No abstract

The developed multisite BBB chip is expected to be used for screening drug by more accurately predicting their permeability through BBB as well as their toxicity.

Neuroinflammation and increased production of tumor necrosis factor (TNF) in the CNS have been implicated in many neurological diseases including white matter disorders periventricular leukomalacia and multiple sclerosis. However, the exact role of TNF in these diseases and how it mediates oligodendrocyte injury remain unclear. Previously, we demonstrated that lipopolysaccharide (LPS) selectively kills oligodendrocyte precursors (preOLs) in a non-cell autonomous fashion through the induction of TNF in mixed glial cultures. Here, we report that activation of oligodendroglial, but not astroglial and microglial, TNFR1 is required for LPS toxicity, and that astrocytes promote TNF-mediated preOL death through a cell contact-dependent mechanism. Microglia were the sole source for TNF production in LPS-treated mixed glial cultures. Ablation of TNFR1 in mixed glia completely prevented LPS-induced death of preOLs. TNFR1-expressing preOLs were similarly susceptible to LPS treatment when seeded into wildtype and TNFR1 Using an in vitro model for inflammatory injury to preOLs, we demonstrated that LPS induces selective preOL death indirectly by activating microglia (Li et al. , 2008. We further identified that peroxynitrite underlies the potent direct killing capability of activated microglia and that astrocytes can shift the killing mechanism to one dependent on TNF signaling (Li et al. 2008). However, neither the cellular source for TNF production nor the TNF receptor mediating preOL death was identified. As primary glial cells all express TNF receptors (Dopp et al. 1997), they can all engage in TNF signaling. Here, we systematically dissected the cellular source for TNF production, determined the TNF receptor required for LPS toxicity, and further identified an essential role for oligodendroglial TNFR1 in TNF-mediated killing of preOLs in mixed glial cultures. Most importantly, we provided the first evidence demonstrating that astrocytes sensitize preOLs to TNF toxicity in a contact-dependent manner. Materials and methodsAnimals and reagents Wildtype B6.129SF2/J and C57BL/6J mice, transgenic eGFP mice (003291, background C57BL/6J), and TNF (background B6.129SF2/J) and TNFR1 (background C57BL/6J) knockout mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Recombinant TNF was obtained from R&D Systems (Minneapolis, MN, USA). Platelet-derived growth factor and basic fibroblast growth factor were from PeproTech (Rocky Hill, NJ, USA). Recombinant green fluorescent protein (GFP) adenovirus was from Gene Transfer Vector Core, University of Iowa. Rabbit polyclonal antibodies against Iba1 were purchased from Wako Chemicals (Richmond, VA, USA). Rat anti-mouse TNF (clone MP6-XT22) was obtained from eBioscience (San Diego, CA, USA). Olig2 antibody was a generous gift from Dr. Richard Lu (University of Texas Southwestern Medical Center). Lactate dehydrogenase (LDH) cytotoxicity kit was from Roche Applied Science (Indianapolis, IN, USA). Unless specified otherwise, all other reagents were from Sigma (St. Lou...

Oligodendroglial injury is a pathological hallmark of many human white matter diseases, including multiple sclerosis and periventricular leukomalacia. Critical regulatory mechanisms of oligodendroglia destruction, however, remain incompletely understood. Ceramide, a bioactive sphingolipid pivotal to sphingolipid metabolism pathways, regulates cell death in response to diverse stimuli and has been implicated in neurodegenerative disorders. We report here that ceramide accumulates in reactive astrocytes in active lesions of multiple sclerosis and periventricular leukomalacia, as well as in animal models of demyelination. Serine palmitoyltransferase, the rate-limiting enzyme for ceramide de novo biosynthesis, was consistently upregulated in reactive astrocytes in the cuprizone mouse model of demyelination. Mass spectrometry confirmed the upregulation of specific ceramides during demyelination and revealed a concomitant increase of sphingosine as well as a suppression of sphingosine-1-phosphate, a potent signaling molecule with key roles in cell survival and mitogenesis. Importantly, this altered sphingolipid metabolism during demyelination was restored upon active remyelination. In culture, ceramide acted synergistically with tumor necrosis factor leading to apoptotic death of oligodendroglia in an astrocyte-dependent manner. Taken together, our findings implicate that disturbed sphingolipid pathways in reactive astrocytes may indirectly contribute to oligodendroglial injury in cerebral white matter disorders.

No abstract

Growth capability of neurons is an essential factor in axon regeneration. To better understand how microenvironments influence axon growth, methods that allow spatial control of cellular microenvironments and easy quantification of axon growth are critically needed. Here, we present a microchip capable of physically guiding the growth directions of axons while providing physical and fluidic isolation from neuronal somata/dendrites that enables localized biomolecular treatments and linear axon growth. The microchip allows axons to grow in straight lines inside the axon compartments even after the isolation; therefore, significantly facilitating the axon length quantification process. We further developed an image processing algorithm that automatically quantifies axon growth. The effect of localized extracellular matrix components and brain-derived neurotropic factor treatments on axon growth was investigated. Results show that biomolecules may have substantially different effects on axon growth depending on where they act. For example, while chondroitin sulfate proteoglycan causes axon retraction when added to the axons, it promotes axon growth when applied to the somata. The newly developed microchip overcomes limitations of conventional axon growth research methods that lack localized control of biomolecular environments and are often performed at a significantly lower cell density for only a short period of time due to difficulty in monitoring of axonal growth. This microchip may serve as a powerful tool for investigating factors that promote axon growth and regeneration.

Recent evidence suggests that the oral drug Fingolimod (FTY720) for relapsing-remitting multiple sclerosis (MS) may act directly on the central nervous system (CNS) and modulate disease pathogenesis and progression in experimental models of MS. However, the specific subtype of sphingosine-1-phosphate (S1P) receptors that mediates the effect of FTY720 on the CNS cells has not been fully elucidated. Here, we report that S1P receptor 1 (S1PR1) is elevated in reactive astrocytes in an autoimmunity independent mouse model of MS and that selective S1PR1 modulation is sufficient to ameliorate the loss of oligodendrocytes and demyelination. The non-selective S1PR modulator, FTY720, or a short-lived S1PR1-specific modulator, CYM5442, was administered daily to mice while on cuprizone diet. Both FTY720- and CYM5422-treated mice displayed a significant reduction in oligodendrocyte apoptosis and astrocyte and microglial activation in comparison to vehicle-treated groups, which was associated with decreased production of proinflammatory mediators and down-regulation of astrocytic S1PR1 protein. Interestingly, S1PR1 modulation during the early phase of cuprizone intoxication was required to suppress oligodendrocyte death and consequent demyelination as drug treatment from 10 days after the initiation of cuprizone feeding was no longer effective. CYM5442 treatment during the brief cuprizone exposure significantly prevented Il-1β, Il-6, Cxcl10, and Cxcl3 induction, resulting in suppression of subsequent reactive gliosis and demyelination. Our study identifies functional antagonism of S1PR1 as a major mechanism for the protective effect of FTY720 in the cuprizone model and suggests pathogenic contributions of astrocyte S1PR1 signaling in primary demyelination and its potential as a therapeutic target for CNS inflammation.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Dysregulation of STAT3, a transcription factor pivotal to various cellular processes including Th17 cell differentiation, has been implicated in MS. Here, we report that STAT3 is activated in infiltrating monocytic cells near active MS lesions and that activation of STAT3 in myeloid cells is essential for leukocyte infiltration, neuroinflammation, and demyelination in experimental autoimmune encephalomyelitis (EAE). Genetic disruption of Stat3 in peripheral myeloid lineage cells abrogated EAE, which was associated with decreased antigen-specific T helper cell responses. Myeloid cells from immunized Stat3 mutant mice exhibited impaired antigen-presenting functions and were ineffective in driving encephalitogenic T cell differentiation. Single-cell transcriptome analyses of myeloid lineage cells from preclinical wild-type and mutant mice revealed that loss of myeloid STAT3 signaling disrupted antigen-dependent cross-activation of myeloid cells and T helper cells. This study identifies a previously unrecognized requisite for myeloid cell STAT3 in the activation of myelin-reactive T cells and suggests myeloid STAT3 as a potential therapeutic target for autoimmune demyelinating disease.