Inhibition of Ferroptotic Toxicity by 4-Hydroxyindole.

To evaluate the effects of 4-HI on ferroptosis, we tested various doses of 4-HI against ferroptosis induced by different agents. In HT-22 cells, we used inducers such as erastin, RSL3, and FINO2. For N27 cells, we utilized RSL3 and FINO2 ( Figure 1A , B ). In our recently published article, we demonstrated that the standard ferroptosis inhibitor, Lip-1, effectively rescues cells from ferroptosis induced by erastin, RSL3, and FINO2. 9 This confirms that these compounds induce ferroptosis in HT-22 and N27 cells. Therefore, we have decided not to include the Lip-1 rescue data in this manuscript. Additionally, we identified the potential antiferroptotic activity of 4-HI in RBE4 cells; however, we were limited to using RSL3 as the ferroptosis inducer because other inducers, such as erastin and FINO2, were ineffective in promoting ferroptosis in this cell type ( Figure 2A , B ). Our results demonstrate that 4-HI protects against ferroptosis across multiple cell lines, including HT-22, N27, and RBE4 ( Figure 1A – C ). The lowest EC50 value of 4-HI was identified as 4.8 μ M against RSL3 toxicity in N27 cells, while the highest EC50 value of 8.8 μ M was observed against RSL3 in HT-22 cells ( Figure 1A , B ). The antiferroptotic activity of 4-HI was further validated through cell death (LDH) and lipid peroxidation (C11-BODIPY) assays. In these experiments, we treated cells with a specific concentration of erastin and RSL3 to induce cell death and lipid peroxidation. The increased release of LDH indicated cell death, while the heightened C11-BODIPY signal reflected lipid peroxidation caused by the ferroptosis inducers erastin and RSL3. Notably, this increase was significantly protected when the cells were treated with 25 μ M of 4-HI ( Figure 1D , E ). We investigated the primary mechanisms of how 4-HI inhibits ferroptosis. Considering the role of hydroxyindoles, such as 3-Hydroxyindole (3-HI), which act as RTAs, we hypothesized that 4-HI protects cells from ferroptosis by functioning as an RTA. First, we confirmed that 4-HI effectively neutralizes ABTS, demonstrating its RTA properties ( Figure 3A ). Next, we explored the RTA effects of 4-HI using another assay in which we employed AAPH to oxidize C11-BODIPY ( Figure 3B ). For comparison, we used Lip-1 as a positive control, which is known to inhibit AAPH-induced C11-BODIPY oxidation ( Figure 3C ). Similar to Lip-1, we found that 4-HI also prevents the oxidation of C11-BODIPY induced by AAPH ( Figure 3B – D ). Notably, 4-HI was observed to be more potent than Lip-1 ( Figure 3D ). Since neither the ABTS nor AAPH-induced C11-BODIPY assays included lipids, we conducted an additional assay using iron to oxidize the lipid arachidonic acid, generating lipid peroxidation products that can enhance C11-BODIPY oxidation. In this assay, we noted that iron combined with arachidonic acid leads to C11-BODIPY oxidation. This oxidation was significantly protected when cotreated with either 4-HI or the standard compound, Lip-1 ( Figure 3E – G ). Nevertheless, 4-HI appeared to be less effective against iron and arachidonic acid-induced C11-BODIPY oxidation compared to Lip-1 ( Figure 3G ). We investigated how structural modifications of 4-HI affect its antiferroptotic activity. 4-HIC is a structural analog of 4-HI, characterized by a carbaldehyde group at the 3-position of the 4-HI structure. This compound is also a plant metabolite found in Capparis spinosa L. and is used in the synthesis of a fluorescent probe. 14 , 15 In our findings, we observed that a concentration of 50 μ M of 4-HIC did not provide protection against toxicity induced by eastin and RSL3 in HT-22 cells ( Figure 4A , B ). We further tested higher concentrations of 4-HIC to see if increased amounts would affect ferroptosis. However, we discovered that even at higher concentrations, 4-HIC did not protect against the toxicity caused by the ferroptosis inducer RSL3 ( Figure 4C ). In addition to the cytotoxicity assay, we assessed whether 4-HIC could inhibit BODIPY oxidation induced by iron and arachidonic acid. Consistent with the cytotoxicity assay results, we found that 4-HIC did not protect against C11-BODIPY oxidation induced by the combination of iron and arachidonic acid ( Figure 4D ).

RPMI 1640 (Cat#MT10041CM) from Corning Inc., USA; penicillin and streptomycin (Cat# 15140122), amphotericin B (Cat#15290026), fetal bovine serum (FBS; Cat#A5256801), phosphate-buffered saline (PBS; Cat#10010023), and Hank’s balanced salt solution (HBSS; Cat# 14–175–079) from Gibco, part of Thermo Fisher Scientific, Waltham, MA, USA; calcein AM (Cat# C1430) from Invitrogen, also part of Thermo Fisher Scientific; 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH; Cat#401560250), ammonium iron(II) sulfate hexahydrate (Iron; Cat#201370250), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Cat#J65535.03), and potassium persulfate (Cat# 202010250) from Thermo Fisher Scientific. Liproxstatin 1 (Lip-1; Cat#S7699), and RSL3 (Cat# S8155) were from Selleck Chemicals LLC, Houston, TX, USA; 4-HI (Cat#sc-sc-216890, purity ≥98%; CAS no. 2380–94–1) was obtained from Santa Cruz Biotechnology, Inc., Dallas, Texas, USA; erastin (Cat#329600), rotenone (Cat#R8875), BODIPY 581/591 C11 (Cat#SML3717) and IGEPAL CA-630 (Cat#I8896) were obtained from Sigma-Aldrich Pty Ltd., an affiliate of Merck KGaA, Darmstadt, Germany. 4-Hydroxyindole-3-carbaldehyde (4-HIC; Cat#AMBH95E088BF, purity 95%; CAS no. 81779–27–3) from Ambeed Inc. (a partner of Sigma), Illinois, USA. Arachidonic acid (Cat#ICN19462510) from MP Biomedicals, CA, US. Cytotoxicity Detection KitPLUS (LDH; Cat#4744926001) was from Roche. Both 4-HI and 4-HIC were dissolved in DMSO to create a stock solution. These compounds were protected from light to ensure their stability, and the final concentration of DMSO in the vehicle was maintained at less than 0.5%. In this study, three immortalized cell lines were used: the HT-22 mouse hippocampal cell line, which was subcloned from the HT-4 cell line; the N27 rat dopaminergic neural cell line, derived from E12 rat mesencephalic tissue; and the RBE4 rat brain endothelial cells. The sources of the N27 and HT-22 cell lines are detailed in a previous article. 9 Dr. Wei Zheng from Purdue University, USA, generously provided the RBE4 cell line. All cell lines were cultured in RPMI 1640 media supplemented with 10% FBS and 1% penicillin-streptomycin. Cell-based assays were conducted using RPMI 1640 media with the same supplements, except for Figure 1D , where the media was additionally supplemented with amphotericin B (an antifungal) at a concentration of 0.5–1 μ g/mL. The calcein AM assay was utilized to assess cell viability after treatment. This assay uses calcein AM, a nonfluorescent compound that can penetrate the membranes of living cells. Once inside the cell, calcein AM is converted by esterases into calcein, which is a fluorescent compound that remains in the cytoplasm. The levels of fluorescence are directly proportional to the number of viable cells. 9 In the experiments, cells were seeded at a density of 1.5 × 10 4 per well in 96-well plates and cotreated with various compounds and ferroptosis inducers for 24 h. After the treatment, the cells were washed with HBSS and then incubated for 30 min to 1 h with a 1 μ M solution of calcein AM in HBSS. Following the incubation, cell viability was measured fluorometrically using a Molecular Devices SpectraMax M2e, with the excitation set at 494 nm and emission at 517 nm. The percentages of viable cells were calculated relative to the control group. The lactate dehydrogenase (LDH) release assay was performed using the Cytotoxicity Detection Kit to measure cell death, as described previously. 9 This assay evaluated LDH levels in culture supernatants, indicating changes in cell permeability. Cells were seeded and treated according to the cell viability protocol. After the treatment, 100 μ L of supernatant was transferred to a 96-well plate and mixed with 100 μ L of assay reagent. This mixture was incubated in the dark for 30 min, and then the absorbance was measured at 492 and 600 nm using a Molecular Devices SpectraMax M2e Reader. DMSO-treated cells released LDH spontaneously, while Triton X-100 resulted in maximum LDH release. The percentage of LDH release was calculated using the following formula: % of LDH release = (experimental value – spontaneous release)/(maximum release – spontaneous release) × 100. Intracellular lipid peroxidation was measured using the fluorescent probe C11-BODIPY 581/591, as previously described. 9 – 12 This probe displays an increase in green fluorescence and a decrease in red fluorescence in response to lipid peroxidation. To conduct this experiment, cells were seeded according to the cell viability experiment and treated simultaneously with the tested compounds and a ferroptosis inducer in a medium containing 2.5 μ M of C11-BODIPY 581/591. After washing the cells with PBS, they were supplemented with additional PBS, and fluorescence was measured using a Synergy H1 microplate reader. Red fluorescence was recorded at 565/600 nm, while green fluorescence was recorded at 477/525 nm. The level of lipid peroxidation was quantified as the ratio of green to red fluorescence. We performed several assays to evaluate the potential RTA activity of the tested compound, including the ABTS assay, AAPH-induced C11-BODIPY oxidation, and iron + AA-induced C11-BODIPY oxidation. The RTA properties of the tested substances were assessed by using the colorimetric ABTS radical scavenging assay. 9 – 12 The procedure involved mixing the ABTS reagent with potassium persulfate to generate free radicals, followed by dilution of the solution to achieve an absorbance of 0.3 to 0.6 at 734 nm. Next, the ABTS reagent (100 μ L) was combined with the sample (100 μ L) in a 96-well microplate and incubated for 7 to 60 min before measuring absorbance at 734 nm using a Molecular Devices SpectraMax M2e. The assay measures the conversion of dark blue ABTS radical cation to colorless ABTS by antioxidants. PBS served as a solvent, while DMSO was used as a control. The formula used to evaluate the ABTS radical scavenging properties of the tested compound is as follows: ABTS radical scavenging (%) = [(Control absorbance – Sample absorbance)/Control absorbance] × 100. AAPH, a free radical initiator, was used to induce the oxidation of C11-BODIPY and evaluate the RTA activity of the tested compound, based on a previously published protocol 13 with slight modifications. A compound with RTA activity can inhibit this oxidation. We prepared solutions of the tested drug at various concentrations, along with C11-BODIPY (2 μ M) and AAPH (20 mM) in PBS. In a 96-well plate, we combined 50 μ L of C11-BODIPY, 50 μ L of AAPH, and 50 μ L of each drug concentration, and then incubated the mixture at room temperature in the dark for 30 min. Fluorescence was measured at 477/525 nm using a Synergy H1 microplate reader. DMSO served as a control. The inhibition of C11-BODIPY oxidation by the tested compound was assessed using the following formula: AAPH-induced C11-BODIPY oxidation (%) = [(Control value – Sample value)/Control value] × 100. Cell-free assays, such as ABTS and AAPH-induced oxidation of C11-BODIPY, can identify potential RTA activity of a tested compound. One limitation of these assays is that they do not require lipids. We recently developed a new assay to address this issue, 12 following previously described protocols. 11 , 13 In the newly developed assay, we use iron to react with AA to produce lipid-derived ROS that oxidize C11-BODIPY. We prepared solutions of the tested drug at various concentrations, C11-BODIPY (2 μ M) and iron (20 μ M) + AA (100 μ M), all dissolved in PBS. We then mixed 50 μ L of the C11-BODIPY solution, 50 μ L of the Iron + AA solution, and 50 μ L of the tested compound at the desired concentration in a 96-well plate. The mixture was incubated for 30 min at 37 °C, protected from light. Following incubation, fluorescence was measured at 477/525 nm (excitation/emission) using a Synergy H1 microplate reader, BioTek Instruments (Winooski, USA). DMSO was used as a control. The inhibition of C11-BODIPY oxidation by the tested compound was assessed using the following formula: Iron + AA-induced C11-BODIPY oxidation (%) = [(Control value – Sample value)/Control value] × 100. Statistical analysis was performed using Microsoft Excel 365 and GraphPad Prism 10. We utilized nonlinear regression with a variable slope model to fit a logistic curve to the dose-response data, allowing us to determine EC 50 values along with 95% confidence intervals. EC50 values were not reported if the effectiveness was below 50%. For comparisons between two groups, we used an unpaired t test, while one-way ANOVA followed by Dunnett’s post hoc analyses was employed for comparisons involving three or more groups. A p-value of less than 0.05 was considered statistically significant.

We demonstrated that 4-HI inhibits ferroptosis by functioning as a ROS scavenger ( Figure 5 ). The unique structural properties of 4-HI may account for its ability to prevent ferroptosis. Notably, an analog of 4-HI, designated as 4-HIC, which contains an additional carbaldehyde group compared to the structure of 4-HI, lacks protective effects against ferroptosis. Overall, the antiferroptotic property makes 4-HI a promising candidate for further study as a potential neuroprotective therapy in the treatment of neurodegenerative diseases.

The role of 4-HI in modulating sensitivity to ferroptosis was unknown. This study has identified a role for 4-HI in protecting cultured cells from ferroptosis. Moreover, we have utilized a new cell-free assay that we developed, which has advanced our understanding of how ferroptosis modulators function. Overall, these technical and theoretical advances enhance our understanding of hydroxyindole compounds, particularly in investigating the role of 4-HI in relation to ferroptosis. The protective effects of 4-HI against various inducers of ferroptosis align with our previous research, 9 which demonstrated that hydroxyindole compounds, such as 3-HI, 6-HI, and 7-HI, can protect cells from ferroptotic toxicity. Although 4-HI was found to be less potent than 3-HI, it was more effective than both 6-HI and 7-HI in HT-22 cells. In addition, 4-HI was found to inhibit amyloid fibril formation and also protect against amyloid β toxicity, 6 but it was unknown how this compound protects against amyloid toxicity. Based on our findings, the RTA mechanism of 4-HI can contribute to its protective effect against amyloid β toxicity, which needs further investigation. In our previous study, we demonstrated that analogs of 4-HI, including 3-HI, 6-HI, and 7-HI, did not protect cells from rotenone-induced nonferroptotic toxicity. 9 This indicates that their protective effect is specific to ferroptosis. Using three different assays, we showed the RTA effect of 4-HI, which most likely explains its protective role against ferroptosis in this study. Two important structural features of 4-HI—the indole ring and the hydroxyl group—are likely contributors to its RTA effect. The indole ring can donate electrons, while the hydroxyl group can provide hydrogen atoms (protons) to free radicals. 16 , 17 Together, these features stabilize free radicals and protect cells from ferroptotic damage. Since 4-HI is less potent than 3-HI in inhibiting ferroptosis, it is possible that changes in the position of the hydroxyl group could affect the antiferroptotic activity of hydroxyindole compounds. While we presented the RTA effect as the primary mechanism for inhibiting ferroptosis by 4-HI, we do not exclude other potential effects, such as iron chelation, that could also contribute to the antiferroptotic effect of 4-HI. To further demonstrate that the structural organization of 4-HI is responsible for its antiferroptotic effect, we tested 4-HIC, an analog of 4-HI that contains an additional functional group: carbaldehyde. The carbaldehyde group is generally considered an electron-withdrawing group, and its presence in an antioxidant molecule can impact the compound’s antioxidant activity. It is possible that the carbaldehyde group reduces the electron-donating ability of the hydroxy group, causing 4-HIC to lose its antioxidant activity. Our study, along with others, demonstrates that 4-HI has three major effects: antiferroptotic, anti-inflammatory, and antiamyloid properties. 1 , 6 , 9 These properties make 4-HI a valuable candidate for neuroprotective therapy for neurodegenerative diseases such as Alzheimer’s disease. In addition, the scaffold of 4-HI could be used for synthesizing a more potent neuroprotective candidate. Our study focused specifically on 4-HI and the compound identified as 4-HIC. However, other compounds and metabolites that share a 4-HI structure could also be investigated for their potential effects on ferroptosis modulation and neuroprotection. One common example of a drug that contains a 4-HI scaffold is psilocin, a tryptamine alkaloid and serotonergic psychedelic. Psilocybin also belongs to the same class as psilocin and is a prodrug of psilocin. Psilocybin can be absorbed into the bloodstream and transported to the liver, where enzymes known as phosphatases break it down into psilocin through a process called dephosphorylation. These two psychedelics were found to have anti-inflammatory activity 18 and enhance neural plasticity. 19 – 21 In addition, psilocybin was found to protect against ischemic brain damage in rats 22 and is currently being tested for treating neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases ( https://clinicaltrials.gov/study/NCT04123314 and NCT06455293 ). It will be interesting to test these psychedelics to see whether they can exert a better effect compared to 4-HI.

4-Hydroxyindole (4-HI) is a chemical compound characterized by a hydroxyl group attached to the fourth position of an indole ring (six-membered benzene ring fused to a five-membered pyrrole ring). It belongs to a class of organic compounds known as hydroxyindoles. Like other hydroxyindole compounds, such as 5-hydroxyindole (5-HI), 4-HI occurs naturally in biological molecules and serves as a building block for pharmaceutical products and industrial polymers. This compound can be found in plants, including Solanum lycopersicum (tomato), and is also produced by gut bacteria. 1 – 3 Moreover, 4-HI is a component of naturally occurring tryptamine alkaloid psilocin, which is known for its psychedelic effects and is present in certain mushrooms, commonly referred to as magic mushrooms. 4 Recently, 4-HI has been identified as a potential biomarker for endometriosis; 1 studies have shown that the level of 4-HI is lower in the stool of women with this condition. Interestingly, 4-HI has been found to inhibit both the onset and progression of endometriosis. 1 Similar to other hydroxyindoles, such as 3-hydroxyindole (3-HI) and 5-HI, 5 , 6 4-HI is also recognized for its protective effects against toxicity. It has been shown to protect dose-dependently (at concentrations of 5, 25, and 50 μ M) against amyloid β -induced toxicity in PC12 cells (a cell line from pheochromocytoma of the rat adrenal medulla), with protective effects comparable to those of 3-HI. 6 Ferroptosis is a type of nonapoptotic cell death caused by phospholipid peroxidation, which has been implicated in neurotoxicity and neurodegeneration. 7 , 8 Identifying molecules that can inhibit ferroptosis is essential for developing potential therapies for neurodegenerative diseases. In our recent study, we demonstrated that hydroxyindole compounds, including 3-HI, 5-HI, 6-hydroxyindole (6-HI), and 7-hydroxyindole, protect neuronal cells from ferroptotic toxicity. 9 Notably, 3-HI was found to have potent protective effects against ferroptosis compared to other hydroxyindole compounds. 9 However, it remains unclear whether 4-HI exhibits similar protective effects against ferroptosis. Given that 4-HI demonstrates protective effects against amyloid β toxicity, it is also uncertain whether 4-HI offers similar protection against ferroptosis-mediated toxicity, as seen with 3-HI. This study aimed to test the hypothesis that 4-HI protects against ferroptosis, akin to other hydroxyindoles such as 3-HI, and aims to elucidate the mechanism by which 4-HI exerts its protective effects against ferroptotic toxicity.