Salicylaldehyde Benzoylhydrazone Protects Against Ferroptosis in Models of Neurotoxicity and Behavioural Dysfunction, In Vitro and In Vivo

Abstract

Metal dyshomeostasis in the brain is a key feature of many neuropathologies, including hypoxic and traumatic injury and chronic conditions such as Alzheimer's and Parkinson's disease. Ferroptosis is a form of cell death driven by the intracellular accumulation of iron. This is primarily characterised by a loss in endogenous antioxidant capacity and uncontrolled lipid peroxidation. Ferroptosis has been reported to underlie the pathology associated with several neurological and neurodegenerative conditions and has, therefore, become an attractive target for therapeutic intervention. Salicylaldehyde benzoylhydrazone (SBH) is a specialised hydrazone agent, known for its antibacterial and anticancer properties. It has robust metal-chelating capacity, with a particular affinity for complexing with iron and copper. The current study sought to investigate the potential of SBH to act as an anti-ferroptotic agent and to alleviate the neurotoxic and dysfunctional consequences of iron overload. We demonstrate that SBH can alleviate the death of HT22 hippocampal neurons, induced by exposure to the iron donor, ferric ammonium citrate (FAC). This was accompanied by a reduction in intracellular iron and lipid peroxidation, and alleviation of hallmark changes in gene expression indicative of ferroptosis. Using FAC-incubated zebrafish larvae as an in vivo model of iron overload, we reveal that SBH can reduce the mortality and toxicity associated with FAC exposure. Moreover, we report a FAC-mediated dysfunction in intrinsic sensorimotor reflex behaviour, which is restored by SBH. Taken together, our findings highlight SBH as an anti-ferroptotic agent and support its further investigation as a potential neurotherapeutic for conditions associated with iron dysregulation.

Keywords: Ferric ammonium citrate; HT22 cells; Iron overload; Lipid peroxidation; Schiff base; Zebrafish larvae.

Fig. 1

SBH significantly alleviates FAC-induced loss of neuronal cell viability and cytotoxicity. Cell viability (A, C, E) and cytotoxicity (B, D, F) were evaluated in HT22 hippocampal neuronal cells exposed to either SBH alone (0–100 µM; A, B), FAC (75 µM; C, D) or TBHP (200 µM; E, F) for 24 h, in the presence of SBH (10 µM) or DMSO as a vehicle control. Data is presented as mean ± SEM. *p < 0.05, ****p < 0.0001, one- or two-way ANOVA followed by Bonferroni post hoc analysis. n = 12 replicates from 4 independent experiments

Fig. 2

SBH attenuates markers of ferroptosis. HT22 hippocampal neurons were exposed to FAC (75 µM) for 24 h, in the presence and absence of SBH (10 µM) or ChB (50 µM). Cell viability was assessed by CCK-8 assay (A). Iron accumulation (B) and lipid peroxidation (C) were determined in HT22 cell protein lysates. The mRNA expression of Gpx4 (D), Hmox1 (E), and Acsl4 (F) was determined as key indicators of ferroptosis. Data is presented as mean ± SEM. ****p < 0.0001, two-way ANOVA and Bonferroni analysis. n = 9 replicates from 3 independent experiments

Fig. 3

FAC induces a loss of morphological and behavioural integrity in zebrafish larvae in vivo. Zebrafish larvae (4 dpf) to FAC (0–600 µM) for 24 h. Individual larvae were assessed for survival (A), incidence of non-lethal malformations (B) and touch startle response (C). Data presented as mean ± SEM (A) or as a proportion of total larvae (B, C). Statistical comparisons were made by one-way ANOVA or Chi-squared analysis. **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 45 larvae from 3 independent experiments

Fig. 4

SBH significantly protects against FAC-induced toxicity in zebrafish larvae in vivo. Zebrafish larvae (4 dpf) were injected i.v. with SBH (10 µM) or vehicle, in the presence and absence of bath-applied FAC (600 µM; 24 h). Larvae were assessed for survival (A), incidence of non-lethal malformations (B) and touch startle response (C). Data presented as mean ± SEM (A) or as a proportion of total larvae (B, C). Statistical comparisons were made by two-way ANOVA or Chi-squared analysis. **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 45 larvae from 3 independent experiments