Uncoupling DAPK1 from NMDA receptor GluN2B subunit exerts rapid antidepressant-like effects

Abstract

Several preclinical studies have reported the rapid antidepressant effects of N-methyl-D-aspartate receptor (NMDAR) antagonists, although the underlying mechanisms are still unclear. Death-associated protein kinase 1 (DAPK1) couples GluN2B subunits at extrasynaptic sites to regulate NMDAR channel conductance. In the present study, we found that chronic unpredictable stress (CUS) induced extracellular glutamate accumulation, accompanied by an increase in the DAPK1-NMDAR interaction, the high expression of DAPK1 and phosphorylated GluN2B at Ser1303, a decrease in phosphorylated DAPK1 at Ser308 and synaptic protein deficits in the rat medial prefrontal cortex (mPFC). CUS also enhanced GluN2B-mediated NMDA currents and extrasynaptic responses that were induced by bursts of high-frequency stimulation, which may be associated with the loss of astrocytes and low expression of glutamate transporter-1 (GLT-1). The blockade of GLT-1 in the mPFC was sufficient to induce depressive-like behavior and cause similar molecular changes. Selective GluN2B antagonist, DAPK1 knockdown by adeno-associated virus-mediated short-hairpin RNA or a pharmacological inhibitor, and the uncoupling of DAPK1 from the NMDAR GluN2B subunit produced rapid antidepressant-like effects and reversed CUS-induced alterations in the mPFC. The inhibition of DAPK1 and its interaction with GluN2B subunit in the mPFC also rescued CUS-induced depressive-like behavior 7 days after treatment. A selective GluN2B antagonist did not have rewarding effects in the conditioned place preference paradigm. Altogether, our findings suggest that the DAPK1 interaction with the NMDAR GluN2B subunit acts as a critical component in the pathophysiology of depression and is a potential target for new antidepressant treatments.

Figure 1

Chronic unpredictable stress increased extracellular glutamate levels and activated extrasynaptic GluN2B-containing NMDARs in the mPFC. (a) Timeline of CUS exposure, behavioral testing and microdialysis (n=4–5 per group). (b) Photograph of microdialysis site in the mPFC. (c) Extracellular glutamate in CUS-exposed rats was significantly higher than that in control rats. The levels of glutamate were measured every 30 min for 3 h after probe equilibration in the mPFC. (d) Timeline of CUS exposure and sample collection (n=8 per group). (e, f) Representative western blots and quantification of fold changes in GLT-1 and GFAP expression in the mPFC relative to control. (g) Representative image of GFAP-labeled astrocytes in the mPFC, counterstained with DAPI. Scale bar=50 μm. (h) Results are expressed as mean±s.e.m. of GFAP-positive cells per mm². n=4 per group. (i) Representative EPSC traces. (j) Plot of the normalized and averaged amplitudes of NMDAR EPSCs that were evoked at 40 Hz in the mPFC at a holding potential of +50 mV in the presence and absence of ifenprodil (n=6 neurons per group). (k) Average decay time constant of NMDAR EPSCs that were evoked by 12 pulses at 40 Hz stimulation in the presence and absence of ifenprodil. The data are expressed as mean±s.e.m., *P<0.05, **P<0.01, compared with control group; ^#P<0.05, compared with CUS+vehicle group. CUS, chronic unpredictable stress; DAPI, 4′,6-diamidino-2-phenylindole; EPSC, excitatory postsynaptic current; GFAP, glial fibrillary acidic protein; GLT-1, glutamate transporter 1; mPFC, medial prefrontal cortex; NMDAR, N-methyl-D-aspartate receptor.

Figure 2

Chronic unpredictable stress increased the expression levels of GluN2B and DAPK1 and their interaction in the mPFC. (a) Timeline of CUS exposure and sample collection (n=8–9 per group). (b) Representative western blots and quantification of fold changes in GluN1, GluN2A, p-GluN2A, GluN2B, p-GluN2B, DAPK1, p-DAPK1, CREB, p-CREB and BDNF in the mPFC relative to control. (c) Co-immunoprecipitation of GluN2B with DAPK1 in the mPFC in control and CUS rats. The quantification analysis revealed a greater interaction between GluN2B and DAPK1 in the mPFC after CUS exposure (n=6 per group). (d) Timeline of surgery, drug microinjection, behavioral testing and sample collection (n=8–9 per group). (e) Microinjection of DHK in the mPFC rapidly decreased sucrose preference but did not affect total fluid consumption. (f) Representative western blots and quantification of fold changes in GluN1, GluN2A, p-GluN2A, GluN2B, p-GluN2B, DAPK1, CREB, p-CREB and BDNF in the mPFC after DHK infusion relative to vehicle. The data are expressed as mean±s.e.m. *P<0.05, **P<0.01, compared with control or vehicle group. BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP response element-binding protein; CUS, chronic unpredictable stress; DAPK1, death-associated protein kinase 1; DHK, dihydrokainate; mPFC, medial prefrontal cortex.

Figure 3

GluN2B-specific antagonist produced rapid antidepressant-like effects and reversed CUS-induced synaptic protein deficits. (a) Timeline of CUS exposure, ifenprodil administration and behavioral testing (n=8–9 per group). (b, c) Acute ifenprodil administration dose-dependently increased sucrose preference in the SPT (b) and reduced immobility time in the FST (c) in CUS-exposed rats. (d) Timeline of CUS exposure, ifenprodil injection and decapitation (n=8–10 per group). (e) Representative western blots and quantification of fold changes in GluN1, GluN2A, p-GluN2A, GluN2B, p-GluN2B, DAPK1, CREB, p-CREB and BDNF in the mPFC 30 min, 1 h and 6 h after ifenprodil administration. (f) Timeline of CUS exposure, ifenprodil injection and decapitation (n=8–9 per group). (g) Representative western blots and quantification of fold changes in GluA1, synapsin I and PSD95 in synaptoneurosomes in the mPFC 6 h after ifenprodil administration. The data are expressed as mean±s.e.m. *P<0.05, **P<0.01, compared with control or control+vehicle group; ^#P<0.05, ^##P<0.01, compared with CUS+vehicle group; n.s., nonsignificant difference. BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP response element-binding protein; CUS, chronic unpredictable stress; DAPK1, death-associated protein kinase 1; FST, forced swim test; mPFC, medial prefrontal cortex; PSD95, postsynaptic density 95; SPT, sucrose preference test.

Figure 4

Knockdown of DAPK1 prevented CUS-induced depressive-like behavior and normalized GluN2B signaling in the mPFC. (a) Representative photographs of the injection sites and coronal brain sections in the mPFC. The figure shows representative micrographs of adeno-associated virus (AAV)-mediated enhanced green fluorescent protein (eGFP; green) after mPFC microinjection. Scale bars=100 μm (low-magnification images) and 50 μm (high-magnification images). (b) DAPK1 expression in the mPFC in rats that were microinfused with AAV-Scramble or AAV-shDAPK1, quantified by western blot. **P<0.01, compared with the AAV-Scramble group. n=6 per group. (c) Timeline of AAV microinjection, CUS exposure and behavioral testing (n=8–18 per group). (d–f) Intra-mPFC microinjection of AAV-shDAPK1 prevented the decrease in sucrose preference (d) and did not affect total fluid consumption (e) in the SPT, and prevented the increase in immobility time in the FST (f) induced by CUS. (g) Representative western blots and quantification of fold changes in GluN1, DAPK1, GluN2B, p-GluN2B, CREB, p-CREB and BDNF in the mPFC after AAV-Scramble or AAV-shDAPK1 microinjection (n=8 per group). The data are expressed as mean±s.e.m. *P<0.05, **P<0.01, compared with control+AAV-Scramble group; ^##P<0.01, compared with CUS+AAV-Scramble group. BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP response element-binding protein; CUS, chronic unpredictable stress; DAPK1, death-associated protein kinase 1; FST, forced swim test; mPFC, medial prefrontal cortex; SPT, sucrose preference test.

Figure 5

Inhibition of GluN2B–DAPK1 interaction produced rapid and sustained antidepressant-like effects. (a) Timeline of surgery, CUS exposure, drug microinjection and behavioral testing (n=10–15 per group). (b, c) Tat-GluN2B_CT rapidly increased sucrose preference in the SPT (b) and decreased immobility time in the FST (c) in CUS rats. (d) Representative GluN2B immunoprecipitation after Tat-GluN2B_CT and Tat-sGluN2B_CT treatment. Administration of Tat-GluN2B_CT led to the dissociation of GluN2B–DAPK1 complexes (n=8 per group). (e) Timeline of surgery, CUS exposure, drug microinjection and behavioral testing 7 days later (n=10–13 per group). (f, g) Tat-GluN2B_CT reversed the decrease in sucrose preference in the SPT (f) and the increase in immobility time in the FST (g) induced by CUS 7 days after administration. **P<0.01, compared with control+Tat-sGluN2B_CT group; ^##P<0.01, compared with CUS+Tat-sGluN2B_CT group. (h) Schematic diagram that illustrates the involvement of GluN2B-containing NMDARs and associated signaling molecules in the mPFC in depressive-like behavior. Chronic unpredictable stress exposure or the blockade of glutamate transporter-1 (GLT-1) impaired glutamate uptake into astrocytes, thus causing extracellular glutamate to accumulate and overflow onto extrasynaptic GluN2B-containing NMDARs. DAPK1 was activated when extracellular glutamate accumulated and phosphorylated GluN2B at Ser1303, leading to calcium influx. The activation of GluN2B by DAPK1 decreased p-CREB and BNDF levels and subsequently induced synaptic protein deficits and depressive-like behavior. Selective GluN2B antagonism, DAPK1 inhibition or uncoupling DAPK1 from the NMDAR GluN2B subunit rapidly rescued depressive-like behavior, followed by the normalization of GluN2B activation and increases in p-CREB and BDNF levels. GluN2B antagonism reversed the synaptic protein deficits that were caused by CUS exposure. BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP response element-binding protein; CUS, chronic unpredictable stress; DAPK1, death-associated protein kinase 1; FST, forced swim test; mPFC, medial prefrontal cortex; NMDAR, N-methyl-D-aspartate receptor; SPT, sucrose preference test.