Rapid mitochondrial dysfunction mediates TNF-alpha-induced neurotoxicity

Inflammatory mechanisms play a crucial role in the pathophysiologic processes after the onset of ischemic stroke (Vila et al. 2000). Ischemic brain injury is complex, and intracellular signaling that regulates innate and adaptive immunity, inflammation, cell death, angiogenesis, and repair processes plays an important role in the initiation, progression, and resolution of ischemia. Initiation of these critical intracellular regulators occurs through rapid cytokine signaling after ischemia, highlighting cytokines as key regulators of stroke damage (Hallenbeck 2002). Tumor necrosis factor alpha (TNF-α) is one of many pro-inflammatory cytokines associated with worsened clinical outcomes after stroke and exacerbations of infarct size in pre-clinical models (Nawashiro et al. 1997; Ormstad et al. 2011). However, our understanding of the pro-inflammatory effects of TNF-α is based primarily on studies of its peripheral actions. TNF-α mRNA increases within 1 h in the ischemic injury core, and the expression of its immunoreactive protein increases within 2–6 h after the onset of ischemia in preclinical models (Botchkina et al. 1997). TNF-α is increased in the serum of stroke patients between 6 and 12 h after symptom onset (Liu et al. 1994; Sotgiu et al. 2006). TNF-α signaling occurs through the TNF-α receptor (TNF-R), where ligand binding recruits adaptor proteins to a core signaling complex allowing for differences in signal transduction depending upon the stimulatory pattern (Wallach et al. 2002). The response to TNF-α is cell type dependent. In bovine and rat oligodendrocytes, TNF-α induces apoptosis, while in astrocytes, TNF-α enhances major histocompatibility complex class II and intracellular adhesion molecule 1 expression (Chao et al. 1995). In primary septo-hippocampal cultures, exposure to 30 ng/mL of TNF-α caused significant cytotoxicity but only with the addition of Actinomycin-D (Zhao et al. 2001). When rat primary cortical neurons are exposed to 10 ng/mL of TNF-α for 24 h, neurite retraction or formation of apoptotic bodies was not observed (Reimann-Philipp et al. 2001). However, in PC12 cells, TNF-α was cytotoxic (Reimann-Philipp et al. 2001). While these studies show neurotoxic effects of TNF-α, cultured embryonic rat hippocampal, septal, and cortical neurons are protected from glucose deprivation-induced injury and excitatory amino acid toxicity by exposure to TNF-α (Cheng et al. 1994). Furthermore, TNF-α can induce the expression of anti-apoptotic proteins B-cell lymphoma 2 and B-cell lymphoma-extra large (Bcl-xl) in hippocampal neurons (Tamatani et al. 1999). These few studies used high concentrations and long periods of exposure to TNF-α. Also, there is a single study of adipocytes that used high concentrations and 4-day exposures to assess mitochondrial function (Chen et al. 2010). In the present study, we tested the hypothesis that acute exposure to TNF-α concentrations seen in serum of stroke patients (Lambertsen et al. 2012; Nayak et al. 2012) causes neuronal cell death through rapid mitochondrial dysfunction. As such, we characterized the effect of acute exposure to low doses of TNF-α in a mouse hippocampal neuronal cell line (HT-22) and mouse primary cortical neurons. A rapid and profound mitochondrial dysfunction was observed after as little as 1.5 h of exposure to TNF-α which preceded cell death. These findings suggest that the damaging effect of TNF-α may be because of the impairment of neuronal mitochondrial function.