Major Facilitator Superfamily Domain-containing 2a Research Articles

P0144 Unveiling the role of the MFSD2A protein in contributing to the resolution of colitis-associated cancer inflammation

Abstract Background The intrinsic connection between inflammation and tumor promotion is well-established in colorectal cancer (CRC), where chronic inflammation, like ulcerative colitis (UC), plays a significant role in intestinal carcinogenesis (1). UC patients are at higher risk of colitis-associated cancer, emphasizing the inflammation-cancer connection. Previous studies from our laboratory revealed that UC patients with active inflammation have impaired production of inflammation-resolving DHA-derived metabolites due to a defect in the endothelial Major Facilitator Superfamily Domain containing 2A (MFSD2A) (2). Given the MFSD2A’s role in reducing colonic inflammation, we hypothesize it also counteracts cancer-associated inflammation. Methods Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with lentiviruses carrying either the GFP-tagged MFSD2A encoding sequence (MFSD2A-OE), or the relative GFP control, or the MFSD2A-targeting shRNAs, or the scramble as control, then analyzed by transcriptomics and lipidomics. A colon-adenocarcinoma cell line (Caco-2) was cocultured with either MFSD2A-OE or GFP CRC HIMEC to evaluate their proliferation rate. An orthotopic model of CRC was performed by intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and CRC HIMEC overexpressing either MFSD2A or GFP as control. Collected tumors were analyzed by flow cytometry and lipidomics (3). Results CRC HIMEC displayed a pro-resolving signature at the lipidomic level, consistently with higher MFSD2A expression as compared to the healthy. However, gene expression analysis shows a pro-inflammatory profile in CRC HIMEC, suggesting that MFSD2A levels alone are insufficient to induce a fully pro-resolving phenotype. We performed loss of function experiments to assess whether the pro-resolving profile in CRC HIMEC depends on MFSD2A. MFSD2A silencing reduces pro-resolving functions, confirming its role in balancing pro-inflammatory and pro-resolving signals in CRC regarding lipid production. Caco-2 co-cultured with MFSD2A-OE CRC HIMEC displayed a decrease in their proliferation rate after 48 and 72 hours of co-culture, indicating that endothelial MFSD2A overexpression reduces tumor cell proliferation. By comparison with animals injected with Caco-2 and GFP HIMEC, mice injected with a Caco-2/MFSD2A-OE CRC HIMEC mixture developed tumors with reduced weight and size, and increased pro-resolving lipid release. Additionally, flow cytometry showed increased M2 macrophage polarization, indicating a shift toward a pro-resolving immune response. Conclusion These data suggested that MFSD2A contrasts cancer-associated inflammation, ensuring the correct balance between pro-inflammatory and pro-resolving milieu.

Dietary sn-2 palmitate influences cognitive behavior by increasing the transport of liver-produced lysophosphatidylcholine VLCPUFAs to the brain

Investigations indicated that sn-2 palmitate have positive effects on brain development, although its mechanism remains largely unexamined. This research delved into how a diet abundant in sn-2 palmitate influenced the cognitive behavior of mice and elucidated the associated mechanisms using metabolomics and lipidomics. The study demonstrated that dietary sn-2 palmitate led to improved working memory and cognition in mice, as well as an increase in brain BDNF concentration when compared to those fed blend vegetable oil (BVO). This was because sn-2 palmitate feeding promoted the synthesis of very long-chain fatty acids (VLCPUFAs) for the lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in the liver. This led to more efficient delivery of VLCPUFAs to the brain, as indicated by elevated concentration of LPC/LPE-VLCPUFAs in the liver and heightened expression of the major facilitator superfamily domain containing 2a (MFSD2A). In essence, this paper offered a potential mechanism by which sn-2 palmitate enhanced mouse neurodevelopment.

Activation of Wnt signaling mitigates blood-brain barrier disruption by inhibiting vesicular transcytosis after traumatic brain injury in mice

Elevated transport of Caveolin-1 (CAV-1) vesicles within vascular endothelial cells constitutes a significant secondary pathogenic event contributing to the compromise of the blood-brain barrier (BBB) post-traumatic brain injury (TBI). While Wnt/β-catenin signaling is recognized for its critical involvement in angiogenesis and the maintenance of BBB integrity, its influence on vascular endothelial transcytosis in the aftermath of TBI is not well-defined. This study aims to elucidate the impact of Wnt/β-catenin signaling on cerebrovascular vesicular transcytosis following TBI. In this experiment, adult male wild-type (WT) C57BL/6 mice underwent various interventions. TBI was induced utilizing the controlled cortical impact technique. Post-TBI, mice were administered either an inhibitor or an agonist of Wnt signaling via intraperitoneal injection. Recombinant adeno-associated virus (rAAV) was administered intracerebroventricularly to modulate the expression of the CAV-1 inhibitory protein, Major facilitator superfamily domain-containing 2a (Mfsd2a). This research utilized Evans blue assay, Western blot analysis, immunofluorescence, transmission electron microscopy, and neurobehavioral assessments. Post-TBI observations revealed substantial increases in macromolecule (Evans blue and albumin) leakage, CAV-1 transport vesicle count, astrocyte end-feet edema, and augmented aquaporin-4 (AQP4) expression, culminating in BBB disruption. The findings indicate that Wnt signaling pathway inhibition escalates CAV-1 transport vesicle activity and aggravates BBB compromise. Conversely, activating this pathway could alleviate BBB damage by curtailing CAV-1 vesicle presence. Post-TBI, there is a diminution in Mfsd2a expression, which is directly influenced by the modulation of WNT signals. Employing a viral approach to regulate Mfsd2a, we established that its down-regulation undermines the protective benefits derived from reducing CAV-1 transport vesicles through WNT signal enhancement. Moreover, we verified that the WNT signaling agonist LiCl notably ameliorates neurological deficits following TBI in mice. Collectively, our data imply that Wnt/β-catenin signaling presents a potential therapeutic target for safeguarding against BBB damage and enhancing neurological function after TBI.

Optimal timing for drug delivery into the hippocampus by focused ultrasound: A comparison of hydrophilic and lipophilic compounds

AimsPrevious studies have reported that focused ultrasound (FUS) helps modulate the blood-brain barrier (BBB). These studies have generally used the paracellular pathway owing to tight junction proteins (TJPs) regulation. However, BBB transport pathways also include diffusion and transcytosis. Few studies have examined transcellular transport across endothelial cells. We supposed that increased BBB permeability caused by FUS may affect transcytosis. We investigated drug delivery through transcytosis and paracellular transport to the brain after BBB modulation using FUS. Main methodsFUS and microbubbles were applied to the hippocampus of rats, and were euthanized at 1, 4, 24, and 48 h after sonication. To investigate paracellular transport, we analyzed TJPs, including zona occludens-1 (ZO-1) and occludin. We also investigated caveola-mediated transcytosis by analyzing caveola formation and major facilitator superfamily domain-containing 2a (Mfsd2a) levels, which inhibit caveola vesicle formation. Key findingsOne hour after FUS, ZO-1 and occludin expression was the lowest and gradually increased over time, returning to baseline 24 h after FUS treatment. Compared with that of TJPs, caveola formation started to increase 1 h after FUS treatment and peaked at 4 h after FUS treatment before returning to baseline by 48 h after FUS treatment. Decreased Mfsd2a levels were observed at 1 h and 4 h after FUS treatment, indicating increased caveola formation. SignificanceFUS induces BBB permeability changes and regulates both paracellular transport and caveola-mediated transcytosis. However, a time difference was observed between these two mechanisms. Hence, when delivering drugs into the brain after FUS, the optimal drug administration timing should be determined by the mechanism by which each drug passes through the BBB.

Effect of high-fat diet on the fatty acid profiles of brain in offspring mice exposed to maternal gestational diabetes mellitus.

Fatty acids play a critical role in the proper functioning of the brain. This study investigated the effects of a high-fat (HF) diet on brain fatty acid profiles of offspring exposed to maternal gestational diabetes mellitus (GDM). Insulin receptor antagonist (S961) and HF diet were used to establish the GDM animal model. Brain fatty acid profiles of the offspring mice were measured by gas chromatography at weaning and adulthood. Protein expressions of the fatty acid transport pathway Wnt3/β-catenin and the target protein major facilitator superfamily domain-containing 2a (MFSD2a) were measured in the offspring brain by Western blot. Maternal GDM increased the body weight of male offspring (P < 0.05). In weaning offspring, factorial analysis showed that maternal GDM increased the monounsaturated fatty acid (MUFA) percentage of the weaning offspring's brain (P < 0.05). Maternal GDM decreased offspring brain arachidonic acid (AA), but HF diet increased brain linoleic acid (LA) (P < 0.05). Maternal GDM and HF diet reduced offspring brain docosahexaenoic acid (DHA), and the male offspring had higher DHA than the female offspring (P < 0.05). In adult offspring, factorial analysis showed that HF diet increased brain MUFA in offspring, and male offspring had higher brain MUFA than female offspring (P < 0.05). The HF diet increased brain LA in the offspring. Male offspring had higher level of AA than female offspring (P < 0.05). HF diet reduced DHA in the brains of female offspring. The brain protein expression of β-catenin and MFSD2a in both weaning and adult female offspring was lower in the HF + GDM group than in the CON group (P < 0.05). Maternal GDM increased the susceptibility of male offspring to HF diet-induced obesity. HF diet-induced adverse brain fatty acid profiles in both male and female offspring exposed to GDM.

P073 Enhancing the MFSD2A protein for the treatment of colitis-associated cancer: novel insights for pathogenesis and treatment

Abstract Background The intrinsic connection between inflammation and tumor promotion is well characterized and is a key pathogenic event in patients with colorectal cancer (CRC). A small fraction of patients with CRC suffers from genetic predisposition. However chronic inflammation, such as ulcerative colitis (UC), increases the risk of intestinal carcinogenesis, thus strengthening the connection between these conditions. A few years ago, our laboratory described that the intestinal endothelium isolated from actively inflamed UC patients displayed defective production of inflammation-resolving DHA-derived metabolites, by comparison with healthy controls and tissues in remission. We also reported that the Major Facilitator Superfamily Domain containing 2A (MFSD2A) participated in the production of the inflammation-resolving DHA-derived metabolites by the intestinal endothelium of patients with UC in remission, ameliorating colonic inflammation. Therefore, we hypothesized that the endothelial MFSD2A also has a role in preventing and/or counteracting cancer-associated inflammation. Methods Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with either MFSD2A protein-encoding lentiviral vectors (MFSD2A) or GFP as control, or MFSD2A targeting shRNA or its scramble, and were analyzed by lipidomics. Furthermore, Caco-2 cells co-cultured with HIMEC-MFSD2A were analyzed by FACS, evaluating their proliferation. Moreover, an orthotopic model of CRC was made intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and HUVEC overexpressing MFSD2A or GFP as control. Mice were gavaged with DHA-ethyl-ester or PBS. FACS analysis was performed on tumor and colonic samples. Results Lipidomic analysis revealed that the loss of MFSD2A in CRC is detrimental because there is a reduced production of pro-resolving lipids confirming that, as for UC, MFSD2A is important for the maintenance of an adequate balance between pro-resolving and pro-inflammatory phenotype. Moreover, enhancing MFSD2A expression at endothelial level limits Caco-2 cell proliferation, supporting its beneficial role. In addition, DHA administration in mice ameliorated clinical symptoms of inflammation upon intestinal endothelial MFSD2A overexpression, supporting the beneficial role of MFSD2A in vivo. Conclusion Tumor-associated inflammation remains a crucial hallmark of CRC. Our study seeks to identify the role of MFSD2A in the resolution phase of inflammation, pointing out alternative therapies for CRC treatment. These data suggested that MFSD2A might contrast the tumor-associated inflammation and ensure the correct balance between pro-inflammatory and pro-resolving milieu.

Investigation of the Binding Interaction of Mfsd2a with NEDD4-2 via Molecular Dynamics Simulations.

Major facilitator superfamily domain-containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine cotransporter that plays an important role in maintaining the integrity of the blood-brain barrier and neurological function. Abnormal degradation of Mfsd2a often leads to dysfunction of the blood-brain barrier, while upregulation of Mfsd2a can retrieve neurological damage. It has been reported that Mfsd2a can be specifically recognized and ubiquitinated by neural precursor cell-expressed developmentally downregulated gene 4 type 2 (NEDD4-2) ubiquitin ligase and finally degraded through the proteasome pathway. However, the structural basis for the specific binding of Mfsd2a to NEDD4-2 is unclear. In this work, we combined deep learning and molecular dynamics simulations to obtain a Mfsd2a structure with high quality and a stable Mfsd2a/NEDD4-2-WW3 interaction model. Moreover, molecular mechanics generalized Born surface area (MM-GBSA) methods coupled with per-residue energy decomposition studies were carried out to analyze the key residues that dominate the binding interaction. Based on these results, we designed three peptides containing the key residues by truncating the Mfsd2a sequences. One of them was found to significantly inhibit Mfsd2a ubiquitination, which was further validated in an oxygen-glucose deprivation (OGD) model in a human microvascular endothelial cell line. This work provides some new insights into the understanding of Mfsd2a and NEDD4-2 interaction and might promote further development of drugs targeting Mfsd2a ubiquitination.

Knockdown of Placental Major Facilitator Superfamily Domain Containing 2a in Pregnant Mice Reduces Fetal Brain Growth and Phospholipid Docosahexaenoic Acid Content.

Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid critical for fetal brain development that is transported to the fetus from the mother by the placenta. The lysophosphatidylcholine (LPC) transporter, Major Facilitator Superfamily Domain Containing 2a (MFSD2a), is localized in the basal plasma membrane of the syncytiotrophoblast of the human placenta, and MFSD2a expression correlates with umbilical cord blood LPC-DHA levels in human pregnancy. We hypothesized that placenta-specific knockdown of MFSD2a in pregnant mice reduces phospholipid DHA accumulation in the fetal brain. Mouse blastocysts (E3.5) were transduced with an EGFP-expressing lentivirus containing either an shRNA targeting MFSD2a or a non-coding sequence (SCR), then transferred to pseudopregnant females. At E18.5, fetuses were weighed and their placenta, brain, liver and plasma were collected. MFSD2a mRNA expression was determined by qPCR in the brain, liver and placenta and phospholipid DHA was quantified by LC-MS/MS. MFSD2a-targeting shRNA reduced placental mRNA MFSD2a expression by 38% at E18.5 (n = 45, p < 0.008) compared with SCR controls. MFSD2a expression in the fetal brain and liver were unchanged. Fetal brain weight was reduced by 13% (p = 0.006). Body weight, placenta and liver weights were unaffected. Fetal brain phosphatidyl choline and phosphatidyl ethanolamine DHA content was lower in fetuses with placenta-specific MFSD2a knockdown. Placenta-specific reduction in expression of the LPC-DHA transporter MFSD2a resulted in reduced fetal brain weight and lower phospholipid DHA content in the fetal brain. These data provide mechanistic evidence that placental MFSD2a mediates maternal-fetal transfer of LPC-DHA, which is critical for brain growth.

MFSD2A potentiates gastric cancer response to anti-PD-1 immunotherapy by reprogramming the tumor microenvironment to activate T cell response.

The efficacy of anti-programmed cell death protein 1 (PD-1) immunotherapy in various cancers, including gastric cancer (GC), needs to be potentiated by more effective targeting to enhance therapeutic efficacy or identifying accurate biomarkers to predict clinical responses. Here, we attempted to identify molecules predicting or/and promoting anti-PD-1 therapeutic response in advanced GC (AGC). The transcriptome of AGC tissues from patients with different clinical responses to anti-PD-1 immunotherapy and GC cells was analyzed by RNA sequencing. The protein and mRNA levels of the major facilitator superfamily domain containing 2A (MFSD2A) in GC cells were assessed via quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Additionally, the regulation of anti-PD-1 response by MFSD2A was studied in tumor-bearing mice. Cytometry by Time-of-Flight, multiple immunohistochemistry, and flow cytometry assays were used to explore immunological responses. The effects of MFSD2A on lipid metabolism in mice cancer tissue and GC cells was detected by metabolomics. Higher expression of MFSD2A in tumor tissues of AGC patients was associated with better response to anti-PD-1 immunotherapy. Moreover, MFSD2A expression was lower in GC tissues compared to adjacent normal tissues, and its expression was inversely correlated with GC stage. The overexpression of MFSD2A in GC cells enhanced the efficacy of anti-PD-1 immunotherapy in vivo by reprogramming the tumor microenvironment (TME), characterized by increased CD8+ T cell activation and reduced its exhaustion. MFSD2A inhibited transforming growth factor β1 (TGFβ1) release from GC cells by suppressing cyclooxygenase 2 (COX2)-prostaglandin synthesis, which consequently reprogrammed TME to promote anti-tumor T cell activation. MFSD2A potentially serves as a predictive biomarker for anti-PD-1 immunotherapy response in AGC patients. MFSD2A may be a promising therapeutic target to potentiate the efficacy of anti-PD-1 immunotherapy by reprogramming the TME to promote T cells activation.

Substrate binding-induced conformational transitions in the omega-3 fatty acid transporter MFSD2A

Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na+-dependent uptake of ω−3 fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na+. Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na+ -bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a “trap-and-flip” mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle.

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

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.

Selective sphingosine-1-phosphate receptor 1 modulator attenuates blood–brain barrier disruption following traumatic brain injury by inhibiting vesicular transcytosis

BackgroundTraumatic brain injury (TBI) provokes secondary pathological damage, such as damage to the blood–brain barrier (BBB), ischaemia and inflammation. Major facilitator superfamily domain-containing 2a (Mfsd2a) has been demonstrated to be critical in limiting the increase in BBB vesicle transcytosis following brain injury. Recent studies suggest that a novel and selective modulator of the sphingosine-1-phosphate receptor 1 (S1P1), CYM-5442, maintains the integrity of the BBB by restricting vesicle transcytosis during acute ischaemic stroke. In the current study, we investigated whether CYM-5442, evaluated in a short-term study, could protect the brains of mice with acute-stage TBI by reversing the increase in vesicle transport due to reduced Mfsd2a expression after TBI.MethodsWe used the well-characterized model of TBI caused by controlled cortical impact. CYM-5442 (0.3, 1, 3 mg/kg) was intraperitoneally injected 30 min after surgery for 7 consecutive days. To investigate the effect of CYM-5442 on vesicle transcytosis, we downregulated and upregulated Mfsd2a expression using a specific AAV prior to evaluation of the TBI model. MRI scanning, cerebral blood flow, circulating blood counts, ELISA, TEM, WB, and immunostaining evaluations were performed after brain injury.ResultsCYM-5442 significantly attenuated neurological deficits and reduced brain oedema in TBI mice. CYM-5442 transiently suppressed lymphocyte trafficking but did not induce persistent lymphocytopenia. After TBI, the levels of Mfsd2a were decreased significantly, while the levels of CAV-1 and albumin were increased. In addition, Mfsd2a deficiency caused inadequate sphingosine-1-phosphate (S1P) transport in the brain parenchyma, and the regulation of BBB permeability by Mfsd2a after TBI was shown to be related to changes in vesicle transcytosis. Downregulation of Mfsd2a in mice markedly increased the BBB permeability, neurological deficit scores, and brain water contents after TBI. Intervention with CYM-5442 after TBI protected the BBB by significantly reducing the vesicle transcytosis of cerebrovascular endothelial cells.ConclusionIn addition to transiently suppressing lymphocytes, CYM-5442 alleviated the neurological deficits, cerebral edema and protective BBB permeability in TBI mice by reducing the vesicle transcytosis of cerebrovascular endothelial cells.

The Role of Major Facilitator Superfamily Domain-Containing 2a in the Central Nervous System.

Major facilitator superfamily-domain containing 2a (Mfsd2a) is selectively expressed in vascular endotheliocytes and plays a crucial role in maintaining the integrity of the blood‒brain barrier and the transport of docosahexaenoic acid. It is currently recognized as the only molecule that inhibits endocytosis mediated by caveolae in brain endothelial cells. Mfsd2a gene knockout leads to an increase in the permeability of the blood-brain barrier from embryonic stages to adulthood while maintaining the normal pattern of the vascular network. In Mfsd2a knockout mice, the docosahexaenoic acid content is significantly reduced and associated with neuron loss, resulting in microcephaly and cognitive impairment. Based on the role of Mfsd2a in the central nervous system, it has been preliminarily suggested as a potential therapeutic target for drug delivery to the central nervous system. This paper reviews the current progress in Mfsd2a research and summarizes the physiological functions of Mfsd2a in the central nervous system and its role in the occurrence and development of a variety of neurological diseases.

Na+-coupled lipid release by MFSD2A transporter

Major facilitator superfamily domain containing 2A (MFSD2A) is a membrane protein that is highly enriched within endothelial cells of the blood-brain and blood-retinal barriers where it mediates Na+-dependent uptake of the ω-3 fatty acid docosahexaenoic acid (DHA) in the form of lysophosphatidylcholine (LPC-DHA) into the brain and eyes, respectively. MFSD2A shares the 12 transmembrane domain architecture with other MFS family members. However, the molecular details of the transport process remain unknown, because unlike most MFS proteins, MFSD2A transports lysolipids rather than water-soluble molecules, suggesting that molecular mechanism of MFSD2A-mediated transport can be different and more akin to that of flippases.

Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability.

The low level of transcytosis is a unique feature of cerebrovascular endothelial cells (ECs), ensuring restrictive blood-brain barrier (BBB) permeability. Major facilitator superfamily domain-containing 2a (MFSD2A) is a key regulator of the BBB function by suppressing caveolae-mediated transcytosis. However, the mechanisms regulating MFSD2A at the BBB have been barely explored. Here, we show that cerebrovascular EC-specific deletion of Pten (phosphatase and tensin homolog) results in a dramatic increase in vesicular transcytosis by the reduction of MFSD2A, leading to increased transcellular permeability of the BBB. Mechanistically, AKT signaling inhibits E3 ubiquitin ligase NEDD4-2-mediated MFSD2A degradation. Consistently, cerebrovascular Nedd4-2 overexpression decreases MFSD2A levels, increases transcytosis, and impairs BBB permeability, recapitulating the phenotypes of Pten-deficient mice. Furthermore, Akt deletion decreases phosphorylated NEDD4-2 levels, restores MFSD2A levels, and normalizes BBB permeability in Pten-mutant mice. Altogether, our work reveals the essential physiological function of the PTEN/AKT/NEDD4-2/MFSD2A axis in the regulation of BBB permeability.

Structural basis of omega-3 fatty acid transport across the blood-brain barrier.

Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources1-6. This nutrient is transported across the blood-brain and blood-retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na+-dependent manner7,8. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na+-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na+-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.

Novel neuroclinical findings of autosomal recessive primary microcephaly 15 in a consanguineous Iranian family

Major facilitator superfamily domain-containing 2A (MFSD2A) is required for brain uptake of Docosahexaenoic acid and Lysophosphatidylcholine, both are essential for the normal neural development and function. Mutations in MFSD2A dysregulate the activity of this transporter in brain endothelial cells and can lead to microcephaly. In this study, we describe an 11-year-old male who is affected by autosomal recessive primary microcephaly 15. This patient also shows severe intellectual disability, recurrent respiratory and renal infections, low birth weight, and developmental delay. After doing clinical and neuroimaging evaluations, due to heterogeneity of neurogenetic disorders, no narrow clinical diagnosis was possible, therefore, we utilized targeted-exome sequencing to identify any causative genetic factors. This revealed a homozygous in-frame deletion (NM_001136493.1: c.241_243del; p.(Val81del)) in the MFSD2A gene as the most likely disease-susceptibility variant which was confirmed by Sanger sequencing. Neuroimaging revealed lateral ventricular asymmetry, corpus callosum hypoplasia, type B of cisterna magna, and widening of Sylvian fissures. All of these novel phenotypes are associated with autosomal recessive primary microcephaly-15 (MCPH15). According to the genotype-phenotype data, p.(Val81del) can be considered a likely pathogenic variant leading to non-lethal microcephaly. However, further cumulative data and molecular approaches are required to accurately identify genotype-phenotype correlations in MFSD2A.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the tunicamycin transporter (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Maternal diet high in Omega-3 fatty acids upregulate genes involved in neurotrophin signalling in fetal brain during pregnancy in C57BL/6 mice

Neurotrophins play a critical role in the development, maintenance, and proper function of the brain. We investigated the effects of maternal diet high in omega (n)-3 polyunsaturated fatty acids (PUFA) on fatty acids composition and the gene expression of neurotrophins in fetal brain at different gestation stages. Female C57BL/6 mice (7-weeks old, n = 8/group) were fed a diet containing high, low or very low n-3 PUFA (9, 3 or 1% w/w, respectively), with an n-6:n-3 PUFA of 5:1, 20:1 and 40:1, respectively, for two weeks before mating and throughout pregnancy. Animals were sacrificed during pregnancy at gestation day 12.5 and 18.5 to determine placental and fetal-brain fatty acids composition. The gene expressions of endothelial lipase (EL) and plasma membrane fatty acid-binding protein (FABPpm) were measured in the placenta, while major facilitator superfamily domain-containing 2a (Mfsd2a), brain-derived neurotrophic factor (BDNF), tropomyosin-receptor kinase (TrK)-B, and cAMP response element-binding protein (CREB) were measured in fetal-brain, using qPCR. The protein expression of phosphorylated CREB (pCREB) was determined using ELISA. The high n-3 PUFA diet increased the mRNA expression of EL, FABPpm, and Mfsd2a at both gestation days, compared to other groups. Docosahexaenoic acid (DHA) and total n-3 PUFA were significantly higher in the high n-3 PUFA group, compared to the other groups at both gestation days. The high n-3 PUFA diet also increased the mRNA expressions of BDNF, TrKB and CREB, as well as the protein concentration of pCREB as gestation progressed, compared to the other groups. Our findings show for the first time that maternal diet high in n-3 PUFA increased the mRNA expression of Mfsd2a, which correlated with an increase in DHA accretion in the fetal-brain. A diet high in n-3 PUFA increased neurotrophin signalling in fetal-brain as gestation progressed, demonstrating the importance of n-3 PUFA during brain development.

Mfsd2a and Spns2 are essential for sphingosine-1-phosphate transport in the formation and maintenance of the blood-brain barrier.

To maintain brain homeostasis, a unique interface known as the blood-brain barrier (BBB) is formed between the blood circulation and the central nervous system (CNS). Major facilitator superfamily domain-containing 2a (Mfsd2a) is a specific marker of the BBB. However, the mechanism by which Mfsd2a influences the BBB is poorly understood. In this study, we demonstrated that Mfsd2a is essential for sphingosine-1-phosphate (S1P) export from endothelial cells in the brain. We found that Mfsd2a and Spinster homolog 2 (Spns2) form a protein complex to ensure the efficient transport of S1P. Furthermore, the S1P-rich microenvironment in the extracellular matrix (ECM) in the vascular endothelium dominates the formation and maintenance of the BBB. We demonstrated that different concentrations of S1P have different effects on BBB integrity. These findings help to unravel the mechanism by which S1P regulates BBB and also provide previously unidentified insights into the delivery of neurological drugs in the CNS.