Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.

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Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.

BackgroundCardiomyopathy and distal symmetrical polyneuropathy (DSPN), including sensory and autonomic dysfunction, often co-occur in diabetic mellitus (DM) patients. However, the temporal relationship and progression between these two complications has not been investigated. Using a streptozotocin DM animal model that develops insensate neuropathy, our aim was to examine in parallel the development of DSPN and DM-associated changes in cardiac structure and function as well as potential mechanisms, such as autonomic dysfunction, evaluated by changes in urinary and myocardial norepinephrine content and myocardial neuronal markers.MethodsSensory neuropathy was measured by behavioral tests using Von Frey filaments and Hargreaves methods. Echocardiography was used to evaluate myocardial structure and function. Autonomic function was evaluated by measuring urinary and myocardial norepinephrine (NE) levels by enzyme-linked immunosorbent assay and high-performance liquid chromatography/mass spectrometry. Quantitative immunohistochemistry was used to measure the myocardial neuronal markers, calcitonin gene-related peptide (CGRP) and general neuronal protein gene product 9.5 (PGP 9.5).ResultsThe DM group developed tactile and thermal insensate neuropathy 4–5 weeks after DM onset. Cardiovascular changes were found between 4 and 12 weeks after DM onset and included bradycardia, diastolic and systolic dysfunction and cardiac dilation. There was a 2.5-fold reduction in myocardial NE levels and a 5-fold increase in urinary NE levels in the DM group. Finally, there was a 2.3-fold increase in myocardial CGRP levels in the DM group and no change in PGP9.5 levels.ConclusionsCardiovascular structural and functional changes developed early in the course of DM and in combination with insensate neuropathy. In parallel, signs of cardiac autonomic dysfunction were also found and included decreased myocardial NE levels and altered CGRP levels. These results may indicate the need for early cardiovascular evaluation in DM patients with insensate neuropathy.