In vitro reactive nitrating species toxicity in dissociated spinal motor neurons from NFL (−/−) and hNFL (+/+) transgenic mice

Abstract

We utilized fetal spinal motor neurons isolated from either NFL (−/−) or hNFL (+/+) transgenic mice to determine whether the loss of the low molecular weight neurofilament protein (NFL) places spinal motor neurons at a greater risk for cell death triggered by reactive nitrating species (RNS). After 21 days in serum‐free, antibiotic‐free medium, both the NFL (−/−) and hNFL (+/+) motor neurons developed neurofilamentous aggregates. Cultures were then exposed to nitric oxide (100 μM NOC 5, 100 μM NOC 12, or 2 mM sodium nitroprusside) or to peroxynitrite (250 mM SIN‐1) for varying intervals. NFL (−/−) cultures demonstrated extensive numbers of apoptotic neurons within six hours and complete cell loss by 24 hours in response to NOC 5 and NOC 12. In contrast, apoptosis was only observed in the motor neurons derived from control (C57bl/6) or hNLF (+/+) mice at 24 hours. In response to 2 mM sodium nitroprusside, necrosis was induced in all cells within 60 minutes. In response to 250 mM SIN‐1, both C57bl/6 and hNFL (+/+) cells survived to six hours with only minimal evidence of degeneration while NFL (−/−) motor neurons were necrotic by 60 minutes. These observations suggest that NFL deficient motor neurons are at an enhanced risk of cell death mediated by RNS.

• amyotrophic lateral sclerosis
• neurofilament
• nitric oxide
• peroxynitrite
• superoxide dismutase