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Optogenetics based discovery of new pathways towards stem-cell mediated myelin repair

Periodic Reporting for period 1 - STEM ZAP (Optogenetics based discovery of new pathways towards stem-cell mediated myelin repair)

Reporting period: 2016-04-06 to 2018-04-05

This project aimed to identify novel molecular mechanisms involved in the process of myelination, which could be used to promote myelin repair in the central nervous system (CNS). This work is both timely and important as injury to myelin, the insulating material that ensures efficient electrical signal transmission in the brain, causes neurological disability in an estimated 405,000 Multiple Sclerosis (MS) patients in the EU at a cost of €14.6 billion per year to the economy. Therefore, new therapies that repair damaged myelin are an important clinical ambition, whose resolution could deliver enormous health and economic benefits across Europe.

CNS Myelin is formed from oligodendrocytes (OL) that develop (differentiate) from specialized precursor cells known as OL precursor cells (OPC). This process involves the activation of pro-developmental genes, which could, if they were identified, be targeted to promote myelin repair. In this project we aimed to use a new technology known as optogenetics to stimulate OPC and induce them to develop into OL. Optogenetics involves the use of specialized proteins (Channel Rhodopsin 2 aka ChR2) derived from green algae that, when stimulated with blue light, enables positive currents to flow into the host cell. The positive current depolarizes the cell membrane leading to powerful changes in the cells activity. In some precursor cells membrane depolarization promotes differentiation, thus we reasoned that membrane depolarization would prove a useful means to maximize the development of OPC and identify new genes involved in myelin formation. The main objectives of this work we therefore to:

1. Genetically engineer OPC to produce ChR2 molecules (ChR2-OPC) and test the ability of membrane depolarization (via blue light stimulation) to promote the differentiation of these cells into oligodendrocytes.

2. Use a sequencing approach to identify the genes that are activated in ChR2-OPC following membrane depolarization.

3. Test a set of these newly identified genes in laboratory experiments designed to test OPC diffentiation and myelination.

4. Study post mortem MS brain tissue to determine if there are links between the newly identified genes and OPC located in damaged and diseased areas of the brain (lesions). OPC often fail to differentiate in areas with MS damage, thus the new OPC genes, if found to be altered in lesions, may provide interesting new targets for further research.
In the first year the work was focused on establishing a new protocol to generate OPC from neural stem cells, and the generation of viral vectors encoding ChR2. We had anticipated that this task would require 3 months of work, but in reality the task was more complicated and beset by technical difficulties. Ultimately the Experienced Researcher (Dr Otsu) succeeded in establishing an effective protocol capable of producing OPC. However, the scale of work required for this optimization, and the necessary characterization that was then performed to guarantee the nature of the cells produced, proved greater than expected. Importantly, this process of optimization and characterization has formed the greater part of the work carried out in this project, and has lead to important new discoveries on OL biology (discussed later).

During the first year Dr Otsu also began production of lentiviral vectors encoding ChR2. Two vectors were generated via a commercial service and used successfully to produce a number of lines of stem-cell derived OPC (ChR2-OPC). The expression of ChR2 proteins was analysed in these lines using fluorescence microscopy (to detect YFP reporter tags) and immunofluorescent (IF) staining to detect ChR2 proteins. All lines exhibited appropriate expression of ChR2 protein, albeit at rather low levels. One ChR2-OPC line was investigated functionally using calcium imaging. Blue light stimulation was found to increase the concentration of intracellular Ca2+ suggesting that the ChR2 proteins are functional.

Work in the second year was mainly focused on further optimization of the stem-cell derived OPC protocol and a deep characterization of the cells properties including transcriptional profile, ability to generate OL and myelin, and their lineage plasticity (ability to generate other types of neural cells). This work has provided a number of interesting and unexpected discoveries that are the subject of a manuscript (now in preparation). For more details please see Progress beyond the state of the art.

Dissemination and exploitation

At the end of the project (24 months) work establishing and investigating the new stem-cell derived OPC lines was complete and a manuscript reporting this work is now under submission.

During the project Dr Otsu also developed expertise in the production of primary OPC since these were required to validate the new stem-cell derived OPC lines. His skills in primary OPC contributed to another project in the lab that has now been published:

Begum G, Otsu M, Ahmed U, Ahmed Z, Stevens A, Fulton D. (2018) NF-Y-dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage. Glia. Epub ahead of print, DOI: 10.1002/glia.23446.

Dr Otsu also presented the work described above at two international conferences:
(1) The XIII European Meeting on Glial Cells in Health and Disease, July 8-11 2017, Edinburgh, UK; (2) British Neuroscience Association Festival of Neuroscience, 10-13 April 2017, Birmingham, UK.
The stem cell derived OPC system represents an important advance for the field because standard methods for OPC isolation from primary tissues tend to generate cultures rich in later stage OPC, but lack more immature stages. Therapies aiming to promote myelin formation and repair via actions on OPC will are likely to influence all stages of OPC differentiation, yet currently the field lacks a suitable model to analyse earlier stages of OPC differentiation. We believe the new system will fill this gap and enable researchers in the field to undertake a finer analysis of OPC differentiation.

This system has also produced measurable impact for the Fulton group. First, the method has been instrumental in a recent successful bid for funding where the cells will be used as a screening tool in a new metabolomics/lipidomic study. Second, it has been incorporated into a recent grant proposal that offers further employment and training opportunities for Dr Otsu, and scientific and clinical impact for wider audiences.
The project has also delivered benefits to the wider community via engagement and dissemination activities undertaken by Drs Fulton and Otsu. Specifically the team were involved in science communication at a recent event (Fun and Brains) at the Library of Birmingham. Dr Otsu assisted at the Build a Brain event where children and adults assembled different types of neurons from reclaimed junk materials while learning fundamental neuroscience from the presenters (see images on the STEM-ZAP project page).
Schematic describing the new stem cell-derived OPC system.