Axonal damage in the making: Neurofilament phosphorylation, proton mobility and magnetisation transfer in multiple sclerosis normal appearing white matter

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Abstract

Aims

Multiple sclerosis (MS) leaves a signature on the phosphorylation and thus proton binding capacity of axonal neurofilament (Nf) proteins. The proton binding capacity in a tissue is the major determinant for exchange between bound and free protons and thus the magnetisation transfer ratio (MTR). This study investigated whether the MTR of non-lesional white matter (NLWM) was related to the brain tissue concentration of neurofilament phosphoforms.

Methods

Unfixed post-mortem brain slices of 12 MS patients were analysed using MTR, T1 at 1.5 T. Blocks containing NLWM were processed for embedding in paraffin and inspected microscopically. Adjacent tissue was microdissected, homogenised and specific protein levels were quantified by ELISA for the Nf heavy chain (NfH) phosphoforms, glial fibrillary acidic protein (GFAP), S100B and ferritin.

Results

Averaged hyperphosphorylated NfH (SMI34) but not phosphorylated NfH (SMI35) levels were different between individual patients NLWM. The concentration of hyperphosphorylated NfH-SMI34 correlated with T1 (R = 0.70, p = 0.0114) and — inversely — with MTR (R =−0.73, p = 0.0065). NfH-SMI35 was not correlated to any of the MR indices.

Conclusions

Post-translational modifications of axonal proteins such as phosphorylation of neurofilaments occur in NLWM and may precede demyelination. The resulting change of proton mobility influences MTR and T1. This permits the in vivo detection of these subtle tissue changes on a proteomic level in patients with MS.

Highlights

□ Neurofilaments are phosphorylated □ Protein phosphorylation competes with the free proton binding capacity □ Magnetisation transfer depends on magnetisation exchange between macromolecular bound and free protons □ In multiple sclerosis the phosphorylation status of neurofilaments is changed in otherwise normal appearing axons □ In vivo assessment of early axonal pathology is possible using magnetisation transfer

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