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Apolipoprotein M-bound sphingosine-1-phosphate regulates blood-brain barrier paracellular permeability and transcytosis

The blood-brain barrier (BBB) is formed by the endothelial cells lining cerebral microvessels. Here, we report that the BBB permeability is modified by apolipoprotein M (apoM)-bound sphingosine 1-phosphate (S1P). We used two-photon microscopy to monitor changes in BBB permeability in apoM-deficient mice (apoM−/−), showing significant increases in paracellular BBB permeability to small molecules without structural changes in junctional complexes between endothelial cells. Lack of apoM-bound S1P increased vesicle-mediated transfer of albumin across endothelium of brain pial and penetrating arterioles, whereas transcytosis in capillaries and venules remained unchanged. S1PR1 agonist SEW2871 rapidly normalized BBB permeability along both the paracellular and transcellular routes in apoM−/− mice. Thus, apoM-bound S1P maintains low paracellular BBB permeability for small molecules in all cerebral microvessels and low levels of adsorptive transcytosis in penetrating arterioles. Modulation of apoM/S1P-dependent signaling may be a novel strategy for the protection of brain endothelial cells to preserve the BBB function.

. 23 Depending on the carrier (i.e., apoM or albumin), S1P can preferentially activate 24 S1PR1 or S1PR2 and S1PR3, which exert opposite effects (20). For example, S1PR1 25 stimulation inhibits neuroinflammation, whereas S1PR2 and S1PR3 stimulation 26 activates pro-inflammatory signaling cascades (21-23). The onset and severity of 27 sepsis and neuroinflammation (e.g., after brain trauma, ischemic stroke, or in brain 28 cancer) correlate with reduced apoM-S1P plasma concentrations (24). Notably, 29 these disorders are also associated with a loss of BBB integrity (6,7,10). 30 Lack of apoM impairs S1PR1 signaling and compromises the endothelial barrier in 31 the lungs and brown adipose tissue (19,25,26), but the brain effects have remained 32 unexplored. One study showed that conditional knock-out of S1PR1 increases the 33 blood sample was collected via the arterial catheter (ABL blood gas analyzer, 23 Radiometer) and the respiration rate and volume adjusted if necessary. Two-photon imaging was performed using an SP5 upright laser scanning microscope 5 (Leica Microsystems) equipped with a MaiTai Ti:Sapphire laser (Millennia Pro,6 Spectra Physics) using the 20x 1.0 NA water-immersion objective. The emitted light 7 was split by an FITC/TRITC filter and collected by separate multi-alkali detectors 8 after 525-560 nm and 560-625 nm band pass filters (Leica Microsystems). 9 10 Monitoring paracellular permeability 11 The excitation wavelength for FITC-dextran, Alexa Fluor 488, and NaFluo was 850 12 nm (14 mWatt output power at the sample). Data were collected as 16-bit image 13 hyperstacks at 512 x 512 pixel resolution (775 μm x 775 μm) in bidirectional 14 scanning mode with a Z-step size of 5 µm (depth span = 114 µm) and 1-minute time 15 interval between Z-stacks. The total recoding time for each fluorophore was 30 min. 16 The recordings were exported to ImageJ (NIH, version 1.48u4) and Z-projected 17 using maximal signal intensity projection. Regions of interest (ROIs) were chosen 18 throughout the brain parenchyma at all locations deprived of vasculature for the 19 measurement of fluorescent intensity. An increase in fluorescent intensity over the 20 baseline (first projected Z-stack in time-lapse recording) in the parenchyma 21 indicated crossing of the blood-circulating fluorophore into the brain. 22

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Monitoring transcellular transport 24 The animals were injected intravenously with BSA-Alexa 488. The excitation 25 wavelength was 870 nm (17 mWatt output power at the sample). Data were 26 collected as 16-bit hyperstacks at 2048 x 2048 pixel resolution (386.66 μm x 386.66 27 μm) in bidirectional scanning mode and triple frame averaging with a Z-step size of 28 2.50 µm, depth span 144 µm, and 7.5-minute interval between Z-stacks for a total 29 recording time of 120 min. The data were exported to ImageJ and Z-projected using 30 maximal signal intensity projection. Based on morphology and location, the vessels 31 were classified as pial arterioles or venules, penetrating arterioles, post-capillary 32 venules, ascending venules, or capillaries. To determine the vesicle surface density, 1 the vessel surface area was calculated based on vessel diameter and length of vessel 2 from which the BSA-Alexa 488 signal was counted. 3 4 Transmission electron microscopy 5 The mice were anesthetized with bolus i.p. injections of xylazine (10 mg/kg BW) 6 and ketamine (60 mg/kg BW), then perfused with Karnovsky fixation (2% 7 glutaraldehyde and 4% paraformaldehyde). Brains were harvested and the cortex 8 cut into 1 mm cubes and left for further fixation in 2% glutaraldehyde in 0.05 M 9 phosphate buffer for 7 days. For transmission electron microscopy (TEM) staining, 10 the samples were washed three times for 15-20 min in 0.12 M cacodylate buffer, 11 followed by post-fixation in 1% osmium tetroxide (OsO4) and 0.05 M (1.5%) 12 potassium ferricyanide (K3FeCn6) in 0.12 M cacodylate buffer for 1 hour at room 13 temperature. Next, the samples were washed three times for 15 -20 min in ddH2O 14 and then dehydrated in an ethanol gradient (70%, 96%, and 3 x 15 min in absolute 15 ethanol). Infiltration was performed with propylene oxide twice for 15 min, 16 followed by incubation for 20 -40 min in a resin epon/propylene oxide (Bie & 17 Berntsen) gradient (1:3, 1:1, 3:1) and pure resin for 2 hours. Samples were 18 embedded in resin at 60°C for 24 hours. Sections were cut at a thickness of 70 nm 19 (Leica EM UC7), placed on copper grids (Leica), and stained with lead citrate (Leica 20 Ultrostain II, Leica Microsystems) for 3 min and uranyl acetate (Leica Ultrostain I, 21 Leica Microsystems) for 10 min using an automatic contrasting system (Leica EM 22 AC20, Leica Microsystems). Imaging was performed using a CM100 transmission 23 electron microscope (Phillips/FEI, ITEM software) at 9700x -17500x and 46000-24 60000x magnification. Two samples from each mouse were imaged, and 20-30 25 capillaries from each sample were chosen randomly. ImageJ was used for the 26 measurement of BBB morphology. 27 28 Drug administration 29 SEW2871 (Sigma-Aldrich) was diluted in PBS (to 0.1 mg/ml in 1% DMSO) and 30 administered as a single i.p. bolus injection (10 µg/g BW) 150 min prior to two-31 photon imaging or perfusion fixation. 32 33 Statistical analysis 1 Statistical analysis was performed using Prism 7 (v7.0b, Graphpad) and R software 2 (v3.5.1). The data were tested for normal distribution with Pearson's normality test 3 and either an unpaired two-tailed Student's t-test with Welch's correction (normally 4 distributed data) or Mann-Whitney test (non-normally distributed data) was used. 5 The TEM data underwent logarithmic transformation and was evaluated using one-6 way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. 7 The sample size has been selected basing on our previous experiments using two-8 photon in vivo microscopy (28), in vivo electrophysiology (29) and TEM (19). The 9 data consisted only from biological replicates, the number of independent biological 10 replicates (N) is provided in results section and/or on figures. All analyses were 11 performed in randomized order, with TEM analysis performed blinded to 12 experimental conditions. No data has been excluded from analysis. To assess the BBB with all structural constituents, including blood flow, we used 19 two-photon fluorescence imaging in vivo in transgenic apoM-deficient mice (apoM -/-20 ) mice (19). Following preparative surgery (Fig. 1a), the brain was imaged through 21 an acute craniotomy over the somatosensory cortex (Fig. 1b). 22 The integrity of the BBB was assessed after an i.v. bolus injection of sodium 23 fluorescein (NaFluo, 0.365 kDa). The fluorescence signal from blood-circulating 24 fluorophore and brain parenchyma was simultaneously measured for 30 min in a 25 series of Z-stacks of relatively large brain volume (750 μm x 750 μm x 114 μm) that 26 contained pial and penetrating microvessels, including capillaries (Fig. 1c). 27 Subsequently, each Z-stack underwent dimensionality reduction, i.e., maximum 28 intensity projection along the Z (depth) axis (Fig. 1d). 29 The morphology of the vasculature delineated by blood-circulating NaFluo did not 30 differ between WT and apoM -/mice ( Fig. 1e-f). We detected progressive 31 accumulation of NaFluo in the brain parenchyma in both WT and apoM-deficient 32 mice. However, apoM -/mice exhibited considerably higher levels of fluorophore 33 accumulation in the brain compared to WT mice already at 15 min after injection, 1 with a clear difference at the 30-minute time-point ( Fig. 1e-f, Video 1). This 2 indicates that the absence of the apoM induces an increase in the paracellular 3 permeability of the BBB. 4 5 S1PR1 stimulation restores BBB permeability characteristics for small molecules 6 7 As apoM is the main carrier for S1P in the blood stream and S1P bound to apoM 8 primarily activates S1PR1 on the endothelial cells (16, 20), we tested whether the 9 increase in BBB permeability due to the lack of apoM was a consequence of the 10 reduction of circulating S1P and S1PR1 hypostimulation. Animals were injected 150 11 min prior to imaging with a selective S1PR1 agonist, SEW2871. Compared to apoM -/-12 mice, the SEW2871-treated animals did not exhibit increased permeation of NaFluo 13 into the brain (Fig. 1g, Video 1), suggesting that the S1PR1 agonist rescued the 14 abnormal apoM -/-BBB phenotype. This result indicates that the apoM-dependent 15 S1PR1 signaling deficiency is the primary cause underlying increased BBB 16 permeability. 17 18 Extent of BBB permeability induced by S1PR1 signaling deficiency 19 20 We next examined whether the BBB also exhibited increased permeability towards 21 other molecules of different sizes and chemical properties. Each animal in the WT, 22 apoM -/-, and apoM -/-SEW2871-treated group was injected at 30-minute intervals 23 with fluorophores of decreasing molecular size, starting with 10 kDa FITC-dextran, 24 followed by 0.643 kDa Alexa Fluor 488 and 0.365 kDa NaFluo (Fig. 2a). After each 25 injection, we imaged the same brain volume over time, with subsequent 26 dimensionality reduction of the Z-stacks. 27 To compare animal groups, we measured the changes in fluorescence signal over 28 time from ROIs placed in the brain parenchyma and vasculature (n=7 mice in each 29 group). For both tracer molecules, Alexa Fluor 488 and NaFluo, in all animal groups, 30 we detected fluorescence increase over time in the parenchyma (Fig. 2b), and the 31 effect was persistent without the signal reaching saturation (Fig. 2c). In contrast, 32 there was no increase in brain fluorescence for FITC-dextran ( Fig. 2b-c). To better 33 characterize the fluorescence increase over time, we calculated the area under the 1 curve (AUC) of the imaging traces, i.e., the cumulative effect of the fluorescence 2 increase for duration of 30 min after fluorophore injection (Fig. 2d). We observed no 3 differences in BBB permeability between WT and apoM -/mice for 10 kDa FITC-4 dextran (WT 0.0018±0.0014 min vs. apoM -/-0.066±0.035 min, p=0.1199), but the 5 BBB in apoM -/mice was significantly more permeable to small molecules, such as Importantly, for all fluorophores and all investigated animal groups, the decrease in 19 fluorescence signal in vessels (i.e., clearance of the fluorophore from the blood 20 circulation) was the same during the whole imaging span (Fig. 2d). In addition, we 21 found no differences in mean arterial blood pressure between animal groups before 22 (pre-imaging)  were not caused by different kinetics of fluorophore clearance from the blood 26 stream or differences in blood pressure. 27 These results show that apoM shortage causes S1PR1 signaling deficiency, 28 increasing the BBB permeability towards small molecules (~0.3-0.7 kDa), and that 29 the effect can be reversed by S1PR1 stimulation. 30 31 Deficit in S1P/S1PR1 signaling does not alter tight junction ultrastructure 32 33 Increases in the BBB permeability towards small molecules may be indicative of 1 defective structural elements that restrict paracellular diffusion across the BBB, i.e. 2 morphological barriers, including junctional complexes (6-8). Therefore, we next 3 assessed the ultrastructure of the BBB using TEM. We analyzed brain microvessels 4 (< 6 µm) cross-sections from WT, apoM -/-, and SEW2871-treated apoM -/mice. In 5 each vessel, we measured the thickness of the endothelium, basement membrane 6 (both n=104 vessels; 4 mice), tight junctional complexes, and the endothelial cleft, 7 the percentage coverage of endothelial cell contact sites by junctional complexes (all 8 n=164, 4 mice), and the endothelial cell area (n=104 vessels, 4 mice) (Fig. 3, Fig. S1). 9 The width, length, and coverage of junctional complexes were the same for all three 10 groups of mice ( Fig. 3a-b, Fig. S1a), suggesting that the compromised BBB was not 11 caused by a structural defect, and that the rescue effect of SEW2871 was not 12 mediated by alteration of the morphology of the BBB structural components. 13 However, we detected an ~10% increase in the thickness of the endothelium in S1P-14 signaling-deficient mice compared to controls (apoM -/-0.078±0.0026 μm vs. WT 15 0.071±0.0025 µm; p<0.05) and the rescue group (apoM -/-+SEW2871 0.070±0.0025 16 μm; p<0.05; Fig. 3c). Notably, the stimulation of S1PR1 in apoM -/mice decreased 17 the endothelial cell area (5.80±0.37 vs. 4.24±0.23 μm 2 ; p<0.001), with a strong trend 18 for the cell area in apoM -/being larger than WT (5.80±0.37 vs. 4.78±0.24 μm 2 ; 19 p=0.068; Fig. 3d). These changes were relatively small, and they are unlikely to 20 underlie the increased BBB permeability for small molecules. Altogether, our results 21 suggest that the increased permeability observed in apoM -/mice is not mediated by 22 ultrastructural changes at the level of the junctional complexes. 23 24 ApoM / S1PR1 signaling deficit increases transcellular transport 25 26 Next, we examined whether lack of apoM leads to increased transcytosis in the BBB. 27 The animals were injected with BSA-Alexa 488 to monitor vesicular transport across 28 the endothelium (9) and 65 kDa TRITC-dx to delineate the vessel lumen. Following 29 the injections, the animals were continuously imaged for 120 min in hyperstack 30 mode with subsequent dimensionality reduction (as in Fig. 1d). In all animal groups, 31 and as early as 30 min post-injection, we detected BSA-Alexa 488-containing 32 vesicles at the BBB interface (Fig. 4). Compared to WT animals, apoM -/mice 33 exhibited increased albumin in endothelial cells, indicating that vesicular transport 1 was increased in the absence of apoM. Furthermore, the treatment of apoM -/-2 animals with SEW2871 reversed the observed effect, suggesting that the same 3 apoM-dependent mechanism underlies the previously observed increase in 4 permeability for small fluorescent tracers, as well as elevated transcytosis. 5 Noteworthy, in apoM -/mice, we observed an increase in the presence of cells 6 containing vesicular albumin in the brain parenchyma (Fig 4). The size of the cell 7 bodies corresponded to activated (phagocytic) microglia (30), and their presence 8 was consistent with ongoing neuroinflammation during S1PR1 hypostimulation 9 (21-23). 10 11 S1P signaling deficit elevates transcytosis only in arterioles 12 13 Next, we assessed whether the increase in transcellular transport caused by the 14 S1PR1-signaling deficiency was the same for all vessel types. The number of vesicles 15 was quantified over time for each individual vessel type: pial arterioles, penetrating 16 arterioles, capillaries, post-capillary venules, ascending venules, and pial venules (n 17 = 5 mice). The data are expressed as the vesicle surface density, i.e., the number of 18 vesicles per square micrometer of vessel surface area (Fig. 5a). In all experimental 19 groups, the distribution of BSA-Alexa 488-positive vesicles was dependent on vessel 20 type and diameter. At 120 min post-injection, the highest vesicle density in WT mice 21 was observed in capillaries and gradually decreased with vessel diameter, 22 regardless of the vessel type (i.e., arterioles or venules) (Fig 5a). In apoM -/mice, the 23 deficiency in S1PR1 signaling selectively increased transcytosis in pial and 24 penetrating arterioles, but not in venules and capillaries. The effect was most 25 pronounced in penetrating arterioles, where the vascular vesicle density in apoM -/-26 mice was 10-fold higher than in WT mice (apoM -/-0.30±0.071 vs. WT 0.038±0.009 27 vesicles/100 μm 2 ; p<0.01), and three times higher than for pial arterioles (apoM -/-28 0.099±0.0023 vs. WT 0.035±0.0011 vesicles/100 μm 2 ; p<0.05; Fig. 5b). The level of 29 transcytosis did not change in apoM -/mice in the capillaries, venules, or pial veins 30 (p=0.2306, p=0.3562, and p=0.9546, respectively). 31

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Understanding the influence of blood-borne signaling molecules on brain endothelial 22 cells is critical for an understanding the BBB function. We report that lack apoM-23 bound S1P increased the BBB permeability for small molecules without changes in 24 size, structure, magnitude, or subcellular localization of tight junction complexes. 25 Furthermore, we report an up to 10-fold increase in adsorptive transcytosis in the 26 endothelial cells of pial and penetrating arterioles, but not in venules and capillaries. 27 Thus, ApoM modulates the flux of small and large molecules to different extents in 28 different vascular segments. Finally, the increased BBB permeability for small 29 molecules and transcytosis-mediated large proteins could be rescued by selective 30 stimulation of the S1PR1. 31 From among the exogenous tracers typically used in BBB paracellular permeability 32 studies, we chose three molecules with different sizes, NaFluo and Alexa Fluor 488 33 that readily diffuse via the BBB, and FITC-dextran, which is reported to have low 1 BBB permeability (31, 32). The loss of BBB integrity in apoM -/mice was observed 2 for small tracer molecules <0.7 kDa, but no significant leak was observed for 10 kDa 3 dextran, which is in accordance with previous findings in S1PR1-knock-out mice 4 (27). However, in contrast to Yanagida and colleagues (27), we show that it is not 5 the complete absence of brain endothelial S1PR1 signaling, but a lack of a specific 6 fraction of S1P circulating in the blood (i.e., apoM-bound S1P) that induces a 7 significant increase in BBB permeability. This occurred despite the presence of 8 active S1PR1 and the remaining S1P circulating in the blood bound to albumin or in 9 an unassociated form in apoM -/mice (19). 10 S1PR1 is implicated in the maintenance of BBB integrity by stabilization of the actin 11 cytoskeleton that anchors junctional complexes at the endothelial membrane cell 12 contact sites (15, 33). The increased permeability along the paracellular pathway 13 occurs most commonly in response to severe damage (34-36) and is typically 14 reported to be secondary to the ultrastructural abnormalities of junctional 15 complexes, such as truncated morphology during acute BBB breakdown, e.g. in 16 stroke, sepsis, and brain tumors (6,7,9), and in diabetes (8). While S1PR1-knock-17 out abolishes the normal structure of tight junctions and the composition of proteins 18 (27), we show that the increases in BBB permeability caused by apoM-S1P signaling 19 deficiency can occur without reduced cytoskeletal association or alteration of the 20 barrier ultrastructure. This finding is in agreement with another study that reported 21 a lack of ultrastructural changes in lung endothelium in the absence of apoM, 22 whereas the cellular barrier function was impaired (37). Notably, a significant 23 increase in paracellular permeability without apparent changes in junctional 24 complex morphology has been observed by others following osmotic shock (34) and 25 in claudin-5-deficient mice (38). The loss of BBB integrity in apoM -/mice appeared 26 to be to a lesser extent than in acute pathology (e.g., traumatic brain injury or 27 ischemic stroke), as the BBB permeability towards 10 kDa dextran did not increase 28 in apoM -/mice (10, 39). Yet, even with is morphology intact, the BBB with a 29 persistent increase in permeability may still contribute to brain pathology (40). 30 Though paracellular permeability restriction is typically viewed as the main 31 functional constituent of BBB function, the low prevalence of transcytosis is equally 32 important to preservation of the brain microenvironment (9-12). Recently, Mfsd2a 33 was identified as a critical protein in the suppression of transcytosis. Similar to 1 apoM -/-, a lack of Mfsd2a leads to an increase in vesicular transport across the 2 endothelium that compromises the BBB without structurally opening the 3 paracellular route (41) or changing junctional protein composition (42). 4 Furthermore, the increase in BBB permeability can occur via both the transcellular 5 and paracellular routes, but at different time points; the increase in transcytosis rate 6 precedes paracellular leakage in stroke (9), and selective dysregulation of the 7 transcellular pathway can occur while the tight junctions are preserved (12). Thus, 8 the intact morphology of junctional complexes, as shown here, is not necessarily 9 indicative of preserved BBB properties. Noteworthy, the lack of apoM-S1P lead to an 10 increased rate of transcytosis, though only in pial and penetrating arterioles, which 11 could be explained by non-homogeneous expression of S1PR1, which is higher in 12 arteriole endothelial cells than the endothelium of capillaries and venules (43). 13 Different G protein-coupled receptors are activated downstream of S1PR1 and 14 S1PR2 (44, 45), which results in opposite regulatory outcomes, such as in 15 neuroinflammation (21-23). The S1P bound to ApoM primarily activates S1PR1, 16 which underlies the preservation of endothelial barrier integrity in the kidneys and 17 lungs (19,25). Conversely, activation of S1PR2 has been implicated as a major factor 18 in the induction of vascular permeability following stroke (46). In a healthy brain, 19 regardless of the carrier, S1PR1 and S1PR2 are constantly stimulated by blood-20 circulating S1P, which is suggested to underlie the homeostatic regulation of 21 endothelial integrity (15, 16). We show that reduction of apoM upstream of S1PR1 22 activation is a clinically relevant mechanism that may shift the balance towards 23 S1PR2 activation, resulting in increased BBB permeability. Consequently, the 24 observed impairment of the BBB cannot simply be linked to a reduction in the 25 amount of circulating S1P, but more specifically to the lack of apoM-bound S1P in 26 favor of albumin-bound S1P in the blood. This notion is further supported by the 27 result of the selective stimulation of S1PR1, which restored the BBB permeability in 28 apoM -/mice to the level of healthy controls. 29

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In summary, ApoM-associated S1P is critical for maintaining low paracellular and 31 transcellular permeability at the BBB. The pronounced increase in transcytosis in 32 apoM -/mice stresses the importance of considering the development of protective 33 strategies for this pathway in order to regulate the immediate microenvironment of 1 brain cells and reduce secondary neural damage in both acute and chronic 2 neurological conditions. 3 4 Acknowledgements 5 We thank the Core Facility for Integrated Microscopy at University of Copenhagen, 6 especially Klaus Qvortrup, for sample preparation for TEM. We thank Micael 7 Lønstrup from the Department of Neuroscience at University of Copenhagen for 8 technical guidance in the animal surgeries. This work was supported by the 9 Lundbeck Foundation, the Danish Council for Independent Research (Medical 10 Sciences), and a Nordea Foundation Grant to the Center for Healthy Aging.

SUPPLEMENTARY VIDEOS LEGENDS 35
Video 1: Lack of apoM leads to a reversible increase in BBB paracellular permeability.

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Accumulation of NaFluo in brain parenchyma presented as fluorescence increase relative to the 37 baseline. Fluorophore injections occurred 1 minute before the first recorded imaging frame.

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Video 2: WT mice BBB paracellular permeability towards NaFluo, Alexa488, and 10kDa 40 FITC-dx. Accumulation of fluorophores in brain parenchyma presented as fluorescence increase 41 relative to the baseline. For each time-lapse recording, the respective fluorophore was injected 1 42 minute before the first recorded imaging frame.

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Video 3: apoM-/-mice BBB paracellular permeability increase towards NaFluo, Alexa488 45 but not 10kDa FITC-dx. Accumulation of fluorophores in brain parenchyma presented as fluorescence increase relative to the baseline. For each time-lapse recording, the respective 1 fluorophore was injected 1 minute before the first recorded imaging frame.

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Video 4: apoM-/-SEW2781-treated mice exhibit normal BBB paracellular permeability 4 towards NaFluo, Alexa488 and 10kDa FITC-dx. Accumulation of fluorophores in brain 5 parenchyma presented as fluorescence increase relative to the baseline. For each time-lapse 6 recording, the respective fluorophore was injected 1 minute before the first recorded imaging 7 frame.