AimsAxonal pathology extends to the axonal cytoarchitecture leaving its signature on axoskeletal proteins. This study investigated whether neurofilament (NfH) phosphorylation would relate to the dynamics of axonal pathology in multiple sclerosis (MS).MethodsNfH phosphoforms (SMI32, SMI34, SMI35) were quantified by ELISA from microdissected samples of control and MS brain and spinal cord. Individual axons were analysed by electron microscopy, densitometrically and morphologically in adjacent tissue sections. Experiments were carried out pre- and post enzymatic dephosphorylation.ResultsIn control tissue a rostro-caudal gradient of NfH indicated an increase in axonal density from the brain gray matter towards the spinal cord. The highest levels of phosphorylated and hyperphosphorylated NfH were found in acute lesions of brain and spinal cord, in contrast to chronic lesions where levels were lower than in white matter, consistent with axonal loss. Dephosphorylated NfH was higher, but less densly packed in MS white matter axons compared to control tissue.ConclusionsThe findings suggest that a less organised/compact axoskeleton or impaired axonal transport may represent an early sign of axonal pathology within the normal appearing white matter in MS. Subsequently a proportional increase of dephosphorylated NfH, aberrant phosphorylation and/or aggregation may occur whilst the protein is transported through the white matter towards the MS plaque, where hyperphosphorylated NfH dominates.

The in vitro activity of gelatinase B, an enzyme whose appearance in the cerebrospinal fluid is associated with inflammatory diseases of the central nervous system, was dose-dependently inhibited by the antirheumatic D-penicillamine. Inhibition of gelatinase B in electrophoretically pure preparations and in cell culture supernatants and human body fluids was obtained at dosages reached in the circulation of patients treated with a peroral dosis of 750 mg D-penicillamine per day. In mice, developing acute demyelination, D-penicillamine significantly reduced the mortality and morbidity rates of experimental allergic encephalomyelitis (EAE). In chronic relapsing EAE in Biozzi AB/H mice, an animal model for relapses in multiple sclerosis (MS), it attenuated the exacerbations, even when the treatment was started after the primary full-blown disease had developed. We infer protease inhibition as the mechanism of action of D-penicillamine and suggest that its use may be effective as peroral treatment for MS.

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