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. 2019 Apr 11;15:39–54. doi: 10.1016/j.isci.2019.04.016

Clostridium perfringens Epsilon Toxin Compromises the Blood-Brain Barrier in a Humanized Zebrafish Model

Drew Adler 1,2, Jennifer R Linden 1, Samantha V Shetty 1, Yinghua Ma 1, Monika Bokori-Brown 3, Richard W Titball 3, Timothy Vartanian 1,4,∗
PMCID: PMC6487375  PMID: 31030181

Summary

Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor.

Subject Areas: Pathogenic Organism, Vascular Remodeling, Molecular Mechanism of Behavior, Model Organism

Graphical Abstract

graphic file with name fx1.jpg

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Highlights

  • •

    ProETX binds specifically to hMAL in live humanized zebrafish

  • •

    hMAL expression in zebrafish confers susceptibility to ETX-mediated BBB breakdown

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    Live imaging reveals ETX-mediated edema, hMAL downregulation, stenosis, and stasis

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    Antibody neutralization abrogates ETX-mediated vascular pathology

Pathogenic Organism; Vascular Remodeling; Molecular Mechanism of Behavior; Model Organism

Introduction

The blood-brain barrier (BBB) separates the complex molecular and cellular environment of blood from the refined and acellular environment of cerebrospinal fluid. This highly active functional and physical barrier maintains homeostatic protein and electrolyte concentrations and regulates mononuclear cell count in the human brain by material exclusion, passive transport, and active transport. However, the selectivity of the BBB can be altered dramatically by pathologic processes within the central nervous system (CNS) or factors originating in blood. Furthermore, pharmacologically modulating the BBB to deliver therapeutics into the CNS or repairing the damaged BBB remains major goals in biotechnology. To better understand the molecular mechanisms of a healthy and diseased BBB, we need techniques to visualize and quantify human BBB function in real time.

Clostridium perfringens epsilon toxin (ETX) is a member of the aerolysin family of pore-forming toxins (Cole et al., 2004). One of the most potent bacterial toxins, ETX is considered a potential bioterrorism weapon (Anderson and Bokor, 2012) and is widely known as a causative agent of enterotoxemia and an associated severe multifocal CNS white matter disease in ruminants (Finnie, 2003, Finnie, 2004, Uzal, 2004, Uzal et al., 2002, Uzal et al., 2004). Most of the research on ETX has been conducted in veterinary contexts, whereas a few ETX-mediated diseases have been described in humans (Mantis, 2005). ETX has been proposed as a causative agent in the human demyelinating disease, multiple sclerosis (MS) (Murrell et al., 1986), a hypothesis that has been supported by recent studies (Rumah et al., 2013, Rumah et al., 2017, Linden et al., 2015, Wagley et al., 2018). ETX appears to enter the CNS via opening of the BBB (Finnie, 2003). ETX causes a severe reduction in the integrity of BBB vessels (Finnie, 2004, Finnie et al., 2014, Morgan et al., 1975, Nagahama and Sakurai, 1991, Worthington and Mulders, 1975, Zhu et al., 2001), subsequently leading to ETX entry into brain parenchyma (Soler-Jover et al., 2007). However, the mechanism by which it crosses the BBB remains unknown.

ETX's specificity for particular cell types (Bokori-Brown et al., 2011, Dorca-Arevalo et al., 2008, Linden et al., 2015) and its inability to interact with membrane models devoid of protein (Manni et al., 2015) suggests that the toxin acts through a protein receptor. Although the receptor for ETX remains unknown, a few candidate proteins have been identified (Fennessey et al., 2012, Linden et al., 2015, Radaram et al., 2011). The ETX receptor resides on the apical surface (Petit et al., 2003) of detergent-resistant membranes, or lipid rafts, of susceptible cells (Gil et al., 2015, Miyata et al., 2002). ETX binding and cytotoxicity are dependent on the expression of the tetraspanin myelin and lymphocyte protein (MAL) (Rumah et al., 2015). Known to play a role in the formation, stabilization, and maintenance of lipid rafts (Anton et al., 2011, Ramnarayanan and Tuma, 2011, Schaeren-Wiemers et al., 2004, Zhou et al., 2012), MAL is a particularly attractive receptor candidate for ETX as it is expressed in the distal collecting duct of the kidney and myelin of the CNS (Schaeren-Wiemers et al., 1995), two well-defined targets of ETX (Nagahama and Sakurai, 1991, Rumah et al., 2015, Soler-Jover et al., 2007, Tamai et al., 2003). Importantly, studies have also shown MAL to be specifically enriched in mouse brain endothelium (Daneman et al., 2010, Tam et al., 2012, Zhang et al., 2014) and human primary brain endothelial cells (ECs) (Urich et al., 2012). We previously demonstrated that ETX (1) specifically binds to CHO-expressing MAL, (2) specifically kills CHO cells expressing MAL, (3) co-localizes with MAL, (4) does not bind to vasculature or myelin of MAL-deficient mice (MAL−/−), and (5) does not cause in vitro demyelination in MAL−/− cerebellar slice culture (Rumah et al., 2015, Linden et al., 2015). Recently, these results have been confirmed, and co-immunoprecipitation experiments exhibiting an association between ETX and human MAL has been achieved, indicating that MAL may be the ETX receptor or at minimum, a crucial component of the ETX pore complex (Blanch et al., 2018).

ETX is released as a 32.9-kDa protoxin (pETX) (Hunter et al., 1992) with approximately 1/1,000th the activity of its activated form (Minami et al., 1997). pETX is subsequently cleaved and activated to more potent forms by extracellular serine proteases (such as trypsin and chymotrypsin) or lambda toxin, a thermolysin-like metalloprotease (Hunter et al., 1992, Minami et al., 1997). pETX and active ETX share essentially identical specificity and affinity for their receptor (Buxton, 1976, Dorca-Arevalo et al., 2008, Gil et al., 2015) as indicated by the protoxin's ability to block active ETX activity and their shared binding to both lipid rafts and identical cell types. Thus pETX is a useful tool for assessing ETX's binding kinetics while maintaining the integrity of the tissue to which it localizes. Unlike pETX, active toxin induces pore formation after binding to a receptor, leading to cell death (Robertson et al., 2011). Pore formation by active ETX is a result of toxin monomer recruitment into homoheptamers on the cell surface, generating a pre-pore complex (Robertson et al., 2011). The pre-pore complex subsequently inserts itself into the lipid bilayer of susceptible cells forming a 0.5- to 1.0-nm pore (Miyata et al., 2001, Miyata et al., 2002, Petit et al., 1997). Pore formation in the BBB is thought to initiate an alteration of vascular selectivity and structure (Popoff, 2011). In contrast, protoxin fails to generate oligomers likely due to the steric inhibitory effects of its C terminus in complex formation (Miyata et al., 2001). Studies that have investigated ETX-mediated cellular and molecular pathology have used in vitro techniques and ETX-sensitive cell lines or ex vivo histology. Our understanding of ETX-mediated BBB permeability has been hindered by the lack of a live, in vivo model system allowing for real-time exploration of ETX mechanism and activity in an intact microenvironment.

To study ETX-induced BBB permeability in vivo, in real time, we developed an hMAL-expressing zebrafish model system. Zebrafish are particularly ideal for studying in vivo BBB integrity and local pathology owing to their small size, low cost, large production of offspring, frequent breeding, and most of all, translucent larval stage, which allows for the visualization of reporter genes in live organisms (Eliceiri et al., 2011, Kim et al., 2017). Transgenic lines have been generated that highlight ECs under the FLI1 enhancer/promoter and allow for the precise study of vasculature (Lawson et al., 2001, Lawson and Weinstein, 2002). Recent studies into zebrafish vascularization have demonstrated the formation of a BBB parallel to that of mammals, with restrictiveness down to 500-Da molecules as early as 3 days post fertilization (DPF) (Fleming et al., 2013, Jeong et al., 2008). In addition, known mammalian BBB tight junction proteins including ZO-1 and claudin-5 have been detected in zebrafish (Jeong et al., 2008, Xie et al., 2010). Utilizing these discoveries and innovations, scientists have used zebrafish to examine the effects of various bacterial and viral infections as well as toxic insults on the integrity of circulatory system and BBB (Bolcome et al., 2008, Ludwig et al., 2011, Mostowy et al., 2013, Takaki et al., 2013). These findings encourage testing ETX-induced barrier dysfunction in this unexploited model organism.

Although zebrafish express MAL, we have previously demonstrated that CHO cells expressing zebrafish MAL (zMAL) do not bind ETX (Rumah et al., 2015). However, this does not exclude the possibility that zebrafish may have another ETX receptor distinct from MAL. In this study, we confirm that zebrafish neurovasculature does not bind ETX and that the introduction of hMAL into EC results in specific, high-affinity binding of protoxin. We further define the specific requirement of MAL for ETX toxicity by showing that BENE, a tetraspanin protein in the MAL family, fails to bind pETX. We provide kinetic evidence for the rapid effect of epsilon toxin on BBB permeability, blood vessel stenosis, focal perivascular edema, and hMAL receptor downregulation in a living organism. Last, we demonstrate that an anti-ETX neutralizing antibody inhibits any evidence of ETX vascular pathology in hMAL-expressing fish. Overall, this gain-of-function study further demonstrates the necessity of MAL for ETX's action, provides indication of ETX-mediated pathology in a humanized model of the BBB, and more generally, elucidates a precise timeline for ETX action in an intact microenvironment.

Results

Wild-Type Zebrafish Do Not Bind ETX in Vasculature

The first step of establishing the suitability of the zebrafish model for ETX toxicity studies was to examine whether the vasculature of wild-type (WT) zebrafish has affinity for ETX. Although it has previously been reported that ETX does not bind the zMAL (Rumah et al., 2015), it is possible that zebrafish express a specific ETX receptor other than MAL. To show that zebrafish lack specific binding sites for ETX, immunohistochemistry was conducted on zebrafish cryosections (Figure 1).

Figure 1.

Figure 1

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pETX Binds to WT Mouse, but Not WT Zebrafish Neurovasculature

(A) Diagram of mouse and zebrafish sagittal sections with the cerebellum and the region of imaging (blue box) highlighted. (B) Slices from both organisms were probed with pETX and incubated with rabbit anti-ETX primary antibody followed by cy3 anti-rabbit secondary/BSL1. DAPI mounting media in blue highlights nuclei, BSL1 in green highlights vasculature, pETX is in red, “Merge” photo identically stacks all three colored pictures. pETX co-localized with BSL1-highlighted vasculature (white arrows) in all areas of each section of mouse positive controls, as indicated in the yellow vessel in the “Merge” picture, but could not be detected in any zebrafish slice. Toxin also bound strongly to the granular layer of mouse positive controls (blue arrows). Central photos taken at 40× magnification with the same exposure time for each condition. ML, molecular layer; GL, granular layer. Scale bars, 50 μm.

Histological sections of adult (8- to 16-week-old) mouse brain and adult (17 months post fertilization) zebrafish brain were generated. Sagittal sections were incubated with pETX and immunostained for toxin binding using a high-affinity monoclonal antibody against epsilon toxin. Immunohistochemistry revealed that the cerebellar molecular layer of the zebrafish did not bind pETX (Figure 1). In comparison, pETX specifically binds to myelin of the mouse granular layer and localizes to mouse BBB microvessels, highlighted by BSL1 staining (Lonchamp et al., 2010, Linden et al., 2015). Importantly, pETX fails to bind to zebrafish cerebellar vasculature.

Larval Zebrafish Bind ETX Specifically to hMAL-Expressing Blood Vessels

Because zebrafish fail to bind pETX, we used the model organism in gain-of-function experiments to determine if cell-specific expression of hMAL conferred specific ETX binding and if this gene would be sufficient to induce hallmarks of ETX-mediated, vascular pathology. To examine whether ETX binds to hMAL-expressing zebrafish vasculature, zebrafish zygotes were injected with hMAL fused to a GFP reporter (hMAL-GFP) under the FLI1 endothelial promoter/enhancer to generate transient expression (Figure 2A). Fish that successfully expressed the reporter construct were subsequently injected with Alexa 647-conjugated pETX at 3–4 DPF to look for binding. These preparations were compared with fish injected with plasmids containing a receptor within the same proteolipid family, hBENE-GFP, under the same promoter, to both control for possible non-MAL contingent binding and examine the specificity of the ETX-MAL interaction. Confocal microscopy revealed binding of pETX to vessels expressing hMAL-GFP and undetectable binding, beyond levels of circulating unbound toxin, in hBENE-GFP controls (Figure 2B).

Figure 2.

Figure 2

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pETX Binds to hMAL-GFP Expressed in Zebrafish ISVs in a Time-Dependent Manner

(A) Schematic depicting zebrafish transformation strategy, toxin injection site, and microscopic focus. Plasmids containing hMAL-GFP or hBENE-GFP were co-injected with tol2 transposase into the cell of 1-cell-stage zebrafish embryos. Positive fish 3–4 days post fertilization (DPF) were injected with Alexa 647-tagged pETX into the duct of Cuvier for systemic infection. The blue box indicates the region imaged in (B).

(B) The 40× confocal z stack images of zebrafish ISVs in hMAL-GFP-expressing fish versus hBENE-GFP-expressing control fish. Scale bar, 25 μm. ISV, intersegmental blood vessel; DLAV, dorsal longitudinal anastomotic vessel.

(C) Quantitative metric of co-localization, “Percent of ETX Material Co-localized in GFP Vessel,” indicates the percentage of pETX voxels co-localized in hMAL-GFP fish ISVs (n = 6 fish) versus hBENE-GFP control ISVs (n = 7 fish) above the automatically set threshold at 1 h post injection. Data are represented as mean ± SEM. Statistical analysis: unpaired, two-tailed Student's t test (**p < 0.01).

(D) Equal volumes of GFP along the walls of ISVs were rendered into Imaris surfaces, and sum GFP and 647 intensities within the surface were tracked over the course of ∼90 min at 30-s intervals per fish. The ratio of 647-pETX/GFP was plotted over time for hMAL and hBENE transients. Individual ratios of every fish at each time point are represented by opaque circles, and the average ratio at each time point is represented by the darker line.

(E) Snapshot images of 647-pETX or 647-H149A pETX and “merged” images with receptor at three time points post injection. Blue arrow highlights toxin accumulation. Scale bar, 20 μm.

(F) Flow cytometry analysis of pETX or H149A pETX binding to hMAL, rMAL, or GFP CHO cells.

See also Videos S1, S2, and S3. Statistical analysis: two-way ANOVA with Tukey’s test (∗∗∗∗p < 0.0001).

Analysis of hMAL-GFP- and hBENE-GFP-expressing vessels revealed significant differences in co-localization with Alexa 647-pETX. Co-localization was quantitatively assessed by first setting a GFP threshold to specify EC localization and then determining the percentage of total Alexa 647 pETX signal specifically associated with these GFP voxels (Figure 2C, Student's t test, p = 0.0028). To assess the pETX binding kinetics, zebrafish expressing hMAL-GFP or hBENE-GFP were subjected to time-lapse imaging of intersegmental vessels (ISVs) strongly expressing hMAL-GFP or hBENE-GFP. Results indicate that hMAL-GFP binds pETX in a time-dependent manner, as exhibited by Alexa 647 pETX intensity increasing over time within hMAL-GFP vessels, with peak binding occurring 54 min post injection (MPI), whereas pETX-647 intensity decreasing over time within hBENE-GFP vessels and no degree of convincible binding being apparent (Figures 2D and 2E, respectively). Time-lapse videos evidence pETX moving from the lumen to areas of hMAL-GFP in the wall of ISV vessels over time (Video S1) and not in hBENE-GFP control vessels (Video S2). To control for non-specific protein-protein interactions, we utilized the pETX H149A mutant, previously shown to have reduced activity to MDCK cells and toxicity in mice (Oyston et al., 1998) while maintaining structural similarities to the WT toxin (Bokori-Brown et al., 2013). pETX H149A was fluorescently tagged and injected into fish transiently expressing hMAL-GFP under control of the FLI1 promoter. Two of four fish showed subtle accumulation of fluorescent pETX H149A in hMAL-expressing vessels indicating low affinity to hMAL in a live, in vivo context (Figure 2E and Video S3). In contrast, by flow cytometry the H149A mutant toxin shows no detectable binding to CHO cells expressing hMAL (Figure 2F).

Video S1. Alexa-Tagged pETX Has Affinity for hMAL-GFP ISVs, Related to Figure 2

A representative time-lapse video of tagged pETX accumulating at the edge of an hMAL-GFP vessel. The first half of the video just shows the red pETX channel, whereas the second half is a repeat with both the red pETX channel and the green hMAL channel.

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Video S2. Alexa-Tagged pETX Has No Affinity for hBENE-GFP ISVs, Related to Figure 2

A representative time-lapse video of tagged pETX failing to accumulate at a hBENE-GFP vessel. The first half of the video just shows the red pETX channel, whereas the second half is a repeat with both the red pETX channel and the green hBENE channel.

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Video S3. Alexa-Tagged H149A Mutant pETX Shows Subtle Affinity for hMAL-GFP ISVs, Related to Figure 2

A representative time-lapse video of tagged pETX accumulating at the edge of an hMAL-GFP vessel. The first half of the video just shows the red pETX channel, whereas the second half is a repeat with both the red H149A mutant pETX channel and the green hMAL channel.

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Active ETX Causes Leakage of the Blood-Brain Barrier in Fish Expressing hMAL

Having established pETX's specific affinity for hMAL and its binding kinetics in a live in vivo context, experiments were then conducted to assess the impact of hMAL on BBB leakage after being challenged with activated toxin (Figures 3A and 4A). Confocal microscopy revealed progressive leakage of a 2,000-kDa rhodamine dextran dye co-injected with activated ETX from 15 MPI to 2 h post injection (HPI) only in hMAL-expressing fish (n = 6. Figure 3C and Video S4) and not in controls expressing hBENE (n = 5, Figure 3B and Video S5). Furthermore, fish expressing hMAL co-injected with H149A mutant ETX also failed to show evidence of vascular leak (n = 4, Figure S3A). In both hBENE and H149A mutant ETX controls, toxin is confined inside blood vessels; this is particularly salient when looking at Imaris surface renderings of the receptor and toxin at 2 HPI (Figures 3Bʹ and S3B) as opposed to hMAL fish injected with active toxin (Figure 3C′). A quantitative assessment of dextran leakage, represented as extracellular/intracellular dextran (“E/I” see Transparent Methods Equation 1), confirms a significant difference in leakage between hMAL + active ETX conditions and hBENE + active ETX and hMAL + H149A pETX controls at 2 HPI (Figure 3D, two-way ANOVA with Tukey's test, p ≤ 0.0001, 0.0006) and a significant difference within the hMAL + active ETX group between time point 15 MPI and 2 HPI and 1 HPI and 2 HPI (Figure 3D, two-way ANOVA with Tukey's test, p ≤ 0.0001, 0.0006). This finding indicates that a progressive reduction in BBB integrity is robustly apparent only in hMAL-expressing fish treated with active ETX.

Figure 3.

Figure 3

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Co-injection of Activated ETX and 2,000-kDa Rhodamine Dextran Reveals Pronounced BBB Leakage in hMAL-Expressing Fish

(A) Schematic depicting experimental procedure.

(B) The 40× confocal z stacks of the vasculature in the anterior dorsal quadrant of the brain in a representative hBENE-GFP transiently expressing control zebrafish. hBENE-GFP receptor is in green, the 2,000-kDa rhodamine dextran is in red, “merge” images show both channels, and “Merge Macro” images show full-brain neurovasculature. The blue box in “Merge Macro” highlights the area focused in for the other images; 15 min, 1 h, and 2 h indicate the time z stack imaging commenced after toxin or dextran injection. Scale bar, 50 μm in “Merge Macro” and 30 μm in the rest of the images.

(C) Same as (B) but in an hMAL-GFP-expressing fish. (B′) Imaris “surface” rendering of hBENE-GFP in green and rhodamine dextran in red used for quantification of GFP sum intensity and total “intravascular” rhodamine sum intensity within the lumen of blood vessels at 2 h post injection. (C′) Same as (B′) but in hMAL-GFP fish.

(D) Quantification of dextran leakage ascertained by determining extravascular dextran sum intensity divided by intravascular sum intensity (“dextran E/I”) at 15 min, 1 h, and 2 h post injection. Data included from experiment using hBENE-GFP fish injected with active ETX (n = 5), hMal-GFP fish injected with active ETX (n = 6), and hMal-GFP fish injected with mutant control H149A pETX (n = 4).

Data are represented as mean ± SEM. Statistical analysis: two-way ANOVA with Tukey’s test (***p < 0.001, ****p < 0.0001). See also Figure S3 and Videos S4 and S5.

Figure 4.

Figure 4

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Within Individual hMAL-GFP-Expressing Fish, Dextran Leaks Markedly More in Volumes of the Brain Populated by Receptor-Expressing Vessels than in Areas Without Receptor Expression

(A) Schematic of injection and imaging strategy.

(B) Confocal z stack of a representative fish transiently expressing hMAL-GFP (green) injected with active toxin and 2,000-kDa rhodamine dextran (red) and an adjacent image of just the red dextran channel. Because of the chimeric nature of expression most fish only expressed receptor in discrete parts of the brain, and as depicted, often in half of the brain. White arrows indicate regions of evident leakage. Blue and orange boxes represent the brain quadrants quantified in (C).

(C) Quantification of E/I ratio at 2 HPI between volumes of the brain that have high levels of dextran expression (blue) and those with little dextran expression (orange) for each fish in each condition: hBENE transients injected with active ETX (n = 5/side of brain), hMAL transients injected with active ETX (n = 6/side of brain), hMAL transients injected with H149A mutant pETX (n = 4/side of brain). Significant difference between these two sides of the brain are only seen in the hMAL + active ETX condition (two-way ANOVA with Tukey's test, p = 0.0008). Data are represented as mean ± SEM.

Video S4. ETX Causes Stark Leakage in hMAL-GFP Neurovasculature, Related to Figures 3 and 4

A representative confocal z stack traversing ventrally through the neuravasculature of an hMAL-GFP-expressing fish. All evident leakage appears adjacent to hMAL-GFP vessels. The red channel represents 2.000-kDa rhodamine dextran. The green channel represents hMAL-GFP. The gray background is differential interference contrast imaging.

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Video S5. ETX Causes No Leakage in hBENE-GFP Neurovasculature, Related to Figure 3

A representative confocal z stack traversing ventrally through the neuravasculature of a hBENE-GFP-expressing fish. All red dextran appears confined in blood vessels. The red channel represents 2.000-kDa rhodamine dextran. The green channel represents hBENE-GFP. The gray background is differential interference contrast imaging.

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Although the FLI1 endothelial promoter/enhancer restricts transgene expression to ECs, the fraction of ECs within an individual fish that expressed the relevant transgene was between 40% and 70%. Incomplete expression within the endothelial lineage often manifested itself as only one-half of the brain expressing the transgene, thus providing us with a novel internal control. Within individual fish an area with high receptor expression was compared with an identical area on the opposite half of the brain with minimal or no observable transgene expression at 2 HPI (Figures 4B and 4C). Corroborating the finding that hMAL's expression is a requisite for BBB disruption, at this time point a significant difference in dextran leakage was only seen between these two halves (receptor expressing and receptor absent) in hMAL + active ETX conditions (Figure 4C, two-way ANOVA with Tukey's test, p = 0.0008).

Active ETX Causes Stenosis of BBB Vessels, Perivascular Edema, and Blood Flow Cessation in hMAL Transients

Besides evident leakage, confocal images were examined for the presence of other previously described ETX-induced vascular pathologies and whether hMAL was required for the presence of such phenomena. One previously described finding—vascular lumen compression (Finnie, 1984a, Sakurai et al., 1983)—was evident in the majority of assessed hMAL-expressing vessels (Figure 5A). Assessment of cerebral central arteries (CCtAs) indicated a progressive decrease in vessel lumen diameter between 15 MPI and 2 HPI in hMAL-expressing vessels challenged with active ETX (n = 6, Figure 5B, two-way ANOVA with Tukey's test, p = 0.0193) but not in hBENE + active ETX (n = 5) or hMAL + H149A pETX controls (n = 3). Significant difference in CCtA lumen diameter started at 1 HPI between hMAL + active ETX and hBENE and H149A controls (two-way ANOVA with Tukey's Test, p = 0.0272, 0.0104). In addition, perivascular edema, a hallmark of both ETX toxicity and MS, was evident in two hMAL-expressing fish (Figure 5C and Video S6) and not in controls (Video S7). In both cases, the locations of edema corresponded directly to regions of dextran leakage and all occurred in vessels with robust receptor expression as early as 15 MPI. Curiously, these areas of acute edema were preferentially seen in vessels traversing through the cerebellum. At 50 MPI, 10-s rapid time-lapse videos of blood flowing through cerebral vessels corroborates this conjecture and indicates the necessity of hMAL, for in hMal-GFP vessels, blood flow appears to completely terminate (Video S8), whereas in both identical, non-hMAL-expressing vessels within the same fish and within all vessels in control fish, blood flow appears unperturbed (Videos S9 and S10).

Figure 5.

Figure 5

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Active ETX Leads to Vessel Stenosis and Perivascular Edema

(A) Zoomed-in confocal images of cerebral central arteries (CCtAs) in hBENE-GFP- and hMAL-GFP-expressing fish 15 min, 1 h, and 2 h post injection with active toxin. Green channels depict receptor expression in these vessels, gray channel depicts differential interference contrast images, “merge” overlays both images. Scale bar, 10 μm.

(B) Quantitative comparison of CCtA vessel diameter over time for each condition; hMAL CCtA exposed to active ETX are significantly narrower than hBENE CCtAs exposed to ETX or hMAL CCtAs exposed to H149A mutant pETX starting at 1 HPI and showed significant decrease from 15 MPI to 2 HPI (two-way ANOVA with Tukey's test, *p < 0.05, **p < 0.01). Data are represented as mean ± SEM.

(C) Confocal differential interference contrast macroscopic z-slice of zebrafish brain exhibiting perivascular edema in an hMAL-GFP-expressing fish and (C′) zoomed in single vessel.

(D) Image (C) with merged hMAL-GFP and dextran channels and (D′) merged, zoomed-in single vessel. Scale bar, 25 μm for full image and 10 μm for zoomed-in vessel.

See also Figure S3 and Videos S6, S7, S8, S9, and S10.

Video S6. ETX Causes Perivascular Edema in hMAL-GFP Neurovessels, Related to Figures 5 and S7

A representative z stack using the differential interference contrast channel (same stack as video S7) reveals edema in select hMAL-expressing blood vessels. The red arrow highlights edema.

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Video S7. ETX Causes No Perivascular Edema in hBENE-GFP Neurovessels, Related to Figures 5 and S8

A representative z stack using the differential interference contrast channel (same stack as video S8) reveals no edema in any hBENE-expressing blood vessels.

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Video S8. ETX Terminates Blood Flow in hMAL-GFP Neurovessels, Related to Figure 5

A representative comparison of blood vessels at the midline the brain of an hMAL-GFP fish evidences continued blood flow in a blood vessel without recognizable hMAL-GFP expression and stopped blood flow in an hMAL GFP vessel after injection of ETX. Red represents 2,000-kDa rhodamine dextran. Green represents hMAL-GFP. The gray background is differential interference contrast imaging. Video taken at 50 MPI.

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Video S9. ETX Fails to Terminate Blood Flow in hBENE-GFP Neurovessels, Related to Figure 5

A representative comparison of blood vessels at the midline the brain of an hBENE-GFP fish evidences blood flow in both a blood vessel without recognizable hBENE-GFP expression and in an hBENE-GFP vessel after injection of ETX. Red represents 2,000-kDa rhodamine dextran. Green represents hBENE-GFP. The gray background is differential interference contrast imaging. Video taken at 50MPI.

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Video S10. H149A Mutant pETX Fails to Terminate Blood Flow in hMAL-GFP Neurovessels, Related to Figures 7 and S3

A representative comparison of blood vessels at the midline the brain of an hMAL-GFP fish evidences blood flow in both a blood vessel without recognizable hMAL-GFP expression and in an hMAL-GFP vessel after injection of H149A pETX. Red represents 2,000-kDa rhodamine dextran. Green represents hMAL-GFP. The gray background is differential interference contrast imaging. Video taken at 50MPI.

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Active ETX Causes hMAL Downregulation

An unforeseen finding revealed in this in vivo preparation was the distinct recycling of the hMAL receptor when challenged with active ETX. By 15 MPI, hMAL-GFP expression appears punctate along vessel edges when compared with hBENE-GFP (Figure 6A). Comparison of hMAL-GFP vessels challenged with active ETX versus H149A mutant pETX reveals that this punctate appearance is mediated by active toxin and is not simply due to a difference between hMAL and hBENE receptor expression, for hMAL vessels challenged with mutant toxin appear indistinguishable from hBENE-expressing vessels challenged with active toxin. Furthermore, images and quantitative analyses reveal that overall receptor protein levels progressively decrease over time in hMAL + active ETX conditions and that there is a significant percent decrease in GFP intensity, and these decreases are seen between both hMAL + active ETX (n = 6) and hBENE + active ETX (n = 5) and hMAL + H149A pETX (n = 4) starting at 1 HPI (Figure 6B, two-way ANOVA with Tukey's test, p = 0.0029, 0.0107).

Figure 6.

Figure 6

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ETX Leads to hMAL Receptor Reduction and Recycling

(A) Representative confocal images of equal volumes of neurovasculature expressing either hBENE-GFP or hMAL-GFP challenged with either active toxin or H149A pETX at 15 MPI, 1 HPI, and 2 HPI. Scale bars, 30 μm.

(B) Percent change in receptor-GFP sum intensity for each condition using 15 MPI as the starting point. Significant percent change in receptor-GFP intensity reached between hMAL + active ETX and controls starting at 1 HPI (two-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001). Data are represented as mean ± SEM.

Neutralizing Anti-ETX Antibody Inhibits ETX Toxicity

To determine the specificity of ETX cytotoxicity in vivo, ETX was pre-incubated with a neutralizing monoclonal antibody, JL008, against ETX before injection into fish expressing hMAL-GFP (n = 4) (Linden et al., 2018). Confocal image analysis of identical experimental parameters revealed dextran well confined within vessels, normal CCtA lumen diameter, and steady blood flow through hMAL-GFP vessels (Video S11). Quantitative analysis confirmed significant difference between neutralized and active ETX E/I at 2 HPI (two-way ANOVA with Tukey's test, p = 0.0003) and no significant difference with H149A pETX mutant (Figure 7A). There was also no significant difference in dextran leakage between areas of the brain with hMAL expression and areas without (Figure 7B). CCtA lumen diameter was also significantly different in animals treated with neutralized ETX compared with active ETX (Figure 7C, two-way ANOVA with Tukey's test, p = 0.0004). Percent change in hMAL-GFP reduction between 15 MPI and 2 HPI was also significant in animals treated with neutralized ETX compared with active ETX (Figure 7D, two-way ANOVA with Tukey's test, p = 0.0047). Thus in all metrics of toxicity, active ETX was effectively neutralized with the neutralizing anti-ETX antibody. Taken together, these data indicate (1) the specificity of ETX-induced pathology and (2) the potential use of this antibody as treatment for ETX-induced disease.

Figure 7.

Figure 7

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Antibody Neutralization of ETX Eliminates All Metrics of Toxicity

(A and B) (A) Quantitative comparison of dextran leakage (Dextran) in hMAL-GFP-expressing fish injected with neutralized ETX (n = 4), active ETX (n = 6), or H149A pETX (n = 4) from 15 MPI to 2 HPI and (B) dextran leakage between two identical areas of the brain, one with high receptor expression and one with minimal expression.

(C) Quantitative comparison of CCtA lumen diameter in vessels expressing hMAL-GFP treated with the same three preparations.

(D) Quantitative comparison of % change in hMAL-GFP intensity for each treatment using 15 MPI as the baseline.

All statistical analyses using two-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are represented as mean ± SEM. Supporting images in Figures 3 and S2.

Video S11. Antibody Neutralization of ETX Restores Blood Flow in hMAL-GFP Neurovessels, Related to Figures 7 and S2

A representative comparison of blood vessels at the midline of the brain of an hMAL-GFP fish evidences blood flow in both a blood vessel without recognizable hMAL-GFP expression and in an hMAL-GFP vessel after injection of antibody-neutralized ETX. Red represents 2,000-kDa rhodamine dextran. Green represents hMAL-GFP. The gray background is differential interference contrast imaging. Video taken at 50 MPI.

Download video file (991.2KB, mp4)

Discussion

This study, taken as a whole, has provided evidence of real-time ETX binding, receptor downregulation, and BBB disruption via live imaging in a whole animal. Developments in techniques for zebrafish models, including readily available gateway-cloning constructs, reliable injection protocols, and their ease of use in confocal microscopy, have allowed here for precise scrutiny of ETX's action within an intact microenvironment. The organism, when exposed to ETX, gives insights into the sufficiency of the MAL receptor to induce hallmarks of ETX-mediated vascular pathology, for zebrafish only exhibit significant BBB leakage, neurovascular compression, blood-flow cessation, perivascular edema, and receptor downregulation when the hMAL gene is expressed. The presence of transiently expressing mosaic fish, as opposed to stable transgenic fish, further affirms the sufficiency of the receptor, for within individual fish only vascular segments with evident receptor expression demonstrated pathology.

Time-lapse imaging allowed for real-time examination of the binding kinetics of pETX to hMAL with peak binding happening approximately 1 h after introduction of toxin into the intravascular space. Peak toxin binding corresponds to the first indication of significant neurovascular changes after ETX treatment. This time point corresponds with the first observations of endothelial damage in mice injected with sublethal doses of activated ETX (Finnie, 1984a) and changes in endothelial barrier antigen after pETX treatment (Zhu et al., 2001)—a further confirmation of the suitability of the zebrafish model for such studies. Certainly, this temporal sequence of vascular pathology would change at different concentrations of toxin; previous studies have shown noticeable leakage of various macromolecules as early as 30 s post intravenous injection using higher, lethal concentrations and tracing much smaller macromolecules (Morgan et al., 1975, Nagahama and Sakurai, 1991, Worthington and Mulders, 1975). Furthermore, although statistical significance was only reached at 1 h, gross imaging suggests leakage and vascular changes as early as 15 MPI. Contrary to previous studies reporting no ETX-induced leakage of large (38.5 kDa) dextrans in in vitro polarized MCDK cells (Petit et al., 1997) or BSA using post mortem histopathology (Soler-Jover et al., 2007), this study exhibits drastic leakage of a much larger 2,000-kDa dextran that aggregates at particular hMAL-expressing vessels and diffuses over time. This stark difference in dextran size leakage, between our and previous studies, could be due to developmental and evolutionary differences between the BBB in the zebrafish and rodents. Although the time point chosen (6–7 DPF) is beyond the origin of tight junction protein expression in zebrafish (Xie et al., 2010), and is later than similar toxicological studies (Bolcome et al., 2008, Zhang et al., 2015), the zebrafish BBB does become increasingly selective beyond 6 DPF, as it begins to inhibit larger dextrans (Fleming et al., 2013). Thus an assessment of ETX-induced leakage past 7 DPF is a possibility for future experimentation, although this later time point requires stable expression of constructs. Leakage of the large dextran could also be due to ectopic overexpression of the hMAL protein, although gross image analysis indicated that the areas with the most abundant hMAL expression were not necessarily the regions that had the most leakage, rather minimal MAL expression was all that was needed for local leakage to be observed. The most likely reason the toxin elicited leakage of such a large tracer was the still relatively high dosage of ETX injected in this study. The ETX dose used in this study most likely led to receptor saturation and maximal pore formation, resulting in massive BBB leakage. The possibility also exists that leakage could be due to cytotoxicity of ECs upon exposure to ETX. Although in vitro work indicates that hMAL-expressing CHO cells are highly resistant to death upon exposure to high concentrations of ETX (data not shown), further tests are required to rule this out in an intact microenvironment.

The H149A mutant pETX was also assessed for its relevance as a control for both binding and toxicity experiments. Our studies indicate similar binding affinities of H149A and WT ETX to MDCK cells (data not shown), and previous studies indicate similar structure of the mutant and WT protoxin (Bokori-Brown et al., 2013) while exhibiting reduced toxicity (Oyston et al., 1998). Binding of pETX to hMAL cells and tissue presented here are less conclusive. Although fluorescent co-localization over time in hMAL-expressing fish is certainly reduced when compared with WT pETX, there was clear evidence of a small degree of mutant protoxin accumulation in two of four hMAL ISVs. In contrast, flow cytometry revealed no significant binding of mutant toxin to hMAL, yet significant binding to rMAL. There are many reasons that potentially explain this discrepancy. First, the toxin likely exhibits different binding affinities to different orthologs of MAL. Also, in vitro cell-based assays using cell lines may not provide the optimal microenvironment for hMAL-H149ApETX binding. ACHN cells, for instance, show negligible suseptibilty to H149A-ETX, even though MDCK cells are suseptible (Bokori-Brown et al., 2013). Possible co-receptors, not expressed in ACHN cells, but expressed in vivo in vasculature, may act in concert with MAL to form a suitable H149ApETX-binding site. Alternatively, as the mutant is already recognized to be 67-fold less toxic in mice (Oyston et al., 1998), in vitro assays may have been insufficient at detecting mutant protoxin binding to hMAL, for we show that WT protoxin is at baseline bound to less than 50% of hMAL-CHO cells. Our live in vivo assay may have been more sensitive and thus revealed modest mutant accumulation. Regardless of the difference between in vivo and in vitro binding of H149ApETX, this mutant proves a suitable control for toxicity studies, as results here indicated no evidence of H149ApETX-mediated vascular pathology despite binding in two of our four fish.

This study confirms previous findings of ETX-induced BBB vessel stenosis (Finnie, 1984b), perivascular edema (Buxton, 1976, Fernandez-Miyakawa et al., 2008, Freedman et al., 2016, Uzal et al., 2004), and subsequent decreased blood-flow rate (Sakurai et al., 1983). Sakurai et al. (1983) hypothesized that their finding of decreased blood-flow rate in mice injected with active ETX was due to a vascular “pressor” activity of the toxin, for no change in heart rate was observed. Later the same group identified a degree of ETX-mediated aortic constriction, yet affirmed that the most likely cause of ETX-mediated elevated blood pressure was a constriction of resistant arteries (Nagahama and Sakurai, 1992). In our present study, the vessel assessed for changes in diameter (the CCtA) acts as a resistance artery as indicated by its ensheathment by smooth muscle cells (Ando et al., 2016) and its expression of a protein (Yap1) that allows for the change of vessel diameter in response to blood flow (Nakajima et al., 2017). Thus by using in vivo imaging, our work confirms the idea that ETX-mediated changes in blood-flow rate are due to its vasoconstrictive capabilities. Furthermore, this study presents real-time imaging of blood flow after ETX injection and indicates a complete halt in blood flow within hMAL-expressing vessels. Such a drastic change in flow suggests that at high doses, ETX-mediated neuropathology may include hypoxia or ischemia. This finding is contrary to an earlier Doppler-based analysis focused on the hippocampus using lower dosages than were used in this study (Miyamoto et al., 1998). Taken together, ETX at low doses appears to lead to leakage of moderate (up to ∼38 kDa) tracers (Petit et al., 2003), endothelial blebbing, and perivascular swelling (Finnie, 1984b) while maintaining steady blood flow (Miyamoto et al., 1998). In contrast, ETX at high concentrations, such as the one used in this study, leads to leakage of much larger (as much as 2,000 kDa) dextrans, stark blood vessel compression, and a termination blood flow.

Recently, from a western blot analysis of epsilon-treated human lymphocyte lysates, it has been shown that MAL and epsilon toxin co-immunoprecipitate (Blanch et al., 2018). This suggests that MAL and epsilon toxin exit together in a protein complex and proposes that there may be a direct protein-protein interaction between the two, although this has not yet been proven. Our study corroborates this finding in vivo, for we observed co-localization of pETX and hMAL-GFP in live zebrafish. In addition, we detected a downregulation in hMAL-GFP fluorescence after ETX treatment, raising the possibility that ETX binding to MAL results in internalization and degradation. We also confirmed previous gain-of-function assays demonstrating that MAL is required for both ETX binding and toxicity (Linden et al., 2015). Such downregulation has been reported before in vitro using the rodent ortholog of the MAL protein (rMAL) and active toxin (Rumah et al., 2015), although this similar finding with hMAL extends its relevance to human models. Moreover, the exemplification of such a stark phenomenon in an intact microenvironment adds biological significance. Taken together, these data strongly suggest that MAL is the receptor for ETX.

The ETX-specific monoclonal antibody, JL008, completely abrogated all analyzed metrics of ETX-mediated pathology. This both suggests that JL008 is of use as a control for further ETX studies and shortlists it as a potential treatment for ETX intoxication among other tested ETX-neutralizing antibodies (McClain and Cover, 2007, Sakurai et al., 1983). This finding, however, was generated when ETX was incubated for an hour before injection into the live organism. Further tests should be conducted to examine if injection of JL008 after ETX can mitigate the toxin's effects in vivo and generate both a time course and a dose course necessary for prevention of ETX-induced pathology. The zebrafish model is already gaining traction as a platform for drug discovery and screening (Kari et al., 2007, MacRae and Peterson, 2015, Parng et al., 2002, Serbedzija et al., 1999, Taylor et al., 2010). Generation of a transgenic endothelial-specific hMAL-expressing fish line would make this model an ideal candidate for screening anti-ETX treatments via high throughput drug discovery assays.

The use of zebrafish would be ideal to answer a final question regarding the ETX-mediated MS hypothesis: does ETX cause demyelination once in the CNS? Regarding such an assay of demyelination, previous developmental studies have confirmed that zebrafish have robust myelin with oligodendrocytes and Schwann cells, which appear at relatively similar time points as mice, and express many of the same protein markers (Ackerman and Monk, 2016), Recently, the organism has been presented as an option for MS modeling after successful demyelinating results using the canonical lysolecithin (Preston and Macklin, 2015) and experimental allergic encephalomyelitis (EAE) (Kulkarni et al., 2017) MS models. We have previously reported that demyelination occurs in WT mouse models and not in MAL−/− challenged with ETX, yet we have also indicated higher-affinity binding of ETX to rMAL than hMAL and a greater degree of death in rMAL-CHO than in hMAL-CHO cells (Linden et al., 2015). Thus, although it seems reasonable, it is unclear if ETX leads to demyelination in humans. If it does, the toxin concentration necessary to initiate such CNS insults remains unexplored. Humanized models, such as the one presented in this study, can provide these necessary, important insights. The zebrafish is a particularly apt option, for as we have shown here, unlike murine models, it is not normally susceptible to ETX. A particularly compelling follow-up study would be to generate fish that express two reporter-tagged hMAL constructs (using two different reporters) simultaneously in ECs and oligodendrocytes and observe if both BBB penetration and demyelination occur, and if so the kinetics of such pathology. This study has the potential to offer the most comprehensive simulation of ETX-MAL-mediated MS as it may occur in an intact microenvironment.

The body evidence indicating human susceptibility to ETX is limited; however, there are reports of human infection with ETX expressing C. perfringens toxinotypes and associated disease. One case report has evidenced a patient with a gangrenous ilium and has isolated ETX secreting C. perfringens (type D) (Gleeson-White and Bullen, 1955). In another report ETX was directly isolated from the blood serum of a patient with ankylosing spondylitis (Kohn and Warrack, 1955). In addition, two studies have isolated C. perfringens type D from patients with gas gangrene (Morinaga et al., 1965) (Miller et al., 2004). A growing body of evidence has implicated ETX in MS with C. perfringens type B (ETX secreting) isolated from a patient with MS and antibodies against the toxin enriched in patient serum (Rumah et al., 2013, Wagley et al., 2018). ETX is known to bind to human renal cell lines (Ivie et al., 2011, Fernandez Miyakawa et al., 2011, Shortt et al., 2000) and cauda equina myelin (Dorca-Arevalo et al., 2008). However, no report has yet shown whether the toxin binds to human BBB. This study's evidence of an association between ETX and hMAL, the interaction's consequential numerous effects on neurovasculature, and MAL's known expression in human BBB (Urich et al., 2012) gives clues as to how ETX might act when circulating through the human bloodstream.

Limitations of the Study

We do not know if there is direct protein-protein interaction between ETX and hMAL or whether there might be other proteins involved in the complex. It is also still unclear whether ETX would still cause the dramatic consequences revealed here at later time points in development as the BBB fully matures or whether decreased concentrations of ETX would elicit the same findings. Another limitation to the generalizability of this study is the mode of infection (intravenous), which fails to address ETX's transportation from the gut to the bloodstream. We were able to neutralize the effect of the toxin by incubating it, before injection, with a custom monoclonal antibody, yet from a therapeutic standpoint it remains to be seen if the antibody could still mitigate ETX toxicity after the toxin is already in the bloodstream. Finally, it is still unclear what occurs to brain parenchyma after BBB leakage commences.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

We would like to thank the Fetcho lab for providing laboratory space, abundant Casper zebrafish fish, consultation, and continual support. In particular J. Fetcho's mentorship and B. Miller's technical expertise were invaluable to the reality of this project. This work was funded in part by the National Institute of Neurological Disorders and Stroke (1R01NS104350, 1R21MS106581) and the Wilma S. and Laurence A. Tisch Foundation.

Author Contributions

Conceptualization. D.A., J.R.L., Y.M., and T.V.; Methodology. D.A., J.R.L., and S.V.S.; Analysis. D.A.; Resources, M.B.-B. and R.T.; Supervision, J.R.L. and T.V.; Writing, D.A.

Declaration of Interests

Current US Patent No. 9758573, “Methods to Protect Against and Treat Multiple Sclerosis.” Inventors: T.V., Rasheed K. Rumah, J.R.L., Y.M., Vincent A. Fischetti.

Published: May 31, 2019

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2019.04.016.

Supplemental Information

Document S1. Transparent Methods and Figures S1–S3
mmc1.pdf (636.1KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Alexa-Tagged pETX Has Affinity for hMAL-GFP ISVs, Related to Figure 2

A representative time-lapse video of tagged pETX accumulating at the edge of an hMAL-GFP vessel. The first half of the video just shows the red pETX channel, whereas the second half is a repeat with both the red pETX channel and the green hMAL channel.

Download video file (3.4MB, mp4)
Video S2. Alexa-Tagged pETX Has No Affinity for hBENE-GFP ISVs, Related to Figure 2

A representative time-lapse video of tagged pETX failing to accumulate at a hBENE-GFP vessel. The first half of the video just shows the red pETX channel, whereas the second half is a repeat with both the red pETX channel and the green hBENE channel.

Download video file (2.4MB, mp4)
Video S3. Alexa-Tagged H149A Mutant pETX Shows Subtle Affinity for hMAL-GFP ISVs, Related to Figure 2

A representative time-lapse video of tagged pETX accumulating at the edge of an hMAL-GFP vessel. The first half of the video just shows the red pETX channel, whereas the second half is a repeat with both the red H149A mutant pETX channel and the green hMAL channel.

Download video file (1.1MB, mp4)
Video S4. ETX Causes Stark Leakage in hMAL-GFP Neurovasculature, Related to Figures 3 and 4

A representative confocal z stack traversing ventrally through the neuravasculature of an hMAL-GFP-expressing fish. All evident leakage appears adjacent to hMAL-GFP vessels. The red channel represents 2.000-kDa rhodamine dextran. The green channel represents hMAL-GFP. The gray background is differential interference contrast imaging.

Download video file (1.5MB, mp4)
Video S5. ETX Causes No Leakage in hBENE-GFP Neurovasculature, Related to Figure 3

A representative confocal z stack traversing ventrally through the neuravasculature of a hBENE-GFP-expressing fish. All red dextran appears confined in blood vessels. The red channel represents 2.000-kDa rhodamine dextran. The green channel represents hBENE-GFP. The gray background is differential interference contrast imaging.

Download video file (1.2MB, mp4)
Video S6. ETX Causes Perivascular Edema in hMAL-GFP Neurovessels, Related to Figures 5 and S7

A representative z stack using the differential interference contrast channel (same stack as video S7) reveals edema in select hMAL-expressing blood vessels. The red arrow highlights edema.

Download video file (1.5MB, mp4)
Video S7. ETX Causes No Perivascular Edema in hBENE-GFP Neurovessels, Related to Figures 5 and S8

A representative z stack using the differential interference contrast channel (same stack as video S8) reveals no edema in any hBENE-expressing blood vessels.

Download video file (1.2MB, mp4)
Video S8. ETX Terminates Blood Flow in hMAL-GFP Neurovessels, Related to Figure 5

A representative comparison of blood vessels at the midline the brain of an hMAL-GFP fish evidences continued blood flow in a blood vessel without recognizable hMAL-GFP expression and stopped blood flow in an hMAL GFP vessel after injection of ETX. Red represents 2,000-kDa rhodamine dextran. Green represents hMAL-GFP. The gray background is differential interference contrast imaging. Video taken at 50 MPI.

Download video file (744KB, mp4)
Video S9. ETX Fails to Terminate Blood Flow in hBENE-GFP Neurovessels, Related to Figure 5

A representative comparison of blood vessels at the midline the brain of an hBENE-GFP fish evidences blood flow in both a blood vessel without recognizable hBENE-GFP expression and in an hBENE-GFP vessel after injection of ETX. Red represents 2,000-kDa rhodamine dextran. Green represents hBENE-GFP. The gray background is differential interference contrast imaging. Video taken at 50MPI.

Download video file (930.9KB, mp4)
Video S10. H149A Mutant pETX Fails to Terminate Blood Flow in hMAL-GFP Neurovessels, Related to Figures 7 and S3

A representative comparison of blood vessels at the midline the brain of an hMAL-GFP fish evidences blood flow in both a blood vessel without recognizable hMAL-GFP expression and in an hMAL-GFP vessel after injection of H149A pETX. Red represents 2,000-kDa rhodamine dextran. Green represents hMAL-GFP. The gray background is differential interference contrast imaging. Video taken at 50MPI.

Download video file (941.8KB, mp4)
Video S11. Antibody Neutralization of ETX Restores Blood Flow in hMAL-GFP Neurovessels, Related to Figures 7 and S2

A representative comparison of blood vessels at the midline of the brain of an hMAL-GFP fish evidences blood flow in both a blood vessel without recognizable hMAL-GFP expression and in an hMAL-GFP vessel after injection of antibody-neutralized ETX. Red represents 2,000-kDa rhodamine dextran. Green represents hMAL-GFP. The gray background is differential interference contrast imaging. Video taken at 50 MPI.

Download video file (991.2KB, mp4)
Document S1. Transparent Methods and Figures S1–S3
mmc1.pdf (636.1KB, pdf)

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