Additive neuroprotective effects of a histone deacetylase inhibitor and a catalytic antioxidant in a transgenic mouse model of amyotrophic lateral sclerosis

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Abstract

ALS is a devastating neurodegenerative disorder for which no effective treatment exists. Multiple molecular mechanisms are involved in the pathogenesis. We tested the catalytic antioxidant AEOL 10150, the histone deacetylase inhibitor phenylbutyrate (PBA), and the combination of PBA and AEOL 10150 in the G93A transgenic mouse model, administered from disease onset. AEOL 10150 alone improved motor function and extended survival by 11%, PBA alone significantly improved motor function and extended survival by 13%. PBA and AEOL 10150 together increased survival by 19%. Increased histone acetylation was confirmed by Western blot. Quantitative real-time RT-PCR analysis revealed upregulation of compounds capable of protecting cells against oxidative stress and apoptosis. Markers of oxidative damage were reduced in the lumbar spinal cord as compared to vehicle administration. These results suggest that agents inhibiting apoptosis and blocking oxidative stress show efficacy in treating mutant-SOD1-associated ALS and that a combination of agents targeting different disease mechanisms may exert additive therapeutic effects.

Introduction

ALS is a fatal neurodegenerative disorder leading to rapidly progressive paralysis due to motor neuron loss in the brain and spinal cord. 10% of cases are familial; 10–20% of these are due to mutations in the superoxide dismutase (SOD) 1 gene (Rosen et al., 1994). Mice overexpressing a human SOD1 mutation, where a glycine is substituted by an alanine at amino acid position 93, show a phenotype closely mimicking human ALS, and serve as a widely used animal model for the disease (Gurney, 1994, Ripps et al., 1995). The precise mechanisms by which the SOD mutations cause motor neuron degeneration have still not been elucidated.

Oxidative stress is an important factor involved in chronic neurodegeneration. Since the discovery of the SOD1 mutations in familial ALS, it has been hypothesized that the resulting toxic gain-of-function of the SOD1 enzyme may lead to Cu-dependent pro-oxidant reactions. Regardless of the precise mechanisms, or whether oxidative injury is primary or secondary, it is clear that oxidative damage markers are increased in both transgenic mouse models and in human ALS. In the G93A mouse model as well as in human familial and sporadic ALS, an increase of markers of oxidative damage (protein carbonyls, nuclear DNA 8-hydroxy-2′-deoxyguanosine (OH8dG) levels, 3-nitrotyrosine and 3-nitro-4-hydroxyphenylacetic acid, malondialdehyde, heme oxygenase-1) has repeatedly been demonstrated (Ferrante et al., 1997a, Ferrante et al., 1997b, Beal et al., 1997). In the G93A mouse model, several compounds, among them antiinflammatory and antioxidative agents, are effective in slowing the neurodegenerative phenotype (Klivenyi et al., 1999, Klivenyi et al., 2004, Zhu et al., 2002, Drachman et al., 2002, Wu et al., 2003, Kiaei et al., 2005, Crow et al., 2005).

Low molecular weight porphyrins represent a novel method to reduce oxidative stress in neurodegenerative disorders. Originally developed as manganese SOD-mimetics, manganese and iron porphyrins react with peroxynitrite and decompose it catalytically (Crow, 2000). Both iron and manganese porphyrins require exogenous reductants such as ascorbate or glutathione for the catalytic cycle, but this requirement is not limiting in vivo as the rates of re-reduction are extremely fast (Lee et al., 1997) and these reductants are present in mammalian tissues (Ferrer-Sueta et al., 1999, Crow, 2000). AEOL 10150 is a manganese porphyrin which possesses important properties. The stably bound central manganese ion can cycle between Mn(III) and Mn(IV) states allowing it to catalytically decompose potentially injurious oxidants such as peroxynitrite, the diffusion limited reaction product of superoxide and nitric oxide (Crow, 1999, Crow, 2000). Intraperitoneal and subcutaneous administration of AEOL 10150 started at disease onset was recently shown to be neuroprotective in the G93A ALS transgenic mouse model (Crow et al., 2005).

Transcriptional dysregulation occurs in human sporadic ALS as well as in the G93A mouse model (Olsen et al., 2001, Malaspina and de Belleroche, 2004, Ishigaki et al., 2002, Yoshihara et al., 2002). Histone deacetylases (HDAC) catalyze the deacetylation of lysine residues in the amino terminals of the highly conserved core histones H2A, H2B, H3, and H4. The posttranscriptional acetylation of lysine residues in the amino terminals of the core histones leads to a neutralization of the positive charge, and opening of the DNA conformation which allows access of transcription factors to target genes. Therefore, acetylation of histones correlates with transcriptional activity and determines specific temporal and spatial gene expression patterns (Pogo et al., 1966, Grunstein, 1997).

In the G93A-ALS-mouse model, the HDAC inhibitors sodium butyrate and phenylbutyrate (PBA) were administered starting at 21 days of age (∼ 70 days prior to disease onset). The best neuroprotective effect was with PBA 400 mg/kg/day which prolonged survival by 21.9% (Ryu et al., 2005). This was correlated with increased expression of nuclear factor-κB (NF-κB) and increased Bcl-2 expression accompanied by reduced cytochrome c and caspase expression.

We investigated in two studies whether either the Mn (III) porphyrin AEOL 10150 (2.5 mg/kg/day) or PBA (400 mg/kg/day), or the combination was effective if administered after the appearance of motor dysfunction in ALS mice, which more closely mimics the clinical situation, and therefore may be an important predictor of efficacy in human ALS.

Section snippets

Transgenic mice

G93A transgenic familial ALS mice (high copy number; B6SJL-Tg(SOD1-G93A)1Gur/J)) (Gurney, 1994) were obtained from Jackson Laboratory (Bar Harbor, ME, U.S.A). We maintained the transgenic G93A hemizygotes by mating transgenic males with B6SJLF1/J hybrid females. Transgenic offspring were genotyped by PCR assay of DNA obtained from tail tissue. First, 35 G93A transgenic mice were randomly assigned to control (vehicle) (n = 17) or AEOL 10150 2.5 mg/kg/day (Aeolus pharmaceuticals) (n = 19) groups.

Results

Systemic administration of AEOL 10150 at a dose of 2.5 mg/kg/day, started at disease onset (which occurred between 85 and 98 days of age with no significant difference between all vehicle- and drug-treated groups), led to a significant increase of survival from 132 ± 5 to 147 ± 15 days (11%) (P < 0.005, Mantel–Cox log-rank test) (Figs. 1a, b). Intraperitoneal administration of phenylbutyrate (PBA) at a dose of 400 mg/kg/day led to a significant increase in survival from 126 ± 4 days to 143 ± 9

Discussion

There is ample evidence that oxidative stress is an important pathogenetic factor in ALS and other neurodegenerative disorders (Ferrante et al., 1997a, Ferrante et al., 1997b, Beal, 2002, Beal et al., 1997). In the G93A ALS mouse model, AEOL 10150 was effective in prolonging survival and inhibiting neuronal loss when administered at symptom-onset by 11% as compared to the control group. Treatment with AEOL 10150 led to a decrease in malondialdehyde and 3-nitrotyrosine immunostaining in the

Acknowledgments

This work was supported in part by grants from Muscular Dystrophy Association and the Amyotrophic Lateral Sclerosis Association, by NIH grants to MFB and MK and by a grant of the Deutsche Forschungsgemeinschaft to SP.

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