# Microglial SLC25A28 Knockout Mitigates Spinal Cord Injury in Mice by Inhibiting Heme Synthesis and Subsequent NOX2 Activation

## TL;DR

Disabling a specific protein in microglia reduces spinal cord injury in mice by limiting harmful inflammation and oxidative stress.

## Contribution

Identifies SLC25A28 as a novel regulator of microglial neuroinflammation via mitochondrial iron and heme metabolism.

## Key findings

- SLC25A28 knockout reduced spinal cord edema, barrier disruption, and motor deficits in mice.
- SLC25A28 deficiency suppressed mitochondrial iron accumulation and NOX2-mediated oxidative stress.
- Pharmacological inhibition of heme synthesis or NOX2 reversed the effects of SLC25A28 overexpression.

## Abstract

Microglial overactivation‐driven neuroinflammation exacerbates secondary damage after spinal cord injury (SCI), but the role of mitochondrial iron metabolism in this process is not well understood. This study investigates the function of the mitochondrial iron transporter solute carrier family 25 member 28 (SLC25A28) in post‐SCI neuroinflammation.

Microglia‐specific SLC25A28 knockout (A28‐MGKO) mice were generated by crossing SLC25A28flox/flox mice with Cx3cr1‐CreERT2 mice and subjected to clip‐compression spinal cord injury (SCI) at the T9 level. Motor recovery was evaluated using the Basso Mouse Scale (BMS), while histological and biochemical assessments including hematoxylin–eosin and Nissl staining, Iba1 immunohistochemistry, Evans blue permeability, and tissue water content were performed to evaluate lesion severity, neuronal survival, microglial activation, and blood–spinal cord barrier integrity. In vitro, primary microglia isolated from A28‐MGKO mice and BV2 cells with SLC25A28 overexpression were used to investigate mitochondrial iron homeostasis, heme biosynthesis, and NOX2‐mediated oxidative stress. Mitochondrial iron content was quantified using a ferrozine‐based assay and Mito‐FerroGreen staining, while ROS production, cytokine release, and inflammatory signaling were analyzed by fluorescence imaging, ELISA, and Western blotting under pharmacological modulation of heme synthesis and NOX2 activity.

We found that SLC25A28 deficiency reduced spinal cord edema, blood‐spinal cord barrier disruption, and motor deficits. Mechanistically, SLC25A28 knockout suppressed mitochondrial iron accumulation, inhibited heme synthesis, and reduced NOX2‐mediated oxidative stress. However, SLC25A28 overexpression enhanced mitochondrial iron overload and NOX2‐driven inflammation, which could be reversed by pharmacological blockade of NOX2 or heme synthesis. Restoration of heme synthesis in A28‐MGKO microglia attenuated the anti‐inflammatory effects of SLC25A28 knockout.

These findings demonstrate that microglial SLC25A28 regulates neuroinflammation and functional recovery after SCI by promoting mitochondrial iron‐dependent heme synthesis and NOX2 activation. Targeting the SLC25A28–heme–NOX2 axis may provide a novel therapeutic approach for SCI.

Microglial SLC25A28 knockout reduces spinal cord injury by inhibiting mitochondrial iron accumulation and heme synthesis, leading to decreased NOX2 activation and neuroinflammation. This study reveals the SLC25A28‐heme‐NOX2 axis as a key regulator of microglial‐driven secondary damage after SCI.