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Neuregulin 1 regulates amyloid precursor protein cell surface expression and non-amyloidogenic processing
Journal of Pharmacological Sciences xxx (2018) 1e8
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Neuregulin 1 regulates amyloid precursor protein cell surface expression and non-amyloidogenic processing Young-Jung Kim a, 1, Ji-Young Yoo a, 1, Ok-Soon Kim a, Han-byeol Kim a, Junghwa Ryu b, Hye-Sun Kim b, Jun-Ho Lee c, Hong-Il Yoo a, Dae-Yong Song a, Tai-Kyoung Baik a, **, Ran-Sook Woo a, * a b c
Department of Anatomy and Neuroscience, College of Medicine, Eulji University, Daejeon, 34824, Republic of Korea Department of Pharmacology and Biomedical Sciences, College of Medicine, Seoul National University, 103 Daehakro, Jongro-gu, Seoul, Republic of Korea Department of Emergency Medical Technology, Daejeon University, Daejeon, 34520, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2018 Received in revised form 6 May 2018 Accepted 17 May 2018 Available online xxx
The amyloid precursor protein (APP) is a key molecule in Alzheimer's disease. The prevailing view is that APP is initially transported to the plasma membrane as a full-length protein. Its localization at the cell surface can trigger downstream signaling and APP cleavage. Our previous work has shown that Neuregulin 1 (NRG1) has neuroprotective effects in an Alzheimer's disease model. In the present study, we examine whether NRG1 signaling is involved in APP expression and non-amyloidogenic processing in neuronal cells. Here we show that NRG1 increased the cell surface expression of APP without changing the total amount of APP mRNA or protein expression in SH-SY5Y cells and in rat primary cortical neurons. In addition, NRG1 significantly increased the levels of the secreted form of APP, sAPPa, in the conditioned media but did not change the expression of ADAM10 on the cell surface or in the cell lysates. Furthermore, we found that the protein level of NRG1 was reduced in the hippocampus of Alzheimer's disease (AD) patients. Our results demonstrate that NRG1 increased APP expression on the cell surface and sAPPa secretion into the media of neuronal cell cultures. Taken together, these results suggest a role for NRG1 in non-amyloidogenic processing. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Amyloid-precursor protein Neuregulin 1 Secreted APPa Non-amyloidogenic processing ADAM10
1. Introduction Amyloid precursor protein (APP) is a key molecule in Alzheimer's disease (AD) by the generation of its metabolites.1 APP is translated in the endoplasmic reticulum (ER) where it forms stable dimers which are transported through the secretory
Abberivations: AD, Alzheimer’s disease; APP, amyloid precursor protein; EGF, epithelial growth factor; ER, endoplasmic reticulum; NRG1, neuregulin; sAPPa, secreted APPa; sAPPb, secreted APPb. * Corresponding author. Department of Anatomy and Neuroscience, College of Medicine, Eulji University, 143-5, Yongdu-Dong, Jung-Gu, Daejeon, Republic of Korea.Tel. þ82 42 259 1623, Fax. þ82 42 259 1629. ** Corresponding author. Department of Anatomy and Neuroscience, College of Medicine, Eulji University, Republic of Korea. Tel. þ82 42 259 1621; Fax: þ82 42 259 1629. E-mail addresses: [email protected] (T.-K. Baik), [email protected] (R.-S. Woo). Peer review under responsibility of Japanese Pharmacological Society. 1 These authors contributed equally to this work.
pathway via the Golgi apparatus to the cell surface.2 APP is proteolyzed by one of two mutually exclusive pathways. The amyloidogenic pathway involves sequential cleavages of APP initially by b-secretase, which leads to the production of the Ab peptide. Non-amyloidogenic APP processing at the cell surface by a-secretase cleavage is considered the major pathway, resulting in the shedding of the Secreted APPa (sAPPa) ectodomain.3 Furthermore, lowered levels of CSF sAPPa have been correlated with poor memory performance.4 Several studies have shown that sAPPa levels are decreased in the cerebrospinal fluid of people with either familial5 or sporadic6 AD. Activation of protein kinase C increases sAPPa secretion by mechanisms involving the formation and release of secretory vesicles from the TGN, thus enhancing APP trafficking to the cell surface.7 There is a large body of evidence that implicates sAPPa in the neuroprotection against excitotoxicity, growth factor deprivation, proteasomal stress and traumatic brain injury in vivo.8
https://doi.org/10.1016/j.jphs.2018.05.004 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004
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Table 1 Information on age-matched, nondemented control subjects and AD patients: Information on Braak & Braak stages, CDR score and Reisberg scale of AD patients and agematched nondemented control subjects is shown. Diagnosis
Sex
Age
Braak & Braak Stage
Region
Non-demented control Non-demented control Non-demented control Non-demented control Alzheimer's disease Alzheimer's disease Alzheimer's disease Alzheimer's disease
M M M M M M M M
64 79 79 83 74 74 74 71
0 2 2 1 5 6 6 6
Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus
CDR stage
Reisberg scale (1e7)
4 3 7 3
Fig. 1. Effect of NRG1 on the subcellular localization of APP in neuronal cells. (A) Subcellular localization of APP in SH-SY5Y cells. SH-SY5Y cells were treated with 5 nM NRG1 for 48 h, and subcellular fractionation followed by immunoblotting was performed with APP (6E10) and actin. (B) Quantification analysis of the data in A. The values of densitometry are shown as the ratio of the control group, n ¼ 6, **P < 0.01. Statistical analysis was performed by t-test. (C) SH-SY5Y cells were treated with 5 nM NRG1 for different periods of time (12, 24, 48 h). Effect of NRG1 on total APP levels in SH-SY5Y cells; n ¼ 8 (D) Quantification analysis of the data in C. (E) qRT-PCR was performed to examine APP expression in cultured primary cortical neurons after treatment with 5 nM NRG1; n ¼ 5.
Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004
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2. Materials and methods 2.1. Reagents and antibodies The NRG1 protein that was used in this study is a recombinant polypeptide that contains the entire EGF domain of the b-type NRG1 from Prospec-Tany TechnoGene (East Brunswick, NJ, USA). The following antibodies were supplied by Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA): ErbB4 (sc-283), mouse IgG (sc2025), rabbit IgG (sc-66931), b-actin (sc-47778), HRP-conjugated anti-rabbit IgG (sc-2004), HRP-conjugated anti-mouse IgG (sc2005) and HRP-conjugated anti-goat IgG (sc-2020). Other antibodies included secreted APPb (sAPPb)-WT (MBS492139; MyBio Source, San Diego, CA, USA), 6E10 (MAB348; Millipore Corporation, Chemicon, MA, USA), 22C11 (#803002; Biolegend, San Diego, CA, USA), ADAM10 (ab1997, Abcam, Cambridge, United Kingdom) glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Ab FRONTIER, Seoul, Korea) and NRG1 (MABN42; Millipore, Darmstadt, Germany). 2.2. Cell culture
Fig. 2. Effect of NRG1 on APP cell surface expression in primary cortical neurons. (A) Primary cortical neurons were treated with 5 nM NRG1 for 48 h. The cell surface expression of APP was measured by biotinylation followed by batch extraction and analysis by Western blotting. (B) Quantification analysis of the data in A. NRG1 increased the cell surface expression of APP, as shown by the densitometric value compared with cell surface control group, n ¼ 8, *P < 0.05. Statistical analysis was performed by t-test.
Primary cortical neurons were cultured as previously described.22 Briefly, the cerebral cortex was removed from SpragueeDawley rat embryos (E18) and dissociated by gentle trituration in PBS (Gibco, Carlsbad, CA, USA). Cells were seeded on poly-L-lysine-coated 6-well plates and cultured in Neurobasal media (Gibco). Experiments were performed 14 days after seeding (DIV14). SH-SY5Y cells were obtained from the Korean cell line bank (Seoul, Korea) and grown in Dulbecco's Modified Eagle's Medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS and a penicillin-streptomycin-amphotericin B mixture (Invitrogen) at 37 C and 5% CO2. 2.3. Subcellular fractionation
In addition to the neuroprotective functions of sAPPa, APP has been implicated in several biological processes including cell adhesion, gene transcription, neuronal differentiation, neuronal migration, neurite outgrowth, and synaptogenesis.9 APP has been shown to be highly expressed in the brain during developmental stages and is expressed throughout the brain in adulthood. Interestingly, this increase in APP at the cell surface is required for reelin-induced neurite outgrowth suggesting that APP at the cell surface triggers some signaling in hippocampal neurons.10 Animals lacking APP show a reduction in synaptic markers, which is correlated with learning and memory dysfunction.11 Both full-length APP and its cleavage products have multiple roles in neuronal motility,12 including the control of neuronal migration in the developing brain.13 Neuregulin 1 (NRG1) is a large family of epithelial growth factor (EGF)-domain containing trophic factors that play an important role in neuronal development and neuronal circuits.14e16 Recent biochemical studies have shown that NRG1 can be neuroprotective for cortical neurons, motor neurons and dopaminergic neurons.17,18 In a previous study, we found that pretreatment with NRG1 prevented Ab1-42-induced impairment of LTP. This LTP-restoring action of NRG1 was disappeared by blocking ErbB4 receptors which is one of its receptor.19 Moreover, we found that NRG1 has neuroprotective effects induced by Swedish APP, Ab or C-terminal fragments of APP via the ErbB4 receptor.20,21 It has been shown that the PI3K pathway is involved in NRG1-mediated rescue of neurotoxicity induced by Ab1-42. These results suggest that NRG1 signaling may be involved in the pathophysiology of AD. In this study, we investigated whether NRG1 regulate nonamyloidogenic APP processing in neuronal cells.
Subcellular fractionation was performed as previously described.23 In brief, cells were lysed with a subcellular fractionation buffer, and cell lysates were centrifuged at 750 g for 10 min. The supernatant was removed and centrifuged at 10,000 g for 10 min. The supernatant, which was the cytosolic and membrane fraction, was transferred to a new tube. The supernatant was recentrifuged at 100,000 g for 1 h. After ultra-centrifugation, the supernatant was the cytoplasmic fraction. The pellet was washed with fractionation buffer and resuspended in nuclear buffer; this was the membrane fraction. 2.4. Biotinylation The biotinylation assay was performed as previously described.15 In brief, cells were rinsed three times with PBS containing 0.1 mM calcium and 1.0 mM magnesium (PBS Ca/Mg). The cells were then incubated with 1 mg/ml sulfo-NHS-SS-biotin in PBS Ca/Mg for 20 min at 4 C with gentle shaking. The biotin solution was removed, and the plates were washed twice with PBS Ca/Mg containing 100 mM glycine. The plates were then incubated in PBS Ca/Mg glycine for 30 min at 4 C with gentle shaking to quench the nonreacted biotin. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Lysates were centrifuged at 16,500 g for 30 min at 4 C, and the cellular debris was removed. Aliquots of the lysate were further incubated with an equal volume of a 50% slurry of avidin beads for 1 h and centrifuged at 16,500 g for 10 min at 4 C. To release the biotinylated proteins, the beads were incubated with SDS sample buffer for 30 min at 37 C. All of the preserved samples were analyzed by Western blotting.
Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004
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Fig. 3. NRG1 increases the fluorescent intensity of cell surface APP. Confocal microscopy analysis of non-permeabilized SH-SY5Y cells or primary cortical neurons exposed to vehicle or 5 nM NRG1 for 48 h. Staining for APP (green) and Hoechst (blue) was performed. (A) Effect of NRG1 on cell surface APP expression in SH-SY5Y cells. Scale bar, 20 mm. (B) Bar graph summarizing the APP fluorescence data in SH-SY5Y cells. The values of densitometry are shown as ratios of the control group, n ¼ 6, *P < 0.05. (C) Effect of NRG1 on cell surface APP expression in primary cortical neurons. Scale bar, 20 mm. (D) Quantification analysis of the data in C; n ¼ 6, *P < 0.05. Statistical analysis was performed by t-test.
2.5. Quantitative real-time PCR Total RNA was isolated from cells with TRIzol (Ambion, Carlsbad, California, USA). The following primers were used for PCR: APP (S): 50 -TGC CCA TTT CCA GAA AGC C-30 , (AS): 50 -CGG CGG TCA TTG AGC ATG GC-3’; and GAPDH (S): 50 -GTG GAC CTC ATG GCC TAC AT-30 , (AS): 50 -TGT GAG GGA GAT GCT CAG TG-3’. All of the primers were designed with the help of primer 3 programs according to the known or predicted rat sequences reported in GenBank. Quantitative real-time PCR was performed using the Bio-Rad Bio-Plex System with 2X QuantiTect SYBR Green PCR Master Mix with 0.5 mM primer, 20 ng cDNA and nuclease-free water as described in the manufacturer's protocol (Finzymes).
For all of the probands, each cycle consisted of a denaturation step at 95 C for 10 s, followed by separate annealing (20 s) and extension (30 s) steps at a temperature that was characteristic for each proband. Fluorescence was monitored at the end of each extension step. The specificity of each PCR product was verified by performing dissociation reaction plots. The levels of mRNA are expressed as the threshold cycle (CT), and the comparative threshold cycle (DCT) analysis method was used. Data were normalized to GAPDH mRNA. The normalized copy number was calculated as follows: (copy number of mRNA of interest/copy number of GAPDH) x 1000. The relative expression was calculated by the comparative threshold cycle method via 2DDCT. Real-time PCR data presented here had a p < 0.05 in Student's t test.
Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004
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Fig. 4. NRG1 increases the secretion of sAPPa. (A) Primary cortical neurons were treated with 5 nM NRG1 for 24 h or 48 h, and conditioned media were analyzed using Western blotting to detect sAPPa, sAPPb and total sAPP. (B) Quantification analysis of sAPPa expression data in A. Significant differences were detected between the 24 h and the 48 h NRG1 treatment groups, n ¼ 6, #P < 0.05. The values of densitometry are shown as the ratio to the control group at each time point, n ¼ 6, *P < 0.05. (C) Quantification analysis of sAPPb expression data in A. The values of densitometry are shown as ratios of the 24 h control group, n ¼ 6. (D) Quantification analysis of total sAPP expression data in A. Significant differences were detected between the 24 h and the 48 h NRG1 treatment groups, n ¼ 6, #P < 0.05. The values of densitometry are shown as ratios of the 24 h control group (oneway ANOVA, n ¼ 6, *P < 0.05). (E) Primary cortical neurons were treated with 5 nM NRG1 for 48 h. The cell surface expression of ADAM10 was measured by biotinylation followed by batch extraction and analysis by Western blotting. (F) Quantification analysis of the data in E; n ¼ 8.
2.6. Immunostaining Immunostaining of rat cortical neurons (E18, DIV14) and SHSY5Y cells were performed as previously described.22 Briefly, neurons were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 20 min. After washing, cells were incubated in buffer containing an antibody against APP (22C11; 1:100) at 4 C overnight followed
by incubation with FITC-conjugated AffiniPure goat anti-mouse IgG (1:400; Jackson ImmunoResearch Laboratories, Inc.) in buffer for 2 h at room temperature. Nuclei were counterstained with Hoechst stain (10 mM in PBS) for 30 min. Stained cells were mounted in Vectorshield (Vector Laboratories) and observed under an LSM 510 Meta System microscope (Zeiss LSM 510 laser scanning microscope, Carl Zeiss, Germany).
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2.7. Western blotting Samples were resolved via SDS-PAGE and then transferred to a nitrocellulose membrane, which was blocked with TBS that contained 5% BSA and 0.05% Tween 20 for 1 h. The membrane was then incubated overnight at 4 C with primary antibodies and developed with horseradish peroxidase-conjugated secondary antibodies and enhanced using a chemiluminescence system (Amersham Pharmacia, CA, USA).
2.8. AD brains Brain tissue from AD patients aged 64e83 years and agematched, nondemented control subjects were obtained from the Netherlands Brain Bank (Amsterdam, The Netherlands). Cases with Braak & Braak stages V to VI were classified as AD, whereas stages 0 to II were classified as controls. Additional clinical information of AD patients including clinical dementia rating (CDR) and Reisberg score are shown in Table 1. All experimental procedures were carried out according to ‘the Guidelines of the Ethics Committee at Seoul National University’.
2.9. Statistical analysis Data are presented as the mean ± SEM of three or more independent experiments. For multiple group comparisons, statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Student's paired ttest was used for comparisons of the means between two groups of cells in a single experiment. Values of p < 0.05 were considered significant.
3. Results 3.1. NRG1 affects APP localization First, we examined the effect of NRG1 on the expression of APP. NRG1 showed no effect on the total protein levels of APP in SHSY5Y cells (Fig. 1AeD); moreover, the total protein levels of APP were not affected in a time-dependent manner (Fig. 1C and D). In addition, we checked the mRNA level of APP by performing qRTPCR after treating SH-SY5Y cells (data not shown) and primary cortical neurons with 5 nM NRG1. NRG1 did not affect the mRNA levels of APP in the cells (Fig. 1E). However, as shown in Fig. 1A and B, NRG1 increased the amount of APP in the cell membrane fractions (control, 1.00 ± 0.17; NRG1, 3.13 ± 0.33; n ¼ 6; Fig. 1A and B). The membrane expression of APP is important for its cleavage and signaling in cells.
3.2. NRG1 increases the cell surface expression of APP However, the technique used for cellular fractionation does not allow a complete separation of the cellular membranes from other intracellular compartments, thus it was necessary to confirm that the cell surface proteins were biotinylated, isolated with avidin beads, and immunoblotted for APP. NRG1 treatment increased the cell surface expression of APP in primary cortical neurons at 48 h without changing the total amount of APP (Fig. 2A and B). The quantification demonstrated that NRG1 significantly increased the cell surface levels of APP (control, 1 ± 0.29; NRG1, 2.53 ± 0.38; n ¼ 8; Fig. 2A and B). These results indicate that NRG1 regulates APP expression at the neuronal cell surface.
3.3. NRG1 enhances the fluorescent intensity of cell surface APP in non-permeabilized cells To verify these results at the cell surface, we fluorescently stained the extracellular domain of APP with the N-terminal anti-APP antibody in non-permeabilized SH-SY5Y cells or primary cortical neurons. The results are shown in Fig. 3A and B. NRG1 enhanced the labeling of APP at the cell surface of SH-SY5Y cells (non-permeabilized control, 1 ± 0.12; NRG1, 2.73 ± 0.27; n ¼ 6; Fig. 3A and B). Moreover, NRG1 also significantly increased the neuronal cell surface immunoreactivity of APP in comparison to that in the control primary cortical neurons (non-permeabilized control, 1 ± 0.17; NRG1, 2.33 ± 0.18; n ¼ 6; Fig. 3C and D). These data confirm that NRG1 increases APP expression at the neuronal cell surface. 3.4. NRG1 regulates APP processing We next confirmed the effects of NRG1 on the levels of secreted APP variants (sAPPa, sAPPb and total sAPP) in the conditioned media of primary cortical neurons. Under these conditions, Western blot analysis revealed that NRG1 induced an approximate 2-fold increase in the levels of sAPPa at 24 and 48 h (Fig. 4A and B). We also examined total sAPP levels (normalized to total protein) in the conditioned media and found similarly increased levels of sAPPa after 24 and 48 h of NRG1 treatment (Fig. 4A and D). However, sAPPb levels did not change in the same conditions (Fig. 4A and C). In addition, we next set out to determine the effect of NRG1 on a-secretase levels in the primary cortical neurons. Increasing sAPPa levels in the media did not affect the protein levels of ADAM10 at the cell surface or in the total lysate (Fig. 4E and F). 3.5. NRG1 is reduced in the brains of AD patients We evaluated NRG1 protein levels in the hippocampus of AD brains and age-matched nondemented control subjects by using Western blotting with an NRG1 antibody in which the epitope is the extracellular domain of NRG1. The protein level of the mature form of NRG1 (approximately 29 kDa) in the hippocampus of AD brains (74, 74, 74 and 71 years old) showed a significant decrease compared with those of the age-matched, nondemented control subjects (64, 79, 79 and 83 years old, Fig. 5A). The NRG1/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratios in the hippocampus of AD patients were 29.81 ± 1.11% of the corresponding ratios in hippocampal lysates from nondemented control subjects (**p < 0.01, Fig. 5B).” 4. Discussion Although the physiological role of APP is not yet fully understood several recent studies have suggested an important function for APP and its cleavage products at synapses. In the non-amyloidogenic pathway, APP is cleaved by a-secretases to produce sAPPa, which is neuroprotective and important for neuronal plasticity.24,25 Therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of AD.26 NRG1 is a trophic factor thought to play a role in nervous system development and the maintenance of brain circuits. Several lines of evidence have demonstrated that NRG1 protects neurons against neurotoxic stimuli under various conditions including an ischemic insult.27 Recently, we reported that NRG1 attenuated cognitive impairments in Tg2576 mice, an animal model of AD. In addition, NRG1 rescued the reduction in the number of dendritic spines detected in the brains of Tg2576 mice.28 These observations indicate that NRG1
Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004
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Fig. 5. NRG1 is reduced in human AD brains (A) NRG1 protein levels in the hippocampus of age-matched, nondemented control subjects (64, 79,79 and 83 years old) and AD patients (74, 74, 74 and 71 years old) were examined by Western blot analysis. (B) NRG1 protein in the hippocampus of the AD brains showed a significantly lower level than that of the age-matched, nondemented control subjects. The NRG1/GAPDH ratio in the hippocampus from AD patients was 29.81 ± 1.11% of the corresponding ratios in hippocampal lysates from nondemented control subjects (**p < 0.01 by Student's t-test, Fig. 5B). Data are presented as the mean ± SEM.
could play a critical role in regulating brain circuitry in connection with AD-related molecules. The goal of the present study was to determine whether NRG1 signaling is involved in the regulation of APP. We have used several independent approaches to demonstrate that NRG1 regulates the subcellular localization of APP in SH-SY5Y cells and primary cortical neurons. The total levels of APP mRNA and protein expression were not changed in a time-dependent manner. Interestingly, NRG1 increased the amount of APP in the cell membrane fraction. Since we found an increase in the levels of APP in the cell membrane fraction, we speculate that NRG1 might play a role in trafficking APP to the cell membrane. These cellular membrane fraction may include the plasma membrane and ER membranes. Therefore, to investigate whether NRG1 directly affects APP translocation to the plasma membrane, we examined APP levels at the cell surface in the NRG1-treated primary cortical neurons or SH-SY5Y cells. We observed that NRG1 treatment increased cell surface expression of APP via biotinylation strategies and immunocytochemistry in non-permeabilized SH-SY5Y cells or primary cortical neurons. These results suggest that NRG1 regulates the expression of APP at the cell surface. APP is synthesized in the ER and transported to both axons and dendrites.29 APP is cleaved both in the neuronal soma and in neurites. Following N- and o-glycosylation, nascent APP travels towards the cell surface followed by internalization into endosomes where it can become the substrate for b-site-APP cleaving enzyme 1 cleavage. In contrast, APP is processed by a-secretase at the cell surface.30
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We measured the effects of NRG1 on the levels of secreted APP (sAPPa, sAPPb and total sAPP) in the conditioned media of primary cortical neurons. NRG1 increased in the levels of sAPPa in the media but did not affect the protein levels of ADAM10 in the cells. Previously, it was reported that sAPPa has the capacity to contribute to normal memory function, which is supported by findings that exogenous administration of sAPPa or its small peptide fragments enhances memory performance in mice.31 The acute synaptic function of endogenous sAPPa in the adult brain was shown in APP/APLP2 cDKO mice.32 sAPPa peptide rescued the severe LTP deficit in acute brain slices of the mutants, indicating that the soluble ectodomain acts on a rapid time-scale.33 Therefore, we propose that the advantageous effects of NRG1 involve protection against AD as well as enhancing neuronal normal activity in the brain. The expression of neurotrophic factors decrease with increasing age, which is a risk factor of many brain diseases.34 In this study, we found a reduction in mature NRG1 protein levels in the hippocampus of AD patients. NRG1 is cleaved extracellularly by proteases including BACE1 into soluble, mature NRG1 (29e45 kDa).35,36 We found that the approximately 29-kDa-sized NRG1 metabolite was reduced in the hippocampus of AD brains when compared with age-matched, nondemented control subjects (Fig. 5). This 29-kDa-sized fragment represents a proteolytic fragment of the amino-terminal domain of NRG1 type III. Therefore, the decrease in NRG1 may likely involve APP processing. Moreover, APP is extensively post-translationally modified by glycosylation and phosphorylation at several residues,37 but further study is needed to clarify the effects of NRG1 on protein modification. Further investigations of the role of NRG1 in APP regulation might facilitate a better understanding of brain function and disorders. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was partly supported by a grant from the Ministry of Education, Science, Technology funded by the National Research Foundation of Korea (NRF; No. NRF-2016R1A2B4010574) and the Institute for Information & communications Technology Promotion (IITP) grant funded by the Korea government (MSIP; No. B01321510010003003, Next Imaging System XIS). References 1. Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev. 2001;81(2):741e766. 2. Isbert S, Wagner K, Eggert S, et al. APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms. Cell Mol Life Sci. 2012;69(8):1353e1375. 3. Sisodia SS. Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A. 1992;89(13):6075e6079. 4. Almkvist O, Basun H, Wagner SL, Rowe BA, Wahlund LO, Lannfelt L. Cerebrospinal fluid levels of alpha-secretase-cleaved soluble amyloid precursor protein mirror cognition in a Swedish family with Alzheimer disease and a gene mutation. Arch Neurol. 1997;54(5):641e644. 5. Lannfelt L, Basun H, Wahlund LO, Rowe BA, Wagner SL. Decreased alphasecretase-cleaved amyloid precursor protein as a diagnostic marker for Alzheimer's disease. Nat Med. 1995;1(8):829e832. 6. Post A, Ackl N, Rucker M, et al. Toward a reliable distinction between patients with mild cognitive impairment and Alzheimer-type dementia versus major depression. Biol Psychiatr. 2006;59(9):858e862. 7. Koo EH, Squazzo SL. Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J Biol Chem. 1994;269(26):17386e17389. 8. Kogel D, Deller T, Behl C. Roles of amyloid precursor protein family members in neuroprotection, stress signaling and aging. Exp Brain Res. 2012;217(3e4): 471e479.
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9. Aydin D, Weyer SW, Muller UC. Functions of the APP gene family in the nervous system: insights from mouse models. Exp Brain Res. 2012;217(3e4): 423e434. 10. Hoe HS, Lee KJ, Carney RS, et al. Interaction of reelin with amyloid precursor protein promotes neurite outgrowth. J Neurosci. 2009;29(23): 7459e7473. 11. Dawson GR, Seabrook GR, Zheng H, et al. Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein. Neuroscience. 1999;90(1):1e13. 12. Young-Pearse TL, Chen AC, Chang R, Marquez C, Selkoe DJ. Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1. Neural Dev. 2008;3:15. 13. Rice HC, Townsend M, Bai J, et al. Pancortins interact with amyloid precursor protein and modulate cortical cell migration. Development. 2012;139(21): 3986e3996. 14. Mei L, Nave KA. Neuregulin-ERBB signaling in the nervous system and neuropsychiatric diseases. Neuron. 2014;83(1):27e49. 15. Yu HN, Park WK, Nam KH, et al. Neuregulin 1 controls glutamate uptake by upregulating excitatory amino acid Carrier 1 (EAAC1). J Biol Chem. 2015;290(33): 20233e20244. 16. Corfas G, Roy K, Buxbaum JD. Neuregulin 1-erbB signaling and the molecular/ cellular basis of schizophrenia. Nat Neurosci. 2004;7(6):575e580. 17. Li BS, Ma W, Jaffe H, et al. Cyclin-dependent kinase-5 is involved in neuregulindependent activation of phosphatidylinositol 3-kinase and Akt activity mediating neuronal survival. J Biol Chem. 2003;278(37):35702e35709. 18. Ricart K, Pearson Jr RJ, Viera L, et al. Interactions between beta-neuregulin and neurotrophins in motor neuron apoptosis. J Neurochem. 2006;97(1):222e233. 19. Min SS, An J, Lee JH, et al. Neuregulin-1 prevents amyloid beta-induced impairment of long-term potentiation in hippocampal slices via ErbB4. Neurosci Lett. 2011;505(1):6e9. 20. Woo RS, Lee JH, Kim HS, et al. Neuregulin-1 protects against neurotoxicities induced by Swedish amyloid precursor protein via the ErbB4 receptor. Neuroscience. 2012;202:413e423. 21. Ryu J, Yu HN, Cho H, et al. Neuregulin-1 exerts protective effects against neurotoxicities induced by C-terminal fragments of APP via ErbB4 receptor. J Pharmacol Sci. 2012;119(1):73e81. 22. Woo RS, Li XM, Tao Y, et al. Neuregulin-1 enhances depolarization-induced GABA release. Neuron. 2007;54(4):599e610. 23. Huang Z, Cheng Y, Chiu PM, et al. Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties. Oncogene. 2012;31(32):3709e3720.
24. Turner PR, O'Connor K, Tate WP, Abraham WC. Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory. Prog Neurobiol. 2003;70(1):1e32. 25. Ring S, Weyer SW, Kilian SB, et al. The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci. 2007;27(29):7817e7826. 26. Yang X, Sheng W, He Y, et al. Secretory phospholipase A2 type III enhances alpha-secretase-dependent amyloid precursor protein processing through alterations in membrane fluidity. J Lipid Res. 2010;51(5):957e966. 27. Guo WP, Wang J, Li RX, Peng YW. Neuroprotective effects of neuregulin-1 in rat models of focal cerebral ischemia. Brain Res. 2006;1087(1):180e185. 28. Ryu J, Hong BH, Kim YJ, et al. Neuregulin-1 attenuates cognitive function impairments in a transgenic mouse model of Alzheimer's disease. Cell Death Dis. 2016;7:e2117. 29. DeBoer SR, Dolios G, Wang R, Sisodia SS. Differential release of beta-amyloid from dendrite-versus axon-targeted APP. J Neurosci. 2014;34(37): 12313e12327. 30. Thinakaran G, Koo EH. Amyloid precursor protein trafficking, processing, and function. J Biol Chem. 2008;283(44):29615e29619. 31. Bour A, Little S, Dodart JC, Kelche C, Mathis C. A secreted form of the betaamyloid precursor protein (sAPP695) improves spatial recognition memory in OF1 mice. Neurobiol Learn Mem. 2004;81(1):27e38. 32. Hick M, Herrmann U, Weyer SW, et al. Acute function of secreted amyloid precursor protein fragment APPsalpha in synaptic plasticity. Acta Neuropathol. 2015;129(1):21e37. 33. Taylor CJ, Ireland DR, Ballagh I, et al. Endogenous secreted amyloid precursor protein-alpha regulates hippocampal NMDA receptor function, long-term potentiation and spatial memory. Neurobiol Dis. 2008;31(2):250e260. 34. Belrose JC, Masoudi R, Michalski B, Fahnestock M. Increased pro-nerve growth factor and decreased brain-derived neurotrophic factor in non-Alzheimer's disease tauopathies. Neurobiol Aging. 2014;35(4):926e933. 35. Loeb JA, Susanto ET, Fischbach GD. The neuregulin precursor proARIA is processed to ARIA after expression on the cell surface by a protein kinase Cenhanced mechanism. Mol Cell Neurosci. 1998;11(1e2):77e91. 36. Fleck D, Voss M, Brankatschk B, et al. Proteolytic processing of neuregulin 1 type III by three intramembrane-cleaving proteases. J Biol Chem. 2016;291(1): 318e333. 37. Muresan V, Muresan Z. Is abnormal axonal transport a cause, a contributing factor or a consequence of the neuronal pathology in Alzheimer's disease? Future Neurol. 2009;4(6):761e773.
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Neuregulin 1 regulates amyloid precursor protein cell surface expression and non-amyloidogenic processing Young-Jung Kim a, 1, Ji-Young Yoo a, 1, Ok-Soon Kim a, Han-byeol Kim a, Junghwa Ryu b, Hye-Sun Kim b, Jun-Ho Lee c, Hong-Il Yoo a, Dae-Yong Song a, Tai-Kyoung Baik a, **, Ran-Sook Woo a, * a b c
Department of Anatomy and Neuroscience, College of Medicine, Eulji University, Daejeon, 34824, Republic of Korea Department of Pharmacology and Biomedical Sciences, College of Medicine, Seoul National University, 103 Daehakro, Jongro-gu, Seoul, Republic of Korea Department of Emergency Medical Technology, Daejeon University, Daejeon, 34520, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2018 Received in revised form 6 May 2018 Accepted 17 May 2018 Available online xxx
The amyloid precursor protein (APP) is a key molecule in Alzheimer's disease. The prevailing view is that APP is initially transported to the plasma membrane as a full-length protein. Its localization at the cell surface can trigger downstream signaling and APP cleavage. Our previous work has shown that Neuregulin 1 (NRG1) has neuroprotective effects in an Alzheimer's disease model. In the present study, we examine whether NRG1 signaling is involved in APP expression and non-amyloidogenic processing in neuronal cells. Here we show that NRG1 increased the cell surface expression of APP without changing the total amount of APP mRNA or protein expression in SH-SY5Y cells and in rat primary cortical neurons. In addition, NRG1 significantly increased the levels of the secreted form of APP, sAPPa, in the conditioned media but did not change the expression of ADAM10 on the cell surface or in the cell lysates. Furthermore, we found that the protein level of NRG1 was reduced in the hippocampus of Alzheimer's disease (AD) patients. Our results demonstrate that NRG1 increased APP expression on the cell surface and sAPPa secretion into the media of neuronal cell cultures. Taken together, these results suggest a role for NRG1 in non-amyloidogenic processing. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Amyloid-precursor protein Neuregulin 1 Secreted APPa Non-amyloidogenic processing ADAM10
1. Introduction Amyloid precursor protein (APP) is a key molecule in Alzheimer's disease (AD) by the generation of its metabolites.1 APP is translated in the endoplasmic reticulum (ER) where it forms stable dimers which are transported through the secretory
Abberivations: AD, Alzheimer’s disease; APP, amyloid precursor protein; EGF, epithelial growth factor; ER, endoplasmic reticulum; NRG1, neuregulin; sAPPa, secreted APPa; sAPPb, secreted APPb. * Corresponding author. Department of Anatomy and Neuroscience, College of Medicine, Eulji University, 143-5, Yongdu-Dong, Jung-Gu, Daejeon, Republic of Korea.Tel. þ82 42 259 1623, Fax. þ82 42 259 1629. ** Corresponding author. Department of Anatomy and Neuroscience, College of Medicine, Eulji University, Republic of Korea. Tel. þ82 42 259 1621; Fax: þ82 42 259 1629. E-mail addresses: [email protected] (T.-K. Baik), [email protected] (R.-S. Woo). Peer review under responsibility of Japanese Pharmacological Society. 1 These authors contributed equally to this work.
pathway via the Golgi apparatus to the cell surface.2 APP is proteolyzed by one of two mutually exclusive pathways. The amyloidogenic pathway involves sequential cleavages of APP initially by b-secretase, which leads to the production of the Ab peptide. Non-amyloidogenic APP processing at the cell surface by a-secretase cleavage is considered the major pathway, resulting in the shedding of the Secreted APPa (sAPPa) ectodomain.3 Furthermore, lowered levels of CSF sAPPa have been correlated with poor memory performance.4 Several studies have shown that sAPPa levels are decreased in the cerebrospinal fluid of people with either familial5 or sporadic6 AD. Activation of protein kinase C increases sAPPa secretion by mechanisms involving the formation and release of secretory vesicles from the TGN, thus enhancing APP trafficking to the cell surface.7 There is a large body of evidence that implicates sAPPa in the neuroprotection against excitotoxicity, growth factor deprivation, proteasomal stress and traumatic brain injury in vivo.8
https://doi.org/10.1016/j.jphs.2018.05.004 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004
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Table 1 Information on age-matched, nondemented control subjects and AD patients: Information on Braak & Braak stages, CDR score and Reisberg scale of AD patients and agematched nondemented control subjects is shown. Diagnosis
Sex
Age
Braak & Braak Stage
Region
Non-demented control Non-demented control Non-demented control Non-demented control Alzheimer's disease Alzheimer's disease Alzheimer's disease Alzheimer's disease
M M M M M M M M
64 79 79 83 74 74 74 71
0 2 2 1 5 6 6 6
Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus Hippocampus
CDR stage
Reisberg scale (1e7)
4 3 7 3
Fig. 1. Effect of NRG1 on the subcellular localization of APP in neuronal cells. (A) Subcellular localization of APP in SH-SY5Y cells. SH-SY5Y cells were treated with 5 nM NRG1 for 48 h, and subcellular fractionation followed by immunoblotting was performed with APP (6E10) and actin. (B) Quantification analysis of the data in A. The values of densitometry are shown as the ratio of the control group, n ¼ 6, **P < 0.01. Statistical analysis was performed by t-test. (C) SH-SY5Y cells were treated with 5 nM NRG1 for different periods of time (12, 24, 48 h). Effect of NRG1 on total APP levels in SH-SY5Y cells; n ¼ 8 (D) Quantification analysis of the data in C. (E) qRT-PCR was performed to examine APP expression in cultured primary cortical neurons after treatment with 5 nM NRG1; n ¼ 5.
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2. Materials and methods 2.1. Reagents and antibodies The NRG1 protein that was used in this study is a recombinant polypeptide that contains the entire EGF domain of the b-type NRG1 from Prospec-Tany TechnoGene (East Brunswick, NJ, USA). The following antibodies were supplied by Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA): ErbB4 (sc-283), mouse IgG (sc2025), rabbit IgG (sc-66931), b-actin (sc-47778), HRP-conjugated anti-rabbit IgG (sc-2004), HRP-conjugated anti-mouse IgG (sc2005) and HRP-conjugated anti-goat IgG (sc-2020). Other antibodies included secreted APPb (sAPPb)-WT (MBS492139; MyBio Source, San Diego, CA, USA), 6E10 (MAB348; Millipore Corporation, Chemicon, MA, USA), 22C11 (#803002; Biolegend, San Diego, CA, USA), ADAM10 (ab1997, Abcam, Cambridge, United Kingdom) glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Ab FRONTIER, Seoul, Korea) and NRG1 (MABN42; Millipore, Darmstadt, Germany). 2.2. Cell culture
Fig. 2. Effect of NRG1 on APP cell surface expression in primary cortical neurons. (A) Primary cortical neurons were treated with 5 nM NRG1 for 48 h. The cell surface expression of APP was measured by biotinylation followed by batch extraction and analysis by Western blotting. (B) Quantification analysis of the data in A. NRG1 increased the cell surface expression of APP, as shown by the densitometric value compared with cell surface control group, n ¼ 8, *P < 0.05. Statistical analysis was performed by t-test.
Primary cortical neurons were cultured as previously described.22 Briefly, the cerebral cortex was removed from SpragueeDawley rat embryos (E18) and dissociated by gentle trituration in PBS (Gibco, Carlsbad, CA, USA). Cells were seeded on poly-L-lysine-coated 6-well plates and cultured in Neurobasal media (Gibco). Experiments were performed 14 days after seeding (DIV14). SH-SY5Y cells were obtained from the Korean cell line bank (Seoul, Korea) and grown in Dulbecco's Modified Eagle's Medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS and a penicillin-streptomycin-amphotericin B mixture (Invitrogen) at 37 C and 5% CO2. 2.3. Subcellular fractionation
In addition to the neuroprotective functions of sAPPa, APP has been implicated in several biological processes including cell adhesion, gene transcription, neuronal differentiation, neuronal migration, neurite outgrowth, and synaptogenesis.9 APP has been shown to be highly expressed in the brain during developmental stages and is expressed throughout the brain in adulthood. Interestingly, this increase in APP at the cell surface is required for reelin-induced neurite outgrowth suggesting that APP at the cell surface triggers some signaling in hippocampal neurons.10 Animals lacking APP show a reduction in synaptic markers, which is correlated with learning and memory dysfunction.11 Both full-length APP and its cleavage products have multiple roles in neuronal motility,12 including the control of neuronal migration in the developing brain.13 Neuregulin 1 (NRG1) is a large family of epithelial growth factor (EGF)-domain containing trophic factors that play an important role in neuronal development and neuronal circuits.14e16 Recent biochemical studies have shown that NRG1 can be neuroprotective for cortical neurons, motor neurons and dopaminergic neurons.17,18 In a previous study, we found that pretreatment with NRG1 prevented Ab1-42-induced impairment of LTP. This LTP-restoring action of NRG1 was disappeared by blocking ErbB4 receptors which is one of its receptor.19 Moreover, we found that NRG1 has neuroprotective effects induced by Swedish APP, Ab or C-terminal fragments of APP via the ErbB4 receptor.20,21 It has been shown that the PI3K pathway is involved in NRG1-mediated rescue of neurotoxicity induced by Ab1-42. These results suggest that NRG1 signaling may be involved in the pathophysiology of AD. In this study, we investigated whether NRG1 regulate nonamyloidogenic APP processing in neuronal cells.
Subcellular fractionation was performed as previously described.23 In brief, cells were lysed with a subcellular fractionation buffer, and cell lysates were centrifuged at 750 g for 10 min. The supernatant was removed and centrifuged at 10,000 g for 10 min. The supernatant, which was the cytosolic and membrane fraction, was transferred to a new tube. The supernatant was recentrifuged at 100,000 g for 1 h. After ultra-centrifugation, the supernatant was the cytoplasmic fraction. The pellet was washed with fractionation buffer and resuspended in nuclear buffer; this was the membrane fraction. 2.4. Biotinylation The biotinylation assay was performed as previously described.15 In brief, cells were rinsed three times with PBS containing 0.1 mM calcium and 1.0 mM magnesium (PBS Ca/Mg). The cells were then incubated with 1 mg/ml sulfo-NHS-SS-biotin in PBS Ca/Mg for 20 min at 4 C with gentle shaking. The biotin solution was removed, and the plates were washed twice with PBS Ca/Mg containing 100 mM glycine. The plates were then incubated in PBS Ca/Mg glycine for 30 min at 4 C with gentle shaking to quench the nonreacted biotin. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Lysates were centrifuged at 16,500 g for 30 min at 4 C, and the cellular debris was removed. Aliquots of the lysate were further incubated with an equal volume of a 50% slurry of avidin beads for 1 h and centrifuged at 16,500 g for 10 min at 4 C. To release the biotinylated proteins, the beads were incubated with SDS sample buffer for 30 min at 37 C. All of the preserved samples were analyzed by Western blotting.
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Fig. 3. NRG1 increases the fluorescent intensity of cell surface APP. Confocal microscopy analysis of non-permeabilized SH-SY5Y cells or primary cortical neurons exposed to vehicle or 5 nM NRG1 for 48 h. Staining for APP (green) and Hoechst (blue) was performed. (A) Effect of NRG1 on cell surface APP expression in SH-SY5Y cells. Scale bar, 20 mm. (B) Bar graph summarizing the APP fluorescence data in SH-SY5Y cells. The values of densitometry are shown as ratios of the control group, n ¼ 6, *P < 0.05. (C) Effect of NRG1 on cell surface APP expression in primary cortical neurons. Scale bar, 20 mm. (D) Quantification analysis of the data in C; n ¼ 6, *P < 0.05. Statistical analysis was performed by t-test.
2.5. Quantitative real-time PCR Total RNA was isolated from cells with TRIzol (Ambion, Carlsbad, California, USA). The following primers were used for PCR: APP (S): 50 -TGC CCA TTT CCA GAA AGC C-30 , (AS): 50 -CGG CGG TCA TTG AGC ATG GC-3’; and GAPDH (S): 50 -GTG GAC CTC ATG GCC TAC AT-30 , (AS): 50 -TGT GAG GGA GAT GCT CAG TG-3’. All of the primers were designed with the help of primer 3 programs according to the known or predicted rat sequences reported in GenBank. Quantitative real-time PCR was performed using the Bio-Rad Bio-Plex System with 2X QuantiTect SYBR Green PCR Master Mix with 0.5 mM primer, 20 ng cDNA and nuclease-free water as described in the manufacturer's protocol (Finzymes).
For all of the probands, each cycle consisted of a denaturation step at 95 C for 10 s, followed by separate annealing (20 s) and extension (30 s) steps at a temperature that was characteristic for each proband. Fluorescence was monitored at the end of each extension step. The specificity of each PCR product was verified by performing dissociation reaction plots. The levels of mRNA are expressed as the threshold cycle (CT), and the comparative threshold cycle (DCT) analysis method was used. Data were normalized to GAPDH mRNA. The normalized copy number was calculated as follows: (copy number of mRNA of interest/copy number of GAPDH) x 1000. The relative expression was calculated by the comparative threshold cycle method via 2DDCT. Real-time PCR data presented here had a p < 0.05 in Student's t test.
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Fig. 4. NRG1 increases the secretion of sAPPa. (A) Primary cortical neurons were treated with 5 nM NRG1 for 24 h or 48 h, and conditioned media were analyzed using Western blotting to detect sAPPa, sAPPb and total sAPP. (B) Quantification analysis of sAPPa expression data in A. Significant differences were detected between the 24 h and the 48 h NRG1 treatment groups, n ¼ 6, #P < 0.05. The values of densitometry are shown as the ratio to the control group at each time point, n ¼ 6, *P < 0.05. (C) Quantification analysis of sAPPb expression data in A. The values of densitometry are shown as ratios of the 24 h control group, n ¼ 6. (D) Quantification analysis of total sAPP expression data in A. Significant differences were detected between the 24 h and the 48 h NRG1 treatment groups, n ¼ 6, #P < 0.05. The values of densitometry are shown as ratios of the 24 h control group (oneway ANOVA, n ¼ 6, *P < 0.05). (E) Primary cortical neurons were treated with 5 nM NRG1 for 48 h. The cell surface expression of ADAM10 was measured by biotinylation followed by batch extraction and analysis by Western blotting. (F) Quantification analysis of the data in E; n ¼ 8.
2.6. Immunostaining Immunostaining of rat cortical neurons (E18, DIV14) and SHSY5Y cells were performed as previously described.22 Briefly, neurons were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 20 min. After washing, cells were incubated in buffer containing an antibody against APP (22C11; 1:100) at 4 C overnight followed
by incubation with FITC-conjugated AffiniPure goat anti-mouse IgG (1:400; Jackson ImmunoResearch Laboratories, Inc.) in buffer for 2 h at room temperature. Nuclei were counterstained with Hoechst stain (10 mM in PBS) for 30 min. Stained cells were mounted in Vectorshield (Vector Laboratories) and observed under an LSM 510 Meta System microscope (Zeiss LSM 510 laser scanning microscope, Carl Zeiss, Germany).
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2.7. Western blotting Samples were resolved via SDS-PAGE and then transferred to a nitrocellulose membrane, which was blocked with TBS that contained 5% BSA and 0.05% Tween 20 for 1 h. The membrane was then incubated overnight at 4 C with primary antibodies and developed with horseradish peroxidase-conjugated secondary antibodies and enhanced using a chemiluminescence system (Amersham Pharmacia, CA, USA).
2.8. AD brains Brain tissue from AD patients aged 64e83 years and agematched, nondemented control subjects were obtained from the Netherlands Brain Bank (Amsterdam, The Netherlands). Cases with Braak & Braak stages V to VI were classified as AD, whereas stages 0 to II were classified as controls. Additional clinical information of AD patients including clinical dementia rating (CDR) and Reisberg score are shown in Table 1. All experimental procedures were carried out according to ‘the Guidelines of the Ethics Committee at Seoul National University’.
2.9. Statistical analysis Data are presented as the mean ± SEM of three or more independent experiments. For multiple group comparisons, statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Student's paired ttest was used for comparisons of the means between two groups of cells in a single experiment. Values of p < 0.05 were considered significant.
3. Results 3.1. NRG1 affects APP localization First, we examined the effect of NRG1 on the expression of APP. NRG1 showed no effect on the total protein levels of APP in SHSY5Y cells (Fig. 1AeD); moreover, the total protein levels of APP were not affected in a time-dependent manner (Fig. 1C and D). In addition, we checked the mRNA level of APP by performing qRTPCR after treating SH-SY5Y cells (data not shown) and primary cortical neurons with 5 nM NRG1. NRG1 did not affect the mRNA levels of APP in the cells (Fig. 1E). However, as shown in Fig. 1A and B, NRG1 increased the amount of APP in the cell membrane fractions (control, 1.00 ± 0.17; NRG1, 3.13 ± 0.33; n ¼ 6; Fig. 1A and B). The membrane expression of APP is important for its cleavage and signaling in cells.
3.2. NRG1 increases the cell surface expression of APP However, the technique used for cellular fractionation does not allow a complete separation of the cellular membranes from other intracellular compartments, thus it was necessary to confirm that the cell surface proteins were biotinylated, isolated with avidin beads, and immunoblotted for APP. NRG1 treatment increased the cell surface expression of APP in primary cortical neurons at 48 h without changing the total amount of APP (Fig. 2A and B). The quantification demonstrated that NRG1 significantly increased the cell surface levels of APP (control, 1 ± 0.29; NRG1, 2.53 ± 0.38; n ¼ 8; Fig. 2A and B). These results indicate that NRG1 regulates APP expression at the neuronal cell surface.
3.3. NRG1 enhances the fluorescent intensity of cell surface APP in non-permeabilized cells To verify these results at the cell surface, we fluorescently stained the extracellular domain of APP with the N-terminal anti-APP antibody in non-permeabilized SH-SY5Y cells or primary cortical neurons. The results are shown in Fig. 3A and B. NRG1 enhanced the labeling of APP at the cell surface of SH-SY5Y cells (non-permeabilized control, 1 ± 0.12; NRG1, 2.73 ± 0.27; n ¼ 6; Fig. 3A and B). Moreover, NRG1 also significantly increased the neuronal cell surface immunoreactivity of APP in comparison to that in the control primary cortical neurons (non-permeabilized control, 1 ± 0.17; NRG1, 2.33 ± 0.18; n ¼ 6; Fig. 3C and D). These data confirm that NRG1 increases APP expression at the neuronal cell surface. 3.4. NRG1 regulates APP processing We next confirmed the effects of NRG1 on the levels of secreted APP variants (sAPPa, sAPPb and total sAPP) in the conditioned media of primary cortical neurons. Under these conditions, Western blot analysis revealed that NRG1 induced an approximate 2-fold increase in the levels of sAPPa at 24 and 48 h (Fig. 4A and B). We also examined total sAPP levels (normalized to total protein) in the conditioned media and found similarly increased levels of sAPPa after 24 and 48 h of NRG1 treatment (Fig. 4A and D). However, sAPPb levels did not change in the same conditions (Fig. 4A and C). In addition, we next set out to determine the effect of NRG1 on a-secretase levels in the primary cortical neurons. Increasing sAPPa levels in the media did not affect the protein levels of ADAM10 at the cell surface or in the total lysate (Fig. 4E and F). 3.5. NRG1 is reduced in the brains of AD patients We evaluated NRG1 protein levels in the hippocampus of AD brains and age-matched nondemented control subjects by using Western blotting with an NRG1 antibody in which the epitope is the extracellular domain of NRG1. The protein level of the mature form of NRG1 (approximately 29 kDa) in the hippocampus of AD brains (74, 74, 74 and 71 years old) showed a significant decrease compared with those of the age-matched, nondemented control subjects (64, 79, 79 and 83 years old, Fig. 5A). The NRG1/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratios in the hippocampus of AD patients were 29.81 ± 1.11% of the corresponding ratios in hippocampal lysates from nondemented control subjects (**p < 0.01, Fig. 5B).” 4. Discussion Although the physiological role of APP is not yet fully understood several recent studies have suggested an important function for APP and its cleavage products at synapses. In the non-amyloidogenic pathway, APP is cleaved by a-secretases to produce sAPPa, which is neuroprotective and important for neuronal plasticity.24,25 Therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of AD.26 NRG1 is a trophic factor thought to play a role in nervous system development and the maintenance of brain circuits. Several lines of evidence have demonstrated that NRG1 protects neurons against neurotoxic stimuli under various conditions including an ischemic insult.27 Recently, we reported that NRG1 attenuated cognitive impairments in Tg2576 mice, an animal model of AD. In addition, NRG1 rescued the reduction in the number of dendritic spines detected in the brains of Tg2576 mice.28 These observations indicate that NRG1
Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004
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Fig. 5. NRG1 is reduced in human AD brains (A) NRG1 protein levels in the hippocampus of age-matched, nondemented control subjects (64, 79,79 and 83 years old) and AD patients (74, 74, 74 and 71 years old) were examined by Western blot analysis. (B) NRG1 protein in the hippocampus of the AD brains showed a significantly lower level than that of the age-matched, nondemented control subjects. The NRG1/GAPDH ratio in the hippocampus from AD patients was 29.81 ± 1.11% of the corresponding ratios in hippocampal lysates from nondemented control subjects (**p < 0.01 by Student's t-test, Fig. 5B). Data are presented as the mean ± SEM.
could play a critical role in regulating brain circuitry in connection with AD-related molecules. The goal of the present study was to determine whether NRG1 signaling is involved in the regulation of APP. We have used several independent approaches to demonstrate that NRG1 regulates the subcellular localization of APP in SH-SY5Y cells and primary cortical neurons. The total levels of APP mRNA and protein expression were not changed in a time-dependent manner. Interestingly, NRG1 increased the amount of APP in the cell membrane fraction. Since we found an increase in the levels of APP in the cell membrane fraction, we speculate that NRG1 might play a role in trafficking APP to the cell membrane. These cellular membrane fraction may include the plasma membrane and ER membranes. Therefore, to investigate whether NRG1 directly affects APP translocation to the plasma membrane, we examined APP levels at the cell surface in the NRG1-treated primary cortical neurons or SH-SY5Y cells. We observed that NRG1 treatment increased cell surface expression of APP via biotinylation strategies and immunocytochemistry in non-permeabilized SH-SY5Y cells or primary cortical neurons. These results suggest that NRG1 regulates the expression of APP at the cell surface. APP is synthesized in the ER and transported to both axons and dendrites.29 APP is cleaved both in the neuronal soma and in neurites. Following N- and o-glycosylation, nascent APP travels towards the cell surface followed by internalization into endosomes where it can become the substrate for b-site-APP cleaving enzyme 1 cleavage. In contrast, APP is processed by a-secretase at the cell surface.30
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We measured the effects of NRG1 on the levels of secreted APP (sAPPa, sAPPb and total sAPP) in the conditioned media of primary cortical neurons. NRG1 increased in the levels of sAPPa in the media but did not affect the protein levels of ADAM10 in the cells. Previously, it was reported that sAPPa has the capacity to contribute to normal memory function, which is supported by findings that exogenous administration of sAPPa or its small peptide fragments enhances memory performance in mice.31 The acute synaptic function of endogenous sAPPa in the adult brain was shown in APP/APLP2 cDKO mice.32 sAPPa peptide rescued the severe LTP deficit in acute brain slices of the mutants, indicating that the soluble ectodomain acts on a rapid time-scale.33 Therefore, we propose that the advantageous effects of NRG1 involve protection against AD as well as enhancing neuronal normal activity in the brain. The expression of neurotrophic factors decrease with increasing age, which is a risk factor of many brain diseases.34 In this study, we found a reduction in mature NRG1 protein levels in the hippocampus of AD patients. NRG1 is cleaved extracellularly by proteases including BACE1 into soluble, mature NRG1 (29e45 kDa).35,36 We found that the approximately 29-kDa-sized NRG1 metabolite was reduced in the hippocampus of AD brains when compared with age-matched, nondemented control subjects (Fig. 5). This 29-kDa-sized fragment represents a proteolytic fragment of the amino-terminal domain of NRG1 type III. Therefore, the decrease in NRG1 may likely involve APP processing. Moreover, APP is extensively post-translationally modified by glycosylation and phosphorylation at several residues,37 but further study is needed to clarify the effects of NRG1 on protein modification. Further investigations of the role of NRG1 in APP regulation might facilitate a better understanding of brain function and disorders. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was partly supported by a grant from the Ministry of Education, Science, Technology funded by the National Research Foundation of Korea (NRF; No. NRF-2016R1A2B4010574) and the Institute for Information & communications Technology Promotion (IITP) grant funded by the Korea government (MSIP; No. B01321510010003003, Next Imaging System XIS). References 1. Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev. 2001;81(2):741e766. 2. Isbert S, Wagner K, Eggert S, et al. APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms. Cell Mol Life Sci. 2012;69(8):1353e1375. 3. Sisodia SS. Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A. 1992;89(13):6075e6079. 4. Almkvist O, Basun H, Wagner SL, Rowe BA, Wahlund LO, Lannfelt L. Cerebrospinal fluid levels of alpha-secretase-cleaved soluble amyloid precursor protein mirror cognition in a Swedish family with Alzheimer disease and a gene mutation. Arch Neurol. 1997;54(5):641e644. 5. Lannfelt L, Basun H, Wahlund LO, Rowe BA, Wagner SL. Decreased alphasecretase-cleaved amyloid precursor protein as a diagnostic marker for Alzheimer's disease. Nat Med. 1995;1(8):829e832. 6. Post A, Ackl N, Rucker M, et al. Toward a reliable distinction between patients with mild cognitive impairment and Alzheimer-type dementia versus major depression. Biol Psychiatr. 2006;59(9):858e862. 7. Koo EH, Squazzo SL. Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J Biol Chem. 1994;269(26):17386e17389. 8. Kogel D, Deller T, Behl C. Roles of amyloid precursor protein family members in neuroprotection, stress signaling and aging. Exp Brain Res. 2012;217(3e4): 471e479.
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Please cite this article in press as: Kim Y-J, et al., Neuregulin 1 regulates amyloid precursor protein cell surface expression and nonamyloidogenic processing, Journal of Pharmacological Sciences (2018), https://doi.org/10.1016/j.jphs.2018.05.004