Contributions
Abstract: P447
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Microbiology and Virology
Background: Clostridium perfringens Epsilon toxin (ETX) is widely known as the causative agent of multifocal CNS white matter disease in ruminants. Recently, ETX has been implicated as a potential actor in Multiple Sclerosis (MS) pathogenesis. The toxin appears to enter the CNS by targeting the blood-brain barrier (BBB), reducing its integrity, before entering into brain parenchyma and targeting oligodendrocytes. However, the mechanism by which the toxin opens the BBB, the kinetics of its action, and its effects in a live organism are still unknown.
Objective: To generate a humanized zebrafish BBB model expressing the Myelin and Lymphocyte Receptor Protein (hMAL) in order to: 1) establish the kinetics of real-time, in vivo ETX-MAL binding, 2) determine if ETX requires MAL to induce neurovascular pathology, 3) assess whether ETX can be neutralized using an antibody.
Methods: Zebrafish cerebellar sections were examined for ETX vascular binding via immunohistochemistry and were compared to mouse WT controls. Zebrafish zygotes were injected with hMAL-GFP or tetraspanin receptor control, hBENE-GFP, under an endothelial promoter. GFP Positive fish were injected intravenously 3-4 days post fertilization (DPF) with Alexa-tagged pro-ETX (pETX). GFP vessels were live imaged and evaluated for toxin-receptor colocalization using confocal microscopy. At 6-7 DPF other hMAL or hBENE fish were co-injected with active ETX or H149A mutant ETX plus dextran dye to look for BBB disruption and vascular pathology using confocal full-brain z-stacks. Experiments were repeated after ETX neutralization with JL008 antibody. Images were quantified using Imaris.
Results: WT zebrafish showed no affinity for ETX, but fish which expressed hMAL exhibited pETX binding in a time dependent manner. Activated toxin in hMAL endothelial-expressing fish initiated dramatic BBB disruption demonstrated by causal leakage of a 2,000 kDa dextran, hMAL receptor recycling, vessel narrowing, and focal perivascular edema when compared to receptor or toxin controls. Incubation of active ETX with JL008 antibody abrogated all assessed vascular pathology.
Conclusion: ETX requires endothelial expressed MAL protein to elicit BBB pathology and can be blocked by neutralizing antibody. This finding adds to the body of evidence implicating the ETX paradigm in MS by providing the first visualization of the toxin's action in a live organism and strengthens the proposal that MAL is the ETX receptor with a new BBB model.
Disclosure: Drew Adler: nothing to disclose, Jennifer Linden: nothing to disclose, Samantha Shetty: nothing to disclose, Richard Titball: nothing to disclose, Monika Bokori-Brown: nothing to disclose, Yinghua Ma: nothing to disclose, Joseph Fetcho: nothing to disclose, Tim Vartanian: consulting for Biogen, Novartis, Genzyme, and speaking for Acorda
Abstract: P447
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Microbiology and Virology
Background: Clostridium perfringens Epsilon toxin (ETX) is widely known as the causative agent of multifocal CNS white matter disease in ruminants. Recently, ETX has been implicated as a potential actor in Multiple Sclerosis (MS) pathogenesis. The toxin appears to enter the CNS by targeting the blood-brain barrier (BBB), reducing its integrity, before entering into brain parenchyma and targeting oligodendrocytes. However, the mechanism by which the toxin opens the BBB, the kinetics of its action, and its effects in a live organism are still unknown.
Objective: To generate a humanized zebrafish BBB model expressing the Myelin and Lymphocyte Receptor Protein (hMAL) in order to: 1) establish the kinetics of real-time, in vivo ETX-MAL binding, 2) determine if ETX requires MAL to induce neurovascular pathology, 3) assess whether ETX can be neutralized using an antibody.
Methods: Zebrafish cerebellar sections were examined for ETX vascular binding via immunohistochemistry and were compared to mouse WT controls. Zebrafish zygotes were injected with hMAL-GFP or tetraspanin receptor control, hBENE-GFP, under an endothelial promoter. GFP Positive fish were injected intravenously 3-4 days post fertilization (DPF) with Alexa-tagged pro-ETX (pETX). GFP vessels were live imaged and evaluated for toxin-receptor colocalization using confocal microscopy. At 6-7 DPF other hMAL or hBENE fish were co-injected with active ETX or H149A mutant ETX plus dextran dye to look for BBB disruption and vascular pathology using confocal full-brain z-stacks. Experiments were repeated after ETX neutralization with JL008 antibody. Images were quantified using Imaris.
Results: WT zebrafish showed no affinity for ETX, but fish which expressed hMAL exhibited pETX binding in a time dependent manner. Activated toxin in hMAL endothelial-expressing fish initiated dramatic BBB disruption demonstrated by causal leakage of a 2,000 kDa dextran, hMAL receptor recycling, vessel narrowing, and focal perivascular edema when compared to receptor or toxin controls. Incubation of active ETX with JL008 antibody abrogated all assessed vascular pathology.
Conclusion: ETX requires endothelial expressed MAL protein to elicit BBB pathology and can be blocked by neutralizing antibody. This finding adds to the body of evidence implicating the ETX paradigm in MS by providing the first visualization of the toxin's action in a live organism and strengthens the proposal that MAL is the ETX receptor with a new BBB model.
Disclosure: Drew Adler: nothing to disclose, Jennifer Linden: nothing to disclose, Samantha Shetty: nothing to disclose, Richard Titball: nothing to disclose, Monika Bokori-Brown: nothing to disclose, Yinghua Ma: nothing to disclose, Joseph Fetcho: nothing to disclose, Tim Vartanian: consulting for Biogen, Novartis, Genzyme, and speaking for Acorda