Transient receptor potential vanilloid subtype 1 mediates cell death of mesencephalic dopaminergic neurons in vivo and in vitro

Abstract

Intranigral injection of the transient receptor potential vanilloid subtype 1 (TRPV1; also known as VR1) 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, 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 N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (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-Asp-Glu-Val-Asp-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonist (6aR)-trans 3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d] pyran-9-methanol (HU210) also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by AM251 and BAPTA/AM. The CAP-, AEA-, or HU210-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210- or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross talk between TRPV1 and CB1 receptors. To our knowledge, this study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons. Our findings suggest that these two types of receptors, TRPV1 and CB1, may contribute to neurodegeneration in response to endogenous ligands such as AEA.

Figure 1.

A, Rat SN sections were immunostained simultaneously with antibodies to TRPV1 (green) and TH (red), and the two were merged (yellow). TRPV1 was detected in TH-i.p. neurons as well as non-TH-i.p. neurons (arrowheads). Cells in neuron-enriched mesencephalic cultures were immunostained with antibody to TH alone (B; red) or together with antibody to TRPV1 (C; green) or CB1 (D; red). Both TRPV1 and CB1 were detected in TH-i.p. and non-TH-i.p. neurons (arrowheads). E, The colocalization of TRPV1 and CB1 receptors in neuron-enriched mesencephalic cultures. F-I, Cultures were treated with vehicle (F, G) or 50 μm capsaicin (H, I) for 24 h and stained with calcein-acetoxymethyl ester (green for live cells; F, H) or ethidium homodimer-1 (red for dead cells; G, I). Scale bars: A, 100 μm; B-E, 30 μm; F-I, 50 μm.

Figure 2.

Neurotoxicity induced by CAP or AEA in neuron-enriched mesencephalic cultures. Cultures were treated with 10-100 μm CAP or 1-10 μm AEA for 24 h. Where indicated, cells were pretreated with 10 μm CZP or 5 μm AM251 for 5 min before treatment with 100 μm CAP or 10 μm AEA for 24 h. In all cultures, cell death was assessed as LDH released into the bathing media (A) and by counting the number of TH-i.p. cells (D). TH immunostaining of cultures treated with vehicle (B) or 50 μm CAP (C) is shown. Scale bar: B, C, 100 μm. E, Number of TH-i.p. neurons in neuron-enriched mesencephalic cultures treated with CAP or AEA in either the absence or presence of TRPV1 antagonist iodo-RTX. Cultures were pretreated with 1 μm iodo-RTX for 5 min before treatment with 50 μm CAP or 5 μm AEA for 24 h. F, Reduction in the number of NeuN-i.p., GABA-i.p., and TH-i.p. neurons in cultures treated with 50 μm CAP or 5 μm AEA for 24 h. All values represent the mean ± SEM of triplicate cultures in four separate platings. ^*p < 0.001 compared with control; ^**p < 0.05 and ^§p < 0.001 compared with cells treated with CAP or AEA; ^‡p < 0.001 compared with cells treated with 50 μm CAP.

Figure 3.

Changes of R (F340/380) fluorescence in fura-2-loaded cultured mesencephalic neurons. [Ca²⁺]_i was measured in cultures treated with 50 μm CAP (left panel, arrowhead) or 5 μm AEA (right panel, arrowhead) in the presence (A, B) or absence (C, D) of 1.8 mm extracellular calcium. E-J, Response to 50 μm CAP or 5 μm AEA (arrowhead) of cultures pretreated with 10 μm CZP (E, F, arrow) or 5 μm AM251 (I, J, arrow) in the presence of 1.8 mm extracellular calcium, or cultures treated with 5 μm BAPTA/AM plus 50 μm CAP (G) or 5 μm AEA (H). Data were averaged from 20-25 randomly selected cells for each condition.

Figure 4.

A-C, Mitochondrial disruption in neuron-enriched mesencephalic cultures treated with 50 μm CAP in the presence of 1.8 mm extracellular calcium. D-G, Inhibition of CAP-induced mitochondrial disruption by pretreatment with 10 μm CZP (D, E) or cotreatment with 5 μm BAPTA/AM (F, G). Each colored arrow indicates the same cells. H-J, Localization of cytochrome c (green) immunoreactivity and Mito-Tracker (red) in untreated cells (H) and cells treated with 50 μm CAP (I) or 5 μm AEA (J) for 12 h. K, Western blot analysis of cytochrome c (Cyto-c) levels after treatment of cells for 6 h with CAP or AEA. Con, Nontreated controls; Cyto, cytosolic fraction; Mito, mitochondrial fraction. L, z-DEVD-fmk attenuates CAP- or AEA-induced cell death. M, N, Immunochemical localization of cleaved caspase-3 (red) in TH-i.p. (green) and non-TH-i.p. (arrowheads) neurons in untreated cultures (M) and cultures treated with CAP or AEA (N). ^*p < 0.001 compared with untreated controls; ^**p < 0.01 and ^§p < 0.001 compared with culture treated with 100 μm CAP or 10 μm AEA. Scale bars: (in A) A-G, 40 μm; (in H) H-J, 20 μm; (in M) M, N, 30 μm.

Figure 5.

A, Neurotoxicity induced by HU210 on TH-i.p. neurons in neuron-enriched mesencephalic cultures. Cultures were treated with 1-3 μm HU210 for 24 h. Where indicated, cells were pretreated with 10 μm CZP or 5 μm AM251 for 5 min before treatment with 3 μm HU210 or cotreated with 5 μm BAPTA/AM for 24 h. Death of TH-i.p. neurons was assessed by counting the number of TH-i.p. cells. All values represent the mean ± SEM of triplicate cultures in four separate platings. ^*p < 0.001 compared with control; ^**p < 0.05 and ^§p < 0.001 compared with cells treated with 3 μm HU210. B-G, Changes of R (F340/380) fluorescence in fura-2-loaded cultured mesencephalic neurons. [Ca²⁺]_i was measured in cultures treated with 3 μm HU210 (arrowhead) in the presence (B) or absence (C) of 1.8 mm extracellular calcium. Changes of R in cultures pretreated with 5 μm AM251 (D, arrow) or 10 μm CZP (E, arrow) or cotreated with 5 μm BAPTA/AM with 3 μm HU210 in the presence of 1.8 mm extracellular calcium (F) are shown. G, The effect of thapsigargin on the Ca²⁺ influx induced by HU210 in the presence of extracellular Ca²⁺. Data were averaged from 20-25 randomly selected cells for each condition.

Figure 6.

CAP-induced neurotoxicity in the SN of rat brains. Animals were administered a unilateral injection of 500 pmol of CAP in 3 μl of PBS containing 14% ethanol (C, D, G, H, K, L, O, P) or vehicle (A, B, E, F, I, J, M, N) into the SN and killed 7 d later. Brain tissues were stained with cresyl violet (A-D) and immunostained with antibody to NeuN (E-H), GAD (I-L), or TH (M-P). Q-T, CZP alters the effect of CAP on TH-i.p. neurons in the SN. Coinjection of 500 pmol of CAP plus 500 pmol of CZP rescued TH-i.p. neurons, as evidenced by TH immunocytochemistry (Q, R), whereas CZP alone had no effect (S, T). The arrowhead indicates syringe track. Dotted lines indicate SNpc (where dopaminergic neurons were degenerated). Scale bars: (in S) M, O, Q, S, 300 μm; (in A) A, C, E, G, I, K, 200 μm; (in T) B, D, F, H, J, L, N, P, R, T, 50 μm. U, Number of TH-i.p. neurons in the SN treated with CAP in the absence or presence of CZP. Animals receiving intranigral CAP injection (500 pmol) with or without administration of CZP (500 pmol) were killed 7 d after injection. Brain tissues were cut, and every sixth serial section was immunocytochemically stained with antibodies against TH. TH-i.p. neurons were counted using a stereological technique in the whole SN. Six to eight animals were used for each experimental group. ^*p < 0.01, significantly different from contralateral side; ^§p < 0.01, significantly different from ipsilateral side treated with CAP only.