BDNF protects neurons form glucose deprevation

BDNF protects neurons form glucose deprevation - J Neural...

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Unformatted text preview: J Neural Transm (1998) 105:rain-derived neurotrophic factor B 905–914 905 Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis L. Tong and R. Perez-Polo Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX, U.S.A. Received December 17, 1997; accepted May 19, 1998 Summary. In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage. Keywords: Apoptosis, BDNF, neurotrophin, hypoglycemia. Introduction Neuronal death, during development or after injury, can be the end point of two distinct processes: apoptosis and necrosis. Apoptosis is an endogenous cell suicide mechanism, which can be selectively triggered by cells in response to various stimuli (Wyllie et al., 1980; Raff, 1992). In general, apoptosis can be distinguished from necrosis by the following events: 1) During the early stages of apoptosis there is condensation and fragmentation of nuclear chromatin, accompanied by a marked decline in total cell volume and general compaction of cellular organelles; (2) during the later stages of the process there are changes in both nucleus and cytoplasm that give rise to small cell bodies surrounded by intact cell membrane (Raff, 1992). There are aspects of cell death observed during neuronal development that are regulated by neurotrophins such as NGF, BDNF, and NT-3, which 906 L. Tong and R. Perez-Polo support the survival of specific neuronal populations (Oppenheim, 1991; Snider, 1994). Furthermore, several neuronal populations retain their dependence on neurotrophic factors well into maturity (Misko et al., 1987; Rich et al., 1987; Raff et al., 1993). Not surprisingly, neurotrophic factors promote neuronal cell survival in a variety of in vitro and in vivo models of neuronal injury (Misko et al., 1987; Mattson et al., 1993). For example, NGF and bFGF protect cultured hippocampal and cortical neurons against glucose deprivation-induced damage (Cheng and Mattson, 1991). Hypoglycemic brain damage is a major component of stroke related to ischemia (Auer and Siesjö, 1988). Cerebellar granule neuronal cultures have been used to study the mechanisms underlying apoptosis in the CNS. Given that cerebellar granule cells express TrkB, the signal-transducing BDNF-specific receptor, and that BDNF also enhances the survival of granule cells in cultures of embryonic cerebella (Segal et al., 1992), we assessed the effects of three neurotrophins on cerebellar granule cell survival after an induced hypoglycemic injury. Materials and methods Materials Mouse 2.5S NGF was prepared by the method of Mobley et al. (1976). The In situ DNA fragmentation detection assay kit was purchased from Oncor, Inc. (Gaitherburg, USA). Other chemicals were purchased from Sigma (St. Louis, MO, U.S.A.). The BDNF and NT-3 used were kind gifts from Regeneron (Regeneron, NY, USA). Cell culture and experimental treatments Cultures enriched in granular neurons were obtained from dissociated cerebella of 8-dayold rats as described by Levi et al. (1984). Following preparation, cells were plated in basal Eagle’s medium (BME) supplemented with 10% fetal calf serum, 25 mM KCl, 2 mM glutamine, and 100 µg/ml gentamycin on dishes coated with poly-L-lysine. Cells were plated at a density of 3 105/cm2. Cytosine arabinofuranoside (AraC, 10 µM) was added to the culture medium 18–22 h after platinst to select against dividing nonneuronal cells. Viability assay The number of living cerebellar granule neurons was determined by the trypan blue exclusion method. Cells grown in 24-well culture dishes were counted at 200 magnification with phase-contrast microscopy and with bright-field examination after trypan blue staining (0.1%, 5 min). Cell counts for each experimental condition were performed on six randomly chosen fields per well and three wells per experiment. Statistical comparisons were made using ANOVA followed by Fisher’s test. The criteria for significance were set at p 0.05. Apoptosis assay Fragmentation of DNA was examined using a procedure described by Hockenberry et al. (1990). Samples of DNA (10–30 µg) present in cytoplasm were electrophoresed in 1% agarose gel, stained with ethidium bromide, and photographed by UV transillumination. DNA fragmentation was also measured by quantitation of cytosolic oligonucleosomebound DNA using a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) Brain-derived neurotrophic factor 907 kit (Boehringer-Mannheim, Germany) following the manufacturer’s instructions. Briefly, DNA was isolated from cytoplasm and incubated with precoated anti-histone antibodies. During the first incubation step, the nucleosomes contained in the sample bind via their histone components to immobilized anti-histone antibodies. In a second incubation step, anti-DNA-peroxidase reacts with the DNA-component of the nucleosome. After removal of the unbound peroxidase conjugated by a washing step, the amount of peroxidase retained in the immunocomplex is measured using the reference wavelengths 405 and 490 nm, respectively, with ABTS (2,2 -azino-di-[3-ethylbenzthiazoline sulfonate]) as substrate. Statistical comparisons were made using ANOVA followed by Fisher’s test. The criteria for significance were set at p 0.05. For nuclei condensation assays, cells were washed with PBS, stained with the DNA specific fluorochrome propidium iodide (PI), and examined under a fluorescence microscope (X400) as described elsewhere (Wei et al., 1994). In situ DNA fragmentation was detected using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method (Gavrieli et al., 1992). Cultures were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) (pH7.4) for 1 h. Cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min and processed. Residues of digoxigenin nucleotide are enzymatically added to DNA. The anti-digoxigenin antibody is then used for the detection (peroxidase reaction) of labeled 3 -OH ends that are abundant in cells containing fragmented DNA. Results Glucose deprivation induces apoptotic cell death in cerebellar granule cells In the initial series of experiments, the viability of cultured rat cerebellar granule neurons after glucose deprivation was measured. Cerebellar granule neurons were incubated in a glucose-free RPMI medium for increasing periods of time. Following treatment with glucose deprivation, culture medium was replaced with medium containing glucose. Cell viability was measured 24 h following the glucose deprivation period using the trypan blue exclusion assay. In line with the observations by Facci et al. (1990), glucose deprivation for 4 h significantly reduced cell survival (Fig. 1). As shown in Fig. 2, 24 h after 4 h exposure to glucose-free medium, most of the neuronal somata and neurite networks had degenerated. To examine whether apoptosis was induced by glucose deprivation, we examined the DNA from neurons after glucose deprivation. As shown in Fig. 3, glucose deprivation induced DNA fragmentation as detected using an internucleosomal DNA ELISA assay. Furthermore, DNA fragmentation was also detected in neurons as early as 2 h after glucose deprivation as demonstrated by the observation of specific in situ labeling of DNA breaks in nuclei (Fig. 4). BDNF protects cells from cerebellar granule neurons from glucose deprivation-induced cell death We examined the ability of NGF, BDNF, and NT-3 to support the survival of granule neurons in culture after glucose deprivation. These factors were chosen because they are synthesized in the cerebellum and have been reported to promote survival of other neuronal populations (Snide, 1994). As shown in Fig. 5, NGF as well as NT-3 failed to rescue cells from death caused by glucose deprivation (Fig. 5). In contrast, BDNF had a protective effect on neuronal 908 L. Tong and R. Perez-Polo Fig. 1. Time-course of cytotoxic effects of glucose deprivation on cerebellar granule neurons. Granule neurons were incubated in the glucose-free ( ) or glucose-containing ( ) BME medium for different periods of time. Following treatment with glucose deprivation, culture medium was replaced with medium containing glucose. Cell viability was measured 24 h following the glucose deprivation period using trypan blue exclusion assay. Data are the means of triplicate cultures SEM • survival (Fig. 5). To further assess the protective effect of BDNF on granule neurons, cells were exposed to various concentrations of BDNF and cell viability was measured 24 h after glucose deprivation. As shown in Fig. 6, maximum protection was achieved at a BDNF concentration of 100 ng/ml with a half-maximal effect at 10 ng/ml. To determine whether granule neurons respond directly to BDNF, we analyzed the induction of the c-Fos protein in these neurons. Immunocytochemical staining of the cultures demonstrated that a relatively large number of neurons displayed c-Fos-immunoreactivity in response to BDNF treatment (Fig. 7). Discussion Hypoglycemic neuronal cell death has been found to exhibit some elements of necrosis and is largely mediated by excitotoxic activation of glutamate receptors (Wieloch, 1985). While there remains a debate in the literature as to whether excitotoxicity results in apoptosis (Ignatowicz et al., 1991; Koh et al., 1995; Ankardrone et al., 1995), increasing evidence suggests that excitotoxicity induces neuronal apoptosis under specific conditions relevant to the strength and duration of the injurious events (Bonfoco et al., 1995) and the state of mitochondrial function (Ankarcrona et al., 1995). It has been reported that the exposure of cortical neurons to relatively brief exposures of NMDA or low concentrations of NMDA induces a delayed form of neurotoxicity characterized by its apoptotic features and that intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage as Brain-derived neurotrophic factor 909 Fig. 2. Glucose deprivation induces cell death of cerebellar granule neurons in culture as shown by phase-contrast microscopy. Cells were incubated in the glucose-containing (A) or glucose-free (B) BME medium for 6 h. Following treatment with glucose deprivation, culture medium was replaced with medium containing glucose. Typical fields ( 200) were taken 24 h after glucose deprivation for 6 h characterized by acute cellular swelling and lysis (Bonfoco et al., 1995). It has also been reported that hypoglycemic neuronal primary cultures can undergo apoptosis when glutamate receptors are blocked (Gwag et al., 1995). In cerebellar granule cells, there is a subpopulation of neurons that display collapsed mitochondrial membrane potential and necrotic death during and shortly after exposure to glutamate. Those neurons that recover mitochondrial potential and energy levels can then undergo apoptosis (Ankarcrona et al., 1995). In the present study, we demonstrate that there is glucose deprivationinduced apoptotic cell death in cerebellar granule cells, as demonstrated by 910 L. Tong and R. Perez-Polo Fig. 3. Glucose deprivation induces DNA fragmentation of cerebellar granule neurons in culture as measured by DNA fragmentation ELISA assays. DNA fragmentation was assessed after 2, or 4 h glucose deprivation ( ) or in the presence ( ) glucose (10 mM). The data was expressed as O.D. as described in Materials and methods. Error bars represent SEM (n 6). * P 0.02 compared to cultures deprived of glucose • TUNEL and DNA fragmentation assays. Our results favor the hypothesis that cell death due to glucose deprivation has a significant apoptotic component. The mechanism of hypoglycemia-induced neuronal cell death is not clear. Cheng et al. (1993) have shown that in hippocampal neurons at early times after the induction of hypoglycemia in vitro there is induced calcium current inhibition and a reduction in the intracellular free calcium levels ([Ca2 ]I) without any morphological signs of neuronal damage; while at later times hypoglycemia induces a large increase in [Ca2 ]I levels with concomitant neuronal damage. These observations are consistent with the hypothesis that calcium plays a critical role in apoptosis and cytotoxicity. In a variety of experimental systems, a perturbation of intracellular Ca2 homeostasis due to increased Ca2 influx and/or inhibition of Ca2 extrusion has been found to be an early event associated with apoptosis (Martin et al., 1994). Recently, c-myc expression has been suggested as a necessary component of glucose-free medium-induced apoptosis in multidrug-resistant human breast carcinoma cells (Lee et al., 1997). There is evidence that BDNF is a survival factor for cerebellar granule neurons. For example, BDNF treatment of primary cultures of cerebellar Fig. 4. DNA nick end labeling of fragmented nuclear DNA in control (A) and glucosedeprived (B and C) granule neurons. Cells were collected at 4 h after glucose deprivation. Arrowheads indicate fragmented nuclei. Magnification: A and B, 100; C, 400 Fig. 7. Fos immunoreactivity of cultured cerebellar granule neurons in response to BDNF. Neuronal cultures were processed, then stained with anti-Fos antibody as described in Materials and Methods. Neurons were exposed to no additive (A) or BDNF (50 ng/ml) (B) for 2 h and then fixed and stained with anti-fos antibody Fig. 7 911 Fig. 4 Brain-derived neurotrophic factor 912 L. Tong and R. Perez-Polo Fig. 5. Effects of BDNF on the survival of cultured cerebellar granule neurons after glucose deprivation. NGF, BDNF, and NT-3 were added at concentration of 50 ng/ml. Cell viability was measured 24 h following the glucose deprivation for 4 h using trypan blue exclusion assay and expressed as percentage of control. Data are the means of triplicate cultures SEM. * P 0.02 compared to cultures deprived of glucose in the absence of BDNF Fig. 6. Dose response of BDNF action on cell suvival after glucose deprivation in granule neurons. Cells were exposed to various concentrations of BDNF. Cell viability was measured 24 h following the glucose deprivation for 4 h using trypan blue exclusion assay and expressed as percentage of control. Data are the means of triplicate cultures SEM Brain-derived neurotrophic factor 913 granule cells enhances their survival under low K conditions (Kubo et al., 1995; Suzuki and Koike, 1997) in agreement with observations of protection cerebellar granule neurons by BDNF (Koi et al., 1994). BDNF also induces sprouting of granule neurons and significantly protects them against neurotoxicity induced by exposure to 1 mM glutamate (Lindholm et al., 1993). Grafting of P3 hypothyroid rats with cell lines expressing high levels of BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15 (Neveu and Arenas, 1996). We found BDNF to promoted cell survival under glucose-free conditions. In summary, our findings suggest that neuronal death due to glucose deprivation has a significant apoptotic component and that BDNF can protect against this hypoglycemic damage. Acknowledgements Thanks to K. Werrbach-Perez for excellent technical assistance and D. Masters for manuscript preparation. Work supported in part by a grant to L.T. from the Kempner Fund and the UTMB Sealy Center on Aging. 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