Inhibition of superoxide dismutase selectively suppresses growth of rat spinal motor neurons: Comparison with phosphorylated neurofilament-containing spinal neurons

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Inhibition of superoxide dismutase selectively suppresses growth of rat spinal motor neurons: Comparison with phosphorylated neurofilament-containing spinal neurons Risa Isonaka⁎, Hiromi Hiruma, Takashi Katakura, Tadashi Kawakami Department of Physiology, Kitasato University School of Medicine 1-15-1, Kitasato, Minami-ku, Sagamihara, 252-0374, Japan

A R T I C LE I N FO

AB S T R A C T

Article history:

Amyotrophic lateral sclerosis (ALS) is characterized by selective degeneration of motor

Accepted 22 September 2011

neurons. The reason why only motor neurons are targeted is unknown. Since ALS has

Available online 29 September 2011

been linked to mutations in Cu/Zn superoxide dismutase (SOD1), oxidative stress is regarded as a major cause of ALS. We hypothesized that motor neurons are more

Keywords:

susceptible to oxidative stress than other neurons. To test our hypothesis, we

Non-phosphorylated neurofilament

investigated differences in neurite growth between motor and non-motor neurons under

Phosphorylated neurofilament

SOD1 inhibition. Spinal motor neurons were identified by immunocytochemistry using

Cu/Zn superoxide dismutase

anti-non-phosphorylated neurofilament (NF) antibody (SMI-32). Other neurons immunore-

Spinal neuron

active to an antibody against phosphorylated NF (SMI-31) were used as a control. Cultured rat spinal neurons were treated with the SOD1 inhibitor diethyldithiocarbamate (DDC). SMI-32-immunoreactive neurons were more sensitive to the growth inhibitory effects of DDC than SMI-31-immunoreactive neurons. Such inhibition was blocked by the antioxidants, L-ascorbic acid, L-histidine, astaxanthin, α-tocopherol, and β-carotene. The results suggested that spinal motor neurons are more vulnerable to oxidative stress than other neurons, which may explain in part the selective degeneration of motor neurons in ALS. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with devastating symptoms. Patients with ALS gradually lose the ability to control their muscles, although consciousness remains unaffected due to the selective nature of motor neuron degeneration. Why only motor neurons are targeted is mysterious, and until this question is answered, the pathogenetic mechanism of ALS remains unclear.

Mutations of the gene encoding Cu/Zn superoxide dismutase (SOD1), which protects cells against free radicals, have been identified in familial ALS patients (Rosen et al., 1993), implicating reduced SOD1 activity in SOD1-associated familial ALS (Bowling et al., 1995). Using biochemical analysis, Ferrante et al. (1997) showed increased oxidative damage in familial and sporadic ALS. Oxidative stress is the presumptive major cause in ALS (Barber and Shaw, 2010; Barber et al., 2006). However, oxidative stress should affect not only motor neurons, but also

⁎ Corresponding author. Fax: + 81 42 778 9841. E-mail address: [email protected] (R. Isonaka). Abbreviations: ALS, Amyotrophic lateral sclerosis; SOD1, Cu/Zn superoxide dismutase; NF, neurofilament; DDC, diethyldithiocarbamate; PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.09.046

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other neurons and cells. Motor neurons in the spinal cord are rich in non-phosphorylated neurofilament (NF) (Tsang et al., 2000) and antibodies to such NFs can be used to identify spinal motor neurons (Carriedo et al., 1995, 1996; Gotow and Tanaka, 1994). Mutant SOD1 transgenic mice (Morrison et al., 1996) and ALS patients (Tsang et al., 2000) show low immunoreactivity for non-phosphorylated NF in the ventral horn of the spinal cord (due to a decline in the number of neurons) compared with the normal control. Thus, non-phosphorylated NF could be used as a marker for the vulnerable population of neurons, since the percentage of these immunoreactive neurons is significantly lower than Nissl-stained cells (Morrison et al., 1996). We hypothesized that spinal motor neurons containing non-phosphorylated NF are more vulnerable to oxidative stress than neurons containing phosphorylated NF. To test this hypothesis, SOD1 activity was inhibited using diethyldithiocarbamate (DDC) and neurite growth was compared between phosphorylated NF- and non-phosphorylated NFcontaining spinal neurons.

not significantly different from those measured in the absence of antioxidant in both the SMI-31 (+) and SMI-32 (+) neurons (Figs. 3A–E, gray bar). Neurons pretreated for 24 h with an antioxidant alone and subsequently treated for 72 h with an antioxidant plus DDC (100 nM) had significantly longer neurite lengths in SMI-31 (+) and SMI-32 (+) neurons treated with L-ascorbic acid, L-histidine, astaxanthin, or α-tocopherol, compared to the mean length of SMI-31 (+) and SMI-32 (+) neurons treated with DDC alone (Figs. 3A–D, diagonal lines gray bar). In contrast, the effect of β-carotene was limited to SMI32 (+) neurons (Fig. 3E, diagonal line gray bar). As shown in Fig. 1E-I, treatment with a combination of an antioxidant plus DDC protected the neurite against DDC-induced damage in SMI-32 (+) neurons (Fig. 1A). These results indicate that antioxidants alone had no effects on neurite growth, whereas they protect against the growth inhibitory effects of DDC in both SMI-31 (+) and SMI-32 (+) neurons.

3. 2.

Results

2.1. Effects of DDC on neurite growth of SMI-31 (+) and SMI-32 (+) neurons As shown in Figs. 1A–D, both SMI-31 (+) and SMI-32 (+) neurons (phosphorylated NF- and non-phosphorylated NFcontaining neurons) were shorter after 72-h treatment with DDC, compared with the control. Incubation with 100 nM DDC induced damage of neurite of SMI-32 (+) neurons (Fig. 1A). The same was true for incubation with 1000 nM DDC for both SMI-31 (+) and SMI-32 (+) neurons. However, the cell bodies of neurons treated with DDC at any concentration were not morphologically different from those of the control neurons. Quantitative analysis confirmed that neurite length under either 100 nM or 1000 nM DDC was significantly less than the respective control in SMI-31 (+) neurons (Fig. 2A). Similarly, neurite length in SMI-32 (+) neurons was less than the control during culture with DDC at all concentrations (Fig. 2B). These results suggest that SOD1 inhibition suppresses neurite growth of both the SMI-31 (+) and SMI-32 (+) neurons. Comparison of the response to DDC between SMI-31 (+) and SMI-32 (+) neurons showed significantly shorter normalized neurite lengths of DDC-treated SMI-32 (+) neurons compared with DDC-treated SMI-31 (+) neurons at all concentrations (Fig. 2C). These results indicate a higher sensitivity of SMI-32 (+) neurons to SOD1 inhibition than SMI-31 (+) neurons, resulting in remarkable inhibition of neurite growth.

2.2. Effects of antioxidants on DDC-induced suppression of neurite growth Next, we investigated the effects of antioxidants on the DDCinduced neurite growth inhibition. In the absence of DDC (0 nM DDC), the neurite lengths after treatment for 96 h (24-h pretreatment plus 72-h treatment period) with each antioxidant (L-ascorbic acid [1 mM], L-histidine [1 mM], astaxanthin [100 nM], α-tocopherol [1 mM], or β-carotene [1 mM]) were

Discussion

In the present study, we examined neurite growth in phosphorylated NF (SMI-31)-immunoreactive and non-phosphorylated NF (SMI-32)-immunoreactive spinal neurons after SOD1 inhibition. DDC inhibited neurite growth of both phosphorylated NFand non-phosphorylated NF-containing neurons. In general, SOD1 plays a vital role in defending cells against damage caused by accumulation of superoxide anion radicals (O2−), which are produced during normal cellular metabolic processes (Fridovich, 1975). Excess free radicals may therefore lead to oxidative stress and cell damage. Our findings indicate that endogenous oxidative stress increased with DDC treatment and that it inhibited neurite growth. A previous study also highlighted the important role of SOD1 in neurons particularly with chronic inhibition of SOD1, causing apoptotic degeneration of cultured rat spinal neurons (Rothstein et al., 1994). The main finding of the present study was that DDC treatment inhibited neurite growth of non-phosphorylated NFpositive neurons more profoundly than phosphorylated NFpositive neurons. These results indicate higher susceptibility of the neurites of non-phosphorylated NF-positive neurons, relative to that of phosphorylated NF, to morphological damage induced by SOD1 inhibition, suggesting that endogenous oxidative stress is more likely to affect non-phosphorylated NFpositive neurons than phosphorylated NF-positive neurons. Thangavel et al. (2009) suggested that non-phosphorylated NFs in temporal cortical regions were vulnerable to early degeneration in Alzheimer's disease, which may be caused at least in part by oxidative stress (Markesbery and Carney, 1999; Smith et al., 2000). The above report indicates degeneration of nonphosphorylated NF during the pathological process, lending support to our finding of the high vulnerability of nonphosphorylated NF-positive neurons to oxidative stress than phosphorylated NF-positive neurons. Oxidative damage plays important roles in ALS (Barber and Shaw, 2010) and in the pathogenesis of neuronal degeneration in spinal cord (Xu et al., 2009). Although the exact mechanism for the vulnerability of motor neurons to oxidative stress-related damaged is not fully known, several scenarios have been proposed. Since motor neurons are large

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Fig. 1 – Photomicrographs of cultured rat spinal neurons. Non-phosphorylated neurofilaments were stained with an anti-SMI-32 antibody (A, B, E-I), and phosphorylated neurofilaments were stained with an anti-SMI-31 antibody (C, D). SMI-32 (+) (A, B) and SMI-31 (+) (C, D) spinal neurons with (A, D) or without (B, C) the SOD1 inhibitor DDC at 100 nM for 72 h. (E-I) SMI-32 (+) neurons with an antioxidant plus 100 nM DDC treatment for 72 h. (E) His, L-histidine; (F) AA, L-ascorbic acid; (G) AX, astaxanthin; (H) α-Toc, α-tocopherol; and (I) β-Car, β-carotene. Scale bar, 100 μm.

cells with long axons, they have high energy demands from mitochondria. Mitochondrial respiration is therefore more active in motor neurons than in other neurons, and hence excessive endogenous oxidative stress in motor neurons (Shaw and Eggett, 2000). In this regard, changes in mitochondria have been described in motor neurons of mutant SOD1 mice and guinea pig as a model for ALS (Kong and Xu, 1998; Xu et

al., 2009), and they are likely to be the trigger and the target in motor neuron death (Duffy et al., 2011; Dupuis et al., 2004). These findings suggest that motor neurons are easily affected by SOD1 mutation-induced oxidative stress. The results of the present study indicate that the vulnerability to SOD1 inhibition of non-phosphorylated NF-positive spinal neurons, presumed to be motor neurons, is a possible

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Fig. 2 – Effects of SOD1 inhibition on neurite growth of phosphorylated neurofilament (SMI-31)- and non-phosphorylated neurofilament (SMI-32)-immunoreactive spinal neurons. (A, B) The mean length of neurites of SMI-31 (+) and SMI-32 (+) neurons after treatment for 72 h with DDC at 0 nM (control), 1 nM, 10 nM, 100 nM and 1000 nM. (C) Comparison of the normalized lengths of neurites of SMI-31 (+) and SMI-32 (+) neurons. The length of neurites of DDC-treated neurons measured at 72 h was normalized to that of control neurons. Data are mean ± SEM of >60 independent samples in each group. *p < 0.05, **p < 0.005, and ***p < 0.0005 (vs. control), Steel test. #p < 0.05, ##p < 0.005, and ###p < 0.0005 (vs. SMI-31 (+) neurons), Mann–Whitney U test.

mechanism for the selective degeneration of spinal motor neurons in ALS. Finally, we examined whether antioxidants could rescue neurons from the neurite growth inhibition induced by inhibition of SOD1 activity. The reduced neurite growth was rescued by all antioxidants tested in this study, L-ascorbic acid, Lhistidine, astaxanthin, α-tocopherol, and β-carotene in nonphosphorylated NF-positive neurons. Thus, these antioxidants may be effective in preventing damage of non-phosphorylated NF-containing motor neurons exposed to oxidative stress. Several reports have described the beneficial effects of antioxidants in familial ALS-linked mutated SOD1 transgenic mice. Mice treated with a high dose of ascorbate before the onset of ALS survived significantly longer than untreated control mice (Nagano et al., 2003), and α-tocopherol significantly delayed the onset of clinical disease in familial ALS mice (Gurney et al., 1996). On the other hand, the direct effect of histidine, astaxanthin, and β-carotene on ALS model mice has rarely been investigated. However, it is reported that astaxanthin could be useful in the prevention and treatment of manganese SOD-deficient mice, a model of Parkinson's disease (Tsuji et al., 2007), which may be due to oxidative stress (Jenner, 2003; Surendran and Rajasankar, 2010). Although treatment of ALS patients with antioxidants has been reported in numerous clinical studies and the therapies are still

ongoing, the therapeutic value of these compounds remains unclear (Orrell et al., 2008). The present study thus provides an evidence base for the relevant clinical trials. In conclusion, the present study indicated that neurite growth of non-phosphorylated NF-positive spinal neurons, presumed to be of the motor type, is more sensitive to the inhibitory effects of SOD1 inhibition than phosphorylated NFpositive spinal neurons. Antioxidants blocked the SOD1 inhibitor-induced suppression of neurite growth. These results may explain the selective degeneration of spinal motor neurons in response to oxidative stress produced by SOD1 inhibition in ALS. Based on these findings, antioxidants might be of help in rescuing motor neuron injury caused by oxidative stress, though confirmation of this action requires further studies.

4.

Experimental procedures

4.1.

Cell culture

The experimental protocols were approved by the Animal Experimentation and Ethics Committee of Kitasato University School of Medicine, which conformed to the National Institutes

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Fig. 3 – Effects of antioxidants on neurite growth of phosphorylated neurofilament (SMI-31)- and non-phosphorylated neurofilament (SMI-32)-positive spinal neurons. The neurons were cultured with 1 mM L-ascorbic acid (AA) (A), 1 mM L-histidine (His) (B), 100 nM astaxanthin (AX) (C), 1 mM α-tocopherol (α-Toc) (D), or 1 mM β-carotene (β-Car) (E) for 24 h and then treated with a combination of DDC at 100 nM and each antioxidant for 72 h. Data are mean length (A–E) of neurites of SMI-31 (+) and SMI-32 (+) neurons after treatment with or without 100 nM DDC and/or antioxidants. The ordinate represents the length of neurites (μm). Data are mean ± SEM of >60 independent samples in each group. *p < 0.05, **p < 0.005, and ***p < 0.0005 (vs. non-antioxidant-treated neurons), Steel test.

of Health guidelines on the ethical use of animals. On day 20 of pregnancy (~1 day before birth), Wistar rats (Oriental Yeast Co, Tokyo, Japan) were anesthetized with pentobarbital sodium (50 mg/kg) and embryos were removed. The embryonic spinal cords were dissected out and immediately immersed in icecold Leibovitz's L-15 medium (Life Technologies, Carlsbad, CA) containing 0.5 mM L-glutamine (Wako, Osaka, Japan). These tissues were then incubated for 15 min at 37 °C in Ca2+, Mg2+-free phosphate buffered saline (PBS) (Life Technologies) containing 0.2 mg/ml DL-cysteine hydrochloride (Sigma-Aldrich, St. Louis, MO), 0.2 mg/ml bovine serum albumin (Sigma-Aldrich), 5 mg/ ml D-(+)-glucose (Wako), 0.55 mg/ml papain (Worthington Biochemical Corporation, Freehold, NJ), and 0.01% DNase (Takara Bio Inc, Shiga, Japan). Fetal calf serum (MP Biomedicals, Morgan, Irvine, CA) was added (30% v/v of PBS) at the end of the incubation to inhibit enzyme activity. Cells were then dissociated by trituration using fire-polished pipettes (0.5-mm inner diameter). Subsequently, the isolated cells were plated onto poly-Llysine-coated glass coverslips (30 × 40 mm, 50 μm-thickness) and cultured in a medium comprising neurobasal medium (Life Technologies), 0.5 mM L-glutamine, and 2% B-27 supplement (Life Technologies) at 37 °C in a humidified 5% CO2 atmosphere.

4.2.

Solutions and drugs

The SOD1 inhibitor sodium N, N-diethyldithiocarbamate trihydrate (DDC, Wako) was initially dissolved in water at a high concentration (1 mM), and thereafter diluted with culture medium at concentrations of 1 nM, 10 nM, 100 nM and 1000 nM. L-ascorbic acid, (±)-α-tocopherol nicotinate (both from Sigma-Aldrich), L-histidine, astaxanthin, and β-carotene (both from Wako) were used as antioxidants. L-ascorbic acid and Lhistidine were initially dissolved in water, while astaxanthin, α-tocopherol, and β-carotene were further dissolved in dimethyl sulfoxide (DMSO, Wako). All antioxidant solutions were diluted with culture medium just prior to the experiments. The final concentration of DMSO was 0.01%, which is reported to have no effect on neurite growth (Hiruma et al., 2003; Tolkovsky et al., 1990). The final concentrations were 1 mM for L-ascorbic acid, 1 mM for L-histidine, 100 nM for astaxanthin, 1 mM for αtocopherol, and 1 mM for β-carotene. The pH of all solutions was 7.3–7.4. The concentration selected for each antioxidant was deemed the minimum required to achieve the maximum effect on DDC-induced inhibition of neurite growth, as determined in preliminary experiments. In these experiments, the concentrations used were 1000 nM to 3 mM (L-ascorbic

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acid, L-histidine, α-tocopherol, and β-carotene) and 10 nM to 1000 nM (astaxanthin). The experiments served to determine the effect of each concentration in >30 independent samples on neurite length in SMI-31 (+) and SMI-32 (+) neurons, and the effects were compared (data not shown).

groups were tested by the Mann–Whitney U test. A p value less than 0.05 denoted the presence of a statistically significant difference.

Acknowledgments 4.3.

Drug treatment

The cells were incubated in the culture medium for 24 h and subsequently treated with DDC (0 nM, 1 nM, 10 nM, 100 nM and 1000 nM) for 72 h. In experiments with antioxidants, the cells were incubated in the culture medium containing an antioxidant for 24 h and then treated with a combination of DDC (100 nM) and the same antioxidant for a further 72 h. These experiments were carried out using DDC at 100 nM, since the neurite growth in both SMI-31 (+) and SMI-32 (+) neurons were significantly inhibited by 100 nM DDC. 4.4.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 10 min and then treated with a protein-blocking agent (Immunon, Pittsburgh, PA) to block non-specific binding sites. The cells were then incubated with mouse anti-phosphorylated NF antibody (1:1000; SMI-31; Convance, Princeton, NJ) or mouse anti-nonphosphorylated NF antibody (1:1000; SMI-32; Convance) for 1 h, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (1:50; Organon Teknika Corp, Durham, NC) for 1 h. The immunostained cells were washed with PBS. Then, the coverslip with the cells was attached to the underside of a thin chamber (0.5mm thickness), and the topside of the chamber was covered with another coverslip. The chamber was filled with PBS and then placed on and examined with a Zeiss Axiovert 135 TV fluorescence microscope equipped with a 450– 490 nm excitation filter and a 515–565 nm emission filter. The photomicrographs were taken by digital camera (AxioCam MRm, Carl Zeiss) driven by software AxioVision 4.7.2 (Carl Zeiss). All immunocytochemistry steps were conducted at 24 °C. 4.5.

Measurement and analysis of neurite length

The length of neurites in SMI-31-immunoreactive (+) and SMI32-immunoreactive (+) neurons at 72 h after treatment with DDC was measured using NIH ImageJ software version 1.44 (http://rsb.info.nih.gov/ij/index.html) from the captured images. The selection of 72 h was based on a series of preliminary experiments that provided the best reproducibility compared with other time periods, with respect to time, morphology of neurites and cultured neurons (e.g., overlapping, identification of neurites). The neurite length in DDC-treated neurons was compared with the control (DDC 0 nM-treated) neurons by the Steel test for nonparametric multiple comparisons. The neurite lengths of SMI-31 (+) and SMI-32 (+) neurons, and the mean (±SEM) length of neurites in DDC-treated neurons were normalized to those of control neurons and presented as percentages, because there was a significant difference in the mean length between SMI-31 (+) and SMI-32 (+) neurites in control neurons. Differences between the two

This work was supported by a grant from the Kitasato University School of Medicine, Japan.

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