Address correspondence and reprint requests to Kenneth M. Johnson, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031, USA. E-mail: email@example.com
Neurodegeneration induced by the NMDA receptor antagonist, phencyclidine (PCP), has been used to model the pathogenesis of schizophrenia in the developing rat. Acute and sub-chronic administration of PCP in perinatal rats results in different patterns of neurodegeneration. The potential role of an alteration in the membrane expression of NMDA receptors in PCP-induced degeneration is unknown. Acute PCP treatment on postnatal day 7 increased membrane levels of both NMDA receptor subunit 1 (NR1) and NMDA receptor subunit 2B (NR2B) proteins in the frontal cortex; conversely, NR1 and NR2B protein levels in the endoplasmic reticulum fraction were decreased. Acute PCP administration also resulted in increased membrane cortical protein levels of post-synaptic density-95, as well as the activation of calpain, which paralleled the observed increase in membrane expression of NR1 and NR2B. Further, administration of the calpain inhibitor, MDL28170, prevented PCP-induced up-regulation of NR1 and NR2B. On the other hand, sub-chronic PCP treatment on postnatal days 7, 9 and 11 caused an increase in NR1 and NR2A expression, which was accompanied by an increase in both NR1 and NR2A in the endoplasmic reticulum fraction. Sub-chronic PCP administration did not alter levels of post-synaptic density-95 and had no effect on activation of calpain. These data suggest that increased trafficking accounts for up-regulation of cortical NR1/NR2B subunits following acute PCP administration, while increased protein synthesis likely accounts for the increased expression of NR1/NR2A following sub-chronic PCP treatment of the developing rat. These results are discussed in the context of the differential neurodegeneration caused by acute and subchronic PCP administration in the developing rat brain.
Over activation of NMDA receptors (NMDARs) has been shown to cause damage that will eventually kill neurons in a process known as excitotoxicity (Lynch and Guttmann 2002). It is generally agreed that NMDARs produce excitotoxicity through increases in Ca2+ influx leading to intracellular Ca2+overload (Garthwaite and Garthwaite 1986; Lei et al. 1992; Abdel-Hamid and Tymianski 1997). Other studies have shown that NMDAR-mediated neurotoxicity involves increased superoxide formation and the activation of the proteolytic enzyme caspase-3 resulting in increased nuclear factor-κB (NF-κB) nuclear transport, mechanisms that are activated by excessive intracellular Ca2+ levels (Takadera et al. 1999; Qin et al. 2000; McInnis et al. 2002), most likely secondary to increased NMDAR function and support the hypothesis that PCP-induced neurotoxicity may be mediated by increases in the NMDAR density (Wang et al. 2005). Thus, further investigation of PCP-induced regulation of the NMDAR in conjunction with neurotoxicity may shed light on the molecular mechanisms underlying the psychotomimetic properties of PCP, particularly those mediating the schizophrenia-like symptoms following perinatal administration (Wang et al. 2001).
Therefore, the purpose of the present study was to characterize the effect of both acute (PN7) and sub-chronic (PN7, 9, and 11) PCP treatment on the regulation of the NMDAR subunits (NR1, NR2A, and NR2B) as well as PSD-95, a member of the NMDAR post-synaptic density complex, in the frontal cortex in order to determine the possible role of NMDAR trafficking and expression in PCP-induced neurodegeneration and later behavioral deficits.
Materials and methods
Timed, day 14 pregnant female Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington, MA, USA). The dams were housed individually with a regular 12 h light-dark cycle (lights on 0700, off at 1900) with food and water ad libitum. Following parturition, male and female pups from four dams were combined and randomly cross-fostered to one of the four lactating dams. Each litter consisted of ten to twelve pups. All experiments were conducted in accordance with the National Institutes of Health and the Institutional Animal Care and Use Committee.
Phencyclidine was acquired from the National Institute on Drug Abuse (NIDA, Rockville, MD, USA) and dissolved in 0.9% NaCl. Calpain inhibitor III (MDL28170) (10 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 1% dimethyl sulfoxide. Doses were chosen based on prior experiments that addressed PCP-induced regulation of the NMDAR (Wang et al. 2001) and our own work with the compounds. Injections were administered i.p. or s.c. in 1 mL/kg of vehicle.
Male and female rat pups were treated on either PN7 (acute) or on PN 7, 9, and 11 (sub-chronic) with 10 mg/kg subcutaneous (s.c.) PCP or saline. In the acute studies, pups were killed by decapitation on PN7 at 0, 4, 8, or 24 h following saline/vehicle (control, time = 0 h), PCP/vehicle treatment. In the acute MDL28170 studies, pups were treated with PCP on PN7 or post-treatment with MDL28170 (10 mg/kg, i.p., every 2 h for 8 h) and then killed by decapitation 24 h following saline/vehicle, PCP/vehicle, MDL28170/vehicle, MDL28170+PCP/vehicle treatment. In the sub-chronic studies, pups were killed by decapitation 24 h following the last injection of the aforementioned drug regimens on PN7, 9 and 11. For both acute and sub-chronic biochemical studies, the frontal cortex was dissected as described below and used for western blot analysis.
Equal amounts of protein were separated on 10% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) using SDS-PAGE with a MES-SDS running buffer system, pH 7.4. Following electrophoresis (110 V for 2 h), proteins were transferred to polyvinylidene difluoride membranes (0.2 μm) in a Mini Electrotransfer Unit (Bio-Rad, Hercules, CA, USA) overnight. The membrane was blocked in 5% non-fat milk, followed by incubation with the primary antibody in 1% milk for 2 h at 25°C. Following washes (3 × 10 min) in Tris Buffered Saline +0.1% Tween 20 (TBST), the membrane was incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at 25°C. Analysis was carried out using the enhanced chemiluminescence plus western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA). The bands corresponding to the various proteins of interest were scanned and densitometrically analyzed by using an automatic imaging analysis system (Alpha Innotech Corporation, San Leandro, CA, USA). All quantitative analyses were normalized to β-actin [after stripping (Reblot mild; Chemicon International, Temecula, CA, USA)].
The monoclonal anti-NR1, anti-NR2A, anti-NR2B, and anti-protein disulfide isomerase (PDI) were purchased from BD Biosciences (San Jose, CA, USA). The monoclonal anti-PSD-95 and anti-αII spectrin antibodies were purchased from Chemicon International. Primary antibody dilution was 1 : 500–1 : 1000. Secondary antibodies were purchased from Zymed (Invitrogen Corporation, Carlsbad, CA, USA) and used at a concentration of 1 : 5000.
Time-dependent effects of acute PCP administration on membrane bound protein expression of NMDAR subunits in the frontal cortex
It has been reported that neurotoxicity caused by non-competitive NMDAR antagonists is dependent on development with the most vulnerable time existing in the early postnatal period in rats (Ikonomidou et al. 1999; Wang and Johnson 2005, 2007). In order to ascertain the effects of acute PCP (10 mg/kg) treatment on the expression of NMDAR subunits, membrane protein from the frontal cortex was extracted on PN 7 at 0, 4, 8 and 24 h after saline or PCP treatment. Saline treated animals killed immediately after administration (t = 0 h) on PN7 served as the control in the biochemical studies of the time course of acute PCP effects. Figure 1a shows representative western blots of NR1 and NR2B from the frontal cortex of acute PCP or saline treated perinatal rats at all time points examined on PN7. Quantitative analysis revealed that acute PCP treatment produced a 3-fold increase in membrane bound NR1 and NR2B subunits in the frontal cortex at 8 h and this persisted for 24 h (Fig. 1b). The calpain-degraded NR2B breakdown product, molecular weight 140 kDa, was increased 80% following acute PCP treatment (saline 0.485 ± 0.161; PCP 0.868 ± 0.216, n = 6 for both groups, p < 0.05 paired t-test). There was no effect of acute PCP treatment on membrane bound NR2A protein levels in the frontal cortex (Fig. 1b).
To investigate the effects of sub-chronic PCP administration on the concentration of the membrane NR1, NR2A, and NR2B in the frontal cortex, rat pups were treated with either saline or PCP on PN7, 9, and 11 and were killed on PN12 (24 h following the last injection). Cortical membrane bound protein extracts were then subjected to western blot analysis. Figure 2a shows representative western blots of NR1 and NR2A subunits from the frontal cortex. Sub-chronic PCP treatment caused a 3-fold increase in membrane NR1 and a 10-fold increase in NR2A protein levels in the frontal cortex with no effect on NR2B protein expression (Fig. 2b).
Mechanism of PCP-induced regulation of the NMDAR
To determine the possible mechanism by which PCP regulates the expression of cortical NMDAR subunits, protein residing in the ER fraction was isolated from saline and acute PCP or sub-chronic PCP treated animals and subjected to western blot analysis. To verify that the ER fraction was successfully isolated, we probed for PDI (protein disulfide isomerase, an ER housekeeping protein); no changes in ER PDI protein levels were measured following acute or sub-chronic PCP administration and PDI was not detected in the membrane protein fraction (data not shown). Acute PCP treatment produced a decrease in NR1 and NR2B protein levels in the ER fraction of the frontal cortex on PN7 at 24 h following administration (Fig. 3). Conversely, sub-chronic PCP treatment produced an increase in NR1 and NR2A protein levels in the ER fraction measured 24 h following the last of the three injections (Fig. 3). These results indicate that acute PCP administration may cause an increase in trafficking of the NR1 and NR2B subunits from intracellular compartments to the membrane, while sub-chronic PCP treatment induces new synthesis of the NR1 and NR2A subunits. In addition, PSD-95 protein expression levels in the membrane fraction of frontal cortex were also increased following 8 h of PCP treatment and remained elevated 24 h after treatment (Fig. 4b). However, following sub-chronic PCP administration, the β-actin normalized values for cortical membrane PSD-95 protein (0.973 ± 0.214, n = 4) were not significantly different from saline treated animals (0.717 ± 0.0781, n = 4, p =0.306, Fig. 4b). Figure 4a shows representative western blots of cortical PSD-95 protein expression from acute and sub-chronic PCP treated animals.
To further define the mechanism of PCP-induced regulation of the NMDAR, we examined the role of calpain (calcium-dependent neutral cysteine protease). Cleavage of the cytoskeletal proteins αII spectrin by calpain between Tyr1176 and Gly1177 produces two αII spectrin specific breakdown products (SBP) of molecular weights 150 kDa and 145 kDa, which are calpain-specific spectrin breakdown products and antibodies to these fragments are available commercially; therefore, one can indirectly measure calpain activation through western blot analysis of SBPs (Nath et al. 1996; Bi et al. 1997; Wu and Lynch 2006; Zhou and Baudry 2006). Acute PCP treatment resulted in an increase in the αII-spectrin calpain SBP, while sub-chronic PCP treatment had no effect on αII-spectrin calpain SBP expression levels (Fig. 5b). To further clarify the role of calpain in PCP-induced trafficking of the NMDAR subunits, we treated animals following acute PCP administration with the selective calpain inhibitor, MDL28170 (10 mg/kg, every 2 h for 8 h, i.p.). MDL28170 is a potent calpain inhibitor (Ki = 10 nmol/L in vitro) that has been shown to cross the blood brain barrier following systemic administration (Li et al. 1998). Animals were killed 24 h after the PCP injection and western blot analysis of cortical membrane NR1 and NR2B was performed. PCP administration resulted in an up-regulation of both NR1 and NR2B, which was blocked by post-treatment with MDL28170 (Fig. 6). These data lend support for the role of calpain activation in acute PCP-induced changes in the trafficking of NR1/NR2B.
The present study demonstrated that acute PCP treatment on PN7 increased the levels of membrane NR1 and NR2B protein expression in the frontal cortex at 8 h and 24 h following PCP administration. Further, analysis of the cortical ER fraction demonstrated a decrease in NR1 and NR2B protein following acute PCP administration. To understand the mechanism by which the up-regulation of NR1 and NR2B occurs, we first examined PCP-induced changes in the expression of PSD-95. The membrane associated guanylate kinase family of proteins, specifically PSD-95, play key roles in NMDAR trafficking to the membrane (Lu et al. 2000; Dong et al. 2004; Suvarna et al. 2005), clustering at the membrane, and stabilization of the NMDAR complex and organization of the post-synaptic architecture (Yamada et al. 1999; Lu et al. 2000; Mauceri et al. 2007; Wu et al. 2007). Acute PCP administration produced an increase in membrane cortical protein levels of PSD-95, which paralleled the observed increase in membrane NR1 and NR2B expression in the frontal cortex. Therefore, increased trafficking of assembled NMDAR from the ER to the membrane in conjunction with intact PSD-95 following acute PCP administration would account for the apparent up-regulation of the receptor in the frontal cortex.
Wu et al. (2005) suggested that after over-activation of NMDARs, calpain will cleave and degrade the NMDAR, resulting in down-regulation of its function and protection of the neuron from excitotoxic cell death; on the other hand, it is possible that following acute PCP administration the protective effects of calpain are abolished; therefore, the cell death cascade is activated. Based on functional studies using I125-MK-801 binding to NR1/NR2B, it has been suggested that in excitotoxic settings, calpain cleavage of NR2B creates novel, active fragments (140, 130, and 115–120 kDa) which remain on the extracellular surface and function similar to the native receptor (Liao et al. 2001; Simpkins et al. 2003). Taken together, our results support the possibility that cortical neurotoxicity measured on PN7 in perinatal rats is because of a hyper-functional NR1/NR2B complex; however, this interpretation is complicated by recent time course studies from this laboratory. It has been shown that following a single injection of PCP on PN7, caspase-3 is activated by 2 h in the frontal cortex; furthermore, TUNEL staining appears 6 h after treatment, peaks at 16 h and is cleared by 24 h (Wang and Johnson 2005, 2007). This study reports that the time course of up-regulation of NR1 and NR2B in the frontal cortex following acute PCP treatment occurs after neuronal neurodegeneration has been initiated (Wang and Johnson 2005, 2007), suggesting that the up-regulation of NR1 and NR2B must also arise in surviving neurons where up-regulation potentially could play a compensatory role in these neurons. Nonetheless, it is also possible that NR1 and NR2B up-regulation plays a significant role in neurotoxicity following acute administration of PCP.
Sub-chronic PCP treatment of pups resulted in increased expression of NR1 mRNA and polypeptide in the frontal cortex (Wang et al. 2001). In addition to up-regulation of NR1, the current study found that sub-chronic PCP administration resulted in increased expression of membrane NR2A in the frontal cortex, with no change in NR2B levels. Furthermore, analysis of the cortical ER protein fraction revealed increased NR1 and NR2A protein levels following sub-chronic PCP treatment, suggesting that up-regulation may be because of increased synthesis of these subunits. While the mechanism underlying this up-regulation is unknown, it is known that the NR1 gene contains a binding site for the transcription factor NF-κB (Liu et al. 2004). Further, it has been reported that binding to the NR1 NF-κB site up-regulates the NR1 promotor and subsequent transcription of the gene through interactions with Sp1/Sp3 factors. This is relevant in that this laboratory previously reported that sub-chronic PCP treatment increased nuclear translocation of NF-κB (Wang et al. 2001). Therefore, PCP-induced activation of NF-κB may signal increased synthesis of the NR1 subunit, resulting in the observed up-regulation of the protein. These data along with the neurotoxic effects of sub-chronic PCP treatment in vivo, support the possibility that up-regulation of NR1 and NR2A in the frontal cortex may be because of an increase in new protein synthesis.
We suggest that the mechanism of regulation of the NMDAR is treatment-dependent. This is supported by our observation of the lack of effect of sub-chronic PCP administration on the levels of PSD-95 and calpain activation. Disruption of the NR2A-PSD-95 interface by either palmitoylation of PSD-95 or elimination of the PSD-95 binding site on NR2A leaves NR2A susceptible to cleavage by calpain (Dong et al. 2004; Wu and Lynch 2006). Alternatively, it has been suggested that PSD-95 may also be a substrate for calpain and it has been shown that NMDA treatment of organotypic hippocampal cultures produced activation of calpain and the subsequent cleavage of PSD-95 (Lu et al. 2000). Truncation of PSD-95 would then lead to an unstable NR1/NR2A/PSD-95 complex in the membrane and cause the internalization and down-regulation of the assembled NMDAR (Dong et al. 2004).
It is reasonable then to predict that the up-regulation of NR1/NR2B following acute PCP administration is because of activation of calpain-mediated trafficking, while up-regulation of NR1/NR2A following sub-chronic PCP administration is because of an increase in protein synthesis as well as a stable NR1/NR2A/PSD-95 complex and the subsequent forward trafficking of this complex into the synapse. These conclusions are consistent with recent studies suggesting that calpain mediated regulation of the NMDAR is dependent on the NR2 subunit present (2A resistant, 2B sensitive) as well as the developmental age and brain region examined (Dong et al. 2006). Furthermore, it has recently been reported that NMDA-induced protein synthesis is dependent on the presence of the NR2A, but not the NR2B subunit (Tran et al. 2007). In summary, this study provides evidence that two distinct mechanisms are likely involved in the differential regulation of NMDAR subunits following perinatal acute and sub-chronic PCP administration. These data are consistent with the hypothesis that changes in NMDAR density may play a role in PCP-induced neurotoxicity in the frontal cortex in the developing rat.
This work was supported by NIH grants F31 DA-022824 to NCA, RO1s DA-02073 & MH-63871 to KMJ and a NIDA pre-doctoral training grant T32 DA-07287. The authors would like to thank Yan Xia for her thoughtful discussions and comments. The authors would also like to thank Benjamin B. Whiddon for his contribution to this study as well as the Summer Undergraduate Research Program at UTMB for his support.