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Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression

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Abstract

The p38 mitogen-activated protein kinase (p38MAPK) is activated via phosphorylation in neurones and glial cells by a variety of stimuli including oxidative stress, excitotoxicity, and inflammatory cytokines. Activated p38MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide, thus contributing to neurodegeneration. We investigated the expression and distribution of p38MAPK in the spinal cord of transgenic mice expressing a superoxide dismutase 1 mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis (ALS). Accumulation of p38MAPK was found by immunoblotting in the spinal cord of G93A mice during the progression of disease, but no changes were detected in its mRNA levels. Immunostaining for phosphorylated p38MAPK in lumbar spinal cord sections of SOD1G93A mice at the presymptomatic and early stages of disease showed an increased labeling in motor neurones that colocalized with phosphorylated neurofilaments in vacuolized perikarya and neurites, as detected by confocal microscopy. As the disease progressed, activated p38MAPK also accumulated in hypertrophic astrocytes and reactive microglia, as demonstrated by colocalization with GFAP and CD11b immunostaining, respectively. These data suggest that activation of p38MAPK in motor neurons and then in reactive glial cells may contribute, respectively, to the development and progression of motor neuron pathology in SOD1G93A mice.

Introduction

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease causing progressive loss of motor neurones in the brain and spinal cord, leading to muscular weakness, paralysis, and early death. The cause of motor neurone death is still unknown; however, the discovery that about one-fifth of the familial forms of ALS are due to mutations in the Cu/Zn superoxide dismutase (SOD1) gene provided new insight on the disease process Rosen et al 1993, Shaw et al 1998. Familial and sporadic forms have the same clinical and pathological profiles, and may therefore share common biochemical mechanisms. ALS is regarded a multifactorial disease which may involve a common cascade of events leading to motor neurone toxicity. Oxidative stress (Ferrante et al., 1997), glutamate-induced excitotoxicity (Rothstein, 1995), and inflammatory processes (McGeer and McGeer, 2002) are indicated as potential causes of ALS.

These pathological factors may lead to cell death through various intracellular signalling pathways, including the mitogen activated protein kinase (MAPK) pathways (Mielke and Herdegen, 2000). MAPKs are a family of related serine/threonine kinases that integrate various signals directing cellular responses to proliferative cues or stressful stimuli. The MAPK family includes extracellular signal-regulated kinases (ERKs), c-Jun-N-terminal kinase or stress activated protein kinase (JNK/SAPK), and p38MAP kinase (p38MAPK). Activation of p38MAPK is often correlated with neuronal degeneration Horstmann et al 1998, Skaper and Walsh 1998 and recently it has been shown to occur in neurodegenerative diseases such as Alzheimer’s and other tau-related diseases Atzori et al 2001, Zhu et al 2000.

The p38MAPK is implicated in various functions such as phosphorylation of cytoskeletal proteins and biosynthesis of cytokines and nitric oxide Mielke and Herdegen 2000, Ono and Han 2000), mechanisms supposed to play a role in the motor neurone degeneration in ALS Cleveland and Rothstein 2001, Julien 2001; Rowland and Shneider, 2001). In order to determine whether p38MAPK pathway is involved in motor neurone degeneration, we examined the expression and distribution of activated p38MAPK in the spinal cord of transgenic mice carrying the human mutant SOD1 with the G93A mutation (SOD1G93A), a mouse model of familial ALS (Gurney et al., 1994), during the progression of the disease. We focused our attention on p38MAPK activation not only in motor neurones but also in astrocytes and microglia. Using confocal microscopy we showed that p38MAPK was initially activated only in the spinal motor neurones of transgenic mice at the presymptomatic stage of the disease and colocalized with phosphorylated neurofilaments in the cell bodies and proximal neurites. As the disease progressed, the astrocytes and microglial cells also showed a progressive activation of this kinase.

Section snippets

P38MAPK levels progressively increase in the spinal cord of SOD1G93A mice

Immunoblot analysis of p38MAPK and its phosphorylated (activated) form (p38MAPK-P) in spinal cord extracts from control and transgenic mice revealed a single band of about 40 kDa corresponding to the band obtained from human monocytes stimulated with fMLP and used as positive control (Fig. 1A).

An antibody selectively recognizing p38MAPK-P did not reveal any signal in the spinal cord extracts from nontransgenic mice (Fig. 1A). By contrast, a moderately intense band was found in spinal cord

Discussion

The present study reports a progressive accumulation of p38MAPK in the spinal cord of SOD1G93A mice that parallels motor neurone degeneration. Accumulation of the protein was likely due to its reduced degradation rather than to increased synthesis, as we did not detect changes in the levels of its mRNA. It is noteworthy that accumulation of p38MAPK was associated to activation of the kinase, as revealed by the increased p38MAPK-P immunostaining which occurred first in the motor neurones, before

Animals

Female transgenic mice were maintained at a temperature of 21 ± 1°C with relative humidity 55 ± 10% and 12 h of light. Food (standard pellets) and water were supplied ad libitum. Transgenic mice originally obtained from Jackson Laboratories and expressing a high copy number of mutant human SOD1 with a Gly-93-Ala substitution (SOD1G93A) or wild-type human SOD1 (SOD1WT) mice were bred and maintained on a C57BL/6 mice strain at the Consorzio Mario Negri Sud, S. Maria Imbaro (CH), Italy. Transgenic

Acknowledgements

We are grateful to Dr. M.Rattray for valuable comments on the manuscript. We also thank Dr. Sozzani of the Department of Immunology of the Mario Negri Institute for kindly providing the lysates from human monocytes treated with f-MLP and Dr. Sara. Vignati for kindly providing the antibody and protocol for Akt immunoblotting. The financial support of Telethon, Italy (Grants GP0222Y01 to A.M. and C.B.), the MND Association (UK), and the Fondazione Monzino (Italy) is gratefully acknowledged.

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