Nerve growth factor alters microtubule targeting agent-induced neurotransmitter release but not MTA-induced neurite retraction in sensory neurons.

Peripheral neuropathy is a dose-limiting side effect of anticancer treatment with the microtubule-targeted agents (MTAs), paclitaxel and epothilone B (EpoB); however, the mechanisms by which the MTAs alter neuronal function and morphology are unknown. We previously demonstrated that paclitaxel alters neuronal sensitivity, in vitro, in the presence of nerve growth factor (NGF). Evidence in the literature suggests that NGF may modulate the neurotoxic effects of paclitaxel. Here, we examine whether NGF modulates changes in neuronal sensitivity and morphology induced by paclitaxel and EpoB. Neuronal sensitivity was assessed using the stimulated release of calcitonin gene-related peptide (CGRP), whereas morphology of established neurites was evaluated using a high content screening system. Dorsal root ganglion cultures, maintained in the absence or presence of NGF, were treated from day 7 to day 12 in culture with paclitaxel (300nM) or EpoB (30nM). Following treatment, the release of CGRP was stimulated using capsaicin or high extracellular potassium. In the presence of NGF, EpoB mimicked the effects of paclitaxel: capsaicin-stimulated release was attenuated, potassium-stimulated release was slightly enhanced and the total peptide content was unchanged. In the absence of NGF, both paclitaxel and EpoB decreased capsaicin- and potassium-stimulated release and the total peptide content, suggesting that NGF may reverse MTA-induced hyposensitivity. Paclitaxel and EpoB both decreased neurite length and branching, and this attenuation was unaffected by NGF in the growth media. These differential effects of NGF on neuronal sensitivity and morphology suggest that neurite retraction is not a causative factor to alter neuronal sensitivity.

Paclitaxel alters the evoked release of calcitonin gene-related peptide from rat sensory neurons in culture.

Peripheral neuropathy (PN) is a debilitating and dose-limiting side effect of treatment with the chemotherapeutic agent, paclitaxel. Understanding the effects of paclitaxel on sensory neuronal function and the signaling pathways which mediate these paclitaxel-induced changes in function are critical for the development of therapies to prevent or alleviate the PN. The effects of long-term administration of paclitaxel on the function of sensory neurons grown in culture, using the release of the neuropeptide calcitonin gene-related peptide (CGRP) as an endpoint of sensory neuronal function, were examined. Dorsal root ganglion cultures were treated with low (10 nM) and high (300 nM) concentrations of paclitaxel for 1, 3, or 5 days. Following paclitaxel treatment, the release of CGRP was determined using capsaicin, a TRPV1 agonist; allyl isothiocyanate (AITC), a TRPA1 agonist; or high extracellular potassium. The effects of paclitaxel on the release of CGRP were stimulant-, concentration-, and time-dependent. When neurons were stimulated with capsaicin or AITC, a low concentration of paclitaxel (10nM) augmented transmitter release, whereas a high concentration (300 nM) reduced transmitter release in a time-dependent manner; however, when high extracellular potassium was used as the evoking stimulus, all concentrations of paclitaxel augmented CGRP release from sensory neurons. These results suggest that paclitaxel alters the function of sensory neurons in vitro, and suggest that the mechanisms by which paclitaxel alters neuronal function may include functional changes in TRP channel activity. The described in vitro model will facilitate future studies to identify the signaling pathways by which paclitaxel alters neuronal sensitivity.

Ret-dependent and Ret-independent mechanisms of Gfl-induced sensitization.

BACKGROUND: The GDNF family ligands (GFLs) are regulators of neurogenic inflammation and pain. We have previously shown that GFLs increase the release of the sensory neuron neuropeptide, calcitonin gene-related peptide (CGRP) from isolated mouse DRG. RESULTS: Inhibitors of the mitogen-activated protein kinase (MAPK) pathway abolished the enhancement of CGRP release by GDNF. Neurturin-induced enhancement in the stimulated release of CGRP, used as an indication of sensory neuronal sensitization, was abolished by inhibition of the phosphatidylinositol-3 kinase (PI-3K) pathway. Reduction in Ret expression abolished the GDNF-induced sensitization, but did not fully inhibit the increase in stimulus-evoked release of CGRP caused by neurturin or artemin, indicating the presence of Ret-independent GFL-induced signaling in sensory neurons. Integrin ß-1 and NCAM are involved in a component of Ret-independent GFL signaling in sensory neurons. CONCLUSIONS: These data demonstrate the distinct and variable Ret-dependent and Ret-independent signaling mechanisms by which GFLs induce sensitization of sensory neurons. Additionally, there is a clear disconnect between intracellular signaling pathway activation and changes in sensory neuronal function.