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Increased GADD34 in oligodendrocytes in Alzheimer’s disease
Neuroscience Letters 602 (2015) 50–55
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Increased GADD34 in oligodendrocytes in Alzheimer’s disease Yasuyuki Honjo a,b , Takashi Ayaki b , Takami Tomiyama c , Tomohisa Horibe a , Hidefumi Ito d , Hiroshi Mori e , Ryosuke Takahashi b , Koji Kawakami a,∗ a
Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Japan Department of Neurology, Graduate School of Medicine, Kyoto University, Japan Department of Neurology and Neuroscience, Osaka City University Medical School, Japan d Department of Neurology, Graduate School of Medicine, Wakayama Medical University, Japan e Department of Clinical Neuroscience, Osaka City University Medical School, Japan b c
h i g h l i g h t s • GADD34 was increased in neurons and oligodendrocytes in human AD brains. • GADD34 was significantly increased in the early stage of APP transgenic mice. • GADD34 and GST--immunopositive oligodendrocytes were co-localized in the AD.
a r t i c l e
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Article history: Received 13 May 2015 Received in revised form 21 June 2015 Accepted 29 June 2015 Available online 2 July 2015 Keywords: Alzheimer’s disease Oligodendrocytes GADD34 ER stress
a b s t r a c t Alzheimer’s disease (AD) is characterized by the accumulation of amyloid- (A) and abnormally phosphorylated tau which contribute to endoplasmic reticulum (ER) stress. Previous studies demonstrated that A and a truncated fragment of A induced death of oligodendrocytes in vitro. In addition, a tripletransgenic AD mouse model exhibits significant region-specific alterations in myelination patterns at time points preceding the appearance of A accumulation. The growth arrest and DNA damage protein (GADD) 34 is up-regulated in response to ER stress and regulates subunit of protein phosphatase 1 (PP1) complex that dephosphorylates eukaryotic translation initiator factor 2␣ (elF2␣). Thus, GADD34 is known as an ER stress regulator or ER stress marker. In a recent study, GADD34 was induced in the spinal cord glial cells of an amyotrophic lateral sclerosis (ALS) mouse model. It is interesting that reduced GADD34 delayed the onset of ALS and prolonged the survival period in the mouse model. In this study, we have demonstrated that GADD34 was increased in neurons of human AD brains. Additionally, this finding was also observed in oligodendrocytes in human AD brains. Furthermore, we showed that the expression levels of GADD34 in neurons and oligodendrocytes were significantly increased in the early stage of AD in the mouse model. As oligodendrocytes were more affected in the early stages of AD in this experimental model, ER stress of A oligomers may be more related to oligodendrocytes than to neurons. These results suggest that GADD34 could be a therapeutic target for preventing ER stress in neuronal cells in AD. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease, but there is currently no effective treatment available because the etiology or mechanism of AD is still unclear. The neurodegenerative diseases are characterized by the accumulation of aggregated unfolded proteins and these accumulated proteins con-
∗ Corresponding author at: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Yoshida Konoecho, Sakyoku, Kyoto 606-8501, Japan. Fax: +81 75 753 4469. E-mail address: [email protected] (K. Kawakami). http://dx.doi.org/10.1016/j.neulet.2015.06.052 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
tribute to endoplasmic reticulum (ER) stress [1]. ER stress signaling, otherwise known as the unfolded protein response, is triggered by an increased load of misfolded proteins in the organelle [2]. In AD, the aggregated unfolded proteins are characterized by the accumulation of amyloid- (A) and abnormally phosphorylated tau [3]. At that time, the dysfunction of oligodendrocytes due to A was reported [4–8]. A and a truncated fragment of A induced oligodendrocytes death of in vitro in a dose-dependent manner with similar potencies [4]. A-induced oligodendrocyte death was accompanied by nuclear DNA fragmentation, mitochondrial dysfunction, and cytoskeletal disintegration. The presence of both A-induced activation of redox-sensitive transcription factors and oxidative stress in A-mediated oligodendrocytes suggest
Y. Honjo et al. / Neuroscience Letters 602 (2015) 50–55
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Table 1 Clinical profiles of all AD patients and control Case
Sex
Age (years)
PMI (h)
Case of death
Co-morbidities
Control 1 2 3 4 5
M F M M M
62 91 94 62 63
3 NA NA NA NA
Pancreas cancer NA NA Heart failure Heart failure
Chronic hepatitis None Chronic heart failure Arrhythmia Chronic heart failure
84 89 94 77 67
4 2.5 15 1.5 18
Heart failure Sepsis Renal failure Pneumonia Leukemia
Anemia Chronic heart failure Hypertension Chronic hepatitis None
Alzheimer disease (AD) 1 F 2 F 3 F 4 F 5 M
Clinical stage
Break stage
Advanced Advanced Advanced Advanced Advanced
VI V VI VI VI
M = male, F = female, Age = age at death, PMI = post-mortem interval, NA = not available.
that oxidative injury contributes to A cytotoxicity in oligodendrocytes [4]. The presenilin 1 (M146 V) mutation predisposes mouse oligodendrocyte precursor cells to A-induced alterations in cell differentiation in vitro. Furthermore, a myelination defect and myelin basic protein distribution subcellular mislocalization triggered by presenilin 1 (M146 V) have been previously reported [5]. AD has not been widely considered a disease of white matter, but recent evidence suggests the existence of abnormalities in myelination patterns and myelin attrition in AD brains [6–8]. The growth arrest and DNA damage protein (GADD) 34 is a multifunctional protein up-regulated in response to cellular stress and is believed to mediate DNA repair and restore protein [9–12]. ER stress activates PKR-like ER kinase (PERK), which coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2␣ (eIF2␣). GADD34 can regulate subunit of PP1 complex that dephosphorylates eIF2␣. In addition, GADD34 can medicate DNA repair and restore protein synthesis [9,12]. Thus, GADD34 is believed to be an ER stress regulator or ER stress marker. Previous studies indicated that GADD34 was increased in ischemic neurons [10–12]. Furthermore, GADD34 was increased in the spinal cord glial cells in an ALS mouse [13,14]. Interestingly, this phenomenon was observed early after detectable onset of the disease, and reduced levels of active GADD34 markedly ameliorated disease [14]. However, reports of GADD34 in AD brain are limited. In this study, we revealed that GADD34 was increased in neurons and oligodendrocytes of human AD brains. Similar increases were also observed in the brain of amyloid precursor protein (APP) transgenic mice in the early stages of pathological onset of AD. 2. Materials and methods 2.1. Postmortem human brain Postmortem brain specimens from five patients with AD (67–94 years old) and five without AD (normal control brains; 62–94 years old) were utilized in this study as shown in Table 1. This study was approved by the ethics committee (approval number E1758) of Kyoto University. Patient diagnosis was determined by pathological examination. Specimens from the hippocampus and frontal lobe were obtained from the autopsied brains of control and AD patients. Brains were fixed in 10% neutral formalin at room temperature. Paraffin-embedded tissue blocks were prepared and cut into 6-mthick sections as previously described [15,16]. 2.2. Transgenic mouse brain The transgenic mouse model of A oligomers (E693 delta mutation in APP) has been previously described [17,18]. Briefly, mouse
brains were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (5 m thick) [17,18]. 2.3. Immunohistochemistry Immunohistochemical staining was performed as previously described [15,16]. Immunohistochemical staining for GADD34 was performed using polyclonal rabbit anti-GADD34 antibody purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), as reported previously [10,12]. We counted the number of GADD34immunopositive neurons in the frontal lobe (layer II–VI) and detate gyrus of hippocampal formation. In addition, we counted the number of GADD34-immunopositive oligodendrocytes in the white matter. 2.4. Double staining of GADD34 and a marker of oligodendrocytes in tissue sections from human and transgenic mouse brain samples To confirm the anatomical relationship between GADD34 and oligodendrocytes, we performed double-staining studies using mouse anti-GST- (marker of mature oligodendrocytes) and rabbit anti-GADD34 antibodies as described previously [15,16]. Double staining was performed using mouse anti-GST- antibody purchased from Vector Laboratories (Burlingame, CA, USA). Approximately, 200 GST--immunopositive oligodendrocytes in the immunostained sections in white matter of the frontal lobe were selected from three patients with AD. The number of GADD34-immunopositive oligodendrocytes in the selected GST--immunopositive oligodendrocytes was then counted for each patient. In the transgenic mice, approximately, 200 GST-immunopositive oligodendrocytes in white matter were also selected from the five animals and counted for each mouse. 2.5. Statistical analysis Statistical analysis of the quantitative data was performed using Student’s t-test, and significance was set at P < 0.01 or P < 0.05. 3. Results 3.1. GADD34-immunopositive neuronal cells in human control brains We first investigated the immunohistochemical expression of GADD34 protein in control brain using anti-GADD34 antibody from rabbit antiserum. In the control specimens, some neurons were immunopositive for the anti-GADD34 antibody. GADD34 immunoreactivity was typically observed in the neuronal bodies
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Y. Honjo et al. / Neuroscience Letters 602 (2015) 50–55
Fig. 1. GADD34-immunopositive neuronal cells in human brain. (A) A small number of neurons in the frontal lobe of control human brain were immunopositive for GADD34. (B) Many anti-GADD34-antibody-immunopositive neurons in the frontal lobe of AD brain were observed. (C) A small number of oligodendrocytes were also GADD34 immunopositive in white matter of control human brain. (D) Many oligodendrocytes were also GADD34 immunopositive in white matter of AD brain. (A), (C) Control brain, (B), (D) AD brain. Scale bars: A and B = 50 m, C and D = 10 m.
and dendrites, but nuclei were not stained. The neurons in the frontal lobe were immunostained by the anti-GADD34 antibody (Fig. 1A). Anti-GADD34 antibody-immunopositive neurons were found in the hippocampus and frontal lobe. In addition, oligodendrocytes in white matter were also anti-GADD34 antibody immunopositive (Fig. 1C).
number of GADD34-immunopositive neurons was quantified and found to be significantly increased in AD brains (Fig. 3A).
3.2. GADD34-immunopositive neurons were increased in AD brains
Immunohistochemical double staining of GADD34 and GST showed that the number of oligodendrocytes labeled by antibodies against GST- was greater than the number of GADD34immunopositive oligodendrocytes. In addition, anti-GADD34 and anti-GST--immunopositive oligodendrocytes were co-localized in the AD brains (Fig. 2A–C). We confirmed this finding in the all three AD patients.
In the tissue sections from patients with AD, we found that the number of GADD34-immunopositive neurons was increased (Fig. 1B). This phenomenon was observed in all patients with AD and was found in both the hippocampus and frontal lobe. The
3.3. Double staining of GADD34 and a marker of mature oligodendrocytes in AD brains
Fig. 2. Double staining of GADD34 and a marker of oligodendrocytes (GST-) in AD brains.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Anti-GADD34 antibody immunostaining (green). (B) Anti-GST- antibody immunostaining (red). (C) Merged image. Scale bar: 10 m.
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and 24-month-old mice and this tendency was also observed in 4-month-old animals (Fig. 4A and B). Of interest, the number of GADD34-immunopositive oligodendrocytes in white matter was significantly increased in 8-, 24-, and even in 4-month-old mice (Fig. 4C). The animals displayed age-dependent accumulation of intraneuronal A oligomers from 8 months but no extracellular amyloid deposits even at 24 months.
4. Discussion
Fig. 3. Number of GADD34-immunopositive neuronal cells was increased in AD brains. (A) The numbers of anti-GADD34-antibody-immunopositive neurons were increased in the frontal lobe and hippocampus of AD brain compared with control human brain. The y-axis represents the number of GADD34-immunopositive neurons/0.45 mm2 . (B) The proportion of GADD34-immunopositive oligodendrocytes in white matter of the frontal lobe of AD brain was larger than in control human brain. The y-axis represents the proportion of GADD34-/GST--immunopositive oligodendrocytes. Statistical analysis of the quantitative data was performed using Student’s t-test. The results are presented as means ± SD. * P < 0.01.
3.4. GADD34-immunopositive oligodendrocytes were increased in AD brains Approximately, 200 GST--immunopositive oligodendrocytes in the immunostained sections of white matter of the frontal lobe were selected from three patients with AD. The proportions of GADD34- and GST--immunopositive oligodendrocytes in AD brains were larger than in control brains (Fig. 1D). The proportions of GADD34- and GST--immunopositive oligodendrocytes in AD brains were quantified and found to be significantly larger than in control brains (Fig. 3B). 3.5. GADD34-immunopositive neuronal cells were increased in the early stage of disease in the transgenic mouse model The number of GADD34-immunopositive neurons was significantly increased in the cerebral cortex and hippocampus in 8-
Soluble oligomers of A are believed to be a cause of synaptic and cognitive dysfunction in the early stages of AD [17–20]. This is based primarily on experimental evidence that natural and synthetic A oligomers impair synaptic plasticity and memory [17,20–22]. Furthermore, many published studies have supported this conclusion by indicating a correlation between soluble A levels and synaptic impairment in humans as well as in animal models of AD [17,23,24]. We previously reported that in APP transgenic mice expressing the E693 delta mutation which showed that AD is caused by enhanced A oligomerization without fibrillization [17,18]. Oligomer formation of A in the transgenic mice was confirmed by immunoprecipitation and western blot analysis. The animals displayed age-dependent accumulation of intraneuronal A oligomers from 8 months but no extracellular amyloid deposits even at 24 months. Hippocampal synaptic plasticity and memory were impaired at 8 months, at which time levels of the presynaptic marker synaptophysin began to decrease [17,18]. In the present study, we used this transgenic mouse to explore ER stress of early-stage AD. The mouse showed accumulation of A oligomers in ER in hippocampal neurons of 22-month-old mice [18]. We believe that this phenomenon is ubiquitously found in the neuronal cells. Although AD is classically thought of as a gray matter disease, white matter lesion pathology has been widely reported in AD. The white matter lesion pathology includes not only secondary changes due to degeneration of neurons but also primary change. A 1–40 and a truncated fragment, A 25–35, induced death of oligodendrocytes in vitro in a dose-dependent manner with similar potencies [4]. In addition, the triple-transgenic AD mouse model, which harbors the human amyloid precursor protein Swedish mutant transgene, presenilin knock-in mutation, and tau P301L mutant transgene, exhibits significant region-specific alterations in myelination patterns at time points preceding the appearance of amyloid accumulation [5,7,8]. The underlying cause of the observed white matter changes in early AD brains suggests that the disease is related to oligodendrocyte dysfunction. In this study, we showed increased GADD34 in oligodendrocytes in the early stages of AD in APP transgenic mice. We believe that ER stress due to A oligomers could affect oligodendrocytes in the early stages of AD. In this study, GADD34 was increased in neurons and oligodendrocytes depend on the age of mouse except for neurons in the cortex. The number of GADD34-immunopositive neurons in the cortex seems decreased among controls at 8 months old. We think this phenomenon is not significant. But oligodendrocytes may be affected ER stress more in the hippocampus or another region than in the cortex at 8 months old. In addition, ER stress may not increase linearly in the cortex. GADD34 recruits the PP1 to dephosphorylate the translation initiation factor eIF2␣, which reverses the shutoff of protein synthesis initiated by ER stress [9,25,26]. GADD34 knockout mouse showed no abnormalities during fetal development or in early adult life [27]. However, in GADD34−/− mouse embryonic fibroblasts, recovery from shutoff of protein synthesis was delayed when the fibroblasts were exposed to ER stress [27]. ER stress stimuli induced expressions of binding Ig protein (Bip) and C/EBP homologous protein (CHOP) in embryonic fibroblasts of wild-type mice. These expressions were strongly reduced
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Fig. 4. Number of GADD34-immunopositive neuronal cells was increased in the early stage of disease in the transgenic mouse. (A,B) The numbers of anti-GADD34-antibody-immunopositive neurons were increased in the cerebral cortex and hippocampus of AD mouse brain compared with control mouse brain. The y-axis represents the number of GADD34-immunopositive neurons/0.11 mm2 . (C) The proportion of GADD34-immunopositive oligodendrocytes in white matter of AD mouse brain was increased compared to control mouse brain (n = 5). The y-axis represents the proportion of GADD34-/GST--immunopositive oligodendrocytes. Statistical analysis of the quantitative data was performed using Student’s t-test. The results are presented as means ± SD. * P < 0.01, ** P < 0.05.
in GADD34−/− mouse embryonic fibroblast, which suggests that GADD34 up-regulates Bip and CHOP [27]. Thus, GADD34 acts as a sensor of ER stress stimuli and aids recovery of cells from shutoff of protein synthesis. A study of the effects of GADD34 in ischemic human brain was previously reported [10]. Extensive ischemic damage was found to correlate with significantly elevated GADD34 immunostaining in the CA1 layer of the hippocampus. The upregulation of GADD34 in response to global ischemia in the human brain may have the potential to influence cell survival [10]. In the present study, we revealed that GADD34 was increased in human AD brains as well as in APP transgenic mice in the early stages of disease. Thus, we believe that ER stress due to soluble oligomers of A could have the potential to influence cell survival and neuronal cell degeneration. 5. Conclusions In summary, we have demonstrated that GADD34 was increased in neurons and oligodendrocytes in human AD brains. Furthermore, we showed that the expression level of GADD34 was significantly increased in the early stage of disease in the APP transgenic mice compared with control mice. We speculate that GADD34 may be increased in the early stage of pathology of AD due to ER stress. We showed that GADD34 was significantly increased in oligodendrocytes in 4-month-old APP transgenic mice, but nonsignificantly increased in neurons of 4-month-old animals. The animals displayed age-dependent accumulation of intraneuronal A oligomers from 8 months but no extracellular amyloid deposits
even at 24 months. This result may suggest that oligodendrocytes were more affected than neurons in the early stages of AD. In addition, ER stress of A oligomers may be more related to oligodendrocytes than neurons. Conflict of interest The authors report no conflicts of interest. Acknowledgments We thank Aya Torisawa, Mitsuko Tachi, Maiko Yamada (Department of Pharmacoepidemiology, Kyoto University), and Akiko Yoshida (Department of Neurology, Kyoto University) for excellent technical assistance. This study was supported by Soshinkai Nagaokakyo Hospital, the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Health, Labour, and Welfare, Japan. References [1] D. Lindholm, H. Wootz, L. Korhonen, ER stress and neurodegenerative diseases, Cell Death Differ. 13 (2006) 385–392. [2] K.M. Doyle, D. Kennedy, A.M. Gorman, S. Gupta, S.J. Healy, A. Samali, Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders, J. Cell Mol. Med. 15 (2011) 2025–2039. [3] M. Goedert, S.S. Sisodia, D.L. Price, Neurofibrillary tangles and beta-amyloid deposits in Alzheimer’s disease, Curr. Opin. Neurobiol. 1 (1991) 441–447.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Increased GADD34 in oligodendrocytes in Alzheimer’s disease Yasuyuki Honjo a,b , Takashi Ayaki b , Takami Tomiyama c , Tomohisa Horibe a , Hidefumi Ito d , Hiroshi Mori e , Ryosuke Takahashi b , Koji Kawakami a,∗ a
Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Japan Department of Neurology, Graduate School of Medicine, Kyoto University, Japan Department of Neurology and Neuroscience, Osaka City University Medical School, Japan d Department of Neurology, Graduate School of Medicine, Wakayama Medical University, Japan e Department of Clinical Neuroscience, Osaka City University Medical School, Japan b c
h i g h l i g h t s • GADD34 was increased in neurons and oligodendrocytes in human AD brains. • GADD34 was significantly increased in the early stage of APP transgenic mice. • GADD34 and GST--immunopositive oligodendrocytes were co-localized in the AD.
a r t i c l e
i n f o
Article history: Received 13 May 2015 Received in revised form 21 June 2015 Accepted 29 June 2015 Available online 2 July 2015 Keywords: Alzheimer’s disease Oligodendrocytes GADD34 ER stress
a b s t r a c t Alzheimer’s disease (AD) is characterized by the accumulation of amyloid- (A) and abnormally phosphorylated tau which contribute to endoplasmic reticulum (ER) stress. Previous studies demonstrated that A and a truncated fragment of A induced death of oligodendrocytes in vitro. In addition, a tripletransgenic AD mouse model exhibits significant region-specific alterations in myelination patterns at time points preceding the appearance of A accumulation. The growth arrest and DNA damage protein (GADD) 34 is up-regulated in response to ER stress and regulates subunit of protein phosphatase 1 (PP1) complex that dephosphorylates eukaryotic translation initiator factor 2␣ (elF2␣). Thus, GADD34 is known as an ER stress regulator or ER stress marker. In a recent study, GADD34 was induced in the spinal cord glial cells of an amyotrophic lateral sclerosis (ALS) mouse model. It is interesting that reduced GADD34 delayed the onset of ALS and prolonged the survival period in the mouse model. In this study, we have demonstrated that GADD34 was increased in neurons of human AD brains. Additionally, this finding was also observed in oligodendrocytes in human AD brains. Furthermore, we showed that the expression levels of GADD34 in neurons and oligodendrocytes were significantly increased in the early stage of AD in the mouse model. As oligodendrocytes were more affected in the early stages of AD in this experimental model, ER stress of A oligomers may be more related to oligodendrocytes than to neurons. These results suggest that GADD34 could be a therapeutic target for preventing ER stress in neuronal cells in AD. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease, but there is currently no effective treatment available because the etiology or mechanism of AD is still unclear. The neurodegenerative diseases are characterized by the accumulation of aggregated unfolded proteins and these accumulated proteins con-
∗ Corresponding author at: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Yoshida Konoecho, Sakyoku, Kyoto 606-8501, Japan. Fax: +81 75 753 4469. E-mail address: [email protected] (K. Kawakami). http://dx.doi.org/10.1016/j.neulet.2015.06.052 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
tribute to endoplasmic reticulum (ER) stress [1]. ER stress signaling, otherwise known as the unfolded protein response, is triggered by an increased load of misfolded proteins in the organelle [2]. In AD, the aggregated unfolded proteins are characterized by the accumulation of amyloid- (A) and abnormally phosphorylated tau [3]. At that time, the dysfunction of oligodendrocytes due to A was reported [4–8]. A and a truncated fragment of A induced oligodendrocytes death of in vitro in a dose-dependent manner with similar potencies [4]. A-induced oligodendrocyte death was accompanied by nuclear DNA fragmentation, mitochondrial dysfunction, and cytoskeletal disintegration. The presence of both A-induced activation of redox-sensitive transcription factors and oxidative stress in A-mediated oligodendrocytes suggest
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Table 1 Clinical profiles of all AD patients and control Case
Sex
Age (years)
PMI (h)
Case of death
Co-morbidities
Control 1 2 3 4 5
M F M M M
62 91 94 62 63
3 NA NA NA NA
Pancreas cancer NA NA Heart failure Heart failure
Chronic hepatitis None Chronic heart failure Arrhythmia Chronic heart failure
84 89 94 77 67
4 2.5 15 1.5 18
Heart failure Sepsis Renal failure Pneumonia Leukemia
Anemia Chronic heart failure Hypertension Chronic hepatitis None
Alzheimer disease (AD) 1 F 2 F 3 F 4 F 5 M
Clinical stage
Break stage
Advanced Advanced Advanced Advanced Advanced
VI V VI VI VI
M = male, F = female, Age = age at death, PMI = post-mortem interval, NA = not available.
that oxidative injury contributes to A cytotoxicity in oligodendrocytes [4]. The presenilin 1 (M146 V) mutation predisposes mouse oligodendrocyte precursor cells to A-induced alterations in cell differentiation in vitro. Furthermore, a myelination defect and myelin basic protein distribution subcellular mislocalization triggered by presenilin 1 (M146 V) have been previously reported [5]. AD has not been widely considered a disease of white matter, but recent evidence suggests the existence of abnormalities in myelination patterns and myelin attrition in AD brains [6–8]. The growth arrest and DNA damage protein (GADD) 34 is a multifunctional protein up-regulated in response to cellular stress and is believed to mediate DNA repair and restore protein [9–12]. ER stress activates PKR-like ER kinase (PERK), which coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2␣ (eIF2␣). GADD34 can regulate subunit of PP1 complex that dephosphorylates eIF2␣. In addition, GADD34 can medicate DNA repair and restore protein synthesis [9,12]. Thus, GADD34 is believed to be an ER stress regulator or ER stress marker. Previous studies indicated that GADD34 was increased in ischemic neurons [10–12]. Furthermore, GADD34 was increased in the spinal cord glial cells in an ALS mouse [13,14]. Interestingly, this phenomenon was observed early after detectable onset of the disease, and reduced levels of active GADD34 markedly ameliorated disease [14]. However, reports of GADD34 in AD brain are limited. In this study, we revealed that GADD34 was increased in neurons and oligodendrocytes of human AD brains. Similar increases were also observed in the brain of amyloid precursor protein (APP) transgenic mice in the early stages of pathological onset of AD. 2. Materials and methods 2.1. Postmortem human brain Postmortem brain specimens from five patients with AD (67–94 years old) and five without AD (normal control brains; 62–94 years old) were utilized in this study as shown in Table 1. This study was approved by the ethics committee (approval number E1758) of Kyoto University. Patient diagnosis was determined by pathological examination. Specimens from the hippocampus and frontal lobe were obtained from the autopsied brains of control and AD patients. Brains were fixed in 10% neutral formalin at room temperature. Paraffin-embedded tissue blocks were prepared and cut into 6-mthick sections as previously described [15,16]. 2.2. Transgenic mouse brain The transgenic mouse model of A oligomers (E693 delta mutation in APP) has been previously described [17,18]. Briefly, mouse
brains were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (5 m thick) [17,18]. 2.3. Immunohistochemistry Immunohistochemical staining was performed as previously described [15,16]. Immunohistochemical staining for GADD34 was performed using polyclonal rabbit anti-GADD34 antibody purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), as reported previously [10,12]. We counted the number of GADD34immunopositive neurons in the frontal lobe (layer II–VI) and detate gyrus of hippocampal formation. In addition, we counted the number of GADD34-immunopositive oligodendrocytes in the white matter. 2.4. Double staining of GADD34 and a marker of oligodendrocytes in tissue sections from human and transgenic mouse brain samples To confirm the anatomical relationship between GADD34 and oligodendrocytes, we performed double-staining studies using mouse anti-GST- (marker of mature oligodendrocytes) and rabbit anti-GADD34 antibodies as described previously [15,16]. Double staining was performed using mouse anti-GST- antibody purchased from Vector Laboratories (Burlingame, CA, USA). Approximately, 200 GST--immunopositive oligodendrocytes in the immunostained sections in white matter of the frontal lobe were selected from three patients with AD. The number of GADD34-immunopositive oligodendrocytes in the selected GST--immunopositive oligodendrocytes was then counted for each patient. In the transgenic mice, approximately, 200 GST-immunopositive oligodendrocytes in white matter were also selected from the five animals and counted for each mouse. 2.5. Statistical analysis Statistical analysis of the quantitative data was performed using Student’s t-test, and significance was set at P < 0.01 or P < 0.05. 3. Results 3.1. GADD34-immunopositive neuronal cells in human control brains We first investigated the immunohistochemical expression of GADD34 protein in control brain using anti-GADD34 antibody from rabbit antiserum. In the control specimens, some neurons were immunopositive for the anti-GADD34 antibody. GADD34 immunoreactivity was typically observed in the neuronal bodies
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Fig. 1. GADD34-immunopositive neuronal cells in human brain. (A) A small number of neurons in the frontal lobe of control human brain were immunopositive for GADD34. (B) Many anti-GADD34-antibody-immunopositive neurons in the frontal lobe of AD brain were observed. (C) A small number of oligodendrocytes were also GADD34 immunopositive in white matter of control human brain. (D) Many oligodendrocytes were also GADD34 immunopositive in white matter of AD brain. (A), (C) Control brain, (B), (D) AD brain. Scale bars: A and B = 50 m, C and D = 10 m.
and dendrites, but nuclei were not stained. The neurons in the frontal lobe were immunostained by the anti-GADD34 antibody (Fig. 1A). Anti-GADD34 antibody-immunopositive neurons were found in the hippocampus and frontal lobe. In addition, oligodendrocytes in white matter were also anti-GADD34 antibody immunopositive (Fig. 1C).
number of GADD34-immunopositive neurons was quantified and found to be significantly increased in AD brains (Fig. 3A).
3.2. GADD34-immunopositive neurons were increased in AD brains
Immunohistochemical double staining of GADD34 and GST showed that the number of oligodendrocytes labeled by antibodies against GST- was greater than the number of GADD34immunopositive oligodendrocytes. In addition, anti-GADD34 and anti-GST--immunopositive oligodendrocytes were co-localized in the AD brains (Fig. 2A–C). We confirmed this finding in the all three AD patients.
In the tissue sections from patients with AD, we found that the number of GADD34-immunopositive neurons was increased (Fig. 1B). This phenomenon was observed in all patients with AD and was found in both the hippocampus and frontal lobe. The
3.3. Double staining of GADD34 and a marker of mature oligodendrocytes in AD brains
Fig. 2. Double staining of GADD34 and a marker of oligodendrocytes (GST-) in AD brains.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (A) Anti-GADD34 antibody immunostaining (green). (B) Anti-GST- antibody immunostaining (red). (C) Merged image. Scale bar: 10 m.
Y. Honjo et al. / Neuroscience Letters 602 (2015) 50–55
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and 24-month-old mice and this tendency was also observed in 4-month-old animals (Fig. 4A and B). Of interest, the number of GADD34-immunopositive oligodendrocytes in white matter was significantly increased in 8-, 24-, and even in 4-month-old mice (Fig. 4C). The animals displayed age-dependent accumulation of intraneuronal A oligomers from 8 months but no extracellular amyloid deposits even at 24 months.
4. Discussion
Fig. 3. Number of GADD34-immunopositive neuronal cells was increased in AD brains. (A) The numbers of anti-GADD34-antibody-immunopositive neurons were increased in the frontal lobe and hippocampus of AD brain compared with control human brain. The y-axis represents the number of GADD34-immunopositive neurons/0.45 mm2 . (B) The proportion of GADD34-immunopositive oligodendrocytes in white matter of the frontal lobe of AD brain was larger than in control human brain. The y-axis represents the proportion of GADD34-/GST--immunopositive oligodendrocytes. Statistical analysis of the quantitative data was performed using Student’s t-test. The results are presented as means ± SD. * P < 0.01.
3.4. GADD34-immunopositive oligodendrocytes were increased in AD brains Approximately, 200 GST--immunopositive oligodendrocytes in the immunostained sections of white matter of the frontal lobe were selected from three patients with AD. The proportions of GADD34- and GST--immunopositive oligodendrocytes in AD brains were larger than in control brains (Fig. 1D). The proportions of GADD34- and GST--immunopositive oligodendrocytes in AD brains were quantified and found to be significantly larger than in control brains (Fig. 3B). 3.5. GADD34-immunopositive neuronal cells were increased in the early stage of disease in the transgenic mouse model The number of GADD34-immunopositive neurons was significantly increased in the cerebral cortex and hippocampus in 8-
Soluble oligomers of A are believed to be a cause of synaptic and cognitive dysfunction in the early stages of AD [17–20]. This is based primarily on experimental evidence that natural and synthetic A oligomers impair synaptic plasticity and memory [17,20–22]. Furthermore, many published studies have supported this conclusion by indicating a correlation between soluble A levels and synaptic impairment in humans as well as in animal models of AD [17,23,24]. We previously reported that in APP transgenic mice expressing the E693 delta mutation which showed that AD is caused by enhanced A oligomerization without fibrillization [17,18]. Oligomer formation of A in the transgenic mice was confirmed by immunoprecipitation and western blot analysis. The animals displayed age-dependent accumulation of intraneuronal A oligomers from 8 months but no extracellular amyloid deposits even at 24 months. Hippocampal synaptic plasticity and memory were impaired at 8 months, at which time levels of the presynaptic marker synaptophysin began to decrease [17,18]. In the present study, we used this transgenic mouse to explore ER stress of early-stage AD. The mouse showed accumulation of A oligomers in ER in hippocampal neurons of 22-month-old mice [18]. We believe that this phenomenon is ubiquitously found in the neuronal cells. Although AD is classically thought of as a gray matter disease, white matter lesion pathology has been widely reported in AD. The white matter lesion pathology includes not only secondary changes due to degeneration of neurons but also primary change. A 1–40 and a truncated fragment, A 25–35, induced death of oligodendrocytes in vitro in a dose-dependent manner with similar potencies [4]. In addition, the triple-transgenic AD mouse model, which harbors the human amyloid precursor protein Swedish mutant transgene, presenilin knock-in mutation, and tau P301L mutant transgene, exhibits significant region-specific alterations in myelination patterns at time points preceding the appearance of amyloid accumulation [5,7,8]. The underlying cause of the observed white matter changes in early AD brains suggests that the disease is related to oligodendrocyte dysfunction. In this study, we showed increased GADD34 in oligodendrocytes in the early stages of AD in APP transgenic mice. We believe that ER stress due to A oligomers could affect oligodendrocytes in the early stages of AD. In this study, GADD34 was increased in neurons and oligodendrocytes depend on the age of mouse except for neurons in the cortex. The number of GADD34-immunopositive neurons in the cortex seems decreased among controls at 8 months old. We think this phenomenon is not significant. But oligodendrocytes may be affected ER stress more in the hippocampus or another region than in the cortex at 8 months old. In addition, ER stress may not increase linearly in the cortex. GADD34 recruits the PP1 to dephosphorylate the translation initiation factor eIF2␣, which reverses the shutoff of protein synthesis initiated by ER stress [9,25,26]. GADD34 knockout mouse showed no abnormalities during fetal development or in early adult life [27]. However, in GADD34−/− mouse embryonic fibroblasts, recovery from shutoff of protein synthesis was delayed when the fibroblasts were exposed to ER stress [27]. ER stress stimuli induced expressions of binding Ig protein (Bip) and C/EBP homologous protein (CHOP) in embryonic fibroblasts of wild-type mice. These expressions were strongly reduced
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Fig. 4. Number of GADD34-immunopositive neuronal cells was increased in the early stage of disease in the transgenic mouse. (A,B) The numbers of anti-GADD34-antibody-immunopositive neurons were increased in the cerebral cortex and hippocampus of AD mouse brain compared with control mouse brain. The y-axis represents the number of GADD34-immunopositive neurons/0.11 mm2 . (C) The proportion of GADD34-immunopositive oligodendrocytes in white matter of AD mouse brain was increased compared to control mouse brain (n = 5). The y-axis represents the proportion of GADD34-/GST--immunopositive oligodendrocytes. Statistical analysis of the quantitative data was performed using Student’s t-test. The results are presented as means ± SD. * P < 0.01, ** P < 0.05.
in GADD34−/− mouse embryonic fibroblast, which suggests that GADD34 up-regulates Bip and CHOP [27]. Thus, GADD34 acts as a sensor of ER stress stimuli and aids recovery of cells from shutoff of protein synthesis. A study of the effects of GADD34 in ischemic human brain was previously reported [10]. Extensive ischemic damage was found to correlate with significantly elevated GADD34 immunostaining in the CA1 layer of the hippocampus. The upregulation of GADD34 in response to global ischemia in the human brain may have the potential to influence cell survival [10]. In the present study, we revealed that GADD34 was increased in human AD brains as well as in APP transgenic mice in the early stages of disease. Thus, we believe that ER stress due to soluble oligomers of A could have the potential to influence cell survival and neuronal cell degeneration. 5. Conclusions In summary, we have demonstrated that GADD34 was increased in neurons and oligodendrocytes in human AD brains. Furthermore, we showed that the expression level of GADD34 was significantly increased in the early stage of disease in the APP transgenic mice compared with control mice. We speculate that GADD34 may be increased in the early stage of pathology of AD due to ER stress. We showed that GADD34 was significantly increased in oligodendrocytes in 4-month-old APP transgenic mice, but nonsignificantly increased in neurons of 4-month-old animals. The animals displayed age-dependent accumulation of intraneuronal A oligomers from 8 months but no extracellular amyloid deposits
even at 24 months. This result may suggest that oligodendrocytes were more affected than neurons in the early stages of AD. In addition, ER stress of A oligomers may be more related to oligodendrocytes than neurons. Conflict of interest The authors report no conflicts of interest. Acknowledgments We thank Aya Torisawa, Mitsuko Tachi, Maiko Yamada (Department of Pharmacoepidemiology, Kyoto University), and Akiko Yoshida (Department of Neurology, Kyoto University) for excellent technical assistance. This study was supported by Soshinkai Nagaokakyo Hospital, the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Health, Labour, and Welfare, Japan. References [1] D. Lindholm, H. Wootz, L. Korhonen, ER stress and neurodegenerative diseases, Cell Death Differ. 13 (2006) 385–392. [2] K.M. Doyle, D. Kennedy, A.M. Gorman, S. Gupta, S.J. Healy, A. Samali, Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders, J. Cell Mol. Med. 15 (2011) 2025–2039. [3] M. Goedert, S.S. Sisodia, D.L. Price, Neurofibrillary tangles and beta-amyloid deposits in Alzheimer’s disease, Curr. Opin. Neurobiol. 1 (1991) 441–447.
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