Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis

The intraneuronal aggregation of phosphorylated high-molecular-weight neuro filament protein (NFH) in spinal cord motor neurons is considered to be a k ey pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyperphosphory lation of NFH, we have purified and characterized NFH from the cervical spi nal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X-100 insoluble protein fractions, with respect to migration patterns on 2D -iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phos phatase mediated dephosphorylation, or the rate of calpain-mediated proteol ysis. The rate of calpain-mediated proteolysis was unaffected by either exh austive NFH dephosphorylation or by the addition of calmodulin to the react ion. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated th at all the phosphorylated residues found in ALS NFH were-also found to be p hosphorylated in normal human NFH samples. Hence, we have observed no diffe rence in the physicochemical properties of normal and ALS NFH extracted fro m cervical spinal cords, suggesting that the perikaryal aggregation of high ly phosphorylated NF in ALS neurons reflects the aberrant somatotopic local ization of normally phosphorylated NFH.