Development of a high-throughput drug screening platform for oligodendrocyte myelination (for progressive multiple sclerosis)
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Date
20/03/2020Author
Kim, Jee Soo
Metadata
Abstract
Aim
As part of the broad strategy in Edinburgh and beyond to discover new treatments for
progressive Multiple Sclerosis (MS), the aims for my PhD project were: (1) to address the lack
of an in vitro phenotypic drug screening platform that is able to fully recapitulate myelin
sheath formation, with the long-term goal being to enhance the discovery of pro
remyelination therapies in progressive MS, and target the poor drug discovery rates in brain
disorders which is partially due to poor disease modelling, and (2) the exploration of non
linear optical imaging microscopy techniques that targets both the resolution and speed to
study myelination.
Background
In multiple sclerosis, an inflammatory autoimmune process destroys the oligodendrocytes
that provide neuronal support by forming multi-layered compact myelin sheaths around
axons, leading to neurodegeneration. Although there are drugs available to suppress the
inflammatory attack to limit the formation of demyelinated lesions, no treatments currently
exist to promote the regeneration of these myelin sheaths once the damage has occurred.
Cell based screens have formed an important part of the strategy used to discover such
regenerative drugs. The majority of published cell-based phenotypic drug screens to target
repair have focused on the differentiation of oligodendrocyte precursor cells into
oligodendrocytes rather than their ability to form mature protective myelin sheaths.
However, in many MS lesions, pre-myelinating oligodendrocytes are present, and studies on
oligodendrocyte biology show that differentiation and myelination are regulated by distinct
mechanisms. There is therefore a need for novel drug screens that target the later
myelinating stages of oligodendrocyte development.
Results
Using a 3D microfibre system for in vitro myelin sheath formation (described by Bechler,
Byrne and ffrench-Constant (2015)), I first asked whether compounds that had been
v
identified as increasing differentiation in conventional 2D culture systems enhanced
myelination in the 3D cultures. While Benztropine and Clemastine showed an increase in the
number of MBP+ (differentiated) oligodendrocytes in the 2D system, consistent with
previous publications, no increase in myelin sheath formation was seen with any of the drugs
in the 3D system, highlighting the potentially important differences between differentiation
and myelin sheath formation for drug discovery.
Next I developed a multiwell plate based assay to allow 3D myelination assays to be used for
drug screening. Using electrospinning to produce PLA microfibres, I was able to develop and
optimise a technique to insert and suspend the fibres across the bottom of a 96-well plate
that can be incorporated into an automated pipeline for high-throughput drug screening. The
3D myelin sheaths could be imaged using the Leica SP8 confocal system with the
MatrixScreener extension and the Opera Phenix high-content screening system.
Finally, I addressed the problem of imaging myelin in such screens. CARS was shown to be
able to preferentially detect myelin sheaths in fixed and live slices in regions and time-points
of varying myelin densities. As the development of new myelin sheaths requires the
formation of lipid and therefore the incorporation of hydrogen, the consumption of D2O (heavy water) with a 2H atom allowed the non-invasive labelling and detection of myelin
sheaths using SRS. Future experiments will allow us to confirm whether this deuterium
detection is preferential and/or specific to new myelin sheaths.
Significance
With only about 10% of drugs that enter Phase I trials successfully launching into clinics, there
is an important need for more effective drug screens that better model disease-relevant
processes and so reduce late-stage failures. The high-throughput-compatible 96-well plate
with suspended PLA microfibres that combines the recent progresses in 3D cellular model
systems with bioengineering and the recent advances in high content imaging systems may
give us the opportunity to more accurately model these disease-relevant structures in vitro,
and therefore improve drug discovery for regenerative therapies in multiple sclerosis and
other myelin diseases.
The research on Raman-based label-free imaging of myelin sheaths is not only applicable for
imaging myelin sheaths in the context of drug validation, but could be important for live imaging of brain slices and detection of newly-formed myelin sheaths without the need for
complex and expensive transgenic animals.