Selective loss of neurofilament proteins after exposure of differentiated human IMR-32 neuroblastoma cells to oxidative stress

BRAIN RESEARCH Brain Research 738 (1996) 162-166

Short communication

Selective loss of neurofilament proteins after exposure of differentiated human IMR-32 neuroblastoma cells to oxidative stress M.R. Cookson “b’*, N.M. Thatcher a, P.G. Ince “C,P.J. Shaw b aMRC Neut-ochemicalPathology Unit, Newcastle General Hospital, Newcastle upon Tyne, NE46BE,

UK

b Department of Neurology, University of Newcastle, Newcastle upon Tyne, UK c Department of Neuropathology, University of Newcastle, Newcastle upon Tyne, UK Accepted 13 August 1996

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Millimolarconcentrationsof ascorbatein the presenceof iron can causeneuronalcell death.This studyshowsthat the humanneuronal cell line IMR-32is sensitiveto ascorbateand that cytotoxicitycan be blockedby the antioxidantenzymesCu/Zn-superoxidedismutase and catalase.There was a selectiveloss of neurofilamentproteinsafterexposureto 5 or 10mM ascorbate,as assessedby immunostaining and by Westernblotting.Loss of actin or tubulinwas not seen, suggestingthat loss of neurofilamentsis a sensitiveand selectivemarker for free radical damagein these cells. Keywords: IMR-32; Cell culture; Neurotlament; SM131; Oxidative stress; Ascorbate

Free radical mediated damage to neurones has been implicated to be involved in the pathophysiology of a number of neurodegenerative diseases [2,5,11,14]. For example, in motor neurone disease (MND) indices of oxidative damage to proteins are increased in the spinal cord and frontal cortex compared to controls [4,12]. However, the precise protein targets of oxidative damage in the CNS are at present uncertain. Neurofilaments (NFs), the intermediate filaments expressed exclusively in neurones, might represent an important potential target for free radical damage because of their abundance in motor neurones and their long biological half-life. Furthermore, the high Iysine content of NFs may increase their potential for oxidative modifications [14]. In some neurodegenerative disorders, including MND and Parkinson’s disease, there is evidence that cytoskeletal abnormalities and/or dysfunction in neurofilament metabolism may occur [6,7,9,15]. It has been reported that exposure to high concentrations of ascorbate induces free radical mediated cell death in primary cultured neurones [8] and in other cells [3]. Iron/ascorbate exposure in chick retinal cells has been shown to affect NMDA-evoked GABA release and lipid peroxidation [1]. Although it has antioxidant properties,

* Corresponding author. Fax: +44 (191) 2725291.

ascorbate may be pro-oxidative in the presence of metal ions, especially iron and possibly copper, because of its ability to reduce them and to participate in free radical generation by the Haber-Weiss cycle ([3] and references therein). Several oxygen free radical species may be produced via a series of reactions, including H20Z, OH “ and O;. . This iron/ascorbate system has also been used to induce oxidative damage, with formation of carbonyl groups, to purified neurofilaments in vitro [17]. To investigate the effects of free radicals on NFs in whole cells in culture, we have used a human neuroblastoma cell line, IMR-32 [16]. These cells have several useful properties which were exploited: they are of human origin, they can be grown in large quantities for protein studies, differentiated and maintained over several weeks [10] and, as illustrated below, express large amounts of NFs. The aims of this work were: (i) to develop an experimental model of oxidative damage to NF proteins within whole cells; (ii) to investigate the relative sensitivity of NF proteins as a measure of free radical damage; and (iii) to identify any changes in the cytoskeletal organisation of NFs and cell morphology in the presence of oxidative stress. IMR-32 cells were plated at a density of approximately 2–3 X 104 cells per cmz into either 8 well chamber slides (Nunc) for staining or T75 flasks (Falcon) for protein

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MR. Cookson et aL/Brain Research 738 (1996) 162-166

extractions. The cells were differentiated for 7–10 days in serum supplemented media [10] containing 1 mM dibutyryl cyclic AMP and 3.75 PM bromodeoxyuridine, which induces a neuronal phenotype as described previously [10]. Exposure to ascorbate was performed in media without serum for 24 h. Ascorbate was added to the media to a final concentration of 0.5 to 10 mM, and neutralised to pH 7.4 where required. The media contained 5 KM iron as FeCl~ in all cases. Cell viability was assessed by staining for 10 min at 37°C using fluorescein diacetate (FDA) and propidium iodide (PI), both at a final concentration of 10 KM in phosphate buffered saline (PBS), pH 7.4 [13]. In some experiments, antioxidant enzymes were added concomitantly with ascorbate to prevent cell death. Copper/zinc superoxide dismutase (Cu/Zn-SOD; Sigma) was used at a concentration of 50 ~g ml–1 (300 U ml–1 ). Catalase was a human recombinant enzyme (Calbiochem) and was used at a concentration of 100 ~g ml-’ (5000 U ml-l). Cytochrome C (Sigma) was used at a final concentration of 0.1 mM. Neurofilament organisation within the cells was assessed by staining with the monoclinal antibody SM131 (Sternberger Monoclonals) which recognises phosphorylated NFs, particularly the 200-kDa heavy neurofilarrtent protein, NF-H. Cells were exposed to ascorbate, washed in cold PBS then fixed in 490 paraformaldehyde in PBS for 1 h at 4°C. They were then permeabilised with PBS plus 10% goat serum and O.lYOTriton X 100 then stained with SM131, diluted 1:1000 in the same buffer, followed by goat anti-mouse IgG conjugated to fluorescein (1:150). To estimate the total amount of NF proteins within the cell, extracts were prepared by sonication in 50 mM Tris buffer, pH 7.4, in the presence of the protease inhibitors phenylmethylsulfonylfluoride, benzamidine and leupeptin, and the protein content measured using a Coomassie blue assay against BSA as a standard. Samples were diluted to known concentrations and 10 ~g aliquots of total protein were separated using SDS-PAGE(7.5Y0 acrylamide gels for neurofilaments, 107ogels for actin and tubulin) and blotted to Immobilon PVDF membranes. Neurofilament proteins were visualized by incubation with a 1:1000 dilution of either SM131 or SM132 (which recognises non-phosphorylated NFs) in PBS plus 0.0590 Tween-20 and 5% skimmed milk, followed by peroxidase labelled secondary antibody (1:1000) and the blots were then developed using enhanced cherniluminescence substrate and ECL-max film, according to the manufacturer’s instructions (Amersham). The neurofilament band at 200 kDa (heavy neurofilament, NF-H) was quantified using a Lynx densitometer (Applied Imaging). Changes in actin and tubulin were evaluated by substituting monoclinal antibodies to these proteins (Sigma) in the above method at a dilution of 1:500. Ascorbate caused cell death in a dose-dependent manner, at concentrations of 1–10 mM, which could be prevented by adding antioxidant enzymes or cytochrome C

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(Fig. 1). Ascorbate reduced the percentage of live cells counted over 8 fields in duplicate cultures from 9370 in control cells to 78Y0at 2.5 mM and 6670 at 5 rnM. The changes in the proportion of live cells were highly significant by chi-square analysis (P < 0.001). The total number of live cells fell from an average of 155 i- 2.8 in control cultures to 62.3 * 3.4 after treatment with 5 mM ascorbate (P= 0.006 by t-tests). Catalase, Cu/Zn-SOD and cytochrome C were all effective in protecting the cells against ascorbate-induced damage as described for pancreatic cells [3], thus implicating free radicals in the toxicity associated with ascorbate (Fig. 1). Untreated, differentiated IMR-32 cells extend long cellular processes as previously described [10], which were full of SM131-positive (i.e., phosphorylated) filaments as shown in Fig. 2. An antibody against non-phosphorylated neurofilament proteins (SM132) labelled filaments around the nucleus rather than in the cell processes (data not shown). After exposure to 5 mM ascorbate, these cellular processes were reduced in diameter and developed multiple bleb-like varicosities along them, evident in unstained cells (Fig. 2b). Staining for SM131 revealed abnormalities of NF organisation within the varicosities and reduced staining in the remainder of the cell processes (Fig. 2). This suggested that there may be changes in the net amount of NFs present within these cells. Total protein 250

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Fig. 1. Ascorbate toxicity and protection by antioxidant enzymes. IMR-32 cells were exposed to 2.5 or 5 mM ascorbate (Asc) for 24 h in serum free media. Cell viability was assessed by counting fluorescein diacetate (FDA) positive (live) and total cells in four fields in each of two replicate cultures (n = 8). Antioxidant enzymes were added in some experiments simultaneously with ascorbate, either Cu/Zn superoxide dismutase (SOD), catalase or cytochrome C (Cyt-C). Each bar shows the mean number of cells counted per field under the conditions described, error bars show the S.E.M. Shaded portion of each bar shows the number of live cells per field. Changes in the number of live cells were assessed by ** P <0.01 versus untreated cultures: a, two tailed t-tests: “, P < 0 P <0.001 versus an equivalent concentration of ascorbate for antioxidant treatments. These results are representative of duplicate experiments.

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Fig. 2. Morphological effects of free radical exposure. IMR-32 cells were grown and differentiated as described in the text and exposed to 10 mM ascorbate for 24 h (B, D) or left untreated (A, C). Unstained, living cells (A, B) have long cellular processes which are evenly thick along their length in control cells (A) but are reduced in diameter and contain many varicosities in ascorbate treated cells (B, arrows). SM131 staining (C, D) reveals that the distribution of NFs within the cell is also affected: the catibre of most of the processes is markedly reduced with small varicosities along them (arrows). The nuclear staining with SM131 is likely to be due to cross-reactivity of this antibody with phosphorylated nuclear proteins, such as histones. Scale bars in a and c = 50 mm for each pair of photographs,

recovered from triplicate flasks of cells was reduced in a dose-dependent manner (Fig. 3) to 72% of control at 5 mM ascorbate (P = 0.04 by ANOVA). However, quantification of NF-H immunoreactivity in the same samples (Fig. 3), when corrected for this reduction in total protein, showed of . control 5values using 7 that NF-H was reduced to 4 SM131 (P= 0.004) and to 58% of control using SM132 (P= 0.03). That these two antibodies gave similar results suggests that these changes are not likely to be due to alterations in the phosphorylation status of NFs, but more probably to changes in the net amount of NFs within the cells. To confirm that changes in NFs are a specific alteration in these cells, the same samples were probed for actin and tubulin. No loss of immunoreactivity for actin was seen

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after exposure to 5 or 10 mM ascorbate, where the amount of SM131 reactivity was greatly reduced (Fig. 3). Similarly, tubulin immunoreactivity was seen to be maintained in samples from cells exposed to 5 or 10 mM ascorbate. Therefore, loss of NF protein is a specific marker for free radical induced damage in this cell line. The differences between the effects of free radicals on the different types of cytoskeletal proteins is interesting. Possible interpretation of this observation include: (i) of the three major cytoskeletal systems, intermediate filaments are most readily damaged by free radicals; or (ii) some inherent characteristic(s) of NFs makes them particularly sensitive to this type of damage. As discussed in the introduction, NFs may have a high sensitivity to free radical damage because of their long half-life and high lysine content [14]. It would

M.R. Cookson et al./Brain Research 738 (1996) 162-166

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IAscorbate] (mM) Fig. 3. Effects of free radical exposure on total protein content and specific cvtoskeletal proteins. (A) Western blots of protein extracts from IMR32 cells were probed with antibodies against phosphorylated neurofilaments (SM131) actin or tubulin as indicated. Samples (from left to right as indicated) were from control cells or from cells exposed to 2, 5 or 10 mM ascorbate for 24 h. The samples used for each of the blots were from 10 p,g aliquots from the same extracts from one experiment. Blots are representative of triplicate samples, and of two experiments. Indicated molecular weights were calculated by comparison with biotinylated molecular weight standards mn on the same gels. (B) Total protein (circles) recovered from flasks of cells was quantified at different concentrations of ascorbate (24 h exposure) and was reduced in a dose dependent mmner. Changes in the 200 ma NF-H were qu~tified on a Per mg protein basis usirw either SM131 (open or SM132 (closed squares) monoelonal antibodies. These two antibodies gave different levels of . squares) . immunoreactivity and so are expressed as percentages of untreated controls. ‘, P < 0.05: * *, P <0.01 by ANOVA, n = 3 in alicases.

be of interest in this context to measure the sensitivity of purified NFs and other intermediate filament proteins, actin and tubulin to free radical damage in a cell-free system, to evaluate whether the proteins themselves are differentially sensitive. Data on the ability of ascorbate in the presence of iron to oxidise NF proteins has already been reported [17]. The 10SSof NF immunoreactivity in our cell culture system may reflect either an increased metabolism of these proteins under oxidative stress or a down-regulation of transcription or translation of the NF gene. Decreased NF mRNA expression has been described in Parkinson’s disease [6] and MND [7], where oxidative stress has been implicated. If NFs are a specific target for free radical damage, then cells which express large amounts of these proteins, such as motor neurones, may be differentially vulnerable to the cumulative effects of oxidative stress. In summary, we have shown that: (i) ascorbate in the presence of iron causes cell death in human neuroblastoma cells; (ii) cell death is mediated by a free radical-mediated mechanism; and (iii) that NF proteinsare sensitive targets for oxidative injury in these cells, as indicated by changes in both the total amount of immunoreactive NFs in the cell and their organisation into intermediate filaments. This system could be used to generate free radicals within cell types that are vulnerable to degenerative pathology, to

further examine the biochemical changes associated with exposure to free radicals.

A The authors would like to thank Mr. David Hughes and Ms. Debbie Jones for their assistance with the IMR-32 cell cultures and Mr. Arthur Oakley for his photographic assistance. P.J.S. is supported by the Wellcome Trust as a Senior Fellow in Clinical Science.

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