TRP channels and white matter function in health and disease

To allow nerve cells to transmit information rapidly over long distances, oligodendrocytes wrap myelin sheaths around their axons. Myelin damage, which is prevalent in many neurological diseases, is a major social and economic cost to society. But we still do not fully understand the processes that lead to myelin damage.
In my laboratory, we focus on the mechanisms that lead to myelin damage during times of energy deficiency. It is known that a rise in intracellular calcium concentration can damage myelin and therefore we study the membrane channels that allow this to occur. We can quantify cell death and myelin damage by looking at myelin ultrastructure with electron microscopy, and staining for cell death with markers. Using these techniques we have discovered that myelin can be damaged during simulated stroke by a protein known as TRPA1. This is interesting because TRPA1 is known to be involved in inflammation and pathology elsewhere in the body, and I hypothesise that TRPA1 plays a major role as a conduit for Ca2+-induced myelin damage in response to multiple pathways that produce substances that can activate TRPA1. In order to investigate this hypothesis, I will address the following aims.

AIM 1 - Generate and characterize oligodendrocyte-specific TRPA1 knockout mice.
In order to study the role of TRPA1 specifically within cells of the oligodendrocyte lineage, we will generate inducible, conditional TRPA1 knockout mice. TRPA1 will only be removed in the oligodendrocyte lineage cells of these mice when they ingest tamoxifen. We can compare these mice with the global TRPA1 knockout mice, to determine whether the myelination differences observed are due to TRPA1 within the oligodendrocyte lineage or other cells. These new mice will allow us to better elucidate the mechanism of action in the following aims.

AIM 2 - Determine whether TRPA1 is a valuable drug target in disease.
Following from my work in brain slices, I will look at the effect of drug-induced inhibition and genetic removal of TRPA1 during: (a) ischaemia-induced loss of white matter function in optic nerves; (b) an in vivo mouse model of stroke caused by blocking a major artery in the brain; and (c) two in vivo mouse models of myelin damage observed in multiple sclerosis, caused by injecting a bacterial toxin into the spinal cord or ingestion of cuprizone. I hypothesise that agents which cause myelin damage, do so via activation of TRPA1, and therefore, that block of TRPA1 or genetic knockout will decrease the myelin damage caused in these models.

AIM 3 Investigating the mechanism of TRPA1-mediated white matter damage
I have uncovered an interesting mechanism triggered by application of ischaemic solution, that involves activation of TRPA1. Specifically, changes in extracellular potassium concentrations that occur in ischaemia lead to acidification of oligodendrocytes, and this acid then activates TRPA1. My preliminary evidence suggests that TRPA1 activation then in turn inhibits the cells' permeability to potassium ions. I now aim to investigate this mechanism further and to determine what role each part plays in damaging oligodendrocytes. I will use the inducible, conditional TRPA1 knockouts, image intracellular ion concentrations while patch-clamping the oligodendrocytes, and perform electron microscopy to look for detrimental ultrastructural changes to myelin. Specifically, I will ask the following questions.
3.1 How does raised [K+]o acidify oligodendrocytes in ischaemia?
3.2 How does TRPA1 activation inhibit oligodendrocyte K+ channels?

These experiments will allow us to define important biological processes involved in demyelination and will provide us with novel opportunities to discover targets for developing treatments to prevent myelin damage. Furthermore, this grant will give me the opportunity to build up a laboratory and to hire a much needed research associate.

The importance of myelin is highlighted by the severity of disabilities observed in diseases in which it is damaged, such as Multiple Sclerosis (MS). I have shown, by simulating ischaemia in fresh rodent brain slices, that Ca2+-evoked myelin decompaction can be mediated by TRPA1 channels. TRPA1 channels were identified because the Ca2+ influx was inhibited by specific TRPA1 blockers and by global genetic knockout. It is vital that we increase our understanding of the role it plays in demyelinating diseases, as TRPA1 expression is heterogeneous, and many pathological substances activate it.

Project aims:

AIM 1 Generate and characterize oligodendrocyte-specific TRPA1 knockout mice.
Using inducible TRPA1 knockout mice (Sox10-CreERT2 x floxed TRPA1), we will investigate the role of TRPA1 specifically in oligodendrocytes lineage cells in myelination and myelin pathology. We will use these mice in the following aims.

AIM 2 Determine whether TRPA1 is a valuable drug target in demyelinating diseases.
2.1 Does oligodendrocyte TRPA1 activation impede white matter function in optic nerve ischaemia?
2.2 Does TRPA1 also cause myelin damage in white matter stroke in vivo?
We will cause stroke in mice in vivo by occluding the middle cerebral artery with a filament.
2.3 Do agents that cause myelin damage in other diseases, do so via oligodendrocyte TRPA1 activation and/or upregulation?
By patch-clamping oligodendrocytes, immunostaining, immunoblotting and qPCR, we will test whether endogenously- and exogenously- derived TRPA1 agonists raise [Ca2+]i in myelin, increase TRPA1 expression or cause cell death.
2.4 Does TRPA1 activation cause myelin damage in a model of MS?
In collaboration with Kenneth Smith we will use the LPS injection mouse model of MS, which results in type III lesion formation.

AIM 3 Investigating the mechanism of TRPA1-mediated white matter damage through patch-clamping and imaging intracellular ions with ion sensitive dyes.

As mentioned in the 'academic beneficiaries' section, there is the potential for far reaching benefits from this work. But this project can also have other less obvious but still important social and economic effects.
Within the laboratory, the research assistant and I will train many students in the experimental techniques. Currently we have one BSc student, two masters students and a visiting PhD student, and there will be a continuous throughput. These students will benefit from learning the techniques and using the state of the art equipment that is currently being acquired. This training will aid them in their career progression.
The chronic and progressive nature of demyelinating diseases such as multiple sclerosis has negative effects on health-, social-, and work-related issues for the patient and family, causing significant socioeconomic burden. The cost to the NHS for MS alone is £2.3billion per annum, but the loss of productivity and decrease in employment is another significant (~£2billion) economic factor. Recent changes to existing therapies have greatly improved the treatment of immune-mediated demyelination that occurs in multiple sclerosis, by decreasing the immune attack on the brain, but there are still questions of how myelin damage initially occurs and spreads in MS and also in other demyelinating diseases.

Therefore, this work has the potential to impact:
(1) Patients by potentially decreasing the severity of disease, improving their health and increasing their productivity.
(2) Neuroscientists by deciphering new mechanisms for oligodendrocyte function in health and disease.
(3) Pharmaceutical agencies by discovering new drug targets for demyelinating diseases.
(4) The economy by potentially reducing the healthcare burden of demyelinating diseases and allowing patients to return to work.
(5) My group and I by providing me with the platform to develop my independent academic researcher position with a specialisation in clinically related studies, as described in detail in my pathway to impact statement.

I will influence the impact of this fellowship on my career development and on society by:
(1) Publishing my data in publicly accessible, high impact journals with a wide audience.
(2) Presenting my data to neurologists and neuroscientists at relevant seminars and conferences.
(3) Disseminating my data to the general public as detailed in the 'communications plan'.
(4) When appropriate, talking with clinicians and industry to develop clinical trials.
(5) Ensuring that my group is open about our research focus, providing detailed methods and giving access to the raw data when possible.
(6) Attending a laboratory management training course at the beginning of the fellowship.

Please see the 'Pathway to Impact' statement for more information.