Structure–Activity Analysis of Dynorphin A Toxicity in Spinal Cord Neurons: Intrinsic Neurotoxicity of Dynorphin A and Its Carboxyl-Terminal, Nonopioid Metabolites

Abstract

Dynorphin A [dynorphin A (1–17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both κ opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through κ opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure–activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing κ opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (≥1 μM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 μM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1–17) > dynorphin A (1–13) ≅ dynorphin A (2–13) ≅ dynorphin A (13–17) (least toxic) > dynorphin A (1–5) ([Leu5]-enkephalin) or dynorphin A (1–11). Dynorphin A (1–5) or dynorphin A (1–11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3–13), dynorphin A (6–17), and dynorphin A (13–17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 μM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13–17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3–13) and (13–17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.