Iron-handling solute carrier SLC22A17 as a blood–brain barrier target after stroke

The pathophysiology of ischemic stroke is complex and multifactorial, involving various forms of cell death such as apoptosis, autophagy, and necrosis. A recent study suggests that oxidative and inflammatory stress can induce ferroptosis, a specialized form of cell death characterized by the accumulation of lipid peroxides dependent on intracellular iron overload (Li and Jia, 2023). Although clinical trials have explored iron chelators—agents that bind to free iron and reduce its availability—as potential therapies for ischemic stroke to mitigate ferroptosis, a recent mechanistic study has focused on neuronal cells (Hanafy et al., 2019). Brain endothelial cells, which are essential for iron transport and homeostasis in the brain, particularly after stroke, express various iron transporters such as transferrin receptors, divalent metal transporter 1, and ferroportin to manage iron levels (Chen et al., 2022). This suggests that ferroptosis may play a significant role in brain vascular injury. Our recent study identified the iron-handling solute carrier protein solute carrier family 22 member 17 (SLC22A17) as part of the cell-death protein–protein interaction network, specifically linked to ferroptosis and uniquely enriched in the brain based on human tissue–specific expression profiling (Li et al., 2024). Re-analysis of genome-wide association studies indicated a potential association between SLC22A17 and large vascular stroke. SLC22A17 functions as a high-affinity receptor for lipocalin 2, a gene implicated in ferroptosis-related mechanisms associated with hypoxic-ischemic brain damage. Previous findings reveal that SLC22A17 modulates the expression of the proapoptotic protein Bcl-2-like protein 11, thereby influencing apoptosis through its regulatory effects on intracellular iron concentration (Devireddy et al., 2005). This highlights significant crosstalk among various cell death pathways. Under normal conditions, SLC22A17 is primarily localized in neurons. However, its expression significantly increases in brain endothelial cells during the acute phase of both human and rodent stroke tissue. This upregulation coincides with elevated expression of ferroptosis markers such as acyl-CoA synthetase long chain family member 4 and prostaglandin-endoperoxide synthase 2 in brain endothelial cells. Concurrently, levels of tight junction proteins zonula occludens-1 (ZO-1) and Claudin-5 in cerebral microvessels are reduced, indicating compromised blood–brain barrier (BBB) integrity. In an in vitro BBB injury model, the iron chelator deferoxamine protects trans-endothelial integrity (Rand et al., 2021), suggesting that early induction of ferroptosis in brain microvessels may contribute to BBB dysfunction during the early phases following stroke. In our in vitro BBB injury model, silencing SLC22A17 protected against tumor necrosis factor alpha (TNF-α)–induced human brain endothelial permeability (Li et al., 2024). Furthermore, knocking down SLC22A17 effectively suppressed TNF-α–induced upregulation of ferroptosis and decreased levels of tight junction proteins. However, beyond the BBB-specialized tight junctions that prevent paracellular leakage, brain endothelial cells also limit transcellular transport (transcytosis) through specific transporter–mediated vesicle trafficking, which is dysregulated after stroke injury or inflammatory stress. Whether SLC22A17 silencing also affects transcytosis pathways requires further investigation. Additionally, iron-transport-related proteins do not function in isolation. Therefore, it is crucial for future research to explore whether knocking out SLC22A17 will lead to compensatory changes in other transport proteins. For example, the transferrin receptor, identified from our cell-death protein–protein interaction network, is a transmembrane protein mainly responsible for transporting transferrin-bound iron into cells and is a key component of iron metabolism. Assessing the impact of such changes on post-stroke BBB dysfunction will provide a more comprehensive understanding of iron’s role in BBB function. Our investigation unveils a novel facet of SLC22A17 dynamics under inflammatory stress, showing its intracellular translocation to the nucleus. This discovery suggests that SLC22A17 may play a regulatory role in gene expression. Notably, through chromatin immunoprecipitation assays following TNF-α stimulation, we identified SLC22A17 binding to the promoters of Claudin-5 and ZO-1, indicating a potential transcriptional repression mechanism. Similar to the dual function of ferritin, which has been traditionally considered a cytoplasmic iron storage protein, ferritin undergoes nuclear localization in response to inflammatory stimuli, specific stages of cell development, and chemokine induction (Alkhateeb and Connor, 2010). Specifically, H-ferritin shows nuclear localization in hepatocytes, corneal epithelial cells, cancer cells, and developing neurons (Alkhateeb and Connor, 2010). These observations suggest new roles for ferritin beyond iron storage, such as regulating iron accessibility to nuclear components, protecting DNA from iron-induced oxidative damage, and transcriptional regulation (Alkhateeb and Connor, 2010). The nuclear localization of SLC22A17 implies its capacity to dynamically respond to cellular cues. Thus, further elucidation of SLC22A17’s role as a transcriptional repressor of tight junction genes promises significant insights into its regulatory mechanisms. Finally, we conducted loss-of-function experiments to assess the impact of blocking SLC22A17 on BBB function after stroke. Intracerebroventricular injection of SLC22A17 siRNA efficiently reduced SLC22A17 expression in both whole-brain homogenates and the striatum, concomitant with the increased levels of ZO-1 and Claudin-5 (Li et al., 2024). Following middle cerebral artery occlusion surgery, mice with down-regulated SLC22A17 showed increased expression of ZO-1 and Claudin-5 in the post-ischemic brain, correlating with reduced BBB permeability as indicated by 10-kDa dextran vascular leakage and decreased brain edema measured by water content. While SLC22A17 is predominantly expressed in neurons under normal conditions, potentially influencing BBB permeability regulation, knockdown experiments in human neurons revealed no significant impact on neuronal viability under normal conditions or oxygen-glucose deprivation-induced cell death (Li et al., 2024). Recent evidence suggests SLC22A17 expression on macrophages (Czech et al., 2024), prompting further investigation into its expression on microglia. Additionally, SLC22A17 can be induced in reactive astrocytes under specific conditions (Bi et al., 2013). Our siRNA-mediated knockdown of SLC22A17 was not endothelial cell-specific, underscoring the need for further exploration to determine whether interfering with SLC22A17 expressed in astrocytes or microglia contributes to BBB function recovery. Future studies employing cell-specific knockout models will be crucial to delineate the specific roles of SLC22A17 across different cell types. While targeting SLC22A17 effectively reduces BBB permeability and brain edema following cerebral focal ischemia, we did not observe a significant difference in infarct size. The improvement in BBB function post–ischemic stroke suggests a restoration of its protective role, thereby mitigating the risk of additional brain damage. However, this enhancement may not directly correlate with changes in the initial size of stroke-induced brain injury, influenced by multifaceted factors and processes. Furthermore, our study’s primary observation time points were at 6 and 24 hours post-stroke, lacking longer-term data to explore the sustained therapeutic effects of targeting SLC22A17 on BBB permeability after cerebral ischemia. Therefore, future research should include longer-term experiments to comprehensively evaluate the sustained effects of targeted SLC22A17 therapy. Establishing whether improved BBB function contributes to long-term neurological recovery will be pivotal for advancing translational efforts. Additionally, our proof-of-concept study utilized male mice to enhance statistical power. Notably, females typically have lower peripheral iron levels compared to males. A neuroimaging study has also revealed differences in intracerebral iron content between genders (Wade et al., 2019). Therefore, the impact of gender differences on iron-handling pathways within the brain cannot be overlooked. Hence, future investigations should consider incorporating both male and female mice to more accurately assess the potential and limitations of SLC22A17 as a therapeutic target. In our study, we focused on the transcriptional regulation of tight junctions by SLC22A17. However, proper tight junction distribution is essential for the formation of a continuous and cohesive barrier, effectively preventing the paracellular passage of molecules (Halder et al., 2023). Mislocalization or improper assembly can lead to a compromised BBB, even if protein expression levels are optimal. Thus, the functional integrity of the BBB relies on a delicate balance between adequate protein expression and precise protein distribution. It remains unclear whether the restored expression of Claudin-5 and ZO-1, following SLC22A17 silencing, ensures the proper localization necessary for maintaining barrier integrity. Furthermore, factors such as diabetes and obesity are closely linked to an increased risk of stroke. A genome-wide association study examined single nucleotide polymorphisms in individuals with overweight or obesity (body mass index > 23 kg/m2) compared to those with normal body mass index and in women with low basal metabolic rate < 1426.3 kcal/day) compared to high basal metabolic rate (Lee et al., 2016). This study identified associations of SLC22A17 with obesity in these populations, suggesting the potential contribution of SLC22A17 to stroke risk and potential preventive strategies. In summary, the iron-handling solute carrier SLC22A17 significantly contributes to BBB damage following ischemic stroke. By regulating tight junction proteins in brain endothelial cells, SLC22A17 exacerbates BBB permeability and cerebral edema (Figure 1). Thus, targeting SLC22A17 offers a promising approach to mitigate BBB damage and enhance recovery in ischemic stroke patients, warranting further investigation into its therapeutic potential.Figure 1: Iron-handling solute carrier SLC22A17 as a blood–brain barrier target after stroke.Under normal conditions, SLC22A17 is primarily expressed in neurons. However, following ischemic stroke, there is a significant upregulation of SLC22A17 in cerebral microvascular endothelial cells. This upregulation leads to the repression of tight junction proteins ZO-1 and Claudin-5 by binding to their promoter regions, resulting in decreased expression of tight junction proteins, thereby increasing the permeability of the brain endothelial barrier. Ultimately, this process impairs the function of the blood–brain barrier. Created with Figdraw (www.figdraw.com). SLC22A17: Solute carrier protein solute carrier family 22 member 17; ZO-1: zonula occludens-1.Open peer reviewer:Shiwani Limbu, University of California Merced, USA. Additional file:Open peer review report 1.P-Reviewer: Limbu S; C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y