Tuberous Sclerosis Complex Activity is Required to Control Neuronal Stress Responses in an mTOR-Dependent Manner

doi:10.1523/JNEUROSCI.0778-09.2009

J Neurosci. Author manuscript; available in PMC 2009 November 6.

Published in final edited form as:

J Neurosci. 2009 May 6; 29(18): 5926–5937.

doi: 10.1523/JNEUROSCI.0778-09.2009

PMCID: PMC2691854

NIHMSID: NIHMS115539

Tuberous Sclerosis Complex Activity is Required to Control Neuronal Stress Responses in an mTOR-Dependent Manner

Alessia Di Nardo,¹ Ioannis Kramvis,¹ Namjik Cho,¹ Abbey Sadowski,¹ Lynsey Meikle,² David J. Kwiatkowski,² and Mustafa Sahin¹

¹ The F.M. Kirby Neurobiology Center, Department of Neurology, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA

² Division of Translational Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

Corresponding Author: Mustafa Sahin, M.D., Ph.D. Department of Neurology, Children’s Hospital, 300 Longwood Avenue CLSB 13074, Boston, MA 02115, Ph: 617-919-4518, Fax: 617-730-0242, Email: mustafa.sahin/at/childrens.harvard.edu

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Abstract

Tuberous Sclerosis Complex (TSC) is a neurogenetic disorder caused by loss-of-function mutations in either the TSC1 or TSC2 genes and frequently results in prominent CNS manifestations including epilepsy, mental retardation, and autism spectrum disorder. The TSC1/TSC2 protein complex plays a major role in controlling the Ser/Thr kinase mTOR, which is a master regulator of protein synthesis and cell growth. In this study, we show that endoplasmic reticulum (ER) stress regulates TSC1/TSC2 complex to limit mTOR activity. In addition, Tsc2-deficient rat hippocampal neurons and brain lysates from a Tsc1-deficient mouse model both demonstrate elevated ER and oxidative stress. In Tsc2-deficient neurons, the expression of stress markers such as CHOP and HO-1 is increased, and this increase is completely reversed by the mTOR inhibitor rapamycin both in vitro and in vivo. Neurons lacking a functional TSC1/TSC2 complex have increased vulnerability to ER stress-induced cell death via the activation of the mitochondrial death pathway. Importantly, knockdown of CHOP reduces oxidative stress and apoptosis in Tsc2-deficient neurons. These observations indicate that ER stress modulates mTOR activity through the TSC protein complex and that ER stress is elevated in cells lacking this complex. They also suggest that some of the neuronal dysfunction and neurocognitive deficits seen in TSC patients may be due to ER and oxidative stress, and therefore potentially responsive to agents moderating these pathways.

Keywords: CHOP, ER stress, oxidative stress, rapamycin, Hippocampus, RNA interference

Introduction

TSC is an autosomal dominant disorder characterized by the growth of benign tumors called hamartomas in multiple organs including the brain (Crino et al., 2006). TSC patients suffer from epilepsy, autism and developmental delay. Within the central nervous system, TSC is associated with cortical tubers, made up of giant cells, dysmorphic neurons and astrocytes. TSC is caused by mutations in either the TSC1 or TSC2 genes. Proteins encoded by TSC1 or TSC2 genes interact with each other to form the TSC1/TSC2 complex. One of the major cellular functions of the TSC1/TSC2 complex is to limit protein synthesis and regulate cell size by inhibiting the Rheb-mTOR pathway (Kwiatkowski and Manning, 2005). Mutations in either TSC1 or TSC2 lead to constitutive activation of mTOR, which phosphorylates substrates such as S6 kinase (S6K) and 4E-BP1, ultimately increasing protein synthesis.

Recently, embryonic fibroblasts and kidney tumors from Tsc2 deficient mice were shown to have increased endoplasmic reticulum (ER) stress (Ozcan et al., 2008). ER stress can be caused by excessive protein synthesis, perturbation in calcium homeostasis, or nutrient deprivation (Ron and Walter, 2007). Under normal conditions, the ER stress sensor GRP-78 has an inhibitory role on the effectors (PERK, ATF6 and IRE1) of the Unfolded Protein Response (UPR), which is the cellular response to ER stress (Dorner et al., 1992; Liu et al., 2000). Upon ER overload, GRP78 releases its inhibition of PERK, ATF6 and IRE1 (Mori, 2000) and activates the UPR. The UPR leads to three distinct specific cascades: (1) The PERK/eIF2α pathway reduces protein synthesis by inhibiting translation; (2) the ATF6 pathway activates transcription of chaperone proteins increasing folding capacity; (3) the IRE/XBP-1 pathway promotes proteosome-dependent protein degradation to remove proteins from the ER (Bertolotti et al., 2000; Mori, 2000; Liu et al., 2003; Rutkowski and Kaufman, 2004). Ultimately, the UPR response either results in the successful elimination of ER overload, or if unsuccessful, in ER stress-induced cell death via caspase activation and induction of the pro-apoptotic transcription factor CHOP, (C/EBP homologous protein, GADD153) (Oyadomari and Mori, 2004).

While ER stress has been demonstrated in Tsc-deficient mouse embryonic fibroblasts and kidney tumors (Ozcan et al., 2008), it remains unclear whether TSC-deficiency leads to ER stress in neurons, what role mTOR pathway plays in neuronal stress response, and whether similar dysfunctions are present in seizure-models of Tsc in vivo. To address these questions, we investigated the role of the TSC1/TSC2 complex during ER stress in greater detail and examined the effects of TSC-deficiency on neuronal stress pathways. We demonstrate that TSC2 is initially inactivated in neurons during ER stress, and later activated, as part of an apparent regulatory mechanism to limit mTOR activity. Lack of a functional TSC1/TSC2 complex abolishes this regulation, resulting in increased ER stress and vulnerability to neuronal damage. Furthermore, Tsc-deficient neurons have increased accumulation of reactive oxygen species (ROS) and oxidative stress. Similar dysfunctions were identified in TSC brain lesions in vivo, identifying a new role for the TSC1/TSC2 complex in the neuronal stress response.

Material and Methods

Animals

All experimental procedures were performed in compliance with animal protocols approved by the IACUC at Children’s Hospital, Boston. The Tsc1^c/⁻ SynCre⁺ mice used in this study were previously described (Meikle et al., 2007). For rapamycin treatment, mice were injected intraperitoneally at 6mg/kg every other day from P9 to P33. Mice subjected to the On/Off treatment were on rapamycin treatment (6mg/Kg) every other day from P9 to P30 followed by no treatment until P45 (On/Off) (Meikle et al., 2008).

Neuronal cultures

Neuronal cultures were prepared as previously published (Sahin et al., 2005). Briefly, hippocampi from 18-day-old rat embryos (Charles River CD1) were isolated under the microscope and collected in Hank’s Balanced Salt Solution containing 10mM MgCl₂, 1mM kynurenic acid, 10mM HEPES and penicillin/streptomycin. After 5min dissociation at 37C in 30u/ml of papain (Worthington) neurons were mechanically triturated and plated in Neurobasal medium containing B27 supplement, 2 mM L-glutamine and penicillin/streptomycin (Invitrogen). For biochemical analysis, cells were plated at 1 × 10⁶cells/well onto six-well plates coated with 20 μg/ml poly-D-lysine (PDL) and 2.5× 10⁶cells/plate for immunofluorescent studies onto PDL-laminin-coated glass coverslips in 24-well plates.

Lentivirus infection

Viral stocks for lentiviral infection were prepared as previously described (Mostoslavsky et al., 2005), except that the four packaging vectors (kindly provided by Dr. R C. Mulligan) were cotransfected into HEK293 T cells with the plasmid to be co-expressed using Lipofectamine 2000 according to the manufacturer’s instructions. Viral particles were collected 48hrs and 72hrs after transfection and filtered though a 0.45 μm membrane. Hippocampal neurons were infected at 1 day in vitro (1DIV) in the presence of polybrene at 0.6 μg/ml. Six hours after infection the virus-containing medium was replaced by fresh NB/B27 medium. After infection, neurons were kept in culture for an additional 10 days. Control ShRNAi construct against the luciferase gene (here referred as GL3-Sh) was previously described (Flavell et al., 2006). The sequence for Tsc2 gene targeting was the following: 5′-GGTGAAGAGAGCCGTATCACA-3′.

Semi-quantitative and real-time qPCR

Total RNA was prepared with an RNAeasy KIT (Qiagen) following manufacturer’s instructions and quantified by a spectrophotometer. A total of 2 micrograms of polyA mRNA was used for reverse transcription using the SuperScript RT system (Invitrogen). Semi-quantitative PCR reactions were performed using Taq Polymerase (Perkin Elmer). Quantification of the semi-quantitative PCR was performed by densitometry scans and values were normalized against total β-actin. Real-time PCRs were performed using SYBG Green PCR master Mix (Applied Biosystem). All qPCR reactions were performed in triplicate and normalized against GAPDH. Analysis was performed using 7300 System SDS Software on a 7300 Real Time PCR System. The sequences of the primers for both semiquantitative and qPCRs are listed in the Supplementary Material. In all cases, data were expressed as means of at least three independent experiments +/− SE. Statistical analysis was performed by unpaired two-tailed Student’s t-test and considered significant at p <0.05.

CHOP knockdown

CHOP Sh-RNAi (CHOP-Sh in the text) and control CHOP RNAi (CHOP-C in the text) were purchased from Sigma and the sequences are below.

CHOP-Sh: 5′-GAAACGAAGAGGAAGAATCA-3′

CHOP-C: 5′-CGGAAGTGTACCCAGCACC-3′

Antibodies and Reagents

Antibodies used for this study included: rabbit polyclonal anti-phospho-S6 (Ser234/Ser235) (cat. no. 2211), mouse monoclonal anti-total S6 (cat. no. 2317), rabbit polyclonal anti-phospho-Akt (Ser473) (cat. no. 9271), rabbit polyclonal anti-S6K (cat. no. 9202), rabbit polyclonal anti-phospho-S6K (Thr389) (cat. no. 9234), rabbit polyclonal anti-Tsc1 (cat. no. 4906), rabbit polyclonal anti-Tsc2 (Thr1462) (cat. no. 3611), (all from Cell signaling Technology); rabbit polyclonal anti-Tsc2 (sc-893), mouse monoclonal anti-GADD153 (CHOP) (cat. no. sc-7351), goat polyclonal anti-Akt (cat. no. sc-1618) (all from Santa Cruz Biotechnology); rabbit polyclonal anti-GRP78 (cat. no. SPA-826) and mouse monoclonal anti-HO-1 (cat. no. OSA-110) from Stressgen. HRP-conjugated secondary antibodies were from VWR.

Western Blot

Details can be found in the Supplementary Material.

ER stress induction

Thapsigargin and Tunicamycin were purchased from Sigma and used at a final concentration of 0.5 μM and 4 μg/ml respectively. Stocks of drugs were made in DMSO and freshly diluted in NB media at 20x of the final concentration before performing each experiment. The same amount of DMSO was used as vehicle-only control. Before ER stress induction, NB/B27 media was replaced with NB in the presence of penicillin/streptomycin for 4hrs, and drugs were then added for an additional 3, 6, and 24 hrs. When included, rapamycin was used for 24hrs in NB media at a final concentration of 20nM.

Apoptosis quantification

The number of apoptotic cells was determined by Hoechst staining and Trypan blue exclusion test. E17 rat neurons were plated on coverslips at a density of 10×10⁴cells/ml and infected with lentivirus as described above. After 10DIV, neurons were left untreated or treated for ER stress induction. For Hoechst quantification, neurons were fixed and stained with 5 μg/ml Hoechst (Molecular probe) for 5min at RT. Neurons were then washed in PBS, mounted and analyzed with a Leica DM RXA microscope equipped with epifluorescence. Apoptotic nuclei were counted under a 20x objective and expressed as the percentage of the total number of infected cells in the same field. Data are expressed as means from at least three different experiments +/− SE, and statistical analysis was performed by Student t-test. For trypan blue exclusion test, neurons were harvested as described in flow cytometric analysis and resuspended in a 0.2% trypan blue solution (Sigma) prepared in HBSS for 5min at RT. Apoptotic cells were evaluated under bright field microscopy by counting non-viable cells (dye-positive) and viable cells (dye-negative) on hemocytometer fields.

For quantification of cell death at the single cell level, the number of apoptotic cells was determined by counting cleaved caspase 3 (Cell Signaling cat. #9664) positively stained neurons after immunofluorescent microscopy using a 20x objective. Data were expressed as a percentage of the total number of infected cells. The experiment was performed in triplicate and at least 300 cells/experiment were counted. Statistical analysis was performed by unpaired two-tailed Student’s t-test and considered significant at p <0.05.

Immunocytochemical analysis

Details can be found in the Supplementary Material.

Mitochondrial ROS

Rat hippocampal neurons were cultured in NB media for 24 hrs followed by incubation with 100nM MitoTracker® Red CM-H₂XRos dye (Molecular Probes) for 30min before being processed for immunofluorescence and stained with Hoechst. Oxidative stress was quantified by counting the number of MT-Red labeled cells under an epifluorescent microscope with a rhodamine filter, and expressed as the average percentage of MT-Red labeled cells from three independent experiments.

Flow Cytometric Analysis

Neurons cultured in NB media for 24 hrs were harvested by 5min incubation at 37C with 15U/ml of papain (Worthington) made in Hank’s Balanced Salt Solution containing 10mM MgCl₂, 1mM kynurenic acid, 10mM HEPES and penicillin/streptomycin. Before dissociation a solution of 7mg/ml trypsin inhibitor (Sigma) was added to stop the reaction. Neurons were then collected by centrifugation, washed and resuspended in NB media at 2× 10⁶cells/ml. Neurons were divided into two aliquots which were incubated in the absence or presence of 100nM MitoTracker® Red CM-H₂XRos dye (Molecular Probes). After 20min at 37C, neurons were collected by centrifugation, rinsed and fixed in 4%PFA made in PBS for 15min at RT. After fixation, neurons were washed and resuspended in 200 μl for analysis. Flow cytometric analysis was performed with Dako Cytomation MoFlo equipped with Spectra-physics laser model 177 with an emission at 488 and a strength of 100mWatts. Data were analyzed with Summit 4.3 software (Dako, Colorado). Gating was performed prior to the collection of data to remove apoptotic cells and cellular debris. Mean fluorescence intensity (MFI) of MitoTracker® Red CM-H₂XRos dye (MT-Red) was calculated by subtracting for each sample the FACS measurement obtained in the absence of the dye (background) to the measurement obtained in the presence of the dye.

RESULTS

TSC and mTOR are dynamically regulated under ER stress

Since some of the most severe manifestations of TSC disease are in the CNS, we investigated the role of the TSC1/TSC2 complex in the neuronal response to ER stress. We treated rat hippocampal neurons with two widely used ER stress inducers: the ER-Ca²⁺-ATPase blocker Thapsigargin (Tg) and the N-glycosylation inhibitor Tunicamycin (Tn) (Li et al., 2000; Urano et al., 2000). To determine the optimal doses for these ER stress-inducing chemicals in neurons, we performed dose/response curves (0.1 μM – 5 μM for Tg; 1 μg/ml – 12 μg/ml, for Tn) using wild-type rat hippocampal neurons and assessed cell death as the outcome. Expression of UPR-regulated genes GRP78 and CHOP confirmed ER stress induction already at the lowest concentrations used for both drugs (Suppl. Fig. 1A–C). As expected, cell death assessed by Hoechst staining (Suppl. Fig. 1D,E) and Trypan blue exclusion assay (Suppl. Fig. 1F) showed that the percentage of apoptotic neurons increased in a dose-dependent manner for both Tg and Tn. For the purpose of this study, we decided to use 0.5 μM for Tg and 4 μg/ml for Tn since at these doses we observed a robust ER-stress induced UPR activation and ER-stress induced cell death of at least 50–60% neuronal cells.

When assessing the effect of ER stress on the Akt/mTOR pathway, we found a response that varied with duration of treatment. Tg treatment led to an initial activation of mTOR as evidenced by increased phosphorylation of S6 ribosomal protein (phospho-S6 Ser235/236) (Figure 1A, D). In contrast, longer exposure to Tg (24hrs) correlated with a progressive decrease in Akt activity (phospho-Akt Ser473) and in the phosphorylation of Tsc2 at Thr1462 (Figure 1A, C, E), a known Akt phosphorylation site (Inoki et al., 2002; Potter et al., 2002). Thr1462 phosphorylation is known to inhibit TSC complex activity (Inoki et al., 2002; Manning et al., 2002). Accordingly, we observed inhibition of the downstream mTOR pathway, as indicated by decreased phosphorylation of S6 at 24 hours (Figure 1A, D). ER stress was confirmed by the time-dependent increase in the expression of the UPR-regulated gene, GRP78. Prolonged ER stress (24hrs) correlated with expression of the pro-apoptotic UPR regulated gene, CHOP, and with apoptosis as shown by activated (cleaved) caspase 3. Tunicamycin treatment, an alternative method of inducing ER stress, produced similar results in neurons (Figure 1B–E). To investigate whether regulation of TSC1/TSC2 complex activity under ER stress also occurred in non-neuronal cells, we treated Hek293T and mouse embryonic fibroblasts (MEFs) with ER stress inducers. We found that ER stress also induced transient mild activation of Akt, phosphorylation of Tsc2, and phosphorylation of S6, followed by inhibition of this pathway at 24 hours (Suppl. Fig 2A–B). These data indicate that activity of the TSC1/TSC2 complex is dynamically regulated in cells undergoing ER stress and may mediate the modulation of mTOR activity in response to ER stress.

Figure 1

Regulation of the Tsc1/Tsc2 complex by ER stress

Loss of Tsc correlates with an mTOR-dependent ER stress response in neurons

Having identified dynamic regulation of the TSC1/TSC2 complex activity under ER stress, we hypothesized that neurons lacking TSC activity would display features of ER stress at baseline due to constitutive activation of mTOR and high levels of protein synthesis. Therefore, we investigated ER stress response in neurons after RNAi-mediated knockdown of the Tsc2 gene. Rat hippocampal neurons were infected after 1 day in vitro (DIV) with a lentivirus expressing Tsc2 gene plasmid-based short hairpin RNA (Tsc2-Sh) or a control lentivirus expressing shRNA against the luciferase gene (GL3-Sh). Since we have previously shown that knockdown of Tsc2 produces the same morphological and biochemical changes as Tsc1 knockout in dissociated cells (Choi et al., 2008), we used Tsc2 knockdown neurons as an in vitro model to examine neuronal ER stress.

In Tsc2 knockdown neurons, there was a significant increase (3.5 fold) in the mRNA of CHOP as assessed by real-time qRT-PCR (Figure 2A). Also upregulated were CHOP’s upstream regulator ATF4 (2.5 fold) (Fawcett et al., 1999; Harding et al., 2000; Ma et al., 2002) and to some extent (1.5 fold) ER stress sensor GRP78. Treatment of Tsc2 knockdown hippocampal neurons with rapamycin reduced CHOP, ATF4 and GRP78 mRNA levels to baseline, indicating that the increase was mTOR-dependent. Consistent with the increased CHOP mRNA levels in the Tsc2-deficient hippocampal cultures, immunocytochemical analysis revealed that in the Tsc2-Sh infected cultures 13.3% of neurons had CHOP nuclear expression and 6.1% had CHOP cytosolic expression compared to 0.7% CHOP nuclear expression and 0.1% CHOP cytosolic expression for control infected neurons (Figure 2B–C).

Figure 2

Regulation of UPR genes in Tsc-deficient neurons at baseline and after ER stress induction

CHOP is a pro-apoptotic transcription factor that is expressed and translocated into the nucleus under ER stress (Oyadomari and Mori, 2004). Since Tsc2 knockdown neurons demonstrated elevated CHOP expression at baseline, we investigated its expression and localization in Tsc-deficient neurons treated with Tg. Exposure to Tg significantly increased CHOP protein nuclear expression in Tsc2-deficient neurons compared to control cultures as assessed by immunocytochemical analysis (Figure 2D–E). Together, these data show that loss of a functional TSC1/TSC2 complex in hippocampal neurons lowers the threshold for activation of UPR-regulated genes.

Tsc2-deficient neurons show increased basal and ER-stress induced cell death

While ER stress-activated signaling is a protective cellular response to reduce ER load, prolonged ER stress often leads to cell death by apoptosis (Rao et al., 2001). To assess the effects of activating UPR response, we treated Tsc2 knockdown and control neuronal cultures with the ER stress inducers Tg or Tn for 3, 6 and 24 hours. Knockdown of Tsc activity was confirmed by the reduced Tsc2 protein level and constitutively high S6 and S6K phosphorylation (Figure 3A–B). Exposure to either Tg or Tn induced cellular ER stress as seen by the gradual increase in the expression of the ER stress sensor GRP78. Western blot analysis showed that Tsc2 knockdown neurons had a small but significant increase in the levels of CHOP protein expression at baseline compared to control-infected neurons, which was consistent with the observed increased basal transcription of CHOP mRNA. No differences were observed in the baseline and in the ER stress induced GRP78 levels between the control and Tsc-deficient neurons (Suppl. Fig. 3A–B).

Figure 3

Lack of Tsc activity correlates with increased CHOP expression

The effects of ER stress induction on neuronal viability were then monitored using an antibody for cleaved (active) caspase 3 (cc3) on western blots. In control neurons, Tg or Tn treatments induced cleavage of caspase 3 only after 24hrs while in Tsc2-deficient neurons, cc3 was already detectable at baseline and further increased shortly after (3 hrs) ER stress-induction (Figure 3A–B). Similarly, as assessed by Hoechst staining, a higher proportion of Tsc2 knockdown neurons showed a significant increase in apoptotic nuclei at baseline and after short Tg (3hrs) (Figure 3C) or Tn (3hrs and 6hrs) treatment (Figure 3E). Similar results were obtained when assessing baseline cell death by Trypan Blue exclusion assay for Tg (Figure 3D) and Tn (Figure 3F). To further confirm induction of apoptosis in Tsc2-knockdown cells, we assessed cytoplasmic levels of cytochrome c, a marker of early apoptosis (Ferri and Kroemer, 2001; Rao et al., 2001). Consistent with the higher level of baseline apoptosis, we detected cytochrome c release in the cytosolic fraction of Tsc2-deficient neurons only (Figure 3G). Densitometric quantification of cytosolic and mitochondrial cytocrome c levels was performed on three independent experiments and revealed a 4.9 fold increase in the cytosolic cytochrome release in Tsc2 deficient neurons compared to controls (*p<0.01 by t-test). In cells under ER stress, the Inositol Requiring Enzyme 1 (IRE1) pathway is responsible for the alternative splicing of the XBP-1 transcript (Lee et al., 2002). Interestingly, in comparison to neurons infected with control virus, Tsc2-deficient neurons had a more robust ER stress-induced activation of the IRE1 pathway upon both Tg and Tn treatment, as shown by XBP-1 splicing (Suppl. Fig. 4A–B). Taken together, these findings suggest that lack of Tsc activity in cultured hippocampal neurons correlates with increased ER-stress induced cell death via activation of the mitochondrial death pathway.

Tsc-deficient neurons have increased oxidative stress and undergo cell death via a CHOP-dependent mechanism

CHOP is a pro-apoptotic transcription factor that promotes apoptosis by modulating the expression of proteins that regulate cell survival and death pathways (Oyadomari and Mori, 2004). In particular, CHOP has been found to affect expression and localization of Bcl2 family members and influence the cellular redox status (McCullough et al., 2001; Marciniak et al., 2004). To determine which of these CHOP targets were affected in Tsc-deficient neurons, we compared the mRNA levels of survival and oxidative stress regulated genes by qRT-PCR (Figure 4A). Tsc2 knockdown did not change the expression of the prosurvival factor bcl2 or of the cellular antioxidant defense gene thioredoxin 2 (Trx-2). Instead, we observed a significant mTOR-dependent increase in the expression (3.5 fold) of the antioxidant enzyme heme oxygenase-1 (HO-1) and of the ER oxidoreductase enzyme ERO1α. HO-1 protein levels were also increased in Tsc2 deficient neurons, and upon rapamycin treatment this increase was blocked (Figure 4B–C). However, under the same conditions, rapamycin treatment was not sufficient to prevent cell death.

Figure 4

Lack of Tsc activity correlates with mTOR dependent oxidative stress response A

HO-1 is a member of the heat shock family (Hsp32) and its expression is induced when cells experience oxidative stress (Takahashi et al., 2004) whereas the ERO1α is a glycosylated flavoenzyme implicated in oxidative protein folding and ROS production in the ER by promoting disulphide bond formation (Harding et al., 2003; Sevier and Kaiser, 2008). Therefore, we asked whether Tsc-deficiency would induce increased production of reactive oxygen species (ROS). Control and Tsc2-deficient rat hippocampal neurons were treated in culture with MitoTracker Red CM-H₂XRos dye (MT-Red) (Figure 4D,E). MT-Red generates fluorescence only upon oxidation by superoxide produced by mitochondria (Kim et al., 2002). Quantification of MT-Red labeled cells under an epifluorescent microscope was performed from three independent experiments and revealed 2.6 fold increase in the percentage of Tsc2-deficient positive neurons compared to control-infected cells (control 14.3% +/− 2.5% vs Tsc2 deficient neurons 37.4% +/− 1.4%; n=250 cells/experiment, *p<0.001 by t-test). MT-Red accumulation, as assessed by FACS analysis, revealed a shift of the MT-Red fluorescence distribution inTsc2-Sh cultures indicating significantly increased fluorescence mean intensity (FMI) (Figure 4F). A 1.7 fold increase in FMI of MT-Red was found in Tsc2-Sh cultures compared to control (*p<0.05 by t-test). The higher levels of ROS and increased HO-1 expression after Tsc2 silencing indicate a critical role for Tsc in regulating oxidative stress response in neurons.

Tsc-deficient neurons undergo cell death via a CHOP-dependent mechanism

The identification of increased cell death and ROS production in Tsc-deficient neurons led us to ask whether CHOP activity was necessary for these effects. To investigate this question, we silenced CHOP expression using RNAi. Rat hippocampal neurons were first infected with either GL3-Sh virus or Tsc2-Sh virus and after 6 days they were re-infected with lentiviral vectors expressing either a CHOP-Sh or control CHOP-C constructs. GL3-Sh and Tsc2-Sh infected neuronal cultures were then either left untreated or treated with Tg for ER stress induction. CHOP-Sh RNAi efficiently reduced baseline and Tg-induced CHOP expression at both the RNA and the protein level in Tsc-deficient neurons and in Tg-treated GL3-Sh cultures (Figure 5A–B). We found that, compared to CHOP-C virus, CHOP-Sh virus efficiently reduced the baseline and the ER stress-induced HO-1 expression at both the mRNA and protein levels in Tsc2-deficient neurons (Figure 5C). Consistent with its role in promoting ERO1α activation (Marciniak et al., 2004), CHOP knockdown reduced ERO1α expression in Tsc2 deficient neurons (Suppl. Fig. 5). A significant reduction was also identified in the oxidative stress response of Tsc2-deficient cultures infected with CHOP-Sh compared to those infected CHOP-C virus (Figure 5D). Quantification of MT-Red positive neurons was performed on IF images from three independent experiments after incubation with 100nM MitoTracker Red CM-H₂XRos (at least 200 cells/experiment; Tsc2-Sh cultures infected with CHOP-C virus 36.9 +/− 2.0 vs Tsc2-Sh cultures infected with CHOP-C virus 17.0 +/− 5.0, *p< 0.05 by t-test). Most importantly, CHOP silencing reduced cell death in Tsc2-deficient neurons at by both cleaved caspase staining both by western blotting and at the single cell level (Figure 5B,C,E,F). Together, these data indicate that in the setting of Tsc-deficiency, CHOP upregulation plays a major role in both oxidative stress response and cell death induction.

Figure 5

Silencing of CHOP expression in GL3-Sh and Tsc2-Sh neurons

Previous studies have shown that loss of TSC1/TSC2 complex activity correlates with an mTOR-dependent negative feedback on the PI3K/Akt pathway, which in turn results in reduced Akt activation (Zhang et al., 2006). To investigate whether a similar inhibition was occurring in Tsc-deficient neurons, we analyzed Akt at the phospho-Ser473 activation site using western blot. When compared to control cultures, Tsc2-Sh infected neurons had indeed lower basal levels of Akt activation which did not change after CHOP-Sh RNAi (Figure 5B). These data indicate that reduced Akt activity in Tsc2 deficient neurons is CHOP-independent, and although downregulated Akt might contribute to increased apoptosis in Tsc-deficient neurons, knockdown of CHOP alone is sufficient to reduce both cell death and oxidative stress.

In vivo identification of stress response in brains from Tsc1^c/−SynCre⁺ mice and in the tuber of a TSC patient

Our data demonstrated increased oxidative stress in Tsc2-deficient hippocampal cultures in vitro. To determine whether a similar stress response occurs in vivo, we assessed levels of expression for CHOP and HO-1 in total brain lysates from Tsc1^c/−SynCre⁺ mice (neuronal Tsc1 knockout) (Meikle et al., 2007). Tsc1^c/−SynCre⁺ mice experience near complete loss of Tsc1 expression in neurons, and display neurological decline with median survival of 35 days. Both the neurological abnormalities and the median survival are markedly improved when mutant mice are treated with rapamycin from P7 to P33 (Meikle et al., 2008). To investigate ER stress responses in vivo, we used Tsc1^c/−SynCre⁺ mice either untreated or treated with rapamycin. Both CHOP and HO-1 protein levels were increased in Tsc1^c/−SynCre⁺ brain lysates and were reduced in mice treated with rapamycin (Figure 6A–B). Interestingly, Tsc1^c/−SynCre⁺ mice taken off rapamycin after 3 weeks of treatment showed recurrence of HO-1 expression (Suppl. Fig. 6).

Figure 6

Upregulation of stress responses in the brain of Tsc-deficient mice

To extend these findings at the cellular level, immunohistochemical (IHC) analysis for HO-1 was performed on control and Tsc1^c/−SynCre⁺ brains. Co-staining with phospho-S6 antibody was used to identify neurons with increased mTOR activity. Phospho-S6 positive dysplastic cells identified in the hippocampus and the red nucleus of Tsc1^c/−SynCre⁺ brains were also positive for HO-1 (Figure 6C,D). Furthermore, rapamycin treatment decreased phospho-S6 staining and HO-1 expression in Tsc1^c/−SynCre⁺ brains to levels comparable to controls (Figure 6D).

To determine whether our findings could be extended to human TSC disease, we performed IHC analysis on sections from a tuber of a 4-year-old TSC patient. Giant cells with increased mTOR activity were identified in the human tuber by phospho-S6 antibody staining (Figure 6E). CHOP and HO-1 co-labeling was observed in 44% (80 out of 115 counted) and 57% (70 out of 123 counted) of the phospho-S6 positive cells, respectively. No CHOP or HO-1 staining was observed in the peri-tuber brain regions (Figure 6F) or in the brain of a non-TSC patient with focal dysplasia (Figure 6G). In agreement with previous reports, balloon cells that are typically found in focal dysplasias showed some phospho-S6 and SMI311 staining (Lurton et al., 2002; Baybis et al., 2004). Together these findings strongly suggest that the ER and oxidative stress responses identified in vitro in the Tsc2 silenced hippocampal neurons are also present in vivo in both the Tsc knockout mouse model and in the human TSC brain.

Discussion

Despite recent progress identifying the genetic mutations and the signaling pathways associated with TSC pathology, the pathogenesis of the diverse neurological symptoms present in this disease remain poorly understood, and treatments are elusive. Here, we demonstrate that Tsc-deficiency correlates with the upregulation of specific stress-related cellular responses both in vitro and in vivo (summarized in Figure 6H). First, we detected ER overload and oxidative damage in Tsc2-deficient hippocampal neurons, in brains from Tsc1^c/⁻SynCre⁺ mice and in human TSC tissue. Second, we demonstrated that these cellular abnormalities are the consequence of constitutive mTOR activation since rapamycin treatment abolished stress responses both in vitro and in vivo. Third, we showed that neuronal stress responses in vitro increased vulnerability to cell death via activation of the mitochondrial death pathway and that silencing CHOP reduced apoptosis. The identification of similar stress responses in primary rodent hippocampal neurons with nearly complete Tsc2 gene silencing and in the human TSC brain highlight the damaging neuronal responses that result from mTOR hyperactivity.

We have recently shown that components of the TSC/mTOR pathway are differentially localized during the development of neuronal polarity, as defined by the elaboration of a single neuron and multiple dendrites (Choi et al., 2008). This fine regulation of TSC activity in neurons during the neuronal polarization process, together with the identification of multiple axon formation in neurons lacking Tsc, indicates a critical role for the TSC/mTOR pathway in axonal specification and connectivity. These findings, along with the identification of a critical role for TSC pathway in dendritic structure (Tavazoie et al., 2005), have highlighted the neuronal defects contributing to the neurological symptoms. In vivo studies using knockout mice have indeed shown that loss of TSC in neurons correlates with anatomical brain abnormalities and neurological defects (Meikle et al., 2007).

In previous reports, loss of TSC1/TSC2 complex has been implicated in increased ER stress in MEFs from Tsc1 and Tsc2 knockout mice (Ozcan et al., 2008). While MEFs display increased expression of the ER stress chaperone, GRP78, and activation of the PERK signaling pathway, we did not detect any changes in GRP78 protein level or PERK activation in neurons (Figure 3A, Suppl. Fig. 4, and data not shown). Such differences may represent cell type specific responses. Our study suggests that neurons lacking Tsc have a basal activation of CHOP via the canonical ATF4 pathway (Fawcett et al., 1999; Harding et al., 2000; Ma et al., 2002). Since many extrinsic factors such as hypoxia, hypoglycemia, and exposure to natural and experimental toxins can lead to ER stress (Koumenis, 2006; Zhang and Kaufman, 2006), Tsc-deficient neurons are more likely to be vulnerable to such insults.

Implications of ER stress for neurological manifestations of TSC

Epilepsy is by far the most common medical condition associated with TSC, occurring in 80–90% of patients. The relationship between ER stress and epilepsy is just starting to be investigated. For example, kainate-induced seizures in rats and depolarization in cultured rat hippocampal neurons lead to ER stress (Sokka et al., 2007). Moreover, increased UPR has been observed in hippocampi resected from patients with temporal lobe epilepsy (Yamamoto et al., 2006). Finally, ER overload due to abnormal trafficking of mis-folded proteins has been proposed to occur in several epilepsy-related “channelopathies” (Hirose, 2006). Together, these findings indicate that seizures can exacerbate ER stress and underlying ER stress could potentially contribute to seizures by mis-folding of synaptic proteins. This is particularly important in TSC disease because the vast majority of patients experience seizures and many of the cases are medically-intractable. A better understanding of the relationship between the TSC/mTOR pathway, ER stress and seizures may help to uncover novel therapies for intractable epilepsy in patients.

Implications of oxidative damage in Tsc-mutant brains

Accumulating evidence has revealed a cross-talk between ER and oxidative stress responses, such that excessive ROS production can contribute to UPR induction and vice-versa (Yokouchi et al., 2008). For instance, UPR-regulated genes can create an imbalance in the cellular redox status and release free radicals such as superoxide anions, leading to damage of ER-resident proteins (Verkhratsky and Petersen, 2002). The combined cellular insult that may arise from ER and oxidative stress has been proposed to further contribute to cell death by increasing the accumulation of ROS (Haynes et al., 2004). Our identification of an altered redox balance in Tsc-deficient neurons is consistent with previous reports of increased basal and growth factor-stimulated ROS in Tsc2^−/− MEFs (Finlay et al., 2005). In Tsc-deficient neurons, we found that silencing of CHOP was sufficient to reduce the oxidative stress response thus indicating a tight-linked connection between these two cellular stress pathways.

The brain is highly sensitive to oxidative stress, which has been correlated with the pathogenesis of several neurological disorders (Reynolds et al., 2007). In particular, the cellular toxicities resulting from oxidative stress, such as massive calcium overload, energy depletion and ROS production, are thought to affect neuronal function by lowering the cellular capacity to respond to stress. A number of studies have suggested that while the response to stress offers homeostatic control of cellular function, a prolonged stress response itself can be toxic (Kaufman, 2002). For example, the expression of HO-1, the rate-limiting enzyme for the degradation of the heme, is overall considered to be beneficial (Schipper, 2004b). However, excessive HO-1 can also contribute to the increase of carbon monoxide (CO) and/or free iron levels, which can have toxic effects on mitochondrial function (Ryter and Tyrrell, 2000; Baranano and Snyder, 2001; Schipper, 2004a). Expression of HO-1 in phospho-S6-positive ectopic neurons of Tsc1^c/⁻SynCre⁺ mice and in the dysplastic cells of human tubers indicates a concomitant increase in mTOR activity and occurrence of oxidative stress in vivo. Importantly, mTOR inhibition in vivo reduced CHOP and HO-1 expression. mTOR inhibitors have already been successfully used in several brain specific TSC mouse models and shown to efficiently improve survival and neurological phenotypes (Ehninger et al., 2008; Meikle et al., 2008; Zeng et al., 2008). These results together with our study addressing the molecular targets affected by rapamycin treatment in vivo provide new insights into the cellular basis of the neuronal dysfunction in TSC.

In the future, it will be important to investigate the damaging effects that could arise from HO-1 overproduction in the TSC brain, such as iron deposition or CO accumulation (Patel et al., 1996). For instance, a highly detrimental effect of iron deposition is the accumulation of free iron in the mitochondria as a result of increased oxidative stress. This would eventually cause mitochondrial dysfunction and energy production failure, affecting several ATP-dependent processes such as the uptake of excitotoxic neurotransmitters (Beal, 1998; Trushina and McMurray, 2007). Such neuronal insults can contribute to glutamate excitotoxicity, which has been implicated in the etiology of seizure-related disorders (Patel, 2002; Schipper, 2004b). In addition, excess CO production could cause dysfunction in synaptic plasticity and consequently lead to defects in cognitive development (Stevens and Wang, 1993; Zhuo et al., 1993). Similarly, the increased ERO1α expression identified in Tsc-deficient neurons could potentially exacerbate neuronal function by enhancing ROS production (Harding et al., 2003; Marciniak et al., 2004). Therefore, the combination of ER and oxidative stress detected in Tsc-deficient neurons may contribute not only to epilepsy but also to neurodevelopmental disabilities in TSC patients.

TSC1/TSC2 as key regulators of cellular stress responses

Here we demonstrate a dynamic regulation of TSC1/TSC2 complex activity downstream of the PI3K/Akt pathway in cells under ER stress. We found that under short exposure to ER stress, both Akt and mTOR are active while the TSC1/TSC2 complex is inhibited. In contrast, under persistent ER stress, mTOR is inhibited in a Tsc-dependent manner. When cells undergo ER stress, the UPR is initially activated as part of a cellular protective mechanism to circumvent ER overload and re-establish proper ER function (Zhang and Kaufman, 2006). However, if ER stress persists, it results in cell death. Thus, cellular fate under ER stress is a balance between survival and apoptotic signals. Previous studies in cell lines from breast, lung and prostate cancer have shown that the PI3K/Akt pathway can be differentially regulated to modulate cellular ER stress (Hu et al., 2004; Hosoi et al., 2007). We now show the TSC1/TSC2 plays a crucial role in the regulation of ER stress by PI3K/Akt. It is generally thought that the PI3K/Akt pathway is regulated by extrinsic signals such as the activation of growth factor receptor tyrosine kinases. Our data indicate that cells under stress can intrinsically modulate the Akt/TSC/mTOR pathway. Recent work has shown that neuronal injury, such as axotomy, can suppress mTOR and decrease protein synthesis (Park et al., 2008). Based on our findings, one possible mechanism underlying this effect may be through ER-stress mediated regulation of Akt and TSC. Exploring the cell-intrinsic upstream mechanisms regulating Akt/TSC/mTOR pathway will be important in understanding the biology of cellular stress and TSC disease. This has implications not only for TSC but also for the spectrum of neurological disorders in which either genetic mutations or environmental insults perturb ER function.

Supplementary Material

Supp1

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Supp2

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Acknowledgments

The authors thank Paul Rosenberg, Zhigang He and members of the Sahin lab for critical reading of the manuscript,, Umut Ozcan and Brendan Manning for helpful discussions, Elizabeth Boush and Karin Hoffmeister for help with FACS analysis, Lihong Bu and the Children’s Hospital Boston Mental Retardation and Developmental Disabilities Research Center for technical assistance (supported by P01HD18655). This work was supported by the National Institutes of Health grants R01 NS058956 (to MS) and P01 NS024279 (to DJK), the Hearst Fund (to ADN), the Manton Foundation and the Children’s Hospital Boston Translational Research Program (to MS), and the Tuberous Sclerosis Alliance (to LM and MS).

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