Fig 1.
Mal and Cav1 gene expression is enriched in brain endothelial cells.
(A) Heatmap showing the enrichment of cell type marker genes in the TRAP IPs from oligodendrocytes, endothelial cells, pericytes, astrocytes, L5b pyramidal cells, and inhibitory interneurons compared to whole cortex input. Gray boxes indicate a failure to detect (ND) the gene in the data set. (B, C) Mean ± SEM of the ratio of Mal (B) and Cav1 (C) expression (log2 fold change of RPKMs) in TRAP IP mRNAs compared to whole cortex input mRNAs in each of the CNS cell types from A. Dotted lines indicate 2-fold enrichment (positive value) or depletion (negative value).
Fig 2.
Epsilon toxin binds to the microvasculature of the CNS and requires expression of MAL.
Wild type mice expressing Mal+/+ or mice deficient in Mal-/- were intravenously injected with Alexa Fluor 594 conjugated ETX (ETX-594) for ten minutes then perfused with PBS to remove unbound toxin. ETX binding to microvasculature was evaluated in brain or spinal cord cryosections and whole mounts of retina and optic nerve. FITC-BSL1 was used to identify microvasculature. ETX-594 bound to the microvasculature of the brain including areas near the corpus callosum (white-dotted line) and the cerebellum (white matter tracts denoted by white line, WM) as well as the spinal cord (thoracic segment), retina and optic nerve (full thickness of optic nerve denoted by white dashed lines). ETX-594 binding was not detected in Mal-/- mice.
Fig 3.
Epsilon toxin does not bind to the microvasculature of other peripheral organs.
Wild type mice expressing Mal+/+ or mice deficient in Mal-/- were intravenously injected ETX-594 for ten minutes then perfused with PBS to remove unbound toxin. ETX binding to microvasculature was evaluated in tissue cyrosections. FITC-BSL1 was used to visualize microvasculature. ETX bound to the epithelial cells of renal tubules of Mal+/+ mice (white arrows) but not the glomerular capillaries (asterisks). In Mal-/- mice, ETX, can be seen accumulating in unidentified renal structures (white arrow heads). ETX is also observed binding to the microvasculature of the intestines in Mal+/+ mice (white arrows), but not Mal-/- mice. ETX binding was not observed in the spleen, lung, liver or heart of Mal+/+ or Mal-/- animals.
Fig 4.
High magnification of Mal+/+ and Mal-/- intravenously injected with ETX-594.
Wild type mice expressing Mal+/+ or mice deficient in Mal-/- were intravenously injected with Alexa Fluor 594 conjugated ETX (ETX-594) for ten minutes then perfused with PBS to remove unbound toxin. ETX binding to kidney tissue was evaluated by fluorescent microscopy. White arrows indicate ETX-594 binding to renal tubules. White asterisks indicate accumulation of ETX-594 in unknown renal compartment.
Fig 5.
Expression of MAL is necessary for ETX induced BBB permeability.
Mal+/+ and Mal-/- mice were injected via IP with 5ng of active ETX per gram of body weight; saline treated animals were used as controls. After 1 hour, all animals were intravenously injected with FITC-Na for ten minutes then sacrificed. Brains were harvested and the amount of FITC-Na per gram of brain was calculated using spectrometry. Results are normalized to individual genotype controls and expressed as FITC-Na extravasation (% CT). Results expressed as Mean ± SEM, *p<0.05 determined by ANOVA, n = 3–5 mice per group. (B) Alternatively, ETX or saline treated Mal+/+ and Mal-/- mice were intravenously injected with 70kDA FITC-dextran for ten minutes one our after treatment. Brains were harvested, cryoprotected, and sectioned. 70kDA FITC-dextran extravasation into the brain parenchyma was determined by fluorescent microscopy. Dashed line identifies corpus callosum (CC). OB, olfactory bulb. CB, Cerebellum. Sections are approximately 1 to 1.5 millimeters to the left of the mid-sagittal plane. (C) Higher magnification of pericallosal white matter in ETX treated Mal+/+ mice identify ovoid shaped lesions (white ovals) perpendicular to corpus callosum (CC, width denoted with white dotted lines). White arrows point to shadows in FITC-dextran halos presumed to be central veins. (D) Sagittal sections of perfused control or ETX treated Mal+/+ stained for endogenous mouse IgG (red). FITC-BSL1 (green) was used to identify microvasculature. Dashed lines identify width of corpus callosum (CC). In control treated mice, limited IgG extravasation was observed, even around large vessels (white arrows), presumed to be deep medullary veins. In ETX treated mice, IgG extravasation is very prominent, especially around large vessels (white arrowheads), presumed to be deep medullary veins.
Fig 6.
CAV1 expression is necessary for ETX induced BBB permeability but not ETX binding to brain microvasculature.
(A) To determine if CAV1 was necessary for ETX binding to brain microvasculature, cerebellum cryosections from Cav1+/+ and Cav1-/- were probed with epsilon protoxin (proETX). ProETX binding was detected by and anti-ETX antibody (red). FITC-BSL1 was used to identify vasculature (green). Note that proETX is observed binding to the microvasculature in the molecular layer (ML) as well as myelin in the granular layer (GL) in both Cav1+/+ and Cav1-/- mice. (B) Higher magnification images of areas in white boxes in image A. (C) Quantification of proETX binding to vasculature in cerebellum gray matter in Cav1+/+ and Cav1-/- mice. proETX fluorescence was normalized to BSL1 fluorescence. Results expressed as Mean ± STDEV, *p value determined by T-Test, n = 4–7. D) To evaluate CAV1’s role in ETX induced BBB permeability, Cav1+/+ Cav1-/- were treated with or without 5ng of ETX per gram of body weight for up to 180 minutes. Mice were perfused with PBS, brains harvested, and cryosectioned. BBB permeability was accessed by immunohistochemistry staining for endogenous mouse IgG in the pericollosal white matter of sagittal sections. Results are normalized to individual genotype controls and expressed as IgG Extravasation (% CT). Results expressed as Mean ± STDEV, * p < 0.05 determined by ANOVA, n = 2–3 mice per group. (E) Representative images from ETX treated Cav1+/+ and Cav1-/- treated mice. Note in ETX treated Cav1+/+ mice, endogenous IgG (red) can be observed leaking for large medullary veins (white arrows) and smaller capillaries (white arrow heads). FITC-BSL1 (green) was used to identify vasculature.
Fig 7.
ETX treatment causes a down regulation in claudin-5 staining.
Mice were treated with 5ng of ETX per gram body weight for one hour and then perfused with PBS. Saline treated mice were used as controls. (A) Cyrsosections were evaluated for tight junction markers including claudin-5, ZO-1, and VE-cadherin. FITC-BSL1 (green) was used to visualize vasculature. (B) Extensive reduction in claudin-5 staining was also observed in ETX treated animals compared to controls. Sagittal sections of cortical matter. (C) Down regulation of claudin-5 was also confirmed in-vitro. Primary mouse BEC were treated with or without 50nM ETX for two hours and then stained for claudin-5. Under control conditions, claudin-5 is found at cell-cell junctions (white arrows). When treated with ETX, claudin-5 treatment significantly decreases and becomes perinuclear.
Fig 8.
ETX treatment causes an increase in caveolae dependent BBB permeability and internalization.
(A) Wild type mice were treated with 5ng ETX per gram of body weight for 1 hour. CAV1 staining was evaluated by IHC. White asterisks denote normal CAV1 staining in various sized blood vessels. In ETX treated mice, aberrant CAV1 staining was observed in some small capillaries as punctate staining (white arrow). In some medium sized vessels, CAV1 staining appeared basally located (white arrow head). In some large vessels, a dramatic increase in CAV1 staining was observed (red asterisk). (B) After ETX treatment, mice were intravenously injected with BSA-594 and then perfused. Coronal sections in control treated mice revealed BSA-594 (red) confined to vasculature lumen (white arrow head). In ETX treated mice, BSA-594 extravasation was observed accumulating in the corpus collosum (CC, thickness denoted by white dotted line) and as halos surrounding smaller vessels (white arrows). PDGF receptor-beta (green) was used to identify pericytes/vasculature. (C) Retinal whole mounts of control and ETX treated mice reveal increased internalization of BSA-594 by retinal endothelial cells after ETX treatment (white arrows). FITC-BSL1 (green) was used to identify vasculature. (D) Primary BEC were treated with our without 50nM ETX for 2 hours in the presence BSA-594 and CAV1 staining was evaluated by ICC. Internalized BSA-594 was observed in perinuclear organelles in ETX treatment cells (red arrows). In control treated cells, CAV1 staining can be observed extending all the way to cell-to-cell contacts (white arrows). After ETX treatment, CAV1 is absent from cell-to-cell contacts (white arrow heads). (E) and (F) Fluorescent quantification of CAV1 and BSA-594, respectively. Results expressed as Mean ± STDEV, p values determined by T-Test, n = 4.
Fig 9.
ETX treatment causes caveolae formation.
(A) SEM of primary BEC treated with or without 50nM of ETX for 1 hour. White arrows point to apical surface invaginations. (B) Higher magnification of surface invaginations on ETX treated BEC. (C) Vertical TEM sections of primary BEC treated with or without 50nM ETX for 2 hours. White arrows point to apical surface invaginations morphologically similar to caveolae. (D) Quantification of the number of caveolae per um of cell surface in control or ETX treated cells. Results expressed as Mean ± STDEV, *p<0.003 determined by T-Test, n = 3. (E) and (F) additional micrographs of BEC treated with 50nM ETX for 2 hours. Black arrows denote caveolae fusing into other internalized caveolae or endosomes. In some cells, large MVB are observed.
Fig 10.
ETX treatment causes vacuolation in primary BEC without cell death.
(A) In vitro ETX treatment of primary BEC revealed large, perinuclear vacuole formation (white arrows) compared to untreated controls. (B) Quantification of the number of vacuolated cells in control or ETX treated BEC after two hours. Results expressed as Mean ± STDEV, *p<0.001 determined by T-Test, n = 4. (C) Cell death was evaluated in BEC after 4 hours of ETX treatment at indicated doses. Cells were treated, trypsinized, and evaluated for cell death via PI inclusion by flow cytometry. Results are expressed as the number of PI positive cells by the total number of cells (% cell death). Results expressed as Mean ± STDEV, *p<0.05 versus untreated control, determined by ANOVA, n = 3. (D) To determine if macropinocytosis, dynamin, clathrin-coated pits, or caveolae are necessary for ETX induced vacuolation, BEC were pretreated with nocadozale (Noc), Pitstop (PS), MiTMAB (MM), or filipin (Fil), respectively, for 30 minutes prior to ETX treatment. Results are expressed as the number of vacuolated cells compared to the number of vacuolated cells when treated with ETX alone (% of ETX Alone). Results expressed as mean ± STDEV of at least two separate experiments performed in triplicate. *p<0.01 versus untreated control, determined by ANOVA. (E) Live imaging of BEC cells treated with 50nM of ETX for 4 hours. PI was used to identify dead cells (red arrows). Note that vacuolated cells (white arrows) are not PI positive. (F) Quantification of the percent of cells that are PI positive only (PI+), contain vacuoles only (vacuoles), or both (PI+Vacuoles). Results expressed as mean ± STDEV, *p<0.01 determined by ANOVA, n = 5–6. (G) and (H) BEC were isolated from WT, Mal-/-, or Cav1-/- mice, and incubated with indicated ETX doses for 4 hours, and cell vacuolation or cell death were evaluated by live cell imaging, respectively. Cell death was expressed as the number of PI positive cells compared to untreated controls (% CT). Results are the mean ± STDEV. *p<0.01 determined by ANOVA versus WT, n = 5–6. (I) Evaluation of ETX oligomerization in primary BEC via western blot. BEC were treated with 50nM of ETX for indicated time points and whole cell lysates were evaluated. CHO cells expressing MAL (rMAL) treated with or without 25nM ETX for 30 minutes were used as positive and negative controls, respectively. ETX oligomers are approximately 150kDa. (J) Densitometry readings of ETX oligomers in BEC normalized to ETX oligomer detected in ETX treated CHO cells. Results are the mean ± STDEV. *p<0.05 determined by ANOVA versus 0 min, n = 2.
Fig 11.
ETX treatment causes formation of RAB7+ endosomes and MVB.
(A) Primary BEC were treated with 50nM ETX for 2 hours. Cells were stained with anti-EEA1, RAB7, or LAMP1. White arrows point to vacuolated BEC. (B) Fluorescence intensity analysis of EEA1, RAB7, and LAMP1 with and without ETX treatment. Fluorescence was normalized to untreated controls and expressed as Fold Change. Results are the mean ± STDEV. *p<0.05 determined by T-Test, n = 3–6. Changes in EEA1 (C) and RAB7 (D) staining intensity was also evaluated at indicated timepoints after 50nM ETX treatment. Fluorescence was normalized to untreated controls (0 min) and expressed as Fold Change. Results are the mean ± STDEV. *p≤0.05 determined by ANOVA versus 0 min, n = 3–6 (E) Horizontal TEM sections near the basal side in control or ETX BEC reveal ETX induced formation of numerous MVB. No increase in lysome (L) numbers was observed. Mitochondria (M) in ETX treated cells appear rounded and swollen compared to the elongated mitochondria in control treated cells.
Fig 12.
Proposed mechanism of ETX induced transcytosis.
(1) ETX binds to MAL and oligomerizes, (2) causing recruitment of CAV1. (3) Recruitment of CAV1 results in increased caveolae formation and internalization and is dependent on dynamin. (4) Internalized caveolae, containing various blood-borne material, rapidly fuse with EEA1+ early endosomes, which traffic contents to RAB7+ late endosomes, (5) ultimately forming MVB. We propose that MVB then fuse with the basal membrane of the endothelial cells, releasing their contents into the brain parenchyma.