Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

RU Laboratory

Fischetti Laboratory

Abstract

After nearly two hundred years of scientific inquiry, the cause of Multiple Sclerosis (MS) remains unknown. Although generally considered an autoimmune disease, recent pathological findings have challenged the longstanding autoimmune view of MS. Indeed, lesions that are just hours past their initial onset display evidence of cellular degeneration in the absence of an inflammatory infiltrate. This seminal finding begs the fundamental question, if immune cells do not participate in early tissue damage in MS, what is the mysterious triggering agent? In the first half of this thesis, I will propose that a soluble bacterial toxin, Clostridium perfringens epsilon toxin (ETX), may be the long sought environmental trigger for MS. ETX is a neurotoxin with a unique tropism for the brain tissues that are specifically damaged during acute MS exacerbations, the blood-brain barrier and the myelin sheath. Using a Western blot seroreactivity assay, I have identified a 10-fold increase in anti-ETX seroreactivity when comparing MS patient samples to that of controls. Furthermore, PCR analysis of bacterial cultures from patient stool samples has yielded the discovery of the first human known to harbor C. perfringens type B, an ETX secreting bacterium that is not part of the normal human microbiota. Intriguingly, this individual was found to be in the early stages of developing MS. I have also determined that MS iv patients are 2X less likely to harbor the human commensal C. perfringens type A, which has been shown to outcompete toxin-producing C. perfringens strains, e.g. type B, within a shared ecological niche. Therefore, commensal type A may protect against type B dysbiosis and may protect against developing MS. Although the ETX/MS hypothesis is obscure at best, ETX’s extreme potency has driven interest in identifying its cellular receptor. Indeed, ETX is the third most potent toxin known to man, following only Clostridium botulinum and tetanus toxins in lethality. For this reason, it is considered a legitimate bioterrorism threat. Despite decades of research, the ETX receptor is still yet to be identified. In the second half of this thesis, I will propose that the tetraspan proteolipid, Myelin and Lymphocyte protein (MAL) is a bona fide ETX receptor. Recombinant expression of MAL by an ETX resistant Chinese Hamster Ovary (CHO) cell line confers both ETX binding and susceptibility. Additionally, probing tissues harvested from MAL knockout (KO) mice with fluorescently labeled ETX demonstrates a complete abolishment of ETX binding. Finally, MAL KO mice display a remarkable level of ETX resistance even when exposed to 1000X of our experimentally determined LD50 for wild type animals. Taken together, these data suggest that MAL is both necessary and sufficient for ETX binding and toxicity. In summary, I have generated data suggesting that an increased exposure to C. perfringens epsilon toxin may exist in the MS population when compared to controls. This may be important, as ETX specifically targets the blood-brain barrier and the myelin sheath, both of which degenerate during acute MS exacerbations. To my knowledge, ETX is unlike any other candidate MS trigger, v as it provides a clear mechanism for how blood-brain barrier disruption and demyelination may occur during an MS relapse. I have also identified Myelin and Lymphocyte protein, as a functional ETX receptor. Identifying the mechanism by which ETX targets cells may help counteract its use as a biological weapon. Furthermore, this discovery may open new avenues for translational research, e.g. novel methods for targeting and bypassing the blood-brain barrier; a major hurdle to drug delivery in the central nervous system.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/555

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