CHOP is involved in neuronal apoptosis induced by neurotrophic factor deprivation

FEBS Letters 580 (2006) 3462–3468

CHOP is involved in neuronal apoptosis induced by neurotrophic factor deprivation Seiji Tajiria,b, Shigetoshi Yanob,*, Motohiro Moriokab, Jun-ichi Kuratsub, Masataka Moria, Tomomi Gotoha,* a

Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan b Department of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan Received 10 March 2006; revised 28 April 2006; accepted 6 May 2006 Available online 15 May 2006 Edited by Vladimir Skulachev

Abstract Neurotrophic factors are essential for the survival of neurons. We found that the endoplasmic reticulum (ER) stressC/EBP homologues protein (CHOP) pathway to be activated during neurotrophic factor deprivation-induced apoptosis in PC12 neuronal cells and in primary cultured neurons, and this apoptosis was suppressed in the neurons from chop/ mice. In addition, we found that CHOP is expressed in the subventricular zone (SVZ) and striatum of the young adult mouse brain. The number of apoptotic cells in the SVZ decreased in chop/ mice. These results indicate that the ER stress-CHOP pathway plays a role in neuronal apoptosis during the development of the brain.  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; C/EBP homologues protein; Endoplasmic reticulum stress; Neurotrophic factor; Subventricular zone

1. Introduction Apoptosis, observed during either synaptogenesis or neurogenesis in the paranatal or postnatal stage of the mammalian brain, is thought to be induced by competition for the limited amount of neurotrophic factors, which are essential for survival [1–3]. The precise control of the neuronal cell number is critical for the morphological development, and also the normal function of the adult brain [4]. However, the mechanisms of those neuronal apoptosis are still not well known. Nerve growth factor (NGF) is needed for the differentiation and the survival of PC12 cells. NGF binds to TrkA receptor on the cell surface, and induces phosphorylation of the receptor. The signal transduction pathway downstream of the TrkA receptor phosphorylation includes the activations of several molecules including MAP kinase. MAP kinase pathway is involved in the NGF-induced differentiation of PC12 cells [5].

* Corresponding authors. Fax: +81 96 373 5145 (T. Gotoh); +81 96 371 8064 (S. Yano). E-mail addresses: [email protected] (S. Yano), [email protected] (T. Gotoh).

Abbreviations: CHOP, C/EBP homologues protein; ER, Endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LDH, lactate dehydrogenase; MAP2, microtubule associated protein 2; NGF, nerve growth factor; SVZ, subventricular zone; TUNEL, terminal transferase-mediated dUTP-biotin nick end labeling

However, the inhibition of the MAP kinase pathway has been reported to not induce apoptosis in differentiated PC12 cells [5] and synaptic neurons [6]. NGF also binds to a low affinity receptor p75NTR while inducing neuronal cell death. This type of neuronal apoptosis is observed during development [7]. The endoplasmic reticulum (ER) is an organelle where newly-synthesized secretory and cell membrane proteins are modified and folded. ER is highly developed in neuronal cells [8]. Protein modification and folding in the ER is impaired under various physiological or pathological conditions [9,10]. Recent studies have revealed that the perturbation of the ER functions, which is called ER stress, activates the ER stress response pathways, including translational attenuation, the induction of ER chaperones such as BiP, and the degradation of unfolded proteins by the system called ERAD, to protect cells [11]. However, when the ER functions are severely impaired, apoptosis occurs. This apoptosis is mediated by factors including C/EBP homologues protein (CHOP)/GADD153 [12]. CHOP is a transcription factor which belongs to the C/ EBP family [13]. CHOP heterodimerizes with other C/EBP family members, and CHOP-C/EBP dimer binds to the CHOP site which is distinct from the C/EBP site, and thus induces apoptosis. The target gene(s) of CHOP, which is directly involved in the apoptosis pathway, is still not known. Therefore, the apoptosis pathway downstream of CHOP is also not well known. CHOP is induced at the transcription level in response to ER stress. We found that the ER stress-CHOP pathway is activated by various stresses including brain ischemia-reperfusion and neuronal hypoxia-reoxygenation [14–17]. We also found that CHOP-induced apoptosis is mediated by the translocation of a proapoptotic Bcl-2 family member Bax from cytosol to mitochondria [18]. The withdrawal of NGF activates the mitochondria-mediated apoptosis pathway in differentiated PC12 neuronal cells [19,20]. Bax has been reported to be involved in the apoptosis of neurons in the adult brain [21–23]. As mentioned above, neuronal apoptosis in the paranatal or postnatal brain is thought to be induced by the competition for limited amounts of neurotrophic factors. We therefore investigated whether neurotrophic factor deprivation-induced apoptosis is mediated by the ER stress pathway including CHOP. We herein report that NGF deprivation in differentiated PC12 cells and neurotrophic factor deprivation in primary cultured neurons activate the ER stress-CHOP-mediated apoptosis. We also describe that the number of apoptotic cells in subventricular zone (SVZ) and striatum is reduced

0014-5793/$32.00  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.05.021

S. Tajiri et al. / FEBS Letters 580 (2006) 3462–3468

in young adult chop/ mice in comparison to wild-type mice. These results suggest that CHOP plays a crucial role in the development and maintenance of the postnatal brain.

2. Materials and methods 2.1. Antibodies Polyclonal antibodies against mouse, human GRP78/BiP, CHOP, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and monoclonal antibody against mouse CHOP were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Monoclonal antibody against mouse neuron-specific microtubule associated protein 2 (MAP2) was obtained from the Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Cell culture and neurotrophic factor deprivation Undifferentiated PC12 cells were cultured at 37 C in Dulbecco’s modified Eagle medium (DMEM) containing 4500 mg/l glucose, supplemented with 4 mM L -glutamine, 5% fetal calf serum (FCS) and 10% horse serum. The cells cultured (1 · 104 cells) were grown until 50% confluent in 60 mm-diameter collagen-coated plates, and were induced to undergo differentiation by treating them with 50 ng/ml NGF (Wako Pure Chemical Industries, Osaka, Japan) in DMEM/F-12 medium with 2% FCS and 0.25% insulin–transferrin–selenium-X supplement (Invitrogen, Carlsbad, CA, USA) for 7 days. Then the cells were cultured in the medium (DMEM/F-12 medium with 2% FCS and 0.25% insulin–transferrin–selenium-X supplement) without NGF for the indicated periods to trigger cell death. Primary cultured cells were prepared from the cortex of embryonic wild-type or chop/ mice at days 16–18 of gestation (E16–18) as described [17]. The cells from cerebral cortexes were suspended in a Neurobasal medium (Invitrogen) with 0.5 mM L -glutamine, 25 lM glutamate, 100 units/ml penicillin, 50 lg/ml streptomycin and 2% B27 supplement containing neurotrophic factors (Invitrogen). The cells were plated at a density of 1 · 105 cells/well on poly-D -lysine-coated dishes (Clontech Laboratories, Inc., Palo Alto, CA, USA) and cultured for 10 days either with cytosine arabinocide (Ara-C). Cell death was then triggered by removal of B27 supplement from the medium. 2.3. Quantitative real-time RT-PCR and semi-quantitative RT-PCR analysis Total RNA from either differentiated PC12 cells or primary cultured cortical neurons from wild-type and chop/ mice was isolated using the acid guanidium thiocyanate–phenol–chloroform extraction procedure [24]. A real-time RT-PCR analysis was performed with iCycler real-time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA) using QuantiTect SYBR Green RT-PCR kit (Qiagen, Hilden, Germany). The amplified products were verified as single expected size bands by agarose gel electrophoresis. The results obtained with 10–105 copies of GAPDH cDNA were used as a standard curve, and all results were standardized to the levels of GAPDH. The primers used for PCR were as follows: sense primer, 5 0 -GAGATTGTTCTGGTTGGCGGATCTACTC-3 0 and antisense primer, 5 0 -CCATATGCTACAGCCTCATCTGGGTT-3 0 for rat BiP (GenBank Accession No. M14050); sense primer, 5 0 -CCTGAAAGCAGAAACCGGTC-3 0 and antisense primer, 5 0 -CCTCATACCAGGCTTCCAGC-3 0 for rat CHOP (GenBank Accession No. NM_024134); sense primer, 5 0 TCTGTATGAGCCCTGAGTCCTACCT-3 0 and antisense primer, 5 0 -GGTCATAAGGTTTGGGTCGAGAACCAC-3 0 for rat ATF4 (GenBank Accession No. NM_024403); sense primer, 5 0 -CCTCTGGAGGTGCCAAGGAG-3 0 and antisense primer, 5 0 -TTGGATCCTGCTGGGCGCTGTGCTGTC-3 0 for rat TrkA (GenBank Accession No. NM_012731) [25]; sense primer, 5 0 -GTCGTGGGCCTTGTGGCC-3 0 and antisense primer, 5 0 -CTGTGAGTTCACACTGGGG-3 0 for rat p75NTR (GenBank Accession No. NM_012610) [25]; sense primer, 5 0 -AAACAGAGTAGCAGCGCAGACTGC-3 0 and antisense primer, 5 0 -GGATCTCTAAAACTAGAGGCTTGGTG-3 0 for mouse XBP1 (GenBank Accession No. NM_013842) [26], sense primer, 5 0 -GAACATCATCCCTGCATCCA-3 0 and antisense primer, 5 0 -CCAGTGAGCTTCCCGTTCA-3 0 for rat GAPDH (GenBank Accession No. NW_047622); sense primer, 5 0 -GAAAGGATGGTTAATGATG-

3463 CTGAGAAG-3 0 and antisense primer, 5 0 -GTCTTCAATGTCCGCATCCTG-3 0 for mouse BiP (GenBank Accession No. NM_022310); sense primer, 5 0 -CATACACCACCACACCTGAAAG-3 0 and antisense primer, 5 0 -CCGTTTCCTAGTTCTTCCTTGC-3 0 for mouse CHOP (GenBank Accession No. NT_081856); sense primer, 5 0 -CAC-GAAATCCAGCAGCAGTGT-3 0 and antisense primer, 5 0 -GGCTGCAAGAATGTAAAGGGG-3 0 for mouse ATF4 (GenBank Accession No. BC085169); sense primer, 5 0 -TGGCACAGTCAAGGCTGAGA3 0 and antisense primer, 5 0 -CTTCTGAGTGGCAGTGATGG-3 0 for mouse GAPDH (GenBank accession number BC083149). All expected PCR products, except for rat and mouse GAPDH, contained plural exons. For a semiquantitative analysis, RT-PCR was performed using the above primer sets and the Superscript One-Step RT-PCR System (Invitrogen). 2.4. Immunoblot analysis Differentiated PC12 cells and primary cultured neurons were homogenized in 50 mM Tris–HCl containing 150 mM NaCl, 1% Triton X-100. After centrifugation, the supernatants were used for an immunoblot analysis. Immunodetection was performed as described [18]. 2.5. Immunocytochemical and immunohistochemical analysis For the immunocytochemical analysis of PC12 cells or primary cultured cortical cells were performed as described [17]. For the immunohistochemical analysis, mice were anesthetized by them giving sodium pentobarbital (50 ml/g, i.p.), then the whole brain was removed. Immunohistochemical analysis using anti-rabbit CHOP polyclonal antibody as the first antibody and the second antibody conjugated with peroxidase (Amersham Pharmacia Biotech) was performed as described [17]. The peroxidase activity was visualized by incubation with a 3,3 0 -diaminobenzidine solution. 2.6. Detection of apoptosis Lactate dehydrogenase (LDH) release from damaged cells was measured as an index of cell destruction, using a LDH cell damage assay kit (Wako Pure Chemical Industries). The disruption of the mitochondrial membrane potential is characteristic of apoptotic cells. To analyze mitochondrial membrane depolarization, the cells were stained with a mitochondrial membrane potential-indicating dye DePsipher (Trevigen Inc., Gaithersburg, MD, USA) as previously described [14]. To analyze the morphological changes of nuclei, the cells were stained with Hoechst dye 33258 as previously described [15]. The detection of apoptosis in vivo was performed by the terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) method using an in situ apoptosis detection kit (Takara, Otsu, Japan) as previously described [17]. TUNEL signals were amplified with Streptavidin Alexa Fluora 488 conjugate (Molecular Probes) using the TSA Biotin System (Perkin–Elmer Life Sciences Inc., Boston, MA, USA). Nuclear staining with propidium iodide (PI) was performed as previously described [17]. 2.7. Statistical analysis Quantitative results were expressed as the means ± S.D. and analyzed using Student’s t-test. The significance in difference was assigned at a level of less than a 5% probability (P < 0.05).

3. Results 3.1. NGF deprivation-induced apoptosis in differentiated PC12 cells Differentiation of PC12 cells were induced by treatment with NGF for 7 days. Differentiation was confirmed by the morphological changes, such as the outgrowth of neurites and the flattening of cell bodies. Twenty-four hours after NGF deprivation from the medium of differentiated cells, morphological apoptotic changes, such as the disappearance of neurites, roundshaped cells, shrunken cells and detachment from the culture bottom, were observed in phase-contrast image (Fig. 1A). LDH release from the cells was used as an index of cell death

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Fig. 1. Induction of NGF deprivation-induced apoptosis in differentiated PC12 cells. (A) Differentiation of PC12 cells were induced by treatment with NGF (50 ng/ml) for 7 days. After differentiation, the cells were cultured with (a) or without (b) NGF for 24 h. Phasecontrast images are shown. Bar, 20 lm. (B) Differentiated PC12 cells were cultured with or without NGF (50 ng/ml), or with tunicamycin (Tm, 1 lg/ml) plus NGF (50 ng/ml) for the indicated periods. The LDH activity of the culture medium was measured, and the results are shown as the means ± S.D. (n = 4). The LDH activity of cell extract before treatment is set at 100%. (C) Differentiated PC12 cells were treated as in (A), and then were stained with a mitochondrial membrane potential-indicating dye DePsipher. The red fluorescence represents the intact potential, while the green fluorescence represents the disrupted potential. The arrowhead indicates a cell negative for membrane potential. Bars, 20 lm. (D) Differentiated PC12 cells were treated as in (A), then were stained with Hoechst dye 33258. The fluorescence images and phase-contrast images of the same fields are shown. The arrowheads indicate cells showing apoptotic changes. Bar, 20 lm.

(Fig. 1B). When the cells were cultured without NGF, LDH release began at 6 h and increased thereafter. LDH release induced by NGF deprivation was delayed compared to the cells treated with an ER stress-inducer tunicamycin, and reached to about 80% of that induced by tunicamycin at 24 h. Apoptosis was confirmed by staining with a mitochondria membrane potential indicating dye (Fig. 1C) and nuclear staining (Fig. 1D). When the cells were cultured without NGF for 24 h, most cells (70%) lost mitochondrial membrane potential, thus indicating cell death. Nuclear condensation, nuclear fragmentation and chromatin condensation, which are characteristic changes of apoptotic cells, were observed in the nuclear staining of the cells cultured without NGF. These results show that NGF deprivation induces apoptosis in differentiated PC12 cells. 3.2. Activation of the ER stress-CHOP pathway by NGF deprivation Next we examined whether the ER stress pathway is activated in NGF deprivation-induced apoptosis (Fig. 2). CHOP mRNA was detected at a low level before deprivation, induced by about 4-fold at 3–6 h, and gradually decreased thereafter

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Fig. 2. Induction of ER stress-associated mRNAs in differentiated PC12 cells treated with the deprivation of NGF. (A) Differentiated PC12 cells were cultured without NGF for the indicated periods on the top, and total RNAs were subjected to the RT-PCR analysis for CHOP, BiP, ATF4 and GAPDH. (B) The results of (A) and two other independent experiments were calculated, and were standardized to the levels of GAPDH. The results at 0 h are set as 1.0, and are shown as the means ± S.D. (C) The differentiated PC12 cells were cultured without NGF for the indicated periods on the top, then the RT-PCR analysis was performed using a primer set encompassing the splicedout region in XBP1 mRNA. The PCR products were resolved on 3% agarose gel to separate the unspliced (XBP1u) and spliced (XBP1s) XBP1 mRNAs. The differentiated PC12 cells treated with tunicamycin (Tm, 1 lg/ml) for 6 h were used as a positive control. (D) A RT-PCR analysis for TrkA, p75NTR mRNAs and GAPDH was performed using total RNAs from differentiated PC12 cells cultured without NGF for the indicated periods. (E) Differentiated PC12 cells were cultured without NGF for the indicated periods on the top, and whole cell extracts were subjected to an immunoblot analysis using antibodies against CHOP (diluted 1000-fold), BiP (diluted 1000-fold) and GAPDH (diluted 1000-fold). Whole cell extracts of PC12 cells treated with tunicamycin (Tm, 1 lg/ml) for 12 h were used as a positive control.

(Fig. 2A and B). BiP, an ER-resident Hsp70 family member, is known to be induced by ER stress. BiP mRNA was present at a low level before deprivation, and it was induced by about 3-fold at 3–6 h, and thereafter it gradually decreased. mRNA for ATF4 which is one of the transcriptional activator of the chop gene, was detected at a low level before deprivation, and it was induced by about 4-fold at 3 h, and then it rapidly decreased thereafter, and finally reached a level close to the control level at 12 h. XBP1 is a member of the CREB/ATF family of transcription factors, which is specifically induced

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by ER stress. Upon ER stress, XBP1 mRNA is subjected to unique and specific splicing by activated IRE1, one of the ER stress sensors on the ER membrane, and it activates the ER stress-response genes [10]. Therefore, the induction and splicing of XBP1 mRNA is a good marker for the activation of the ER stress pathway. As shown in Fig. 2C, the XBP1 unspliced form was detected at a low level, and the spliced form was not detected before the deprivation of NGF. The XBP1 unspliced form was slightly induced at 1 h and then decreased at 2 h. In contrast, the XBP1 spliced form was slightly detected at 1 h and then it increased at 2 h. Fig. 2D shows the expression of mRNAs for TrkA and p75NTR, which are receptors for NGF, to not be greatly influenced by the deprivation of NGF. These results show that NGF deprivation activates the ER stress pathway including CHOP. We next examined whether the ER stress pathway is activated in the NGF deprivation-induced apoptosis at the protein level (Fig. 2). CHOP was barely detected before NGF deprivation, however, it began to increase at 6 h, reached a maximum level at 18 h, then it decreased slightly at 24 h. The induced level at 18 h was about 30% of the level in the cells treated with tunicamycin for 12 h. BiP was detected at a low level before NGF deprivation, but it began to increase at 6 h, and thereafter remained mostly unchanged until 24 h. The induced level at 24 h was about half of the level in the cells treated with tunicamycin for 12 h. These results show that the ER stress pathway is activated in differentiated PC12 cells by NGF deprivation. 3.3. Induction of CHOP-dependent apoptosis by deprivation of neurotrophic factor in primary cultured neuron We next examined whether the deprivation of neurotrophic factor induces apoptosis in primary cultured neurons. Ara-C was added to the medium to remove glial cells [17]. As we mentioned in Section 2, B27 supplement for primary cultured neurons contains several neurotrophic factors (and some radical scavengers). Therefore, we asked whether the deprivation of B27 supplement induces ER stress- and CHOP-mediated apoptosis in primary cultured neurons. The deprivation of B27 supplement for 6 h induced apoptosis in primary cultured neurons (Fig. 3A). In the cells deprived for B27 supplement for 6 h, the disappearance of neurites and cell shrinking was observed in neuron-specific MAP2 staining, while nuclear shrinking and chromatin condensation were observed in nuclear staining. As mentioned above, B27 supplement contains several components. It is not clear which component(s) is needed for the survival of neurons at present. We next asked whether the ER stress pathway is activated in primary cultured neurons by the deprivation of B27 supplement, using a real-time RT-PCR analysis (Fig. 3B). Primary cultured neurons were prepared from wild-type and chop/ mice. In wild-type cells, CHOP mRNA was detected at a low level before deprivation, began to increase at 30 min, reached a maximal level at 1 h, and then decreased at 2 h. BiP mRNA was barely detected before deprivation, began to increase at 30 min, reached a maximal level at 1 h, and then decreased at 2 h. In contrast, the induction of BiP mRNA by B27 supplement deprivation was markedly suppressed in chop/ cells. The induction of BiP mRNA by tunicamycin was also suppressed in chop/ cells. Marciniak et al. [27] reported that CHOP inhibits ER stress-induced attenuation of protein synthesis by dephosphorylation of the a-subunit of translation initiation factor 2 (eIF2a) through the induction of GADD34.

Fig. 3. Induction of ER stress-associated mRNAs and apoptosis in primary cultured neurons treated with B27 supplement-deprivation. (A) Primary cultured neuronal cells from wild-type mice at embryonic 16–18 days were cultured with arabinocide (Ara-C) and B27 supplement for 10 days, then were cultured further for 0 or 6 h without B27 supplement. Next, the cells were fixed and immunostained with antibodies against neuronal cell-specific MAP2 (diluted 1000-fold) and stained with Hoechst dye 33258. The results of each staining and their merged images of the same fields are shown. The arrows show apoptotic cells. Bar, 20 lm. (B) Primary cultured neurons from wildtype (white bars) and chop/ mice (black bars) were cultured with AraC and B27 supplement for 10 days, then were cultured further without B27 supplement for the indicated periods, and total RNAs were subjected to a real time RT-PCR analysis for CHOP and BiP using each specific primer indicated in Section 2. The results from three independent experiments were calculated, and then were standardize to the levels of GAPDH. The results at 0 h in wild-type neurons are set as 1.0, and are shown as the means ± S.D. (C) The primary cultured neurons from wild-type and chop/ mice were cultured with Ara-C and B27 supplement for 10 days, then were cultured without B27 supplement for the indicated periods on the top, and stained with anti-MAP2 antibody (diluted 1000-fold). When the cells were cultured without B27 supplement, a portion of the cells was apparently dead and detached from the dish and the number of attached cells decreased. Bar, 20 lm.

They also showed that the release of translational attenuation by CHOP induction under ER stress conditions leads to the accumulation of high molecular weight protein complex in ER, and enhances ER stress. This may be the reason why the activation of the ER stress pathway upstream of CHOP was partially suppressed in chop/ mice. These results show that the deprivation of neurotrophic factor(s) activates the ER stress pathway including CHOP. We next examined whether neurotrophic factor deprivationinduced apoptosis in primary cultured neurons depends on CHOP. As shown in Fig. 3C, wild-type cells became roundshaped and then lost most neurites at 3 h. Six hours after deprivation, most cells were detached from the dishes and the remaining cells had obviously shrunken. These morpholog-

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ical changes suggested apoptosis. In the case of chop/ cells, some cells still preserved neurites and many more attached cells were observed at 6 h after deprivation. These results show that the apoptosis induced by the neurotrophic factor deprivation in primary cultured neuron is CHOP-dependent. 3.4. Induction of apoptosis by CHOP in neurons of SVZ and striatum of young adult mice In the brain, an excess number of neurons are produced during development, and those excess neurons later disappear through apoptosis, due to competition for limited amounts of neurotrophic factors [2,3]. Recently, it was revealed that neuronal progenitor cells exist in the SVZ of the cerebrum even in adult mice [28]. These progenitor cells always need stimulation by neurotrophic factors for their survival. As mentioned above, neurotrophic factor deprivation induces ER stress-mediated apoptosis in primary cultured neuronal cells. Therefore, we investigated whether the apoptosis of neuronal progenitor cells and/or striatum neurons in adult mice is mediated by the ER stress-CHOP pathway. Immunostaining of CHOP was performed in the SVZ and striatum of young adult (P14) mice (Fig. 4). Many neuronal cells, whose nuclei were positive for CHOP, were observed in the SVZ and striatum of wild-type mice (Fig. 4A and B). Most CHOP-positive cells showed nuclear shrinking, thus suggesting apoptosis. In chop/ mice, no such cells were observed (Fig. 4C and D). We next examined apoptosis using the TUNEL method (Fig. 5). In the SVZ of wild-type mice, many TUNEL-positive cells were detected, and these cells showed nuclear shrinking in

Fig. 5. Suppression of apoptosis of the SVZ in chop/ mice. (A) In the SVZ of wild-type and chop/ mice brain at postnatal day 21, apoptosis was detected with the TUNEL method. The magnified images of the squares are also shown. The results of TUNEL staining, nuclear staining with propidium iodide (PI) and their merged images of the same fields are shown. Bars, 20 lm. (B) TUNEL-positive cells in the SVZ of wild-type and chop/ mice in each hemisphere were counted and were shown as the means ± S.D. (*; P < 0.05, n = 4). (C) The total brain weight of male wild-type and chop/ mice, female wild-type and chop/ mice were measured on postnatal day 14 and 42, and the results are shown as the means ± S.D. (n = 7).

PI staining (Fig. 5A). In contrast, TUNEL-positive cells were only rarely observed in the SVZ of chop/ mice. The number of TUNEL-positive cells per cerebral hemisphere in chop/ mice was about 25% of that in the wild-type mice (Fig. 5B). The brain weight was higher in chop/ mice than in wild-type animals (Fig. 5C). These results strongly suggest that the apoptosis of neuronal cells in adult brain is mediated by the ER stress-CHOP pathway, thus supporting the idea that apoptosis is involved in the development or maintenance of the brain after birth by eliminating any excess neurons.

Fig. 4. Expression of CHOP in the SVZ and the striatum in the mouse brain at postnatal day 14. Coronal slices (5 lm) of wild-type (A and B) and chop/ mice (C and D) brain at postnatal day 14 were immunostained with anti-mouse CHOP monoclonal antibody (diluted 200-fold). Representative photographs of the SVZ (A and C) and the striatum (B and D) are shown. Magnified images of the squares are also shown. The arrowheads show cells positive for CHOP. Bar, 20 lm.

4. Discussion In this study, we found that NGF deprivation induces ER stress-mediated apoptosis in differentiated PC12 neuronal cells. We also found that the removal of B27 supplement from the medium induces CHOP-dependent apoptosis in primary cultured neurons. B27 supplement is used as an alternative

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for fetal calf serum, and it contains several hormones (corticosterone, progesterone, triodo-1-thyronine and insulin), several radical scavengers and some other components [29]. It is not known about the anti-ER stress-induced apoptosis effects of these hormones in neuronal cells at present. There have been several reports regarding the mechanism of NGF deprivation-induced apoptosis [5,30]. We need to investigate which composition(s) of B27 supplement is crucial for the survival. Reactive oxygen species can be the inducer of ER stress [27]. Therefore, we also need to consider about the anti-apoptotic functions of radical scavengers. Deckwerth and Johnson [31] reported that glucose uptake in differentiated PC12 cells falls rapidly to 35% of the control level within 6 h after NGF deprivation. A decrease in the glucose uptake can disturb protein modification and cause ER stress. Then the expression of ER stress-related molecules including CHOP is induced [32,33]. In fact, we found that chop/ mouse-derived neurons are resistant to neurotrophic factor deprivation-induced apoptosis. Aoki et al. [34] showed that BiP mRNA is induced by NGF deprivation in PC12 cells. Under our conditions, neurotrophic factor deprivation-induced apoptosis was not completely suppressed in the chop/ primary cultured neurons. At least three pathways of ER stress-induced apoptosis are present. They are the CHOP pathway, the TRAF2-ASK1JNK pathway and the caspase 12 pathway [10]. We speculate that other two pathways except the CHOP pathway are involved in the late apoptosis of chop/ neurons. The precise mechanism of how neurotrophic factor deprivation induces ER stress remains to be investigated. We also found that CHOP is expressed in SVZ and the striatum of young adult mice, and that the nuclei of CHOPexpressing showed apoptotic changes. Apoptosis was suppressed in the SVZ of chop/ mice. From our present results, we speculate that the ER stress-CHOP pathway is therefore involved in the development and/or maintenance of the brain after birth, by its role in the removal of excess neurons. Acknowledgments: We thank Dr. Shizuo Akira (Osaka University, Japan) for the chop/ mice. We thank our colleagues for valuable suggestions and discussions. We also thank R. Shindo and M. Obata for technical assistance, and Y. Indo for secretarial assistance. This work was supported in part by Grants-in-Aid (Nos. 14037257 and 17390096 to M.M., 16590233 to T.G. and 16591448 to S.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant (to T.G.) from the Inamori Foundation.

References [1] Kuan, C.Y., Roth, K.A., Flavell, R.A. and Rakic, P. (2000) Mechanisms of programmed cell death in the developing brain. Trends Neurosci. 23, 291–297. [2] Levi-Montalcini, R. and Angeletti, P.U. (1968) Nerve growth factor. Physiol. Rev. 48, 534–569. [3] Alvarez-Buylla, A., Garcia-Verdugo, J.M. and Tramontin, A.D. (2001) A unified hypothesis on the lineage of neural stem cells. Nat. Rev. Neurosci. 2, 287–293. [4] Gould, E., McEwen, B.S., Tanapat, P., Galea, L.A. and Fuchs, E. (1997) Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J. Neurosci. 17, 2492–2498. [5] Yao, R. and Cooper, G.M. (1995) Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267, 2003–2006. [6] Greenlund, L.J., Deckwerth, T.L. and Johnson Jr., E.M. (1995) Superoxide dismutase delays neuronal apoptosis: a role for

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[7] [8]

[9]

[10] [11] [12] [13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

reactive oxygen species in programmed neuronal death. Neuron 14, 303–315. Frade, J.M., Rodriguez-Tebar, A. and Barde, Y.A. (1996) Induction of cell death by endogenous nerve growth factor through its p75 receptor. Nature 383, 166–168. Majdan, M., Lachance, C., Gloster, A., Aloyz, R., Zeindler, C., Bamji, S., Bhaker, A., Belliveau, D., Fawcett, J., Miller, F.D. and Barker, P.A. (1997) Transgenic mice expressing the intracellular domain of the p75 neurotrophin receptor undergo neuronal apoptosis. J. Neurosci. 17, 6988–6998. Kaufman, R.J., Scheuner, D., Schro¨der, M., Shen, X., Lee, K., Liu, C.Y. and Arnold, S.M. (2002) The unfolded protein response in nutrient sensing and differentiation. Nat. Rev. Mol. Cell Biol. 3, 411–421. Oyadomari, S., Araki, E. and Mori, M. (2002) Endoplasmic reticulum stress-mediated apoptosis in pancreatic b-cells. Apoptosis 7, 335–345. Yoshida, H., Matsui, T., Hosokawa, N., Kaufman, R.J., Nagata, K. and Mori, K. (2003) A time-dependent phase shift in the mammalian unfolded protein response. Dev. Cell 4, 265–271. Ferri, K.F. and Kroemer, G. (2001) Organelle-specific initiation of cell death pathways. Nat. Cell Biol. 3, E255–E263. Ron, D. and Habener, J.F. (1992) CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 6, 439–453. Oyadomari, S., Takeda, K., Takiguchi, M., Gotoh, T., Matsumoto, M., Wada, I., Akira, S., Araki, E. and Mori, M. (2001) Nitric oxide-induced apoptosis in pancreatic b cells is mediated by the endoplasmic reticulum stress pathway. Proc. Natl. Acad. Sci. USA 98, 10845–10850. Gotoh, T., Oyadomari, S., Mori, K. and Mori, M. (2002) Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. J. Biol. Chem. 277, 12343–12350. Oyadomari, S., Koizumi, A., Takeda, K., Gotoh, T., Akira, S., Araki, E. and Mori, M. (2002) Targeted disruption of the Chop gene delays endoplasmic reticulum stress-mediated diabetes. J. Clin. Invest. 109, 525–532. Tajiri, S., Oyadomari, S., Yano, S., Morioka, M., Gotoh, T., Hamada, J-I., Ushio, Y. and Mori, M. (2004) Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ. 11, 403–415. Gotoh, T., Terada, K., Oyadomari, S. and Mori, M. (2004) hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOPinduced apoptosis by inhibiting translocation of Bax to mitochondria. Cell Death Differ. 11, 390–402. Putcha, G.V., Deshmukh, M. and Johnson Jr., E.M. (1999) BAX translocation is a critical event in neuronal apoptosis: regulation by neuroprotectants, BCL-2, and caspases. J. Neurosci. 19, 7476– 7485. Vyas, S., Juin, P., Hancock, D., Suzuki, Y., Takahashi, R., Triller, A. and Evan, G. (2004) Differentiation-dependent sensitivity to apoptogenic factors in PC12 cells. J. Biol. Chem. 279, 30983–30993. Deckwerth, T.L., Elliott, J.L., Knudson, C.M., Johnson Jr., E.M., Snider, W.D. and Korsmeyer, S.J. (1996) BAX is required for neuronal death after trophic factor deprivation and during development. Neuron 17, 401–411. Lindsten, T., Golden, J.A., Zong, W.X., Minarcik, J., Harris, M.H. and Thompson, C.B. (2003) The proapoptotic activities of Bax and Bak limit the size of the neural stem cell pool. J. Neurosci. 23, 11112–11119. Sun, W., Winseck, A., Vinsant, S., Park, O.H., Kim, H. and Oppenheim, R.W. (2004) Programmed cell death of adultgenerated hippocampal neurons is mediated by the proapoptotic gene Bax. J. Neurosci. 24, 11205–11213. Chomczynski, P. and Sacchi, N. (1997) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Ann. Biochem. 162, 156–159. Gascon, E., Vutskits, L., Zhang, H., Barral-Moran, M.J., Kiss, P.J., Mas, C. and Kiss, J.Z. (2005) Sequential activation of p75 and TrkB is involved in dendritic development of subventricular zone-derived neuronal progenitors in vitro. Eur. J. Neurosci. 21, 69–80.

3468 [26] Calfon, M., Zeng, H., Urano, F., Till, J.H., Hubbard, S.R., Harding, H.P., Clark, S.G. and Ron, D. (2002) IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92–96. [27] Marciniak, S.J., Yun, C.Y., Oyadomari, S., Nvoa, I., Zhang, Y., Jungreis, R., Nagata, K., Harding, H.P. and Ron, D. (2004) CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev. 18, 3066– 3077. [28] Alvarez-Buylla, A. and Garcia-Verdugo, J.M. (2002) Neurogenesis in adult subventricular zone. J. Neurosci. 22, 629–634. [29] Brewer, G.J., Torricelli, J.R., Evege, E.K. and Price, P.J. (1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576.

S. Tajiri et al. / FEBS Letters 580 (2006) 3462–3468 [30] Chao, M.V. and Bothwell, M. (2002) Neurotrophins: to cleave or not to cleave. Neuron 33, 9–12. [31] Deckwerth, T.L. and Johnson Jr., E.M. (1993) Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor. J. Cell Biol. 123, 1207–1222. [32] Kaufman, R.J. (2002) Orchestrating the unfolded protein response in health and disease. J. Clin. Invest. 110, 1389–1398. [33] Oyadomari, S. and Mori, M. (2004) Roles of CHOP/ GADD153 in endoplasmic reticulum stress. Cell Death Differ. 11, 381–389. [34] Aoki, T., Koike, T., Nakano, T., Shibahara, K., Kondo, S., Kikuchi, H. and Honjo, T. (1997) Induction of Bip mRNA upon programmed cell death of differentiated PC12 cells as well as rat sympathetic neurons. J. Biochem. (Tokyo) 121, 122–127.