APT1-Mediated Depalmitoylation Regulates Hippocampal Synaptic Plasticity

Abstract

Palmitoylation may be relevant to the processes of learning and memory, and even disorders, such as post-traumatic stress disorder and aging-related cognitive decline. However, underlying mechanisms of palmitoylation in these processes remain unclear. Herein, we used acyl-biotin exchange, coimmunoprecipitation and biotinylation assays, and behavioral and electrophysiological methods, to explore whether palmitoylation is required for hippocampal synaptic transmission and fear memory formation, and involved in functional modification of synaptic proteins, such as postsynapse density-95 (PSD-95) and glutamate receptors, and detected if depalmitoylation by specific enzymes has influence on glutamatergic synaptic plasticity. Our results showed that global palmitoylation level, palmitoylation of PSD-95 and glutamate receptors, postsynapse density localization of PSD-95, surface expression of AMPARs, and synaptic strength of cultured hippocampal neurons were all enhanced by TTX pretreatment, and these can be reversed by inhibition of palmitoylation with palmitoyl acyl transferases inhibitors, 2-bromopalmitate and N-(tert-butyl) hydroxylamine hydrochloride. Importantly, we also found that acyl-protein thioesterase 1 (APT1)-mediated depalmitoylation is involved in palmitoylation of PSD-95 and glutamatergic synaptic transmission. Knockdown of APT1, not protein palmitoyl thioesterase 1, with shRNA, or selective inhibition, significantly increased AMPAR-mediated synaptic strength, palmitoylation levels, and synaptic or surface expression of PSD-95 and AMPARs. Results from hippocampal tissues and fear-conditioned rats showed that palmitoylation is required for synaptic strengthening and fear memory formation. These results suggest that palmitoylation and APT1-mediated depalmitoylation have critical effects on the regulation of glutamatergic synaptic plasticity, and it may serve as a potential target for learning and memory-associated disorders.SIGNIFICANCE STATEMENT Fear-related anxiety disorders, including post-traumatic stress disorder, are prevalent psychiatric conditions, and fear memory is associated with hyperexcitability in the hippocampal CA1 region. Palmitoylation is involved in learning and memory, but mechanisms coupling palmitoylation with fear memory acquisition remain poorly understood. This study demonstrated that palmitoylation is essential for postsynapse density-95 clustering and hippocampal glutamatergic synaptic transmission, and APT1-mediated depalmitoylation plays critical roles in the regulation of synaptic plasticity. Our study revealed that molecular mechanism about downregulation of APT1 leads to enhancement of AMPAR-mediated synaptic transmission, and that palmitoylation cycling is implicated in fear conditioning-induced synaptic strengthening and fear memory formation.

Keywords: APT1; PSD-95; fear conditioning; glutamate receptors; palmitoylation; synaptic plasticity.

Figure 1.

Synaptic scaling increases palmitoylation and surface expression of glutamatergic receptors in vitro. A, Schematic diagram of the experiment. Hippocampal neuron cultures, application of TTX (day 14-16) and 2-BP or NtBuHA (day 16), and differential testing approaches for palmitoylation. B, Western blotting analysis of streptavidin-labeled protein from cultured neurons treated with TTX and 2-BP. TTX increased global palmitoylation, which was prevented by 2-BP. C, Representative (left lane; calibration: 10 pA, 500 ms) and average (right lane) traces of mEPSC recordings from cultured neurons. D, Cumulative probabilities and average mEPSC amplitudes showing that 2-BP abolished the increase of the mEPSC amplitude induced by TTX. E, Cumulative probabilities and average mEPSC frequencies showing that TTX and 2-BP had no effect on frequency of mEPSC recording from hippocampal neuron cultures. Data are mean ± SEM. F, G, Western blots of surface-expressing GluA1-2 and NMDARs from cultured neurons (F), and Western blots of total expression of GluA1-2 and NMDARs from the same samples of the cultured neurons (G), with β-actin as internal reference. H, Quantification results of membrane surface expression (biotin-linked aggregated subunits) of glutamatergic receptors, showing that TTX increased the surface level of GluA1-2, not NMDARs, and 2-BP or NtBuHA abolished the increase. Data are mean ± SEM. One-way ANOVA (D,E); two-way ANOVA (G). n = 12, 12, 13 per group (D,E); n = 4 per group (B,G). *p < 0.05, **p < 0.01 versus Ctrl group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus TTX group. For detailed statistical analyses, see Table 1. Detailed information about antibodies used for Figure 1 and other figures in this study are shown in Extended Data Figure 1-1. Original images of Western blots for Figure 1 are shown in Extended Data Figure 1-2.

Figure 2.

Homeostatic scaling facilitates palmitoylation and function of glutamate receptors. A, B, Western blotting analysis showing that TTX increased PSD-95 and GluA1 palmitoylation, which was reversed by 2-BP or NtBuHA. C-E, Western blot analysis of streptavidin-labeled protein from cultured neurons treated with TTX and 2-BP. TTX significantly increased the palmitoylation of GluA2 (C), GluN2A (D), and GluN2B (E), which was reversed by NtBuHA or 2-BP. F, Cell lysates from cultured neurons were analyzed by coimmunoprecipitation using antibody against stargazin, then subjected to Western blotting analysis, and IgG was used as the isotype control. TTX promoted the association between PSD-95 and stargazin, which was canceled by 2-BP or NtBuHA. Data are mean ± SEM. Two-way ANOVA was used, and n = 4 per group for all (A-F). *p < 0.05, **p < 0.01, ***p < 0.001 versus Ctrl group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus TTX group. For detailed statistical analyses, see Table 1. Original images of Western blots for Figure 2 are shown in Extended Data Figure 2-1.

Figure 3.

ABE method is reliable for palmitoylation detection, and 2-BP has no effect on basal palmitoylation of PSD-95. A, Palmitoylation of PSD-95 in cultured hippocampal neurons. B, C, Palmitoylation of GluA1 and GluA2 in cultured hippocampal neurons. D, E, Palmitoylation of GluN2A and GluN2B in cultured hippocampal neurons. F, G, Western blotting analysis of lysates prepared from cultured neurons treated with vehicle or 2-BP. Data show that treatment with 2-BP dose did not decrease the palmitoylation of PSD-95 on a basal level. Data are mean ± SEM. Student's t test was used, and n = 4 per group. For detailed statistical analyses, see Table 1.

Figure 4.

Synaptic strength is regulated by inhibition of depalmitoylation. A, Representative (left lane; calibration: 10 pA, 500 ms) and average (right lane) traces of mEPSCs recorded from cultured neurons transfected with LV-eGFP, LV-shAPT1, and LV-shPPT1. B, C, Cumulative probabilities and average mEPSC amplitudes and frequencies showing that knockdown of APT1, not PPT1, significantly enhanced the mEPSC amplitude and frequency. D, Representative (left lane; calibration: 10 pA, 500 ms) and average (right lane) traces of mEPSCs recorded from cultured neurons treated with vehicle or ML348. E, F, Cumulative probabilities and average mEPSC amplitudes and frequencies illustrating that ML348-treated neurons had a significant overall increase in mEPSC amplitude and frequency. G, Traces of paired pulse-evoked EPSCs from cultured neurons treated with vehicle or ML348 (left), and statistical analysis result of paired-pulse facilitation ratio (PPR), indicating that ML348 increased the PPR (right). Calibration: 50 pA, 25 ms. H, Traces of AMPAR- and NMDAR-meditated EPSCs from cultured neurons treated with vehicle or ML348 (left), and statistical analysis of AMPAR/NMDAR EPSC amplitude ratio validating ML348 elevated the AMPA/NMDA ratio (right). Calibration: 100 pA, 20 ms. Data are mean ± SEM. One-way ANOVA (B,C), Student's t test (others). n = 71, 37, 51 per group (B,C); n = 39, 31 per group (E,F); n = 6 per group (G,H). *p < 0.05, **p < 0.01, ***p < 0.001 versus LV-eGFP or Veh group. ^#p < 0.05, ^##p < 0.01 for LV-shPPT1 versus LV-shAPT1 group. For detailed statistical analyses, see Table 1.

Figure 5.

2-BP can reverse the effect of APT1 knockdown on mEPSCs. A, Representative (left lane; calibration: 10 pA, 500 ms) and average (right lane) traces of mEPSCs from cultured neurons transfected with LV-eGFP or LV-shAPT1, or treated with 2-BP. B, C, Cumulative probabilities and average mEPSC amplitudes and frequencies showing that knockdown of APT1 significantly enhanced the mEPSC amplitude and frequency, whereas these were reversed by 2-BP. Data are mean ± SEM. B, C, One-way ANOVA was used, and n = 11-15 per group from 5 rats. **p < 0.01, ***p < 0.001 versus LV-eGFP + Veh group. ^##p < 0.01 versus LV-shAPT1 + Veh group. For detailed statistical analyses, see Table 1.

Figure 6.

Silencing APT1 increases palmitoylation of glutamate receptors and PSD-95. A, Western blotting analysis of APT1 from cultured neurons treated with LV-eGFP and LV-shAPT1. Data show that the expression of APT1 was dramatically declined after knockdown with transfection. B-D, Western blotting analysis from cultured neurons transfected with LV-eGFP or LV-shAPT1. Data show that LV-shAPT1 had a promoting effect on the palmitoylation of PSD-95 (B), GluA1 (C), and GluA2 (D). E, F, Western blotting analysis of lysates prepared from cultured neurons treated with LV-eGFP or LV-shAPT1. Knockdown of APT1 upregulated the palmitoylation of GluN2A and GluN2B. G, Western blotting analysis from cultured neurons transfected with LV-eGFP and LV-shAPT1. Data show that treatment with LV-shAPT1 increased the surface level of GluA1-2, but without effect on the surface expression of NMDARs. Data are mean ± SEM. Student's t test was used, and n = 4 per group for all. *p < 0.05, **p < 0.01, ***p < 0.001 versus LV-eGFP group. For detailed statistical analyses, see Table 1. Original images of Western blots for Figure 6 are shown in Extended Data Figure 6-1.

Figure 7.

Downregulation of APT1 activity increases palmitoylation of glutamate receptors and PSD-95. A, B, Western blotting analysis results from cultured neurons treated with vehicle and ML348. Data show that ML348 had a promoting effect on the palmitoylation of PSD-95 (A) and GluA1 (B). C-E, Western blotting analysis of lysates prepared from cultured neurons treated with vehicle or ML348. Data show that treatment with ML348 increased the palmitoylation of GluA2 and GluN2A-B. F, Western blotting analysis from cultured neurons treated with vehicle or ML348. Results showed that ML348 increased the surface level of GluA1-2, but without influence on the surface expression of NMDARs. Data are mean ± SEM. Student's t test was used, and n = 4 per group for all. *p < 0.05, **p < 0.01, ***p < 0.001 versus Veh group. For detailed statistical analyses, see Table 1. Original images of Western blots for Figure 7 are shown in Extended Data Figure 7-1.

Figure 8.

Silencing PPT1 has no effects on the palmitoylation of glutamate receptors and PSD-95. A, Western blotting analysis of PPT1 from cultured neurons treated with LV-eGFP and LV-shPPT1. Data showing the expression of PPT1 were significantly decreased. B-D, Western blotting results of lysates prepared from cultured neurons treated with LV-eGFP or LV-shPPT1. Results indicate that knockdown of PPT1 did not alter the palmitoylation of PSD-95 and GluA1-2. E-G, Western blotting analysis of lysates prepared from cultured neurons treated with LV-eGFP or LV-shPPT1. Knockdown PPT1 did not affect the palmitoylation of GluN2A-B and had no effect on the surface expression of GluA1-2 and NMDARs. Data are mean ± SEM. n = 4 per group for all. ***p < 0.001 versus LV-eGFP group (Student's t test). For detailed statistical analyses, see Table 1. Original images of Western blots for Figure 8 are shown in Extended Data Figure 8-1.

Figure 9.

Palmitoylation is involved in fear conditioning-induced hippocampal synaptic strengthening. A, Timelines of the animal experiments with fear-conditioned rats. B, Western blotting analysis of streptavidin-labeled protein from the hippocampal tissue of the control or fear-conditioned (FC) rats. FC increased global palmitoylation level. C, Representative (left lane; calibration: 10 pA, 500 ms) and average (right lane) traces of mEPSC recordings from rat hippocampal slices at 2 h after fear conditioning training. D, Cumulative probabilities and average mEPSC frequencies showing that FC enhanced the frequency of mEPSC on hippocampal slices, which could be abolished by 2-BP treatment. E, Cumulative probabilities and average mEPSC amplitudes showing that 2-BP abolished the increase of mEPSC amplitude induced by FC. F, Time course of the SC-CA1 LTP induced by HFS in hippocampal slices from different groups, showing LTP recording before and 60 min after the application of HFS. Calibration: 2 mV, 10 ms. G, Histogram illustrating the mean increase in fEPSPs slopes averaged from the last 15 min in each group and showing 2-BP significantly attenuated FC-induced LTP enhancement with no obvious effect on LTP of control group. H, The freezing behavior of rats tested on day 3 was significantly reduced by 2-BP treatment compared with the vehicle, indicating that inhibition of palmitoylation impairs the formation of fear memory. I, Time course of SC-CA1 LTP in hippocampal slices from different groups, showing LTP recording before and 60 min after application of HFS. Calibration: 2 mV, 10 ms. J, Histogram illustrating the mean increase in fEPSPs slopes averaged from last 15 min in each group and showing that ML348 had no significant influence on LTP of both control and fear conditioning groups. K, The freezing behavior of rats tested on day 3 was slightly facilitated by ML348 treatment compared with the vehicle (p = 0.06), indicating that inhibition of APT1 promotes the formation of fear memory. Data are mean ± SEM. Two-way ANOVA (D,E,G,J); one-way ANOVA (H,K). n = 6 per group for (D,E,H,K); n = 4 for (G,J). *p < 0.05, **p < 0.01 versus Ctrl or Veh group. ^##p < 0.01, ^###p < 0.001 versus FC + Veh group. For detailed statistical analyses, see Table 1. Original images of Western blots for Figure 9 are shown in Extended Data Figure 9-1.

Figure 10.

Schematic illustration of palmitoylation cycling in hippocampal synaptic plasticity. During synaptic scaling, the activity of PATs is upregulated, which promotes the palmitoylation modification, translocation, and stabilization of synaptic proteins (PSD-95, glutamate receptors) in postsynaptic density. Knockdown with shRNA or inhibiting the activity of depalmitoylatase APT1 with ML348 can largely simulate the effect of synaptic scaling. Fear conditioning-induced enhancement in synaptic strength was also attenuated by palmitoylation inhibitor 2-BP.