Neurofilament metabolism in sporadic amyotrophic lateral sclerosis.

Although the role of intraneuronal neurofilamentous aggregates in the pathogenesis of ALS is unknown, their presence forms a key neuropathological hallmark of the disease process. Conversely, the experimental induction of neurofilamentous aggregates in either neurotoxic or transgenic mice gives rise to motor system degeneration. To determine whether alterations in the physiochemical properties of NF are present in sporadic ALS, we purified NF subunit proteins from cervical spinal cord of ALS and age-matched control patients. The cytoskeleton-enriched, Triton X-100 insoluble fraction was further separated into individual NF subunits using hydroxyapatite HPLC. We observed no differences between control and ALS in the characteristics of NFH, including migration patterns on 2D-IEF, sensitivity to E. coli, alkaline phosphatase mediated dephosphorylation, peptide mapping, or proteolysis (calpain, calpain/calmodulin mediated, phosphorylated or dephosphorylated NFH). NFL showed no differences in 2D-IEF migration patterns, peptide mapping, or the extent of NFL nitrotyrosine immunoreactivity in either the Triton soluble or insoluble fractions. The latter observation demonstrated that NFL nitration is a ubiquitous occurrence in neurons and suggests that NFL might function as a sink for free reactive nitrating species. In contrast to the lack of differences in the post-translational processing of NF in ALS, we did observe a selective suppression of NFL steady state mRNA levels in the limb innervating lateral motor neuron column of ALS. This occurred in the absence of modifications in NFH, NFM or neuronal nitric oxide synthase (Type I NOS; nNOS) steady state mRNA levels. Coupled with previous observations of nNOS immunoreactivity co-localizing with NF aggregates in ALS motor neurons, this suggests activation of the nNOS enzyme complex in ALS, which would be predicted to contribute directly to the generation of reactive nitrating species. Given this, the isolated suppression of NFL steady state mRNA levels in ALS may indicate that ALS motor neurons are at an intrinsic deficit in the ability to buffer free reactive nitrating species.