Cannabinol inhibits oxytosis/ferroptosis by directly targeting mitochondria independently of cannabinoid receptors

Abstract

The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway can be used to identify novel drug candidates for the treatment of age-associated neurodegenerative diseases that act by preserving mitochondrial function. Previously, we identified cannabinol (CBN) as a potent neuroprotector. Here, we demonstrate that not only does CBN protect nerve cells from oxytosis/ferroptosis in a manner that is dependent on mitochondria and it does so independently of cannabinoid receptors. Specifically, CBN directly targets mitochondria and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis. These protective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP-activated protein kinase (AMPK) signaling. Together, our data highlight the potential of mitochondrially-targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction as well as opportunities for the discovery and development of future neurotherapeutics.

Keywords: AMPK; Aging; Antioxidant defense; Cannabinoid; Mitochondrial dysfunction; Neurodegenerative disease; Neurotherapeutics; Oxytosis/ferroptosis.

Figure 1.

Total synthesis of cannabinol (CBN) and its CNS druglike properties. Reagents and conditions: (a) n-butylamine, toluene, reflux; (b) iodine, toluene, reflux, 76%. MW, molecular weight; TPSA, topological polar surface area; HBD, hydrogen bond donor; HBA, hydrogen bond acceptor; cLogD, calculated distribution coefficient; cLogP, calculated partition coefficient; pKa, acid dissociation constant.

Figure 2.

CBN inhibits oxytosis/ferroptosis in HT22 cells. Representative micrographs of HT22 cells following treatment for 16 hr: (A) 0.2% ethanol treatment as vehicle control, (B) 5 μM CBN treatment, (C) 5 mM glutamate treatment, (D) 50 nM RSL3 treatment, (E) 5 μM CBN pretreatment for 1 h followed by 5 mM glutamate treatment, (F) 5 μM CBN pretreatment for 1 h followed by 50 nM RSL3 treatment. Micrographs show the representative morphological characteristics of the cell cultures under a given condition of 16 experimental replicates. Scale bar = 100 μm. (G) Cells were pretreated with varying concentrations of CBN for 1 hr followed by 5 mM glutamate treatment and incubation for 16 hr. (H) Cells were pretreated with varying concentrations of CBN for 1 hr followed by 50 nM RSL3 treatment and incubation for 16 hr. The results are presented as the percentage of the neuroprotective activity relative to control (100%) and Glu/RSL3 (0%). The neuroprotection curves were analyzed by four-parameter regression. (I) Cytotoxicity assessment of CBN in HT22 cells. Cells were treated with increasing concentrations of CBN or 0.2% ethanol vehicle and incubated for 16 hr. Data are the mean of 8–16 replicates per condition ± SD.

Figure 3.

CBN prevents oxidative stress induced during oxytosis/ferroptosis in HT22 cells. (A) Cellular ROS levels upon different treatment conditions in the cells for 16 hr. Data were normalized to total protein/well and are the mean of 16 replicates per condition ± SD. (B) Mitochondrial ROS levels upon different treatments in the cells for 16 hr. Data were normalized to total protein/well and are the mean of 16 replicates per condition ± SD. (C) Cellular lipid peroxidation levels upon different treatment conditions of the cells for 16 hr. Data were normalized to total protein/well and are the mean of 12 replicates per condition ± SD. (D) Time course of lipid peroxidation levels following different treatment conditions in a cell-free system. Data are the mean of 4 replicates per condition ± SD. (E) Trolox equivalent antioxidant capacities (TEAC) following different treatment conditions in a cell-free system. Data are the mean of 8 replicates per condition ± SD. The results are presented as the percentage of the TEAC relative to Trolox (100%). (F) Iron (II) binding capacities following different treatment conditions in a cell-free system. Data are the mean of 8 replicates per condition ± SD. The results are presented as the percentage relative to the maximum (vehicle without iron, 100%) and minimum (vehicle with iron only, 0%) Fe²⁺ binding capacity. (G) Total GSH levels upon different treatment conditions of the cells for 16 hr. Data are the mean of 4 replicates per condition ± SD. (H) Western blot data of Nrf2, ATF4, HO-1, SOD2, GPX4, HSP60, and actin (n = 3–6). Protein levels were measured upon different treatment conditions of the cells for 16 hr. (I) Densitometric quantification of the Western blots. Data were normalized to actin and are the mean ± SD. All data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 relative to vehicle control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to the 50 nM RSL3 treatment; ns, not significant.

Figure 4.

CBN prevents Ca²⁺ influx induced by oxytosis/ferroptosis in HT22 cells. (A) Cellular Ca²⁺ levels upon different treatment conditions of the cells for 16 hr. Data were normalized to total protein/well and are the mean of 16 replicates per condition ± SD. (B) Mitochondrial Ca²⁺ levels upon different treatment conditions of the cells for 16 hr. Data were normalized to total protein/well and are the mean of 16 replicates per condition ± SD. (C) Western blot data of MCU and actin (n = 3–6). Protein levels were measured following different treatments of the cells for 16 hr. (F) Densitometric quantification of the Western blots. Data were normalized to actin and are the mean ± SD. All data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 relative to vehicle control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to the 50 nM RSL3 treatment.

Figure 5.

CBN preserves mitochondrial bioenergetics following induction of oxytosis/ferroptosis. (A) Mitochondrial oxygen consumption rate (OCR) profiles in HT22 cells after different treatments for 16 hr. Data were normalized to total protein/well and are the mean of 20 replicates per condition ± SD. (B) Graphs for basal respiration, maximal respiration, and ATP production in HT22 cells. (C) Mitochondrial oxygen consumption rate (OCR) profiles in primary cortical neurons after different treatments for 16 hr. Data were normalized to total protein/well and are the mean of 20 replicates per condition ± SD. (D) Graphs for basal respiration, maximal respiration, and ATP production in primary cortical neurons. (E) Mitochondrial membrane potential in HT22 cells after different treatments for 4 hr. Data are the mean of 8–12 replicates per condition ± SD. (F) Relative mtDNA copy number in HT22 cells after different treatments for 16 hr. Data are the mean of 3 replicates per condition ± SD. (G) Western blot data of ETC complex proteins (ATP5A, MTCO1, UQCRC2, SDHB, NDUFB8), TOM20, VDAC, and actin (n = 3–6). Protein levels were measured following different treatments of the cells for 16 hr. (H) Densitometric quantification of the Western blots. Data were normalized to TOM20 and are the mean ± SD. All data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 relative to vehicle control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to the 50 nM RSL3 treatment; ns, not significant.

Figure 6.

CBN stimulates mitochondrial biogenesis following induction of oxytosis/ferroptosis in HT22 cells. Representative fluorescent images of HT22 cells following treatment for 16 hr: (A) 0.2% ethanol treatment as vehicle control, (B) 5 μM CBN treatment, (C) 50 nM RSL3 treatment, (D) 5 μM CBN pretreatment for 1 hr followed by 50 nM RSL3 treatment. Mitochondria stained with MitoTracker (red); nuclei stained with Hoechst 33342 (blue). Enlarged images from the boxed areas are indicated. The micrographs show representative morphological characteristics of the cells under the different conditions with 4 experimental replicates per condition. Scale bar = 200 μm. (E) Relative quantification of mitochondrial mass with MitoTracker in HT22 cells following different treatment conditions for 16 hr. Data were normalized to total protein/well and are the means of 16 replicates per condition ± SD. (F) Protein levels of VDAC relative to actin were measured following the different treatments of the HT22 cells for 16 hr using immunoblotting. Data are the means of 3–6 replicates per condition ± SD. (G) Western blot data of SIRT1, pAMPKα (Thr172), total AMPKα, PGC-1α, NRF1, TFAM, and actin (n = 3–6). Protein levels were measured following different treatments of the cells for 16 hr. (H) Densitometric quantification of the Western blots. Data were normalized to actin and are the mean ± SD. All data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 relative to vehicle control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to the 50 nM RSL3 treatment; ns, not significant.

Figure 7.

CBN modulates mitochondrial fusion/fission dynamics following induction of oxytosis/ferroptosis in HT22 cells. Representative fluorescent images of HT22 mt-GFP cells following treatment for 16 hr: (A) 0.2% ethanol treatment as vehicle control, (B) 5 μM CBN treatment, (C) 50 nM RSL3 treatment, (D) 5 μM CBN pretreatment for 1 hr followed by 50 nM RSL3 treatment. Enlarged images from the boxed areas are indicated in the bottom panels. The micrographs show representative images of the morphological characteristics of the cells under the different conditions with 4 experimental replicates per condition. Scale bar = 10 μm. (E) Mitochondrial network morphology analyses on micrographs of HT22 cells after different treatments: (E) mitochondrial footprint, (F) mitochondrial branch length, (G) mitochondrial summed branch lengths, (H) mitochondrial network branches. Data are the mean of 20–25 cells per condition ± SD. (I) & (J) Western blot data of OPA1, MFN2, DRP1, MFF, and actin (n = 36). Protein levels were measured following the different treatments of the cells for 16 hr. (K) Densitometric quantification of the Western blots. Data were normalized to actin and are the mean ± SD. All data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 relative to vehicle control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to the 50 nM RSL3 treatment; ns, not significant.

Figure 8.

CBN requires functional mitochondria for protection against oxytosis/ferroptosis in HT22 cells. (A) & (B) Representative fluorescent images of HT22 mt-GFP WT and mt-GFP/mCherry-Parkin cells following the different treatment conditions. GFP-labeled mitochondria (green), mCherry-Parkin (red). FCCP-induced mitophagy in mt-GFP/mCherryParkin cells but not in mt-GFP cells. The micrographs show representative images of the morphological characteristics of the cells under the different conditions with 12 experimental replicates per condition. Scale bar = 50 μm. (C), (D) & (E) DNA-based cell viability analysis of HT22 mt-GFP or mt-GFP/mCherry-Parkin cells following the different treatment conditions. Data are the mean of 8–12 replicates per condition ± SD. All data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. ####p < 0.0001 relative to vehicle control; ****p < 0.0001 relative to the 50 nM RSL3 treatment; ^^^^p < 0.0001 relative to the 5 μM FCCP treatment; ^●●●●p < 0.0001 relative to the FCCP (5 μM) + RSL3 (50 nM) treatment; ns, not significant.

Figure 9.

Proposed neuroprotective mechanisms of CBN against oxytosis/ferroptosis through directly targeting mitochondria to restore multiple key parameters of mitochondrial function. Oxytosis/ferroptosis induced by RSL3 (red arrows) causes an increase in mitochondrial oxidative stress and calcium overload, disturbance of mitochondrial membrane potential, and decrease in mitochondrial bioenergetics, biogenesis, and fusion/fission dynamics. CBN protects neuronal cells through counteracting all these six aspects of mitochondrial dysfunction (green arrows).