Mfsd2a attenuated hypoxic-ischemic brain damage via protection of the blood–brain barrier in mfat-1 transgenic mice

Cellular and Molecular Life Sciences (2023) 80:71 https://doi.org/10.1007/s00018-023-04716-9 Cellular and Molecular Life Sciences ORIGINAL ARTICLE Mfsd2a attenuated hypoxic‑ischemic brain damage via protection of the blood–brain barrier in mfat‑1 transgenic mice Xiaoxue Li1,2 · Yumeng Zhang1,2 · Jianghao Chang1,2 · Chenglin Zhang1,2 · Lin Li1,2 · Yifan Dai1,2,3,4 · Haiyuan Yang1,2,3 · Ying Wang1,2,3 Received: 22 November 2022 / Revised: 10 January 2023 / Accepted: 1 February 2023 © The Author(s) 2023 Abstract Previous studies have shown that mfat-1 transgenic mice have protective effects against some central nervous system (CNS) disorders, owing to the high docosahexaenoic acid (DHA) content enriched in their brains. However, whether this protective effect is connected to the blood–brain barrier (BBB) remains unclear. This study aims to investigate the mechanisms of the protective effect against hypoxic-ischemic brain damage (HIBD) of mfat-1 transgenic mice. mfat-1 mice not only demonstrated a significant amelioration of neurological dysfunction and neuronal damage but also partly maintained the physiological permeability of the BBB after HIBD. We initially showed this was associated with elevated major facilitator superfamily domain-containing 2a (Mfsd2a) expression on the BBB, resulting from more lysophosphatidylcholine (LPC)DHA entering the brain. Wild-type (WT) mice showed a similar Mfsd2a expression trend after long-term feeding with an LPC-DHA-rich diet. Knockdown of Mfsd2a by siRNA intra-cerebroventricular (ICV) injection neutralized the protective effect against HIBD-induced BBB disruption in mfat-1 mice, further validating the protective function of Mfsd2a on BBB. HIBD-induced BBB high permeability was attenuated by Mfsd2a, primarily through a transcellular pathway to decrease caveolae-like vesicle-mediated transcytosis. Taken together, these findings not only reveal that mfat-1 transgenic mice have higher expression of Mfsd2a on the BBB, which partly sustains BBB permeability via vesicular transcytosis to alleviate the severity of HIBD, but also suggest that dietary intake of LPC-DHA may upregulate Mfsd2a expression as a novel therapeutic strategy for BBB dysfunction and survival in HIBD patients. Keywords NormFinder · LC–MS/MS · Hepatocytes · PLA1 · TEM · Apoptosis Introduction * Yifan Dai * Haiyuan Yang * Ying Wang 1 Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing 211166, China 2 Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China 3 Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China 4 State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 211166, China Hypoxic-ischemic (HI) brain damage (HIBD) occurs when cerebral blood flow is decreased or suspended, resulting in partial or complete hypoxia of brain tissues [1]. In neonates and adults, brain injury produced by HI is recognized as a devastating incident that frequently leads to death or profound long-term neurological morbidity [2]. Perinatal asphyxia is the leading cause of HIBD in neonates, affecting three out of every 1000 term newborns [3]. Adults have seen an increase in the prevalence of brain ischemia in recent years due to cardiac arrest or cerebrovascular illness, with subsequent hypoxia caused by decreased blood flow [4]. As a result of the preceding, HIBD has emerged as one of the most severe public health issues. While studies on HIBD are becoming increasingly in-depth, the underlying mechanisms are challenging to decipher and remain largely elusive. Meanwhile, there are also no FDA-approved 13 Vol.:(0123456789) 71 Page 2 of 16 pharmacotherapies for the treatment of HIBD [5]. Therefore, it is urgent to explore an effective way to prevent or alleviate HIBD. The blood–brain barrier (BBB) is a selectively semipermeable boundary formed by central nervous system (CNS) vascular endothelial cells (ECs) [6]. Two primary mechanisms of ECs maintain the low permeability of the BBB: (i) by suppressing vesicular trafficking or transcytosis (transcellular pathway) [7]; (ii) by limiting intercellular transit via specialized tight junctions (TJ) complexes (paracellular pathway). BBB breakdown has been linked to various acute and chronic CNS diseases, highlighting the possible deleterious implications of BBB disruption on brain function [8]. Major facilitator superfamily domain-containing 2a (Mfsd2a) expresses predominantly in the cerebral microvessel endothelium and serves as the primary membrane transporter for lysophosphatidylcholine (LPC)-DHA uptake [9]. Additionally, Mfsd2a is a crucial regulator of BBB function [10], and it is unknown whether Mfsd2a contributes to BBB disruption following HI. Previous studies have established that fat-1 transgenic mice can protect against various CNS disorders [11]. The mfat-1 (mammalian fat-1, thereafter called mfat-1) transgenic mice, overexpressing a codon-optimized ω-3 desaturase derived from C. elegans, can synthesize ω-3 polyunsaturated fatty acids (PUFAs) from ω-6 PUFAs, thereby maintaining a low and well-balanced ratio of ω-6/ω-3 PUFAs in their tissues [12]. It is worth mentioning that the brain of mfat-1 mice has a significantly higher level of total DHA, the main component of ω-3 PUFAs [13, 14]. DHA cannot be de novo synthesized in the brain and must be imported across the BBB from plasma [15, 16]. In the liver, some enzymes, especially hepatic lipase, can act preferentially on phospholipids containing DHA at the sn-2 position, resulting in LPC-DHA [17]. Mfsd2a-deficient mice show a noticeable drop in cerebral LPC-DHA levels [18], indicating that Mfsd2a is the primary transporter of LPC-DHA into the brain [9]. Furthermore, increasing the expression of genes encoding fatty acid transporters increases PUFA uptake [19–21]. Hence, we speculated that when more LPCDHA derived from plasma was required for transport into the brain of mfat-1 mice, the expression of Mfsd2a protein on the BBB would increase accordingly. Currently, few studies have described the molecular mechanism underlying mfat-1 transgenic mice's resistance to HIBD. In our study, we found that mfat-1 mice partially maintained BBB physiological function following HIBD. We showed that the Mfsd2a expression on the BBB was upregulated in both WT mice fed with LPCDHA-rich diet and mfat-1 mice, mainly associated with more LPC-DHA entering the brain. In addition, we proved that the elevated Mfsd2a in mfat-1 mice could attenuate HIBD-induced BBB breakdown primarily through the 13 X. Li et al. transcellular pathway to reduce caveolae-like vesiclesmediated transcytosis. These findings revealed the protective effect and underlying molecular mechanisms of mfat-1 transgenic mice against HIBD, which will provide insight in (...truncated)