MULTIPLE SCLEROSIS

Field of the Invention

This invention relates to a novel therapeutic approach for treating demyelinating diseases such as multiple sclerosis (MS) and for treating conditions associated with white matter

ischaemia.

Background of the Invention

Oligodendrocytes create and maintain the myelin sheaths wrapped around the axons of neuronal cells. Myelination is needed to enable efficient transmission of electrical impulses (action potentials) along neuronal axons. Hence, oligodendrocytes are crucial for brain function. In demyelinating diseases such as MS, damage to the myelin sheath of neurons impairs the

propagation of action potentials in the affected nerves.

Action potential propagation through myelinated axons is also impaired in ischaemia (1) . This had been thought to reflect a rundown of ion gradients across the axonal membrane, but impulse propagation only partly recovers on readmitting oxygen and glucose to the tissue to restore ion pumping (1) .

Electron microscopy (2) and imaging of dye-filled

oligodendrocytes (3) show that ischaemia evokes Ca2+-dependent damage to the capacitance-reducing myelin sheaths, suggesting that irreversible myelin damage underlies much of the loss of action potential propagation in ischaemia. Myelin is damaged in a Ca2+-dependent manner in ischaemia, abolishing action potential propagation (1), (2) . This has been attributed to glutamate release activating Ca2+-permeable NMDA receptors (2)-(4). MS is a demyelinating disease which involves both the loss of oligodendrocytes and damage to myelin sheaths. The aetiology of MS involves both genetic and environmental factors. In MS, the oligodendrocytes themselves demyelinate and die by immune attack. All current disease-modifying drugs target the immune system in order to decrease disease progression, but they are still relatively unsuccessful and have side effects caused by the diminished immune system.

The environmental factors known to increase the incidence or severity of MS, include smoking (causing increases in CNS concentrations of nitric oxide, nicotine, carbon monoxide, hydrogen-cyanide) , surgery (the use of volatile anaesthetics such as sevofluorane) , viral infection (causing increases in CNS concentrations of TNF and INF-γ) and bacterial infections (causing increases in CNS concentrations of LPS, TNF and INF-γ) .

Environmental factors that decrease the incidence of MS include latitude (vitamin D production) and caffeine consumption. Vitamin D decreases brain nitric oxide concentrations.

In many demyelinating diseases such as adrenoleukodystrophy, Refsum disease and dysmyelinating leukodystrophy, fatty acid metabolism or toxicity causes dysmyelination and/or

demyelination . Many long chain fatty acids are TRPA1 agonists.

Summary of the Invention

The present inventors have found that oligodendrocytes express TRP channels that are activated by intracellular protons, including TRPA1 channels, and have found that these channels are activated during pathology and lead to demyelination. The inventors have also found that TRPA1 channels generate -70% of the ischaemia-evoked intracellular Ca2+ concentration ([Ca2+]i) rise, and that TRPA1 antagonists reduce ischaemic damage to myelin. TRPA1 channels had not previously been implicated in oligodendrocyte pathology. Therefore this represents a

completely new therapeutic target for demyelinating diseases such as MS. Accordingly, in a first aspect, this invention provides an agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel for use in a method of treatment or prophylaxis of a demyelinating or dysmyelinating disease or a failure of myelin to form in a subject, the method comprising administering the TRP blocker or desensitizer to the subject. In most embodiments, the TRP channel is activated by intracellular protons.

In most embodiments, the TRP channel is Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1 (TRPA1) . In most embodiments, the agent that blocks or

desensitizes a Transient Receptor Potential (TRP) channel is an antagonist. In most embodiments, the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is a TRPA1 antagonist. Hence, in some embodiments, the invention provides a TRPA1 antagonist for use in a method of treatment or prophylaxis of a demyelinating or dysmyelinating disease or a failure of myelin to form in a subject, the method comprising administering the TRPA1 antagonist to the subject.

In some embodiments, the demyelinating disease is multiple sclerosis (MS) . In some embodiments, the demyelinating disease or the MS is associated with brain hypoxia and/or white matter ischaemia and/or infection (including infection of a mother bearing a foetus, for example in conditions leading to

periventricular leukoencephalopathy, cerebral palsy and related conditions) . In some embodiments, the demyelinating disease is associated with an autoimmune disease. In some embodiments, the demyelinating disease is caused by a genetic factor impairing fatty acid synthesis for myelin production. In some embodiments, the demyelinating disease is associated with fatty acid

accumulation harming oligodendrocytes. In some embodiments, the demyelinating disease a leukoencephalopathy, such as toxic leukoencephalopathy . In some embodiments, the demyelinating disease is caused by a disorder of fatty acid metabolism. In some embodiments, the demyelinating disease is Refsum disease. In some embodiments, the demyelinating disease is adrenoleukodystrophy (ALD) . In some embodiments, the disease is a dysmyelination disease, which is dysmyelinating leukodystrophy. In some embodiments, the disease is progressive multifocal leukoencephalopathy caused by virus or bacterial infection. In some embodiments, the demyelinating disease is acute disseminated encephalomyelitis involving inflammation or bacterial infection. In some embodiments, the demyelinating disease is osmotic demyelination syndrome.

In a second aspect, this invention provides a Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1

(TRPA1) antagonist for use in a method of treatment or

prophylaxis of the deleterious effects of white matter ischaemia and/or conditions associated with white matter ischaemia in a subject, the method comprising administering a TRPA1 antagonist to the subject. Deleterious effects of white matter ischaemia include white matter damage, subcortical white matter lesions

(leukoaraiosis ) , block of action potential signalling within the brain and to the limbs due to loss of oligodendrocytes, which leads to chronic physical and/or mental disability (including paralysis and loss of speech) and sometimes death.

In some embodiments, the white matter ischaemia is associated with a stroke. In other embodiments, the white matter ischaemia is a secondary ischaemia caused by spinal cord injury. In some embodiments, the white matter is damaged by hypoxia associated with multiple sclerosis (MS) . In some embodiments the white matter ischaemia and subsequent demyelination are associated with radiation treatment, chronic hypertensive encephalopathy or Leber's hyperintensive encephalopathy.

While white matter ischaemia is not conventionally classified as a demyelinating disease, demyelination is prevalent, and the finding that TRPA1 antagonists (TRPA1 blockers) can protect against the Ca2+-dependent myelin damage in white matter

ischaemia shows that block of oligodendrocyte TRPAl-containing channels may be useful for reducing myelin loss during the energy deprivation that follows stroke, secondary ischaemia caused by spinal cord injury, hypoxia associated with multiple sclerosis, hypoxia associated with radiation treatment, and ischaemia caused by chronic hypertensive encephalopathy and Leber's hyperintensive encephalopathy .

Hence, the first and second aspects of the invention share a common mechanism of action: TRPAl antagonists target the

underlying pathology of oligodendrocytes. Hence the TRPAl antagonist may reduce oligodendrocyte pathology in the subject. The TRPAl antagonist may reduce oligodendrocyte loss in the subject. The invention is therefore useful for treating, preventing and giving alleviation from demyelinating conditions such as MS; and for treating, preventing and giving alleviation from white matter ischaemia. In light of this disclosure, the skilled person will understand that the TRPAl-containing channels that are blocked according to this invention are present upon (i.e. expressed by) oligodendrocytes, particularly those within the brain of the subject.

In a third aspect, which is related to both the first and second aspects of the invention, the invention provides an agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel for use in a method of treating or preventing an

oligodendrocyte disease in a subject, the method comprising administering the agent to the subject. In most embodiments, the TRP channel is Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1 (TRPAl) . In most embodiments, the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is an antagonist. In most embodiments, the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is a TRPAl antagonist. Hence, in some embodiments, the invention provides a Transient Receptor Potential cation channel subfamily A, member 1 (TRPAl) antagonist for use in a method of treating or preventing an oligodendrocyte disease in a subject, the method comprising administering the TRPAl antagonist to the subj ect .

In some embodiments, the oligodendrocyte disease is associated with oligodendrocyte loss and/or oligodendrocyte demyelination and/or oligodendrocyte necrosis, apoptosis or other modes of death, and/or autoimmune attack directed at oligodendrocytes. I some embodiments, the oligodendrocyte disease is leukodystrophy. The oligodendrocyte disease may be caused by an infection. The infection may be a viral infection or the infection may be a bacterial infection. Alternatively or additionally, the oligodendrocyte disease may have a nutritional or environmental cause .

The subject may be a mammal, e.g. a human subject. In some embodiments, the human subject is a smoker. A smoker can be regarded as a person who inhales tobacco smoke on one or more occasions per week. A smoker may be an active smoker, who intentionally inhales tobacco smoke. Alternatively, a smoker is a passive smoker who inhales tobacco smoke exhaled by an active smoker. In some embodiments, the TRPAl antagonist treats or is prophylactic against the harmful effects of nitric oxide (NO) , nicotine, hydrogen-cyanide (HCN) carbon monoxide (CO) and/or volatile anaesthetics such as sevofluorane . The nitric oxide (NO), nicotine, hydrogen-cyanide (HCN) and/or carbon monoxide (CO) may be inhaled in tobacco smoke. In other embodiments the smoker is a heroin (diamorphine) smoker. The heroin smoker may inhale heroin on one or more occasions per week.

In some embodiments, the TRPAl antagonist is used in a method of a method of preventing intracellular [Ca2+] rise and/or

preventing damage to myelin. In some embodiments, the TRPAl antagonist is used in a method of reducing the intracellular concentration of divalent and trivalent cations including Ca2+, Mg2+, Zn2+, Fe2+ and Fe3+ and/or reducing changes in membrane K+ conductance . In some embodiments, the TRPA1 antagonist protects the subject from the harmful effects of nitric oxide (NO) and/or nitric acid produced by other sources, for example produced by microglia. In some embodiments, the subject has undergone, is undergoing or is about to undergo surgery. The subject may have been treated with, may be being treated with or may be about to be treated with, a volatile anaesthetic, such as sevofluorane , e.g. as part of a surgical procedure. In some embodiments, the volatile anaesthetic (such as sevofluorane) acts on TRPA1. The TRPA1 antagonist of the invention can be used as a preventative measure in association with surgery in order to stop the possibility of patients subsequently getting MS evoked by the anaesthesia.

In some embodiments, the invention provides a method of treatment of demyelinating diseases, dysmyelinating diseases or diseases in which myelin fails to form, the method comprising administering a drug that blocks one or more downstream effectors of the calcium that enters through a Transient Receptor Potential (TRP) channel. The TRP channel may be TRPA1.

In some embodiments, the invention provides a method of treatment of a demyelinating diseases, dysmyelinating diseases or diseases in which myelin fails to form, the method comprising

administering a drug that modulates TRPA1 or that modulates interactions of TRPA1 with other proteins, molecules, TRP channels, receptors or receptor subunits .

The subject may be suffering from an autoimmune condition. In some embodiments, the subject has elevated estrogen levels.

In some embodiments, the subject may be suffering from an infection. The infection may be a viral infection or the infection may be a bacterial infection. The bacterial infection may result in LPS-mediated stimulation of TRPAl-containing channels. The infection may trigger an immune response. The infection may trigger an autoimmune response. The immune/autoimmune response may be mediated by Thl cells and/or mediated by Thl7 cells. The immune/autoimmune response may be mediated by TNF, IFN (e.g. I N-gamma) , IL-l-beta and/or other immunostimulatory cytokines.

The subject may be suffering from an autoimmune response to myelin debris. This autoimmune response may be mediated by Thl cells and/or mediated by Thl7 cells. This autoimmune response may be mediated by TNF, IFN (e.g. IFN-gamma), IL-l-beta and/or other immunostimulatory cytokines.

In a further aspect (related to the first aspect, above) , a method of treating or preventing a demyelinating disease in a subject is provided, the method comprising administering an agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel to the subject. Preferably, the TRP channel is activated by intracellular protons. In most embodiments, the TRP channel is Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1 (TRPA1) . In most embodiments, the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is an antagonist. In most embodiments, the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is a TRPA1 antagonist. In some embodiments, the

demyelinating disease is multiple sclerosis (MS) . In some embodiments, the MS is associated with brain hypoxia and/or white matter ischaemia. In some embodiments, the demyelinating disease is associated with an autoimmune disease.

In another aspect (related to the second aspect, above) , a method of treating or preventing white matter ischaemia in a subject is provided, the method comprising administering a TRPA1 antagonist to the subject. In some embodiments, the white matter ischaemia is associated with a stroke. In other embodiments, the white matter ischaemia is a secondary ischaemia caused by spinal cord injury. In some embodiments, the white matter ischaemia is caused by hypoxia associated with multiple sclerosis (MS) . In some embodiments, the method of treating or preventing white matter ischaemia is specifically a method of preventing damage to myelin .

Demyelination may be caused by inflammation; e.g. due to

neuromyelitis optica, acute disseminated encephalomyelitis (ADEM) or bickerstaff brainstem encephalitis. Demyelination may be caused by viral or bacterial infection; e.g. due to progressive multifocal leukoencephalopathy, HIV, MS or ADEM associated with infection with the Gram-negative bacterium Chlamydia pneumoniae . Demyelination may be caused by acquired metabolic changes; e.g. due to demyelination syndrome (also known as central pontine myelinolysis ) , toxic leukoencephalopathy (e.g. after smoking heroin), injesting paradichlorobenzene or glue sniffing.

Demyelination may be caused by hypoxia/ischaemia; e.g. due to radiotherapy, chronic hypertensive encephalopathy leading to subcortical leukoencephalopathy, stroke, spinal cord injury or possibly in MS (Davies et al . , 2013, which is hereby encorporated by reference) . Demyelination may be caused by compression, e.g. due to trigeminal neuroalgia or spinal cord injury.

Demyelination may be caused by environmental toxicity which are linked to MS. The present invention offers a novel therapeutic approach for these demyelination associated conditions.

In a still further aspect (related to the first aspect, above) , use of a TRPA1 antagonist in the manufacture of a medicament for the treatment or prophylaxis of a demyelinating disease in a subj ect is provided. In a still further aspect (related to the second aspect, above) , use of a TRPA1 antagonist in the

manufacture of a medicament for the treatment or prophylaxis of white matter ischaemia in a subj ect is provided.

In some embodiments of thi s invention, the TRPA1 antagonist is specific for TRPA1 over other TRP channels. In other embodiments of this invention, the TRPA1 antagonist blocks TRPA1 as well as other TRP channels expressed by oligodendrocytes. In some embodiments of this invention, the TRPAl antagonist or TRPAl desensitiser is Hydra HC-030031, Abbott A967079, Glenmark GRC-17536, cannabinoids , gingerol, curcumin, cinnemaldehyde, Novartis compound 31, AMG0902, Novartis AP18, Amgen compound 10, Janssen Compound 43, CHEM-5861528 , resolving D2 , gentamicin, isopentenyl diphosphate (IPP) , TCS 5861528, a fatty acid, amiloride or analogues thereof. In other embodiments, TRPAl channel blockade may be effected by an agent that has some action as a TRPAl agonist with reduced efficacy, such as a partial agonist or a mixed agonist/antagonist, that leads to subsequent blockade of TRPAl, e.g. by desensitization .

Diagnosis and prognosis In another aspect, a method of detecting or prognosing a

demyelinating disease in a subject is provided, the method comprising using a molecule that specifically binds to TRPAl or to its DNA or RNA to determine TRPAl expression level in a subject, the method further comprising comparing the TRPAl level to a control level. In some embodiments, a sample has been obtained from the subject and the TRPAl expression level is determined in said obtained sample. In some embodiments, the sample has been obtained from the CNS or CSF of the subject.

Preferably, the sample comprises oligodendrocytes. The method may comprise determining the TRPAl expression level in (or at the surface of) oligodendrocytes. In other embodiments, the method of detecting or prognosing a demyelinating disease comprises the detection of a labelled molecule that specifically binds TRPAl, which has been administered to the subject.

In a further aspect, the invention provides an in vitro or in vivo method of screening for agents suitable for use in treating demyelinating diseases, dysmyelinating diseases and/or diseases in which myelin fails to form, the method comprising; providing an oligodendrocyte, applying a candidate agent to an

oligodendrocyte and measuring the effects of the candidate agent on expression levels of TRPAl in the oligodendrocyte and/or the rate of ion entry through TRPA1 channels in the oligodendrocyte. The ion may be Ca2+, Mg2+ or any other permeant ion.

Combination therapies

The TRPA1 antagonist can be combined with other agents and therapies, in which two or more agents or therapies are combined, for example, sequentially or simultaneously. The agents may include nutritional supplementation and/or conventional medicines such as beta-interferon, or glatiramer acetate. The therapy or therapies may include surgery.

In some embodiments, the subject may be administered with the TRPA1 antagonist in combination with caffeine. The caffeine may be consumed by the subject by drinking coffee. In some

embodiments, the subject may be administered in combination with vitamin D. The vitamin D may be consumed by the subject in the form of vitamin supplements. The consumption of caffeine and/or vitamin D may be due to advice by a doctor or physician.

The sequential or simultaneous administration of the two or more agents or therapies is further described in the section entitled "Treatment & Prophylaxis".

Pharmaceutical preparations

This invention also provides a pharmaceutical preparation or pharmaceutical composition comprising a TRPA1 antagonist, for use in a method of treating or preventing a demyelinating disease; for use in a method of treating or preventing white matter ischaemia; or for use in a method of treating or preventing an oligodendrocyte disease. The pharmaceutical preparation or composition of the invention may be used according to the embodiments of the invention disclosed herein. The pharmaceutical preparation or composition of the invention may comprise a pharmaceutically acceptable excipient . The pharmaceutical compositions of the present invention are

discussed in more detail below.

Kits

The invention also provides kits. A kit according to the invention may comprise a dry preparation (e.g. lyophilised preparation) of the TRPAl antagonist in a container, optionally with a buffer solution in a second container. The kits may include instructions for dissolving the TRPAl antagonist, and/or mixing the TRPAl antagonist with the buffer, and/or administering the TRPAl antagonist to a subject.

Brief Description of the Figures

Figure 1. Ischaemia evokes an inward current in oligodendrocytes by altering K+ fluxes

a Whole-cell clamped oligodendrocyte; inset shows Alexa dye in processes around an axon, b Ischaemia-evoked inward membrane current in a single cell, c Mean current in 179 control cells, 12 cells exposed to 25 μΜ NBQX and 200 μΜ D-AP5 from before the start of ischaemia, or preloaded (16) for 30 mins with 1 mM PDC . d Current (normalised to interleaved control cells) averaged from 8-10 mins after the start of ischaemia in cells preloaded with PDC, exposed to NBQX and AP5 throughout ischaemia or from 200 sec after ischaemia starts, or exposed to NBQX or AP5 alone or to zero-Ca2+ solution throughout ischaemia. P values (from Mann- Whitney tests) compare with control cells; cell numbers shown on bars, e Effect of Gd3+ on ischaemia-evoked current at 8-10 mins (p=0.83 from Mann-Whitney test) . f I-V relation of 10 cells before and after 5 mins ischaemia (10 mM HEPES internal) . g

Ischaemia-evoked current in 10 cells with 0.5 mM HEPES internal and in 9 cells with 50 mM internal HEPES. h Change of

extracellular K+ concentration ([K+]o) in grey matter (GM, granule cell layer) , and in white matter (WM, different slice) with simultaneously recorded oligodendrocyte current. Figure 2. NMDA does not elevate [Ca2+] i in oligodendrocytes a—b Oligodendrocyte membrane current (lower traces in a) and background-subtracted fluorescent dye ratio (R, see Methods, concentration increases are plotted upwards for all dyes) when measuring [Ca2+] i with Fura-2, [Na+] i with SBFI, and [K+]i with PBFI; 100 μΜ NMDA was applied, or [K+]o was raised from 2.5 to 5 mM, with fluorescence measured in the soma (a) or myelinating processes (b) . Right panels show mean peak fluorescence change normalised to the evoked current (number of cells on bars) . c

NMDA-evoked current compared with simultaneously recorded [K+]o. d Measuring [Ca2+]i with X-Rhod-1, loaded from the pipette or as an acetoxymethyl ester2, reveals no NMDA-evoked [Ca2+] i rise, e Spontaneous [Ca2+] i transients in four myelinating processes of a cell recorded with Fura-2 confirm that the dye is working.

Figure 3. Ischaemia evokes a [Ca2+]i and [Mg2+]i rise gated by internal protons

a Ratiometric Fluo-4 /Alexa-Fluor-594 signals ([Ca2+]i) and membrane potential (Vm) in oligodendrocytes exposed to normal ischaemia (starting at arrow), or ischaemia in zero [Ca2+]o, or with the drugs shown present at these concentrations (μΜ) : AP5 50, MK-801 50, 7-chlorokynurenate (7CK to block the NMDAR glycine site) 100, NBQX 25, GABAzine (Gz, to block GABAARs) 20, TTX (to block Na+ channels) 1, Cd2+ (to block Ca2+ channels) 100. b

Mean data from experiments as in a (cell numbers on bars; p values compare with soma or process control values) . c Data as in a after preloading with PDC or in 0 mM [K+]bath. d Ischaemia- evoked [Mg2+]i rise monitored with Mag-Fluo-4 in normal and Mg2+- free solution, e Ischaemia-evoked [Na+]i change monitored with SBFI in 6 cells, f Ischaemia-evoked [H+] I rise monitored with BCECF with 0.5 mM and high 50 mM HEPES in the cell (p value from Mann-Whitney test) . g [K+]o and oligodendrocyte membrane

potential (Vm) with 2.5 or 0 mM [K+]bath, before (Resting) and during ischaemia (Isch peak), and the change produced by

ischaemia (01sch) . h Effect of removing K+ from the bath solution on [H+] i in control conditions (relative to value at start of K+ removal), and the [H+] i increase evoked by ischaemia (data normalised to value at start of ischaemia) with 2.5 or 0 mM K+ in the bath, i High [HEPES] i blocks the ischaemia-evoked decrease of membrane conductance, j—k Ischaemia-evoked rise of [Ca2+]i (j) and [Mg2+]i (k) are inhibited with 50 mM internal

HEPES. 1—m Applying light to uncage H+ (bars) raises [Ca2+]i (1, an effect reduced by 200 μΜ IPP or 80 μΜ HC-030031) and [Mg2+] i (m) , but not when the caged H+ is omitted from the pipette. P values in 1 are from Mann-Whitney tests.

Figure 4. TRPAl-containing channels mediate ischaemic Ca2+ accumulation and myelin damage

a Change of [Ca2+]i (measured with Fluo-4 and Alexa-Fluor-594 ) in soma (black) or processes (white) evoked by TRPA1/TRPV3 agonists (in mM: menthol 2, vanillin 1, 2-APB 2), TRPV3 agonists (camphor 2, FPP 0.5) and TRPA1 agonists AITC (0.5), FFA (1) and polygodial (0.2) . b The rise of [Ca2+]i evoked by the TRPA1 /TRPV3 agonist carvacrol (2 mM, in 1 μΜ TTX to avoid any effects of altered neuronal spiking) is inhibited by the TRPA1/TRPV3 antagonist isopentyl pyrophosphate (IPP, 200 μΜ, Mann Whitney test on soma data) , the TRPA1 antagonist HC-030031 (80 μΜ) , and by TRPA1 knock-out (Mann- Whitney test on process data) , but not by 50 mM internal [HEPES] (p values compare with soma or process control value) . c—d The ischaemia-evoked [Ca2+]i rise is blocked by Ruthenium Red (RuR, 10 μΜ, measured with Fura-2) (c) , and by La3+ (d, 1 mM, measuredwith Fluo-4/Alexa 594, using HEPES- buffered external solution to avoid precipitation, Mann- Whitney test on soma data) . e Block of the ischaemia-evoked [Ca2+] i rise (measured with Fluo-4) by the TRPA1 /TRPV3 blocker IPP (200 μΜ) and by the TRPA1 blockers HC-030031 (80 μΜ) and A967079 (10 μΜ) . P values from Mann-Whitney tests compare with soma or process control values, f Ischaemia-evoked [Ca2+]i rise in wild-type mice, with TRPA1 knocked out (process data showed the same trend as the soma data but with p=0.07) and with the TRPA1 /TRPV3 blocker IPP (200 μΜ) present in the KO . g Ischaemia-evoked

[Ca2+] i rise in wild-type mice, with TRPV3 knocked out and with the TRPA1 blockers HC- 030031 (80 μΜ) and A967079 (10 μΜ) present in the KO (p values from Mann-Whitney tests) . h Ischaemia-evoked [Ca2+]i rise in the TRPA1 KO with 10 mM and 50 mM [HEPES] in the cell, i Specimen EM pictures showing control optic nerves and myelin decompaction (white arrows) in optic nerves exposed to ischaemia or ischaemia in RuR (10 μΜ) or A967079 (10 μΜ) and HC- 030031 (80 μΜ) applied together for 60 mins (TRPA1 block) . j-m Mean data for lamella separations (j), g ratio (k) , axon diameter (1) and axon vacuoles (m: EM picture shows vacuoles (black arrows) forming within the axon and the periaxonal space during ischaemia) in control, ischaemia alone or ischaemia with RuR (10 μΜ) or with A967079 (10 μΜ) and HC-030031 (80 μΜ) (TRPA1 block) . Numbers on bars are 'images (axons)' . P values for j-m from Mann- Whitney test except 1 from Kolmogorov-Smirnov test.

Figure 5. Schematic showing environmental factors implicated in MS influence TRPA1 receptors

The oligodendrocyte TRPA1 channel described in the present application is linked with the environmental factors implicated in MS : 1: Oligodendrocytes express TRPA1 subunit containing channels, which in other organs are known to function as a molecular integrator of multiple irritants and inflammatory mediators. Synergistic effects of multiple agonists lead to larger currents, pore dilation, lowering of the activation threshold and upregulation of channel expression.

2: Women are three times more likely to get MS than men. One obvious difference is the concentration of oestrogen, which is highest in women between the ages of 11-60 years, the time window within which MS begins. Estrogen increases the expression of TRPA1 and women with endometriosis, who produce too much

oestrogen, have increased incidence of MS. 3: Multiple studies show smoking increases incidence, severity and progression of MS. 4: Smoke contains many harmful components including nitric oxide (NO) , nicotine, hydrogen cyanide (HCN) and carbon monoxide (CO) , that can enter the blood stream and cross the blood-brain barrier. 5: Nicotine reaches a plasma concentration (~300nM) that can activate TRPA1. 6: Nicotine administration to the CNS increases nitric oxide production. 7: The CSF concentrations of NO and its metabolites are greater in MS patients and increase during acute MS relapse. A persistent increase in NO metabolites is associated with increased MS progression. NO and its

metabolites can cause demyelination and oligodendrocyte necrosis. 8: Microglia contribute to the production of NO and its

metabolites. 9: NO and its metabolites can activate TRPA1. 10: LPS (>lug/ml), which is produced by lysis of bacteria, activates TRPA1 independently of TLR4. Co-application of low concentrations of the endogenous agonist HNE with LPS produced a strong

potentiation of TRPA1 activity, resulting in much larger currents than the sum of both the agonist responses alone. 11: HCN and CO reduce the metabolic capacity of the brain making it more ischaemic. Chronic low doses of cyanide cause demyelination. 12: Ischaemia activates oligodendrocyte TRPA1 via intracellular acidification. C02 can activate TRPA1, along with several oxidative stress-related substances. They are also activated directly by hypoxic conditions, and upregulate their expression following an hypoxic episode. 13: Previous exposer to Epstein Barr Virus increases the incidence of MS. 14: Viral attack leads to production of pro-inflammatory cytokines such as TNF, IL-Ιβ and INF-γ, all of which (at lOng/ml, lng/ml and lOng/ml

respectively) can activate TRPA1. Pre-incubation with TNF induces sensitisation and upregulation of TRPA1, increasing the maximum response activated whilst also decreasing the activation

threshold of another agonist (polygodial) by a factor of 50. 15: Surgery before the age of 20 has been linked to increased incidence of MS; volatile anaesthetics used for surgery appear to increase the incidence of MS in nurse anaesthetists, kill oligodendrocytes and activate TRPA1. 16: Vitamin D is suggested to decrease incidence of MS by directly effecting the immune system. 17: Vitamin D can change ion channel expression (i.e. TRPV6 expression, and gene transcription. 18: Vitamin D decreases the production of NO and pro-inflammatory cytokines by microglia. 19: Calcium activates TRPA1, and systemic calcium homeostasis is heavily influenced by vitamin D. 20: Caffeine consumed (via 4-5 cups of coffee per day) has been found to decrease the incidence of MS, and caffeine improves the outcome of EAE (Wang et al . , 2014) . 21: Caffeine crosses the BBB and reaches plasma

concentrations (62μΜ that can partially inhibit TRPA1. 22:

Cannabinoids activate TRPA1 but also desensitise them to noxious stimuli .

Figure 6. Tests for causes of the ischaemia-evoked current a Effect of blocking various putative glutamate release

mechanisms (blocker concentrations given in "Effect of blocking potential glutamate release mechanisms" below) on peak ischaemia- evoked currents measured in the presence of each drug and in interleaved controls. No significant differences were measured (p>0.20) . b Schematic showing effect of ischaemia-evoked decrease in resting conductance (which is dominated by gK, left) and ischaemia-evoked [K+]o rise (right) on oligodendrocyte membrane current. Black lines are control I-V relations. Red lines are I-V relations in ischaemia showing the effect of a conductance decrease (left) or of a positive shift of reversal potential due to [K+]o rising (right) . c Ischaemia-evoked current change for the two mechanisms in b (cf . Fig. lg) . d Sum of currents in c gives an I-V relation with a reversal potential more negative than EK. e Experimentally observed ischaemia-evoked current in 10 oligodendrocytes with 10 mM internal HEPES (difference of curves in Fig. If) . Figure 7. K+ flux changes generate the oligodendrocyte NMDA- evoked current

a—b Extracellular Cs+ (30 mM, replacing Na+) reduces the inward current evoked by 100 μΜ NMDA at -74 mV in oligodendrocytes (a) , but not in hippocampal CA1 pyramidal neurons (b) . c—d

Intracellular MK-801 (1 mM) has no effect on NMDA-evoked currents in oligodendrocytes (c) but blocks them in pyramidal cells (d) , while extracellular MK-801 (50 μΜ) blocks both, e Voltage- dependence of the current evoked in 16 oligodendrocytes by 100 μΜ NMDA and by elevating [K+]o from 2.5 to 5 mM. f Specimen plot of membrane current in an oligodendrocyte versus local [K+]o in response to applying 100 μΜ NMDA or elevating [K+]o from 2.5 to 5 mM. Horizontal cell line shows that if NMDA raises [K+]o to (say) 4.5 mM, the current attributable to the [K+]o rise alone is 51% of the NMDA-evoked current. Mean value in 11 cells was 49% (see "A [K+]o rise and K+ influx mediate much of the response to NMDA" below) .

Figure 8. NMDA-evoked ion concentration changes in neurons differ from those in oligodendrocytes

a Specimen records of cerebellar granule cell membrane current and background-subtracted fluorescent dye ratio (R, see Methods) when measuring [Ca2+]i with Fura-2, [Na+]i with SBFI, and [K+] i with PBFI, when 100 μΜ NMDA was applied, b Mean peak fluorescence changenormalised to evoked current (number of cells on bars; p value from Mann-Whitney test) . Oligodendrocyte data for

comparison are shown in Fig. 2.

Figure 9. Comparison of NMDA-, [K+]bath- and ischaemia-evoked changes of [H+]i

a Measurements of changes of ratio (R, see Methods) of

background-subtracted BCECF fluorescence in oligodendrocytes in response to 100 μΜ NMDA and raising [K+]bath from 2.5 to 5 mM with 0.5 mM internal HEPES, and to ischaemia with 0.5 mM and 50 mM internal HEPES. b-c Effect of pH of 10 mM HEPES internal solution on: (b) baseline ratio of Fluo-4 to Alexa 594

fluorescence (p value from Mann-Whitney test) , and (c) change of ratio when [Ca] was increased by 200 nM.

Figure 10. In situ hybridization data on TRP channel expression a. In situ data for TRPA1 and TRPV3 in the cerebellum of rats and mice show TRPA1 mRNA in white matter (WM) cells in rats and mice (with denser expression in the adjacent granule cell layer, GCL) , but TRPV3 mRNA only in white matter cells in rats. Specimen cells are labelled with white circles, b Higher magnification views of white matter, combining in situ hybridization for TRPA1 and TRPV3 with immunocytochemistry for 01ig2 (to label oligodendrocyte lineage cells) and CC1 (to define myelinating oligodendrocytes) . TRPA1 mRNA is present (in rats and mice) and TRPV3 mRNA is present (in rats but not mice) in myelinating oligodendrocytes (01ig2+, CC1+: arrowheads) and also in some presumed oligodendrocyte precursor cells (01ig2+, CC1-: arrows) . c—d

Quantification of presence of mRNA for TRPAl (c) and TRPV3 (d) in different oligodendrocyte lineage cell classes. Numbers on bars are 'images analysed (cells counted) ' . P value from Mann-Whitney test .

Figure 11. Further evidence for the identity of TRP channels in oligodendrocytes

[Ca2+] i increase (ratio signal from Fluo-4 and Alexa Fluor 594) in oligodendrocyte somata when ischaemia solution was applied with the following drugs present (data normalised to interleaved controls, shown as black bars) : RN-1734 (0.5 mM) , which

blocks20,59 TRPV4 and, less well, TRPV1, TRPV3 and TRPM8 ; a cocktail of blockers inhibiting (see section on "Specificity of drugs acting on TRP channels") TRPP2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPM2, TRPM4, TRPM5, TRPV1, TRPV2 , TRPM7, TRPM8 and TRPP1, as well as the store-operated calcium channel component STIM1 and some voltage-gated calcium channels; blocking voltage-gated Ca2+ channels with 10μΜ benidipine; or blocking reversed Na/Ca exchange with 10μΜ KB-R7943 mesylate (p values, from Mann-Whitney test and t-test as appropriate, were non-significant (p>0.28)) .

Figure 12. Schematic of how oligodendrocyte [Ca2+] i is raised in ischaemia

Run-down of transmembrane ion gradients leads to glutamate transporters (GluT) reversing (1) and releasing glutamate (2) . This depolarizes (and causes a neurotoxic [Ca2+] i rise in) neurons (3) and raises [K+]o (4), causing an inward current (5) through oligodendrocyte K+ channels (KCh) . At the same time, metabolic changes and also the rise of [K+]o lead to a rise in oligodendrocyte [H+]i (6) . This decreases the membrane K+ conductance (either directly or via TRPAl-containing channels opening) which contributes to the inward current generated, and opens TRPAl-containing channels (7) that let Ca2+ and Mg2+ into the cell (8) . The resulting rise of [Ca2+] i damages the myelin. Figure 13. TRPAl mRNA and protein expression by CCl-expressing mature oligodendrocytes

a. In situ hybridisation showing P12 rat cerebellar slices express TRPAl mRNA ² . b. Antibody labelling of TRPAl (Abeam 58844) in P21 rat corpus callosum.

Figure 14. TRPAl agonists evoke currents in oligodendrocytes a, b. Increase in rat oligodendrocyte conductance is evoked when an electrophilic TRPAl agonist is bath-applied.

c. Increase in [Ca ²⁺ ]i evoked by a TRPAl agonist is blocked by the TRPAl antagonist HC 030031.

d. TRPAl agonist evoked increase in [Ca ²⁺ ]i is larger in the presence of an inflammatory cytokine. Figure 15. TRPAl agonist propargyl isothiocyanate (PITC)

preferentially binds to white matter somata

a. P21 Rat coronal brain slices were incubated in PITC for 1 hour. After fixation, the PITC was tagged by Click-IT chemistry to a fluorescent molecule (Chromeo 546) and found to stain somata in the corpus callosum.

b. Negative staining for GFAP indicates that these cells are not astrocytes .

c. Arrows are oligodendrocytes identified by their location, morphology and GFAP negative status.

Detailed Description

The following applications of the present invention are provided by way of example and not limitation. TRPAl Antagonists

Any type of TRPAl antagonist can be used in the methods and formulations of this invention. TRPAl antagonists can be readily identified and characterised using well-known methods in the art, including patch-clamping, ion imaging and binding assays.

TRPAl antagonists inhibit or block the flow of Ca2+ (or other divalent) ions through the TRPAl-containing ion channel. By "inhibit" or "block" is meant the ability to cause an overall decrease preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75%, 85%, 90%, 95%, or greater in Ca2+ ion flow.

The TRPAl antagonist may be specific for TRPAl (meaning that the extent of inhibition or blocking of TRPAl-containing channels is at least 50% greater than, at least 60% greater than, at least 70% greater than, at least 80% greater than, at least 90% greater than or most preferably at least 95% greater than the extent of inhibition or blocking of one or more other ion channels, e.g. those containing TRPV3, TRPC3, TRPC5, TRPC6, TRPC7, TRPV4, TRPV5, TRPV6, TRPM2, TRPM3, TRPM4 , TRPM5 or TRPM8 ) , or it may also act on other TRP channels.

In some embodiments, the TRPAl antagonist inhibits the TRP channels expressed by oligodendrocytes. In some embodiments, the TRPAl antagonist inhibits TRP channels that are activated by intracellular protons, which are expressed by oligodendrocytes.

TRP channel blockade with agents that have some agonist action

As noted in the ^Λ Summary of the Invention' , above, in a first aspect, the invention provides an agent that blocks or

desensitizes a Transient Receptor Potential (TRP) channel. In some embodiments of this invention, an agonist that leads to subsequent desensitization of TRP channels such as TRPAl may be used to desensitize and thus block TRPAl channels. In these embodiments of the invention, a sufficient therapeutic plasma concentration is needed to effect desensitization. The dose of the desensitizing agonist can be chosen accordingly.

Similarly, a TRP channels agonist (such as a TRPAl agonist) with reduced efficacy may be used as a partial agonist or a mixed agonist/antagonist to effect at least partial blockade of TRPAl channels. The agonist may be electrophilic or non-electrophilic . TRPA1 antagonist antibodies

A "blocking" antibody or an antibody "antagonist" is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies completely inhibit the biological activity of the antigen.

Appropriate doses of antibody molecules are well known in the art (Ledermann J. A. et al . (1991) Int. J. Cancer 47: 659-664;

Bagshawe K.D. et al . (1991) Antibody, Immunoconj ugates and

Radiopharmaceuticals 4: 915-922) . Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of an antibody molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody,

fragment) and the nature of any detectable label or other molecule attached to the antibody. A typical antibody dose will be in the range 100 μg to 1 g for systemic applications, and 1 μg to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered. Typically, the antibody will be a whole antibody, e.g. the IgGl or IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be

proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight . The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (see below) so long as they exhibit the desired biological activity. "Antibody fragments" comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.

Oligodendrocyte TRPA1 channels and environmental factors in MS An extensive literature search links the oligodendrocyte TRPA1 channel (newly described in the present application) with the environmental factors implicated in MS. These links are shown in Fig. 5. 1: Oligodendrocytes express TRPA1 subunit containing channels (present disclosure) , which in other organs are known to function as a molecular integrator of multiple irritants and inflammatory mediators (Bautista et al . , 2006) . Synergistic effects of multiple agonists lead to larger currents (Hu et al . , 2010; Brierly et al . , 2011; Lennertz et al . , 2012; Meseguer et al . , 2014; Lowin et al . , 2015), pore dilation (Karashima et al . , 2010), lowering of the activation threshold (Lowin et al . , 2015) and upregulation of channel expression (Takahashi et al., 2011; Meseguer et al . , 2014) . 2: Women are 3 times more likely to get MS than men. One obvious difference is the concentration of estrogen, which is highest in women between the ages of 11-60 years, the time window within which MS begins. Estrogen increases the expression of TRPA1 (Greaves et al . , 2014) and women with endometriosis, who produce too much estrogen, have increased incidence of MS (Mormile and Vittori . , 2014) . 3: Multiple studies show smoking increases incidence (Hernan et al . , 2001), severity and progression of MS (Hernan et al . , 2005) . 4: Smoke contains many harmful components including nitric oxide (NO) , nicotine, hydrogen-cyanide (HCN) and carbon monoxide (CO) , that can enter the blood stream and cross the blood-brain barrier. 5: Nicotine reaches a plasma concentration (~300nM; Russel et al . , 1975) that can activate TRPA1 (Talavera et al . , 2009) . 6:

Nicotine administration to the CNS increases nitric oxide production (Suemaru et al . , 1997; Smith et al . , 1998; Tonnessen et al., 2000) . 7: The CSF concentrations of NO and its metabolites are greater in MS patients and increase during acute MS relapse (Yamashita et al . , 1997; Svenningsson et al . , 1999) . Whilst persistent increases in NO metabolites is associated with increased MS progression (Rejdak et al . , 2004) . NO and its metabolites can cause demyelination and oligodendrocyte necrosis (Mitrovic et al . , 1995, 1996; Smith et al . , 1999) . 8: Microglia contribute to the production of NO and its metabolites (Boje and Arora, 1992) . 9: NO and its metabolites can activate TRPA1

(Miyamoto et al . , 2009) . 10: LPS (>lug/ml) , which is produced by lysis of bacteria, activates TRPA1 independently of TLR4

(Meseguer et al., 2014) . Co-application of low concentrations of the endogenous agonist HNE with LPS produced a strong

potentiation of TRPA1 activity, resulting in much larger currents than the sum of both the agonist responses alone. 11: HCN and CO reduce the metabolic capacity of the brain making it more ischaemic (Lawther and Commins, 1970) . Chronic low doses of cyanide cause demyelination (Smith et al . , 1963; Philbrick et al . , 1979) . 12: Ischaemia activates oligodendrocyte TRPA1 via intracellular acidification (Present disclosure) . C02 can activate TRPA1 (Wang et al . , 2010), along with several oxidative stress-related substances (Andersson et al . , 2008) . They are also activated directly by hypoxic conditions, and upregulate their expression following an hypoxic episode (Takahashi et al . , 2011) . 13: Previous exposure to Epstein Barr Virus increases the incidence of MS (Reviewed by Belbasis et al . , 2015) . 14: Viral attack leads to production of pro-inflammatory cytokines such as TNF, IL-Ιβ and INF-γ, all of which (at lOng/ml, lng/ml and lOng/ml respectively) can activate TRPA1 (Lowin et al . , 2015) . Pre-incubation of TNF induces sensitisation and upregulation of TRPA1, increasing the maximum response activated whilst also decreasing the activation threshold of another agonist

(polygodial) by a factor of 50. 15: Surgery before the age of 20 has been linked to increased incidence of MS (Lunny et al . , 2013); volatile anaesthetics used for surgery appear to increase the incidence of MS in nurse anaesthetists (Landtblom et al . , 2006), kill oligodendrocytes (Brambrink et al . , 2012) and activate TRPA1 (Matta et al . , 2008) . 16: Vitamin D is suggested to decrease incidence of MS by directly affecting the immune system (reviewed by Hayes et al., 2015) . 17: Vitamin D can change ion channel expression (i.e. TRPV6 expression; Taparia et al . , 2005), and gene transcription. 18: Vitamin D decreases the production of NO and pro-inflammatory cytokines by microglia (Lefebvre d' Hellencourt et al . , 2002) . 19: Calcium activates TRPA1 (Doerner et al . , 2007), and systemic calcium homeostasis is heavily influenced by vitamin D. 20: Caffeine consumed (via 4-5 cups of coffee per day) has been found to decrease the incidence of MS (paper in preparation by Dr Ellen Mowry, Johns Hopkins

School of Medicine, Baltimore) , and caffeine improves the outcome of EAE (Wang et al . , 2014) . 21: Caffeine crosses the BBB and reaches plasma concentrations (62μΜ, Birkett and Miners ,'91) that can partially inhibit TRPA1 (Nagatomo and kubo, 2008) . 22: Cannabinoids activate TRPA1 but also desensitise them to noxious stimuli (Akopian et al . , 2008) . TRPA1 is also activated and then desensitised by long-chain fatty acids (Motter and Ahern, 2012. 23: One way for environmental irritants to activate

oligodendrocyte TRPA1 subunit containing receptors is through the nasal cavity where the olfactory tract is susceptible to inhaled compounds that can circumvent the blood brain barrier to enter the CNS (Ilium, 2000) . Neuropathological studies have shown that olfactory bulb/tract demyelination is frequent, can occur early, is highly inflammatory, and is specific to demyelinating disease, especially multiple sclerosis (DeLuca et al . , 2014) .

Demyelinating diseases

Demyelination can be caused by the following altered brain states: 1) inflamma ion, which occurs in MS (Lucchinetti et al . , 2000), neuromyelitis optica (Sharma et al . , 2010), acute

disseminated encephalomyelitis (ADEM; Esposito et al . , 2015) and bickerstaff brainstem encephalitis, ; 2) viral or bacterial infection which occurs in progressive multifocal

leukoencephalopathy (PML, Weiner et al . , 1973), HIV (Manji and Miller, 2000), MS (Cusick et al . , 2013) and ADEM associated with infection with the Gram-negative bacterium Chlamydia

pneumoniae (Heick and Skriver, 2000) . ; 3) acquired metabolic changes causing osmotic demyelination syndrome also known as central pontine myelinolysis (Alleman, 2014) , toxic

leukoencephalopathy (after smoking heroin (Wolters et al . , 1982) , injesting paradichlorobenzene (Weidman et al . , 2015) or glue sniffing (Davies et al . , 2000) ; 4) , hypoxia/ischaemia which occurs after radiotherapy (Greene-Schloesser et al . , 2012) , in chronic hypertensive encephalopathy leading to subcortical leukoencephalopathy (Stoner and Parker, 1991), in stroke

(Pendlebury et al . , 2000), in spinal cord injury (McDonald and Belegu, 2006) and possibly in MS (Davies et al . , 2013) ;

5) compression, which occurs in trigeminal neuroalgia (Love and Coakham, 2001) and spinal cord injury ; 6) environmental

toxicity which (along with genetic factors) is thought to occur in MS (see Fig. 5 and the section on ^Oligodendrocyte TRPAl channels and environmental factors in MS') .

The present disclosure presents oligodendrocyte TRPAl as a new therapeutic target for demyelination in all of these instances because TRPAl elsewhere in the body, or in vitro, is known to be activated by inflammatory cytokines (Lowin et al . , 2015) , by bacterial infection, extracellular hypertonic solution (Zhang et al . , 2008) , morphine (Forster et al . , 2009) i.e. from heroin use, toluene (Taylor-Clark et al . , 2009) i.e. from glue sniffing, hypoxia (Takahashi et al . , 2011) , ischaemia (present disclosure), compression (as they are mechanosensors ; Brierley et al . , 2011), and by the aforementioned environmental factors implicated in MS.

Leukodystrophies (degeneration of white matter (oligodendrocytes) in the brain) , include adrenomyeloneuropathy, Alexander disease, Cerebrotendineous xanthomatosis, hereditary CNS demyelinating disease, Krabbe disease, metachromatic leukodystrophy, Pelizaeus- Merzbacher disease, Canavan disease, leukoencephalopathy with vanishing white matter, adrenoleukodystrophy, Refsum disease and Xenobefantosis . In these diseases oligodendrocyte TRPAl is a new potential therapeutic target because of its newly found location and ability to be activated by many factors occurring in these diseases . Symptoms

Symptoms of demyelinating diseases that may be alleviated by the therapeutic approaches provided in include blurred (or double) vision, ataxia, clonus, dysarthria, fatigue, clumsiness, hand paralysis, hemiparesis, genital anaesthesia, incoordination, paresthesias, ocular paralysis, impaired muscle coordination, weakness (muscle) , loss of sensation, impaired vision,

neurological symptoms, unsteady gait, spastic paraparesis, incontinence, hearing problems, speech problems, diminished memory, dizziness, poorly controlled blood pressure, racing heart beat or palpitations and pain. Other demyelinating conditions and associated risk factors

Demyelination also occurs in toxic leukoencephalopathy which can be caused by smoking heroin (60) . Morphine is a TRPAl agonist and sensitiser (62) and can sensitise TRPAl subunits to other TRPAl agonists in situ when it is applied at concentrations as low as 1 μΜ (Kumazawa et al . , 1989) The peak saliva

concentrations of heroin after smoking a (modest) dose of 10.5mg heroin base, are from 9.6 to 56 micromolar (Jenkins et al . , 1995) . Hence, in some embodiments of this invention, the demyelinating disease is a leukoencephalopathy, such as toxic

leukoencephalopathy. In some embodiments, the toxic

leukoencephalopathy is caused by smoking heroin. Pharmaceutical compositions

The TRPAl antagonists of the present invention may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient . A pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical

composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the TRPAl antagonist, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in

solution. These pharmaceutically acceptable vehicles are well known and will be adapted by the person skilled in the art as a function of the mode of administration of the TRPAl antagonist.

TRPAl antagonists will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the TRPAl antagonist. Thus

pharmaceutical compositions may comprise, in addition to the TRPAl antagonist, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the TRPAl antagonist. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.

In some embodiments, the pharmaceutical preparation will exclude water and/or exclude glycerol and/or exclude sodium azide and/or exclude bovine serum albumin (BSA) and/or exclude phosphates and/or exclude surfactants.

For intra-venous administration, e.g. by injection, the

pharmaceutical composition comprising the TRPAl antagonist may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids;

antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol ; 3'-pentanol; and m- cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine;

monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt- forming counter-ions, such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG) .

A pharmaceutical composition comprising a TRPAl antagonist may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the

condition to be treated.

A TRPAl antagonist as described herein may be used in a method of treatment of the human or animal body, including prophylactic treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset) . The method of treatment may comprise administering a TRPAl antagonist to an individual in need thereof.

Treatment & Prophylaxis

The term "treatment, " as used herein in the context of treating a condition, pertains generally to treatment and therapy of a human, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a

prophylactic measure (i.e., prophylaxis, prevention) is also included . The term "therapeutically-effective amount," as used herein, pertains to that amount of a compound of the invention, or a material, composition or dosage from comprising said compound, which is effective for producing some desired therapeutic effe commensurate with a reasonable benefit/risk ratio, when

administered in accordance with a desired treatment regimen.

Similarly, the term "prophylactically effective amount," as used herein pertains to that amount of a compound of the invention, or a material, composition or dosage from comprising said compound, which is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

"Prophylaxis" in the context of the present specification should not be understood to circumscribe complete success i.e. complete protection or complete prevention. Rather prophylaxis in the present context refers to a measure which is administered in advance of detection of a symptomatic condition with the aim of preserving health by helping to delay, mitigate or avoid that particular condition.

The term "treatment" includes combination treatments and

therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.

For example, as described above, it may be beneficial to combine treatment with a compound as described herein with one or more other (e.g., 1, 2, 3, 4) agents or therapies.

The particular combination would be at the discretion of the physician who would select dosages using his/her common general knowledge and dosing regimens known to a skilled practitioner.

The agents (i.e., a compound as described herein, plus one or more other agents) may be administered simultaneously or

sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required) , the precise dosage regimen being commensurate with the properties of the therapeutic agent ( s ) .

The agents (i.e., a compound as described here, plus one or more other agents) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit,

optionally with instructions for their use. Dosages

Administration is normally in a "therapeutically effective amount", this being sufficient to show benefit to a patient.

Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time- course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of

treatment, e.g. decisions on dosage etc, is within the

responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated.

In the methods of the invention, the dose regimen of the

compounds is preferably such as to achieve a therapeutic plasma concentration. The therapeutic plasma concentration varies by TRPA1 agonist and antagonist and is estimated first by its in vitro EC50 or IC50 and later by its bioavailability, permeability across the blood-brain barrier, body clearance and unbound fraction, and resulting efficacy in the plasma. The known IC50 of some TRPAl antagonists at human TRPAl are HC 030031 (IC50 : 6.2μΜ) , AP18 (IC50 : 3. ΙμΜ) , Jannssen Compound 43 (IC50 : 0.013μΜ) , Amgen Compound 10 (IC50 : 0.17μΜ) , Abbott A-967079 ( IC50 : 0.067μΜ) ,

Novartis Compound 31 ( IC50 : 0.015μΜ) .

Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or clinician.

In general, a suitable dose of the compound may be in the range of about 1 mg to about 200 mg per kilogram body weight of the subject per day e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, etc. mg, but this will vary according to the compound used.

The dosing may be split into loading and maintenance doses.

Optionally a loading dose is between and 5 and 500 mg, e.g.

between 10 and490, 100 and 400, or 200 and 300 mg/kg/day.

Optionally a maintenance dose is between and 1 and 400 mg/kg/day. Preferably a maximum daily maintenance dose is less than 1000 mg/day.

Thus in one embodiment, to achieve a therapeutic concentration, a TRPAl antagonist may be loaded using a dose divided into three equal doses given once daily on 3 consecutive days to achieve a therapeutic drug concentration gradually over the 3 days. For example, a loading dose of about 15 mg/kg (which may be rounded up to the nearest 100 mg) can be used. This could be followed by a daily maintenance dose of a lesser amount once daily. For example, a maintenance dose of about 4 mg/kg (which may be rounded up to the nearest 50 mg, with a maximum of 300 mg) can be used.

In some embodiments, the compound is administered to a human patient according to the approved dosage regime for that drug, but which could be about 50mg, 3 times daily.

In some embodiments, the compound is administered to a human patient according to the following dosage regime: about 100 mg, times daily.

In some embodiments, the compound is administered to a human patient according to the following dosage regime: about 150 mg, times daily.

In some embodiments, the compound is administered to a human patient according to the following dosage regime: about 200 mg, times daily.

Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. TRPA1 antagonists may be delivered orally, through nasal inhalation or by injection. Treatments may be every two to four weeks for subcutaneous administration and every four to eight weeks for intra-venous administration. Treatment may be periodic, and the period between administrations is about two weeks or more, e.g. about three weeks or more, about four weeks or more, or about once a month. Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above .

Examples The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and

description of how to practise the invention, and are not intended to limit the scope of the invention.

EXAMPLE 1: Proton-gated Ca2+-permeable TRP channels mediate myelin damage in ischaemia

The myelin sheaths wrapped around axons by oligodendrocytes are crucial for brain function. Surprisingly, we now show that NMDA

(previously thought to be a mediator of calcium entry into oligodendrocytes) does not raise intracellular Ca2+ in mature oligodendrocytes and that, although ischaemia evokes a glutamate- triggered membrane current (4), this is generated by a rise of extracellular K+ and a decrease of membrane K+ conductance.

Nevertheless, ischaemia raises oligodendrocyte intracellular

[Ca2+] , intracellular [Mg2+] and intracellular [H+] , and

buffering intracellular pH reduces the increases of intracellular

[Ca2+] and intracellular [Mg2+] showing that these are evoked by the rise of intracellular [H+] . The H+-gated intracellular

[Ca2+] elevation is mediated by channels with characteristics of TRPA1, being inhibited by ruthenium red, isopentenyl

pyrophosphate, HC-030031, A967079 or TRPAl-knockout . TRPA1 block reduces myelin damage in ischaemia. These data suggest TRPA1- containing ion channels as a therapeutic target in white matter ischaemia. Furthermore, these channels are known to be activated by a large number of compounds, many of which are factors associated with increased incidence of MS.

Ischaemia blocks action potential propagation through myelinated axons (1) . This might reflect a rundown of ion gradients across the axonal membrane, but impulse propagation only partly recovers on readmitting oxygen and glucose to the tissue to restore ion pumping (1) . Electron microscopy (2) and imaging of dye-filled oligodendrocytes3 show ischaemia-evoked Ca2+-dependent damage to the capacitance-reducing myelin sheaths, suggesting that

irreversible myelin damage underlies much of the loss of action potential propagation. Glutamate receptor block reduces myelin damage and loss of action potential propagation (2) -(7), and glutamate evokes a membrane current in oligodendrocytes mediated by AMPA/kainate and NMDA receptors (4), which have been

identified on oligodendrocytes using immunocytochemistry and electron microscopy (2) -(4) . Thus, the damage occurring to oligodendrocytes is thought to be excitotoxic, resembling that occurring to neurons in ischaemia: a rise of extracellular glutamate concentration (8) caused by reversal of glutamate uptake carriers in oligodendrocytes and axons (9), (10) activates ionotropic receptors that raise (2) oligodendrocyte intracellular Ca2+ and thus damage the cells.

However, although AMPA/KA and NMDA receptors on oligodendrocyte precursors regulate their proliferation and myelination (11),

(12) , these receptors are down-regulated as the cells mature

(13) -(15) .

To test whether oligodendrocyte intracellular Ca2+ ions ([Ca2+]i) is raised by glutamate receptor activation in ischaemia, we characterised membrane current and [Ca2+]i changes evoked in cerebellar white matter oligodendrocytes by ischaemia.

Surprisingly, although ischaemia evokes a glutamate- triggered inward membrane current in oligodendrocytes, this reflects an alteration of [K+] fluxes, while oligodendrocyte [Ca2+]i is raised as a result of an ischaemia-evoked rise of intracellular hydrogen ions ([H+]i) in the oligodendrocyte that gates a TRPA1 channel-mediated Ca2+ influx.

Solution mimicking ischaemia (see ^Methods', below) evoked a slowly increasing inward current in oligodendrocytes (Fig. la, b) , that often had a sharper increase superimposed upon it. The timing of this sharper increase varied between cells, so that when responses in many cells were averaged (Fig. lc) it was not visible as a separate entity. The peak inward current evoked by ischaemia (and its waveform) was not significantly different with Cs+ or K+ as the main pipette cation (209+10 pA, n=179, and 191_+41 pA, n=10, respectively, p=0.67) . When applied from before the onset of ischaemia, NBQX and D-AP5 reduced the ischaemia- evoked current by 66% (after 8-10 mins ischaemia: Fig. lc, d) , while mGluR block had no significant effect (Fig. 6a) . Preloading for 30 mins with the glutamate transport blocker PDC, to prevent ischaemia-evoked glutamate release by reversal of glutamate transporters in the white (9) and grey (16) matter, also greatly reduced the inward current (by 68%, Fig. lc, d) , while blocking other candidate release mechanisms had no significant effect (see Fig. 6a) . Thus, glutamate release by reversed uptake plays a significant role in triggering the ischaemia-evoked current.

Strikingly, however, current flow through glutamate receptors is responsible for only a small fraction of the sustained inward current evoked by ischaemia, since when we applied NBQX and D-AP5 from 200 sec after ischaemia had started there was only a nonsignificant 21% suppression of the ischaemia-evoked inward current (Fig. Id) .

An ischaemia-evoked inward current which is triggered by

glutamate release but then maintained by non-glutamatergic mechanisms is reminiscent of the "Extended Neuronal

Depolarization" (END) that is triggered in neurons by glutamate and contributes to neuronal death (17) . However, the ischaemia- evoked current in oligodendrocytes was not prevented by removal of external Ca2+ and chelation of trace Ca2+ with 50 μΜ EGTA, or by gadolinium (100 μΜ) , which both block the END17 (Fig. Id, e) , indicating that a different mechanism maintains the inward current triggered by glutamate.

Emphasising the difference from neurons, where ischaemia evokes a conductance increase mediated largely by ionotropic glutamate receptors (16), ischaemia decreased the conductance of

oligodendrocytes (Fig. If, g; decreased by 2.110.7 nS near -70 mV in 11 cells using 10 mM, and 2.310.6 nS in 10 cells using 0.5 mM, internal HEPES; see below for results with higher pH buffering) . The suppressed conductance had a reversal potential more negative than the K+ reversal potential (EK=-104 mV) , being -121 mV with 10 mM HEPES (Fig. If, Fig. 6e) and -118 mV with 0.5 mM HEPES (Fig. lg) . This I-V relation is expected if ischaemia decreases the membrane K+ conductance, at the same time as extracellular [K+] ([K+]o) rises (due to Na+/K+ pump inhibition throughout the slice) which shifts EK positive and increases the inward current at all potentials (see Fig. 6b-d and discussed below) . Using insensitive electrodes, we found that [K+]o in the white matter rose slowly initially during ischaemia, but then increased more abruptly (Fig. lh) . The peak rise was 2.3510.13 mM in the white matter (n=12) and 2.4810.35 mM in the adjacent grey matter (n=4) . The [K+]o rise in the grey matter reflects the anoxic

depolarization of neuronsl8. The [K+]o rise in the white matter paralleled the ischaemia-evoked current in oligodendrocytes (Fig. lh) . We estimate that the conductance decrease described above produced 32%, while the [K+]o rise produced 68% of the inward current in oligodendrocytes at -74mV (discussed below) . Thus, changes in K+ fluxes generate the ischaemia-evoked inward current . Part of the NMDA-evoked inward current in oligodendrocytes (4) could also reflect a [K+]o rise. Extracellular Cs+ greatly reduced the NMDA-evoked current while intracellular MK-801 had no effect (Fig. 7a-d) , suggesting that the majority of the NMDA- evoked current is generated by a rise of [K+]o rather than by oligodendrocyte NMDA receptors. By applying NMDA or raised [K+]o solution, and correlating the resulting inward current with the [K+]o rise occurring (see discussion in "A [K+]o rise and K+ influx mediate much of the response to NMDA" below and Fig. 7e- f) , we found that at least 49% of the NMDA-evoked inward current was attributable to the [K+]o rise that it produced. Since mature oligodendrocytes express few NMDA receptors (13) -(15), this presumably reflects NMDA depolarizing neurons or astrocytes in the slice and releasing K+ . The data above challenge the idea (2) -(5) that, during ischaemia, activation of NMDA receptors in mature oligodendrocytes generates a prolonged calcium influx which damages the cells excitotoxically . We therefore investigated the ion concentration changes evoked in oligodendrocytes by activation of NMDA

receptors, using Ca2+-, Na+- and K+-sensitive dyes (Fura-2, SBFI and PBFI respectively) loaded into cells from the patch pipette. When 100 μΜ NMDA was applied to whole-cell clamped cerebellar granule neurons at -74 mV, as expected it evoked an inward current at the resting potential (Fig. 8), and raised [Ca2+] i and

[Na+]i (and also [K+]i, reflecting K+ entry: [K+]pipette was 32.5 mM, so EK>-60mV for [K+]o >3.3 mM; Fig. 8) . In contrast, although oligodendrocytes generated an inward current in response to NMDA, this was associated with no [Ca2+] i or [Na+] i elevation, in fact

[Na+]i decreased substantially after applying NMDA (Fig. 2a) . NMDA raised [K+] i however. Similar ion concentration changes were seen at the soma (Fig. 2a) and in the cells' internodal processes where NMDA receptors are reported to be located (2) -(4)

(Fig. 2b) . Like NMDA, raising [K+]o lowered [Na+]i (Fig. 2a, b) . A likely explanation is that NMDA raises [K+]o (Fig. 2c), which leads to a decrease of [Na+]i via activation of the Na+/K+ pump. The absence of a rise of [Ca2+]i and [Na+]i is surprising if oligodendrocytes express NMDA receptors2-4. We considered the possibility that NMDA receptors might pass ions into a restricted compartment in their myelinating processes, which only certain Ca2+-sensing dyes such as X-Rhod-1 can access, as has been suggested2. However, whether X-Rhod-1 was loaded into the cell as an acetoxymethyl ester2 or from the pipette, we observed no change of [Ca2+] i in the myelinating processes in response to NMDA application (Fig. 2d) . Nevertheless, we could detect rises of oligodendrocyte [Ca2+]i when they did occur, since spontaneous [Ca2+]i rises propagating through myelinating processes were seen in 55% of cells (Fig. 2e) .

To confirm that ischaemia does indeed raise2 oligodendrocyte

[Ca2+]i, we loaded the Ca2+-sensing dye Fluo-4 (together with a reference dye, Alexa Fluor 594, to allow ratiometric imaging) into the cells from a whole-cell pipette (these experiments were carried out in current-clamp mode to allow the oligodendrocyte voltage to change, in case activation of voltage-gated Ca2+ channels contributes to the ischaemic [Ca2+] rise) . Ischaemia increased [Ca2+] i in the oligodendrocyte soma and processes over a period of 10 mins. This rise was abolished if calcium was omitted from the extracellular solution (Fig. 3a, b) , and reduced in the absence of external K+ (Fig. 3c), suggesting that the ischaemia-evoked rise of [K+]o might contribute to evoking calcium entry from the extracellular solution. However, unlike in the earlier report (2), blocking NMDA receptors with MK-801, D- AP5 and 7-chloro-kynurenate, or blocking NMDA and AMPA/KA receptors with NBQX and D-AP5 while also blocking voltage-gated Na+ and Ca+ channels and GABAA receptors, did not prevent the [Ca2+] i rise (Fig. 3a, b) . Similarly, when PDC-preloading was used to reduce glutamate release by transporter reversal, the [Ca2+] rise was not significantly affected (Fig. 3c) .

Similar experiments using the Mg2+-sensitive dye Mag-Fluo-4 revealed that [Mg2+]i also rises in ischaemia (Fig. 3d) . We hypothesised that this might be due to ATP breakdown in

ischaemia, which releases Mg2+ (although ATP was present in the pipette) , but the [Mg2+] i rise was abolished in the absence of extracellular Mg2+ (Fig. 3d) implying that Mg2+ enters across the cell membrane. In contrast, surprisingly, ischaemia did not raise [Na+]i (Fig. 3e) . Thus ischaemia activates a membrane conductance that allows the entry of divalent ions.

In searching for an agent that might decrease membrane K+ conductance and activate a Ca2+-permeable pathway, we measured the pH change evoked by ischaemia in oligodendrocytes using the pH-sensitive dye BCECF. Ischaemia increased [H+] i over a

timescale similar to that on which [Ca2+]i increases (Fig. 3f) . A similar (but smaller) [H+] i rise was evoked by superfusing solution containing elevated [K+]o or NMDA (Fig. 9a) suggesting that the ischaemia-evoked [K+]o rise may partly generate this pH change. To investigate this further, we removed K+ from the superfusion solution, which reduced the [K+]o in the slice from 2.46+0.02 mM (n=13) to 0.99+0.3 mM (n=4) (p=0.001, Mann-Whitney test, Fig. 3g) , and hyperpolarized the resting potential by 7 mV (-84.0+4.7 versus -77.2+4.3 mV, n=4, p=0.002, paired t-test, Fig. 3g) . The [K+]o rise and depolarization evoked by ischaemia were unaffected (Fig. 3g) , but the initial [H+] i and the [H+] i reached during ischaemia were reduced by K+ removal (Fig. 3h) . These data and those in Fig. 3c suggest that the ischaemia-evoked [K+]o rise helps to acidify the cell, which in turn evokes Ca2+ entry.

When cells were clamped with an internal solution containing a high (50 mM) HEPES concentration, the ischaemia-evoked rise of [H+] i was, as expected, greatly reduced (Fig. 3f) . This prevented the decrease of K+ conductance (Figs, lg, 3i), consistent with intracellular acidity suppressing the activity of tonically- active K+ channels (19) . Strikingly, however, buffering the change of intracellular pH also prevented the ischaemia-evoked rise of [Ca2+]i and [Mg2+]i (Fig. 3j , k) , implying that the rise of [H+]i produced by ischaemia activates a pathway allowing these cations to enter the cell. Consistent with this, uncaging of protons in oligodendrocytes evoked a rise of [Ca2+]i and [Mg2+]i in the cell (Fig. 31, m) .

Few channels allow entry of Ca2+ and Mg2+ well compared to Na+, but many of the TRP channel family share this property (20), and TRP channel activation may contribute to ischaemic damage to neurons (21) and astrocytes (22) . Of these channels, only (20), (23) , (24) TRPA1 and TRPV3 are known to be activated by

intracellular H+, so we applied agonists and antagonists of these channels and examined the effect on oligodendrocyte [Ca2+] i (specificity of the TRP agonists and blockers used is discussed below) . The TRPA1 /TRPV3 blocker isopentenyl pyrophosphate (25) (IPP) and the specific26 TRPA1 blocker HC-030031 slowed and reduced the [Ca2+]i rise evoked by uncaging H+ in the cell (Fig. 31) . The TRPA1 /TRPV3 agonists (20) , (27) menthol, vanillin, carvacrol (Cv) and 2-APB all evoked [Ca2+] i rises in

oligodendrocyte somata and myelinating processes, as did the TRPA1 agonists (see ref. (27) and discussed below) AITC,

polygodial and flufenamic acid, while the TRPV3 agonists (20), (27) camphor and farnesyl pyrophosphate (FPP) did not (Fig. 4a, b) , suggesting that TRPAl subunit-including channels contribute to these responses, but that TRPV3 channels are not needed. The carvacrol-evoked rise of [Ca2+]i was reduced by HC-030031 which blocks TRPAl but not TRPV3(26), by the TRPAl /TRPV3 blocker isopentenyl pyrophosphate25 (IPP) , and by TRPAl knock-out (Fig 4b), again implying the involvement of TRPAl channels. It was unaffected by buffering [H+]i (Fig. 4b), consistent with Ca2+ entry via TRPAl being downstream of the [H+] i rise, as seen with H+ uncaging (Fig. 31) .

TRPAl and TRPV3 antibodies appeared to label the myelinating processes and somata of oligodendrocytes in rat, but the

labelling was not significantly different in wild-type mice and mice with TRPAl or TRPV3 knocked-out (data not shown) . We therefore turned to in situ hybridization. A TRPAl probe labelled the cerebellar white matter in both rat and mouse, while a TRPV3 probe labelled rat only (Fig. 10a) . Simultaneous

immunocytochemistry revealed that TRPAl and TRPV3-expressing cells included myelinating oligodendrocytes expressing both 01ig2 and CC1 (Fig. lOb-d) .

Consistent with ischaemia raising [Ca2+] i mainly through

activation of TRPAl rather than TRPV3-containing channels, the general TRP blockers (20), (27) ruthenium red (RuR, 10 μΜ) and La3+ (1 mM) and the TRPAl /TRPV3 blocker25 IPP (200 μΜ) all reduced the rise of [Ca2+]i, as did HC-030031 and A967079, which block TRPAl but not TRPV3 (26) , (28) (Fig. 4c-e) . Knock-out of TRPAl slowed and halved the ischaemia-evoked [Ca2+]i rise, and the TRPA1/TRPV3 blocker IPP produced no further reduction in the knock-out (suggesting no contribution of TRPV3 : Fig. 4f) , while knock-out of TRPV3 had no effect on the [Ca2+] i rise, and applying the TRPAl blocker HC-030031 slowed and halved the rise occurring in the TRPV3 knock-out (Fig 4g) . Blockers of many other TRP channels had no effect (Fig. 11) . These data identify TRPAl as the dominant contributor to the ischaemia-evoked rise of

[Ca2+]i in oligodendrocytes. Nevertheless, the larger (70%) block of the [Ca2+] i rise by the TRPAl blocker HC-030031 than by TRPAl knock-out (50%: Figs. 4e-f) suggests that there may be compensatory upregulation of another Ca2+ entry pathway in the TRPAl knock-out, which normally generates only -30% of the [Ca2+]i rise. Introducing high pH- buffering power solution into the cell essentially abolished the [Ca2+]i rise evoked by ischaemia in the TRPAl knock-out (Fig. 4h) , implying that the residual non-TRPAl Ca2+ entry pathway is also H+-activated. Since the non-specific TRP blockers RuR and La3+ completely abolished the ischaemia-evoked [Ca2+]i rise, these data suggest the presence of another Ca2+-permeable TRP channel (other than TRPAl and TRPV3) which is activated by internal H+ in oligodendrocytes and generates -30% of the ischaemia-evoked [Ca2+]i rise.

To assess the role of TRPAl-containing channels in evoking myelin damage, we exposed rat optic nerves to 60 mins ischaemia. This led to disruption of the myelin sheath (2), (3), which we quantified by counting the number of regions of myelin

decompaction (lamellar separation) per axon cross section (see Methods) . Taking as baseline the level of decompaction which occurs even in control nerves during processing for EM, ischaemia significantly increased decompaction (p=3xl0-8), and ruthenium red or the TRPAl blockers HC-030031 and A967079 (applied

together) reduced this increase by 69% (p=3xl0-5) and 59%

(p=2.0x10-5) respectively (Fig. 4i, j) . Ischaemia did not affect the g ratio of the axons (Fig. 4k, interpolated over

decompactions: see Methods), but increased axon diameter due to swelling (Fig. 41) . It also caused some axon vacuolization (Fig. 4m) : vacuoles were seen in only 4.3% of 2390 control axons, but in 21% of 15976 axons after ischaemia. Vacuolization was not prevented by TRP channel block (Fig. 4m) , suggesting different mechanisms for axon and myelin damage in ischaemia. Our data show that ischaemic damage to oligodendrocytes differs fundamentally from that occurring to neurons (Fig. 12), where [Ca2+] i is raised initially by glutamate- gated receptors and in the longer term by a TRP channel activated by reactive oxygen species21. Contrary to current ideas (2) -(4), ischaemia does not damage oligodendrocytes by activating a Ca2+ influx through ionotropic glutamate receptors in their membranes. Instead, sodium pump inhibition and glutamate release in ischaemia evoke a long-lasting rise of [K+]o that, together with metabolic changes, acidifies the oligodendrocyte cytoplasm and leads to activation of H+-gated TRP channels through which Ca2+ and Mg2+ enter.

In the optic nerve, ischaemia-evoked Ca2+ entry into

oligodendrocytes is reported to be blocked by NMDA receptor antagonists (2), which contradicts our demonstration that NMDA evokes no [Ca2+]i rise in oligodendrocytes (Fig. 2) and that the ischaemia-evoked [Ca2+]i rise is unaffected by NMDA receptor blockers (Fig. 3) . Conceivably, in the optic nerve, NMDA

receptors on astrocytes29 make a greater contribution than in cerebellum to generating the ischaemia-evoked rise of [K+]o and thus the [Ca2+]i rise.

TRPA1 generates -70% of the ischaemia-evoked [Ca2+]i rise, and TRPA1 blockers reduce ischaemic damage to myelin (Fig. 4) . These results suggest block of oligodendrocyte TRPAl-containing channels as a useful strategy for reducing myelin loss during the energy deprivation that follows stroke, secondary ischaemia caused by spinal cord injury or hypoxia associated with multiple sclerosis (30 ) .

Methods Summary

Oligodendrocytes in cerebellar slices were patch-clamped, labelled using antibodies and in situ hybridization, and had their ion concentrations and morphology imaged, as described in the Methods.

Methods

Animals

Experiments used Sprague-Dawley rats or transgenic mice of either sex. Animal procedures were carried out in accordance with the guidelines of the UK Animals (Scientific Procedures) Act 1986 and subsequent amendments. TRPV3 knock-out (KO) mice were obtained from JAX (http://jaxmice.jax.org/strain/010773.html) . TRPA1 KO mice were obtained as a double knock-out with TRPV1 knocked out (kindly provided by John Wood and Jane Sexton) . TRPV1 does not contribute to the ischaemia-evoked [Ca2+]i rise described here because the TRPV1 antagonist20 , 27 capsazepine did not reduce the ischaemia-evoked [Ca2+]I rise in rat oligodendrocytes (Fig. 11) and the TRPV1 agonists20 , 27 capsaicin (10 μΜ) and camphor (2 mM) did not evoke a [Ca2+]i rise (see Specificity of drugs acting on TRP channels and Fig. 4a) . Wild type and (double) KO mice were obtained from a colony obtained by breeding mice doubly

heterozygous for the TRPA1 and TRPV1 knock-outs. The wild-type and KO mice compared share the same doubly heterozygous

grandparents.

Brain slice preparation

Cerebellar slices (225 μιτι thick) were prepared from the

cerebellum of P12 rats in ice-cold solution containing (mM) 124 NaCl, 26 NaHC03, 1 NaH2P04, 2.5 KC1, 2 MgC12, 2-2.5 CaC12, 10 glucose, bubbled with 95% 02/5% C02, pH 7.4, as well as 1 mM Na- kynurenate to block glutamate receptors. Slices were then incubated at room temperature (21- 24 °C) in the same solution until used in experiments. Cerebellar slices from P10-17 mice were prepared in ice-cold solution containing (mM) 87 NaCl, 25

NaHC03, 1.25 NaH2P04, 2.5 KC1, 7 MgC12, 0.5 CaC12, 25 glucose, 75 sucrose, 1 Na-kynurenate and then transferred to the same solution at 27 °C and allowed to cool naturally to room

temperature. Only 1 cell was recorded from in each slice.

Cell identification and electrophysiology

Oligodendrocytes, cerebellar granule cells and hippocampal pyramidal cells were identified by their location and morphology. All cells were whole-cell clamped with pipettes with a series resistance of 8-30 ΜΩ. Electrode junction potentials were compensated. I-V relations were from responses to 200 msec voltage steps. Unless otherwise indicated, cells were voltage- clamped at -74 mV.

External solutions

Slices were superfused with either bicarbonate-buffered solution containing (mM) 124 NaCl, 2.5 KC1, 26 NaHC03, 1 NaH2P04, 2-2.5 CaC12, 1 MgC12, 10 glucose, pH 7.4, bubbled with 95% 02 and 5% C02, or with HEPES-buffered solution containing (mM) 144 NaCl, 2.5 KC1, 10 HEPES, 1 NaH2P04, 2-2.5 CaC12, 1 MgC12, 10 glucose, pH set to 7.3 with NaOH, bubbled with 100% 02. During experiments when NMDA was applied and ion concentration changes were observed with ion sensitive dyes, MgC12 was omitted from the solution to minimize the Mg2+ block. For experiments involving Gd3+ and La3+, the HEPES- based solution was used and NaH2P04 was omitted. To simulate ischaemia we replaced external 02 with N2 , and external glucose with 7 mM sucrose, added 2 mM iodoacetate to block glycolysis, and 25 μΜ antimycin to block oxidative

phosphorylation (31), (4) . All ischaemia experiments were done at 33-36°C, while applications of NMDA and of TRP channel agonists were at 24oC. Control and drug conditions were interleaved where appropriate .

Intracellular solutions

Cells were whole-cell clamped with electrodes containing either Cs- (to improve voltage uniformity) or K-gluconate-based

solution, comprising (mM) 130 Cs-gluconate (or K- gluconate) , 2 NaCl, 0.5 CaC12, 10 HEPES, 10 BAPTA, 2 NaATP, 0.5 Na2GTP, 2 MgCl, 0.5

K-Lucifer yellow, pH set to 7.2 with Cs- or K-OH (all from

Sigma) . The K+-based solution was used for current-clamp

experiments. For Ca2+ imaging experiments, BAPTA was decreased to 0.01 mM and replaced with 10 mM phosphocreatine, added CaC12 was reduced to 10 μΜ, and Lucifer Yellow was replaced with 1 mM Fura- 2, or 200 μΜ Fluo-4 with 50 μΜ Alexa Fluor 594, or 200 μΜ X-Rhod- 1 with 50 μΜ Alexa Fluor 488 (all from Molecular Probes) to allow ratiometric imaging. For imaging pH, Lucifer yellow was replaced with BCECF (96 μΜ) and the HEPES concentration was decreased to 0.5 mM. This [HEPES] was also used for control experiments when examining the effect of 50 mM internal [HEPES] on the ischaemia- evoked current; ischaemia-evoked membrane current changes were indistinguishable when 0.5 and 10 mM HEPES were used (see main text) , presumably because endogenous pH buffering dominates at these low [HEPES] levels. For experiments where the pH-buffering capacity of the internal solution was increased, 68 mM K- gluconate and 50 mM HEPES were used. When uncaging protons, 2 mM 1- (2-nitrophenyl ) ethyl sulphate sodium salt (NPE-caged protons, Tocris) was added to the pipette solution and 10 mM HEPES was replaced by 30 mM Tris to prevent UV light-mediated oxidation (32) of HEPES (and K-gluconate was reduced from 130 to 120 mM) . For Na+ and K+ imaging experiments, Lucifer yellow was replaced with 1 mM of the Na+-sensing dye SBFI tetra- ammonium salt or of the K+-sensing dye PBFI tetra-ammonium salt (Molecular Probes) . In some experiments MK-801 (1 mM) was added to the internal solution to block NMDA receptors, and cells were depolarised to 10 mV for 10 sec intermittently over a 20 min waiting period to facilitate MK-801 block of open channels.

Single cell ion imaging and H+ uncaging

For Fura-2, SBFI and PBFI imaging when applying NMDA or during ischaemia, white matter oligodendrocytes and grey matter granule cells were patch-clamped with pipettes containing a solution as described above, fluorescence was excited sequentially at 340110 nm and 380110 nm, and emitted light was collected at 510120 nm. The ratio (R) of the emission intensities (340 nm/380 nm) , after subtraction of the background intensity averaged over 4 distant areas of the image, was used as a measure of intracellular ion concentration. Increases of ion concentration generated a fall o fluorescence (F) excited at 380 nm and a rise in fluorescence excited at 340 nm, which is plotted as 0R/R in the graphs shown, with R = F340nm/F380nm: an upward deflection corresponds to a rise of concentration of the sensed ion. For Fura-2, SBFI and PBFI, mean values of R before applying NMDA or ischaemia solution were 0.41+0.05 (n=5), 1.68+0.14 (n=13) and 1.86+1.10 (n=8) respectively. Fluo-4 and Alexa Fluor 594 were used in the internal solution to measure [Ca2+] i changes ratiometrically during H+-uncaging and most ischaemia experiments. To measure [Mg2+]i Mag-Fluo-4 was used instead of Fluo-4. Fluo-4 (or Mag-Fluo-4) and Alexa Fluor 594 fluorescence were excited sequentially every 2, 10 or 30 seconds at 488±10nm and 585±10nm, and emission was collected using a triband filter cube (DAPI /FITC/Texas Red, 69002,

Chroma) . The mean ratio of intensities (F488nm/F585nm) before applying NMDA or

ischaemia was 0.81+0.09 (n=16) for Fluo-4 and 0.55+0.04 (n=6) for Mag-Fluo-4. Caged-H+ were uncaged using 380120 nm light for 1 second every 2 seconds (repeated 30 times) interspersed with the above excitation wavelengths. BCECF was imaged every 30 seconds at 400 and 480nm, with emission collected using the above triband filter. The ratio (R) of the emitted light excited by these two wavelengths (F480nm/F400nm) was used as a measure of [H+]i (mean value before ischaemia was 17.6+0.9, n=8) but, since this ratio decreases with increasing [H+]i, when plotting changes in dR/R in Fig. 3e and Fig. 9 we multiplied them by -1 to produce a trace that increased with [H+]i.

During ischaemia, slices swelled at the time of the anoxic depolarisation . When cells were patch-clamped with calcium dyes, the resulting movement of the cell away from the electrode sometimes caused [Ca2+]i oscillations within the cells. These oscillations did not occur if the patch pipette was removed (after 2 minutes to allow dye-filling) before the ischaemic solution was applied. Without the pipette attached to the cell, the time-course of the ischaemia-evoked [Ca2+] i rise was the same as with the electrode attached, but its amplitude was 69% larger (ratio increase 0.21+0.03, n=20 versus 0.12+0.02, n=16) . In some experiments (those in Fig. 4c-g and Fig. 11) we therefore removed the pipette for calcium-imaging. Control experiments were carried out to check whether the ischaemia-evoked change of pH would affect our [Ca2+]i

measurements. The internal solution for Ca2+-sensing was studied in the experimental bath that the slices usually are placed in. The resting ratio of Fluo-4 fluorescence to Alexa 594

fluorescence was not significantly affected by altering the pH of the solution from 7.05 to 6.55, and this also did not affect the change of ratio produced by adding 200 nM Ca2+ to the sensing solution (Fig. 9b, c) . Thus, even a 0.5 unit pH change occurring in the oligodendrocyte would not significantly affect the calcium dye measurements .

AM dye loading

X-Rhod-l-AM (38 μΜ) dye loading with the myelin marker DIOC6 into P12 cerebellar slices was performed as described previously for optic nerves2. Loading times ranged from 1-2 hrs and a de- esterification period of 30 mins at 36°C was allowed before imaging .

Potassium electrodes

Potassium electrodes were made as described33. Electrodes were pulled with a resistance of 4-10 M . Electrode tips were

silanised by heating them to 250 °C for 7 minutes whilst N2 and N, -dimethyltrimethylsilylamine (Fluka) were gassed into the tip from the back of the electrode. The tip was then filled with either 6% valinomycin, 1.5% potassium tetrakis(4- chlorophenyl ) borate (Fluka) and 92.5% 1 , 2-dimethyl-3-nitrobenzene (Fluka) or the pre-made potassium sensitive ionophore I - cocktail B (Fluka) . The electrodes were back- filled with the bicarbonate-buffered external solution mentioned above (2.5 mM K+) , and attached to a sensitive high resistance

electrometer (Model FD 223, World Precision Instruments) . A reference electrode tip was placed less than 5 μιτι away from the K+ electrode tip, and the voltage changes measured by it were subtracted from those measured with the K+ electrode. [K+]o was determined by calibrating each electrode at the end of every experiment with at least 3 different K+ concentrations (1, 2.5, 5, 7.5, 10 or 17.5 mM) . To check for cross-reactivity, the

[NaCl]o was decreased by 60 mM which led to a -2.210.1 mV change in voltage (n=3) , while a pH change from 7.3 to 6.5 led to a 0.4710.22 mV change (n=3) . Both of these changes are much less than the 17.510.5 mV change (n=18) seen in response to an increase of [K+]o from 2.5 to 5 mM (which is consistent with the electrodes used having an average calibration slope of 60.910.9 mV (n=6) per 10-fold change of [K+]o) . Drugs used

Stock solutions of the following drugs were made up in water: NMDA, AP5, NBQX, MK801, 7-CK, TTX, PDC, IPP, CPG, SKF 96365 and RuR. (S) -MCPG and amiloride were made up in external solution. Carvacrol was made up in ethanol . Bicuculline, bumetanide, HC 030031, A967079, flufenamic acid, capsazepine, FTY720-HC1, 2-APB, AITC, RN1734 and ML204 were made up in DMSO. When used, DMSO and ethanol were also added to control solution at the same

concentrations, and did not evoke [Ca2+] i changes at the

concentrations used. Stocks were kept at -20oC apart from carvacrol, menthol, vanillin, AITC, and RuR, which were made up fresh on each day of use. To minimise evaporation of carvacrol, vanillin and menthol, lids were kept on until the solutions were used. Gd3+ and La3+ were applied (as chloride salts) in

bicarbonate- and phosphate-free solution to avoid chelation by these anions (see External Solutions above) .

Immunohistochemical labelling of oligodendrocytes

Cerebellar slices were fixed for 30 mins in 4% PFA, and incubated for 1 to 6 h in 0.1% Triton X-100, 10% goat serum in phosphate- buffered saline at 21°C, then with primary antibody at 4°C overnight with agitation, and then 2 hours or overnight at 24 °C with secondary antibody. Primary antibodies were: anti-CCl

(mouse, 1:300, Calbiochem OP80 monoclonal) and anti-Olig-2

(rabbit, 1:700, Millipore, AB9610 polyclonal) . Secondary

antibodies were: goat anti-rabbit AlexFluor 488 or 568 (Molecular Probes, 1:1000), donkey anti-rabbit AlexFluor 488 (Millipore, 1:1000), and goat anti-mouse Alexa Fluor 568 (Millipore, 1:1000) . In situ hybridization for T PA1 and TRPV3

Solutions used for in situ hybridization were pretreated with 0.1% DEPC. Animals were perfused with PBS followed by 4% PFA. Brains were post-fixed in 4% PFA overnight at 4°C, cryoprotected in 20% sucrose overnight at 4°C and frozen in Tissue-Tek O.C.T. Sections (20 μιτι) collected onto Superfrost Plus microscope slides (VWR International) were hybridized at 65oC overnight with hybridization buffer [50% v/v deionized formamide (Sigma), 10% w/v dextran sulphate (Fluka) , 0.1 mg/ml yeast tRNA (Roche), lx Denhardt's solution (Sigma) and lx "salts" (200 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 7.5, 5 mM NaH2P04, 5 mM Na2HP04)] containing digoxigenin (DIG) -labelled antisense RNA probe

(1:1000) . Sections were washed with a washing solution (50% v/v formamide, lx SSC, 0.1% Tween 20) three times at 65°C for 30 min, followed by two lx MABT (100 mM maleic

acid, 150 mM NaCl, pH 7.5, 0.1% Tween-20) washes at room

temperature for 30 min each. Sections were subsequently blocked with blocking solution [2% w/v Blocking Reagent (Roche

Diagnostics), 10% v/v heat-inactivated sheep serum (Sigma) in lx MABT] for 1 h at room temperature and incubated with anti-DIG antibody conjugated with alkaline phosphatase (AP) (Roche

Diagnostics, 1:1500 in blocking solution) at 4°C overnight.

Sections were then washed in lx MABT 5 times for 20 min each at room temperature, followed by two 5 min washes in staining buffer (100 mM NaCl, 50 mM MgC12, 100 mM Tris-HCl, pH 9.5, 0.1% Tween- 20) . Development was performed at 37 °C for 24-48 hours overnight with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate in freshly prepared staining solution (50% v/v staining buffer, 25 mM MgC12, 5% w/v polyvinyl alcohol) . Sections were washed in PBS and immunohistochemistry was performed as described above. The plasmids used to generate RNA probes were: IMAGE clone

40129486 for Trpal (linearized with Clal and transcribed with T3 RNA polymerase) and IMAGE clone 40047664 for Trpv3 (linearized with Xhol and transcribed with SP6 RNA polymerase) . In situ hybridization was repeated using at least three animals for each probe . Quantifying myelin decompaction during chemical ischaemia using electron microscopy

For chemical ischaemia experiments, optic nerves were dissected from P28 Sprague- Dawley rats and incubated for 1 hour at 36°C in either control or ischaemic solution with and without the

TRPA1/V3 channel blocker ruthenium red (10 μΜ) or the combined presence of the TRPA1 blockers HC-030031 (80 μΜ) and A967079 (10 μΜ) . The optic nerves were then immersion fixed in 2%

paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer overnight. All samples were then post-fixed in 1% OsO4/0.1 M cacodylate buffer (pH 7.3) at 3°C for 2 hours before washing in 0.1 M cacodylate buffer (pH 7.3) . The samples were dehydrated in a graded ethanol-water series at 3°C and infiltrated with Agar 100 resin mix. The nerve was then cut transversely at the midpoint, blocked out and hardened. Ultra-thin sections were taken, 300 microns from the cut end of the middle of the nerve, on a Reichert Ultracut S microtome. Sections were collected on 300 mesh copper grids and stained with lead citrate. The sections were imaged using a Joel 1010 transition electron microscope and a Gatan Orius camera.

In 3 out of 4 experiments the experimenter was blinded to the drug condition prior to imaging (all 4 experiments gave similar results) . One section was used from each nerve and eight 21.5 μιη x 17.3 μιη images were collected at x8000 magnification, four from the peripheral borders of the nerve at 0°, 90°, 180° and 270° positions on the section, and four covering the central portion of the nerve. In all experiments the image identities were then blinded prior to analysis, and the number of large separations of lamellae (decompaction) was counted. Decompaction was defined as a visible white inter-lamellar gap being present between at least 2 normal lamellae. Regions of decompaction were normally

separated from each other by an area of compact myelin, but when most of the myelin surrounding an axon had separated lamellae, decompacted regions were counted at 0.5 micron intervals around the sheath. The number of decompacted regions was normalised to the number of axons per image. Some decompaction occurred even in control nerves as a result of the processing for electron microscopy, so we assessed drug block of decompaction by

quantifying the ischaemia-evoked increase in decompactions seen without and with the drug present. Myelin g ratios were

calculated as the square root of the ratio of the area of the axon to the area of the axon plus myelin sheath. When drawing lines around the axon and sheath, areas of decompaction were ignored, i.e. we interpolated the lines from regions that were not decompacted. Axon diameter was calculated as (4. (axon area) /n) 0.5. Axon vacuolisation was defined as the inclusion of one or more large (>0.1 μιτι) empty membrane bound (often circular) organelles within the axon or periaxonal space (Fig. 4m) which may reflect rearrangement of internal axonal membranes or be formed from inclusion of myelin membranes into the axon (Fig. 4m) .

Statistics

Data are presented as mean+s.e.m. Experiments were carried out on brain slices from at least 3 animals on at least 3 separate days, except for a few experiments using expensive drugs which were done on only 2 days. Only 1 cell was recorded from in each slice, so the numbers of cells given are also the numbers of slices. P values are from two tailed Student's t-tests (for normally distributed data, assessed using Shapiro-Wilk tests) or Mann-Whitney U tests (for non- normally distributed data) . Normally distributed data were tested for equal variance (p<0.05, unpaired F-test) and homo- or heteroscedastic t-tests were chosen accordingly. P values in the text are from unpaired t-tests unless

otherwise stated. When small sample sizes (n<4) achieved p<0.05, analysis of sample and effect size typically demonstrated a power for detecting the observed effect of 80-99% (mean 92%), with two exceptions: the process data in Fig. 4c (power 78%) and the soma data in Fig. 4h (power 75%) . For multiple comparisons within one experiment

(usually one figure panel, but measurements of [Ca2+]i in somata and processes were treated as separate experiments even when plotted in the same

figure panel) , P values were corrected using a procedure equivalent to the Holm-Bonferroni method (for N comparisons, the most significant p value is multiplied by N, the 2nd most significant by N-l, the 3rd most significant by N-2, etc.; corrected p values are significant if they are less than 0.05) . All statistical analysis was conducted using OriginLab software.

Effect of blocking potential glutamate release mechanisms

In contrast to the suppressive effect of blocking glutamate release by reversed uptake, blocking other candidate mechanisms for glutamate release (from before the onset of ischaemia) did not affect the inward current significantly. Fig. 6a shows that there was no significant effect, when compared with interleaved control cells, on the peak ischaemia-evoked current of

application of: 50 μΜ bumetanide to inhibit NKCC1 transporters and prevent swelling of astrocytes leading to glutamate

release34; 100 μΜ CdC12 to prevent exocytotic glutamate release driven by Ca2+ entry through voltage-gated Ca2+ channels35, 36; 100 μΜ lanthanum (LaC13) to block glutamate release through gap junctional hemichannels37 ; 10 μΜ BBG to block P2X7 receptors38; 100 μΜ amiloride to block ASIC channels which may be activated by the acid shift occurring in ischaemia39, 40 ; 1 μΜ TTX to block action potential driven glutamate release35, 36; 100 μΜ

bicuculline to block GABAA receptors, 1 mM MCPG to block mGluRs, or 50 μΜ CPG ( (S ) -4-carboxyphenylglycine ) to block glutamate release by cystine-glutamate exchange (41), (42).

Contribution of [K+] o rise and gK decrease to the ischaemia- evoked inward current

We assumed that the ischaemia-evoked current reflects the sum of the current produced by a rise of [H+] i suppressing a mainly K+- specific ohmic conductance with a conductance of 2.3 nS (the slope of the 0.5 mM HEPES line in Fig. lg; the change of current this produces is schematised in Fig. 6b left panel), and a voltage-independent inward current of -58 pA (the mean value of the 50 mM HEPES data in Fig. lg) produced by the rise of [K+]o shifting the Nernst potential for K+ (the change of current this produces is schematised in Fig. 6b right panel) . Together, these changes are expected to generate an ischaemia-evoked current which shows a conductance decrease and an apparent reversal potential more negative than the original value of EK (Fig. 6c-d) as is seen experimentally (Fig. 6e) . For a mean input resistance of 148+23 M in 10 cells, this analysis implies that, on average, ischaemia reduced the resting conductance of the cell by 34%. Since the total inward current generated at -74 mV by the conductance decrease and the [K+]o rise was 85 pA (Fig. If) and 58 pA of this was generated by the [K+]o rise, it follows that the conductance decrease generated 27 pA of inward current. Thus, the conductance decrease produced 32%, and the [K+]o rise produced 68% of the inward current at -74mV (at more positive potentials the contribution of the conductance decrease will be larger) .

Ά [K+] o rise and K+ influx mediate much of the response to NMDA

Since both ischaemia and application of glutamate or NMDA are expected to depolarize cells and release [K+]o, we compared the effects of 100 μΜ NMDA application to those of a small elevation of [K+]o. The oligodendrocyte response to a 2.5 mM elevation of [K+]o was strikingly similar to the response to NMDA, with a decrease of [Na+]i and an elevation of [K+]i (Fig. 2a, b) and no significant change in [Ca2+] i (-0.001+0.010 change in R, p=0.36) . The I-V relation of both the NMDA and K+-evoked currents

decreased towards zero above +25 mV (Fig. 7e) . Consistent with a [K+]o rise-evoked K+ influx mediating a substantial part of the NMDA-evoked current, applying 30 mM external Cs+ to block oligodendrocyte K+ channels reduced the NMDA-evoked current in oligodendrocytes by 88% (p=0.03, 7 cells), while having no effect on the NMDA-evoked current in hippocampal pyramidal cells (Fig.

7a, b) . Conversely, including 1 mM MK-801 in the pipette to block NMDA receptors from the inside43 (and waiting 20 mins to allow the drug to diffuse through the cell) reduced the NMDA-evoked current in pyramidal cells by 88% (p=0.003, n=6) , while having no effect on that in oligodendrocytes (Fig. 7c, d) . Although this lack of effect might theoretically reflect the NMDA receptors in oligodendrocytes having an unusual subunit composition4 which is not susceptible to block by internal MK-801, this is not likely because they are blocked by external MK-801 (Fig. 7c) .

Using an extracellular K+-sensitive microelectrode to assess the [K+]o rise occurring near the patch-clamped oligodendrocyte when NMDA or an increase in [K+]o were applied (Fig. lh) , we found that 100 μΜ NMDA raised the local [K+]o by 1.5210.17 mM (n=19), while switching from 2.5 mM to 5 mM [K+] in the bath superfusate raised the local [K+]o by 2.32+0.18 mM near 21 cells. Plots of the membrane current change against local [K+]o during these responses (Fig. 7f) in 11 cells showed that the inward membrane current increased approximately linearly with the rise of [K+]o, with a slope that was 51% smaller (p=0.03) when increasing [K+] in the superfusate (-61.2119.3 pA/mM) than when applying NMDA (- 124.0146.1 pA/mM) . These data suggest that a rise of [K+]o is responsible for 49% of the inward current, although the

percentage could be significantly larger since the

oligodendrocyte membrane is presumably closer than the K+-sensing electrode to the source of K+ when NMDA is applied than when the superfusate [K+] is raised.

Specificity of drugs acting on TRP channels

Both TRPA1 and TRPV3 have previously been suggested to play a role in the response of the CNS to ischaemia (44), (45) .

Unfortunately the pharmacology of TRP channels is complex, with many drugs acting non-specifically on different channels. Our conclusion that TRPA1 is the major contributor to raising [Ca2+] i in ischaemia is based on the following logic.

(1) The ischaemia-evoked [Ca2+]i rise is abolished by removal of external Ca2+ and so reflects entry through an ion channel (Fig. 3a) . (2) The general TRP channel blockers ruthenium red and La3+ block the ischaemia-evoked [Ca2+]i rise (Fig. 4c-d) , suggesting the involvement of a TRP channel .

(3) Other known intracellular targets of ruthenium red, such as ryanodine receptors and the mitochondrial uniporter, are unlikely to explain the actions of ruthenium red because, as stated above, the [Ca2+]i rise reflects Ca2+ entry from the extracellular space.

(4) Voltage-gated Ca2+ channels, which are also targeted by ruthenium red and La3+, are not the source of the ischaemia- evoked [Ca2+]i rise since neither Cd2+ (Fig, 3b) nor benidipine (Fig. 11) blocked the rise, and these channels are not Mg2+- permeable while ischaemia evokes entry of Mg2+ through the H+- gated channel (Fig. 3d, k) .

(5) The ischaemia-evoked [Ca2+]i rise is abolished (Fig.

3j ) by reducing the ischaemia-evoked rise of [H+]i with a pH buffer (Fig. 3f) , and so reflects entry through a H+-gated channel .

(6) Of the TRP channels, only TRPA1 and TRPV3 are currently known to be activated by intracellular protons (20), (23), (24) .

(7) Drugs that activate TRPA1 but not TRPV3 (polygodial, AITC, FFA) , or that activate both TRPA1 and TRPV3 (menthol, vanillin, carvacrol, 2-APB) , but not drugs that activate TRPV3 and not TRPA1 (FPP, camphor), elevate [Ca2+] i (Fig. 4a, b) . The most specific TRPA1 agonist is polygodial which does not act (46) on TRPV1, TRPV2, TRPV3, TRPV4 or TRPM8 , followed by AITC (allyl- isothiocyanate ) which also acts (20) on TRPV1. Flufenamic acid activates TRPA1 but inhibits TRPV1 and TRPV347 and also acts on (20), (27), (47) -(49) TRPC3, TRPC5, TRPC6, TRPC7, TRPV4, TRPV5, TRPV6, TRPM2, TRPM3, TRPM4 , TRPM5 and TRPM8. Carvacrol activates TRPA1 and TRPV3, and inhibits TRPM7 (20) , (27) . Farnesyl

pyrophosphate (FPP) activates TRPV3 but does not activate TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 (25), (50) . Camphor activates TRPV3, activates TRPV1 and TRPM8 , and inhibits TRPA1 (20), (27) . Agonists for other TRPs (including 10 μΜ capsaicin (20), (27) for TRPV1, 5 cells, not shown) did not raise [Ca2+]i detectably. Thus, activation of TRPAl, but not of TRPV3, raises oligodendrocyte [Ca2+]i.

(8) The [Ca2+]i rise evoked by carvacrol (a TRPAl and TRPV3 agonist, and TRPM7 blocker (20), (27) is blocked by the

TRPAl /TRPV3 antagonist (25) isopentenyl pyrophosphate (IPP) , by the specific TRPAl antagonist (51) HC-030031, and by TRPAl knock-out (Fig. 4b) . HC-030031 blocks TRPAl without affecting TRPVl, TRPV3, TRPV4, TRPM8 or 48 other receptors, transporters and enzymes (51) . Thus, the blocker pharmacology matches that of the activator pharmacology.

(9) Isopentenyl pyrophosphate and HC-030031 also reduce the [Ca2+] i rise evoked by uncaging of protons in

oligodendrocytes (Fig. 31), implying that TRPAl is the main contributor to this response.

(10) The specific TRPAl blockers (51), (52) HC-030031 and

A967079 greatly reduced the ischaemia-evoked [Ca2+]i rise (Fig. 4e) . HC-030031 blocks TRPAl without affecting TRPVl, TRPV3, TRPV4, TRPM8 or 48 other receptors, transporters and enzymes (51) . A967079 has minimal activity at 89 different G-protein- coupled receptors, enzymes, transporters, and ion channels including other TRP channels (52) .

(11) TRPAl knock-out, but not knock-out of TRPV3, slowed and greatly reduced the ischaemia-evoked [Ca2+]i rise (Fig. 4f, g) . To check for mistakes in our understanding, we applied the TRPAl /TRPV3 blocker IPP in the TRPAl KO and (as expected) it had no effect, and we applied the TRPAl blocker HC-030031 in the TRPV3 KO and (as expected) it greatly reduced the ischaemia- evoked [Ca2+]i rise.

(12) Myelin damage was reduced by the specific TRPAl blockers HC-030031 and A967079 (Fig. 4i-j) .

(13) The only TRP channel previously reported to be present in oligodendrocytes is TRPM3, which acts as a sphingosine-gated Ca2+-channel (53) . However, when we applied sphingosine (50 μΜ) it evoked no significant increase in [Ca2+]i (0R/R =

0.014+0.012, n=5, p=0.16) .

(14) The ischaemia-evoked [Ca2+]i increase was not

inhibited (p>0.53, Fig. 11) by agents blocking TRP channels other than TRPAl, as follows: flufenamic acid (250 μΜ) which blocks (27), (54) TRPP2, TRPC3, TRPC5, TRPC7, TRPM2 , TRPM4 and TRPM5;

ML204 (20 μΜ) which blocks (20), (55) TRPC4 and TRPC5; SKF-96365 (100 μΜ) which blocks (27), (56), (57) TRPV2 , TRPC3, TRPC6, TRPC7 and TRPP1, as well as the store-operated calcium channel

component STIMl and some voltage-gated calcium channels;

capsazepine (CPZ, 100 μΜ) which blocks (20) TRPV1 and TRPM8;

FTY720 (1 μΜ) which blocks (58) TRPM7; RN-1734 which blocks (59)

TRPV4.

Example 2: Further characterisation of TRPAl expression and activation on oligodendrocytes

As described herein and in Additional Reference 1 (Hamilton et al . , 2016), inhibition of TRPAl channels on oligodendrocytes with HC-030031, A967079 or ruthenium red decreases myelin damage caused by ischaemia. As TRPAl is activated by inflammatory mediators, this demonstrates for the first time that

oligodendrocytes are vulnerable to TRPAl agonists, and that TRPAl is a therapeutic target in neurodegerative diseases. The present Example further characterises the expression of TRPAl by

oligodendrocytes, the TRPAl-mediated current and the TRPAl evoked intracellular Ca ²⁺ concentration rise. TRPAl expression on oligodendrocytes

Both mRNA expression TRPAl and protein expression on

oligodendrocytes is demonstrated. TRPAl protein expression is detected using labelled with antibodies to TRPAl (Abeam 58844) (Fig. 13) . TRPAl appears on oligodendrocyte somata and myelin.

Blockade of currents across oligodendrocyte by TRPAl antagonists and Inhibition of oligodendrocyte potassium conductance by non- electrophlllc TRPAl agonists

Different TRPAl agonists have different effects on

oligodendrocytes depending on the site at which the agonist activates TRPAl (Fig. 14) . Electrophilic TRPAl agonists evoke an increase in oligodendrocyte conductance that can be inhibited by a TRPAl antagonist (Fig. 14a) . However non-electrophilic TRPAl agonists evokes an immediate inhibition of oligodendrocyte potassium conductance, such as occurs in ischaemia (Hamilton et al . , 2016) . TRPAl agonists can evoke a sustained [Ca ²⁺ ]i increase or initiate oscillations that are inhibited by the TRPAl

antagonist HC-030031 (Fig. 14c) . Fig. 14d provides an indication that TRPAl-evoked Ca ²⁺ increases can be augmented by co-application of inflammatory cytokines.

Fluorescently labelled TRPAl agonist bind oligodendrocyte somata in the corpus callosum

As shown in Figure 15, the isothiocyanate based TRPAl agonist propargyl isothiocyanate preferentially binds to oligodendrocyte cell bodies within the corpus callosum (Fig. 15b) . The

oligodendrocytes were identified by morphology, location in the white matter, and GFAP negative status (Fig. 15b and c) . This is further evidence that TRPAl agonists can preferentially act upon oligodendrocytes in the brain.

References :

All publications, patent and patent applications cited herein or filed with this application, including references filed as part of an Information Disclosure Statement are incorporated by reference in their entirety. Akopian, A.N., Ruparel, N.B., Patwardhan, A., and Hargreaves,

K.M. (2008) . Cannabinoids Desensitize Capsaicin and Mustard Oil Responses in Sensory Neurons via TRPAl Activation. J.

Neurosci. 28, 1064-1075.

Alleman, A.M. (2014) . Osmotic Demyelination Syndrome: Central Pontine Myelinolysis and Extrapontine Myelinolysis . Seminars in Ultrasound, CT and MRI 35, 153-159.

Andersson, D.A., Gentry, C., Moss, S., and Bevan, S. (2008) . Transient Receptor Potential Al Is a Sensory Receptor for Multiple Products of Oxidative Stress. J. Neurosci. 28, 2485- 2494.

Bautista, D.M., Jordt, S.-E., Nikai, T., Tsuruda, P.R., Read, A. J., Poblete, J., Yamoah, E.N., Basbaum, A.I., and Julius, D.

(2006) . TRPAl Mediates the Inflammatory Actions of

Environmental Irritants and Proalgesic Agents. Cell 124, 1269- 1282.

Belbasis, L., Bellou, V., Evangelou, E., Ioannidis, J. P. A., and Tzoulaki, I. (2015) . Environmental risk factors and multiple sclerosis: an umbrella review of systematic reviews and metaanalyses. The Lancet Neurology 14, 263-273.

Birkett, D.J., and Miners, J.O. (1991) . Caffeine renal clearance and urine caffeine concentrations during steady state dosing. Implications for monitoring caffeine intake during sports events. Br J Clin Pharmacol 31, 405-408.

Boje, K.M., and Arora, P.K. (1992) . Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Research 587, 250-256.

Brambrink, A.M., Back, S.A., Riddle, A., Gong, X., Moravec, M.D., Dissen, G.A., Creeley, C.E., Dikranian, K.T., and Olney, J.W. (2012) . Isoflurane-induced apoptosis of oligodendrocytes in the neonatal primate brain. Ann Neurol. 72, 525-535.

Brierley, S.M., Castro, J., Harrington, A.M., Hughes, P. A., Page, A. J., Rychkov, G.Y., and Blackshaw, L.A. (2011) . TRPAl contributes to specific mechanically activated currents and sensory neuron mechanical hypersensitivity. The Journal of Physiology 589, 3575-3593.

Copeland, K.W., Boezio, A. A., Cheung, E., Lee, J., Olivieri, P., Schenkel, L.B., Wan, Q., Wang, W., Wells, M.C., Youngblood,

B., et al . (2014) . Development of novel azabenzofuran TRPAl antagonists as in vivo tools. Bioorganic & Medicinal Chemistry Letters 24, 3464-3468.

Cusick, M.F., Libbey, J.E., and Fujinami, R.S. (2013) . Multiple sclerosis: autoimmunity and viruses. Current Opinion in

Rheumatology 25, 496-501.

Davies, A.L., Desai, R.A., Bloomfield, P.S., Mcintosh, P.R., Chappie, K.J., Linington, C, Fairless, R., Diem, R., Kasti, M. , Murphy, M.P., et al . (2013) . Neurological deficits caused by tissue hypoxia in neuroinflammatory disease. Ann.

Neurol. 74, 815-825.

Davies, M.B., Weatherby, S.J., Haq, N. , and Ellis, S.J. (2000) . A multiple-sclerosis-like syndrome associated with glue- sniffing. J R Soc Med 93, 313-314.

Doerner, J.F., Gisselmann, G., Hatt, H., and Wetzel, C.H. (2007) .

Transient Receptor Potential Channel Al Is Directly Gated by Calcium Ions. J. Biol. Chem. 282, 13180-13189.

Esposito, S., Di Pietro, G.M., Madini, B., Mastrolia, M.V. , and Rigante, D. (2015) . A spectrum of inflammation and

demyelination in acute disseminated encephalomyelitis (ADEM) of children. Autoimmunity Reviewsl4, 923-929.

Forster, A.B., Reeh, P.W., Messlinger, K., and Fischer, M.J.

(2009) . High concentrations of morphine sensitize and activate mouse dorsal root ganglia via TRPV1 and TRPA1 receptors .

Molecular Pain 5, 17.

Greaves, E., Grieve, K., Home, A.W., and Saunders, P.T.K.

(2014) . Elevated peritoneal expression and estrogen regulation of nociceptive ion channels in endometriosis. J. Clin.

Endocrinol. Metab. 99, E1738-E1743.

Greene-Schloesser, D., Robbins, M.E., Peiffer, A.M., Shaw, E.G., Wheeler, K.T., and Chan, M.D. (2012) . Radiation-induced brain injury: A review. Front Oncol 2.

Hayes, C.E., Hubler, S.L., Moore, J.R., Barta, L.E., Praska,

C.E., and Nashold, F.E. (2015) . Vitamin D actions on CD4+ T cells in autoimmune disease. Front. Immunol. 6, 100.

Heick, A., and Skriver, E. (2000) . Chlamydia pneumoniae- associated ADEM. Eur. J. Neurol. 7, 435-438.

Hernan, M.A., Oleky, M.J., and Ascherio, A. (2001) . Cigarette

Smoking and Incidence of Multiple Sclerosis. Am. J. Epidemiol. 154, 69-74.

Hernan, M.A., Jick, S.S., Logroscino, G. , Olek, M.J., Ascherio, A., and Jick, H. (2005) . Cigarette smoking and the progression of multiple sclerosis. Brain 128, 1461-1465.

Hu, H., Tian, J., Zhu, Y., Wang, C, Xiao, R., Herz, J.M., Wood, J.D., and Zhu, M.X. (2010) . Activation of TRPAl Channels by Fenamate Non-steroidal Anti-inflammatory Drugs. Pflugers Arch 459, 579-592.

Karashima, Y., Prenen, J., Talavera, K., Janssens, A., Voets, T . , and Nilius, B. (2010) . Agonist-Induced Changes in Ca2+

Permeation through the Nociceptor Cation Channel TRPAl .

Biophys J 98, 773-783.

Landtblom, A.-M., Tondel, M. , Hjalmarsson, P., Flodin, U., and Axelson, 0. (2006) . The risk for multiple sclerosis in female nurse anaesthetists: a register based study. Occup Environ Med

63, 387-389.

La ther, P.J., and Commins, B.T. (1970) . Cigarette Smoking and

Exposure to Carbon Monoxide. Annals of the New York Academy of

Sciences 174, 135-147.

Lefebvre d' Hellencourt , C, Montero-Menei , C.N., Bernard, R. , and

Couez, D. (2003) . Vitamin D3 inhibits proinflammatory

cytokines and nitric oxide production by the EOC13 microglial cell line. J. Neurosci. Res. 71, 575-582.

Lennertz, R.C., Kossyreva, E.A., Smith, A.K., and Stucky, C.L.

(2012) . TRPAl Mediates Mechanical Sensitization in Nociceptors during Inflammation. PLoS ONE 7, e43597.

Lowin, T., Spath, T., and Straub, R.H. (2015) . Pro-inflammatory cytokines up-regulate and sensitize metabotropic and

ionotropic cannabinoid receptors in rheumatoid arthritis and osteoarthritis synovial fibroblasts. Brain, Behavior, and

Immunity 49, Supplement, e3-e4.

Love, S., and Coakham, H.B. (2001) . Trigeminal neuralgia.

Brain 124, 2347-2360.

Lucchinetti, C., Briick, W. , Parisi, J., Scheithauer, B.,

Rodriguez, M., and Lassmann, H. (2000) . Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination . Ann. Neurol. 47, 707-717.

Lunny, C., Knopp-Sihota, J. A., and Fraser, S.N. (2013) . Surgery and risk for multiple sclerosis: a systematic review and meta- analysis of case-control studies. BMC Neurology 13, 41.

Manji, H., and Miller, R.F. (2000) . Progressive multifocal

leucoencephalopathy : progress in the AIDS era. J Neurol Neurosurg Psychiatry 69, 569-571.

Matta, J. A., Cornett, P.M., Miyares, R.L., Abe, K. , Sahibzada, N. , and Ahern, G.P. (2008) . General anesthetics activate a nociceptive ion channel to enhance pain and inflammation. PNAS 105, 8784-8789.

McDonald, J.W., and Belegu, V. (2006) . Demyelination and

Remyelination after Spinal Cord Injury. Journal of

Neurotrauma 23, 345-359.

Meseguer, V., Alpizar, Y.A., Luis, E., Tajada, S., Denlinger, B., Fajardo, 0., Manenschijn, J. -A., Fernandez-Pena, C, Talavera,

A., Kichko, T., et al . (2014) . TRPAl channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins. Nat Commun 5, 3125.

Mitrovic, B., Parkinson, J., and Merrill, J.E. (1996) . Anin

VitroModel of Oligodendrocyte Destruction by Nitric Oxide and

Its Relevance to Multiple Sclerosis. Methods 10, 501-513.

Miyamoto, T., Dubin, A.E., Petrus, M.J., and Patapoutian, A.

(2009) . TRPVl and TRPAl Mediate Peripheral Nitric Oxide- Induced Nociception in Mice. PLoS ONE 4, e7596.

Mormile, R., and Vittori, G. (2014) . Endometriosis and

susceptibility to multiple sclerosis: is there any absolute truth? European Journal of Obstetrics & Gynecology and

Reproductive Biology 179, 253.

Motter, A.L., and Ahern, G.P. (2012) . TRPAl is a polyunsaturated fatty acid sensor in mammals. PLoS ONE 7, e38439.

Philbrick, D.J., Hopkins, J.B., Hill, D.C., Alexander, J.C., and Thomson, R.G. (1979) . Effects of prolonged cyanide and thiocyanate feeding in rats. J Toxicol Environ Health 5, 579- 592.

Rejdak, K., Eikelenboom, M.J., Petzold, A., Thompson, E.J.,

Stelmasiak, Z., Lazeron, R.H.C., Barkhof, F., Polman, C.H., Uitdehaag, B.M.J. , and Giovannoni, G. (2004) . CSF nitric oxide metabolites are associated with activity and progression of multiple sclerosis. Neurology 63, 1439-1445.

Russell, M.A., Wilson, C, Patel, U.A., Feyerabend, C, and Cole, P.V. (1975) . Plasma nicotine levels after smoking cigarettes with high, medium, and low nicotine yields. Br Med J 2, 414- 416.

Pendleburyab, S.T., Lee, M.A., Blamire, A.M., Styles, P., and

Matthews, P.M. (2000) . Correlating magnetic resonance imaging markers of axonal injury and demyelination in motor impairment secondary to stroke and multiple sclerosis. Magnetic Resonance

Imaging 18, 369-378.

Sharma, R. , Fischer, M.-T., Bauer, J., Felts, P. A., Smith, K.J.,

Misu, T., Fujihara, K. , Bradl, M., and Lassmann, H. (2010) .

Inflammation induced by innate immunity in the central nervous system leads to primary astrocyte dysfunction followed by demyelination. Acta Neuropathol 120, 223-236.

Smith, A.D., Duckett, S., and Waters, A.H. (1963) .

NEUROPATHOLOGICAL CHANGES IN CHRONIC CYANIDE INTOXICATION.

Nature 200, 179-181.

Smith, D.A., Hoffman, A.F., David, D.J., Adams, C.E., and

Gerhardt, G.A. (1998) . Nicotine-evoked nitric oxide release in the rat hippocampal slice. Neuroscience Letters 255, 127-130. Smith, K.J., Kapoor, R., and Felts, P. A. (1999) . Demyelination:

The Role of Reactive Oxygen and Nitrogen Species. Brain

Pathology 9, 69-92.

Stoner, J.D., and Parker, J.C. (1991) . The effects of

hypertension on the nervous system. Ann. Clin. Lab. Sci. 21,

147-152.

Suemaru, K., Kawasaki, H., Gomita, Y., and Tanizaki, Y. (1997) .

Involvement of nitric oxide in development of tail-tremor induced by repeated nicotine administration in rats. European Journal of Pharmacology 335, 139-143.

Svenningsson, A., Petersson, A.-S., Andersen, 0., and Hansson, G.K. (1999) . Nitric oxide metabolites in CSF of patients with MS are related to clinical disease course. Neurology 53, 1880-

1880.

Takahashi, N., Kuwaki, T., Kiyonaka, S., Numata, T., Kozai, D. , Mizuno, Y., Yamamoto, S., Naito, S., Knevels, E., Carmeliet, P., et al . (2011) . TRPA1 underlies a sensing mechanism for 02. Nat Chem Biol 7, 701-711.

Taylor-Clark, T.E., Kiros, F., Carr, M.J., and McAlexander, M.A.

(2009) . Transient Receptor Potential Ankyrin 1 Mediates Toluene Diisocyanate-Evoked Respiratory Irritation. Am J Respir Cell Mol Biol 40,

Talavera, K., Gees, M. , Karashima, Y., Meseguer, V.M.,

Vanoirbeek, J.A.J. , Damann, N. , Everaerts, W. , Benoit, M. , Janssens, A., Vennekens, R., et al . (2009) . Nicotine activates the chemosensory cation channel TRPA1. Nat Neurosci 12, 1293- 1299.

Taparia, S., Fleet, J.C., Peng, J.-B., Wang, X.D., and Wood, R.J.

(2005) . 1 , 25-Dihydroxyvitamin D and 25-hydroxyvitamin D— mediated regulation of TRPV6 (a putative epithelial calcium channel) mRNA expression in Caco-2 cells. Eur J Nutr 45, 196- 204.

Tonnessen, B.H., Severson, S.R., Hurt, R.D., and Miller, V.M.

(2000) . Modulation of nitric-oxide synthase by nicotine. J. Pharmacol. Exp. Ther. 295, 601-606.

Wang, T., Xi, N. , Chen, Y., Shang, X., Hu, Q., Chen, J., and

Zheng, R. (2014) . Chronic caffeine treatment protects against experimental autoimmune encephalomyelitis in mice: Therapeutic window and receptor subtype mechanism. Neuropharmacology 86, 203-211.

Wang, Y.Y., Chang, R.B., and Liman, E.R. (2010) . TRPA1 Is a

Component of the Nociceptive Response to C02. J. Neurosci. 30, 12958-12963.

Weidman, E.K., Tsiouris, A.J., and Heier, L.A. (2015) . Toxic

encephalopathy due to paradichlorobenzene toxicity: a case report and review of imaging characteristics. Clinical

Imaging 39, 1095-1098.

Weiner, L.P., Johnson, R.T., and Herndon, R.M. (1973) . Viral

Infections and Demyelinating Diseases. New England Journal of Medicine 288, 1103-1110.

Wolters, E.C., van Wijngaarden, G.K., Stam, F.C., Rengelink, H., Lousberg, R.J., Schipper, M.E., and Verbeeten, B. (1982) .

Leucoencephalopathy after inhaling "heroin" pyrolysate.

Lancet 2, 1233-1237.

Yamashita, T., Ando, Y., Obayashi, K., Uchino, M., and Ando, M.

(1997) . Changes in nitrite and nitrate (N02-/N03-) levels in cerebrospinal fluid of patients with multiple sclerosis. Journal of the Neurological Sciences 153, 32-34.

Numbered references

1. Stys, P.K., Ransom,, B.R., Waxman, S.G. & Davis, P.K. Role of extracellular calcium in anoxic injury of central white matter. Proc. Natl. Acad, Sci. USA 87, 4212-4216 (1990) .

2. Micu, I. et al . NMDA receptors mediate calcium

accumulation in myelin during chemical ischaemia. Nature 439, 988-992 (2006) .

3. Salter, M.G. & Fern, R. NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury. Nature 438, 1167-1171 (2005) .

4. Karadottir, R. , Cavelier, P., Bergersen, L.H. &

Attwell, D. NMDA receptors are expressed in

oligodendrocytes and activated in ischaemia. Nature 438, 1162-1168 (2005) .

5. Bakiri, Y., Hamilton, N.B., Karadottir, R. & Attwell, D. Testing NMDA receptor block as a therapeutic strategy for reducing ischaemic damage to CNS white matter. Glia 56,

233- 240 (2008) .

6. McCarran, W.J. & Goldberg, M.P. White matter axon vulnerability to AMPA/kainate receptor-mediated ischemic injury is developmentally regulated. J. Neurosci . 27, 4220- 4229 (2007) .

7. Agrawal, S.K. & Fehlings, M.G. Role of NMDA and non- NMDA ionotropic glutamate receptors in traumatic spinal cord axonal injury. J. Neurosci . 17, 1055-1063 (1997) .

8. Sarrafzadeh, A.S., Kiening, K.L., Callsen, T.A. &

Unterberg, A.W. Metabolic changes during impending and manifest cerebral hypoxia in traumatic brain injury. Brit. J. Neurosurg. 17, 340-346 (2003) .

9. Li, S., Mealing, G.A., Morley, P. & Stys, P.K. Novel injury mechanism in anoxia and trauma of spinal cord white matter: glutamate release via reverse Na+-dependent glutamate transport. J Neurosci 19, RC16 (1999) .

10. Back, S.A. et al . Hypoxia-ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter. J. Cereb. Blood Flow Metab. 27, 334-347 (2007) .

11. Yuan, X., Eisen, A.M., McBain, C.J. & Gallo, V. A role for glutamate and its receptors in the regulation of oligodendrocyte development in cerebellar tissue slices. Development 125, 2901-2914 (1998) .

12. Lundgaard, I . et al . Neuregulin and BDNF induce a switch to NMDA receptor-dependent myelination by

oligodendrocytes. PLoS Biol. 11, el001743 (2013) .

13. Cahoy, J.D. et al . A transcriptome database for

astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J.

Neurosci. 28, 264- 278 (2008) .

14. De Biase, L.M., Nishiyama, A. & Bergles, D.E.

Excitability and synaptic communication within the

oligodendrocyte lineage. J. Neurosci. 30, 3600-3611 (2010) .

15. Kukley, M. , Nishiyama, A. & Dietrich, D. The fate of synaptic input to NG2 glial cells: neurons specifically downregulate transmitter release onto differentiating oligodendroglial cells. J. Neurosci. 30, 8320-8331 (2010) .

16. Rossi, D.J., Oshima, T. & Attwell, D. Glutamate

release in severe brain ischaemia is mainly by reversed uptake. Nature 403, 316-321 (2000) .

17. Limbrick, D.D. Jr., Sombati, S. & DeLorenzo, R.J.

Calcium influx constitutes the ionic basis for the

maintenance of glutamate-induced extended neuronal

depolarization associated with hippocampal neuronal death. Cell Calcium 33, 69-81 (2003) .

18. Hamann, M. , Rossi, D.J., Mohr, C, Andrade, A.L. &

Attwell, D. The electrical response of cerebellar Purkinje neurons to simulated ischaemia. Brain 128, 2408-2420

(2005) .

19. Lesage, F. et al . TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. EMBO

J. 15, 1004-1011 (1996) .12

0. Nilius, B. & Szallasi, A. Transient receptor potential channels as drug targets: from the science of basic research to the art of medicine. Pharmacol. Rev. 66, 676- 814 (2014) .

21. Aarts, M. et al . A key role for TRPM7 channels in

anoxic neuronal death. Cell 115, 863- 877 (2003) .

22. Butenko, 0. et al . The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia . PLoS One 7, e39959 (2012) .

23. Wang, Y.Y., Chang, R.B. & Liman, E.R. TRPA1 is a

component of the nociceptive response to C02. J. Neurosci .

30, 12958-12963 (2010) .

24. Cao, X., Yang, F., Zheng, J. & Wang, K. Intracellular proton-mediated activation of TRPV3 channels accounts for the exfoliation effect of alpha-hydroxyl acids on

keratinocytes . J. Biol. Chem. 31, 25905-25916 (2012) .

25. Bang, S., Yoo, S., Yang, T.J., Cho, H. & Hwang, S.W.

Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels. Pain 152, 1156-1164 (2011) .

26. McNamara, C.R. et al . TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. U.S.A. 104, 13525-13530

(2007) .

27. Clapham, D.E. Snapshot: Mammalian TRP channels. Cell

129, 220.el-2 (2007) .

28. Chen, J. et al . Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation. Pain, 152, 1165- 1172 (2011) .

29. Hamilton, N.B. et al . Mechanisms of ATP- and

glutamate-mediated calcium signaling in white matter astrocytes. Glia 56, 734-749 (2008) .

30. Davies, A.L. et al . Neurological deficits caused by tissue hypoxia in neuroinflammatory disease. Ann. Neurol. 74, 815-825 (2013) .

31. Allen, N.J., Karadottir, R. & Attwell, D. A

preferential role for glycolysis in preventing the anoxic depolarization of rat hippocampal area CA1 pyramidal cells. J. Neurosci. 25, 848-859 (2005) .

32. Keynes, R.G., Griffiths, C. & Garth aite, J.

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments.

Biochem. J. 369, 399-406 (2003) .

33. Marcaggi, P., Jeanne, M. & Coles, J.A. Neuron-glial

trafficking of NH ⁺ and K ⁺ : separate routes of uptake into glial cells of bee retina. Eur. J. Neurosci. 19, 966-976 (2004) .

34. Wilke, S., Thomas, R., Allcock, N. Fern, R. Mechanism of acute ischemic injury of oligodendroglia in early myelinating white matter: the importance of astrocyte injury and glutamate release. J. Neuropathol . Exp. Pathol. 63, 872-881 (2004) .

35. Kukley, M. , Capetillo-Zarate, E. & Dietrich, D.

Vesicular release of glutamate from axons in white matter. Nat. Neurosci. 10, 311-320 (2007) .

36. Ziskin, J.L., Nishiyama, A., Rubio, M. , Fukaya, M. & Bergles, D.E. Vesicular release of glutamate from

unmyelinated axons in white matter. Nat. Neurosci. 10, 321-

330 (2007) .

37. Ye, Z.C., Wyeth, M.S., Baltan-Tekkok, S. & Ransom, B.R. Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J. Neurosci. 23, 3588-3596 (2003) .

38. Domercq, M. et al . P2X7 receptors mediate ischemic damage to oligodendrocytes. Glia 58, 730-740 (2010)

39. Friese, M.A. et al . Acid-sensing ion channel-1

contributes to axonal degeneration in autoimmune

inflammation of the central nervous system. Nat. Med. 13,

1483-1489 (2007) .

40. Feldman, D.H. et al . Characterization of acid-sensing ion channel expression in oligodendrocyte-lineage cells. Glia 56, 1238-1249 (2008) .

41. Cavelier, P. & Attwell, D. Tonic release of glutamate by a DIDS-sensitive mechanism in rat hippocampal slices. J. Physiol. 564, 397-410 (2005) .

42. Domercq, M. et al . System xc- and glutamate

transporter inhibition mediates microglial toxicity to oligodendrocytes. J. Immunol. 178, 6549-6556 (2007) .

43. Li, Y.H., Han, T.Z. & Meng, K. Tonic facilitation of glutamate release by glycine binding sites on presynaptic NR2B-containing NMDA autoreceptors in the rat visual cortex. Neurosci Lett. 432, 212-216 (2008) .

44. Sasaki, A. et al . A mouse model of peripheral

postischemic dysesthesia: involvement of reperfusion- induced oxidative stress and TRPAl channel. J. Pharmacol. Exp. Ther. 351, 368-375 (2014) .

45. Moussaieff, A. et al . Protective effects of incensole acetate on cerebral ischemic injury. Brain Res. 1443, 89-97 (2012) .

46. Escalera, J., von Hehn, C.A., Bessac, B.F., Sivula, M.

& Jordt, S.E. TRPAl mediates the noxious effects of natural sesquiterpene deterrents. J. Biol. Chem. 283, 24136-24144 (2008) .

47. Hu, H. et al . Activation of TRPAl channels by fenamate nonsteroidal anti-inflammatory drugs. Pflugers Arch 459, 579-592 (2010) .

48. Albert, A. P., Pucovsky, V., Prest ich, S.A. & Large, W.A. TRPC3 properties of a native constitutively active Ca2+-permeable cation channel in rabbit ear artery

myocytes. J. Physiol. 571, 361-369 (2006) .

49. Klose, C. et al . Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3. Br. J. Pharmacol. 162, 1757-1762 (2011) .

50. Bang, S., Yoo, S., Yang, T.J., Cho, H. & Hwang,

S.W. Farnesyl pyrophosphate is a novel pain-producing molecule via specific activation of TRPV3. J. Biol. Chem. 285, 19362-19371 (2010) .

51. Eid, S.R. et al . HC-030031, a TRPAl selective

antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Molec. Pain 4, 48 (2008) . 52. McGaraughty, S. et al . TRPA1 modulation of spontaneous and mechanically evoked firing of spinal neurons in uninjured, osteoarthritic , and inflamed rats. Molec. Pain 6, 14 (2010) .

53. Hoffmann, A. et al . TRPM3 is expressed in sphingosine- responsive myelinating oligodendrocytes. J. Neurochem. 114, 654-665 (2010) .

54. Ullrich, N.D. et al . Comparison of functional

properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice. Cell Calcium 37, 267-278 (2005) .

55. Miller M et al . Identification of ML204, a novel

potent antagonist that selectively modulates native

TRPC4/C5 ion channels. J. Biol. Chem. 286, 33436-33446 (2011) .

56. Clapham, D.E., Julius, D., Montell, C. & Schultz, G.

International union of pharmacology. XLIX. Nomenclature and structure-function relationships of transient receptor potential channels. Pharmacol. Rev. 57, 427-450 (2005) .

57. Harteneck, C. & Gollasch, M. Pharmacological

modulation of diacylglycerol-sensitive TRPC3/6/7 channels.

Curr. Pharm. Biotech. 12, 35-41 (2011) .

58. Qin, X. et al . Sphingosine and FTY720 are potent

inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels. Brit. J. Pharmacol. 168, 1294-1312

(2013) .

59. Vincent, F. et al . Identification and characterization of novel TRPV4 modulators Biochem. Biophys . Res. Commun. 389, 490-494 (2009) .

60. Wolters, E.C., van Wijngaarden, G.K., Stam, F.C.,

Rengelink, H., Lousberg, R.J., Schipper, M.E., and

Verbeeten, B. (1982) . Leucoencephalopathy after inhaling "heroin" pyrolysate. Lancet 2, 1233-1237.

61. Weidman, E.K., Tsiouris, A.J., and Heier, L.A. (2015) .

Toxic encephalopathy due to paradichlorobenzene toxicity: a case report and review of imaging characteristics. Clinical

Imaging 39, 1095-1098. 62. Forster, A.B., Reeh, P.W., Messlinger, K., and

Fischer, M.J. (2009) . High concentrations of morphine sensitize and activate mouse dorsal root ganglia via TRPV1 and TRPA1 receptors. Molecular Pain 5, 17.

63. Kumaza a, T . , Mizumura, K. , Sato, J., and Minaga a, M.

(1989) . Facilitatory effects of opioids on the discharges of visceral nociceptors. Brain Research 497, 231-238.

Additional References

1. Nicola B. Hamilton, Karolina Kolodziej czyk, Eleni

Kougioumtzidou, and David Att ell (2016) . Proton-gated Ca2+- permeable TRP channels damage myelin in conditions mimicking ischaemia. Nature, 2016 January 28; 529(7587) : 523-527.