De-palmitoylation by N-(tert-Butyl) hydroxylamine inhibits AMPAR-mediated synaptic transmission via affecting receptor distribution in postsynaptic densities

Abstract

Aims: Palmitoylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) subunits or their "scaffold" proteins produce opposite effects on AMPAR surface delivery. Considering AMPARs have long been identified as suitable drug targets for central nervous system (CNS) disorders, targeting palmitoylation signaling to regulate AMPAR function emerges as a novel therapeutic strategy. However, until now, much less is known about the effect of palmitoylation-deficient state on AMPAR function. Herein, we set out to determine the effect of global de-palmitoylation on AMPAR surface expression and its function, using a special chemical tool, N-(tert-Butyl) hydroxylamine (NtBuHA).

Methods: BS³ protein cross-linking, Western blot, immunoprecipitation, patch clamp, and biotin switch assay.

Results: Bath application of NtBuHA (1.0 mM) reduced global palmitoylated proteins in the hippocampus of mice. Although NtBuHA (1.0 mM) did not affect the expression of ionotropic glutamate receptor subunits, it preferentially decreased the surface expression of AMPARs, not N-methyl-d-aspartate receptors (NMDARs). Notably, NtBuHA (1.0 mM) reduces AMPAR-mediated excitatory postsynaptic currents (mEPSCs) in the hippocampus. This effect may be largely due to the de-palmitoylation of postsynaptic density protein 95 (PSD95) and protein kinase A-anchoring proteins, both of which stabilized AMPAR synaptic delivery. Furthermore, we found that changing PSD95 palmitoylation by NtBuHA altered the association of PSD95 with stargazin, which interacted directly with AMPARs, but not NMDARs.

Conclusion: Our data suggest that the palmitoylation-deficient state initiated by NtBuHA preferentially reduces AMPAR function, which may potentially be used for the treatment of CNS disorders, especially infantile neuronal ceroid lipofuscinosis (Batten disease).

Keywords: AMPA receptor; N-(tert-Butyl) hydroxylamine; palmitoylation; postsynaptic density protein 95; stargazin.

Figure 1

The structural formula of NtBuHA and NtBuHA (1.0 mmol/L) remarkably reduced global protein palmitoylation. A, Representative structure of NtBuHA. B, Global protein palmitoylation is detected in the WB with HRP‐streptavidin followed by acyl‐biotin exchange (ABE) protocol. C, The effect of NtBuHA (0, 0.25, 0.5, and 1.0 mmol/L) on global palmitoylated proteins in the hippocampus of mice in vitro. Incubation of hippocampal slices with NtBuHA (1.0 mmol/L) remarkably reduced global protein palmitoylation

Figure 2

NtBuHA decreases the surface expression of AMPARs. A, B, Representative Western blots and statistical histogram showing NtBuHA (1.0 mmol/L) did not affect the total expression of GluA1(A) and GluA2 (B) (n = 4 mice for each group). C, Representative Western blots and statistical histogram showing NtBuHA (1.0 mmol/L) decreased the surface expression of GluA1 (n = 8 mice for each group). D, Representative Western blots and statistical histogram showing NtBuHA (1.0 mmol/L) decreased the surface expression of GluA2 (D) (n = 6 mice for each group). *P < 0.05 and **P < 0.01 vs control. Data are normalized to the untreated control group and presented as mean ± SEM. The data were evaluated by Student's t test. GluA1(S): surface GluA1; GluA1(I): Intracellular GluA1; GluA2(S): surface GluA2; GluA2(I): Intracellular GluA2. The data were evaluated by a two‐sided unpaired Student's t test

Figure 3

NtBuHA exerts little effect on the total expression and surface level of NMDARs. A‐C, Representative Western blots and a statistical histogram showing NtBuHA (1.0 mmol/L) did not affect the total expression of GluN1(A), GluN2A (B), and GluN2B (C) (n = 4 mice for each group). D‐F, Representative Western blots and a statistical histogram showing NtBuHA (1.0 mmol/L) did not affect the surface expression of GluN1 (D), GluN2A (E), and GluN2B (F) (n = 8 mice for each group). *P < 0.05 vs control. Data are normalized to the control group and presented as mean ± SEM. The data were evaluated by Student's t test. GluN1(S): surface GluN1; GluN1(I): Intracellular GluN1; GluN2A(S): surface GluN2A; GluN2A(I): Intracellular GluN2A; GluN2B(S): surface GluN2B; GluN2B(I): Intracellular GluN2B

Figure 4

NtBuHA decreases the amplitude of AMPAR‐mediated mEPSC in the hippocampal neurons. A, Representative mEPSCs recording in the hippocampal pyramidal neurons. Scale bars, 20 pA and 1 s. B‐E, Cumulative probabilities and average mEPSCs frequencies and amplitudes from the control group and NtBuHA group and statistical histogram showing NtBuHA (1.0 mmol/L) decreased the mEPSCs amplitude of hippocampal pyramidal neurons, not frequency (n = 13‐14 cells from 11 to 12 mice). F, The mean rise time and (G) the mean decay time of mEPSCs were shown. No significant difference was observed in the kinetics of the mEPSC waveform between two groups (n = 12 cells from 11 to 12 mice per group). **P < 0.01 vs control. Data are normalized to the control group and presented as mean ± SEM. The data were evaluated by a two‐sided unpaired Student's t test

Figure 5

NtBuHA decreases the palmitoylation of PSD95 and AKAP79/150 in the hippocampus. A, B, Representative Western blot and a statistical histogram showing NtBuHA (1.0 mmol/L) decreased the level of GluA1 (A) and GluA2 (B) palmitoylation (n = 7 mice for each group). C, D, Representative Western blot and a statistical histogram showing NtBuHA (1.0 mmol/L) decreased the level of GluN2A (C) and GluN2B (D) palmitoylation (n = 6 mice for each group). E, Representative Western blot and a statistical histogram showing NtBuHA (1.0 mmol/L) decreased the level of PSD95 palmitoylation (n = 6 ‐ 8 mice for each group). (F) Representative Western blot and a statistical histogram showing NtBuHA (1.0 mmol/L) decreased the level of AKAP150 palmitoylation (n = 8 mice for each group). *P < 0.05 and **P < 0.01 vs control. Data are normalized to control group and presented as mean ± SEM. The data were evaluated by a two‐sided unpaired Student's t test

Figure 6

NtBuHA preferentially reduces the interactions of PSD95 with AMPARs, not NMDARs. A, NtBuHA (1.0 mmol/L) did not change the expression of AMPARs, stargazin and NMDARs (Input). B, C, Representative Western blot and a statistical histogram showing NtBuHA (1.0 mmol/L) disrupted the interactions between PSD95 and AMPARs and stargazin, but not NMDARs (n = 3‐4 mice for each group). *P < 0.05 vs control. Data are normalized to control group and presented as mean ± SEM. The data were evaluated by a two‐sided unpaired Student's t test