Priming of Cultured Neurons with Sabeluzole Results in Long-Lasting Inhibition of Neurotoxin-Induced Tau Expression and Cell Death
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SYNAPSE 26:95–103 (1997) Priming of Cultured Neurons With Sabeluzole Results in Long-Lasting Inhibition of Neurotoxin-Induced Tau Expression and Cell Death DANIELA UBERTI,1 CLAUDIA RIZZINI,1 PAOLA GALLI,1 MARINA PIZZI,1 MARIAGRAZIA GRILLI,1 ANNE LESAGE,2 PIERFRANCO SPANO,1 AND MAURIZIO MEMO1* 1Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, School of Medicine, University of Brescia, Brescia, Italy 2Department of Biomemical Pharmacology, Janssen Research Foundation, Beerse, Belgium KEY WORDS cerebellar granule cells; human neuroblastoma SH-SY5Y; glutamate; doxorubicin; Tau proteins; PCR ABSTRACT Sabeluzole was described to have antiischemic, antiepileptic, and cognitive-enhancing properties, and is currently under development for Alzheimer’s disease. Recently, it was reported that repeated treatments with sabeluzole protect cultured rat hippocampal neurons against NMDA- and glutamate-induced neurotoxicity. We evaluated the possibility that sabeluzole elicits neuroprotection by acting, either directly or indirectly, on tau proteins. We found that repeated treatments during development of primary cultures of cerebellar granule cells with nanomolar concentra- tions of sabeluzole resulted in mature cells that were resistant to the excitotoxicity induced by glutamate. Also, sabeluzole treatment specifically prevented the glutamate- induced increase of tau expression without modifying the basal pattern of expression of tau proteins, as shown by measurement of mRNA and protein levels. In human neuroblastoma cell line SH-SY5Y, differentiated by treatment with retinoic acid, doxorubicin increased tau immunoreactivity, and later induced cell death. Both effects were prevented by sabeluzole. Our data indicate that increased tau expression is a common response to different types of cells to neurotoxic agents, and that sabeluzole- induced neuroprotection is functionally associated with the prevention of the injury- mediated increase of tau expression. Synapse 26:95–103, 1997. r 1997 Wiley-Liss, Inc. INTRODUCTION tau proteins are the major component of neurofibrillary Sabeluzole is an experimental drug originally devel- tangles, one of the hallmarks of the Alzheimer’s disease oped for its antiischemic, antiepileptic (Wauquier et al., brain (Braak et al., 1986). We previously found that inhibition of tau expression by tau oligonucleotide 1986), and cognitive-enhancing properties (Clinke and antisense treatment in primary cultures of cerebellar Sahgal, 1986), and it is currently under development granule cells reduced neural vulnerability to glutamate- for Alzheimer’s disease. Recently, it was reported that induced neurotoxicity (Pizzi et al., 1995a). Increased repeated treatments with sabeluzole protect cultured tau expression may thus be considered one of the rat hippocampal neurons against NMDA- and gluta- crucial steps in the chain of intracellular events, in- mate-induced neurotoxicity (Pauwels et al., 1990). How- duced by glutamate, leading to neural death. ever, the site(s) as well as the mechanism(s) of action of In this report we show that increased tau expression sabeluzole responsible for its neuroprotective proper- is a converging effect induced by a variety of neurotoxic ties are up to now largely unknown. agents, and that the neuroprotective properties of We tested the possibility that sabeluzole may elicit sabeluzole, independent of type of experimental injury, neuroprotection by acting, either directly or indirectly, on tau expression. Tau proteins are cytoskeleton- associated, neural proteins promoting microtubule as- *Correspondence to: Prof. Maurizio Memo, Division of Pharmacology, De- sembly and neurite polarization (Caceres and Kosik, partment of Biomedical Sciences and Biotechnologies, School of Medicine, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy. 1990; Dubrin and Kirschner, 1986). The interest in tau E-mail: [email protected] proteins has recently been increased by the finding that Received 23 May 1996; Accepted 3 October 1996. r 1997 WILEY-LISS, INC. 96 D. UBERTI ET AL. are associated with its capability to prevent such an medium; 24 h later, neurotoxicity was evaluated as increase. percentage of total lactate dehydrogenase (LDH) activ- ity. Extracellular and intracellular LDH activity was MATERIALS AND METHODS measured spectrophotometrically, following NADH- Cell cultures oxidation at 340 nm (Bergmeyer, 1974; Pauwels et al., 1989). Total LDH activity was defined as the sum of Primary cultures of cerebellar granule cells were intracellular and extracellular LDH activity. prepared from 8-day-old Sprague-Dawley rat pups as previously described (Levi et al., 1984). Cells were plated onto poly-1-lysine-coated dishes and cultured in Intracellular calcium concentration basal Eagle’s medium containing 10% heat-inactivated measurement fetal calf serum, 2 mM glutamine, 50 µg/ml gentamicin, Regulation of cytosolic free calcium concentration 5 2 and 25 mM KCl, at a density of 1.5 3 10 cells/cm . was investigated by microfluorimetry in single cerebel- Cytosine arabinoside (100 µM) was added to the cul- lar granule neurons, according to Malgaroli et al. (1987) tures 18 h after seeding to prevent non-neuronal cell with minor modifications (Pizzi et al., 1995a). Briefly, proliferation. Experiments were done after culturing cells were plated onto 100 µg/ml poly-l-lysine-coated the neurons for 12 days in vitro (DIV) unless otherwise glass coverslips and cultured as described above. On indicated. Neuroblastoma cell line SH-SY5Y was rou- DIV 12, cells were loaded with the calcium-sensitive tinely cultured in 1:1 Ham’s F12 Dulbecco’s modified fluorescent dye fura-2, by a 60-min incubation at 37°C Eagle’s medium with 10% fetal calf serum, 2 mM with its acetoxymethyl derivate (4 µM in Krebs-Ringer glutamine, in 120-mm Falcon plastic petri dishes in medium containing 1.3 mg/ml bovine serum albumin, humidified incubators at 37°C with 5% CO . For differ- 2 pH 7.4), and then mounted in a 22-mm holder. Fura-2 entation, cultures were seeded at approximately 105 emission was monitored using an inverted fluorescence cells/dish, and retinoic acid was added to give a final microscope (Nike Diaphot, Ni Ron, Japan) associated concentration of 1025 µM. Medium was changed on with an intensified charge-coupled device camera (Mira- alternate days, and cultures were allowed to differenti- 100 TE; Applied Imaging, Gateshead, UK) which re- ate for at least 3 weeks. corded the 510-nm fluorescence emission in neurons excited through a narrow bandpass filter (340 and 380 Neurotoxicity assay nm). The background was subtracted, and the amount The culture-conditioned media of cerebellar granule of free calcium within the cell bodies was calculated cells were collected, and the cells were washed once from the ratio of 340/380 nm obtained every 3–4 sec. with Locke’s solution (154 mM NaCl, 5.6 mM KCl, 3.6 Calibration was according to external standards of mM NaHCO3, 2.3 mM CaCl2, 5.6 mM glucose, and 5 calcium and fura-2 (Connor, 1986). Fluorescence image mM HEPES, pH 7.4) and exposed to different concentra- acquisition and analysis were performed using the tions of glutamate for 15 min. After this period, cells MIRAcal (Multiple Image Ratioing and Analysis with were washed three times with Locke’s solution contain- Calibration) system by Applied Imaging. ing 1 mM MgSO4 and returned to the original culture- conditioned media. Cells were cultured for an addi- Polymerase chain reaction tional 24 h before intravital staining was performed. To evaluate the acute effect of sabeluzole, 0.001% dimethyl A polymerase chain reaction (PCR)-derived method solfoxide (DMSO) or sabeluzole, prepared from a 100- was used to determinate tau mRNA levels in cerebellar fold concentrated drug solution in 0.1% DMSO, were granule cells (Pizzi et al., 1995b) and in the neuroblas- added 20 min before the glutamate pulse. When the toma SH-SY5Y cell line. Total mRNA was isolated from chronic effect of sabeluzole was investigated, vehicle or the cultures by the method of RNAzol (Chirgwin et al., the drug, prepared as described above, was added to the 1979; Chomczynski and Sacchi, 1987), extracted by cultures on DIV 5 and DIV 8. Cell viability was chloroform-ethanol, and reverse-transcribed with determined by fluorescein diacetate (15 µg/ml) and Moloney murine leukemia virus reverse transcriptase propidium iodite (80 µg/ml) staining as previously in the presence of [32P]dCTP, as recommended by the described (Jones and Senft, 1985). manufacturer (BRL). The resulting cDNA was quanti- Neurotoxicity experiments were also performed on tated by determining the amount of radioactivity incor- differentiated neuroblastoma SH-SY5Y cells. The acute porated into trichloroacetic acid-precipitable nucleic effects of sabeluzole were evaluated by pretreating the acids. PCR was done with Taq polymerase (Perkin- cells with vehicle or the drug 20 min before cell injury, Elmer Cetus) in 100 µl of standard buffer containing 0.5 while chronic effects were studied by adding vehicle or ng cDNA, [32P]dCTP as radioactive tracer, and 1 µM sabeluzole on DIV 14 and DIV 18. On DIV 21, 10 nM specific primers. The following primers were used: doxorubicin, an anthracycline antibiotic that induces 58-AGCCGCCTACAGACTGCCCCTGTG-38, correspond- DNA damage (Cole et al., 1985), was added to the ing to nucleotides 693–717 and referred to as A, and 38- SABELUZOLE INHIBITS CELL DEATH 97 ATTTCTGCTCCATGGTCTGTCTTG-58, corresponding tau, a sequence that appears to be conformationally to nucleotides