A functional proteomic screen of myelination
Lead Research Organisation:
University of Cambridge
Department Name: Pathology
Abstract
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Technical Summary
Myelination represents one of the most spectacular cell-cell interactions in biology. Despite
this, we know very few of the signalling molecules involved in axo-glial adhesion and/or in
ensuring that the number of wraps is precisely related to axon diameter. Identification of
these molecules will provide therapeutic targets for remyelination strategies in Multiple
Sclerosis (MS), the failure of which results in the axon loss and progressive disability that
characterises the disease. As well as representing a major disease burden to sufferers and
their families, the chronic progressive nature of MS causes a significant cost to society –
currently estimated at 5-13 billion euros/annum in the EU. Currently there are no therapies
in clinical use or even in trials to promote remyelination, in large part due to the lack of
targets. We propose a novel approach to the problem by using a combination of
monoclonal antibody and recombinant phage display technology with FALI (fluorophoreassisted
light inactivation), a technique to create acute protein knockdown of antibodybinding
targets by singlet oxygen-mediated protein damage when the fluorophore is
exposed to light. We will generate libraries of monoclonal antibodies or single-chain
variable fragments (scFvs) by immunizing mice with oligodendrocytes, the myelin-forming
cells of the CNS. We will screen these for surface binding to oligodendrocytes, then couple
those that bind to a fluorochrome (FITC) and determine which inhibit myelination following
exposure to 490nm light in a neurone/oligodendrocyte myelinating co-culture assay.
Proteomics will then be used to identify the target antigens for those that inhibit
myelination, so defining signalling molecules for validation by conventional transgenic
techniques. The use of monoclonal antibody technology and FALI provides an unbiased
approach for the identification of key signalling molecules quite distinct from conventional
candidate-driven approaches. It therefore provides a method both to understand this
essential part of CNS development and also to identify entirely new targets for
remyelination therapies that will not be detected by conventionally-funded incremental
research programmes.
this, we know very few of the signalling molecules involved in axo-glial adhesion and/or in
ensuring that the number of wraps is precisely related to axon diameter. Identification of
these molecules will provide therapeutic targets for remyelination strategies in Multiple
Sclerosis (MS), the failure of which results in the axon loss and progressive disability that
characterises the disease. As well as representing a major disease burden to sufferers and
their families, the chronic progressive nature of MS causes a significant cost to society –
currently estimated at 5-13 billion euros/annum in the EU. Currently there are no therapies
in clinical use or even in trials to promote remyelination, in large part due to the lack of
targets. We propose a novel approach to the problem by using a combination of
monoclonal antibody and recombinant phage display technology with FALI (fluorophoreassisted
light inactivation), a technique to create acute protein knockdown of antibodybinding
targets by singlet oxygen-mediated protein damage when the fluorophore is
exposed to light. We will generate libraries of monoclonal antibodies or single-chain
variable fragments (scFvs) by immunizing mice with oligodendrocytes, the myelin-forming
cells of the CNS. We will screen these for surface binding to oligodendrocytes, then couple
those that bind to a fluorochrome (FITC) and determine which inhibit myelination following
exposure to 490nm light in a neurone/oligodendrocyte myelinating co-culture assay.
Proteomics will then be used to identify the target antigens for those that inhibit
myelination, so defining signalling molecules for validation by conventional transgenic
techniques. The use of monoclonal antibody technology and FALI provides an unbiased
approach for the identification of key signalling molecules quite distinct from conventional
candidate-driven approaches. It therefore provides a method both to understand this
essential part of CNS development and also to identify entirely new targets for
remyelination therapies that will not be detected by conventionally-funded incremental
research programmes.
Organisations
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ORCID iD |
| Charles Ffrench-Constant (Principal Investigator) |