Tumor necrosis factor-α elevates neurite outgrowth through an NF-κB-dependent pathway in cultured adult sensory neurons: Diminished expression in diabetes may contribute to sensory neuropathy.

The presence of a proinflammatory environment in the sensory neuron axis in diabetes was tested by measuring levels of proinflammatory cytokines in lumbar dorsal root ganglia (DRG) and peripheral nerve from age matched control and streptozotocin (STZ)-induced diabetic rats. The levels of tumor necrosis factor-α (TNFα) and other cytokines were diminished in lumbar DRG from diabetic animals. Consequently, we tested the hypothesis that TNFα modulated axonal plasticity in adult sensory neurons and posited that impairments in this signal transduction pathway may underlie degeneration in diabetic sensory neuropathy. Cultured adult rat sensory neurons were grown under defined conditions and TNFα caused a dose-dependent 2-fold (P<0.05) elevation in neurite outgrowth. Neurons derived from 3 to 5month STZ-induced diabetic rats exhibited significantly reduced levels of neurite outgrowth in response to TNFα. TNFα enhanced NF-κB activity as assessed using Western blotting and plasmid reporter technology. Blockade of TNFα-induction of NF-κB activation caused inhibition of neurite outgrowth in cultured neurons. Immunofluorescent staining for NF-κB subunit p50 within neuronal nuclei revealed that medium to large diameter neurons were most susceptible to NF-κB inhibition and was associated with decreased neurite outgrowth. The results demonstrating reduced cytokine expression in DRG confirm that diabetic sensory neuropathy does not involve a neuroinflammatory component at this stage of the disease in experimental animal models. In addition, it is hypothesized that reduced TNFα expression in the DRG and possibly associated deficits in anterograde transport may contribute to impaired collatoral sprouting and regeneration in target tissue in type 1 diabetes.

Activation of nuclear factor-kappaB via endogenous tumor necrosis factor alpha regulates survival of axotomized adult sensory neurons.

Embryonic dorsal root ganglion (DRG) neurons die after axonal damage in vivo, and cultured embryonic DRG neurons require exogenous neurotrophic factors that activate the neuroprotective transcription factor nuclear factor-kappaB (NF-kappaB) for survival. In contrast, adult DRG neurons survive permanent axotomy in vivo and in defined culture media devoid of exogenous neurotrophic factors in vitro. Peripheral axotomy in adult rats induces local accumulation of the cytokine tumor necrosis factor alpha (TNFalpha), a potent activator of NF-kappaB activity. We tested the hypothesis that activation of NF-kappaB stimulated by endogenous TNFalpha was required for survival of axotomized adult sensory neurons. Peripheral axotomy of lumbar DRG neurons by sciatic nerve crush induced a very rapid (within 2 h) and significant elevation in NF-kappaB-binding activity. This phenomenon was mimicked in cultured neurons in which there was substantial NF-kappaB nuclear translocation and a significant rise in NF-kappaB DNA-binding activity after plating. Inhibitors of NF-kappaB (SN50 or NF-kappaB decoy DNA) resulted in necrotic cell death of medium to large neurons (> or =40 microm) within 24 h (60 and 75%, respectively), whereas inhibition of p38 and mitogen-activated protein/extracellular signal-regulated kinase did not effect survival. ELISA revealed that these cultures contained TNFalpha, and exposure to an anti-TNFalpha antibody inhibited NF-kappaB DNA-binding activity by approximately 35% and killed approximately 40% of medium to large neurons within 24 h. The results show for the first time that cytokine-mediated activation of NF-kappaB is a component of the signaling pathway responsible for maintenance of adult sensory neuron survival after axon damage.