Multiple sclerosis as a cytodegenerative proteopathy

项目总结/文摘

Project Summary/Abstract MS is traditionally considered to be an autoimmune demyelinating disorder of the CNS. However there are a number of inconsistencies with this “outside-in” hypothesis of MS pathogenesis which lead us to propose instead that MS is primarily an “inside-out” disease, where a primary degeneration mainly targeting the myelin- oligodendrocyte, secondarily entrains an auto-immune reaction in the predisposed host. HYPOTHESIS: MS is a primary protein misfolding disorder targeting the myelinating unit, driven by accumulation of transmissible protein aggregates. Specifically, this proteopathy is underpinned by a pathological prion (in the generic sense) which promotes chronic toxicity of myelin and oligodendrocytes, resulting in disruption of myelin integrity, leading to secondary autoimmunity, thus programming the broad spectrum of inflammatory and progressive MS phenotypes. Aim 1: Intracerebral transmission of MS-like pathology. This aim will conduct a detailed examination of our ability to transmit an MS-like pathology from human brain homogenate (from deceased controls and progressive MS subjects) to wild-type and humanized transgenic mice, via direct i.c. inoculation. This aim will test the hypothesis that human MS brain contains toxic misfolded protein aggregates that can transmit pathology and propagate in the recipient hosts. High- field MRI, (immuno)histology and behavior will be used to monitor developing pathology. We expect to find pathology similar to “normal appearing white matter” abnormalities in human MS brain (subtle myelinopathy, axonal damage, micro- and astrogliosis), and notably, we do expect a significant lymphocytic response indicative of adaptive immunity. Negative controls (non-MS human brain homogenate) will be performed to demonstrate MS specificity. Passaging (ie taking infected transgenic mouse brain homogenate and re-inoculating into naïve mice) will show that the offending agent continues to transmit. Prion protein is the etiological agent originally hypothesized, therefore experiments will be tailored accordingly using PrP transgenics, recognizing that PrP and/or one of its interacting proteins could also be playing a key role. Aim 2: Intraperitoneal transmission of MS-like pathology. While direct i.c. inoculation is the most robust method of transmitting prion-like misfolded proteins, it carries the risk of inducing non-specific pathology, especially in strains that are more susceptible to various insults. This aim will attempt to transmit pathology via a less invasive route using i.p. injections. Similar source material will be used. Advantages include potentially earlier and more sensitive behavioural readouts (locomotor deficits, sensory abnormalities) if the expected myelopathy is induced. Moreover, larger volumes of inocula and more frequent injections are possible with i.p. compared to i.c. injection, potentially increasing the chances of transmission. Terminally, similar (immuno)histology will be performed. We will also examine the spleen for evidence of misfolded protein deposition as a clue to whether this organ participates in processing and amplification of transmissible seeds. Aim 3: Biochemical analysis of human MS brain and passaged mouse CNS. A key observation made during our preliminary biochemical studies showed a striking alteration in the interaction between prion protein and myelin basic protein, one of the major myelin proteins. This aim will explore in greater detail this potentially very important interaction that could be key for maintenance of myelin integrity. Immunoprecipitation, Westerns, LC-MS and biochemical analysis will be performed to probe disruptions in this interaction. Focus will be on citrullination of both proteins as this post- translational modification alters net charge, and therefore the interaction efficiency, exerting a potentially important adverse influence on physiological protein-protein interactions.