Lineage-Specific Metabolic Properties and Vulnerabilities of T Cells in the Demyelinating Central Nervous System.

Multiple sclerosis (MS) is a disease that is characterized by immune-mediated destruction of CNS myelin. Current MS therapies aim to block peripheral immune cells from entering the CNS. Although these treatments limit new inflammatory activity in the CNS, no treatment effectively prevents long-term disease progression and disability accumulation in MS patients. One explanation for this paradox is that current therapies are ineffective at targeting immune responses already present in the CNS. To this end, we sought to understand the metabolic properties of T cells that mediate ongoing inflammation in the demyelinating CNS. Using experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, a well-studied model of MS, we showed that the CD4+ and CD8+ T cells that invade the EAE CNS are highly glycolytic. Elevated glycolytic rates in T cells isolated from the EAE CNS correlate with upregulated expression of glycolytic machinery and is essential for inflammatory responses to myelin. Surprisingly, we found that an inhibitor of GAPDH, 3-bromopyruvic acid (3-BrPa), blocks IFN-γ, but not IL-17A, production in immune cells isolated from the EAE CNS. Indeed, in vitro studies confirmed that the production of IFN-γ by differentiated Th1 cells is more sensitive to 3-BrPa than is the production of IL-17A by Th17 cells. Finally, in transfer models of EAE, 3-BrPa robustly attenuates the encephalitogenic potential of EAE-driving immune cells. To our knowledge, these data are among the first to demonstrate the metabolic properties of T cells in the demyelinating CNS in vivo.

LRP1 expression in microglia is protective during CNS autoimmunity.

Multiple sclerosis is a devastating neurological disorder characterized by the autoimmune destruction of the central nervous system myelin. While T cells are known orchestrators of the immune response leading to MS pathology, the precise contribution of CNS resident and peripheral infiltrating myeloid cells is less well described. Here, we explore the myeloid cell function of Low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor involved in myelin clearance and the inflammatory response, in the context of Multiple sclerosis. Supporting its central role in Multiple sclerosis pathology, we find that LRP1 expression is increased in Multiple sclerosis lesions in comparison to the surrounding healthy tissue. Using two genetic mouse models, we show that deletion of LRP1 in microglia, but not in peripheral macrophages, negatively impacts the progression of experimental autoimmune encephalomyelitis, an animal model of Multiple sclerosis. We further show that the increased disease severity in experimental autoimmune encephalomyelitis is not due to haplodeficiency of the Cx3cr1 locus. At the cellular level, microglia lacking LRP1 adopt a pro-inflammatory phenotype characterized by amoeboid morphology and increased production of the inflammatory mediator TNF-α. We also show that LRP1 functions as a robust inhibitor of NF-kB activation in myeloid cells via a MyD88 dependent pathway, potentially explaining the increase in disease severity observed in mice lacking LRP1 expression in microglia. Taken together, our data suggest that the function of LRP1 in microglia is to keep these cells in an anti-inflammatory and neuroprotective status during inflammatory insult, including experimental autoimmune encephalomyelitis and potentially in Multiple sclerosis.

Neuroprotective targets through which 6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79), a sigma receptor ligand, mitigates the effects of methamphetamine in vitro.

Exposure to high or repeated doses of methamphetamine can cause hyperthermia and neurotoxicity, which are thought to increase the risk of developing a variety of neurological conditions. Sigma receptor antagonism can prevent methamphetamine-induced hyperthermia and neurotoxicity, but the underlying cellular targets through which the neuroprotection is conveyed remain unknown. Differentiated NG108-15 cells were thus used as a model system to begin elucidating the neuroprotective mechanisms targeted by sigma receptor antagonists to mitigate the effects of methamphetamine. In differentiated NG108-15 cells, methamphetamine caused the generation of reactive oxygen/nitrogen species, an increase in PERK-mediated endoplasmic reticulum stress and the activation of caspase-3, -8 and -9, ultimately resulting in apoptosis at micromolar concentrations, and necrotic cell death at higher concentrations. The sigma receptor antagonist, 6-acetyl-3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one (SN79), attenuated methamphetamine-induced increases in reactive oxygen/nitrogen species, activation of caspase-3, -8 and -9 and accompanying cellular toxicity. In contrast, 1,3-di(2-tolyl)-guanidine (DTG), a sigma receptor agonist, shifted the dose response curve of methamphetamine-induced cell death towards the left. To probe the effect of temperature on neurotoxicity, NG108-15 cells maintained at an elevated temperature (40 °C) exhibited a significant and synergistic increase in cell death in response to methamphetamine, compared to cells maintained at a normal cell culture temperature (37 °C). SN79 attenuated the enhanced cell death observed in the methamphetamine-treated cells at 40 °C. Together, the data demonstrate that SN79 reduces methamphetamine-induced reactive oxygen/nitrogen species generation and caspase activation, thereby conveying neuroprotective effects against methamphetamine under regular and elevated temperature conditions.

A 96-well filtration method for radioligand binding analysis of σ receptor ligands.

σ receptors represent a potential drug target for numerous therapeutic indications including cancer, depression, psychostimulant abuse, and stroke. Most published radioligand binding studies for σ receptors utilize a low throughput method employing a "cell harvester." Higher throughput methods are required to facilitate efficient screening of large numbers of novel compounds. In this study, a series of reference compounds was analyzed with a new medium-throughput 96-well filtration method and the results were compared to those obtained using the conventional cell harvester-based method. The 96-well assay utilized rat liver membranes for the determination of both known σ receptor subtypes (σ(1) and σ(2)) because this tissue contains high densities of both subtypes and fulfills criteria required for reliable use with the 96-well format. The new method gave comparable K(i) values for reference ligands analyzed in parallel with samples prepared in rat brain membranes and processed on the traditional cell harvester. For σ(1) receptors, equivalent affinity values were observed for both methods/tissues. For σ(2) receptors, approximately 2-fold higher affinities were observed for most compounds in liver, as compared to brain membranes, but excellent correlation with brain-derived values was maintained. To further demonstrate the utility of the new method it was used to screen a novel series of 2(3H)-benzothiazolone compounds, resulting in the identification of several analogues with nanomolar affinity and greater than 50-fold specificity for σ(1) versus σ(2) receptors.