Histone Modification of Nedd4 Ubiquitin Ligase Controls the Loss of AMPA Receptors and Cognitive Impairment Induced by Repeated Stress

Abstract

Stress and the major stress hormone corticosterone induce profound influences in the brain. Altered histone modification and transcriptional dysfunction have been implicated in stress-related mental disorders. We previously found that repeated stress caused an impairment of prefrontal cortex (PFC)-mediated cognitive functions by increasing the ubiquitination and degradation of AMPA-type glutamate receptors via a mechanism depending on the E3 ubiquitin ligase Nedd4. Here, we demonstrated that in PFC of repeatedly stressed rats, active glucocorticoid receptor had the increased binding to the glucocorticoid response element of histone deacetylase 2 (HDAC2) promoter, resulting in the upregulation of HDAC2. Inhibition or knock-down of HDAC2 blocked the stress-induced impairment of synaptic transmission, AMPAR expression, and recognition memory. Furthermore, we found that, in stressed animals, the HDAC2-dependent downregulation of histone methyltransferase Ehmt2 (G9a) led to the loss of repressive histone methylation at the Nedd4-1 promoter and the transcriptional activation of Nedd4. These results have provided an epigenetic mechanism and a potential treatment strategy for the detrimental effects of chronic stress.

Significance statement: Prolonged stress exposure can induce altered histone modification and transcriptional dysfunction, which may underlie the profound influence of stress in regulating brain functions. We report an important finding about the epigenetic mechanism controlling the detrimental effects of repeated stress on synaptic transmission and cognitive function. First, it has revealed the stress-induced alteration of key epigenetic regulators HDAC2 and Ehmt2, which determines the synaptic and behavioral effects of repeated stress. Second, it has uncovered the stress-induced histone modification of the target gene Nedd4, an E3 ligase that is critically involved in the ubiquitination and degradation of AMPA receptors and cognition. Third, it has provided the epigenetic approach, HDAC2 inhibition or knock-down, to rescue synaptic and cognitive functions in stressed animals.

Keywords: AMPA receptors; E3 ubiquitin ligase; corticosterone; histone deacetylase; histone methyltransferase; histone modification.

Figure 1.

HDAC2 is selectively upregulated by GR activation in PFC of stressed animals. A, Quantitative real-time RT-PCR data on the mRNA level of HDAC family members in PFC from control (con) groups versus rats exposed to 7 d restraint stress (RS). *p < 0.05, t test. B, Immunoblots and quantification analysis of the protein level of HDAC1 and HDAC2 in the nuclear fraction of PFC pyramidal neurons from control (con) versus rats exposed to repeated stress (RS) or acute stress (AS). *p < 0.05, t test. C, Immunoblots and quantification analysis of the protein level of active GR (^S211pGR) in the nuclear fraction of PFC neurons from control (con) versus repeatedly stressed (RS) rats. *p < 0.05, t test. D, ChIP assay data on the binding of pGR to the HDAC2 promoter GRE region in PFC from control versus stressed rats. *p < 0.05, t test. Top, Diagram showing the location of GRE and primers (F, forward; R, reverse). E, Quantitative real-time RT-PCR data on the HDAC2 mRNA level in PFC from control versus repeatedly stressed rats with intraperitoneal injection of the GR antagonist RU486 or vehicle. *p < 0.05, ANOVA.

Figure 2.

HDAC inhibitors block the reducing effect of repeated stress or CORT on AMPAR-EPSC. A, Input/output curves of AMPAR-EPSC evoked by a series of stimulus intensities in PFC pyramidal neurons from control (con) versus repeatedly stressed (RS) rats with the intraperitoneal injection of TSA (0.5 mg/kg, a pan-HDAC inhibitor) or vehicle. **p < 0.01, ANOVA. B, Dot plots showing the amplitude of AMPAR-EPSC evoked by the same stimulus in PFC pyramidal neurons from control versus stressed animals with intraperitoneal injections of TSA, SB (0.4 g/kg, a pan-HDAC inhibitor except for HDAC6), MGCD0103 (15 μg/kg, a selective inhibitor for HDAC1 and HDAC2), or MS-275 (15 μg/kg, a selective inhibitor for HDAC1). Inset, Representative AMPAR-EPSC traces. Scale bars, 50 pA, 20 ms. C, Immunoblots and quantification analysis of the level of acetylated H3 and total H3 in cortical slices either from rats injected intraperitoneally with various agents (vehicle, 15 μg/kg MGCD0103, or 15 μg/kg or 5 mg/kg MS-275) or treated with vehicle versus MS-275 (10 μm, 4 h in vitro). **p < 0.01, *p < 0.05, t test. D, Bar graphs showing the mEPSC amplitude and frequency in cultured cortical neurons treated with CORT (100 nm, 7 d) in the absence or presence of TSA (1 μm), SB (500 μm), tubastatin A (10 μm, an inhibitor for HDAC6), MGCD0103 (500 nm), or MS-275 (600 nm). **p < 0.01, *p < 0.05, ANOVA. Inset, Representative mEPSC traces. Scale bar, 20 pA, 5 s.

Figure 3.

HDAC2 knock-down blocks the effect of prolonged CORT or repeated stress on AMPAR-mediated synaptic responses. A, Western blots showing the expression of HDAC2, HDAC1, and HDAC6 in HEK293 cells transfected with HDAC2 shRNA or a GFP control shRNA. Actin was used as a loading control. B, Immunocytochemical staining and quantification analysis of HDAC2 in cultured PFC neurons infected with HDAC2 shRNA lentivirus or a GFP lentivirus. ***p < 0.001, t test. C, Bar graphs and representative mEPSC traces showing the effect of CORT treatment (100 nm, 7 d) on mEPSC amplitude and frequency in cortical cultures transfected with GFP, HDAC2 shRNA, or HDAC6 shRNA. Scale bar, 20 pA, 5 s. **p < 0.01, ANOVA. D, Image showing the spread of HDAC2 shRNA lentivirus stereotaxically injected to the prelimbic region of PFC. E, Immunoblots and quantification analysis of HDAC2 and HDAC6 expression in rat PFC infected with HDAC2 shRNA lentivirus or GFP lentivirus. ***p < 0.001, t test. F, G, Summarized input/output curves of AMPAR-EPSC in control (con) versus repeatedly stressed (RS) rats with the PFC injection of GFP lentivirus, HDAC2 shRNA lentivirus (F), or HDAC6 shRNA lentivirus (G). Inset, Representative AMPAR-EPSC traces. Scale bars, 50 pA, 20 ms. **p < 0.01, *p < 0.05, ANOVA. H, Summarized input/output curves of NMDAR-EPSC in control (con) versus repeatedly stressed (RS) rats with the PFC injection of GFP or HDAC2 shRNA lentivirus. Inset, Representative NMDAR-EPSC traces. Scale bars, 100 pA, 200 ms. **p < 0.01, ANOVA.

Figure 4.

HDAC2 inhibitors block the reducing effect of repeated stress on the total and surface levels of AMPAR subunits in PFC. A, B, Immunoblots (A) and quantification analysis (B) of the total and surface AMPAR subunits in PFC from control (con) versus repeatedly stressed (RS) rats with the intraperitoneal injection of vehicle, TSA, MGCD0103, or MS-275. **p < 0.01, *p < 0.05, ANOVA.

Figure 5.

HDAC2 inhibition or knock-down blocks the effect of repeated stress on TOR memory. A, Bar graphs showing the DR of TOR tasks in control (con) versus repeatedly stressed (RS) rats with the intraperitoneal injection of vehicle, MGCD0103 (15 μg/kg), or MS-275 (15 μg/kg). *p < 0.05, ANOVA. B, Bar graphs showing the exploration time for novel versus familiar objects in the test trial of TOR tasks in control (con) versus repeatedly stressed animals with the intraperitoneal injection of vehicle, MGCD0103 (15 μg/kg), or MS-275 (15 μg/kg). ***p < 0.001, **p < 0.01, *p < 0.05, t test. C, Bar graphs showing the DR of TOR tasks in control versus repeatedly stressed animals injected with GFP or HDAC2 shRNA lentivirus. **p < 0.01, *p < 0.05, ANOVA. D, Bar graphs showing the exploration time for novel versus familiar objects in the test trial of TOR tasks in control (con) versus repeatedly stressed animals with PFC injection of GFP or HDAC2 shRNA lentivirus. *p < 0.05, t test.

Figure 6.

HDAC2-dependent downregulation of histone methyltransferase Ehmt2 (G9a) mediates the loss of repressive histone methylation at the Nedd4-1 promoter in stressed animals. A, B, Immunoblots and quantification analysis of the protein level of Nedd4-1 in PFC punches from control versus stressed rats with the intraperitoneal injection of MGCD0103 versus vehicle control (A) or the local injection of GFP versus HDAC2 shRNA lentivirus (B). **p < 0.01, *p < 0.05, ANOVA. C, Quantitative real-time RT-PCR data on the mRNA level of Nedd4-1 in PFC from control versus repeatedly stressed rats. **p < 0.01, t test. D, Representative blots and quantification showing the ubiquitination of GluR1 subunits in control (con) versus repeatedly stressed (RS) rats with the intraperitoneal injection of vehicle, MGCD0103, or MS-275. Lysates of PFC slices were immunoprecipitated with anti-GluR1 and then blotted with an ubiquitin antibody. **p < 0.01, ANOVA. E, ChIP assay data showing the H3K9Me2 level at rat Nedd4-1 promoter regions in PFC lysates from control versus repeatedly stressed (RS) rats. *p < 0.05, t test. Top, Diagram showing the location of primers. F, Quantitative real-time RT-PCR data on the mRNA level of Ehmt1 and Ehmt2 in PFC from control versus repeatedly stressed rats without or with the intraperitoneal injection of MGCD0103. *p < 0.05, ANOVA. G, Quantitative real-time RT-PCR data on the mRNA level of Ehmt2 in PFC from control versus repeatedly stressed rats with the local injection of GFP or HDAC2 shRNA lentivirus. **p < 0.01, ANOVA. H, ChIP assay data showing the acetylated histone H3 level at rat Ehmt2 promoter regions in PFC lysates from control versus repeatedly stressed (RS) rats. *p < 0.05, t test. Top, Diagram showing the location of primers.

Figure 7.

A working model showing the epigenetic mechanism of repeated stress. In stressed animals, activated GR binds to GRE of HDAC2 promoter, resulting in HDAC2 upregulation. Consequently, Ehmt2 is suppressed, leading to the loss of repressive histone methylation at the Nedd4 promoter and increased transcription of Nedd4. Nedd4 induces GluR1 ubiquitination and degradation, causing the loss of glutamatergic transmission and PFC-mediated cognitive function. Inhibiting HDAC2 blocks the signaling cascade and detrimental effects of repeated stress.