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Epigenetic regulation of death of crayfish glial cells but not neurons induced by photodynamic impact
Brain Research Bulletin 102 (2014) 15–21
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Epigenetic regulation of death of crayfish glial cells but not neurons induced by photodynamic impact S.A. Sharifulina a , M.A. Komandirov b , A.B. Uzdensky a,b,∗ a b
A.B. Kogan Research Institute for Neurocybernetics, Southern Federal University, Rostov-on-Don 344090, Russia Department of Biophysics and Biocybernetics, Southern Federal University, Rostov-on-Don 344090, Russia
a r t i c l e
i n f o
Article history: Received 23 November 2013 Received in revised form 19 January 2014 Accepted 22 January 2014 Available online 4 February 2014 Keywords: Neuron Glia Epigenetic DNA methylation Histone deacetylation Photodynamic Cell death
a b s t r a c t Epigenetic processes are involved in regulation of cell functions and survival, but their role in responses of neurons and glial cells to oxidative injury is insufficiently explored. Here, we studied the role of DNA methylation and histone deacetylation in reactions of neurons and surrounding glial cells to photodynamic treatment that induces oxidative stress and cell death. Isolated crayfish stretch receptor consisting of a single mechanoreceptor neuron surrounded by glial cells was photosensitized with aluminum phthalocyanine Photosens that induced neuron inactivation, necrosis of the neuron and glia, and glial apoptosis. Inhibitors of DNA methylation 5-azacytidine and 5-aza-2 -deoxycytidine (decitabine) reduced the level of PDT-induced necrosis of glial cells but not neurons by 1.3 and 2.0 times, respectively, and did not significantly influence apoptosis of glial cells. Histone deacetylase inhibitors valproic acid and trichostatin A inhibited PDT-induced both necrosis and apoptosis of satellite glial cells but not neurons by 1.6–2.7 times. Thus, in the crayfish stretch receptor DNA methylation and histone deacetylation are involved in epigenetic control of glial but not neuronal necrosis. Histone deacetylation also participates in glial apoptosis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Photodynamic therapy (PDT) is based on photoinduced generation of strongly cytotoxic singlet oxygen, following oxidative stress and death of stained cells under light exposure in the presence of oxygen. It is currently used in oncology (Agostinis et al., 2011) including treatment of brain tumors (Kostron, 2010; Madsen et al., 2006). However, in the last case not only malignant, but also surrounding normal neurons and glial cells are damaged that can induce unacceptable side effects and neurological disorders. Therefore, PDT effect on the normal nervous tissue should be carefully investigated. Cell reactions to various impacts including PDT and their death are controlled by the complex signaling system that consists of thousands proteins that form the intracellular regulatory network (Buytaert et al., 2007; Gomperts et al., 2009; Uzdensky, 2008). If the regulatory potential of the proteins present in the cell is insufficient,
Abbreviations: DNMT, DNA methyltransferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; PDT, photodynamic therapy. ∗ Corresponding author at: Department of Biophysics and Biocybernetics, Rostov State University, 194/1 Stachky Prospect., Institute of Neurocybernetics, Rostov-onDon 344090, Russia. Tel.: +7 8632 433111; fax: +7 8632 433577. E-mail address: [email protected] (A.B. Uzdensky). 0361-9230/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2014.01.005
additional protein synthesis is stimulated. Gene expression is controlled by transcription factors and epigenetic regulators: DNA methylation/demethylation and histone modifications such as methylation/demethylation, acetylation/deacetylation, and phosphorylation/dephosphorylation, which regulate access of transcription factors and RNA polymerase II to gene promoters. Aberrant DNA methylation and histone modifications are involved in diverse neuronal functions and neurological disorders such as synaptic plasticity and memory formation (Sultan and Day, 2011), Alzheimer, Parkinson, and Huntington diseases (Gebicke-Haerter, 2012; Gray, 2011; Zawia et al., 2009), schizophrenia (Harrison and Dexter, 2013), epilepsy (Hwang et al., 2013), acute and chronic stress (Stankiewicz et al., 2013), and stroke (Hwang et al., 2013). However, there are very few direct data on the involvement of epigenetic processes in neurodegeneration (Chestnut et al., 2011). The role of epigenetic processes in the reactions of the normal nervous tissue to photodynamic treatment remains almost unstudied. Using the proteomic approach we have recently shown that PDT influences the expression of various proteins involved in epigenetic regulation in the murine cerebral cortex such as histone deacetylases HDAC-1 and HDAC-11, transcriptional repressors Kaiso and dimethylated histone H3, transcription factors AP1/c-Jun and FOXC2, phosphorylated histone H2AX involved in DNA repair, importin ␣5/7, protein methyltransferase PRMT5, and some others (Demyanenko et al., 2013). However, the study
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of the complex brain tissue that consists of different cell types including neurons, glial cells, and blood vessels does not provide the information on epigenetic regulation in different cell types. The suitable object for study of epigenetic processes occurring simultaneously in the interacting neuronal and glial cells is the crayfish stretch receptor that consists of a single mechanoreceptor neuron surrounded by glial cells, which form a multilayer envelope around the neuron (Fedorenko and Uzdensky, 2009). The role of diverse signaling and metabolic processes in responses of crayfish neurons and glial cells to photodynamic treatment has been previously studied (Komandirov et al., 2011; Kovaleva et al., 2013; Uzdensky et al., 2005, 2007, 2013). In the present work we studied the role of DNA methylation and histone deacetylation in PDT-induced inactivation and death of the stretch reception neuron and surrounding glial cells. We showed that inhibitors of DNA methylation 5-azacytidine and 5aza-2 -deoxycytidine (decitabine) inhibited PDT-induced necrosis of glial cells but not neurons, whereas inhibitors of histone deacetylase (HDAC) valproic acid and trichostatin A inhibited PDT-induced necrosis and apoptosis of satellite glial cells but not neurons. 2. Materials and methods 2.1. Chemicals The following chemicals were used: DNA methyltransferase inhibitors 5-azacytidine (5-Aza, 10 M) and 5-aza-2 deoxycytidine (decitabine, 10 M); histone deacetylase inhibitors valproic acid, sodium salt (VPA, 0.5 mM) and trichostatin A (TSA, 100 nM); fluorochromes propidium iodide and Hoechst 33342. All chemicals were obtained from Sigma–Aldrich-Rus (Moscow, Russia). Photosensitizer Photosens, a mixture of sulphonated aluminum phthalocyanines, AlPcSn , where mean n = 3.1, was obtained from NIOPIK (Moscow, Russia). 2.2. Crayfish stretch receptor and recording of its firing The crayfishes Astacus leptodactylus from Don River affluences were purchased on the local market. Their abdominal stretch receptors were isolated as described by Florey and Florey (1955). These were placed into a plexiglass chamber equipped with a device for receptor muscle extension and filled with 2 ml of van Harreveld saline (mM: NaCl – 205; KCl – 5.4; NaHCO3 – 0.2; CaCl2 – 13.5; MgCl2 – 5.4; pH 7.2–7.4). Neuron spikes were recorded extracellularly from axons by glass pipette suction electrodes, amplified, digitized by the analog-digital converter L-761 (L-Card, Moscow, Russia), and processed by a personal computer using the homemade software that provided continuous monitoring of firing. Experiments were carried out at 23 ± 4 ◦ C. 2.3. Photodynamic treatment and application of enzyme inhibitors At the beginning of the experiment the initial level of neuronal activity was set near 6–10 Hz by application of the appropriate receptor muscle extension. After 30 min control recording of neuronal activity, Photosens (75 nM) and an inhibitor were added into the chamber with an interval of 3–5 min. After following 30-min incubation, cells were irradiated with the He–Ne laser (633 nm, 0.3 W/cm2 ). A laser beam diameter was of 3 mm, so that the neuronal body and a significant part of axon were irradiated. 30-min light exposure was longer than the duration of bioelectric neuron response measured from the irradiation start to the moment of firing abolition (typically, 10–20 min), which we called the “neuron lifetime”. Photosens and an inhibitor were present in the chamber during and after irradiation. The inhibitor concentration was
usually chosen to be approximately 2 times lower than the predetermined concentration, which disturbed neuron firing in the darkness for 3–4 h.
2.4. Cell death assay In order to visualize dead neurons and glial cells, 20 M propidium iodide and 10–20 M Hoechst 33342 (both from Sigma–Aldrich) were added into the experimental chamber at 8 h postirradiation. This time interval was sufficient for apoptosis development (Uzdensky et al., 2005). Then preparations were washed with van Harreveld saline, fixed with 0.2% glutaraldehyde, repeatedly washed and mounted in glycerol. Fluorescent images were acquired using the fluorescence microscope LumamI3 (LOMO, Sankt-Petersburg, Russia) equipped with a digital photocamera. Propidium iodide, a membrane impermeable fluorochrome, imparts red fluorescence to nuclei of necrotic cells with the compromised plasma membrane. Hoechst 33342 imparts blue fluorescence to the nuclear chromatin. It visualizes intact nuclei of living cells and fragmented nuclei of apoptotic cells (Fig. 1). Nucleus fragmentation is the final stage of apoptosis when the noreturn point has passed. It should be mentioned that other methods for apoptosis evaluation such as caspase activation, cytochrome c release, or annexin V assay, which require observation of the cytoplasm or of the plasma membrane, are not suitable for study of glial cells in the isolated stretch receptor because of their multilayer roulette-like morphology and overlapping of optical images of different glial processes and the neuronal cytoplasm (Fedorenko and Uzdensky, 2009). The red nuclei of necrotic glial cells stained by propidium iodide were counted in the predetermined standard field (100 m × 100 m) around SRN soma so that the neuron nucleus was situated in its center. Fragmented nuclei of apoptotic glial cells were counted around the proximal 2 mm axon fragment where glial apoptosis was more profound than around the neuron body. Their mean number representing the level of glial apoptosis was expressed below as relative units. The one way ANOVA was used for statistical evaluation of the difference between independent experimental groups. Data are presented as mean ± S.E.M.
3. Results Neither Photosens (75 nM), nor laser radiation separately changed significantly neuronal activity and survival after stretch receptor isolation. However, their combined action, i.e. PDT led to inhibition of neuronal activity, necrosis of neurons and glial cells and apoptosis of glial cells (Figs. 2–5). Apoptotic nuclear fragmentation was never observed in neurons as described earlier (Uzdensky et al., 2002, 2005). In the darkness, both inhibitors of DNA methyltransferase 5-azacytidine (10 M) and decitabine (10 M) did not influence significantly the duration of bioelectric neuron response (Figs. 2A and 3A) and the level of neuronal necrosis (Figs. 2B and 3B). However, 5-azacytidine more than 2 times increased the level of glial necrosis (p < 0.05; Fig. 2C) and showed the similar tendency for glial apoptosis (p > 0.05; Fig. 2D). Unlike, decitabine did not demonstrate glia toxicity in the darkness (Fig. 3C and D). 5-Azacytidine and decitabine did not influence the PDTinduced changes in the neuronal activity and necrosis level (Figs. 2A and B, 3A and B). We also did not observe significant effects of these DNA methyltransferase inhibitors on PDT-induced glial apoptosis (Figs. 2D and 3D). However, both of them reduced the levels PDT-induced necrosis of glial cells by 1.3 and 2.0 times (p < 0.05; Fig. 2C and p < 0.01; Fig. 3C, respectively).
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Fig. 1. The example of the effect of decitabine (B), PDT (C and E), and PDT + decitabine (D and F) on nuclear morphology of the stretch receptor neuron and surrounding glial cells. (A) Control. (E) and (F) show fragmented apoptotic nuclei of satellite glial cells in the proximal axon regions. The preparation was fluorochromed with propidium iodide that imparts the red fluorescence to nuclei of necrotic cells and with Hoechst 33342 that imparts blue fluorescence to nuclei of alive or fragmented apoptotic cells. Arrowheads on A–D indicate big neuron nuclei; thin arrows on E and F – fragmented nuclei of apoptotic glial cells. Scale bar 50 m.
Histone deacetylase inhibitors valproic acid, sodium salt (VPA, 0.5 mM) and trichostatin A (TSA, 100 nM) did not influence neuronal and glial necrosis and glial apoptosis in the darkness (Figs. 4 and 5, respectively). However, valproic acid reduced the firing duration of the isolated stretch receptor neuron by about 20% (p < 0.05; Fig. 4A). Both VPA and trichostatin A did not influence the PDT-induced shortening of neuronal firing (Figs. 4A and 5A) and the level of the neuronal necrosis (Figs. 4B and 5B). However, they significantly reduced photoinduced necrosis of glial cells by 2.3 and 1.6 times (p < 0.01; Figs. 4C and 5C, respectively). They also protected glial cells from PDT-induced apoptosis and reduced its level by 2.4 and 2.7 times (p < 0.05; Fig. 4D and p < 0.01; Fig. 5D, respectively). 4. Discussion Methylation of DNA, in which DNA methyltransferase (DNMT) transfers methyl group from S-adenosylmethionine to cytosine in the CpG-dinucleotide, is a major epigenetic process controlling gene transcription. Genes regulated by DNA methylation are involved in the regulation of cell survival, proliferation, and apoptosis (Baylin, 2005; Robertson and Jones, 2000). Neuronal functions and fate in health and disease are also controlled by DNA methylation (Chestnut et al., 2011; Gray, 2011; Hwang et al., 2013; Mehler, 2008; Stankiewicz et al., 2013; Zawia et al., 2009). The present data have shown that DNMT inhibitors 5azacytidine and decitabine protect crayfish glial cells but not neurons from PDT-induced necrosis. One can, therefore, suggest the involvement of DNMT in PDT-induced necrosis of glial cells.
However, the mechanism of action of these drugs may be more complex. DNA methylation of cytosines in the CpG-islands suppresses transcription. In contrast, DNA demethylation mediated by 5-azacytidine or decitabine activates the expression of silenced genes via chromatin decondensation. This allows binding of transcription factors to DNA promoters and reactivates gene expression (Alcazar et al., 2012; Haaf, 1995). For example, decitabine, except inhibition of DNA methyltransferase, induces depletion of DNMT1 and reactivates the expression of tumor suppressor genes such as p16INK4a, p15INK4b, hMLH1, VHL, APC, pRb, and BRCA1 (Alcazar et al., 2012; Karpf and Jones, 2002). Both 5-azacytidine and decitabine demonstrate general cytotoxicity related to DNA methylation. For example, in PC12 cells decitabine significantly enhanced the paraquat-mediated production of reactive oxygen species (ROS) and induced oxidative stress, mitochondrial deficit, cytochrome C release, and apoptosis (Kong et al., 2012). In normal lymphocytes and leukemia cells decitabine as well as HDAC inhibitors butyrate or SAHA stimulated ROS production and induced apoptosis. The combination of these inhibitors exerted the synergistic effect (Brodská and Holoubek, 2011). However the cytotoxic effects of these drugs are not identical. 5-Azacytydine but not decitabine inhibited the proapoptotic protein p53 and activated caspase and induced apoptosis of tumor cells (Venturelli et al., 2013). The enhancement of PDT-induced apoptosis of glial cells in the presence of 5-azacytydine corresponded to this data. However, unlike the pronecrotic effect of 5-azacytidine on crayfish glial cells in the dakness, 5-azacytidine and decitabine exerted the antinecrotic effect on photosensitized glial cells. This is a novel activity of these drugs. It cannot be explained by their effect on
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Fig. 2. (A–D) Effect of DNA methyltransferase inhibitor 5-azacytidine (5-Aza, 10 M) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of 5-azacytidine in the darkness, 3 – PDT, 4 – PDT in the presence of 5-azacytidine. Significant difference from the indicated bars: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 3. (A–D) Effect of DNA methyltransferase inhibitor decitabine (10 M) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of decitabine in the darkness, 3 – PDT, 4 – PDT in the presence of decitabine. Significant difference from the indicated bars: *p < 0.05, **p < 0.01.
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Fig. 4. (A–D) Effect of histone deacetylase inhibitor trichostatin A (TSA, 100 nM) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of trichostatin A in the darkness, 3 – PDT, 4 – PDT in the presence of trichostatin A. Significant difference from the indicated bars: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 5. (A–D) Effect of histone deacetylase inhibitor valproic acid, sodium salt (VPA, 0.5 mM) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of valproic acid, sodium salt in the darkness, 3 – PDT, 4 – PDT in the presence of valproic acid, sodium salt. Significant difference from the indicated bars: *p < 0.05, **p < 0.01, ***p < 0.001.
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PDT-induced ROS production. It may be rather related to restoring of expression of some genes, which are unidentified yet. Acetylation of histones leads to chromatin relaxation, increase in the accessibility of gene promoters to transcription factors, and expression of prosurvival genes. In contrast, histone deacetylatylation inhibits gene expression. Histones acetylation depends on the balance between histone deacetylases and histone acetyltransferases (HATs) activities (Konsoula and Barile, 2012; Struhl, 1998). In neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer and Parkinson diseases this balance is shifted toward histone deacetylation (Harrison and Dexter, 2013; Saha and Pahan, 2006). HDAC inhibitors valproic acid sodium salt, trichostatin A, sodium butirate, and others demonstrate neuroprotective effects in neurodegenerative diseases, intracerebral hemorrhage and ischemic stroke. They also protect neurons from glutamate excitotoxicity and oxidative stress (Biermann et al., 2011; Harrison and Dexter, 2013; Kanai et al., 2004; Zhang et al., 2012). Prosurvival activity of these drugs is associated with histone deacetylase inhibition and transcriptional activation of expression of neuroprotective proteins such as BDNF (brain derived neurotrophic factor), GDNF (glial derived neurotrophic factor), heat shock protein HSP70, synuclein ␣, apoptosis inhibitors Blc-2 and Bcl-XL, etc. (Chuang et al., 2009; Wu et al., 2008; Zhang et al., 2012). Valproic acid sodium salt and trichostatin A are promising for treatment of various neurodegenerative diseases (Harrison and Dexter, 2013; Saha and Pahan, 2006). In our experiments valproic acid sodium salt and trichostatin A suppressed PDT-induced death of glial cells but not neurons. Therefore, the PDT-induced shift of the HAT/HDAC balance toward histone deacetylation is involved in both necrosis and apoptosis of crayfish glial cells. Apoptosis is regulated by the complex cell signaling system, which is controlled by the epigenetic processes. Interesting, in crayfish glial cells not only apoptosis but also necrosis is under the epigenetic control. Further investigations should identify the genes targeted by HDAC inhibitors and the pathways involved in glial protection. The insensitivity of crayfish mechanoreceptor neurons to inhibitors of DNA methylation and histone deacetylation indicates the insignificant role of epigenetic processes in regulation of neuronal survival. Unlike glial cells, the apoptotic nucleus fragmentation was not observed in the crayfish stretch receptor neuron. As shown early, apoptosis of this neuron was not induced by various physical and pharmacological factors. This large neuron, unique in the crayfish nervous system, is necessary for movement control and, therefore, for animal survival. The apoptosis program in this cell has been suggested to be intrinsically blocked (Uzdensky et al., 2002, 2005, 2007), like in many vertebrate adult neurons, which are necessary throughout all organism lifetime, and their premature death can irreversibly disturb organism functions (Benn and Woolf, 2004; Walsh et al., 2004). One can suggest that necrosis of this neuron is not epigenetically regulated as well. The neuroglial interactions may be involved in maintaining of neuron and glial survival (Kolosov and Uzdensky, 2006). Neurotrophic factors (Bian et al., 2012; Komandirov et al., 2011; Lobanov and Uzdensky, 2009), nitric oxide (Kovaleva et al., 2013), or neuromediators such as extra-synaptic glutamate (Gafurov et al., 2002; Rodriguez et al., 2013) are among possible intercellular signals. All of them can induce intracellular pathways that include epigenetic regulation of gene transcription, which modulate cell survival. The nucleus of the stretch receptor neuron is very large. It contains almost completely unpacked euchromatin that shows its high transcriptional activity. Unlike, glial nuclei are much smaller and contain big masses of condensed heterochromatin (Fig. 1 and Fedorenko and Uzdensky, 2009) that indicates silencing of many genes. One can suggest that the potency of epigenetic regulators to stimulate gene transcription may be much
stronger in glial cells than in the neuron, in which genes are already expressed. In conclusion, our data showed that in the photosensitized crayfish stretch receptor epigenetic processes such as DNA methylation and histone deacetylation participate in PDT-induced necrosis of glial cells but not neurons. Histone deacetylation is also involved in regulation of glial apoptosis. Conflict of interests None declared. Acknowledgement The work was supported by the RFBR grant no. 11-04-01476. References Agostinis, P., Berg, K., Cengel, K.A., Foster, T.H., Girotti, A.W., Gollnick, S.O., Hahn, S.M., Hamblin, M.R., Juzeniene, A., Kessel, D., Korbelik, M., Moan, J., Mroz, P., Nowis, D., Piette, J., Wilson, B.C., Golab, J., 2011. Photodynamic therapy of cancer: an update. C. A. Cancer J. Clin. 61, 250–281, http://dx.doi.org/10.3322/caac.20114. Alcazar, O., Achberger, S., Aldrich, W., Hu, Z., Negrotto, S., Saunthararajah, Y., Triozzi, P., 2012. Epigenetic regulation by decitabine of melanoma differentiation in vitro and in vivo. Int. J. Cancer. 131, 18–29, http://dx.doi.org/10.1002/ijc.26320. Baylin, S.B., 2005. DNA methylation and gene silencing in cancer. Nat. Clin. Pract. Oncol. 2 (Suppl. 1), S4–S11. Benn, S.C., Woolf, C.J., 2004. Adult neuron survival strategies—slamming on the brakes. Nat. Rev. Neurosci. 5, 686–700. Bian, Y., Pan, Z., Hou, Z., Huang, C., Li, W., Zhao, B., 2012. Learning, memory, and glial cell changes following recovery from chronic unpredictable stress. Brain Res. Bull. 88, 471–476, http://dx.doi.org/10.1016/j.brainresbull.2012.04.008. Biermann, J., Boyle, J., Pielen, A., Lagrèze, W.A., 2011. Histone deacetylase inhibitors sodium butyrate and valproic acid delay spontaneous cell death in purified rat retinal ganglion cells. Mol. Vision 17, 395–403. Buytaert, E., Dewaele, M., Agostinis, P., 2007. Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim. Biophys. Acta 1776, 86–107. Brodská, B., Holoubek, A., 2011. Generation of reactive oxygen species during apoptosis induced by DNA-damaging agents and/or histone deacetylase inhibitors. Oxid. Med. Cell Longev., 253529, http://dx.doi.org/10.1155/2011/253529. Chestnut, B.A., Chang, Q., Price, A., Lesuisse, C., Wong, M., Martin, L.J., 2011. Epigenetic regulation of motor neuron cell death through DNA methylation. J. Neurosci. 31, 16619–16636, http://dx.doi.org/10.1523/jneurosci.1639-11.2011. Chuang, D.-M., Leng, Y., Marinova, Z., Kim, H.-J., Chiu, C.-T., 2009. Multiple roles of HDAC inhibition in neurodegenerative conditions. Trends Neurosci. 32, 591–601, http://dx.doi.org/10.1016/j.tins.2009.06.002. Demyanenko, S.V., Uzdensky, A.B., Sharifulina, S.A., Lapteva, T.O., Polyakova, L.P., 2013. PDT-induced epigenetic changes in the mouse cerebral cortex: a protein microarray study. Biochim. Biophys. Acta 1840, 262–270, http://dx.doi.org/10.1016/j.bbagen.2013.09.014. Fedorenko, G.M., Uzdensky, A.B., 2009. Ultrastructure of neuroglial contacts in crayfish stretch receptor. Cell Tissue Res. 337, 477–490, http://dx.doi.org/10.1007/s00441-009-0825-7. Florey, E., Florey, E., 1955. Microanatomy of the abdominal stretch receptors of the crayfish (Astacus fluviatilis L.). J. Gen. Physiol. 39, 69–85. Gafurov, B.S., Urazaev, A.K., Grossfeld, R.M., Lieberman, E.M., 2002. Mechanism of NMDA receptor contribution to axon-to-glia signaling in the crayfish medial giant nerve fiber. Glia 38, 80–86. Gebicke-Haerter, P.J., 2012. Epigenetics of schizophrenia. Pharmacopsychiatry 45, S42–S48, http://dx.doi.org/10.1055/s-0032-1304652. Gomperts, B., Kramer, I., Tatham, P., 2009. Signal Transduction. Elsevier, Academic Press, Amsterdam. Gray, S.G., 2011. Epigenetic treatment of neurological disease. Epigenomics 3, 431–450, http://dx.doi.org/10.2217/epi.11.67. Haaf, T., 1995. The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated cellular processes. Pharmacol. Ther. 65, 19–46. Harrison, I.F., Dexter, D.T., 2013. Epigenetic targeting of histone deacetylase: therapeutic potential in Parkinson’s disease? Pharmacol. Ther. 140, 34–52, http://dx.doi.org/10.1016/j.pharmthera.2013.05.010. Hwang, J.Y., Aromolaran, K.A., Zukin, R.S., 2013. Epigenetic mechanisms in stroke and epilepsy. Neuropsychopharmacology 38, 167–182, http://dx.doi.org/10.1038/npp.2012.134. Kanai, H., Sawa, A., Chen, R.W., Leeds, P., Chuang, D.M., 2004. Valproic acid inhibits histone deacetylase activity and suppresses excitotoxicity-induced GAPDH nuclear accumulation and apoptotic death in neurons. Pharmacogenomics J. 4, 336–344. Karpf, A.R., Jones, D.A., 2002. Reactivating the expression of methylation silenced genes in human cancer. Oncogene 21, 5496–5503.
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Research report
Epigenetic regulation of death of crayfish glial cells but not neurons induced by photodynamic impact S.A. Sharifulina a , M.A. Komandirov b , A.B. Uzdensky a,b,∗ a b
A.B. Kogan Research Institute for Neurocybernetics, Southern Federal University, Rostov-on-Don 344090, Russia Department of Biophysics and Biocybernetics, Southern Federal University, Rostov-on-Don 344090, Russia
a r t i c l e
i n f o
Article history: Received 23 November 2013 Received in revised form 19 January 2014 Accepted 22 January 2014 Available online 4 February 2014 Keywords: Neuron Glia Epigenetic DNA methylation Histone deacetylation Photodynamic Cell death
a b s t r a c t Epigenetic processes are involved in regulation of cell functions and survival, but their role in responses of neurons and glial cells to oxidative injury is insufficiently explored. Here, we studied the role of DNA methylation and histone deacetylation in reactions of neurons and surrounding glial cells to photodynamic treatment that induces oxidative stress and cell death. Isolated crayfish stretch receptor consisting of a single mechanoreceptor neuron surrounded by glial cells was photosensitized with aluminum phthalocyanine Photosens that induced neuron inactivation, necrosis of the neuron and glia, and glial apoptosis. Inhibitors of DNA methylation 5-azacytidine and 5-aza-2 -deoxycytidine (decitabine) reduced the level of PDT-induced necrosis of glial cells but not neurons by 1.3 and 2.0 times, respectively, and did not significantly influence apoptosis of glial cells. Histone deacetylase inhibitors valproic acid and trichostatin A inhibited PDT-induced both necrosis and apoptosis of satellite glial cells but not neurons by 1.6–2.7 times. Thus, in the crayfish stretch receptor DNA methylation and histone deacetylation are involved in epigenetic control of glial but not neuronal necrosis. Histone deacetylation also participates in glial apoptosis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Photodynamic therapy (PDT) is based on photoinduced generation of strongly cytotoxic singlet oxygen, following oxidative stress and death of stained cells under light exposure in the presence of oxygen. It is currently used in oncology (Agostinis et al., 2011) including treatment of brain tumors (Kostron, 2010; Madsen et al., 2006). However, in the last case not only malignant, but also surrounding normal neurons and glial cells are damaged that can induce unacceptable side effects and neurological disorders. Therefore, PDT effect on the normal nervous tissue should be carefully investigated. Cell reactions to various impacts including PDT and their death are controlled by the complex signaling system that consists of thousands proteins that form the intracellular regulatory network (Buytaert et al., 2007; Gomperts et al., 2009; Uzdensky, 2008). If the regulatory potential of the proteins present in the cell is insufficient,
Abbreviations: DNMT, DNA methyltransferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; PDT, photodynamic therapy. ∗ Corresponding author at: Department of Biophysics and Biocybernetics, Rostov State University, 194/1 Stachky Prospect., Institute of Neurocybernetics, Rostov-onDon 344090, Russia. Tel.: +7 8632 433111; fax: +7 8632 433577. E-mail address: [email protected] (A.B. Uzdensky). 0361-9230/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2014.01.005
additional protein synthesis is stimulated. Gene expression is controlled by transcription factors and epigenetic regulators: DNA methylation/demethylation and histone modifications such as methylation/demethylation, acetylation/deacetylation, and phosphorylation/dephosphorylation, which regulate access of transcription factors and RNA polymerase II to gene promoters. Aberrant DNA methylation and histone modifications are involved in diverse neuronal functions and neurological disorders such as synaptic plasticity and memory formation (Sultan and Day, 2011), Alzheimer, Parkinson, and Huntington diseases (Gebicke-Haerter, 2012; Gray, 2011; Zawia et al., 2009), schizophrenia (Harrison and Dexter, 2013), epilepsy (Hwang et al., 2013), acute and chronic stress (Stankiewicz et al., 2013), and stroke (Hwang et al., 2013). However, there are very few direct data on the involvement of epigenetic processes in neurodegeneration (Chestnut et al., 2011). The role of epigenetic processes in the reactions of the normal nervous tissue to photodynamic treatment remains almost unstudied. Using the proteomic approach we have recently shown that PDT influences the expression of various proteins involved in epigenetic regulation in the murine cerebral cortex such as histone deacetylases HDAC-1 and HDAC-11, transcriptional repressors Kaiso and dimethylated histone H3, transcription factors AP1/c-Jun and FOXC2, phosphorylated histone H2AX involved in DNA repair, importin ␣5/7, protein methyltransferase PRMT5, and some others (Demyanenko et al., 2013). However, the study
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of the complex brain tissue that consists of different cell types including neurons, glial cells, and blood vessels does not provide the information on epigenetic regulation in different cell types. The suitable object for study of epigenetic processes occurring simultaneously in the interacting neuronal and glial cells is the crayfish stretch receptor that consists of a single mechanoreceptor neuron surrounded by glial cells, which form a multilayer envelope around the neuron (Fedorenko and Uzdensky, 2009). The role of diverse signaling and metabolic processes in responses of crayfish neurons and glial cells to photodynamic treatment has been previously studied (Komandirov et al., 2011; Kovaleva et al., 2013; Uzdensky et al., 2005, 2007, 2013). In the present work we studied the role of DNA methylation and histone deacetylation in PDT-induced inactivation and death of the stretch reception neuron and surrounding glial cells. We showed that inhibitors of DNA methylation 5-azacytidine and 5aza-2 -deoxycytidine (decitabine) inhibited PDT-induced necrosis of glial cells but not neurons, whereas inhibitors of histone deacetylase (HDAC) valproic acid and trichostatin A inhibited PDT-induced necrosis and apoptosis of satellite glial cells but not neurons. 2. Materials and methods 2.1. Chemicals The following chemicals were used: DNA methyltransferase inhibitors 5-azacytidine (5-Aza, 10 M) and 5-aza-2 deoxycytidine (decitabine, 10 M); histone deacetylase inhibitors valproic acid, sodium salt (VPA, 0.5 mM) and trichostatin A (TSA, 100 nM); fluorochromes propidium iodide and Hoechst 33342. All chemicals were obtained from Sigma–Aldrich-Rus (Moscow, Russia). Photosensitizer Photosens, a mixture of sulphonated aluminum phthalocyanines, AlPcSn , where mean n = 3.1, was obtained from NIOPIK (Moscow, Russia). 2.2. Crayfish stretch receptor and recording of its firing The crayfishes Astacus leptodactylus from Don River affluences were purchased on the local market. Their abdominal stretch receptors were isolated as described by Florey and Florey (1955). These were placed into a plexiglass chamber equipped with a device for receptor muscle extension and filled with 2 ml of van Harreveld saline (mM: NaCl – 205; KCl – 5.4; NaHCO3 – 0.2; CaCl2 – 13.5; MgCl2 – 5.4; pH 7.2–7.4). Neuron spikes were recorded extracellularly from axons by glass pipette suction electrodes, amplified, digitized by the analog-digital converter L-761 (L-Card, Moscow, Russia), and processed by a personal computer using the homemade software that provided continuous monitoring of firing. Experiments were carried out at 23 ± 4 ◦ C. 2.3. Photodynamic treatment and application of enzyme inhibitors At the beginning of the experiment the initial level of neuronal activity was set near 6–10 Hz by application of the appropriate receptor muscle extension. After 30 min control recording of neuronal activity, Photosens (75 nM) and an inhibitor were added into the chamber with an interval of 3–5 min. After following 30-min incubation, cells were irradiated with the He–Ne laser (633 nm, 0.3 W/cm2 ). A laser beam diameter was of 3 mm, so that the neuronal body and a significant part of axon were irradiated. 30-min light exposure was longer than the duration of bioelectric neuron response measured from the irradiation start to the moment of firing abolition (typically, 10–20 min), which we called the “neuron lifetime”. Photosens and an inhibitor were present in the chamber during and after irradiation. The inhibitor concentration was
usually chosen to be approximately 2 times lower than the predetermined concentration, which disturbed neuron firing in the darkness for 3–4 h.
2.4. Cell death assay In order to visualize dead neurons and glial cells, 20 M propidium iodide and 10–20 M Hoechst 33342 (both from Sigma–Aldrich) were added into the experimental chamber at 8 h postirradiation. This time interval was sufficient for apoptosis development (Uzdensky et al., 2005). Then preparations were washed with van Harreveld saline, fixed with 0.2% glutaraldehyde, repeatedly washed and mounted in glycerol. Fluorescent images were acquired using the fluorescence microscope LumamI3 (LOMO, Sankt-Petersburg, Russia) equipped with a digital photocamera. Propidium iodide, a membrane impermeable fluorochrome, imparts red fluorescence to nuclei of necrotic cells with the compromised plasma membrane. Hoechst 33342 imparts blue fluorescence to the nuclear chromatin. It visualizes intact nuclei of living cells and fragmented nuclei of apoptotic cells (Fig. 1). Nucleus fragmentation is the final stage of apoptosis when the noreturn point has passed. It should be mentioned that other methods for apoptosis evaluation such as caspase activation, cytochrome c release, or annexin V assay, which require observation of the cytoplasm or of the plasma membrane, are not suitable for study of glial cells in the isolated stretch receptor because of their multilayer roulette-like morphology and overlapping of optical images of different glial processes and the neuronal cytoplasm (Fedorenko and Uzdensky, 2009). The red nuclei of necrotic glial cells stained by propidium iodide were counted in the predetermined standard field (100 m × 100 m) around SRN soma so that the neuron nucleus was situated in its center. Fragmented nuclei of apoptotic glial cells were counted around the proximal 2 mm axon fragment where glial apoptosis was more profound than around the neuron body. Their mean number representing the level of glial apoptosis was expressed below as relative units. The one way ANOVA was used for statistical evaluation of the difference between independent experimental groups. Data are presented as mean ± S.E.M.
3. Results Neither Photosens (75 nM), nor laser radiation separately changed significantly neuronal activity and survival after stretch receptor isolation. However, their combined action, i.e. PDT led to inhibition of neuronal activity, necrosis of neurons and glial cells and apoptosis of glial cells (Figs. 2–5). Apoptotic nuclear fragmentation was never observed in neurons as described earlier (Uzdensky et al., 2002, 2005). In the darkness, both inhibitors of DNA methyltransferase 5-azacytidine (10 M) and decitabine (10 M) did not influence significantly the duration of bioelectric neuron response (Figs. 2A and 3A) and the level of neuronal necrosis (Figs. 2B and 3B). However, 5-azacytidine more than 2 times increased the level of glial necrosis (p < 0.05; Fig. 2C) and showed the similar tendency for glial apoptosis (p > 0.05; Fig. 2D). Unlike, decitabine did not demonstrate glia toxicity in the darkness (Fig. 3C and D). 5-Azacytidine and decitabine did not influence the PDTinduced changes in the neuronal activity and necrosis level (Figs. 2A and B, 3A and B). We also did not observe significant effects of these DNA methyltransferase inhibitors on PDT-induced glial apoptosis (Figs. 2D and 3D). However, both of them reduced the levels PDT-induced necrosis of glial cells by 1.3 and 2.0 times (p < 0.05; Fig. 2C and p < 0.01; Fig. 3C, respectively).
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Fig. 1. The example of the effect of decitabine (B), PDT (C and E), and PDT + decitabine (D and F) on nuclear morphology of the stretch receptor neuron and surrounding glial cells. (A) Control. (E) and (F) show fragmented apoptotic nuclei of satellite glial cells in the proximal axon regions. The preparation was fluorochromed with propidium iodide that imparts the red fluorescence to nuclei of necrotic cells and with Hoechst 33342 that imparts blue fluorescence to nuclei of alive or fragmented apoptotic cells. Arrowheads on A–D indicate big neuron nuclei; thin arrows on E and F – fragmented nuclei of apoptotic glial cells. Scale bar 50 m.
Histone deacetylase inhibitors valproic acid, sodium salt (VPA, 0.5 mM) and trichostatin A (TSA, 100 nM) did not influence neuronal and glial necrosis and glial apoptosis in the darkness (Figs. 4 and 5, respectively). However, valproic acid reduced the firing duration of the isolated stretch receptor neuron by about 20% (p < 0.05; Fig. 4A). Both VPA and trichostatin A did not influence the PDT-induced shortening of neuronal firing (Figs. 4A and 5A) and the level of the neuronal necrosis (Figs. 4B and 5B). However, they significantly reduced photoinduced necrosis of glial cells by 2.3 and 1.6 times (p < 0.01; Figs. 4C and 5C, respectively). They also protected glial cells from PDT-induced apoptosis and reduced its level by 2.4 and 2.7 times (p < 0.05; Fig. 4D and p < 0.01; Fig. 5D, respectively). 4. Discussion Methylation of DNA, in which DNA methyltransferase (DNMT) transfers methyl group from S-adenosylmethionine to cytosine in the CpG-dinucleotide, is a major epigenetic process controlling gene transcription. Genes regulated by DNA methylation are involved in the regulation of cell survival, proliferation, and apoptosis (Baylin, 2005; Robertson and Jones, 2000). Neuronal functions and fate in health and disease are also controlled by DNA methylation (Chestnut et al., 2011; Gray, 2011; Hwang et al., 2013; Mehler, 2008; Stankiewicz et al., 2013; Zawia et al., 2009). The present data have shown that DNMT inhibitors 5azacytidine and decitabine protect crayfish glial cells but not neurons from PDT-induced necrosis. One can, therefore, suggest the involvement of DNMT in PDT-induced necrosis of glial cells.
However, the mechanism of action of these drugs may be more complex. DNA methylation of cytosines in the CpG-islands suppresses transcription. In contrast, DNA demethylation mediated by 5-azacytidine or decitabine activates the expression of silenced genes via chromatin decondensation. This allows binding of transcription factors to DNA promoters and reactivates gene expression (Alcazar et al., 2012; Haaf, 1995). For example, decitabine, except inhibition of DNA methyltransferase, induces depletion of DNMT1 and reactivates the expression of tumor suppressor genes such as p16INK4a, p15INK4b, hMLH1, VHL, APC, pRb, and BRCA1 (Alcazar et al., 2012; Karpf and Jones, 2002). Both 5-azacytidine and decitabine demonstrate general cytotoxicity related to DNA methylation. For example, in PC12 cells decitabine significantly enhanced the paraquat-mediated production of reactive oxygen species (ROS) and induced oxidative stress, mitochondrial deficit, cytochrome C release, and apoptosis (Kong et al., 2012). In normal lymphocytes and leukemia cells decitabine as well as HDAC inhibitors butyrate or SAHA stimulated ROS production and induced apoptosis. The combination of these inhibitors exerted the synergistic effect (Brodská and Holoubek, 2011). However the cytotoxic effects of these drugs are not identical. 5-Azacytydine but not decitabine inhibited the proapoptotic protein p53 and activated caspase and induced apoptosis of tumor cells (Venturelli et al., 2013). The enhancement of PDT-induced apoptosis of glial cells in the presence of 5-azacytydine corresponded to this data. However, unlike the pronecrotic effect of 5-azacytidine on crayfish glial cells in the dakness, 5-azacytidine and decitabine exerted the antinecrotic effect on photosensitized glial cells. This is a novel activity of these drugs. It cannot be explained by their effect on
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Fig. 2. (A–D) Effect of DNA methyltransferase inhibitor 5-azacytidine (5-Aza, 10 M) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of 5-azacytidine in the darkness, 3 – PDT, 4 – PDT in the presence of 5-azacytidine. Significant difference from the indicated bars: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 3. (A–D) Effect of DNA methyltransferase inhibitor decitabine (10 M) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of decitabine in the darkness, 3 – PDT, 4 – PDT in the presence of decitabine. Significant difference from the indicated bars: *p < 0.05, **p < 0.01.
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Fig. 4. (A–D) Effect of histone deacetylase inhibitor trichostatin A (TSA, 100 nM) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of trichostatin A in the darkness, 3 – PDT, 4 – PDT in the presence of trichostatin A. Significant difference from the indicated bars: *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 5. (A–D) Effect of histone deacetylase inhibitor valproic acid, sodium salt (VPA, 0.5 mM) on duration of neuron firing (neuron lifetime), necrosis of neurons and glial cells, and apoptosis of glial cells in the darkness and under photodynamic treatment with 75 nM Photosens. Numbers under the bars indicate the number of the experiment: 1 – control, 2 – effect of valproic acid, sodium salt in the darkness, 3 – PDT, 4 – PDT in the presence of valproic acid, sodium salt. Significant difference from the indicated bars: *p < 0.05, **p < 0.01, ***p < 0.001.
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PDT-induced ROS production. It may be rather related to restoring of expression of some genes, which are unidentified yet. Acetylation of histones leads to chromatin relaxation, increase in the accessibility of gene promoters to transcription factors, and expression of prosurvival genes. In contrast, histone deacetylatylation inhibits gene expression. Histones acetylation depends on the balance between histone deacetylases and histone acetyltransferases (HATs) activities (Konsoula and Barile, 2012; Struhl, 1998). In neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer and Parkinson diseases this balance is shifted toward histone deacetylation (Harrison and Dexter, 2013; Saha and Pahan, 2006). HDAC inhibitors valproic acid sodium salt, trichostatin A, sodium butirate, and others demonstrate neuroprotective effects in neurodegenerative diseases, intracerebral hemorrhage and ischemic stroke. They also protect neurons from glutamate excitotoxicity and oxidative stress (Biermann et al., 2011; Harrison and Dexter, 2013; Kanai et al., 2004; Zhang et al., 2012). Prosurvival activity of these drugs is associated with histone deacetylase inhibition and transcriptional activation of expression of neuroprotective proteins such as BDNF (brain derived neurotrophic factor), GDNF (glial derived neurotrophic factor), heat shock protein HSP70, synuclein ␣, apoptosis inhibitors Blc-2 and Bcl-XL, etc. (Chuang et al., 2009; Wu et al., 2008; Zhang et al., 2012). Valproic acid sodium salt and trichostatin A are promising for treatment of various neurodegenerative diseases (Harrison and Dexter, 2013; Saha and Pahan, 2006). In our experiments valproic acid sodium salt and trichostatin A suppressed PDT-induced death of glial cells but not neurons. Therefore, the PDT-induced shift of the HAT/HDAC balance toward histone deacetylation is involved in both necrosis and apoptosis of crayfish glial cells. Apoptosis is regulated by the complex cell signaling system, which is controlled by the epigenetic processes. Interesting, in crayfish glial cells not only apoptosis but also necrosis is under the epigenetic control. Further investigations should identify the genes targeted by HDAC inhibitors and the pathways involved in glial protection. The insensitivity of crayfish mechanoreceptor neurons to inhibitors of DNA methylation and histone deacetylation indicates the insignificant role of epigenetic processes in regulation of neuronal survival. Unlike glial cells, the apoptotic nucleus fragmentation was not observed in the crayfish stretch receptor neuron. As shown early, apoptosis of this neuron was not induced by various physical and pharmacological factors. This large neuron, unique in the crayfish nervous system, is necessary for movement control and, therefore, for animal survival. The apoptosis program in this cell has been suggested to be intrinsically blocked (Uzdensky et al., 2002, 2005, 2007), like in many vertebrate adult neurons, which are necessary throughout all organism lifetime, and their premature death can irreversibly disturb organism functions (Benn and Woolf, 2004; Walsh et al., 2004). One can suggest that necrosis of this neuron is not epigenetically regulated as well. The neuroglial interactions may be involved in maintaining of neuron and glial survival (Kolosov and Uzdensky, 2006). Neurotrophic factors (Bian et al., 2012; Komandirov et al., 2011; Lobanov and Uzdensky, 2009), nitric oxide (Kovaleva et al., 2013), or neuromediators such as extra-synaptic glutamate (Gafurov et al., 2002; Rodriguez et al., 2013) are among possible intercellular signals. All of them can induce intracellular pathways that include epigenetic regulation of gene transcription, which modulate cell survival. The nucleus of the stretch receptor neuron is very large. It contains almost completely unpacked euchromatin that shows its high transcriptional activity. Unlike, glial nuclei are much smaller and contain big masses of condensed heterochromatin (Fig. 1 and Fedorenko and Uzdensky, 2009) that indicates silencing of many genes. One can suggest that the potency of epigenetic regulators to stimulate gene transcription may be much
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