Effect of nitric oxide and inflammatory mediators on axonal transport

Abstract

Increased production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and reactive oxygen species by glia in the central nervous system (CNS) is a hallmark of a variety of inflammatory and degenerative brain diseases. Axonal transport of mitochondria or synaptic vesicle precursors via the kinesin motor protein is essential to keep the integrity of the axon and synapse. Disturbance of axonal transport and accumulation of axonally transported organelles is an early sign of neuroinflammatory diseases such as multiple sclerosis, brain injury and Alzheimer's disease. We studied whether axonal transport is compromised by acute exposure to inflammatory mediators produced by activated glia. We dissected the transport of the synaptic vesicle precursor proteins synaptophysin and synaptotagmin tagged with green fluorescent protein (synaptophysin-EGFP and synaptotagmin-EGFP). We found that microglia pre-stimulated by inflammatory cytokines to produce nitric oxide (NO) and tumor necrosis factor-α (TNF-α) focally suppressed the axonal motility of labeled vesicle precursors at the point of contact between activated glia and axons, as measured using confocal microscopy of fluorescence recovery after photobleaching (FRAP). We found that direct application of either TNF-α or a short-term NO donor to cultured hippocampal neurons inhibited axonal motility of both synaptophysin-EGFP and synaptotagmin-EGFP. Furthermore, TNF-α inhibited axonal transport of mitochondria in a similar manner. Consistently, TNF-α induced axonal transport impairment normalized again after 6-8 hours of TNF-α application. Inhibition of axonal transport by NO or TNF-α was associated with a significantly increased fraction of immobile particles. Furthermore, inhibition of axonal transport was dependent on phosphorylation of c-jun NH2-terminal kinase (JNK).In particular, we show that both NO and TNF-α stimulated phosphorylation of JNK in axons. In experiments with both genetically labeled mitochondria and synaptic vesicle precursors, we observed that the velocity of the remaining mobile particles was largely unchanged. This suggested a model where TNF-α produced by activated glial cells in inflammatory or degenerative brain diseases acts on axons by inhibition of cargo transport and dissociation kinesin motor protein from microtubules. Confocal microscopy and lifetime-based Förster resonance energy transfer (FRET) analysis were performed on primary cultured neurons to analyse the effect of TNF-α on the axonal mitochondrial transport system. TNF-α induced detachment of the heavy chain kinesin family-5B (KIF5B) protein from tubulin in axons, but not cell bodies as determined by lifetime based FRET analysis. Dissociation of KIF5B from tubulin after TNF-α treatment was also dependent on phosphorylation of JNK. Thus, overt production of inflammatory mediators by activated microglial cells blocks the motility of synaptic vesicle precursors and mitochondria via phosphorlyation of JNK and may cause axonal and synaptic dysfunction in inflammatory and degenerative brain diseases.