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Published in final edited form as: J Neurochem. 2009 Oct 24;112(1):238–245. doi: 10.1111/j.1471-4159.2009.06448.x

ACTIVATION OF PERK KINASE IN NEURAL CELLS BY PROTEASOME INHIBITOR TREATMENT

Le Zhang 1, Philip J Ebenezer 1, Kalavathi Dasuri 1, Annadora J Bruce-Keller 1, Sun Ok Fernandez-Kim 1, Ying Liu 1, Jeffrey N Keller 1,2
PMCID: PMC2882876  NIHMSID: NIHMS155625  PMID: 19860852

Abstract

Inhibition of the proteasome proteolytic pathway occurs as the result of normal aging, as well as in a variety of neurodegenerative conditions, and is believed to promote cellular toxicity in each of these conditions through diverse mechanisms. In the present study we examined whether proteasome inhibition alters the protein kinase (PKR)-like ER kinase (PERK). Our studies demonstrate that proteasome inhibitors induce the transient activation of PERK in both primary rat neurons as well as the N2a neural cell line. Experiments with siRNA to PERK demonstrated that the modulation of PERK was not significant involved in regulating toxicity, ubiquitinated protein levels, or ribosome perturbations in response to proteasome inhibitor treatment. Surprisingly, PERK was observed to be involved in the upregulation of p38 kinase following proteasome inhibitor treatment. Taken together, these data demonstrate the ability of proteasome inhibition to activate PERK and demonstrate evidence for novel cross talk between PERK and the activation of p38 kinase in neural cells following proteasome inhibition. Taken together, these data have implications for understanding the basis by which proteasome inhibition alters neural homeostasis, and the basis by which cell signaling cascades are regulated by proteasome inhibition.

Keywords: aging, neurotoxicity, p38 kinase, proteolysis, ubiquitin

INTRODUCTION

The proteasome is a large intracellular protease that is responsible for a large percentage of overall intracellular proteolysis, including the degradation of the majority of ubiquitinated proteins (Shringarpure et al. 2002; Goldberg 2003). Numerous lines of evidence suggests that the proteasome proteolytic pathway is inhibited during the aging of numerous tissues including the brain (Chondrogianni et al. 2003; Sullivan et al. 2004; Rideout et al. 2001, 2003; Hyun et al. 2003; Li et al. 2008), as well as in a variety of neurodegenerative disorders (Chondrogianni et al. 2005; Keller et al. 2002). The fact that proteasome inhibition is sufficient to induce neuron death and promote a variety of pathogenic processes in neural cells, suggests that proteasome inhibition may serve a role in initiating and promoting neuronal toxicity in both aging and age-related diseases of the brain (Ding and Keller 2003; Ding et al. 2006a, 2007; Keller et al. 2000, 2002; Olanow and McNaught 2006; Halliwell 2002; McNaught 2004; Seo et al. 2004; Sullivan et al. 2004). Additionally, studies have demonstrated an essential role for the proteasome in learning and memory (Chaine et al. 1999; Segref and Hoppe 2009), highlighting the potential role of proteasome inhibition in modulating learning and memory.

Following proteasome inhibition in there is known to be the induction of gross disturbances in protein homeostasis including ribosomal disturbances, and decreases in protein synthesis (Ding et al. 2006a, 2006b; Lam et al., 2007; Zhang et al. 2009;). Proteasome inhibition results in the induction of endoplasmic reticulum (ER) stress, as part of the unfolded protein response (UPR) (Paschen 2004; Kim et al. 2006; Wang and Takahashi 2007). Following the induction of ER-stress, and activation of the UPR, a variety of signal transduction pathways can become selectively activated to promote cell survival as well as cell death. One kinase important to both ER-stress and the UPR, in some experimental settings, is the PKR-like endoplasmic reticulum kinase (PERK) (Kim et al. 2006; Schröder and Kaufman 2006; Raven and Koromilas 2008). Currently, the role of proteasome inhibition as a potential modulator of PERK in neurons has not been elucidated, nor have studies examined the potential contribution of PERK to the downstream effects of proteasome inhibition in neurons.

In the present study we identified that PERK activation occurs in both neural N2a cells as well as primary rat cortical neurons following treatment with proteasome inhibitors. Administration of PERK siRNA resulted in a dramatic downregulation of PERK activation, but did not alter the toxicity of proteasome inhibitors on neural cells. PERK siRNA was not observed to alter the increase in ubiquitinated proteins or ribosomal disturbances observed following proteasome inhibition. PERK siRNA was observed to significantly impair the increase in activation of the stress kinase p38 following proteasome inhibitor treatment. These studies identify for the first time the activation of PERK in neural cells following proteasome inhibition, and identify a novel role for PERK in the activation of p38 following proteasome inhibition.

MATERIALS AND METHODS

Materials

All cell culture supplies were obtained from GIBCO Life Sciences (Gaithersburg, MD, USA). Proteasome inhibitor MG132 and MG115 were obtained from Calbiochem (San Diego, CA). Velcade was purchase from ChemieTek (Indianapolis, IN). Antibodies against PERK, phosphorylated PERK, p38, and phosphorylated p38 were purchased from Cell Signaling Technology (Danvers, MA). The antibody against ubiquitin and β-actin were purchased from Santa Cruz Biotech, Inc. (Santa Cruz, CA). The secondary peroxidase-conjugated goat anti-rabbit IgG (H+L) and peroxidase-conjugated goat anti-mouse IgG (H+L) were purchased from Jackson ImmunoResearch Lab, Inc. (West Grove, PA). Protease inhibitor was purchase from Roche Diagnostics (Indianapolis, IN), phosphatase inhibitor cocktail 1 and phosphatase inhibitor cocktail 2 were purchased from Sigma (St. Louis, MO). All Western-blot supplies and gels were purchased from BIO-RAD (Hercules, CA). Polysome analyzing system was purchased from Brandel (Gaithersburg, MD). All other chemicals were purchased from Sigma (St. Louis, MO).

Cell cultures

N2a cells were cultured in 5% CO2 at 37°C in minimum essential medium (MEM) containing 5% fetal bovine serum, 1% of antibiotic antimycotic and 1 mM pyruvate. All cells were plated in fresh medium one day prior to experimentation and were used at ~70% confluency. Neuronal cultures were established as described previously by our laboratory (Keller et al. 1999a, b). Primary rat cortical neuronal cells were cultured from E18 Sprague-Dawley rats and maintained in 5% CO2 at 37°C in MEM/Neurobasal medium containing 5% fetal bovine serum (heat inactivated), N2 supplement, B27 supplement, and 1% antibiotic. Cells were used in experiments between days 5-8 post plating. All animals were utilized in accordance with IACUC approved protocols at the Pennington Biomedical Research Center.

Analysis of cell survival

Cell survival was determined using MTT reduction as a measure of cell viability as reported previously (Keller et al. 1998).

Western blotting

N2a cells and neurons were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 20% glycerol, 140 mM NaCl, 0.5% Nonidet P-40, 5 mM MgCl2, 0.2 mM EDTA) with protease inhibitors and phsophatase inhibitors. Proteins were separated by electrophoresis and transferred to nitrocellulose membrane. Blots were probed with antibodies and visualized with peroxidase-linked secondary antibodies by using Pierce ECL Western blotting substrate (Pierce, Rockford, IL).

siRNA

The perk siRNA was purchase from Sigma (ID: SASI_Mm01_00138620), the negative control siRNA was purchase from Santa Cruz Biotech. The X-tremeGENE siRNA Transfection Reagent was purchased from Roche Diagnostics. Transfection was performed according to the Roche’s instructions with some minor modifications. Briefly, 8 μg of siRNA and 40 μL transfection reagent were diluted into MEM medium at a final volume of 200 μL and mixed within 5 minutes. The resulting transfection reagent-siRNA mixture was allowed to further complex for 15 - 20 minutes at room temperature and then added to the N2a cells growing in a 75 cm2 cell culture containing 8 ml of N2a culture medium. Following 24 hours of transfection, the same transfection as describe above was repeated using the same N2a cells in order to increase the overall transfection efficiency. Twenty four hours following the second transfection the N2a cells were trypsinized and plated for use in the different experiments outlined in the current study. All siRNA treated N2a cells were collected within 72 hours of trypsinization.

Analysis of ribosomal homeostasis

Ribosome and polyribosome fractions were purified and quantified as described in previous studies from our laboratories and by others (Zhang et al. 2009; Ding et al. 2006b; Zhou et al. 2008). Briefly, N2a cells were cultured in Dulbecco’s modified Eagle’s medium, as highlighted above, in the presence of 10 μM MG132, or to no MG132, for 6 hours. At that time 10 μg/ml cycloheximide was added to the medium to stabilize ribosome and polyribosome complexes. Cells were washed in cold phosphate-buffered-saline solution (supplemented with 10 μg/ml cycloheximide), and then lysed with ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 mM NaCl, 0.4% Nonidet P-40, and 10 μg/ml cycloheximide. The extracts were passed through a 23-gauge needle for proper lysis of cells, incubated for 10 min on ice, and insoluble material was collected by microcentrifugation at 10,000 rpm for 10 min at 4 °C. The resulting supernatant was then applied onto a 15-50% sucrose gradient containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 mM NaCl, 10 μg/ml cycloheximide, and centrifuged for 2 h at 40,000 rpm in a Beckman SW-41Ti rotor. Following centrifugation, the gradients were fractionated by Brandel fractionation system, and the absorbance of RNA at calculated at 254 nm wavelength using an in-line UV monitor.

RESULTS

Proteasome inhibition increases PERK activation in neural cells

In order to begin to elucidate the potential for proteasome inhibition serving as an activator of PERK, we conducted studies in which neural N2a cells were treated with multiple proteasome inhibitors, followed by analysis of phosphorylated PERK levels. The inhibitors chosen included reversible inhibitors MG132 (Ding et al. 2006b) and MG115 (Vinitsky et al. 1992), as well as an irreversible inhibitor of the proteasome, velcade (Adams et al. 2004). All proteasome inhibitors were observed to result in an increase in phosphorylated PERK (Figure 1A), consistent with proteasome inhibition activating PERK in neural cells. We next conducted studies in which we examined whether the activation of PERK could be reversed following removal of proteasome inhibitor. Following a 4 or 8 hour treatment with the reversible proteasome inhibitor MG132 both N2a cells and primary rat cortical neurons were observed to have increased levels of PERK activation (Figure 1B), with washout of the inhibitor reversing the observed increase in PERK activation. Taken together, these data demonstrate that proteasome inhibition increases PERK activation in N2a and primary rat neuron cultures, with activation of PERK following proteasome inhibitor administration apparently requiring the presence of continual proteasome inhibition.

Figure 1. Proteasome inhibition increases PERK phosphorylation.

Figure 1

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Neural N2a cells were treated with multiple proteasome inhibitors (A) and analyzed for the levels of phosphorylated PERK (PERK-P). Increased levels of PERK-P, but not PERK or beta-actin, were observed following proteasome inhibitor treatment. Increased levels of phosphorylated PERK (PERK-P) were observed in neural N2a cells and primary rat cortical neurons following 4 or 8 hour treatment with the reversible proteasome inhibitor MG132 (B), with an overnight washout of the inhibitor decreasing the levels of PERK-P. Data representative of results from 3 separate experiments.

siRNA to PERK decreases the levels of phosphorylated and non-phosphorylated PERK

Next we conducted studies in which neural N2a cells were pre-treated with siRNA to PERK (see Materials and Methods) and then analyzed cells for phosphorylated PERK, total PERK levels, or beta-actin following addition of the reversible proteasome inhibitor MG132. In these studies we observed that cells treated with siRNA exhibited a 85-90% reduction in total PERK, as well as a greater than 75% reduction in phosphorylated PERK following incubation with the proteasome inhibitor MG132 (Figure 2). Interestingly, the ability of siRNA to suppress PERK expression was ameliorated following overnight washout of the siRNA (Figure 2). The siRNA treatment did not significantly induce neuron death within the first 24 hours of treatment (data not shown).

Figure 2. siRNA to PERK decreases the levels of phosphorylated and non-phosphorylated PERK.

Figure 2

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Neural N2a cells were treated with siRNA to PERK overnight and then analyzed for phosphorylated PERK (PERK-P), PERK or beta-actin following addition of the reversible proteasome inhibitor MG132. Data are representative of results from 3 separate experiments.

siRNA to PERK has no effect on neuron death following proteasome inhibitor treatment

Studies then examined the effect of PERK siRNA towards the toxicity of proteasome inhibition. In these studies we observed that PERK siRNA had no significant effect on the neural death observed following incubation with increasing concentrations of the proteasome inhibitor MG132 (Figure 3). These data suggest that PERK activation does not contribute to the acute toxicity of proteasome inhibition in neural cells.

Figure 3. siRNA to PERK has no effect on neuron death following proteasome inhibitor treatment.

Figure 3

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Neural N2a cells were treated with siRNA to PERK overnight and then analyzed for cell survival 24 hours following treatment with increasing concentrations of the reversible proteasome inhibitor MG132. Data representative of results from 3 separate experiments.

siRNA to PERK has no effect on ubiquitinated protein levels following proteasome inhibitor treatment

Neural N2a cells were next treated with siRNA to PERK and then analyzed for ubiquitinated protein and beta-actin levels following treatment with proteasome inhibitor MG132. In this analysis, we observed that MG132 treatment induced an expected increase in the levels of ubiquitinated protein, which was not modulated by the presence of PERK siRNA (Figure 4A). Additional studies examined the effects of PERK siRNA on increased levels of ubiquitinated protein following treatment with MG132, MG115, or velcade (Figure 4B). Perk siRNA was observed to not alter the levels of ubiquitinated protein following treatment with each of these proteasome inhibitors. These data indicate that elevations in ubiquitinated proteins following proteasome inhibition are not grossly altered by PERK activity.

Figure 4. siRNA to PERK has no effect on ubiquitinated protein levels following proteasome inhibitor treatment.

Figure 4

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(A) Neural N2a cells were treated with siRNA to PERK overnight and then analyzed for ubiquitinated protein and beta-actin levels following treatment with proteasome inhibitor MG132. (B) Neural N2a cells were treated with siRNA to PERK overnight and then analyzed for ubiquitinated protein, oxidized protein and beta-actin levels following treatment with proteasome inhibitors MG132, MG115, and velcade. Data representative of results from 3 separate experiments.

siRNA to PERK has no effect on ribosomal disturbances following proteasome inhibitor treatment

Previous studies from our laboratory and others have demonstrated that proteasome inhibition results in impaired protein synthesis and profound ribosomal disturbances (Ding et al. 2006b, 2007; Lam et al., 2007; Zhang et al. 2009). In the next set of experimentation we demonstrated that N2a cells exhibited a significant increase in 80S ribosome complexes, and corresponding decrease in polyribosome levels (Figure 5). These data are consistent with proteasome inhibition potently inhibiting polyribosome stability, and thereby serving as a robust modulator of protein synthesis, where polyribosomes are responsible for the active translation of proteins and polypeptides. Washout of the proteasome inhibitor was observed to promote a restoration in polyribosome levels (Figure 5). Treatment with PERK siRNA had no significant effect on the ribosomal and polyribosomal disturbances observed following proteasome inhibitor treatment (Figure 5).

Figure 5. siRNA to PERK has no effect on ribosomal disturbances following proteasome inhibitor treatment.

Figure 5

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(A) Neural N2a cells were treated for 18 hours with the reversible proteasome inhibitor MG132 (5 μM) and analyzed for levels of ribsosomal complexes (40S,60S,80S) and polyribosomal complexes. Decreased levels of polyribosomes (and expected increase in free 80S ribosomes) were observed following proteasome inhibitor treatment which was largely reversed by washout of the inhibitor. (B) Identical experiments with the inclusion of siRNA gave nearly identical results suggesting PERK activation does not play a role in ribosomal disturbances induced by proteasome inhibition. Data representative of results from 3 separate experiments.

siRNA to PERK decreases activation of p38 kinase following proteasome inhibitor treatment

Lastly our studies examined the relationship between PERK activation and the activation of a vital stress kinase in neural cells following proteasome inhibitor treatment. In these studies there was observed to be a dramatic and reversible increase in the phosphorylation of the stress kinase p38 following proteasome inhibitor treatment (Figure 6), consistent with proteasome inhibition promoting a transient elevation in p38 activity. Interestingly, PERK siRNA was observed to significantly reduce the levels of p38 activation following proteasome inhibitor treatment (Figure 6). Inhibition of p38 activation by siRNA to PERK occurred in response to all 3 proteasome inhibitors (Figure 6). These data not only demonstrate a novel role for PERK in p38 activation following proteasome inhibition, but are one of the first studies to demonstrate a role for PERK in modulating p38 activity in any cell type or experimental condition.

Figure 6. siRNA to PERK decreases activation of p38 kinase following proteasome inhibitor treatment.

Figure 6

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(A) Neural N2a cells were treated with siRNA to PERK overnight and then analyzed for phosphorylated p38 (p38-P) or native p38 levels following treatment with proteasome inhibitor MG132. (B) Neural N2a cells were treated with siRNA to PERK overnight and then analyzed for phosphorylated p38 (p38-P) or native p38 levels following treatment with proteasome inhibitors MG132, MG115, and velcade. (C) Representative graph of the ratio of activated p38 (phosphorylated p38) to the levels of p38. Data expressed in relation to the levels of phosphorylated p38 after 4 hr of MG132 treatment. Data representative of results from 3 separate experiments.

DISCUSSION

The present study demonstrates for the first time that PERK activation occurs in neural cells following proteasome inhibitor treatment. The fact that multiple types of proteasome inhibitors were able to promote PERK activation, and the fact that PERK activation occurred in both N2a and primary rat neurons following proteasome inhibitor treatment, suggest that there the results are not an artifact of a specific proteasome inhibitor or cell type. It is interesting that the activation of PERK is reversible, and suggests that the increased activity of PERK following proteasome inhibition may require continual inhibition of the proteasome. While it is unclear how proteasome inhibition promotes activation of PERK, it is highly likely that it is the result of the ability of proteasome inhibition to promote the UPR, which is a well established pathway for PERK activation in many cell types (Kim et al. 2006; Schröder and Kaufman 2006; Raven and Koromilas 2008). Consistent with this speculation previous studies have reported on the ability of proteasome inhibition to induce ER stress and UPR in a variety of cell types (Szokalska et al. 2009; Pyrko et al. 2007; Obeng et al. 2006; Nawrocki et al. 2005; Kitiphongspattana et al. 2005; Fribley et al. 2004). In future studies it will be important to elucidate the ability of proteasome inhibition to activate other UPR-induced signaling cascades (Raven and Koromilas 2008; Hamanaka et al. 2005).

The majority of work done to date on the role of proteasome inhibition as a mediator of neuropathogenesis has focused on the role of proteasome inhibition as a mediator of increased levels of protein aggregation (Keller et al. 2000, 2002; Dietrich et al., 2003; McNaught 2004; Seo et al. 2004; Chondrogianni et al. 2005). A growing body of work indicates that additional disturbances in protein homeostasis may contribute to the pathogenesis of proteasome inhibition. For example, ribosomal disturbances observed in the present study and previous studies (Ding et al., 2006a, 2006b; Zhang et al. 2009), suggest that proteasome inhibition causes robust and rapid declines in global protein synthesis which may contribute to the deleterious effects of proteasome inhibition. The current study and previous works indicate a role for ER stress and UPR as additional mechanisms by which proteasome inhibition promote neuropathogenesis. Clearly, the effects of proteasome inhibition on the proteome appear to extend to a much larger landscape of effects than just the promotion of protein aggregation.

PERK is known to play a major role in the impairment of protein synthesis following the induction of the UPR (Harding et al., 2000a, 2000b, 2001), as well as mediate a variety of cellular processes (Kim et al. 2006; Schröder and Kaufman 2006; Raven and Koromilas 2008). Because of these established functions we were surprised to see that inhibition of PERK was not observed to significantly modulate cell toxicity, ubiquitinated protein levels, or ribosomal disturbances observed following proteasome inhibition. However, one possibility to explain these findings is that while we were able to reduce the levels of PERK (>75%) we were unable to completely inhibit its expression. These findings therefore raise the possibility that some residual activity of PERK is sufficient to allow for physiologically significant levels of PERK activity. Additionally, the relatively acute nature of the experimentation in the present study, and relatively severe levels of proteasome inhibition in the present study, may have contributed to the apparent lack of PERK involvement in some aspects of proteasome inhibition toxicity within the present study.

In the present study we did observe a significant role for PERK in modulating the activation of p38 in neural cells treated with proteasome inhibitors. These studies are particularly important because they are the first studies to link PERK to the regulation of p38 in any cell type or experimental condition. Many ER transmembrane proteins, including PERK, are activated and mediate their effects via the activation of CHOP (Oyadomari and Mori 2000) and p53 (Raven and Koromilas 2008; Schröder and Kaufman 2006). Because it is known that CHOP and p53 are capable of modulating p38 activity, it is possible that a similar mechanism (involving CHOP or p53) is responsible for PERK-mediated p38 activation in the present study.

Recent studies have demonstrated that in both brain aging and age-related diseases of the brain, where proteasome inhibition is also known to occur, activation of the UPR and PERK is also known to occur. For example, in the rodent brain increases in PERK and UPR have been reported (Hussain and Ramaiah 2007; Naidoo et al. 2008; Paz Gavilán et al. 2006). Similarly, studies have demonstrated in neurons of Alzheimer’s disease (Hol et al. 2006; Hoozemans et al. 2009) and in models of PD (Ryu et al. 2002) that there is evidence for activation of UPR and PERK. Interestingly, PERK positive neurons were observed to be colocalized with tau pathology (Hoozemans et al. 2005, 2009; Unterberger et al. 2006). The data in the present study suggest for the first time that activation of the UPR and PERK in each of these settings may be mediated by proteasome inhibition.

In neurons the stress kinase p38 has been most intensively studied for its role in regulating synaptic plasticity, myelination, and apoptosis (Sugiura et al. 2009; Thomas and Huganir 2004; Haines et al. 2007; Fragoso et al. 2007; Thornton et al. 2008; Han and Sun 2007; Hui et al. 2007; Schröder and Kaufman 2006). Additionally, studies have identified that activation of p38 occurs in AD and contributes to toxicity of AD related stressors (Zhu et al. 2002, 2005). Analysis of aging brain has identified increased levels of p38 during brain aging (Suh 2001; Martin et al. 2002). Our findings are consistent with a role for proteasome inhibition-induced activation of PERK, serving as a potential mediator of increased p38 activation in each of these experimental settings. Chronic activation of p38 may negatively impact multiple aspects of neuronal homeostasis, by serving to disrupt the balance in each of these aspects of neuronal homeostasis. In such a model, neuron death would not necessarily be an endpoint following chronic p38 activation.

While proteasome inhibition is known to be cytotoxic to most cell types, it is well established that there is a tremendous variation in cellular vulnerability to toxicity induced by proteasome inhibitors (Shringarpure et al 2006; Ding et al., 2006a). Additionally, studies have demonstrated that proteasome inhibition is protective/beneficial in some experimental settings (Ding et al. 2006a, 2006b), and is capable of promoting both pro-apoptotic as well as pro-survival pathways in the same population of cells (Yew et al., 2005; Ding et al. 2006b). Intriguingly, studies have demonstrated that in primary rat neurons the toxicity of proteasome inhibition is time dependent (Ding et al. 2006b), requiring greater than 12 hours of proteasome inhibition in order to induce neuron death. Together, these studies raise the possibility that the ability of proteasome inhibition to promote cell death is dependent on the amount of pro-survival signals present in a cell. In this model, potentially deleterious events (protein aggregation, UPR activation, etc) are not capable of inducing neuron death until the pro-survival pathways (new synthesis of heat shock proteins, Bcl-2, etc.) has been surpassed. The current study, indicates that PERK and p38 cross-talk are evident following proteasome inhibition, highlighting the complexity of cell signal transduction which occurs in response to proteasome inhibition. While the activation of signal transduction pathways such as PERK and p38 following proteasome inhibition may not be sufficient to induce acute neuron death, it is possible that their activation is sufficient to promote disturbances in multiple aspects of neuronal homeostasis including neurochemistry and synaptic activity.

Acknowledgements

This work was generously supported by grants from the NIA (AG029885, AG025771) to J.N.K..

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