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. Author manuscript; available in PMC: 2009 Oct 9.
Published in final edited form as: Brain Res. 2008 Aug 7;1234:206–212. doi: 10.1016/j.brainres.2008.07.111

Immortalized cortical neurons expressing caspase-cleaved tau are sensitized to endoplasmic reticulum stress induced cell death

Tori A Matthews-Roberson a,b,1, Rodrigo A Quintanilla c,1, Huiping Ding a,b, Gail VW Johnson a,b,c,*
PMCID: PMC2572685  NIHMSID: NIHMS71958  PMID: 18718455

Abstract

It has been previously reported that an Asp421 cleaved form of tau is toxic when expressed in cells. The purpose of this study was to understand if, and in what manner, the presence of Asp421 cleaved tau in neurons, which is generated by caspase cleavage, might facilitate neuronal death in Alzheimer’s disease (AD). For these studies we used immortalized cortical neurons that inducibly express either a full-length tau isoform (T4) or an isoform that has been pseudo-truncated at Asp421 (T4C3), to mimic caspase-3 cleavage. Neurons expressing either T4 or T4C3 were treated with thapsigargin, a drug, which has been shown to induce endoplasmic reticulum (ER) stress. Following long-term treatment with thapsigargin, cells expressing T4C3 presented with a marked increase in cell toxicity, underscored by differential activation of caspase-3 in comparison with cells expressing T4. Furthermore, we found that an inhibitor of the ERK1/2 signaling pathway, which is upregulated to different extents in each cell type, significantly reduced toxicity in both T4 and T4C3 cells. Our results suggest that the presence of Asp421 cleaved tau may sensitize neurons to ER stressors and possibly potentiate cell death processes during AD progression.

Keywords: Tau, Alzheimer’s disease, Caspase, Endoplasmic stress, Thapsigargin

1. Introduction

Tau is a microtubule-associated protein, which in a hyperphosphorylated form constitutes the major component of neurofibrillary tangles (NFTs) (Grundke-Iqbal et al., 1986; Kosik et al., 1986). NFTs are one of the primary pathophysiological hallmarks of Alzheimer’s disease (AD) and have been suggested to play a major role in facilitating neuronal degeneration (Kosik et al., 1986). Studies of brain tissue from AD patients show that the quantity of abnormal tau protein, as well as, the abundance of NFTs, directly correlates with the severity of impaired cognition (Grober et al., 1999).

Recent studies suggest that caspase mediated cleavage of tau is another posttranslational modification, in addition to phosphorylation, capable of promoting tau tangle formation (Gamblin et al., 2003; Rissman et al., 2004). Tau is an in vitro substrate for caspase-3 and is readily cleaved at Asp421, the apparent in vivo caspase-3 cleavage site (Chung et al., 2001; Fasulo et al., 2000; Gamblin et al., 2003; Rissman et al., 2004). This cleavage event results in a highly fibrillogenic tau isoform which, in in vitro studies, aggregates more readily and to a greater extent than full-length tau while also facilitating aggregate formation of the full-length protein (Gamblin et al., 2003; Rissman et al., 2004). Antibodies that specifically recognize Asp421 truncated tau show that tau cleaved at Asp421, active caspase-3 and fibrillar tau pathologies co-localize in AD patient brains (Gamblin et al., 2003; Rissman et al., 2004). It has also been found in a mouse tauopathy model that the majority of cells with active caspases also have NFTs (Spires-Jones et al., 2008). These results suggest a casual relationship between caspase-3 activation, tau Asp421 cleavage and tangle formation. Further, experiments in cell culture models provide evidence that Asp421 cleaved tau, alone, is toxic to neurons (Chung et al., 2001; Fasulo et al., 2005). Nonetheless, the additional negative affects that Asp421 cleaved tau may have on neuronal health in reference to other AD related stressors (i.e.; ER stress) has not been investigated.

ER stress is likely an important factor involved in facilitating neuronal death in AD. It has been shown that the ER stress response is activated in AD patients (Hoozemans et al., 2005) and mutations commonly associated with familial forms of AD induce ER stress in disease models [for a review see (Yoshida, 2007)]. These findings suggest a strong causal relationship between ER stress and AD and it is highly possible that ER stress initiated by and potentiated by the accumulative nature of aggregate prone proteins associated with AD is one of the mechanisms involved in disease progression. To better understand the role that Asp421 cleaved tau may play in facilitating neuronal death related to ER stress, we examined immortalized cortical neurons that inducibly express either a full-length form of tau (T4) or a tau isoform that has been pseudo-truncated at Asp421 (T4C3), in response to thapsigargin treatment. Thapsigargin is a drug known to induce ER stress following long-term exposure to cells (Shelton et al., 2004). Following treatment, we measured toxicity levels, caspase activation and examined signaling pathways known to be important in deciding neuronal fate following stress conditions.

2. Results

2.1. Tau protein expression levels and baseline toxicity

Tau expression was induced in immortalized cortical neurons (CN) by incubating cells in media containing doxycycline (Dox; 2 μg/mL) for 48 h. In the absence of Dox, inducible cells express minimal amounts of tau, as measured by western blotting (Shelton et al., 2004). Treatment with Dox resulted in a robust increase in tau expression; the levels were approximately equivalent to concentrations seen in rat primary neuronal cortical cultures (data not shown). Following induction, tau levels were comparable in both T4 and T4C3 expressing cells (Fig. 1A). Additionally, when probed with TauC3 antibody, which solely recognizes Asp421 truncated tau (Gamblin et al., 2003), only the T4C3 protein was immunoreactive (Fig. 1A). A lactate dehydrogenase (LDH) assay was used to determine differences in toxicity between cells expressing T4 or T4C3 (Krishnamurthy et al., 2000). Following induction of tau expression, LDH release was slightly but significantly increased in T4C3 cells compared to T4 cells (Fig. 1B).

Fig. 1.

Fig. 1

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Tau protein expression levels and baseline toxicity assay. A) Immortalized cortical neurons (CNs) were grown in media containing Dox (2 μg/mL) for 48 h to induce expression of the tau transgenes. Following induction, lysates were collected and tau levels examined by western blotting. The first lane is of T4 cells and the second lane of T4C3 cells. Total levels of T4 and T4C3 were expressed at comparable levels as shown in the Tau5 blot. The T4C3 protein migrates faster on the blot due to truncation at Asp421. Immunoreactivity of the Asp421 tau specific antibody (TauC3) is only observed in the lane where the T4C3 protein is present. Actin levels are used a loading control. B) Toxicity levels in each cell type were measured following 48 h of induction using LDH release as a readout (mean±SE; n=3 independent experiments). LDH release levels were significantly greater in T4C3 cells compared to T4 cells. (*P<0.05).

2.2. Evaluation of chronic thapsigargin treatments

2.2.1. Toxicity measurements

Following induction, cells were treated with thapsigargin (1 μM) for 0, 6, 12 or 24 h (Fig. 2). Toxicity in cells expressing T4C3 was significantly greater as compared to cells expressing T4 at the 6, 12, and 24 h time points, as measured by LDH release (Fig. 2). The greatest difference in LDH release between T4 and T4C3 expressing cells was observed at 24 h, where the toxicity levels were approximately 30% and 45%, respectively (Fig. 2).

Fig. 2.

Fig. 2

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Cells expressing T4C3 show decreased cell viability in chronic experiments. CNs expressing T4 or T4C3 were treated with thapsigargin (1 μM) for up to 24 h and LDH release was measured as an indicator of cell toxicity. Thapsigargin treatment resulted in significant increases in toxicity levels in T4C3 cells, as compared to T4 cells, at all time points tested with the greatest difference being observed at 24 h.

2.2.2. Caspase activation

Caspase-3, -6, and -9 activity was measured in each cell type following treatment. After thapsigargin treatment, caspase-3 activity was increased in both T4 and T4C3 expressing cells (Fig. 3A). At the 12 and 24 h time points the level of caspase-3 activity was significantly higher in cells expressing T4, as compared to T4C3 expressing cells (Fig. 3A). There were no significant differences in caspase-6 (Fig. 3B) and caspase-9 (Fig. 3C) activity levels between the two cell types.

Fig. 3.

Fig. 3

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Caspases are activated differentially in cells expressing T4 and T4C3. Thapsigargin treatment resulted in an increase in A) caspase-3, B) -6 and C) -9 activities. A) Caspase-3 activity was increased to a greater extent in T4 expressing cells than in T4C3 cells. There was no significant difference between B) caspase-6 and C) caspase-9 activity in T4 and T4C3 cells following thapsigargin treatment. Values are expressed as the mean (±SE) of n=3-4 independent experiments. (*P<0.05; **P<0.01).

2.2.3. T4 and T4C3 protein processing following exposure to thapsigargin

Following thapsigargin treatment, T4 and T4C3 proteins were examined via western blotting. The level of total tau and the Asp421 truncated tau isoform was detected with the Tau5 and TauC3 antibodies, respectively. There was no TauC3 immunoreactivity present in the T4 lane under control conditions; however, TauC3 immunoreactivity was increased in lanes containing T4 protein lysate following 6, 12, and 24 h of thapsigargin treatment (Fig. 4). Following 12 h of treatment, the level of T4C3 protein decreases compared to the 0 and 6 h time points; after 24 h the level of T4C3 was diminished even further, as indicated by both Tau5 and TauC3 immunoreactivity (Fig. 4). Cleaved-poly (ADP-ribose) polymerase (PARP) immunoreactivity increases following treatment in both T4 and T4C3 cells, and Bip/Grp78 immunoreactivity is also upregulated after 12 h of treatment (Fig. 4).

Fig. 4.

Fig. 4

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T4 and T4C3 protein is processed differently following exposure to thapsigargin. The state of the tau protein in T4 and T4C3 cells following thapsigargin treatment was examined. The level of total tau and Asp421 truncated tau isoforms were detected with the Tau5 and TauC3 antibodies, respectively. TauC3 immunoreactivity was not present in the T4 lane under control conditions. However, TauC3 immunoreactivity did increase in T4 lanes following treatment, at all time points examined. During the course of treatment, the level of T4C3 protein decreased and was greatly diminished after 24 h, as indicated by both Tau5 and TauC3 immunoreactivity. Cleaved-PARP immunoreactivity also increased in both T4 and T4C3 cells following treatment, indicating caspase-3 activity. Bip/Grp78 immunoreactivity, a marker for ER stress, showed a robust increase after 12 h treatment. The immunoblots are representative of three separate experiments.

2.3. ERK1/2 activation

2.3.1. A MEK inhibitor decreases thapsigargin-induced cell toxicity in T4 and T4C3 cells

Treating cells with U-0126, an upstream inhibitor of the extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway (Chong et al., 2006), for 2 h prior to the addition of thapsigargin effectively attenuated LDH release in both T4 and T4C3 cells, in a dose dependent manner (Fig. 5A). LDH release in cells treated with U-0126 (40 μM) for 2 h followed by 24 h of thapsigargin treatment was comparable to that of cells under control conditions (Fig. 5A).

Fig. 5.

Fig. 5

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MEK inhibitor U-0126 decreases cell toxicity in T4C3 cells in a dose dependent manner. A) LDH release in response to thapsigargin treatment was significantly attenuated in both T4 and T4C3 cells in a dose dependent manner, following incubation with an upstream inhibitor of the ERK1/2 signaling pathway, compound U-0126, for 2 h. Cells treated with 40 μM U-0126, followed by 24 h of exposure to thapsigargin, exhibited levels of LDH release comparable to those of cells under control conditions. B) The level of phospho-ERK1/2 (P-ERK1/2), the active form of the kinase, was initially increased in both cell types following 0.5 h treatment. P-ERK1/2 was differentially down-regulated in T4 and T4C3 cells. In T4 cells, the levels of P-ERK1/2 declined steadily over the treatment course, while P-ERK1/2 levels remain elevated in T4C3 cells even after 12 h of treatment. Levels of P-ERK1/2 were not detectable by western blotting after 24 h of treatment in either cell type. The results are reported as the mean (±SE) of n=3-4 independent experiments. (*P<0.05; **P<0.01 compared within each treatment group and #P<0.05; ##P<0.01 compared to the control thapsigargin treated T4 or T4C3 cells, respectively).

2.3.2. Activated ERK1/2 levels are down-regulated differentially in T4 and T4C3 cells

The levels of phospho-ERK1/2 (P-ERK1/2), the active form of the kinase, were measured and initially found to be increased in both cell models following 0.5 h of treatment with thapsigargin (Fig. 5B). However, there was a differential down-regulation of P-ERK1/2 between T4 and T4C3 expressing cells. In T4 cells, the levels of P-ERK1/2 showed a steady decline over the course of treatment, while P-ERK1/2 levels in T4C3 cells remained elevated even after 12 h of treatment (Fig. 5B). Levels of P-ERK1/2 were not detectable by western blotting after 24 h of treatment in either cell type (Fig. 5B).

3. Discussion

Previously it was reported that an Asp421 cleaved isoform of tau is toxic when expressed in neurons and a number of other different cell types (Chung et al., 2001; Fasulo et al., 2000; Fasulo et al., 2005). This Asp421 cleaved tau also has an increased aggregative nature and a high propensity for forming tau tangles in vitro (Gamblin et al., 2003; Rissman et al., 2004). We, therefore, considered the idea that the presence of Asp421 cleaved tau might increase susceptibility to an AD relevant stressor and result in increased cell death. ER stress is an expected contributor to AD progression (Shelton et al., 2004; Verkhratsky and Toescu, 2003). Hence, we chose to examine the effects of Asp421 cleaved tau on thapsigargin-induced cell death in a cortical neuronal model. Thapsigargin inhibits calcium uptake into the ER through sarco-endoplasmic reticula calcium-ATPase-type (SERCA) pumps; subsequently the calcium levels in the ER are depleted [for a review see (Paschen and Mengesdorf, 2005)]. Relatively high ER calcium concentrations are necessary for proper ER function (Kuznetsov et al., 1992; Lodish et al., 1992) and long-term treatment with thapsigargin has been shown to result in ER stress and apoptosis (Shelton et al., 2004).

Here we report that a pseudo-Asp421 truncated form of tau is toxic when expressed in immortalized cortical neurons (Fig. 1B). These findings are in agreement with previous reports. We found that chronic treatment with thapsigargin (up to 24 h) resulted in significantly greater toxicity levels in T4C3 cells when compared to cells expressing T4 (Fig. 2). There was also an increase in active caspases-3, -6, and -9 following treatment in both cell lines (Fig. 3A-C); however, despite the greater toxicity in T4C3 cells (Fig. 2), the extent of caspase-3 activation was greater in cells expressing T4 (Fig. 3A). Caspase-6, an executioner caspase in the same class as caspase-3, and caspase-9, an initiator caspase which plays a role in the activation of caspase-3 (Cohen, 1997), were activated to the same extent in both T4 and T4C3 cells (Figs. 3B and C). Active caspase-8 was also measured and found not to increase in either cell type (data not shown). A disconnection between active caspase-3 levels and increased cell toxicity has been previously reported. In studies examining the effects of mitochondria dysfunction in a Huntington’s disease (HD) model, Ruan et. al. observed that cells expressing a mutant form of the huntingtin protein exhibited more cell death in response to treatment with the mitochondrial complex II toxin, 3-nitropropionic acid (3-NP), while the levels of active caspase-3 were higher in cells expressing the wild-type huntingtin protein (Ruan et al., 2004). Finally it should be noted that thapsigargin treatment in our paradigm did not result in significant calpain activation as measured by tau and spectrin cleavage (data not shown). Although increases in cytosolic calcium levels occur following thapsigargin treatment (Paschen and Mengesdorf, 2005), treatment with 1 μM thapsigargin results in increases that are insufficient to activate calpain (Guttmann et al., 1997).

The thapsigargin-induced increase in caspase-3 activity in T4 and T4C3 cells was further evidence by an increase in the presence of Asp421 cleaved tau in T4 cells and an increase in cleaved PARP in both cell types, as measured by western blotting (Fig. 4). PARP is a known substrate of caspase-3 (Rosen and Casciola-Rosen, 1997) and cleavage in both cell types indicates that active caspase-3 is functioning normally in both T4 and T4C3 cells. The fact that immunoreactivity of the Asp421 tau specific antibody (TauC3) increased in T4 cells (Fig. 4) concurrently with active caspase-3 levels (Fig. 3A) supports the role of active caspase-3 in mediating tau cleavage at Asp421. Furthermore, Bip/Grp78 immunoreactivity, a marker of ER stress (Kim et al., 2005), does not increase until 12 h of treatment in both T4 and T4C3 expressing cells (Fig. 4) suggesting that thapsigargin mediated ER stress is being induced at similar rates in both cell types.

Additionally, we investigated whether treatment with a pharmacological inhibitor of the ERK1/2 signaling pathway could reduce the amount of cell toxicity in our experimental models. ERK1/2 signaling has been shown to be highly involved in the stress response mechanisms in neurons (Cheung and Slack, 2004). Treating with U-0126, a MEK pathway inhibitor, which is upstream of ERK1/2 activation (Chong et al., 2006), for 2 h prior to the addition of thapsigargin significantly decreased the level of cell death in both T4 and T4C3 cells in a dose dependent manner (Fig. 5A). Interestingly, when the levels of the active phosphorylated forms of ERK1/2 (i.e.; P-ERK1/2 levels) were measured we found that, although the levels of P-ERK1/2 were increased in both cell types following 0.5 h of treatment, the rate at which P-ERK1/2 levels were down-regulated was different between cell types. The levels of P-ERK1/2 in T4 cells declined steadily over the course of the 24 h treatment, while the levels remained relatively constant in T4C3 cells (Fig. 5B). The duration of ERK1/2 activation in these cells may impact their fate as it has been shown that downstream signaling of ERK1/2 signaling can either potentiate or prevent apoptosis through both caspase-3 dependent and independent mechanisms (Cheung and Slack, 2004).

Recently, multi-photon imaging techniques were used to visualize the brain in a living reversible mouse model of a tauopathy. Spires-Jones et. al. showed that active caspase-3, caspase-cleaved tau and NFTs can be present in neurons concurrently and although only a small percentage of cells imaged were positive for active caspase, the vast majority of these cells also contained NFTs (Spires-Jones et al., 2008). Further, these neurons showed no evidence of TUNEL staining or apoptotic nuclei and suppression of the tau transgene, while stopping ongoing cell death, did not decrease caspase activity (Spires-Jones et al., 2008). Taken together, our caspase-3 results (Fig. 3A) and the data from Spires-Jones et. al. supports the hypothesis that caspase-3 activation alone may not be sufficient to, nor responsible for, inducing acute neuronal death and active caspase-3 may not be the determining factor in deciding cell fate.

Understanding how the presence of Asp421 cleaved tau sensitizes neurons to ER stress and what are the long-term ramifications could be of great importance in understanding the cell death processes in AD progression. Further studies are required to discover the precise mechanism underlying this phenomena and specifically whether, as in the Ruan et. al. studies of the HD model (Ruan et al., 2004), mitochondrial dysfunction might also be involved in the cell death process in our AD model.

4. Experimental procedures

4.1. Cell culture

Immortalized cortical neurons (Bongarzone et al., 1998) expressing inducible full-length (T4) or Asp421 truncated (T4C3) tau were prepared as described previously (Krishnamurthy and Johnson, 2004). Cells were cultured in DMEM with High Glucose (Mediatech) and supplemented with 5% FBS, 0.1% gentamicin, and 4 mM glutamine, penicillin, and streptomycin at 33 °C. In these studies cells were treated with the tetracycline derivative doxycycline (Dox) at a concentration of 2 μg/mL for 48 h to induce tau expression. After 48 h, the induction media was removed from cells and replaced with DMEM/High Glucose containing only 4 mM glutamine and 1% FBS. The cells were moved to a 39 °C incubator 2 h prior to thapsigargin treatment and maintained under these conditions throughout the course of the experiments.

4.2. Immunoblotting

Cells were collected in SDS buffer (0.25 M Tris Cl pH 6.8, 10% glycerol, 2% SDS, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF and 10 μg/ml of leupeptin, aprotinin and pepstatin), sonicated for 5 s on ice and centrifuged. The protein concentration of each sample was determined using the bicinchoninic acid (BCA) assay (Pierce). Equal amounts of proteins were diluted with 2× SDS protein loading buffer (2× SDS buffer, 25 mM dithiothreitol and 0.01% bromophenol blue), incubated in a boiling water bath for 5 min, separated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membrane and blocked in 5% non-fat dry milk in TBST (20 mM Tris Cl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. Subsequently, membranes were probed with the indicated antibodies. Tau5 and TauC3 (from Dr. L. Binder) are phospho-independent tau and Asp421 truncated tau specific antibodies, respectively (Carmel et al., 1996; Johnson et al., 1997). Anti-cleaved PARP, p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (P-ERK1/2) were from Cell Signaling (Beverly, MA, USA), anti-actin is from Chemicon (Billerica, MA, USA) and Bip/Grp78 is from Stressgen (Ann Arbor, MI, USA). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, the blots were developed using enhanced chemiluminescence (Amersham, Piscataway, NJ, USA).

4.3. LDH assay

The release of the intracellular enzyme lactate dehydrogenase (LDH) into the medium was used as a quantitative measurement of cell viability. The measurement of LDH was carried out as described previously (Decker and Lohmann-Matthes, 1988).

4.4. In situ caspase activity

In situ caspase activity was measured using a previously described protocol (Bijur et al., 2000). Briefly, 200 μl of assay buffer (20 mM Hepes, pH 7.5, 10% glycerol, and 2 mM dithiothreitol) containing the fluorogenic peptide substrate for either caspase-3, -6 or -9 (Axxora, San Diego, CA, USA) was added to each well (final concentration of 25 ng/μl) of a 96-well clear bottom plate (Corning). Cell lysate (20 μg of protein) was added to start the reaction. Triplicate measurements were done for each sample. Background fluorescence was measured in wells containing assay buffer, substrate and lysis buffer without the cell lysate. Assay plates were incubated at 37 °C for 1 h and fluorescence was measured on a fluorescence plate reader set at 360 nm excitation and 460 nm emission.

4.5. Statistical analysis

All data is expressed as the mean of at least three independent experiments (±standard error of the mean) unless otherwise stated. Statistical comparisons between treatment groups were performed using Student’s t test. P values are *, # (P<0.05); **, ## (P<0.01).

Acknowledgments

The authors would like to thank Dr. L. Binder for the generous gifts of the tau antibodies. This work is supported by a grant from the Alzheimer’s Disease Association (G.V.W.J), NIH Grant NS051279 (G.V.W.J) and a Merck Graduate Dissertation Fellowship (T.M.R).

Abbreviations

AD

Alzheimer’s disease

NFT

neurofibrillary tangle

ER

endoplasmic reticulum

SERCA

sarco-endoplasmic reticula calcium-ATPase-type pumps

PARP

poly (ADP-ribose) polymerase

ERK

extracellular signal-regulated kinase

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

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