Transient receptor potential vanilloid subtype 1 (TRPV1, also known as VR1) has an oligomeric structure formed by subunits having six transmembrane segments with a pore domain formed by the fifth and sixth transmembrane regions. This receptor is a nonselective cation channel activated by the vanilloids or products of lipoxygenases, and protein kinase C and phospholipase C mediate the sensitization of TRPV1. Moreover, the widespread distribution of TRPV1 including substantia nigra (SN) in the brain has suggested that this receptor plays a significant role in the central nervous system (CNS). However, little is known about toxicity via TRPV1 in the SN.
Intranigral injection of the TRPV1 agonist capsaicin (CAP) into the rat brain, or treatment of rat mesencephalic cultures with CAP, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was ameliorated by the TRPV1 antagonist capsazepine (CZP) or iodo-resiniferatoxin (I-RTX), suggesting the direct involvement of TRPV1 in neurotoxicity. In cultures, both CAP and anandamide (AEA), an endogenous ligand for both TRPV1 and cannabinoid type 1 (CB1) receptors, induced degeneration of DA neurons, increases in intracellular Ca2+ ([Ca2+]i), and mitochondrial damage, which were inhibited by CZP, the CB1 antagonist AM251 or the intracellular Ca2+ chelator BAPTA/AM. We also found that CAP or AEA increased mitochondrial cytochrome c release as well as immunoreactivity to cleaved caspase-3, and that the caspase-3 inhibitor z-DEVD-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonists HU210 or WIN 55,212-2 also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by CZP, AM251 or BAPTA/AM. The CAP-, AEA-, HU210-, or WIN 55,212-2-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210-, WIN 55,212-2 or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross-talk between TRPV1 and CB1 receptors. Moreover, 12-hydroperoxyeicosatetraenoic acid (12-HPETE, known as TRPV1 agonist) produced via activation of CB1 receptors by HU210 or WIN 55,212-2 induced neuronal toxicity via activation of TRPV1 in mesencephalic cultures, and intranigral injection of 12-HPETE into the rat brain also resulted in neuronal cell death.
In addition, this study examined whether microglia express TRPV1 and activation of TRPV1 contributes to cell death of microglia. In cultures, RT-PCR, Western blot analysis and immunocytochemical staining showed that microglia, but not astrocytes, expressing TRPV1 underwent cell death following the treatment with TRPV1 agonist CAP or RTX. Moreover, treatment with CAP or RTX induced cell death of immortalized human microglial cell line HMO6 expressing this receptor. CAP- or RTX-induced cell death of microglia was accompanied by increases in cytosolic Ca2+ concentration in the presence of extracellular Ca2+ and mitochondrial damage as well as mesencephalic neurons. This toxicity was also ameliorated by TRPV1 antagonists or BAPTA/AM, suggesting involvement of increases in cytosolic Ca2+ via influx through the direct activation of TRPV1. Additional study demonstrated that intranigral injection of CAP or 12-HPETE into the rat brain produced cell death of microglia, but not astrocytes in the SN, visualized by immunocytochemistry, and this in vivo effect was ameliorated by CZP or I-RTX, suggesting involvement of TRPV1 in the toxicity. This study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons, microglia express TRPV1, and activation of TRPV1 also mediates cell death of microglia. The findings in this study suggest that these two types of receptors, TRPV1 and CB1 receptors, may contribute to neurodegeneration in response to endogenous ligands such as AEA or 12-HPETE.