Heightened intrathecal release of axonal cytoskeletal proteins in multiple sclerosis is associated with progressive disease and clinical disability

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Abstract

The pathologic basis of disease progression in multiple sclerosis (MS) is thought to involve axonal degeneration, which contributes to the accumulation of neurological disability. Recent reports suggest that intrathecal concentrations of the neurofilament protein in relapsing remitting MS correlate with disease activity and the degree of disability. We sought to investigate the intrathecal levels of other cytoskeletal components of axons, primarily actin, tubulin and the light subunit of neurofilament (NFL) in patients with progressive MS and relevant controls and correlate results with clinical parameters of disease severity. Cerebrospinal fluid (CSF) concentrations of actin, tubulin and NFL were significantly increased in MS patients when compared to corresponding levels in patients with other inflammatory or non-inflammatory neurological diseases. Moreover, the intrathecal release of actin and tubulin, and to a lesser extent NFL, was significantly more marked in patients with primary and secondary progressive MS when compared to patients with relapsing remitting disease and was correlated with clinical disability. Our findings suggest that progressive MS is associated with the heightened intrathecal release of axonal cytoskeletal proteins, and that CSF actin, tubulin and NFL are reliable markers of axonal damage.

Introduction

Multiple sclerosis (MS) is basically an inflammatory demyelinating disease of the central nervous system (CNS) (Antel, 1999). However, there is increasing evidence that axonal degeneration plays an important role in the pathogenesis of neurological disability (reviewed by Silber and Sharief, 1999). Axonal damage is seen in both acute and chronic MS lesions, and this damage appears to correlate with the degree of clinical disability (Trapp et al., 1999). The cause of axonal damage in MS is unknown, but it is likely that a destructive process directed against specific components of the axonal cytoskeleton may contribute to the accumulation of disability (Silber and Sharief, 1999).

The axonal cytoskeleton is a highly regulated system that plays a central role in maintaining the integrity of axons. The physiological functions of axonal cytoskeleton are dependent upon several interconnected filaments that primarily consist of actin microfilaments (6 nm in diameter), neurofilament (10 nm) and microtubules (23 nm) (Fuchs, 1996). Collectively, these proteins control axonal shape and caliber, maintain axonal transport of nutrients and organelles, define specialized membrane domains and regulate growth and focal adhesions Moreau and Way, 1999, Baron et al., 2000, Rando et al., 2000. Actin, one of the main proteins of the cytoskeleton, also plays an important role in axonal growth and guidance, in the formation and elongation of neuritis and in a variety of other biological responses (Doussau and Augustine, 2000). The neurofilament protein is the most abundant cytoskeletal element in large myelinated axons that preserves axonal caliber and transport (Yabe et al., 2001). Tubulin, the primary structural protein of microtubules, comprises between 15% and 20% of cellular protein in the brain (Downing, 2000). It maintains axonal growth and morphology and provides a conduit for fast axonal transport of organelles and proteins (Laferriere et al., 1997).

Preliminary reports suggest that some cytoskeletal proteins may be detected in the cerebrospinal fluid (CSF) from patients with neurodegenerative conditions and that these proteins may serve as useful confirmatory markers of neurodegeneration or progression of CNS disorders (Shaw and Williams, 2000). High levels of actin or its regulatory proteins have been detected in the CSF form patients with Alzheimer's disease Merched et al., 1998, Chauhan et al., 1999. Similarly, the main component of the axonal core, the light subunit of neurofilament (NFL) protein, has been used as a marker for the axonal damage in experimental animal (Karlsson et al., 1991) and human neurodegeneration Eyer et al., 1998, Rosengren et al., 1999. Furthermore, high CSF levels of NFL have been detected in patients with relapsing remitting MS and appear to correlate with clinical disability (Lyke et al., 1998). However, it is currently unknown if CSF cytoskeletal proteins are similarly increased in patients with progressive MS and if so, whether these proteins would correlate with the extent of clinical disability. Thus, we sought to measure the CSF levels of actin, tubulin and NFL in patients with primary and secondary progressive MS and determine whether these levels serve as indicators of neurological dysfunction or clinical disability. Our results indicate that these cytoskeletal proteins are significantly elevated in CSF from patients with progressive MS when compared with relapsing remitting disease or other neurological disorders and that they correlate with neurological dysfunction.

Section snippets

Patients and controls

CSF samples were obtained by lumbar puncture (LP) from 35 MS patients that included 19 with primary or secondary progressive disease (Table 1). All MS patients had clinically definite diseases (Poser et al., 1983), and each patient underwent detailed clinical assessment and scoring of the Expanded Disability Status Scale (EDSS) at the time of LP. We also collected CSF samples from 16 patients with inflammatory and 20 with non-inflammatory neurological disorders to serve as disease controls

Actin concentrations in CSF

We detected low actin reactivity in healthy individuals and in patients with pseudotumor cerebri, peripheral neuropathy and Guillain–Barré syndrome. However, higher CSF levels were observed in patients with MS and those with neurodegenerative or inflammatory CNS disorders. The highest CSF actin levels were observed in patients with progressive MS, and these levels were significantly higher than in patients with relapsing remitting MS or disease controls (Fig. 1). We detected significantly high

Discussion

Our findings provide evidence that actin, tubulin and NFL are released within the intrathecal compartment in patients with MS and also in some patients with neuro-degenerative disorders or parenchymal CNS damage. Moreover, our results indicate that CSF levels of these axonal cytoskeletal proteins are significantly elevated in patients with progressive MS and demonstrate an association with the degree of clinical disability. Taken together, these findings suggest that the CSF levels of actin,

Acknowledgements

We are grateful to Dr. E. Silber for the helpful discussions and assistance with the sample collections and Ms. J. Smalley for the technical advice. This work was supported by a project grant from the Special Trustees of Guy's and St. Thomas' Hospital.

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