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Streptozotocin causes neurotoxic effect in cultured cerebellar granule neurons
Brain Research Bulletin 130 (2017) 90–94
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Streptozotocin causes neurotoxic effect in cultured cerebellar granule neurons Elisaveta E. Genrikhs a , Elena V. Stelmashook a,∗ , Sergey A. Golyshev b , Olga P. Aleksandrova a , Nickolay K. Isaev a,b a
Research Center of Neurology, Volokolamskoe Shosse 80, 125367 Moscow, Russia M. V. Lomonosov Moscow State University, N. A. Belozersky Research Institute of Physico-Chemical Biology, Leninskye gory, 1, b. 40, 119991 Moscow, Russia b
a r t i c l e
i n f o
Article history: Received 15 November 2016 Received in revised form 29 December 2016 Accepted 3 January 2017 Available online 6 January 2017 Keywords: Streptozotocin Cultured neurons Insulin Mitochondrial membrane potential Calcium
a b s t r a c t Streptozotocin (STZ) is a glucosamine-nitrosourea compound used for experimental simulation of sporadic Alzheimer’s disease at intracerebroventricular administration in vivo. The studies of STZ influence on neurons of central nervous system performed on the primary cultures are practically absent. We have shown the application of STZ (1–5 mM) in primary culture for 48 h induced strong dose-dependent death in cultured cerebellar granule neurons. This toxic effect was decreased by pyruvate, insulin partially. Using the indicator Fluo-4 AM for measurements of intracellular calcium ions and tetramethylrhodamine ethyl ester (TMRE) for detection of changes of mitochondrial membrane potential in live cells we have shown that 5 h-exposure to STZ induced intensive increase of Fluo-4 and decrease TMRE fluorescence in neurons. STZ exposure caused considerable ultrastructural alterations in granule neurons: chromatin clumping, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. Probably, STZ significantly impaired glucose metabolism and mitochondrial function that, in turn, resulted in mitochondrial membrane potential damage, excessive calcium overload and neuronal death. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Streptozotocin is a glucosamine-nitrosourea compound, particularly toxic to the insulin-producing beta-cells of the pancreas in mammals, applied in medicine for treating certain cancers of the Islets of Langerhans. Intraperitoneal injections of STZ are used in a medical research to produce Type 1 diabetes in animal model (Szkudelski, 2001). In the field of neuroscience and experimental medicine the intracerebroventricular administration of STZ is applied as experimental simulation of sporadic Alzheimer’s disease in vivo during 20 years by now (Mayer et al., 1990). In this model, deficits in hippocampal synaptic transmission and long-term potentiation (LTP) were observed. The decline in LTP correlated with decreased expression of NMDA-receptor subunits NR2A and NR2B. Similarly, there has been a decrease in the expression of brain derived neurotrophic factor (BDNF) (Shonesy et al., 2012). The STZ-intracerebroventricular treated animals developed insulin resistant brain state associated with memory impairment
∗ Corresponding author at: Research Center of Neurology, bs. Obukha, 5, Brain Research Department, Research Center of Neurology, Moscow, Russia. E-mail address: [email protected] (E.V. Stelmashook). http://dx.doi.org/10.1016/j.brainresbull.2017.01.004 0361-9230/© 2017 Elsevier Inc. All rights reserved.
and progressive cholinergic deficits, glucose hypometabolism, tau-hyperphosphorylation, astrogliosis and amyloid angiopathy, oxidative stress and neurodegeneration that share many features in common with sporadic Alzheimer’s disease in humans (Park, 2011; Salkovic-Petrisic et al., 2013; Ejaz Ahmed et al., 2013). The reduction of glucose utilization occurred owing to the decrease of glycolytic enzymatic activities (Plaschke and Hoyer, 1993). The most part of researches of STZ toxic action in the brain is carried out on the models in vivo. The STZ researches performed on the cultured central nervous system neurons are absent practically. The present study was designed to test the streptozotocin toxic action in primary neuronal cultures in vitro.
2. Materials and methods 2.1. Primary cerebellar neuronal cultures Primary cultures were prepared from the cerebella of 7–8 days old Wistar rats as described elsewhere (Stelmashook et al., 2009). The cerebella were washed with Ca2+ - and Mg2+ -free PBS (Dulbecco) and incubated in the same solution containing 0.05% trypsin and 0.02% EDTA (15 min, 37 ◦ C). After incubation, the tissue was
E.E. Genrikhs et al. / Brain Research Bulletin 130 (2017) 90–94
washed twice in PBS and dissociated by repeated pipetting in a nutrient medium of the following composition: fetal calf serum (10%), minimum essential medium Eagle (90%), glutamine (2 mM), and HEPES (10 mM). After mild centrifugation, the cells were resuspended in the required volume of the nutrient medium containing 25 mM KCl. Cell suspension (0.1 ml) was added to a poly-l-lysinecoated 96-well plate or coverslips in a 40-mm diameter Petri dishes and then kept in a CO2 -incubator (5% CO2 , 36.5 ± 0.5 oC). Cultures maintained up to 3–4 days in vitro, without replacing the medium. All experimental protocols were approved by the Animal Ethics Committee of the Research Center of Neurology of Russian Academy of Medical Sciences (the Protocol Registration number 05/10) and in accordance with Council Directive 2010/63 EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. 2.2. Pharmacological treatment Streptozotocin (1–5 mM), pyruvate (1 mM), insulin (0.01 mM) (final concentration are noted) were supplemented directly to the culture medium on the 2nd day in vitro. Control and treated cultures have been incubated at 5 or 48 h in CO2 -incubator (5% CO2 , 36.5 ± 0.5 ◦ C). 2.3. Assessment of neuronal viability Viability of cerebellar granule neurons (CGNs) was determined as described previously (Stelmashook et al., 2009). After 48 h STZ (1–5 mM) incubation cell cultures were fixed with ethanolformaldehyde-acetic acid (7:2:1) mixture and stained with trypan blue. The percentage of surviving neurons was estimated by counting the intact nuclei of the CGNs in five fields of view. The viability of untreated control cultures was taken as 100%, and the viability of treated cells was counted as a percentage referred to control values. 2.4. Analysis of intracellular Ca2+ After 5 h STZ (3–4.5 mM) incubation cells were loaded with 0.005 mM Fluo-4 AM (Ca2+ detection) for 30 min at 36.5 ± 0.5 ◦ C followed by triple washing in BSS, containing (in mM): NaCl 154, KCl 25, CaCl2 2.3, MgCl2 1, NaHCO3 3.6, Na2 HPO4 0.35, HEPES 10; at pH 7.3. Levels of intracellular Ca2+ were measured by the fluorescence intensity of Fluo-4 on a fluorescence plate reader (CytoFluor II, PerSeptive Biosystems, Framingham, MA, USA) using excitation at 485 nm and maximal emission at 530 nm. Changes in fluorescence were calculated as (F − F0 )/F0 × 100%, where F0 and F are fluorescence of the dye in cell cultures incubated without and with STZ in medium, respectively.
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2.6. Electron microscopy For electron microscopy we used the same volume of cell suspension placed on poly-l-lysine-coated coverslips in a 40-mm diameter Petri dishes (Lozier et al., 2012) which incubated 2–3 days in vitro. After 5 h STZ exposure (4.5 mM) cultures were fixed using 2.5% glutar aldehyde prepared in phosphate buffer (pH 7.2) followed by postfixation with 1% osmium tetroxide, dehydratation in ethanol, en-bloc staining with uranyl-acetate and embedding in Epon 812. Ultrathin sections were prepared on the ReichertJung Ultracut E ultramicrotome, additionally stained with aqueous uranyl-acetate and lead citrate, examined using the transmission electron microscope JEM-1400 (JEOL, Japan) running at 100 kV. 2.7. Statistics The one-way ANOVA with Neuman–Keuls and Bonferroni posttest was used for statistical analysis. Levels of p < 0.05 were considered as statistically significant. The results are represented as means ± SEM. All data were obtained using at least 9–12 different cultures from 3 to 4 independent experiments. 2.8. Reagents Unless otherwise noted, all media and supplements used in experiments were purchased from Biochrom KG Berlin, Germany. Fluo-4 AM and TMRE were from Molecular Probes (USA). Streptozotocin and other reagents were from Sigma Chemicals (Germany). 3. Results 3.1. Streptozotocin toxicity in cerebellar granule neurons Counting of neurons with normal morphology demonstrated that application of STZ (1–5 mM) in culture medium for 48 h with effect from 2.5 mM induced strong dose-dependent CGNs death (Fig. 1). STZ concentrations which cause death more than 50% of neurons have been chosen for further experiments. Under these conditions, the neurons were partially protected from cell death by supplementation of 1 mM pyruvate or 0.01 mM insulin (Fig. 2). 3.2. Intracellular calcium ions level and mitochondrial membrane potential The exposure to STZ (3–4.5 mM) for 5 h induced significantly strong dose-dependent increase of Fluo-4 fluorescence in the CGNs to 22 ± 4–63 ± 10% in comparison with control (Fig. 3A). Under these conditions TMRE fluorescence decreased to 17 ± 4% (Fig. 3B).
2.5. Measurement of mitochondrial membrane potential
3.3. Electron microscopy
Alterations in the mitochondrial membrane potential were analyzed using the mitochondrial membrane potential-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Briefly, following treatment with STZ (3–4.5 mM, 5 h), cells were incubated with 100 nM TMRE in the culture medium at 36.5 ± 0.5 ◦ C for 15 min. Stained cells were then washed triply with balanced salt solution. Positively-charged TMRE as a penetrating cation readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. Fluorescence (excitation at 530 nm, emission at 640 nm) was analyzed on a fluorescence plate reader (CytoFluor II, PerSeptive Biosystems, Framingham, MA, USA). Changes in fluorescence were calculated as described previously.
In an effort to identify and characterise the ultrastructural changes in cerebellar granule neurons induced following treatment with STZ electron microscopy has been used. The ultrastructural study showed that control CGNs cultivated under normal conditions had typical for normal cells mitochondrial ultrastructure: mitochondria were not enlarged, had slightly oval or elongated shape. The cristae were clearly seen, and the mitochondrial matrix had a higher electron density than the surrounding cytoplasm (Fig. 4A). In STZ-treated neurons for 5 h, the mitochondria were enlarged, and the majority of cristae were damaged (Fig. 4B). Pathological signs of STZ-treated CGNs were seen also in some other neuronal compartments: nuclear chromatin was clumped, distinctly expanded perinuclear space and swelling of the granular endoplasmic reticulum were apparent.
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Fig. 1. The influence of STZ in culture medium on cerebellar granule neurons. A–B, cultures were fixed with ethanol–formaldehyde–acetic acid (7:2:1) mixture and stained with trypan blue, (A) control, (B) 4.5 mM STZ. Intact nuclei of CGNs are indicated by arrows. Scale bar 15 m. (C) quantitative evaluation of neuronal survival. *Statistically significant difference from control values (0 mM STZ), p < 0.01.
Fig. 2. Pyruvate and insulin attenuate toxic effect of STZ in cultured cerebellar granule neurons. (A) 1 mM pyruvate – black columns, (B) 0.01 mM insulin – black columns. White bars – in lack of a protector. Quantitative evaluation of neuronal viability. * p < 0.01.
4. Discussion Following the parenteral injection of high doses more 40 mg/kg STZ selectively destroys insulin producing beta cells in the pancreas, causing diabetes type I in adult animals (Szkudelski, 2001). Intracerebroventricular administration of STZ in subdiabetogenic doses (3 mg/kg) to rats or mice is used as experimental simulation of sporadic Alzheimer’s disease in vivo (Ponce-Lopez et al., 2011; Hamidi et al., 2013). Total brain volume is about 769–881 mm3 , and lateral ventricles volume is 2.19–2.39 mm3 (Fabricius et al., 2010). Therefore, local concentration of a streptozotocin in a brain can
reach more than 4 mM after intracerebroventricular administration. Our experiments were carried out on dissociated cell cultures obtained from cerebellum of 7–8 days old rats. Previously this type of culture was used for studying cytotoxicity of nitrosamine (1–2.5 mM), which is a part of a STZ molecule (De la Monte and Tong, 2009). We have shown that application of 2.5 mM and more STZ for 48 h induced strong dose-dependent death in cultured CGNs. STZ concentrations which cause death more than 50% of neurons have been chosen for further experiments. Previously these concentrations of STZ and incubation period were tested in human neuroblastoma cells. Authors showed significant increase in cells
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Fig. 3. The exposure to STZ for 5 h induced the dose-dependent increase of Fluo4 fluorescence in CGNs ([Ca2+ ]i changes) (A) and decrease of TMRE fluorescence (decrease of mitochondrial membrane potential) (B). Changes in fluorescence (Fluo4 or TMRE) were calculated as (F − F0 )/F0 × 100%, where F0 and F are fluorescence of the dye in cell cultures incubated without and with STZ in medium, respectively. *Statistically significant difference from control values (0 mM STZ), p < 0.05.
death following treatment with more 2.5 mM STZ (Plaschke and Kopitz, 2015). Biswas et al. (2016) demonstrated, that 1 mM STZ for 48 h induced decrease of cell viability on 30% (MTT-test) in mouse neuronal N2A cells. In this work the LDH-test has been used also. However authors haven’t made assessment of total activity of enzyme in cell cultures, and the real percentage of the died cells can’t be determined by these data. It is known that STZ can induce G2 arrest in the cell cycle (Johnston et al., 2007). Therefore STZ can be more toxic for N2A cells capable to proliferation, than for the differentiated neurons, for example cerebellar granule neurons. 5 h-exposure of neuronal cultures to STZ didn’t cause cell death, but measurements of intracellular calcium ions using Fluo-4 AM and mitochondrial membrane potential with TMRE demonstrated that intensive increase of Fluo-4 fluorescence and decrease of TMRE fluorescence was observed. Perhaps STZ-induced impaired mitochondrial function can lead to the calcium overload of CGNs. Brain glucose metabolism has been found to be markedly decreased in the STZ-intracerebroventricular treated rats, demonstrated decrease
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ATP and phosphocreatine concentrations as well as the ATP/ADP ratio and glucose utilization in the cerebral cortex (Nitsch and Hoyer, 1991; Duelli et al., 1994). The reduction of glucose utilization occurs owing to the decrease of glycolytic enzymatic activities (Plaschke and Hoyer, 1993). Earlier it has been shown that pyruvate decrease CGNs death under glucose starvation conditions (Stelmashook et al., 2010). Therefore we have suggested that pyruvate as the mitochondrial energy substrate can increase survival of neurons by STZ toxicity, what has been confirmed in our experiments. Intracerebroventricular injection of streptozotocin lead to development of insulin resistance in the brain, at the same time the intracerebroventricular-administration of insulin and its analogues recover STZ-induced cognitive decline in rats (Shingo et al., 2013). Our study demonstrated that the medium supplementation with insulin partially protected CGNs from the STZ toxic action. In addition we have found that mitochondrial ultrastructure were significantly damaged in STZ treated CGNs. It was noted, the mitochondria were enlarged, and the majority of cristae were damaged. Thus, ultrastructural alterations are usually indicative for the organelles disfunctioning. Pathological signs of STZ treated neurons were seen also in some other neuronal compartments: nuclear chromatin was clamped, perinuclear space distinctly broadened and swelling of granular reticulum were apparent. Similar neuronal ultrastructural changes were observed previously in CGNs after calcium overload induced glutamate treatment (Isaev et al., 1996; Isaev et al., 2005). Early it has been shown that isolated brain mitochondria from intracerebroventricular-STZ rats displayed a decrease in pyruvate and ␣-ketoglutarate dehydrogenases and cytochrome C oxidase activities and an increase in the susceptibility to calcium-induced mitochondrial permeability transition (Correia et al., 2013). These deficits in mitochondrial function can limit protective effect of the pyruvate in our experiments.
5. Conclusions STZ exposure of cultured neurons causes significantly disturbance of glucose metabolism and mitochondrial function that, in turn, results in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.
Conflict of interest The authors declare that there is no conflict of interest.
Fig. 4. Transmission electron micrograph of cultured cerebellar granule neurons. (A) Control culture was exposed to the culture medium. Mitochondria and other compartments appear undamaged. (B) cells after 5 h-exposure to 4.5 mM streptozotozin in the culture medium. Note mitochondrial swelling and disruption of the cristae. Clumping of heterochromatin, distinctly broadened perinuclear space and endoplasmic reticulum swelling are seen.
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Acknowledgements This work was done with financial support from the Russian Science Foundation (projects No. 16-15-10108). This work was done with the implementation of equipment received under MSU development program PNR5.13. References Biswas, J., Goswami, P., Gupta, S., Joshi, N., Nath, C., Singh, S., 2016. Streptozotocin induced neurotoxicity involves Alzheimer’s related pathological markers: a study on N2A cells. Mol. Neurobiol. 53, 2794–2806, http://dx.doi.org/10.1007/ s12035-015-9144-z. Correia, S.C., Santos, R.X., Santos, M.S., Casadesus, G., Lamanna, J.C., Perry, G., Smith, M.A., Moreira, P.I., 2013. Mitochondrial abnormalities in a streptozotocin-induced rat model of sporadic Alzheimer’s disease. Curr. Alzheimer Res. 10, 406–419. De la Monte, S.M., Tong, M., 2009. Mechanisms of nitrosamine-mediated neurodegeneration: potential relevance to sporadic Alzheimer’s disease. J. Alzheimers Dis. 17, 817–825, http://dx.doi.org/10.3233/JAD-2009-1098. Duelli, R., Schrock, H., Kuschinski, W., Hoyer, S., 1994. Intracerebroventricular injection of streptocotozin induces discrete local changes in cerebral glucose utilization in rats. Int. J. Dev. Neurosci. 12, 737–743. Ejaz Ahmed, M., Khan, M.M., Javed, H., Vaibhav, K., Khan, A., Tabassum, R., Ashafaq, M., Islam, F., Safhi, M.M., Islam, F., 2013. Amelioration of cognitive impairment and neurodegeneration by catechin hydrate in rat model of streptozotocin-induced experimental dementia of Alzheimer’s type. Neurochem. Int. 62, 492–501, http://dx.doi.org/10.1016/j.neuint.2013.02.006. Fabricius, K., Helboe, L., Steiniger-Brach, B., Fink-Jensen, A., Pakkenberg, B., 2010. Stereological brain volume changes in post-weaned socially isolated rats. Brain Res. 1345, 233–239, http://dx.doi.org/10.1016/j.brainres.2010.05.040. Hamidi, G., Arabpour, Z., Shabrang, M., Rashidi, B., Alaei, H., Sharifi, M.R., Salami, M., Reisi, P., 2013. Erythropoietin improves spatial learning and memory in streptozotocin model of dementia. Pathophysiology 20, 153–158, http://dx. doi.org/10.1016/j.pathophys.2013.01.001. Isaev, N.K., Zorov, D.B., Stelmashook, E.V., Uzbekov, R.E., Kozhemyakin, M.B., Victorov, I.V., 1996. Neurotoxic glutamate treatment of cultured cerebellar granule cells induces Ca2+ -dependent collapse of mitochondrial membrane potential and ultrastructural alterations of mitochondria. FEBS Lett. 392, 143–147. Isaev, N.K., Andreeva, N.A., Stel’mashuk, E.V., Zorov, D.B., 2005. Role of mitochondria in the mechanisms of glutamate toxicity. Biochemistry (Mosc.) 70, 611–618. Johnston, A.P., Campbell, J.E., Found, J.G., Riddell, M.C., Hawke, T.J., 2007. Streptozotocininduces G2 arrest in skeletal muscle myoblasts and impairs muscle growth in vivo. Am. J. Physiol. Cell Physiol. 292, 1033–1040.
Lozier, E.R., Stelmashook, E.V., Uzbekov, R.E., Novikova, S.V., Zorov, S.D., Alieva, I.B., Arbeille, B., Zorov, D.B., Isaev, N.K., 2012. Stimulation of kainate toxicity by zinc in cultured cerebellar granule neurons and the role of mitochondria in this process. Toxicol. Lett. 208, 36–40. Mayer, G., Nitsch, R., Hoyer, S., 1990. Effects of changes in peripheral and cerebral glucose metabolism on locomotor activity, learning and memory in adult male rats. Brain Res. 532, 95–100. Nitsch, R., Hoyer, S., 1991. Local action of the diabetogenic drug streptozotocin on glucose and energy metabolism in rat brain cortex. Neurosci. Lett. 128, 199–202. Park, S.A., 2011. A common pathogenic mechanism linking type-2 diabetes and Alzheimer’s disease: evidence from animal models. J. Clin. Neurol. 7, 10–18, http://dx.doi.org/10.3988/jcn.2011.7.1.10. Plaschke, K., Hoyer, S., 1993. Action of the diabetogenic drug streptozotocin on glycolytic and glycogenolytic metabolism in adult rat brain cortex and hippocampus. Int. J. Dev. Neurosci. 11, 477–483. Plaschke, K., Kopitz, J., 2015. In vitro streptozotocin model for modeling Alzheimer-like changes: effect on amyloid precursor protein secretases and glycogen synthase kinase-3. J. Neural Transm. (Vienna) 122, 551–557, http:// dx.doi.org/10.1007/s00702-014-1319-7. Ponce-Lopez, T., Liy-Salmeron, G., Hong, E., Meneses, A., 2011. Lithium, phenserine, memantine and pioglitazone reverse memory deficit and restore phospho-GSK3 decreased in hippocampus in intracerebroventricular streptozotocin induced memory deficit model. Brain Res. 1426, 73–85, http:// dx.doi.org/10.1016/j.brainres.2011.09.056. Salkovic-Petrisic, M., Knezovic, A., Hoyer, S., Riederer, P., 2013. What have we learnedfrom the streptozotocin-induced animal model of sporadic Alzheimer’s disease, about the therapeutic strategies in Alzheimer’s research. J. Neural Transm. (Vienna) 120, 233–252, http://dx.doi.org/10.1007/s00702-012-08779. Shingo, A.S., Kanabayashi, T., Kito, S., Murase, T., 2013. Intracerebroventricular administration of an insulin analogue recovers STZ-induced cognitive decline in rats. Behav. Brain Res. 241, 105–111, http://dx.doi.org/10.1016/j.bbr.2012. 12.005. Shonesy, B.C., Thiruchelvam, K., Parameshwaran, K., Rahman, E.A., Karuppagounder, S.S., Huggins, K.W., Pinkert, C.A., Amin, R., Dhanasekaran, M., Suppiramaniam, V., 2012. Central insulin resistance and synaptic dysfunction in intracerebroventricular-streptozotocin injected rodents. Neurobiol. Aging 33, 430, http://dx.doi.org/10.1016/j.neurobiolaging.2010.12.002, e5–e18. Stelmashook, E.V., Isaev, N.K., Plotnikov, E.Y., Uzbekov, R.E., Alieva, I.B., Arbeille, B., Zorov, D.B., 2009. Effect of transitory glucose deprivation on mitochondrial structure and functions in cultured cerebellar granule neurons. Neurosci. Lett. 461, 140–144, http://dx.doi.org/10.1016/j.neulet.2009.05.073. Stelmashook, E.V., Novikova, S.V., Isaev, N.K., 2010. Glutamine effect on cultured granule neuron death induced by glucose deprivation and chemical hypoxia. Biochemistry (Mosc.) 75, 1039–1044. Szkudelski, T., 2001. The mechanism of alloxan and streptozotocin action in B cell of the rat pancreas. Physiol. Res. 50, 537–546.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Streptozotocin causes neurotoxic effect in cultured cerebellar granule neurons Elisaveta E. Genrikhs a , Elena V. Stelmashook a,∗ , Sergey A. Golyshev b , Olga P. Aleksandrova a , Nickolay K. Isaev a,b a
Research Center of Neurology, Volokolamskoe Shosse 80, 125367 Moscow, Russia M. V. Lomonosov Moscow State University, N. A. Belozersky Research Institute of Physico-Chemical Biology, Leninskye gory, 1, b. 40, 119991 Moscow, Russia b
a r t i c l e
i n f o
Article history: Received 15 November 2016 Received in revised form 29 December 2016 Accepted 3 January 2017 Available online 6 January 2017 Keywords: Streptozotocin Cultured neurons Insulin Mitochondrial membrane potential Calcium
a b s t r a c t Streptozotocin (STZ) is a glucosamine-nitrosourea compound used for experimental simulation of sporadic Alzheimer’s disease at intracerebroventricular administration in vivo. The studies of STZ influence on neurons of central nervous system performed on the primary cultures are practically absent. We have shown the application of STZ (1–5 mM) in primary culture for 48 h induced strong dose-dependent death in cultured cerebellar granule neurons. This toxic effect was decreased by pyruvate, insulin partially. Using the indicator Fluo-4 AM for measurements of intracellular calcium ions and tetramethylrhodamine ethyl ester (TMRE) for detection of changes of mitochondrial membrane potential in live cells we have shown that 5 h-exposure to STZ induced intensive increase of Fluo-4 and decrease TMRE fluorescence in neurons. STZ exposure caused considerable ultrastructural alterations in granule neurons: chromatin clumping, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. Probably, STZ significantly impaired glucose metabolism and mitochondrial function that, in turn, resulted in mitochondrial membrane potential damage, excessive calcium overload and neuronal death. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Streptozotocin is a glucosamine-nitrosourea compound, particularly toxic to the insulin-producing beta-cells of the pancreas in mammals, applied in medicine for treating certain cancers of the Islets of Langerhans. Intraperitoneal injections of STZ are used in a medical research to produce Type 1 diabetes in animal model (Szkudelski, 2001). In the field of neuroscience and experimental medicine the intracerebroventricular administration of STZ is applied as experimental simulation of sporadic Alzheimer’s disease in vivo during 20 years by now (Mayer et al., 1990). In this model, deficits in hippocampal synaptic transmission and long-term potentiation (LTP) were observed. The decline in LTP correlated with decreased expression of NMDA-receptor subunits NR2A and NR2B. Similarly, there has been a decrease in the expression of brain derived neurotrophic factor (BDNF) (Shonesy et al., 2012). The STZ-intracerebroventricular treated animals developed insulin resistant brain state associated with memory impairment
∗ Corresponding author at: Research Center of Neurology, bs. Obukha, 5, Brain Research Department, Research Center of Neurology, Moscow, Russia. E-mail address: [email protected] (E.V. Stelmashook). http://dx.doi.org/10.1016/j.brainresbull.2017.01.004 0361-9230/© 2017 Elsevier Inc. All rights reserved.
and progressive cholinergic deficits, glucose hypometabolism, tau-hyperphosphorylation, astrogliosis and amyloid angiopathy, oxidative stress and neurodegeneration that share many features in common with sporadic Alzheimer’s disease in humans (Park, 2011; Salkovic-Petrisic et al., 2013; Ejaz Ahmed et al., 2013). The reduction of glucose utilization occurred owing to the decrease of glycolytic enzymatic activities (Plaschke and Hoyer, 1993). The most part of researches of STZ toxic action in the brain is carried out on the models in vivo. The STZ researches performed on the cultured central nervous system neurons are absent practically. The present study was designed to test the streptozotocin toxic action in primary neuronal cultures in vitro.
2. Materials and methods 2.1. Primary cerebellar neuronal cultures Primary cultures were prepared from the cerebella of 7–8 days old Wistar rats as described elsewhere (Stelmashook et al., 2009). The cerebella were washed with Ca2+ - and Mg2+ -free PBS (Dulbecco) and incubated in the same solution containing 0.05% trypsin and 0.02% EDTA (15 min, 37 ◦ C). After incubation, the tissue was
E.E. Genrikhs et al. / Brain Research Bulletin 130 (2017) 90–94
washed twice in PBS and dissociated by repeated pipetting in a nutrient medium of the following composition: fetal calf serum (10%), minimum essential medium Eagle (90%), glutamine (2 mM), and HEPES (10 mM). After mild centrifugation, the cells were resuspended in the required volume of the nutrient medium containing 25 mM KCl. Cell suspension (0.1 ml) was added to a poly-l-lysinecoated 96-well plate or coverslips in a 40-mm diameter Petri dishes and then kept in a CO2 -incubator (5% CO2 , 36.5 ± 0.5 oC). Cultures maintained up to 3–4 days in vitro, without replacing the medium. All experimental protocols were approved by the Animal Ethics Committee of the Research Center of Neurology of Russian Academy of Medical Sciences (the Protocol Registration number 05/10) and in accordance with Council Directive 2010/63 EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. 2.2. Pharmacological treatment Streptozotocin (1–5 mM), pyruvate (1 mM), insulin (0.01 mM) (final concentration are noted) were supplemented directly to the culture medium on the 2nd day in vitro. Control and treated cultures have been incubated at 5 or 48 h in CO2 -incubator (5% CO2 , 36.5 ± 0.5 ◦ C). 2.3. Assessment of neuronal viability Viability of cerebellar granule neurons (CGNs) was determined as described previously (Stelmashook et al., 2009). After 48 h STZ (1–5 mM) incubation cell cultures were fixed with ethanolformaldehyde-acetic acid (7:2:1) mixture and stained with trypan blue. The percentage of surviving neurons was estimated by counting the intact nuclei of the CGNs in five fields of view. The viability of untreated control cultures was taken as 100%, and the viability of treated cells was counted as a percentage referred to control values. 2.4. Analysis of intracellular Ca2+ After 5 h STZ (3–4.5 mM) incubation cells were loaded with 0.005 mM Fluo-4 AM (Ca2+ detection) for 30 min at 36.5 ± 0.5 ◦ C followed by triple washing in BSS, containing (in mM): NaCl 154, KCl 25, CaCl2 2.3, MgCl2 1, NaHCO3 3.6, Na2 HPO4 0.35, HEPES 10; at pH 7.3. Levels of intracellular Ca2+ were measured by the fluorescence intensity of Fluo-4 on a fluorescence plate reader (CytoFluor II, PerSeptive Biosystems, Framingham, MA, USA) using excitation at 485 nm and maximal emission at 530 nm. Changes in fluorescence were calculated as (F − F0 )/F0 × 100%, where F0 and F are fluorescence of the dye in cell cultures incubated without and with STZ in medium, respectively.
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2.6. Electron microscopy For electron microscopy we used the same volume of cell suspension placed on poly-l-lysine-coated coverslips in a 40-mm diameter Petri dishes (Lozier et al., 2012) which incubated 2–3 days in vitro. After 5 h STZ exposure (4.5 mM) cultures were fixed using 2.5% glutar aldehyde prepared in phosphate buffer (pH 7.2) followed by postfixation with 1% osmium tetroxide, dehydratation in ethanol, en-bloc staining with uranyl-acetate and embedding in Epon 812. Ultrathin sections were prepared on the ReichertJung Ultracut E ultramicrotome, additionally stained with aqueous uranyl-acetate and lead citrate, examined using the transmission electron microscope JEM-1400 (JEOL, Japan) running at 100 kV. 2.7. Statistics The one-way ANOVA with Neuman–Keuls and Bonferroni posttest was used for statistical analysis. Levels of p < 0.05 were considered as statistically significant. The results are represented as means ± SEM. All data were obtained using at least 9–12 different cultures from 3 to 4 independent experiments. 2.8. Reagents Unless otherwise noted, all media and supplements used in experiments were purchased from Biochrom KG Berlin, Germany. Fluo-4 AM and TMRE were from Molecular Probes (USA). Streptozotocin and other reagents were from Sigma Chemicals (Germany). 3. Results 3.1. Streptozotocin toxicity in cerebellar granule neurons Counting of neurons with normal morphology demonstrated that application of STZ (1–5 mM) in culture medium for 48 h with effect from 2.5 mM induced strong dose-dependent CGNs death (Fig. 1). STZ concentrations which cause death more than 50% of neurons have been chosen for further experiments. Under these conditions, the neurons were partially protected from cell death by supplementation of 1 mM pyruvate or 0.01 mM insulin (Fig. 2). 3.2. Intracellular calcium ions level and mitochondrial membrane potential The exposure to STZ (3–4.5 mM) for 5 h induced significantly strong dose-dependent increase of Fluo-4 fluorescence in the CGNs to 22 ± 4–63 ± 10% in comparison with control (Fig. 3A). Under these conditions TMRE fluorescence decreased to 17 ± 4% (Fig. 3B).
2.5. Measurement of mitochondrial membrane potential
3.3. Electron microscopy
Alterations in the mitochondrial membrane potential were analyzed using the mitochondrial membrane potential-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Briefly, following treatment with STZ (3–4.5 mM, 5 h), cells were incubated with 100 nM TMRE in the culture medium at 36.5 ± 0.5 ◦ C for 15 min. Stained cells were then washed triply with balanced salt solution. Positively-charged TMRE as a penetrating cation readily accumulates in active mitochondria due to their relative negative charge. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE. Fluorescence (excitation at 530 nm, emission at 640 nm) was analyzed on a fluorescence plate reader (CytoFluor II, PerSeptive Biosystems, Framingham, MA, USA). Changes in fluorescence were calculated as described previously.
In an effort to identify and characterise the ultrastructural changes in cerebellar granule neurons induced following treatment with STZ electron microscopy has been used. The ultrastructural study showed that control CGNs cultivated under normal conditions had typical for normal cells mitochondrial ultrastructure: mitochondria were not enlarged, had slightly oval or elongated shape. The cristae were clearly seen, and the mitochondrial matrix had a higher electron density than the surrounding cytoplasm (Fig. 4A). In STZ-treated neurons for 5 h, the mitochondria were enlarged, and the majority of cristae were damaged (Fig. 4B). Pathological signs of STZ-treated CGNs were seen also in some other neuronal compartments: nuclear chromatin was clumped, distinctly expanded perinuclear space and swelling of the granular endoplasmic reticulum were apparent.
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Fig. 1. The influence of STZ in culture medium on cerebellar granule neurons. A–B, cultures were fixed with ethanol–formaldehyde–acetic acid (7:2:1) mixture and stained with trypan blue, (A) control, (B) 4.5 mM STZ. Intact nuclei of CGNs are indicated by arrows. Scale bar 15 m. (C) quantitative evaluation of neuronal survival. *Statistically significant difference from control values (0 mM STZ), p < 0.01.
Fig. 2. Pyruvate and insulin attenuate toxic effect of STZ in cultured cerebellar granule neurons. (A) 1 mM pyruvate – black columns, (B) 0.01 mM insulin – black columns. White bars – in lack of a protector. Quantitative evaluation of neuronal viability. * p < 0.01.
4. Discussion Following the parenteral injection of high doses more 40 mg/kg STZ selectively destroys insulin producing beta cells in the pancreas, causing diabetes type I in adult animals (Szkudelski, 2001). Intracerebroventricular administration of STZ in subdiabetogenic doses (3 mg/kg) to rats or mice is used as experimental simulation of sporadic Alzheimer’s disease in vivo (Ponce-Lopez et al., 2011; Hamidi et al., 2013). Total brain volume is about 769–881 mm3 , and lateral ventricles volume is 2.19–2.39 mm3 (Fabricius et al., 2010). Therefore, local concentration of a streptozotocin in a brain can
reach more than 4 mM after intracerebroventricular administration. Our experiments were carried out on dissociated cell cultures obtained from cerebellum of 7–8 days old rats. Previously this type of culture was used for studying cytotoxicity of nitrosamine (1–2.5 mM), which is a part of a STZ molecule (De la Monte and Tong, 2009). We have shown that application of 2.5 mM and more STZ for 48 h induced strong dose-dependent death in cultured CGNs. STZ concentrations which cause death more than 50% of neurons have been chosen for further experiments. Previously these concentrations of STZ and incubation period were tested in human neuroblastoma cells. Authors showed significant increase in cells
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Fig. 3. The exposure to STZ for 5 h induced the dose-dependent increase of Fluo4 fluorescence in CGNs ([Ca2+ ]i changes) (A) and decrease of TMRE fluorescence (decrease of mitochondrial membrane potential) (B). Changes in fluorescence (Fluo4 or TMRE) were calculated as (F − F0 )/F0 × 100%, where F0 and F are fluorescence of the dye in cell cultures incubated without and with STZ in medium, respectively. *Statistically significant difference from control values (0 mM STZ), p < 0.05.
death following treatment with more 2.5 mM STZ (Plaschke and Kopitz, 2015). Biswas et al. (2016) demonstrated, that 1 mM STZ for 48 h induced decrease of cell viability on 30% (MTT-test) in mouse neuronal N2A cells. In this work the LDH-test has been used also. However authors haven’t made assessment of total activity of enzyme in cell cultures, and the real percentage of the died cells can’t be determined by these data. It is known that STZ can induce G2 arrest in the cell cycle (Johnston et al., 2007). Therefore STZ can be more toxic for N2A cells capable to proliferation, than for the differentiated neurons, for example cerebellar granule neurons. 5 h-exposure of neuronal cultures to STZ didn’t cause cell death, but measurements of intracellular calcium ions using Fluo-4 AM and mitochondrial membrane potential with TMRE demonstrated that intensive increase of Fluo-4 fluorescence and decrease of TMRE fluorescence was observed. Perhaps STZ-induced impaired mitochondrial function can lead to the calcium overload of CGNs. Brain glucose metabolism has been found to be markedly decreased in the STZ-intracerebroventricular treated rats, demonstrated decrease
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ATP and phosphocreatine concentrations as well as the ATP/ADP ratio and glucose utilization in the cerebral cortex (Nitsch and Hoyer, 1991; Duelli et al., 1994). The reduction of glucose utilization occurs owing to the decrease of glycolytic enzymatic activities (Plaschke and Hoyer, 1993). Earlier it has been shown that pyruvate decrease CGNs death under glucose starvation conditions (Stelmashook et al., 2010). Therefore we have suggested that pyruvate as the mitochondrial energy substrate can increase survival of neurons by STZ toxicity, what has been confirmed in our experiments. Intracerebroventricular injection of streptozotocin lead to development of insulin resistance in the brain, at the same time the intracerebroventricular-administration of insulin and its analogues recover STZ-induced cognitive decline in rats (Shingo et al., 2013). Our study demonstrated that the medium supplementation with insulin partially protected CGNs from the STZ toxic action. In addition we have found that mitochondrial ultrastructure were significantly damaged in STZ treated CGNs. It was noted, the mitochondria were enlarged, and the majority of cristae were damaged. Thus, ultrastructural alterations are usually indicative for the organelles disfunctioning. Pathological signs of STZ treated neurons were seen also in some other neuronal compartments: nuclear chromatin was clamped, perinuclear space distinctly broadened and swelling of granular reticulum were apparent. Similar neuronal ultrastructural changes were observed previously in CGNs after calcium overload induced glutamate treatment (Isaev et al., 1996; Isaev et al., 2005). Early it has been shown that isolated brain mitochondria from intracerebroventricular-STZ rats displayed a decrease in pyruvate and ␣-ketoglutarate dehydrogenases and cytochrome C oxidase activities and an increase in the susceptibility to calcium-induced mitochondrial permeability transition (Correia et al., 2013). These deficits in mitochondrial function can limit protective effect of the pyruvate in our experiments.
5. Conclusions STZ exposure of cultured neurons causes significantly disturbance of glucose metabolism and mitochondrial function that, in turn, results in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.
Conflict of interest The authors declare that there is no conflict of interest.
Fig. 4. Transmission electron micrograph of cultured cerebellar granule neurons. (A) Control culture was exposed to the culture medium. Mitochondria and other compartments appear undamaged. (B) cells after 5 h-exposure to 4.5 mM streptozotozin in the culture medium. Note mitochondrial swelling and disruption of the cristae. Clumping of heterochromatin, distinctly broadened perinuclear space and endoplasmic reticulum swelling are seen.
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