The major brain cholesterol metabolite 24(S)-hydroxycholesterol is a potent allosteric modulator of N-methyl-D-aspartate receptors

Abstract

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are critical to the regulation of excitatory synaptic function in the CNS. NMDARs govern experience-dependent synaptic plasticity and have been implicated in the pathophysiology of various neuropsychiatric disorders including the cognitive deficits of schizophrenia and certain forms of autism. Certain neurosteroids modulate NMDARs experimentally but their low potency, poor selectivity, and very low brain concentrations make them poor candidates as endogenous ligands or therapeutic agents. Here we show that the major brain-derived cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC) is a very potent, direct, and selective positive allosteric modulator of NMDARs with a mechanism that does not overlap that of other allosteric modulators. At submicromolar concentrations 24(S)-HC potentiates NMDAR-mediated EPSCs in rat hippocampal neurons but fails to affect AMPAR or GABAA receptors (GABA(A)Rs)-mediated responses. Cholesterol itself and other naturally occurring oxysterols present in brain do not modulate NMDARs at concentrations ≤10 μM. In hippocampal slices, 24(S)-HC enhances the ability of subthreshold stimuli to induce long-term potentiation (LTP). 24(S)-HC also reverses hippocampal LTP deficits induced by the NMDAR channel blocker ketamine. Finally, we show that synthetic drug-like derivatives of 24(S)-HC, which potently enhance NMDAR-mediated EPSCs and LTP, restore behavioral and cognitive deficits in rodents treated with NMDAR channel blockers. Thus, 24(S)-HC may function as an endogenous modulator of NMDARs acting at a novel oxysterol modulatory site that also represents a target for therapeutic drug development.

Figures

Figure 1.

24(S)-HC and SGE-201 are potent oxysterol positive allosteric modulators of NMDA receptors. A, Natta projection structures for 24(S)-HC, SGE-201, and SGE-301. Note that the key hydroxyl group (denoted in red) in all three structures is on the same carbon relative to the cholesterol backbone. The similarity in the location of the hydroxyl group is further emphasized in the three dimensional stick and ball models below (D-ring and C-17 side chain shown). The 3-α methyl group that distinguishes SGE-201 and SGE-301 is circled in red. B, Effects of endogenous oxysterols and other cholesterol metabolites on NMDA receptor currents. Cultured primary hippocampal neurons were preincubated with 10 μm test compound in 0.5 μm glycine for 90 s, followed by 10 s NMDA (10 μm). The percentage change in NMDA current is plotted. C, Representative traces from B. The red lines represent application of test article (note that the red lines do not encompass the full 90 s of preincubation). The black lines represent the application of NMDA (10 μm, 10 s). Scale bar: vertical = 200 pA, horizontal = 10 s. D, Active oxysterols compared with pregnenolone metabolites. All compounds were tested at 10 μm, 90 s preincubation, with the exception of 24(S)-HC (10 μm, 360 s preincubation), and pregnenolone sulfate (10 and 50 μm, 90 s preincubation). Cholesterol was solubilized in ethanol rather than DMSO. Asterisk represents a significant difference from current induced by NMDA alone (p < 0.05).

Figure 2.

24(S)-HC and SGE-201 are effective at submicromolar concentrations. A, Potentiation of 10 μm NMDA (0.5 μm glycine) by increasing 24(S)-HC concentrations with 40 s oxysterol preapplication in a DIV5 rat hippocampal neuron. B, Potentiation values for 24(S)-HC and for SGE-201 were fit with the Hill equation (solid lines). EC50 estimates were 0.11 μm for SGE-201 (N = 7 cells), and 1.2 μm for 24(S)-HC (N = 5 cells).

Figure 3.

Slow oxysterol reversibility. A, B, NMDA (10 μm) currents were potentiated with 24(S)-HC (2 μm) or with SGE-201 (0.2 μm, red traces) with repeated 20 s preapplication of potentiator between successive NMDA challenges. Following 60 s total oxysterol exposure (three red traces), cells were challenged with saline wash for 80 s (four black traces). There was little reversibility for either drug. Cells were then challenged with γ-cyclodextrin (CDX; 500 μm) wash, before NMDA application in the absence of γ-cyclodextrin (blue traces). A1, B1, Traces show results from representative neurons. A2, B2, Summary plots for five and six cells, respectively. γ-Cyclodextrin extracted SGE-201 potentiation but not 24(S) potentiation. C, D, Oxysterols were premixed with γ-CDX in solution at subsaturating oxysterol concentrations (0.5 μm for each compound mixed with 1 mm γ-cyclodextrin). γ-Cyclodextrin effectively reduced the free concentration of SGE-201, indicated by reduced potentiation, but failed to affect 24(S)-HC potentiation.

Figure 4.

Occlusion studies suggest a unique mechanism for oxysterols versus other lipophilic positive modulators. A, B, Examples of occlusion protocol. Cells were preincubated for >5 min in 10 μm 24(S)-HC then challenged with 10 μm NMDA plus 50 μm PREGS (A) as a representative nonoccluding potentiator or 0.2 μm SGE-201 (B) as a representative occluding potentiator. C, Summary of results from 6 to 10 cells per bar for the four indicated modulators. Arachidonic acid was used at 5 μm. SGE-301 was used at 0.5 μm. Asterisks indicate a significant reduction in potentiation (p < 0.05).

Figure 5.

Potentiation in excised outside-out membrane patches. A, Baseline NMDAR channel activity (black trace) in 300 μm NMDA in an excised outside-out patch from a DIV2 hippocampal neuron was augmented following SGE-201 incubation for 60 s (0.2 μm, red trace). The patch was excised before drug applications. B, The all-points histograms represent 30 s of NMDA-induced channel activity before (black) and following (red) SGE-201 application from the patch represented in A. C, Summary of NPo analysis from eight excised outside-out patches.

Figure 6.

Potentiation of recombinant receptors suggests little or no subunit selectivity. A, HEK-293 cells stably expressing human GluN1–3 and transiently expressing human GluN2A were activated with NMDA (30 μm) and glycine (5.0 μm) (small gray bars). After determining the baseline response to NMDA and glycine, a test compound (as indicated) was added at 0.1 μm (short white bars) or 1 μm (tall white bars). B, The mean (± SEM) percentage potentiation (by 1 μm test compound) above NMDA and glycine alone is plotted. Asterisks denote a significant difference from baseline (p < 0.05). C, Sample traces from HEK cells transiently transfected with GluN1a plus each of the indicated GluN2 subunits. Potentiation of 10 μm NMDA currents (0.5 μm glycine) by 0.2 μm SGE-201 is shown (gray traces of each pair). D, Each subunit combination exhibited significant potentiation by SGE-201 above baseline (asterisks), but no significant difference in potentiation values among subunits was detected.

Figure 7.

Selective potentiation of NMDAR EPSCs. A, Potentiation by 1 μm 24(S)-HC of evoked NMDAR EPSCs isolated with 1 μm NBQX and 10 μm gabazine. Slow onset, slow reversibility, and relative γ-CDX insensitivity paralleled effects on responses to exogenous NMDA. B, Pharmacologically isolated AMPAR EPSCs (10 μm gabazine and 25 μm D-APV) were unaffected. C, SGE-201 (0.2 μm; 90 s) also potentiated peak NMDAR EPSCs with little effect on decay time course of the EPSCs (inset). D, Summary of effects of 1 μm 24(S)-HC on NMDAR EPSCs and AMPAR EPSCs (N = 8 and 7, respectively). AMPARs were statistically unaltered. A summary of 0.2 μm SGE-201 effects is also shown (N = 7 and 6). E, GABAAR IPSCs were statistically unaltered by prolonged 24(S)-HC application (N = 11 for1 μm 24(S)-HC and N = 6 for 1 μm SGE-201).

Figure 8.

Augmentation of LTP by 24(S)-HC. A, P120 hippocampal slices were challenged with a brief HFS that produces STP, but not LTP (open symbols). However, in the presence of SGE-201 the same stimulus effectively induced LTP (closed symbols). Responses in control slices were 91.9 ± 5.6% of baseline EPSP slope at 50% of maximum on the I/O curve, 60 min following HFS. With SGE-201 present, EPSP slopes were increased to 156 ± 10.8% of baseline (p < 0.001, N = 5 slices each). B, Similar experiment with 24(S)-HC. Black symbols represent effect of 1 μm 24(S)-HC (166.1 ± 19.4% of baseline; p < 0.001), whereas 0.1 μm 24(S)-HC did not significantly enhance LTP induction (103.9 ± 9.8% of baseline, p = n.s., N = 5 slices each). Insets for A and B show representative traces. Dashed lines are baseline and solid lines represent traces in the indicated oxysterol 60 min following HFS. Calibration bars: 1 mV, 5 ms.

Figure 9.

SGE-201 and SGE-301 reverse synaptic plasticity deficits following NMDA receptor blockade. A, B, Reversal of ketamine suppression of long-term potentiation (LTP) by 24(S)-HC (A) and SGE-201 (B) in P30 hippocampal slices. Open symbols are baseline response following ketamine administration (1 μm, 30 min preincubation) to 100 Hz × 1 s HFS (vertical arrow). The change in baseline EPSP slope was 93.1 ± 2.3% 60 min following HFS in ketamine-treated control slices. Solid symbols represent the same condition except 0.5 μm 24(S)-HC (A; 131.3 ± 6.7% of baseline, p < 0.001, N = 5) or SGE-201 (B; 129.1 ± 9.2% of baseline, p < 0.001, N = 5) was present; N = 5 each, p = 0.008. Calibration bars: 1 mV, 5 ms.

Figure 10.

SGE-201 and SGE-301 reverse behavioral deficits following NMDA receptor blockade. A, Pharmacokinetic profiles of SGE-201(10 mg/kg, N = 3) and SGE-301(20 mg/kg, N = 2) following acute intraperitoneal administration in the mouse and rat respectively. Inset, Brain concentrations measured at 60 min following compound administration. Note that SGE-301 treatment resulted in a disproportionately higher brain concentration than SGE-201. B, SGE-201 reverses MK-801-nduced deficits in spontaneous alternations in the Y-maze in mice (N = 17–19/group). Percentage alternation was significantly reduced by MK-801 compared with vehicle (#p < 0.0001). SGE-201 restores alternation after MK-801, with increased percentage alternation in the 3 and 10 mg/kg SGE-201 groups compared with MK-801 alone (***p < 0.0005 and *p < 0.05). C, SGE-301 rescues social interaction deficits in PCP-experienced rats (N = 12–15/group). Vehicle or PCP (5 mg/kg, bid, i.p.) was administered twice daily from days 1 to 7. On day 14, SGE-301 was administered 60 min before testing. Time spent in active, nonaggressive social behavior was assessed during a 10 min session. PCP-experienced rats had significantly reduced interaction time compared with vehicle controls (#p < 0.0001). SGE-301 (3 and 10 mg/kg, i.p.) restored social interaction in PCP-experienced rats with significantly increased interaction time (****p < 0.0001 and *p < 0.05) versus PCP + vehicle group. D, SGE-301 rescues novel object recognition in PCP-experienced rats (12–15/group). On day 21, 7 d after social interaction testing, rats were administered SGE-301 intraperitoneally 60 min before object training. After the 30 min retention interval, object recognition was assessed in a 3 min test session. Discrimination ratio (time spent exploring the novel object/time spent exploring both objects during the test session) was calculated, so that a ratio of 0.5 corresponds to equal object preference (chance performance). Vehicle-treated PCP-experienced rats did not exhibit novel object preference and had a significantly reduced discrimination ratio (#p < 0.0001) compared with vehicle-treated rats. SGE-301 (1 and 3 mg/kg) significantly increased the discrimination ratio in PCP-experienced (*p < 0.05 and **p < 0.01vs vehicle + PCP), demonstrating rescue of the object recognition deficit.