Oxidative stress induces axonal beading in cultured human brain tissue

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Oxidative stress induces axonal beading in cultured human brain tissue Ben Roediger and Patricia J. Armati* Neuroscience Unit, School of Biological Sciences A08, University of Sydney, Sydney, NSW, 2006, Australia Received 15 August 2002; revised 16 January 2003; accepted 21 March 2003

Abstract Oxidative stress has been implicated in the pathogenesis of a number of human neurodegenerative disorders of the central nervous system (CNS), including Alzheimer’s disease (AD). To better understand the pathological effects of oxidative stress on CNS neurons we used a primary human brain cell culture model of hydrogen peroxide-induced oxidative stress. Neuronal and astrocytic morphology was visualised by immunofluorescence with antibodies to the neuron-specific microtubule component ␤-tubulin III and against glial fibrillary acidic protein (GFAP), respectively. After exposure to 40 mM H2O2 for 60 –90 min, axonal swelling was observed, which developed into axonal beading after 48 h. No beading was observed in GFAP-positive astrocytes. Despite the concentration of H2O2 used, neurons remained attached to the substratum and showed no signs of apoptosis. This was attributed to the neuroprotective effect of the B-27 medium supplement, which contained antioxidants. The axonal swelling and beading was consistent with a disruption of microtubules by oxidative stress and subsequent hold-up of axonal transport. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Central nervous system; Human brain tissue; Hydrogen peroxide; Neuronal beading; Oxidative stress

Introduction Oxidative stress is defined as a disturbance in the prooxidant-antioxidant balance in favour of the former, leading to potential damage (Sies, 1991). The central nervous system (CNS) seems to be especially vulnerable to oxidative stress on account of its high rate of oxygen utilisation and the fact that neuronal membranes contain a high proportion of oxidation-prone polyunsaturated fatty acids, making them more susceptible to peroxidative damage (Sayre et al., 1999). Also, the brain appears to contain lower levels of molecular antioxidants such as superoxide dismutase and glutathione peroxidase (Halliwell, 1992; Floyd, 1999; Sayre et al., 1999) as well as lower activities of antioxidant enzymes such as catalase (Halliwell, 1992; Ro¨hrdanz et al., 2001). Given the sensitivity of the brain to free radical damage, it is not surprising that oxidative stress has been implicated in a number of human degenerative disorders of * Corresponding author. School of Biological Sciences, Bldg. A 08, Science Road, University of Sydney, Sydney, NSW 2006, Australia. Fax: ⫹612-9351-4119. E-mail address: [email protected] (P.J. Armati).

the CNS, including Parkinson’s disease, amyotrophic lateral sclerosis, and, in particular, Alzheimer’s disease (AD) (Ro¨hrdanz et al., 2001). We are particularly interested in the possible role of oxidative stress in a human brain tissue culture model of relevance to late-onset AD. AD is a progressive degenerative disease of the brain that inexorably leads to severe loss of cognitive function and accounts for over 65% of dementias in the elderly. Although the cause of AD is unknown, numerous studies have implicated a role for oxidative stress in the pathogenesis of the disease (Martins et al., 1986; Pappolla et al., 1992; Good et al., 1996; Pratico` and Delanty, 2000; Ramassamy et al., 2000; Smith et al., 2000b; Aksenov et al., 2001). Although the exact role of oxidative stress in AD remains unknown, the emerging model of the disease is one in which oxidative factors, produced over time in the CNS, gradually lead to a progressive oxidative imbalance with increasing age and hence contribute to the pathogenesis of AD (Smith et al., 2000b; Nunomura et al., 2001). Such an imbalance could be generated through repeated transient increases in reactive oxygen species during events such as trauma and ischemia (Sahuquillo et al., 2001; Gilgun-Sherki

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et al., 2002), known risk factors for late-onset AD (Kalaria, 2000; Nemetz et al., 1999). Understanding the pathological effects of oxidative stress in the CNS and the progression of changes occurring in individual cells, however, cannot be achieved by examination of autopsied brain tissue but, rather, requires the study of living tissue. Given the difficulty of performing such studies in vivo, the use of cell culture models can provide an alternative. Many laboratories have developed cell culture models of oxidative stress; however, most have used cell lines or studied primary brain tissue obtained from mice or rats. Given that AD is a uniquely human disorder, it is important that the effects of oxidative stress on primary human brain tissue be studied and characterised. Most cell culture models of oxidative stress employ hydrogen peroxide (H2O2) as the prooxidant to induce oxidative stress (Morel and Barouki, 1999; Sokolova et al., 2001), and even though it does not contain an unpaired electron and is therefore not a free radical per se, it is capable of altering the intracellular redox state of a cell and causing oxidative damage by its conversion to the highly reactive hydroxyl radical, 䡠OH (Halliwell et al., 2000). H2O2 has also been implicated a major contributor to oxidative stress in vivo, given that it is a naturally occurring, abundant reactant in mitochondria (Kehrer, 2000). Additionally, there is evidence that levels of H2O2 and 䡠OH are elevated during CNS trauma and ischemic events, and thus contribute to the pathogenesis of neurodegenerative disorders such as AD (Lewen et al., 2000; Facchinetti et al., 1998; Halliwell, 1992). Here, we describe a primary human brain cell culture model of hydrogen peroxide-induced oxidative stress, designed to mimic a transiently elevated level of free radicals in vivo, to better understand the effects of oxidative stress on brain cells, particularly the morphological changes in neurons and astrocytes. It is known that oxidative stress can cause the disruption of the cytoskeleton, particularly microtubules (Mirabelli et al., 1989; Rogers et al., 1989). However, the cellular processes following such cytoskeletal alterations are still largely unknown. We thus aimed to study the morphological changes that occurred in human neurons in response to oxidative stress by using noncytotoxic levels of hydrogen peroxide, and to better our understanding of possible functional consequences of oxidative stress in the CNS. To achieve this, cultured human fetal forebrain tissue was exposed to H2O2 for 90 min prior to immunohistochemical analysis.

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Human Ethics Committee approval No. 96/7/7. Brain tissue was transferred to Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) in 50-ml Falcon tubes immediately post termination. Within 2 h of termination, the brain tissue was triturated and plated onto Matrigel-(Becton Dickinson, Franklin Lakes, NJ, USA) coated T25 flasks and maintained in Neurobasal medium (Life Technologies, Rockville, MD, USA) supplemented with 1% B-27 serum-free supplement, ⫹ antioxidants (GibcoBRL), L-glutamine (200 mM; GibcoBRL, Grand Island, NY, USA), penicillin (50 U/ml; GibcoBRL), and streptomycin (50 ␮g/ml; GibcoBRL) (NB ⫹ B-27) at standard culture conditions (SCC) of 37°C in a humidified 5% CO2 environment. Medium was changed every 48 –72 h and prior to experimentation. After 2–3 weeks, medium was removed from flasks and replaced with Hanks’ calcium- and magnesium-free saline (CSL) containing 0.025% trypsin (Sigma) until cells detached from the Matrigel. The cell suspension was then centrifuged at 700g for 4 min, the pellet resuspended in fetal bovine serum (FBS), and washed by centrifugation to inactivate the trypsin. After washing, cells were resuspended in NB ⫹ B-27 and plated onto Matrigel-coated glass coverslips (Lomb Scientific, Taren Point, NSW, Australia) in 24-well plates (Becton Dickinson) at a density of 0.5–2 ⫻ 105 cells/well for immunohistochemical analysis. All cells were grown in NB ⫹ B-27 for a minimum of 7 days at SCC before experimentation. Experimental design For each experiment and control treatment group a minimum of two cultures were examined. All experiments were performed twice. Oxidative stress Cultures were passaged into 24-well plates and grown at SCC for a minimum of 72 h prior to oxidative stress experiments. Acute oxidative stress was induced by the use of 40 mM H2O2, in NB ⫹ B-27. Cell culture medium was replaced with the H2O2 medium and incubated at SCC for 90 min. For recovery experiments, the culture medium was replaced with fresh NB ⫹ B-27 alone after 90-min exposure and incubated at SCC for 4 and 48 h before cells were fixed and analysed by immunohistochemistry. Immunohistochemistry

Materials and methods Human brain tissue culture Human fetal forebrain was obtained from therapeutic terminations following fully informed consent 14 –20 weeks post menstrual in accordance with the University of Sydney

Primary antibodies were mouse anti-human ␤-tubulin III (1/2000) (Promega) to mark neurons and mouse anti-human glial fibrillary acidic protein (GFAP) (1/400) (Sigma) to mark astrocytes. ␤-Tubulin III is a component of microtubules expressed specifically in neurons that is found both within the neuron cell body and its processes (Sullivan and Cleveland, 1986). GFAP is a major subunit of astrocyte

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Fig. 1. Phase-contrast micrographs of untreated central nervous system cells (A) and cells exposed to 100 mM H2O2 for 15 min (B). After 15-min exposure, fewer fine cell processes were observed when compared to untreated cells. Other processes appeared damaged and “grainy” (arrows). Bar ⫽ 50 ␮m.

intermediate filaments that was originally isolated from glial scars and subsequently found to be astrocyte specific (Gaskin and Shelanski, 1976). Secondary antibody was fluorescein isothiocyanate (FITC)-conjugated sheep antimouse (1/100) (Amersham). All antisera were diluted in Tris-buffered saline (TBS) consisting of 50 mM Tris, 150 mM NaCl, pH 7.5. Control treatments consisted of duplicate cultures immunostained with (1) an irrelevant primary antibody for Thy1.1 (2) minus primary antibody. No staining was apparent with either irrelevant primary antibody or minus primary antibody. Cultures were fixed and permeabilised by incubation in cold (4°C) 3.5– 4% paraformaldehyde in TBS for 20 min at room temperature (RT), washed three times in TBS, then incubated in 0.2% Triton X in TBS for 10 min at RT. Cells were then washed three times in TBS and blocked in 1% bovine serum albumin (BSA)/1% FBS/TBS for 1 h at RT. After washing in TBS, cultures were incubated at RT in primary antibody for 1 h, washed again in TBS, and incubated in secondary antibody for 1 h at RT. After washing as before, duplicate coverslips were inverted on microscope slides in 3.5 ␮l of fluorescence anti-fade solution (Vectashield). Light and fluorescence microscopy Cell cultures were visualised by phase-contrast microscopy using an Olympus inverted microscope and images were collected by using a SciTech SPOT digital camera. Fluorescent microscopy was performed with a Nikon

E800 microscope at the Electron Microscope Unit, University of Sydney. Images from FITC-stained cultures were collected by a CCD (Sensicam). Confocal laser scanning microscopy was performed with a Nikon E800 with a Bio-Rad RadiancePLUS confocal unit (Electron Microscope Unit, University of Sydney). All images were collected using Z-series.

Results Previous studies of oxidative stress in the CNS have diluted H2O2 into different types of media such as DMEM or Hanks’ saline, or in buffers such as TBS, phosphatebuffered saline, or HEPES. It is well known that oxidative stress induced by H2O2 is dependent on the media type (Halliwell et al., 2000), since differing media may catalyse the formation of 䡠OH differently, and also contain differing levels of antioxidants. This is the first study to use the antioxidant-rich NB ⫹ B-27. An appropriate concentration of H2O2 to overcome the antioxidants but did not result in immediate cell death and/or detachment needed to be determined. To achieve this, replicate cultures were exposed to concentrations of H2O2 ranging from 100 ␮M to 100 mM, and assessed by phase-contrast microscopy. No morphological changes were detected until the H2O2 concentrations used were in the order of 75–100 mM (Fig. 1). After exposure to oxidative stress, there was some loss of neurite networks, while remaining neurites took on a “grainy” appearance. Although 75–100 mM H2O2 led to cell detachment after an hour of exposure, it remained possible that

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some minor cytoskeletal alterations would occur after exposure to lower concentrations of H2O2. Neurons were therefore examined by immunohistochemistry following exposure to 40 mM H2O2, a concentration at which no morphological changes could be detected by the relatively insensitive technique of phase-contrast microscopy Oxidative stress induces axonal beading in cultured neurons but not astrocytes To examine the effects of acute oxidative stress on human brain cells, cultures were exposed to 40 mM H2O2 for 60 to 90 min, fixed, and immunostained for ␤-tubulin III. ␤-Tubulin III staining, while consistent and linear in untreated neurons (2A), was found to become periodical in the processes of neurons in cultures exposed to 40 mM H2O2 for 60 min (Fig. 2B). This staining became more intense after 90-min exposure (Fig. 2C). The intense ␤-tubulin III staining observed along these neuronal processes was indicative of microtubule disruption. This periodical staining was exhibited as elongated axonal swellings in more than 60% of neurons. No morphological changes of neuronal cell bodies were observed. To examine the morphological changes occurring in neurons during recovery from acute oxidative stress, replicate cultures were exposed to 40 mM H2O2 for 90 min, and their medium replaced with fresh NB ⫹ B-27. Cultures were fixed and immunostained for ␤-tubulin III at 4 and 48 h. After 4-h recovery, there was an approximate 40% increase in the number of axonal swellings. By 48 h there was no increase in the number of swellings; however, the swellings now appeared as prominent, spherical beads almost twice the size of the original swellings. These were clearly observed along most axons (Fig. 2D). ␤-Tubulin III staining was observed to further intensify within these neuronal beads. There was no reduction in bead size during the 48-h recovery period. Throughout the stress and recovery period, no cell loss was observed. Despite extensive axonal beading, the neurons remained attached to the Matrigel substratum, indicating that 40 mM H2O2 in NB ⫹ B-27 was not cytotoxic. To examine morphological changes in astrocytes under the same stress conditions, replicate brain cultures were exposed to 40 mM H2O2 for 90 min and examined by GFAP staining. However, while the occasional detachment of a cell process from the substratum was observed, there was no evidence of cell loss or beading in human astrocytes (Fig. 3B).

Discussion As mentioned earlier, cell culture models provide invaluable information about the effects of oxidative stress on the CNS. Here, we used primary human brain tissue in culture to better understand the effects of oxidative stress of rele-

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vance to human neurodegenerative disorders, in particularly AD. To dissect cellular responses to transient increases in oxidative stress it was important in our model that the oxidative stress inflicted upon the cells did not exceed oxidative defences, or rapid apoptotic death would result, a scenario not seen in AD (Smith et al., 2000a). Previous reports have shown that H2O2 can be cytotoxic at varying concentrations. However, the level of cytotoxicity is dependent upon a number of factors, including the medium type used, incubation period, and whether the neurons are grown in coculture with astrocytes as in our study (Desagher et al., 1996). The apparently high level of H2O2 used in our study to induce morphological changes was most likely due to the presence of the antioxidants in the B-27 supplement used. These antioxidants included vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione. Current studies in our laboratory utilising the same B-27 supplement minus antioxidants, not available at the time of this study, have shown H2O2 is cytotoxic at 100 ␮M to 1 mM concentrations after 30-min exposure (unpublished data). This confirms that the antioxidants in the B-27 supplement are the most probable factors responsible for the high H2O2 concentrations required to cause oxidative stress. Further support is provided by a recent study that showed B-27-supplemented DMEM is as effective as serum-supplemented medium in modifying the effect of H2O2 on murine cortical neurons (Ricart and Fiszman, 2001). Another study has also demonstrated that only nanomolar concentrations of vitamin E are required to exert a neuroprotective effect against H2O2 (Behl, 2000). Thus, to disrupt the prooxidant:antioxidant balance and induce oxidative stress, we found that 40 mM H2O2 was required, a concentration larger than previously reported for other media types. As the brain cells remained attached to the coverslips and did not show signs of blebbing, typically associated with apoptosis (Bellomo and Mirabelli 1992), there was no evidence that 40 mM H2O2 in NB ⫹ B-27 was not cytotoxic after 90-min exposure. While this is the first report of oxidative stress causing neuritic beading in human CNS neurons, previous studies have demonstrated such beading by fluid-percussion injury (Povlishock and Kontos, 1992), oxygen-glucose deprivation and N-Methyl-D-Aspartate receptor overactivation (Park et al., 1996), and administration of the neurotoxin p-bromophenylacetylurea (Jortner et al., 1997). Thus, axonal beading is a morphological change indicative of neuronal injury. The mechanism by which hydrogen peroxide induces such beading remains unknown, but a possible explanation is disruption of the neuronal cytoskeleton, particularly the microtubules. Park et al. (1996) proposed that depolymerisation of microtubules, resulting in an inability of neurons to maintain cell morphology, may explain the beading observed during oxygen-glucose deprivation. However, alterations to microtubules also cause disruption of axonal trans-

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Fig. 2. Representative photomicrographs of ␤-tubulin III-positive neurons in untreated central nervous system cultures (A), cultures exposed to 40 mM H2O2 for 60 min (B) and 90 min (C), and after 48-h recovery (D) from 40 mM H2O2 exposure for 90 min. Axonal swelling (arrows) was observed after exposure for 60 min (B), and became more prominent after 90 min (C). This developed into beading of cell processes during the recovery period (arrowheads), with intense ␤-tubulin III staining within each bead. Bar ⫽ 10 ␮m.

port. Such transport has long been known to be dependent on microtubules and neuronal specific intermediate filaments of the cytoskeleton for transport of organelles— particularly mitochondria, the major suppliers of energy in neurons— by kinesin and dynein motor proteins (Vale et al., 1985; Hirokawa, 1993). Disruption of microtubules will inhibit or halt this transport. Since axons have a high rate of

intracellular trafficking, a disturbance of this transport system by microtubule disruption would result in an accumulation of axoplasmic constituents delivered by fast and slow axoplasmic transport, as thought to occur after diffuse axonal injury (Wilkinson et al., 1999), and thus lead to the neuronal swelling and beading observed in our experiments. Evidence for microtubule disruption in our study is seen

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Fig. 3. Representative confocal photomicrographs of glial fibrillary acidic protein-positive astrocytes in untreated central nervous system cultures (A) and cultures exposed to 40 mM H2O2 for 90 min (B). No beading or cell detachment was observed, although the occasional astrocytic process detached from the substratum (arrow) was observed. Bar ⫽ 10 ␮m.

by the intense accumulation of ␤-tubulin III within the beads (Fig. 2D). Since ␤-tubulin III is a neuronal specific component of the microtubule tracks along which kinesin and dynein travel (Goldstein and Yang, 2000), the intensity of this staining is consistent with fragmentation of the cytoskeleton and disruption of the microtubule array, either directly or indirectly. This is not the first study, however, to implicate the involvement of oxidative stress in the alteration of the axonal cytoskeleton: Neely et al. (1999) demonstrated that the lipid peroxidation product 4-hydroxy-2(E)-nonenal disrupts neuronal microtubules, while Eiserich et al. (1999) found that the oxidative product nitrotyrosine also causes microtubule disassembly. These by-products of oxidative reactions, however, are unlikely to be the only mechanism by which oxidative stress disrupts microtubules, as the process almost undoubtedly involves multiple pathways. Indeed, Ishihara et al. (2000) have recently suggested that the process may also occur as a result of elevated intracellular calcium. A further pathway by which oxidative stress may disrupt microtubule organisation is by phosphorylation of microtubule-associated proteins (MAPs), proteins that bind to and stabilise microtubules against disassembly (Zhu et al., 2001). For example, MAP binding is known to be promoted by dephosphorylation, while phosphorylation allows destabilisation of microtubule arrays (Burns et al., 1984). A scenario in which oxidative stress disturbs MAP stabilisation of microtubules would be consistent with studies of mammalian cells that have been shown to respond to oxi-

dative stress by activating signalling cascades such as c-Jun N-terminal kinase, enzymes that phosphorylate the neuronspecific MAP tau in vitro (Zhu et al., 2001). Or, alternatively, oxidative stress may disrupt the MAP:tubulin ratio somehow, and thus result in disruption of neuronal intracellular transport as observed by Stamer et al. (2002). Of particular interest is that similar morphological changes to those observed in our study have been observed in AD brain: Perry et al. (1991) observed periodic constrictions in some neuropil threads, similar in appearance to beads, that are a characteristic of AD pathology (Braak et al., 1986); Praprotnik et al. (1996) observed swellings in dystrophic neurites of senile plaques were similar to the axonal swellings of experimental traumatic injury, while Velasco et al. (1998) have suggested that the striations in neuropil threads of AD may arise from mechanisms similar to those responsible for beading. This raises the question of whether axonal transport disruption and beading occur in vivo as a result of repeated episodes of oxidative stress and decreasing ability for repair in aging brain. This could be particularly relevant in pathological conditions such as AD. Indeed, recent studies have implicated a role for disrupted axonal transport in AD pathology. Hirai et al. (2001) found an accumulation of mitochondrial DNA in the neurons vulnerable to death in AD and attributed it to a possible reduction in the number of microtubules, which would result in diminished vesicular transport and mitochondrial turnover (Hirai et al., 2001; Perry et al., 2002) In AD again, it has long been known that there are characteristic cytoskeletal alterations in neurons (Brion,

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1992). They have often been stated to be a consequence of ␤-amyloid deposition or neurofibrillary tangle formation. Recent studies, however, have implicated that many of the morphological alterations in AD neurons may in fact be a result of oxidative stress (Praprotnik et al., 1996; Good et al., 1996). Indeed, the observation that cytoskeletal changes tend to occur in AD neurons in the absence of amyloid deposits favours the notion that the pathological process is initiated by intrinsic events, rather than by influences from the surrounding tissue (Braak et al., 1994). Although axonal swelling and beading was observed in over 60% of neurons, some neurons remained unaffected by the H2O2. As the CNS cell cultures used in this study were obtained from trituration of primary human fetal forebrain, there would have been heterogeneous populations of neurons in the cultured tissue. The observed ability of some neurons to withstand oxidative stress while others appeared more affected could be attributed to such regional differences. This could be of particular relevance to AD, where the hippocampus and amygdala are more severely affected than other regions, especially in late-onset AD (Braak and Braak, 1991). This is again relevant to other human neurodegenerative disorders. This study shows further evidence that while astrocytes remained apparently unaffected, oxidative stress disrupts neuronal microtubules and results in disruption of axonal transport.

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