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Neuroprotective effects of diarylpropionitrile against β-amyloid peptide-induced neurotoxicity in rat cultured cortical neurons
Neuroscience Letters 578 (2014) 44–49
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotective effects of diarylpropionitrile against -amyloid peptide-induced neurotoxicity in rat cultured cortical neurons Nirut Suwanna a , Wipawan Thangnipon a,∗ , Rungtip Soi-ampornkul b a b
Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom, Thailand Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
h i g h l i g h t s • • • •
Treatment with DPN alleviated A1–42 -induced cell death and morphological changes. DPN also reduced production of ROS induced by A1–42 . DPN prevents A1–42 -induced toxicity by attenuating apoptosis and inflammation. DPN effects may involve inhibition of JNK and p38 including activation of ERK1/2 and Akt.
a r t i c l e
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Article history: Received 16 December 2013 Received in revised form 2 June 2014 Accepted 12 June 2014 Available online 21 June 2014 Keywords: Alzheimer’s disease -Amyloid peptide Diarylpropionitrile Antioxidant Apoptosis Anti-inflammation
a b s t r a c t Alzheimer’s disease is a major cause of dementia in the elderly that involves a -amyloid peptide (A)-induced cascade of an increase in oxidative damage and inflammation. The present study demonstrated the neuroprotective effects of diarylpropionitrile (DPN), a non-steroidal estrogen receptor  selective ligand, against 10 M A1–42 -induced oxidative stress and inflammation in primary rat cortical cell culture. Pre-treatment with 1–100 nM DPN significantly decreased neuronal cell death by increasing cell viability through a significant attenuation in the reactive oxygen species level, downregulation of pro-apoptotic activated caspase-3 and Bax, and upregulation of anti-apoptotic Bcl-2, thereby mitigating apoptotic morphological alterations. DPN pre-treatment decreased the expression levels of pro-inflammatory cytokines IL-1 and IL-6 through attenuation of A1–42 -induced phosphorylation of mitogen-activated protein kinases JNK and p38. In addition, DPN enhanced ERK1/2 and Akt phosphorylation depressed by A1–42 . These findings suggest that DPN protects neurons from A1–42 -induced neurotoxicity through a variety of mechanisms, ranging from anti-oxidation, anti-apoptosis, through to anti-inflammation. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is characterized by progressive loss of memory and impairment of higher cognitive functions. The pathological hallmarks of human AD brain are the presence of extracellular plaques composed of -amyloid (A) and intracellular neurofibrillary tangles [21]. A1–42 is the form that is prominently increased in brains of AD patients [14]. The mechanisms by which A1–42 induces neurotoxicity have not been completely elucidated, but previous studies on rat cultured cortical neurons
∗ Corresponding author. Tel.: +66 24419003x1203; fax: +66 24419003x1311. E-mail addresses: [email protected], [email protected] (W. Thangnipon). http://dx.doi.org/10.1016/j.neulet.2014.06.029 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
exposed to A1–42 showed apoptotic cell death, as evidenced by both morphological changes and expression of pro-apoptotic proteins, such as activated caspase-3 and Bax, and the elevation of reactive oxygen species (ROS) levels [24,25]. It has been demonstrated that A can increase expression levels of IL-1 and IL-6 in rat cortical neuronal cell cultures [22]. Thus, protection of neurons from A1–42 -induced neurotoxicity with antioxidants and anti-inflammatory compounds, such as N-trans-feruloyltyramine (NTF) [24] and N-benzylcinnamide (PT-3) [25], provides important avenues to ameliorate the pathological effects of A. (DPN) or 2,3-bis(4-hydroxyphenyl)Diarylpropionitrile propionitrile is a non-steroidal selective ligand of estrogen receptor  (ER) with neuroprotective effects in a number of neurological diseases [13]. The treatments with 10 and 100 nM DPN protected rat cultured hippocampal neurons from glucose
N. Suwanna et al. / Neuroscience Letters 578 (2014) 44–49
deprivation-induced cell death [9]. DPN also has an antiinflammatory role in neuroprotection by suppressing IL-1 and IL-6 expressions in mouse brain [2]. In primary rat cultured hippocampal neurons DPN up-regulates expressions of a panel of bioenergetic enzymes and antioxidant proteins [10]. ERK1/2 and Akt signaling cascades play critical roles in the transmission of signals from growth factor receptors to regulate gene expression and prevent apoptosis [18], and these pathways are disrupted by A in rat cultured cortical neurons [4,26]. DPN treatment can increase the phosphorylation of Akt and ERK1/2 in neuronal cultures derived from mouse embryonic stem cells [30]. In our previous studies, we demonstrated that NTF and PT-3 attenuate A1–42 -induced cell death in rat cortical neuronal cultures by inhibiting the generation of ROS, reducing expression of proapoptotic activated caspase-3 and Bax, activating anti-apoptotic protein Bcl-2, reducing expression of pro-inflammatory cytokines IL-1 and IL-6, and attenuating phosphorylation of JNK and p38, proteins associated with pro-inflammatory response [24,25]. As DPN shares some common structural characteristics with NTF, such as phenolic hydroxyl groups which may serve as effective radical scavenging moieties [5], we examined the mechanisms of DPN neuroprotection against the neuronal insults of A1–42 treatment in rat primary neuronal cell cultures.
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Fig. 2. ROS levels in A1–42 -treated cultures were significantly reduced by 0.1–100 nM DPN pre-treatment. Results are expressed as mean ± SEM of three independent experiments. ***p < 0.001 vs. control; ### p < 0.001 vs. A1–42 -treated group.
2. Methods Enriched primary neuronal cell cultures were prepared from cerebral cortices of 10 to 12 Wistar rat fetuses at embryonic day-17 according to the previous procedures [19]. On day 5, cultures were
Fig. 1. DPN prevents morphological alterations from A1–42 -induced toxicity in cultured cortical neurons. Morphologies of control (A), 10 M A1–42 -treated (B), 10 nM DPN pre-treated followed by 10 M A1–42 -treated (C). In the presence of A1–42 (B), neurons exhibited the degenerative findings of cell body shrinkage (arrowheads) and neurite blebbing (arrows) compared to controls (A) and DPN-treated (C). Cell viability of A1–42 -treated cultures determined by MTT assay was significantly increased by 1–100 nM DPN treatment (D). Results are expressed as mean ± SEM of 3 independent experiments. ***p < 0.001 vs. control; # p < 0.05, ### p < 0.001 vs. A1–42 -treated group. Scale bar = 25 m.
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Fig. 4. Pre-treatment with 0.1–100 nM DPN significantly reduced activated IL-1 levels induced by A1–42 treatment on cultured cortical neurons (A). Expression of IL-6 was reduced by pre-treatment with 100 nM DPN (B). Results are expressed as mean ± SEM of 3 independent experiments. *p < 0.05, ***p < 0.001 vs. control; # p < 0.05, ## p < 0.01 vs. A1–42 -treated group.
Fig. 3. A1–42 -stimulated expression of Bax was reduced by pre-treatment with 0.1–100 nM DPN (A). Pre-treatment with 0.1–100 nM DPN increased the expression of Bcl-2 (B) and decreased the expression of activated caspase-3 (C). Results are expressed as mean ± SEM of 3 independent experiments. *p < 0.05, ***p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. A1–42 -treated group.
treated with 0, 0.1, 1, 10 and 100 nM DPN dissolved in absolute ethanol for 1 h prior to incubating for 24 h with a sublethal dose of 10 M oligomeric A1–42 (Keck Biotechnology, USA) [6]. Then cultures were processed for western blot analysis, ROS determination and cell viability evaluation. Morphological changes of neurons were visualized under an inverted phase-contrast microscope. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the absorbance at 570 nm measured with a microplate reader (Molecular Probes, Eugene, OR) [11].
Intracellular ROS levels were determined using 2 ,7 dichlorodihydrofluorescein diacetate (DCF-DA) dye assay [16]. In brief, cells were incubated with 50 M DCFH-DA dissolved in absolute ethanol at 37 ◦ C for 45 min in the dark prior to DPN and A1–42 treatment and DCF fluorescence was quantified using a Multi-Detection microplate reader (BioTek Synergy HT, Winooski, VT) with excitation and emission wavelength of 485 and 530 nm respectively. Equal amounts of protein from cell lysate were separated on 10% SDS-PAGE under reducing condition, and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with 1:500 mouse anti-Bcl-2, -Bax, -IL-6, 1:500 rabbit anti-IL-1 (Santa Cruz), 1:500 rabbit anti-activated caspase-3, phospho-p38, -p38, 1:1000 rabbit anti-phospho-ERK1/2, -ERK1/2, -phospho-JNK, -JNK, -phospho-Akt, -Akt, or 1:2500 rabbit antiactin antibodies (Cell Signaling), and then incubated with a HRP-conjugated secondary antibody (Zymed). The signal was detected with ECL Western blotting substrate (Bio-Rad) and captured on HyperfilmTM (Amersham Pharmacia Biotech). Statistical significance of differences was determined by oneway analysis of variance (ANOVA), followed by SNK post-test. The results are considered statistically significant at p < 0.05. All data are presented as mean ± SEM of 3 independent experiments conducted in triplicate.
N. Suwanna et al. / Neuroscience Letters 578 (2014) 44–49
3. Results Rat primary neuronal cells treated with 10 M A1–42 displayed cell shrinkage and neurite blebbing compared to normal neuronal cells (Fig. 1A and B), and pre-treatment with 10 nM DPN significantly attenuate A1–42 -induced morphological abnormalities (Fig. 1C). In addition, pre-treatment of neuronal cell cultures with 1–100 nM DPN significantly reduced A1–42 -induced neuronal cell death as assessed by MTT assay (Fig. 1D). DPN treatment alone at the test concentrations did not result in cytotoxicity. As expected, ROS production in 10 M A1–42 -treated neuronal cells is significantly increased compared to control (Fig. 2), and pre-treatment with 0.1–100 nM DPN decreased ROS levels in a non-dose response manner. DPN treatment did not significantly alter ROS levels from that of control. Apoptosis was confirmed to be the mechanism of neuronal cell death induced by 10 M A1–42 as evidenced by the elevated levels of Bax (Fig. 3A), and 0.1–100 nM DPN treatment significantly lowered Bax levels in a non-dose dependent manner. Conversely, level of anti-apoptotic Bcl-2 was significantly reduced by A1–42 compared to control and was restored by DPN pre-treatment (Fig. 3B). Levels of pro-apoptotic activated caspase-3 protein were significantly increased in A1–42 -treated cells compared to controls but were significantly attenuated by 0.1–100 nM DPN (Fig. 3C) in a dose-dependent manner. As anticipated, IL-1 and IL-6 expression levels were significantly increased by A1–42 compared to control, and repressed by
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DPN pre-treatment (Fig. 4A and B). A1–42 -induced JNK and p38 phosphorylation were inhibited by DPN pre-treatment. By the same token, inhibition of ERK1/2 and Akt phosphorylation by A1–42 , were antagonized by pre-treatment with DPN (Fig. 5). 4. Discussion A1–42 induces neurotoxicity through an apoptotic event mediated in part by the generation of ROS and the peptide is able to induce expression of pro-inflammatory cytokines via activation of ERK1/2/Akt and JNK/p38 pathways [28]. In the present study we demonstrated that DPN significantly attenuated A1–42 -induced apoptotic cell death of rat primary neuronal cell by decreasing ROS generation with concomitant increase in expression of antiapoptotic Bcl-2 and decrease in pro-apoptotic Bax and activated caspase-3. ROS release from dysfunctional mitochondria is considered as the cause of oxidative stress in AD [21]. A1–42 can initiate the production of ROS leading to inner mitochondrial membrane alterations that result in an opening of the mitochondrial permeability transition pore, promote the loss of the mitochondrial transmembrane potential, and trigger the release of cytochrome c from the inter-membrane space into the cytosol that can further activate caspase-3 [11]. The decline in ROS levels observed with DPN treatment of A1–42 -treated primary cortical neurons is not dose-dependent. Consistent with our findings, Bryant and Dorsa [3] showed that 0.1 nM DPN did not significantly protect rat cultured cortical neurons from glutamate-induced cell death.
Fig. 5. DPN at concentrations of 1–100 nM suppressed the A1–42 -upregulated phosphorylation of JNK (A) and p38 (B). A1–42 -downregulated phosphorylation of ERK (C) and Akt (D) was increased by pre-treatment with 1–100 nM DPN. Results are expressed as mean ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. A1–42 -treated group.
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Similarly, Vallés et al. [27] demonstrated that 0.2 nM estrogen prevented an increase in ROS levels caused by A in rat primary cultured cortical neurons. A is able to potentiate ROS generating capacity of metal ions in vitro by A-metal interaction [1]. As DPN is a phenolic compound, which has been shown to contain metal-binding motif [8], it may attenuate ROS production via its metal chelating capacity at the early onset of AD pathogenesis prior to neuronal cell death. According to Praticò [20], A could increase the generation of ROS and induce oxidative stress at the early stage of AD process. ROS generation-induced by A1–42 treatment is only one of a number of possible mechanisms underlying neuronal cell death and A1–42 can also trigger other cellular responses such as synaptic dysfunction and tau hyperphosphorylation [14]. Therefore, 0.1 nM DPN can attenuate ROS production and the protection from neuronal cell death, which is the consequence of ROS production, was observed at 1–100 nM DPN, as we have shown in this study. A study by Li et al. [15] supported our findings by demonstrating that 1 M of the antioxidant 2-methoxy-6-acetyl-7-methyljuglone (MAM) showed greater effect on ROS production triggered by tert-butyl hydroperoxide (t-BHP) but 2.5 M MAM elicited more pronounced neuroprotection against t-BHP-induced neuronal cell death. Besides its anti-oxidative effects, DPN likely mediates neuroprotection against A1–42 through its direct interaction with ER. De Marinis et al. [7] reported that DPN protects H2 O2 -induced cell death by attenuating activated caspase-3 expression in neuroblastoma SK-N-BE cells, which is similar to our findings. To clarify whether ER plays a significant role in neuroprotective effects of DPN, they demonstrated that (R,R)-THC, a specific ER inhibitor, completely inhibited this protective effect. Furthermore, the effects of DPN on the activation of Akt signaling pathway in rat rostral ventrolateral medulla neurons are significantly inhibited by (R,R)-THC [29]. These above findings lend support to the notion that the effects of DPN on reducing activated caspase-3 level and on activating Akt signaling pathway are through its mediation of ER function and hence the inhibition exerted by (R,R)-THC. The natural estrogen, unlike DPN, can stimulate both ER␣ and ER signaling pathways. Examining the differential effects between DPN and estrogen may lead to better understanding of DPN neuroprotection against A toxicity. A previous study by Spampinato et al. [23] showed that 100 nM DPN caused only a slight protective effect against A25–35 -induced neuronal death. In addition, this study was focused on the neuroprotective effects of estrogens, including DPN, through the mGlu1 receptor. The results showed that ER␣, not ER, interact with mGlu1 receptors to promote neuroprotection. These findings agree with our result of 100 nM DPN preventing A1–42 -induced neuronal cell death. In addition, DPN repressed A1–42 -induced cytokines IL-1 and IL-6 production, and up-regulated ERK1/2/Akt pathway but downregulated that of JNK/p38. While phosphorylation of JNK and p38 is associated with cytotoxicity and pro-inflammatory responses, phosphorylation of ERK promotes cell survival [12]. It is worth noting that ERK1/2 activity is mediated by Ras, whereas the activities of JNK and p38 are Ras-independent [17], suggesting that the effects of DPN are associated with both the Ras-dependent and -independent pathways. Several experiments on rat cultured hippocampal neurons demonstrated that the neuroprotective effects of DPN are mediated through ERK1/2 and PI3K/Akt signaling pathways. ERK inhibitor PD98059 was found to significantly block DPN-induced neuroprotection against glutamate-induced excitotoxicity [31]. Furthermore, PI3K inhibitor, wortmannin, abolished DPN-induced an elevation of the levels of insulin-degrading enzyme (IDE) [33] which is involved in A degradation and reduced IDE expression has been associated with the development of AD [32]. The findings from this study suggest that the mechanisms responsible for the anti-oxidant, anti-apoptotic, and
anti-inflammatory properties of DPN in A1–42 -mediated neuronal cytotoxicity may be associated with inhibition of JNK and p38 phosphorylation, and activation of ERK1/2 and Akt phosphorylation pathways. Acknowledgements This research project was supported by The Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (Grant no. PHD/0354/2550) and the Office of the Higher Education Commission, Ministry of Education, Thailand. We are grateful to Prof. Prapon Wilairat, Mahidol University, for the valuable suggestions that improved the quality of this manuscript. References [1] S.C. Bondy, S.X. Guo-Ross, A.T. Truong, Promotion of transition metal-induced reactive oxygen species formation by beta-amyloid, Brain Res. 799 (1998) 91–96. [2] C.M. Brown, T.A. Mulcahey, N.C. Filipek, P.M. Wise, Production of proinflammatory cytokines and chemokines during neuroinflammation: novel roles for estrogen receptors ␣ and , Endocrinology 151 (2010) 4916–4925. [3] D.N. Bryant, D.M. Dorsa, Roles of estrogen receptors alpha and beta in sexually dimorphic neuroprotection against glutamate toxicity, Neuroscience 170 (2010) 1261–1269. [4] T.J. Chen, D.C. Wang, S.S. Chen, Amyloid- interrupts the PI3K-Akt-mTOR signaling pathway that could be involved in brain-derived neurotrophic factorinduced Arc expression in rat cortical neurons, J. Neurosci. Res. 87 (2009) 2297–2307. [5] J. Clayden, N. Greeves, S. Warren, Organic Chemistry, second ed., Oxford University Press, Oxford, 2012. [6] K.N. Dahlgren, A.M. Manelli, W.B. Stine Jr., L.K. Baker, G.A. Krafft, M.J. LaDu, Oligomeric and fibrillar species of amyloid- peptides differentially affect neuronal viability, J. Biol. Chem. 277 (2002) 32046–32053. [7] E. De Marinis, P. Ascenzi, M. Pellegrini, P. Galluzzo, P. Bulzomi, M.A. Arevalo, L.M. Garcia-Segura, M. Marino, 17-estradiol-a new modulator of neuroglobin levels in neurons: role in neuroprotection against H2 O2 -induced toxicity, Neurosignals 18 (2010) 223–235. [8] M. Guo, C. Perez, Y. Wei, E. Rapoza, G. Su, F. Bou-Abdallah, N.D. Chasteen, Ironbinding properties of plant phenolics and cranberry’s bio-effects, Dalton Trans. 43 (2007) 4951–4961. [9] K. Hernández-Fonseca, L. Massieu, S. García de la Cadena, C. Guzmán, I. Camacho-Arroyo, Neuroprotective role of estradiol against neuronal death induced by glucose deprivation in cultured rat hippocampal neurons, Neuroendocrinology 96 (2012) 41–50. [10] R.W. Irwin, J. Yao, J. To, R.T. Hamilton, E. Cadenas, R.D. Brinton, Selective oestrogen receptor modulators differentially potentiate brain mitochondrial function, J. Neuroendocrinol. 24 (2012) 236–248. [11] K.J. Ivins, J.K. Ivins, J.P. Sharp, C.W. Cotman, Multiple pathways of apoptosis in PC12 cells. CrmA inhibits apoptosis induced by -amyloid, J. Biol. Chem. 274 (1999) 2107–2112. [12] M.R. Junttila, S.P. Li, J. Westermarck, Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival, FASEB J. 22 (2008) 954–965. [13] S. Kumar, R. Patel, S. Moore, D.K. Crawford, N. Suwanna, M. Mangiardi, S.K. Tiwari-Woodruff, Estrogen receptor  ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis, Neurobiol. Dis. 56 (2013) 131–144. [14] F.M. LaFerla, K.N. Green, S. Oddo, Intracellular amyloid- in Alzheimer’s disease, Nat. Rev. Neurosci. 8 (2007) 499–509. [15] Y.B. Li, Z.Q. Lin, Z.J. Zhang, M.W. Wang, H. Zhang, Q.W. Zhang, S.M. Lee, Y.T. Wang, P.M. Hoi, Protective, antioxidative and antiapoptotic effects of 2methoxy-6-acetyl-7-methyljuglone from Polygonum cuspidatum in PC12 cells, Planta Med. 77 (2011) 354–361. [16] J.K. Lin, P.C. Chen, C.T. Ho, S.Y. Lin-Shiau, Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3 -digallate, (−)-pigallocatechin-3-gallate, and propyl gallate, J. Agric. Food Chem. 48 (2000) 2736–2743. [17] Y. Liu, K.Z. Guyton, M. Gorospe, Q. Xu, J.C. Lee, N.J. Holbrook, Differential activation of ERK JNK/SAPK and P38/CSBP/RK MAP kinase family members during the cellular response to arsenite, Free Radic. Biol. Med. 21 (1996) 771–781. [18] J.A. McCubrey, L.S. Steelman, S.L. Abrams, J.T. Lee, F. Chang, F.E. Bertrand, P.M. Navolanic, D.M. Terrian, R.A. Franklin, A.B. D’Assoro, J.L. Salisbury, M.C. Mazzarino, F. Stivala, M. Libra, Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance, Adv. Enzyme Regul. 46 (2006) 249–279. [19] C.J. Pike, D. Burdick, A.J. Walencewicz, C.G. Glabe, C.W. Cotman, Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state, J. Neurosci. 13 (1993) 1676–1687. [20] D. Praticò, Oxidative stress hypothesis in Alzheimer’s disease: a reappraisal, Trends Pharmacol. Sci. 29 (2008) 609–615.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Neuroprotective effects of diarylpropionitrile against -amyloid peptide-induced neurotoxicity in rat cultured cortical neurons Nirut Suwanna a , Wipawan Thangnipon a,∗ , Rungtip Soi-ampornkul b a b
Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom, Thailand Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
h i g h l i g h t s • • • •
Treatment with DPN alleviated A1–42 -induced cell death and morphological changes. DPN also reduced production of ROS induced by A1–42 . DPN prevents A1–42 -induced toxicity by attenuating apoptosis and inflammation. DPN effects may involve inhibition of JNK and p38 including activation of ERK1/2 and Akt.
a r t i c l e
i n f o
Article history: Received 16 December 2013 Received in revised form 2 June 2014 Accepted 12 June 2014 Available online 21 June 2014 Keywords: Alzheimer’s disease -Amyloid peptide Diarylpropionitrile Antioxidant Apoptosis Anti-inflammation
a b s t r a c t Alzheimer’s disease is a major cause of dementia in the elderly that involves a -amyloid peptide (A)-induced cascade of an increase in oxidative damage and inflammation. The present study demonstrated the neuroprotective effects of diarylpropionitrile (DPN), a non-steroidal estrogen receptor  selective ligand, against 10 M A1–42 -induced oxidative stress and inflammation in primary rat cortical cell culture. Pre-treatment with 1–100 nM DPN significantly decreased neuronal cell death by increasing cell viability through a significant attenuation in the reactive oxygen species level, downregulation of pro-apoptotic activated caspase-3 and Bax, and upregulation of anti-apoptotic Bcl-2, thereby mitigating apoptotic morphological alterations. DPN pre-treatment decreased the expression levels of pro-inflammatory cytokines IL-1 and IL-6 through attenuation of A1–42 -induced phosphorylation of mitogen-activated protein kinases JNK and p38. In addition, DPN enhanced ERK1/2 and Akt phosphorylation depressed by A1–42 . These findings suggest that DPN protects neurons from A1–42 -induced neurotoxicity through a variety of mechanisms, ranging from anti-oxidation, anti-apoptosis, through to anti-inflammation. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is characterized by progressive loss of memory and impairment of higher cognitive functions. The pathological hallmarks of human AD brain are the presence of extracellular plaques composed of -amyloid (A) and intracellular neurofibrillary tangles [21]. A1–42 is the form that is prominently increased in brains of AD patients [14]. The mechanisms by which A1–42 induces neurotoxicity have not been completely elucidated, but previous studies on rat cultured cortical neurons
∗ Corresponding author. Tel.: +66 24419003x1203; fax: +66 24419003x1311. E-mail addresses: [email protected], [email protected] (W. Thangnipon). http://dx.doi.org/10.1016/j.neulet.2014.06.029 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
exposed to A1–42 showed apoptotic cell death, as evidenced by both morphological changes and expression of pro-apoptotic proteins, such as activated caspase-3 and Bax, and the elevation of reactive oxygen species (ROS) levels [24,25]. It has been demonstrated that A can increase expression levels of IL-1 and IL-6 in rat cortical neuronal cell cultures [22]. Thus, protection of neurons from A1–42 -induced neurotoxicity with antioxidants and anti-inflammatory compounds, such as N-trans-feruloyltyramine (NTF) [24] and N-benzylcinnamide (PT-3) [25], provides important avenues to ameliorate the pathological effects of A. (DPN) or 2,3-bis(4-hydroxyphenyl)Diarylpropionitrile propionitrile is a non-steroidal selective ligand of estrogen receptor  (ER) with neuroprotective effects in a number of neurological diseases [13]. The treatments with 10 and 100 nM DPN protected rat cultured hippocampal neurons from glucose
N. Suwanna et al. / Neuroscience Letters 578 (2014) 44–49
deprivation-induced cell death [9]. DPN also has an antiinflammatory role in neuroprotection by suppressing IL-1 and IL-6 expressions in mouse brain [2]. In primary rat cultured hippocampal neurons DPN up-regulates expressions of a panel of bioenergetic enzymes and antioxidant proteins [10]. ERK1/2 and Akt signaling cascades play critical roles in the transmission of signals from growth factor receptors to regulate gene expression and prevent apoptosis [18], and these pathways are disrupted by A in rat cultured cortical neurons [4,26]. DPN treatment can increase the phosphorylation of Akt and ERK1/2 in neuronal cultures derived from mouse embryonic stem cells [30]. In our previous studies, we demonstrated that NTF and PT-3 attenuate A1–42 -induced cell death in rat cortical neuronal cultures by inhibiting the generation of ROS, reducing expression of proapoptotic activated caspase-3 and Bax, activating anti-apoptotic protein Bcl-2, reducing expression of pro-inflammatory cytokines IL-1 and IL-6, and attenuating phosphorylation of JNK and p38, proteins associated with pro-inflammatory response [24,25]. As DPN shares some common structural characteristics with NTF, such as phenolic hydroxyl groups which may serve as effective radical scavenging moieties [5], we examined the mechanisms of DPN neuroprotection against the neuronal insults of A1–42 treatment in rat primary neuronal cell cultures.
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Fig. 2. ROS levels in A1–42 -treated cultures were significantly reduced by 0.1–100 nM DPN pre-treatment. Results are expressed as mean ± SEM of three independent experiments. ***p < 0.001 vs. control; ### p < 0.001 vs. A1–42 -treated group.
2. Methods Enriched primary neuronal cell cultures were prepared from cerebral cortices of 10 to 12 Wistar rat fetuses at embryonic day-17 according to the previous procedures [19]. On day 5, cultures were
Fig. 1. DPN prevents morphological alterations from A1–42 -induced toxicity in cultured cortical neurons. Morphologies of control (A), 10 M A1–42 -treated (B), 10 nM DPN pre-treated followed by 10 M A1–42 -treated (C). In the presence of A1–42 (B), neurons exhibited the degenerative findings of cell body shrinkage (arrowheads) and neurite blebbing (arrows) compared to controls (A) and DPN-treated (C). Cell viability of A1–42 -treated cultures determined by MTT assay was significantly increased by 1–100 nM DPN treatment (D). Results are expressed as mean ± SEM of 3 independent experiments. ***p < 0.001 vs. control; # p < 0.05, ### p < 0.001 vs. A1–42 -treated group. Scale bar = 25 m.
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Fig. 4. Pre-treatment with 0.1–100 nM DPN significantly reduced activated IL-1 levels induced by A1–42 treatment on cultured cortical neurons (A). Expression of IL-6 was reduced by pre-treatment with 100 nM DPN (B). Results are expressed as mean ± SEM of 3 independent experiments. *p < 0.05, ***p < 0.001 vs. control; # p < 0.05, ## p < 0.01 vs. A1–42 -treated group.
Fig. 3. A1–42 -stimulated expression of Bax was reduced by pre-treatment with 0.1–100 nM DPN (A). Pre-treatment with 0.1–100 nM DPN increased the expression of Bcl-2 (B) and decreased the expression of activated caspase-3 (C). Results are expressed as mean ± SEM of 3 independent experiments. *p < 0.05, ***p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. A1–42 -treated group.
treated with 0, 0.1, 1, 10 and 100 nM DPN dissolved in absolute ethanol for 1 h prior to incubating for 24 h with a sublethal dose of 10 M oligomeric A1–42 (Keck Biotechnology, USA) [6]. Then cultures were processed for western blot analysis, ROS determination and cell viability evaluation. Morphological changes of neurons were visualized under an inverted phase-contrast microscope. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the absorbance at 570 nm measured with a microplate reader (Molecular Probes, Eugene, OR) [11].
Intracellular ROS levels were determined using 2 ,7 dichlorodihydrofluorescein diacetate (DCF-DA) dye assay [16]. In brief, cells were incubated with 50 M DCFH-DA dissolved in absolute ethanol at 37 ◦ C for 45 min in the dark prior to DPN and A1–42 treatment and DCF fluorescence was quantified using a Multi-Detection microplate reader (BioTek Synergy HT, Winooski, VT) with excitation and emission wavelength of 485 and 530 nm respectively. Equal amounts of protein from cell lysate were separated on 10% SDS-PAGE under reducing condition, and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with 1:500 mouse anti-Bcl-2, -Bax, -IL-6, 1:500 rabbit anti-IL-1 (Santa Cruz), 1:500 rabbit anti-activated caspase-3, phospho-p38, -p38, 1:1000 rabbit anti-phospho-ERK1/2, -ERK1/2, -phospho-JNK, -JNK, -phospho-Akt, -Akt, or 1:2500 rabbit antiactin antibodies (Cell Signaling), and then incubated with a HRP-conjugated secondary antibody (Zymed). The signal was detected with ECL Western blotting substrate (Bio-Rad) and captured on HyperfilmTM (Amersham Pharmacia Biotech). Statistical significance of differences was determined by oneway analysis of variance (ANOVA), followed by SNK post-test. The results are considered statistically significant at p < 0.05. All data are presented as mean ± SEM of 3 independent experiments conducted in triplicate.
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3. Results Rat primary neuronal cells treated with 10 M A1–42 displayed cell shrinkage and neurite blebbing compared to normal neuronal cells (Fig. 1A and B), and pre-treatment with 10 nM DPN significantly attenuate A1–42 -induced morphological abnormalities (Fig. 1C). In addition, pre-treatment of neuronal cell cultures with 1–100 nM DPN significantly reduced A1–42 -induced neuronal cell death as assessed by MTT assay (Fig. 1D). DPN treatment alone at the test concentrations did not result in cytotoxicity. As expected, ROS production in 10 M A1–42 -treated neuronal cells is significantly increased compared to control (Fig. 2), and pre-treatment with 0.1–100 nM DPN decreased ROS levels in a non-dose response manner. DPN treatment did not significantly alter ROS levels from that of control. Apoptosis was confirmed to be the mechanism of neuronal cell death induced by 10 M A1–42 as evidenced by the elevated levels of Bax (Fig. 3A), and 0.1–100 nM DPN treatment significantly lowered Bax levels in a non-dose dependent manner. Conversely, level of anti-apoptotic Bcl-2 was significantly reduced by A1–42 compared to control and was restored by DPN pre-treatment (Fig. 3B). Levels of pro-apoptotic activated caspase-3 protein were significantly increased in A1–42 -treated cells compared to controls but were significantly attenuated by 0.1–100 nM DPN (Fig. 3C) in a dose-dependent manner. As anticipated, IL-1 and IL-6 expression levels were significantly increased by A1–42 compared to control, and repressed by
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DPN pre-treatment (Fig. 4A and B). A1–42 -induced JNK and p38 phosphorylation were inhibited by DPN pre-treatment. By the same token, inhibition of ERK1/2 and Akt phosphorylation by A1–42 , were antagonized by pre-treatment with DPN (Fig. 5). 4. Discussion A1–42 induces neurotoxicity through an apoptotic event mediated in part by the generation of ROS and the peptide is able to induce expression of pro-inflammatory cytokines via activation of ERK1/2/Akt and JNK/p38 pathways [28]. In the present study we demonstrated that DPN significantly attenuated A1–42 -induced apoptotic cell death of rat primary neuronal cell by decreasing ROS generation with concomitant increase in expression of antiapoptotic Bcl-2 and decrease in pro-apoptotic Bax and activated caspase-3. ROS release from dysfunctional mitochondria is considered as the cause of oxidative stress in AD [21]. A1–42 can initiate the production of ROS leading to inner mitochondrial membrane alterations that result in an opening of the mitochondrial permeability transition pore, promote the loss of the mitochondrial transmembrane potential, and trigger the release of cytochrome c from the inter-membrane space into the cytosol that can further activate caspase-3 [11]. The decline in ROS levels observed with DPN treatment of A1–42 -treated primary cortical neurons is not dose-dependent. Consistent with our findings, Bryant and Dorsa [3] showed that 0.1 nM DPN did not significantly protect rat cultured cortical neurons from glutamate-induced cell death.
Fig. 5. DPN at concentrations of 1–100 nM suppressed the A1–42 -upregulated phosphorylation of JNK (A) and p38 (B). A1–42 -downregulated phosphorylation of ERK (C) and Akt (D) was increased by pre-treatment with 1–100 nM DPN. Results are expressed as mean ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. A1–42 -treated group.
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Similarly, Vallés et al. [27] demonstrated that 0.2 nM estrogen prevented an increase in ROS levels caused by A in rat primary cultured cortical neurons. A is able to potentiate ROS generating capacity of metal ions in vitro by A-metal interaction [1]. As DPN is a phenolic compound, which has been shown to contain metal-binding motif [8], it may attenuate ROS production via its metal chelating capacity at the early onset of AD pathogenesis prior to neuronal cell death. According to Praticò [20], A could increase the generation of ROS and induce oxidative stress at the early stage of AD process. ROS generation-induced by A1–42 treatment is only one of a number of possible mechanisms underlying neuronal cell death and A1–42 can also trigger other cellular responses such as synaptic dysfunction and tau hyperphosphorylation [14]. Therefore, 0.1 nM DPN can attenuate ROS production and the protection from neuronal cell death, which is the consequence of ROS production, was observed at 1–100 nM DPN, as we have shown in this study. A study by Li et al. [15] supported our findings by demonstrating that 1 M of the antioxidant 2-methoxy-6-acetyl-7-methyljuglone (MAM) showed greater effect on ROS production triggered by tert-butyl hydroperoxide (t-BHP) but 2.5 M MAM elicited more pronounced neuroprotection against t-BHP-induced neuronal cell death. Besides its anti-oxidative effects, DPN likely mediates neuroprotection against A1–42 through its direct interaction with ER. De Marinis et al. [7] reported that DPN protects H2 O2 -induced cell death by attenuating activated caspase-3 expression in neuroblastoma SK-N-BE cells, which is similar to our findings. To clarify whether ER plays a significant role in neuroprotective effects of DPN, they demonstrated that (R,R)-THC, a specific ER inhibitor, completely inhibited this protective effect. Furthermore, the effects of DPN on the activation of Akt signaling pathway in rat rostral ventrolateral medulla neurons are significantly inhibited by (R,R)-THC [29]. These above findings lend support to the notion that the effects of DPN on reducing activated caspase-3 level and on activating Akt signaling pathway are through its mediation of ER function and hence the inhibition exerted by (R,R)-THC. The natural estrogen, unlike DPN, can stimulate both ER␣ and ER signaling pathways. Examining the differential effects between DPN and estrogen may lead to better understanding of DPN neuroprotection against A toxicity. A previous study by Spampinato et al. [23] showed that 100 nM DPN caused only a slight protective effect against A25–35 -induced neuronal death. In addition, this study was focused on the neuroprotective effects of estrogens, including DPN, through the mGlu1 receptor. The results showed that ER␣, not ER, interact with mGlu1 receptors to promote neuroprotection. These findings agree with our result of 100 nM DPN preventing A1–42 -induced neuronal cell death. In addition, DPN repressed A1–42 -induced cytokines IL-1 and IL-6 production, and up-regulated ERK1/2/Akt pathway but downregulated that of JNK/p38. While phosphorylation of JNK and p38 is associated with cytotoxicity and pro-inflammatory responses, phosphorylation of ERK promotes cell survival [12]. It is worth noting that ERK1/2 activity is mediated by Ras, whereas the activities of JNK and p38 are Ras-independent [17], suggesting that the effects of DPN are associated with both the Ras-dependent and -independent pathways. Several experiments on rat cultured hippocampal neurons demonstrated that the neuroprotective effects of DPN are mediated through ERK1/2 and PI3K/Akt signaling pathways. ERK inhibitor PD98059 was found to significantly block DPN-induced neuroprotection against glutamate-induced excitotoxicity [31]. Furthermore, PI3K inhibitor, wortmannin, abolished DPN-induced an elevation of the levels of insulin-degrading enzyme (IDE) [33] which is involved in A degradation and reduced IDE expression has been associated with the development of AD [32]. The findings from this study suggest that the mechanisms responsible for the anti-oxidant, anti-apoptotic, and
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