Dual neuroprotective signaling mediated by downregulating two distinct phosphatase activities of PTEN

Abstract

The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid and protein phosphatase. We report here that PTEN physically associates with the NR1 and NR2B subunits of NMDA receptors (NMDARs) in rat hippocampus. Downregulating the protein expression of PTEN inhibits the function of extrasynaptic NMDARs and decreases NMDAR surface expression, suggesting a crucial role for endogenous PTEN in the modulation of NMDAR-mediated neuronal function. Reducing PTEN expression also enhances Akt/Bad phosphorylation in hippocampal neurons. Importantly, suppressing lipid and protein phosphatase activity of PTEN, respectively, activates Akt and inhibits extrasynaptic NMDAR activity and thereby protects against ischemic neuronal death in vitro and in vivo. Thus, our study reveals a dual neuroprotective mechanism by which Akt/Bad and extrasynaptic NMDARs are regulated via downregulation of two distinct PTEN phosphatase activities and present the possibility of PTEN as a potential therapeutic target for stroke treatment.

Figure 1.

Physical association between PTEN and NMDARs in rat hippocampus. A, C, Coimmunoprecipitation of hippocampal NR1 and NR2B subunits, but not GluR1 subunit of AMPA receptors, by PTEN Ab. B, D, E, Coimmunoprecipitation of hippocampal PTEN by NR1 and NR2B Ab but not by NR2A Ab. F, In vitro binding assay showing the direct binding of [³⁵S]PTEN to GST-NR1-1a_CT but not NR2A-_CT and NR2B-_CT. GST was used as a control. IP, Immunoprecipitation.

Figure 2.

Suppression of PTEN protein expression by siRNA in cultured hippocampal neurons. A, Representative images of siRNApten-GFP expression in cultured hippocampal neurons 24 hr after transfection. Scale bar, 40 μm. B, An example of PTEN knockdown by siRNApten-GFP but not by SsiRNApten-GFP introduction in cultured hippocampal neurons 24 hr after transfection. Scale bar, 20 μm. C, Time course of siRNA-induced decrease of PTEN expression in cultured hippocampal neurons (n = 12 for each group; ^*p < 0.05). Data are normalized to values from nontransfected neurons.

Figure 3.

Down regulating PTEN inhibits extrasynaptic NMDAR function. A, An example of decreased NMDAR-mediated whole-cell currents in neurons expressing siRNApten-GFP (bottom) but not in neurons expressing SsiRNApten-GFP (top). B, Graph showing peak NMDA currents inhibited by PTEN downregulation (Non-GFP, n = 28; SsiRNApten-GFP, n = 23; siRNApten-GFP, n = 25; ^*p < 0.05). C, I-V relationship in neurons expressing non-GFP and siRNApten-GFP. D, Quantification analyses indicate that PTEN knockdown has no significant effects on NMDAR-mediated mEPSC amplitudes (left, n = 8 for each group) and frequency (right, n = 8 for each group). Data are normalized to non-GFP values. E, Representative traces of NMDA-activated channels recorded in cell-attached patches from neurons expressing SsiRNApten-GFP (left) and siRNApten-GFP (right). F, Effects of PTEN knockdown on NMDA channel open probability (P_o), mean open time (t_o), and channel conductance (γ). Data are normalized to values from neurons expressing SsiRNApten-GFP as control (n = 6; ^*p < 0.05).

Figure 4.

PTEN knockdown reduces surface expression of NMDARs and increases Akt/Bad phosphorylation. A, An example showing decreased surface expression of NMDARs in PTEN-deficient hippocampal neurons. Scalebar, 30 μm. B, Summarized data showing surface expression of NMDARs (Non-GFP, n = 57; SsiRNApten-GFP, n = 69; siRNApten-GFP, n = 65; ^*p < 0.05). C, Graph showing that suppressing PTEN did not alter total NR1 expression in hippocampal neurons (Non-GFP, n = 22; SsiRNApten-GFP, n = 26; siRNApten-GFP, n = 19). D, Downregulation of PTEN reduced the number of functional NMDA channels (N) at the membrane surface. Currents were elicited by application of NMDA (1 μm NMDA with 50 μm glycine) in the continuous presence of the open-channel blocker MK-801 (5 μm) in the neurons expressing SsiRNApten-GFP (top) and the neurons expressing siRNApten-GFP(bottom) at a holding potential of -60 mV. The NMDA inward current increased to a peak value and then decayed exponentially because of MK-801 block of NMDA channels as they opened. The cumulative charge transfer (Q), which is the total current flow during the time interval for complete block by MK-801, was obtained by integration of (Figure legend continues.) (Figure legend continued.) the current trace over time (area indicated by shading). E, Examples showing that both Akt and Bad phosphorylation are increased in neurons expressing siRNApten-GFP. Scale bar, 20 μm. F, Graph plotting the increased phosphorylation levels of Akt and Bad in PTEN-deficient hippocampal neurons (left: Non-GFP, n = 39; siRNApten-GFP, n = 51; ^*p < 0.05; right: Non-GFP, n = 45; siRNApten-GFP, n = 55; ^*p < 0.05). SsiRNApten-GFP transfection has no significant effects on both Akt and Bad phosphorylation (data not shown).

Figure 5.

Akt and NMDARs are, respectively, regulated by PTEN lipid and protein phosphatase activities. A, Representative images showing that Akt phosphorylation is increased in neurons transfected with either C124A-GFP or G129E-GFP. However, transfection of C124A-GFP decreases NR1 surface expression, and expression of G129E-GFP increases NR1 surface expression. Scale bar, 20 μm. B, Summarized data showing surface NR1 expression (Non-GFP, n = 52; GFP, n = 48; C124A-GFP, n = 46; G129E-GFP, n = 61; ^*p < 0.05). C, Examples of responses of NMDA currents to GFP, C124A-GFP, or G129E-GFP expression. D, Graph showing peak NMDA currents inhibited by C124A-GFP transfection but increased by G129E-GFP expression (Non-GFP, n = 21; GFP, n = 27; C124A-GFP, n = 31; G129E-GFP, n = 35; ^*p < 0.05).

Figure 6.

Neuroprotection mediated by PTEN down regulation. A, Left, Examples of PI staining. Scale bar, 25 μm. Right, Summarized data from PI staining (GFP, n = 216; SsiRNApten-GFP, n = 189; siRNA-GFP, n = 207; ^*p < 0.05). B, Top, An example of reduced PTEN expression in the CA1 pyramidal cell layer 7 d after microelectroporation. Bottom, Time course of PTEN expression after microelectroporation of PTEN siRNA-GFP (n = 6 for each group; ^*p < 0.05). Data are normalized to values from nontransfected neurons. C, Left, Examples of Fluoro-Jade B staining of degenerating neurons in the CA1 pyramidal cell layer. Scale bar, 60 μm. Right, Summarized data from Fluoro-Jade B staining (^*p < 0.05, difference from control; ^**p < 0.05, difference from siRNApten expression; data shown are means ± SE of 4 determinations). D, DN-Akt blocks the neuroprotection induced by PTEN downregulation (SsiRNApten-GFP, n = 125; siRNApten-GFP, n = 151; siRNApten-GFP + DN-Akt, n = 116. ^*p < 0.05, difference from SsiRNApten-GFP; ^#p < 0.05, difference from siRNApten-GFP). E, PI labeling showing that ifenprodil treatment significantly reduces OGD-induced neuronal death in neurons expressing GFP (left: control, n = 183; ifenprodil treatment, n = 162; ^*p < 0.05) but does not have an effect on neurons coexpressing C124A-GFP and DN-Akt (right: control, n = 151; ifenprodil treatment, n = 178).