A Clickable Oxysterol Photolabel Retains NMDA Receptor Activity and Accumulates in Neurons

Abstract

Oxysterol analogs that modulate NMDA receptor function are candidates for therapeutic development to treat neuropsychiatric disorders. However, the cellular actions of these compounds are still unclear. For instance, how these compounds are compartmentalized or trafficked in neurons is unknown. In this study, we utilized a chemical biology approach combining photolabeling and click chemistry. We introduce a biologically active oxysterol analog that contains: (1) a diazirine group, allowing for the permanent labeling of cellular targets, and (2) an alkyne group, allowing for subsequent in situ visualization using Cu²⁺ catalyzed cycloaddition of an azide-conjugated fluorophore. The physiological properties of this analog at NMDA receptors resemble those of other oxysterols, including occlusion with other oxysterol-like compounds. Fluorescent imaging reveals that the analog accumulates diffusely in the cytoplasm of neurons through an energy-independent mechanism. Overall, this work introduces a novel chemical biology approach to investigate oxysterol actions and introduces a tool useful for further cell biological and biochemical studies of oxysterols.

Keywords: NMDA receptor; click chemistry; modulation; oxysterol; photolabel.

FIGURE 1

MQ-182 is a potent positive allosteric modulator of NMDARs. (A) Natta projection structure of MQ-182. (B) Effect of MQ-182 on NMDAR currents. Cultured primary hippocampal neurons were incubated in 10 μM MQ-182 and 20 μM D-serine in the nominal absence of Mg²⁺ for 30 s, followed by 5 s NMDA application. The horizontal black bar represents the application of NMDA (10 μM, 5 s) and the red bar represents the application of MQ-182 (10 μM, 45 s). Pre-application was for 30 s before co-application with NMDA. (C) The normalized change in NMDAR current magnitude is plotted. The red symbol denotes the mean potentiation. Asterisk represents a significant increase in NMDAR current magnitude (p = 0.00020, n = 15, one-sample t-test).

FIGURE 2

MQ-182 is effective at sub-micromolar concentrations. (A) Increasing potentiation of NMDAR currents (10 μM NMDA, 20 μM D-serine) by increasing concentrations of MQ-182. Concentrations used were 0.1 nM (red), 1 nM (blue), 0.1 μM (green), 1 μM (purple), pre-applied before agonist application. Dotted line represents magnitude of baseline NMDA current. (B) Potentiation values were fit with the Hill equation (solid line). EC₅₀ of MQ-182 was estimated at 1.2 nM (n = 14). Symbols are color coded as in (A).

FIGURE 3

MQ-182 accelerates rate of memantine block reversal, suggesting that it increases the open-channel probability of the NMDAR. (A) Cells were challenged with 10 μM NMDA (horizontal blue bar, 40 s) and 10 μM memantine (MEM, horizontal red bar, 15 s). (B) Cells were pre-incubated in 10 μM MQ-182 for >5 min and challenged with the same protocol as described in (A). (C) The current relaxation upon memantine removal (red arrows in A) was fitted with a monoexponential curve. Time constant values (τ) from these fits are plotted. Asterisk represents a decrease in tau (p = 0.0017, n = 19 for -MQ-182, n = 18 for +MQ-182, Student’s unpaired t-test).

FIGURE 4

MQ-182 exhibits characteristics of oxysterol potentiation rather than sulfated steroid potentiation. (A–D) Occlusion studies. (A,B) Representative traces of occlusion protocol. Cells were preincubated in MQ-182 for >5 min and then challenged with 10 μM NMDA (5 s, horizontal black bar) and 0.2 μM SGE-201 or 50 μM PS (45 s pre-application). Each pair of black and red traces comes from a different cell. (C,D) Summary data of (A,B). Although SGE-201’s potentiation of NMDARs was decreased by pre-incubation in MQ-182, PS’s potentiation was not (p = 0.78, n = 17 for -MQ-182, n = 15 for +MQ-182). Asterisk represents p = 0.0013, n = 8 (potentiated response vs. baseline), Student’s unpaired t-test. (E–G) Tests of subunit selectivity. (E,F) N2A cells expressing human GluN1a and one of four GluN2 subunits (A–D) were incubated in 10 μM MQ-182 and 20 μM D-serine in the nominal absence of Mg²⁺ for 30 s, followed by 5 s NMDA application. Representative traces are shown for two cells in (E,F). (G) Subunits containing each of the four GluN2 subunits (GluN2A-D) showed potentiation by MQ-182. Dotted line represents magnitude of baseline NMDA current. Asterisks represent a significant increase in NMDAR current magnitude (asterisks: GluN2A, p = 0.00020, n = 8; GluN2B, p = 0.00070, n = 12; GluN2C, p = 0.0032, n = 12; GluN2D, p = 0.0053, n = 8; one-sample paired t-test).

FIGURE 5

The actions of MQ-182 exhibit weak reversibility. (A) Cells were challenged with 10 μM NMDA to establish baseline NMDAR currents. Cells were then challenged with repeated 30 s pre-applications of 0.5 μM MQ-182, a sub-saturating concentration (red traces). Following 60 s total MQ-182 exposure, cells were challenged with 30 s saline wash (black trace). Cells were then challenged with γ-CDX wash (30 s, 500 μM) before application of NMDA in the absence of γ-CDX. (B) Summary plot. The Friedman test, a modified one-way repeated measures ANOVA for non-parametric data, revealed no interaction between the treatment condition (MQ-182, saline wash, γ-CDX wash) and magnitude of NMDAR current (p = 0.37, n = 5). Post hoc Dunn’s multiple comparisons tests revealed no differences between the MQ-182 and saline wash conditions (p = 0.42, n = 5), or between the MQ-182 and γ-CDX wash conditions (p = 0.23, n = 5). (C) Test of CDX/MQ-182 interaction. Cells were first challenged with 10 μM NMDA (black trace), and baseline NMDAR currents were established. Next, they were challenged with a solution of 0.5 μM MQ-182, pre-mixed with 500 μM γ-CDX (red trace). Finally, they were challenged with 0.5 μM MQ-182 without γ-CDX (light blue trace). (D) Comparison of potentiation by MQ-182 alone and MQ-182 pre-mixed with γ-CDX. Dotted line represents magnitude of baseline NMDA current. A Wilcoxon matched-pairs signed rank test showed a difference between conditions. Asterisk represents an increase in potentiation from the MQ-182 + γ-CDX condition to the MQ-182 alone condition (p = 0.0078, n = 8). Dashed line represents baseline NMDA current.

FIGURE 6

Diffuse cytoplasmic labeling of a hippocampal neuron by MQ-182. (A) A hippocampal culture was incubated in 10 μM MQ-182, exposed to 365 nM UV irradiation for 15 min, and processed for click cyto-fluorescence using azide-conjugated AlexaFluor 488. Panels are labeled with the experimental conditions. Scale bar: 15 μm. (B) Comparison of MQ-182 +UV, MQ-182 -UV, and no drug +UV conditions. A two-way ANOVA with experimental condition and day of experiment (represented as separate colored symbols) as the two independent variables revealed a significant difference in fluorescence between experimental conditions [p < 1.0 × 10^-15, n = 35, F_(2,75) = 523.3]. There was also a significant difference between experiment [p = 6.2 × 10^-12, n = 35, F_(6,75) = 16.34] and an interaction between experimental condition and experiment [p < 1.0 × 10^-15, n = 35, F_(12,75) = 16.44], suggesting differences in the degree of overall fluorescence in different experimental runs. Post hoc Bonferroni corrected Student’s unpaired t-tests revealed significant differences between the MQ-182 +UV and MQ-182 -UV conditions [p = 1.1 × 10^-7, n = 35, bottom asterisk) and between the MQ-182 +UV and the no drug +UV conditions (p < 1.0 × 10^-15, n = 35, top asterisk). (C) Comparison of peri-membrane and intracellular fluorescence in the MQ-182 +UV condition. Membrane (L) refers to the left side of the cell membrane as viewed in the z-projection, while Membrane (R) refers to the right side. A two-way ANOVA suggested differences between intracellular and peri-membrane fluorescence [p = 0.0048, n = 35, F_{(2, 56)} = 5.88]. Post hoc Bonferroni corrected Student’s unpaired t-tests revealed differences between the intracellular and left membrane locations (p = 0.019, n = 35, left asterisk) and between the intracellular and right membrane locations (p = 0.0046, n = 35, right asterisk), suggesting somewhat stronger intracellular fluorescence. (D,E) Combined click cyto-fluorescence with immunofluorescence for PDI, an endoplasmic reticulum marker, and giantin, a Golgi-specific protein, show little co-labeling. Scale bar: 2.14 μm. Line scans are shown in (F,G).

FIGURE 7

MQ-182 accumulation in fixed neurons. (A) Neurons were incubated with 10 μM MQ-182 and exposed to the UV irradiation protocol while either alive or fixed in 4% paraformaldehyde. Live cells were then fixed in paraformaldehyde, and all cells were processed for click cyto-fluorescence as previously described. Panels depict live and fixed cells in the MQ-182 +UV condition. Scale bar: 5 μm. (B) Summary of labeling intensity. A linear mixed model revealed no difference in fluorescence between the live and fixed conditions when adjusting for day of experiment and presence of drug/UV [p = 0.1192, n = 20, F_(1,110) = 2.47]. Each independent experiment is denoted with a different color symbol.

FIGURE 8

MQ-182 does not potentiate responses to exogenous GABA. (A) Hippocampal cultures were incubated in 10 μM MQ-182, 25 μM D-APV and 1 μM NBQX for 30 s, followed by 20 s application of 5 μM GABA (black). Baseline GABA_AR currents were established. Cells were then challenged with 30 s pre-incubation of MQ-182, followed by 20 s application of GABA co-applied with MQ-182 (green). Finally, they were challenged with 20 s application of GABA co-applied with 10 μM PS (blue). Representative traces are shown, with peak and steady state areas of the trace circled with colors used for ensuing summaries. (B) Summary of effect of drugs on peak and steady-state GABA current. A two-way repeated measures ANOVA indicated a main effect of drug for PS (p = 0.009) but not for MQ-182 (p = 0.70). For PS there was also a significant interaction between drug and time (p = 0.009), indicating a more prominent effect on steady-state current, as expected from the mechanism of PS modulation of NMDARs (Eisenman et al., 2003).

FIGURE 9

Effect of MQ-182 on sPSCs and mPSCs. (A,B) Effect on AMPAR ESPCs (A) and GABA_AR IPSCs (B). (A) In 10 μM gabazine and 25 μM D-APV to isolate AMPAR EPSCs, baseline sEPSCs were followed by 30 s pre-application of 10 μM MQ-182 and further recording. Because many EPSCs occurred in burst-like clusters in which individual events could not be accurately discerned, currents were integrated to yield total negative-going charge. The total charge of AMPAR EPSCs did not increase. The right plot shows effect of MQ-182 normalized to baseline for individual cells. The red symbol denotes the mean change. ( (B) Hippocampal cultures were exposed to 1 μM NBQX and 25 μM D-APV to isolate GABA_AR IPSCs and the MQ-182 application protocol was repeated. The total charge of GABA_AR IPSCs was increased. Asterisk represents a significant increase in total charge (p = 0.024, n = 8, one-sample t-test). (C,D) Selective potentiation of GABA_AR mIPSCs. AMPAR mEPSCs (C) were isolated with 10 μM gabazine, 25 μM D-APV, and 0.2 μM TTX. Neither the amplitude nor the frequency of the mEPSCs were potentiated by MQ-182. GABA_AR mIPSCs (D) were isolated with 1 μM NBQX, 25 μM D-APV, and 0.2 μM TTX. Both the amplitude and the frequency of GABA_AR mIPSCs were potentiated by 10 μM MQ-182. Left asterisk represents a significant increase in amplitude (p = 0.0011, n = 9, one-sample t-test). Right asterisk represents a significant increase in frequency (p = 0.0034, n = 9, one-sample t-test). (E) Integrated total charge of GABA_AR IPSCs was not potentiated by 1 μM MQ-182. GABA_AR IPSCs were isolated as in (B). Recording in the presence of 1 μM MQ-182 followed a 30 s application. Application of 1 μM MQ-182 did not increase the total negative-going charge of GABA_AR IPSCs (p = 0.49, n = 8, one-sample t-test).)

FIGURE 10

MQ-182 potentiates NMDAR sEPSCS. (A) In 10 μM gabazine and 1 μM NBQX to isolate NMDARs, baseline sEPSCs were followed by 30 s pre-application of 1 μM MQ-182 and further recording. Because the majority of NMDAR sEPSCs did not occur in burst-like clusters, like GABA_AR sIPSCs, individual EPSCs could be detected with confidence. (B) Representative trace of an average waveform of individual EPSCs for an exemplar cell. (C) EPSC decays were fitted with a bi-exponential function. Tau values weighted by the relative amplitudes of the components are plotted. The red symbols denote the mean decay time for each condition. MQ-182 increased the decay time of NMDAR ESPCs. Asterisk represents potentiation (p = 0.018, n = 6, Student’s paired t-test). Amplitude of events was not increased (-1.96 ± 5.92% increase, p = 0.71, n = 6, Student’s paired t-test). Frequency of events was also not increased (-2.65 ± 7.32% increase, p = 0.49, n = 6, Student’s paired t-test).