Receptor-Independent Anti-Ferroptotic Activity of TrkB Modulators

Abstract

Dysregulated brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signalling is implicated in several neurodegenerative diseases, including Alzheimer's disease. A failure of neurotrophic support may participate in neurodegenerative mechanisms, such as ferroptosis, which has likewise been implicated in this disease class. The current study investigated whether modulators of TrkB signalling affect ferroptosis. Cell viability, C11 BODIPY, and cell-free oxidation assays were used to observe the impact of TrkB modulators, and an immunoblot assay was used to detect TrkB expression. TrkB modulators such as agonist BDNF, antagonist ANA-12, and inhibitor K252a did not affect RSL3-induced ferroptosis sensitivity in primary cortical neurons expressing detectable TrkB receptors. Several other modulators of the TrkB receptor, including agonist 7,8-DHF, activator phenelzine sulphate, and inhibitor GNF-5837, conferred protection against a range of ferroptosis inducers in several immortalised neuronal and non-neuronal cell lines, such as N27 and HT-1080 cells. We found these immortalised cell lines lack detectable TrkB receptor expression, so the anti-ferroptotic activity of these TrkB modulators was most likely due to their inherent radical-trapping antioxidant properties, which should be considered when interpreting their experimental findings. These modulators or their variants could be potential anti-ferroptotic therapeutics for various diseases.

Keywords: BDNF; TrkB; agonist; antagonist; ferroptosis; inhibitor; protection.

Figure 1

Activity of TrkB modulators against ferroptosis in mature primary cortical neurons. Immunoblot shows TrkB expression in mature primary cortical neurons (a). GNF-5837 inhibits (while other TrkB modulators do not affect) RSL3-induced ferroptosis in primary cortical neurons (b–e). Cells were co-treated with ferroptosis inducer RSL3 and tested compounds for 24 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n =  9–10 from 2–3 independent experiments. p values were calculated using the two-way ANOVA (Dunnett’s multiple comparisons test, (c) and the unpaired t-test (d), and the significant level is noted as **** p < 0.0001 (vs. control).

Figure 2

Both 7,8-DHF and phenelzine protect ferroptosis in several immortalised cell lines. Efficacy of 7,8-DHF, Lip-1, and DFP against erastin- and RSL3-induced ferroptosis in N27 cells (a,b). Like Lip-1 and DFP, 7,8-DHF significantly prevents ferroptosis in HT-1080 and U-251 cells (c,d). Efficacy of phenelzine against erastin- and RSL3-induced ferroptosis in HT-1080 and N27 cells (e,f). Cells were co-treated with ferroptosis inducers and tested compounds for 24 h. Cell viability was measured by MTT assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n =  12–20 from 3–5 independent experiments. p values were calculated using the two-way ANOVA (Šídák’s multiple comparisons test), and the significant levels are noted as ** p < 0.01 and **** p < 0.0001 (vs. no compound).

Figure 3

Both 7,8-DHF and phenelzine protect against ferroptosis and have RTA properties. Both 7,8-DHF and phenelzine attenuate FINO2- and tBH-induced toxicity (a,b). RTA properties of 7,8-DHF, phenelzine and Lip-1 in cell-free ABTS assay (c). Cells were co-treated with ferroptosis inducers and tested compounds for 24 h, except for tBH, which had a 4 h co-treatment. Cell viability assay was measured by MTT assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n =  9–16 from 3–4 independent experiments. P values were calculated using the two-way ANOVA (Dunnett’s multiple comparisons test), and the significant level is noted as **** p < 0.0001 (vs. control).

Figure 4

GNF-5837 protects ferroptosis in several immortalised cell lines. Comparative anti-ferroptotic activity between GNF-5837 and Lip-1 for erastin- and RSL3-induced ferroptosis following 24 h incubation in different cell lines: N27, HT-22, and HT-1080 cells (a–f). Cell viability was measured by MTT assay. Data points signify mean percentage survival relative to untreated controls ± SEM, n = 11–16 from 3–4 independent experiments.

Figure 5

GNF-5837 protects against ferroptosis likely via its RTA property. Toxicity assay upon 48 h treatment with GNF-5837 in different cell lines (a). GNF-5837 protects against toxicity induced by FINO2, tBH, and iron in N27 cells (b). Like Lip-1, GNF-5837 significantly protects against lipid peroxidation in the C11 BODIPY assay caused by erastin, BSO, and RSL3 in N27 cells (c). RTA property of GNF-5837 and Lip-1 against lipid peroxidation in cell-free MDA assay (d). Cells were co-treated with ferroptosis inducers and tested compounds for 24 h, except for tBH-induced ferroptosis, which had a 4 h co-treatment, and MTT assay measured cell viability. Lipid peroxidation in C11 BODIPY assay is expressed as the ratio of green to red C11 BODIPY fluorescence. Data points signify mean percentage survival relative to respective controls ± SEM (n = 36 for (a), n =  9–12 for (b,c), and n = 6 for (d) from 3–4 independent experiments). P values were calculated using the one-way ANOVA (Tukey’s multiple comparisons test for (b)) and two-way ANOVA (Dunnett’s multiple comparisons test for (c)), and the significant level is noted as **** p < 0.0001 (vs. inducers/control).