Excitotoxic Glutamate Release: Is This What Stymies Oligodendrocytes’ Protective Role in MS?

DeSilva lab pursues mechanisms regulating glutamate in demyelinating diseases

By Tara DeSilva, PhD

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Inflammation and MS

The major hallmark of demyelinating diseases such as multiple sclerosis (MS) is immune cell infiltration into the central nervous system (CNS) resulting in axon-myelin damage and eventual neurodegeneration. Demyelination of the neuronal axon not only slows propagation of nerve impulses, but makes the axon more vulnerable to injury, resulting in motor and visual impairments, bowel and bladder dysfunction, pain sensation and cognitive decline.

Current treatments for MS focus on attenuating immune cell infiltration into the CNS. While these treatments reduce the number of relapses, they do not completely eliminate disease activity in the CNS, so long-term disability is not improved. This underscores the importance of understanding mechanisms of CNS protection as a potential add-on therapeutic strategy for MS.

Evidence for glutamate dysregulation in MS

Our laboratory at the Cleveland Clinic focuses on mechanisms that regulate glutamate as a therapeutic strategy for neuroprotection in MS. Excitotoxicity is a pathological consequence of prolonged activation of glutamate receptors resulting in excessive influx of calcium into the cytosol, leading to cell death. Oligodendrocytes, the cells that make up the myelin sheath, contain glutamate receptors, rendering them potentially vulnerable to excitotoxicity.

Glutamate levels are elevated in the CSF, acute lesions and plasma of patients with MS, and elevated glutamate levels (as measured by MRI) have been associated with decreased brain volume in patients with MS. These findings provide evidence that glutamate homeostasis may be altered, thereby contributing to excitotoxic mechanisms.

Causal link between inflammation and glutamate dysregulation

To address the efficacy of blocking glutamate release in MS, we targeted the system xc transporter as a potential source of glutamate release in experimental autoimmune demyelination (EAE), an animal model of MS.

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System xc functions as an exchanger that imports cystine into the cell for synthesis of glutathione, a major antioxidant, in exchange for release of glutamate. Our group showed that genetic or pharmacologic inhibition of the system xc transporter resulted in abrogation of clinical disease and demyelination in the CNS in both chronic and relapsing-remitting models of EAE (J Immunol. 2015;195:450-463). An interesting finding from this work is that glutamate also regulates immune cell infiltration into the CNS. Mechanisms regulating how immune cells are able to gain access to the CNS are an important area of research interest in neuroinflammatory diseases. Inhibiting system xc transporter function attenuated T cell infiltration into the CNS, which prevented clinical disease presentation and myelin destruction but not T cell activation in the periphery. Further studies identified expression of system xc on peripheral macrophages as well as dendritic cells, suggesting a novel role for glutamate signaling in activating T cell entry into the CNS.

To further explore the hypothesis that excitotoxicity contributes to demyelination independent of immune cell modulation, we performed pharmacologic inhibition of the system xc transporter after immune cell infiltration in the relapsing-remitting model of EAE. Our results provide evidence that myelin-specific T helper cells provoke microglia to release glutamate via the system xc transporter, causing excitotoxic death to mature myelin.

Taken together, these studies support a novel role for the system xc transporter in mediating T cell infiltration into the CNS and in promoting myelin destruction after immune cell infiltration in EAE. These studies led to an important conceptual idea that modulation of immune cell infiltration must be considered when evaluating CNS neuroprotective strategies, resulting in a methodology to differentiate these two important mechanisms, as outlined in a recent paper by our group (J Vis Exp. 2016 Sep 12;[115]:e54348).

Blocking the target for glutamate release prevents myelin and axonal damage

This prompted the next experimental question: “Does inhibiting the target for glutamate on mature myelinating oligodendrocytes prevent demyelination and correspond with improved clinical scores in EAE?” Data being prepared for publication show that mice genetically deficient in AMPA-type glutamate receptors on mature oligodendrocytes have improved clinical scores and reduced myelin degradation compared with littermate controls subjected to EAE. Furthermore, deleting AMPA-type glutamate receptors from myelin also prevents axonal injury and loss as evidenced by 3-D confocal imaging (Figure).

Figure. Reduction of AMPA receptors in myelin attenuates demyelination and axonal damage in EAE. 3-D representations of 15-µm confocal z-stacks of spinal cord sections from littermate control mice (left panel) and AMPA receptor-deficient mice (right panel). Far-red immunofluorescence represents SMI-312 (neurofilament) staining of axons, and green immunofluorescence represents MBP (myelin) staining. Axon swelling and myelin loss are reduced in the AMPA receptor-deficient mice (right panel) compared with controls (left panel). Unpublished data from the DeSilva laboratory.

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To confirm that abrogation of disease activity was CNS-specific, we analyzed CNS-infiltrating immune cells by flow cytometry and demonstrated no changes in AMPA receptor-deficient mice compared with controls. These data, currently being prepared for publication, support the idea that AMPA receptors expressed on myelin mediate damage to both the myelin sheath and the neuronal axon, suggesting the importance of myelin maintenance for axonal health in MS.

Glutamate and regeneration in MS

In MS, new early-stage myelin-producing cells (oligodendrocyte progenitor cells) proliferate but fail to rebuild the insulation around nerve fibers as they would during normal development. Our work suggests that AMPA receptors on oligodendrocyte progenitor cells sense glutamate release from axons as an instructive signal to start myelinating axons. Therefore, excessive glutamate release could negatively regulate differentiation, myelin formation and remyelination in MS.

A perfect storm?

Excitotoxic glutamate release from the system xc transporter may provide the perfect storm in MS by facilitating immune cell infiltration into the CNS to promote excitotoxic death of myelin and inhibit remyelination of newly proliferated oligodendrocyte progenitor cells. Blocking the source of excitotoxic glutamate is a more feasible therapeutic strategy for CNS protection than blocking the target, since AMPA receptor signaling is necessary not only for remyelination but also for synaptic function in neurons. This insight underlies our active pursuit of clinically relevant therapeutic strategies for blocking the system xc transporter to protect and promote regeneration in the CNS in MS.

Dr. DeSilva (desilvt@ccf.org) is an associate staff member in the Department of Neurosciences in Cleveland Clinic’s Lerner Research Institute.