In contrast, TAT-STEP [C/S] blocked DHPG-induced AMPAR endocytosis (). TAT-STEP [C/S] blockade of DHPG-induced internalization of GluR1 and GluR2 was further confirmed in hippocampal neuronal cultures by surface biotinylation ().
Tyrosine phosphorylation of GluR2 after TAT-STEP [C/S] treatment was next examined to explore potential mechanisms. Hippocampal slice lysates were immunoprecipitated with GluR2 antibody and probed with anti-Tyr-P antibody (1:2000, MP Biomedicals, UK). DHPG caused a decrease in the tyrosine phosphorylation of GluR2 in the TAT-Myc treated groups. TAT-STEP [C/S] diminished the DHPG-induced decrease in tyrosine phosphorylation of GluR2 without affecting the total amount of GluR2 ().
DHPG-induced AMPAR endocytosis is abolished in STEP KO mice
We next took advantage of STEP KO mice to determine whether STEP is necessary for DHPG-induced AMPAR endocytosis. We confirmed that DHPG decreased the expression of GluR1 and GluR2 in littermate WT mice in LP1 fractions obtained from hippocampal slices (GluR1, 61 ± 6%, GluR2, 67 ± 10%, n = 5; p < 0.01 and p < 0.05, respectively) (). Baseline synaptic expression of GluR1 and GluR2 was increased in STEP KO mice (GluR1, 147 ± 12%; GluR2, 135 ± 19%; n = 5; p < 0.05 versus STEP WT CTL), indicating a constitutive inhibition of AMPARs trafficking towards the synaptic membranes by STEP. Of note, however, the DHPG-induced endocytosis of these receptors was abolished in the STEP KO mice (GluR1, 138 ± 21%; GluR2, 150 ± 7%; n = 5). Together with the previous observation showing that anisomycin blocked both AMPARs endocytosis and STEP translation, our results suggest that STEP translation machinery is required for AMPA receptor redistribution.
DHPG-induced internalization of GluR1 and GluR2 is absent in STEP KO mice
Immunocytochemical studies confirmed that DHPG increased STEP expression in WT hippocampal cultures (). We next verified that DHPG stimulation led to a decrease in GluR2 surface expression (, top panel). However, DHPG failed to induce GluR2 endocytosis in STEP KO hippocampal cell cultures (, middle panel). We attempted to “rescue” the original endocytosis phenotype by replacing WT TAT-STEP in KO culture; DHPG was again able to induce GluR2 internalization (, bottom panel). Similar results were obtained for GluR1 in hippocampal cell cultures (data not shown). These results suggest that STEP is required for the DHPG-induced AMPAR endocytosis.