T cell infiltration into the central nervous system (CNS) is a significant underlying pathogenesis in autoimmune inflammatory demyelinating diseases. Several lines of evidence suggest that glutamate dysregulation in the CNS is an important consequence of immune cell infiltration in neuroinflammatory demyelinating diseases; yet, the causal link between inflammation and glutamate dysregulation is not well understood. A major source of glutamate release during oxidative stress is the system xc− transporter, however, this mechanism has not been tested in animal models of autoimmune inflammatory demyelination. We find that pharmacological and genetic inhibition of system xc− attenuates chronic and relapsing-remitting experimental autoimmune encephalomyelitis (EAE). Remarkably, pharmacological blockade of system xc− seven days after induction of EAE attenuated T cell infiltration into the CNS, but not T cell activation in the periphery. Mice harboring a Slc7a11 (xCT) mutation that inactivated system xc− were resistant to EAE, corroborating a central role for system xc− in mediating immune cell infiltration. We next examined the role of the system xc− transporter in the CNS after immune cell infiltration. Pharmacological inhibitors of the system xc− transporter administered during the first relapse in a SJL animal model of relapsing-remitting EAE abrogated clinical disease, inflammation, and myelin loss. Primary co-culture studies demonstrate that myelin-specific CD4+ T helper type 1 (Th1) cells provoke microglia to release glutamate via the system xc− transporter causing excitotoxic death to mature myelin-producing OLs. Taken together these studies support a novel role for the system xc− transporter in mediating T cell infiltration into the CNS as well as promoting myelin destruction after immune cell infiltration in EAE.

Glutamate dysregulation occurs in multiple sclerosis (MS), but whether excitotoxic mechanisms in mature oligodendrocytes contribute to demyelination and axonal injury is unexplored. Although current treatments modulate the immune system, long-term disability ensues, highlighting the need for neuroprotection. Glutamate is elevated before T2-visible white matter lesions appear in MS. We previously reported that myelin-reactive T cells provoke microglia to release glutamate from the system xc− transporter promoting myelin degradation in experimental autoimmune encephalomyelitis (EAE). Here, we explore the target for glutamate in mature oligodendrocytes. Most glutamate-stimulated calcium influx into oligodendrocyte somas is AMPA receptor (AMPAR)–mediated, and genetic deletion of AMPAR subunit GluA4 decreased intracellular calcium responses. Inducible deletion of GluA4 on mature oligodendrocytes attenuated EAE and loss of myelinated axons was selectively reduced compared to unmyelinated axons. These data link AMPAR signaling in mature oligodendrocytes to the pathophysiology of myelinated axons, demonstrating glutamate regulation as a potential neuroprotective strategy in MS.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

One of the most important genes up‐regulated in cancer is known as Matrix Metalloproteinase (MMPs). MMP’s are enzymes critical for remodeling the protein matrix, called extracellular matrix (ECM), that surrounds and supports cells. This protein can be secreted or membrane‐bound as it remodels the extracellular environments in healthy individuals. However, in cancerous cells, its overexpression leads to the metastasis of cancer cells. To investigate novel ways to block metastasis, our lab engineered a new cellular Two‐Tag Assay as a tool for drug discovery, enabling the search for inhibitors. The assay, based on a system we developed for HIV and Dengue Virus, was adapted to MMP‐14. It relies on a scaffold composed of two antibody epitopes, HA and FLAG, flanking a protein substrate. Because the FLAG epitope is encoded upstream of the substrate and HA, it is cleaved while HA remains attached to the cell membrane. We then use flow cytometry to monitor the presence of HA and FLAG. Based on the presence of one or two tags, we can determine the robustness of cleavage. Previously, we inserted viral proteases between the tags and showed autocatalytic cleavage or lack of while the protein travels through the classical secretory pathway. The MMP‐14 assay has a consensus substrate of MMP‐14 instead of a viral protease, as well as a cell line obtained through retroviral technology. In addition, we expressed MMP‐14 enzyme in cells expressing substrate, as well as naïve cells. Comparing substrate‐expressing cells with substrate and enzyme expressing cells, we showed that MMP‐14 cleaves the substrate. We performed mixing experiments with substrate‐expressing and enzyme‐expressing cells to specifically corroborate cleavage at the cell surface. Results were corroborated with confocal microscopy and western blotting. Decrease in FLAG expression in the presence of MMP‐14 proved the assay’s ability to discover new inhibitors of MMP‐14. Support or Funding Information This research is supported by fellow researchers in the Wolkowicz Lab and the National Cancer Institute of the National Institutes of Health under award numbers: U54CA132384 & U54CA132379