Contributions
Abstract: P459
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Neurobiology
Background: Fingolimod (FTY720; Gilenya), when phosphorylated, is an S1P analog that has high affinity interactions with four of five S1P receptor (S1PR) subtypes, S1P1,3-5. It has been proposed to work by sequestration of pathogenic lymphocyte subsets in secondary lymphoid organs. However, it also enters the CNS where it can bind to multiple S1PR subtypes on cells from neural and non-neural lineages. In particular, astrocytes show internalization and functional S1P1 loss during fingolimod exposure, which is in part responsible for its efficacy in experimental autoimmune encephalomyelitis (EAE). The present goal was to better understand the astrocyte effects of S1P signaling modulation.
Methods and results: A transgenic mouse line with a tetracycline-suppressible cis element (tTA) and a c-Fos driven nuclear GFP-histone fusion protein was crossed with an astrocyte specific, conditional S1P1-null mouse line. Monophasic EAE was induced, then tTA activated upon onset of EAE signs, allowing c-Fos activated cells to express a semi-permanent GFP signal. Activated astrocytes were visualized in the intact lower spinal cord using tissue clearance techniques that identified columnar structures in the cord"s long axis, particularly in the periphery. Astrocytes were the predominantly activated cell-type. The extent of activation correlated with disease severity as demonstrated by flow cytometry. Fingolimod administration or astrocyte targeted S1P1 removal reduced the number of activated astrocytes. GFP-positive EAE-activated astrocytes were isolated by FACS and processed for RNAseq, revealing differential transcriptional changes.
Conclusions: Astrocyte activation correlates with clinical severity; both are reduced by genetic or pharmacological S1P1 signaling loss in murine EAE. Examination of the lower spinal cord by using tissue clearance technologies during the course of EAE development identified increases in activated astrocytes within lesions that produced discrete 3-dimensional geometries in the spinal cord. Distinct transcriptional changes in isolated astrocytes processed by FACS and RNAseq during EAE were identified and will be discussed.
Disclosure:
JC has received compensation for serving as a consultant or speaker, or having received research funds from AbbVie, Amira Pharmaceuticals, Arena, Biogen Idec, Cellular Bioengineering Inc., GlaxoSmithKline, Johnson & Johnson, Novartis, Pfizer, Ono Pharmaceuticals, and Taisho Pharmaceutical Corporation.
Abstract: P459
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Neurobiology
Background: Fingolimod (FTY720; Gilenya), when phosphorylated, is an S1P analog that has high affinity interactions with four of five S1P receptor (S1PR) subtypes, S1P1,3-5. It has been proposed to work by sequestration of pathogenic lymphocyte subsets in secondary lymphoid organs. However, it also enters the CNS where it can bind to multiple S1PR subtypes on cells from neural and non-neural lineages. In particular, astrocytes show internalization and functional S1P1 loss during fingolimod exposure, which is in part responsible for its efficacy in experimental autoimmune encephalomyelitis (EAE). The present goal was to better understand the astrocyte effects of S1P signaling modulation.
Methods and results: A transgenic mouse line with a tetracycline-suppressible cis element (tTA) and a c-Fos driven nuclear GFP-histone fusion protein was crossed with an astrocyte specific, conditional S1P1-null mouse line. Monophasic EAE was induced, then tTA activated upon onset of EAE signs, allowing c-Fos activated cells to express a semi-permanent GFP signal. Activated astrocytes were visualized in the intact lower spinal cord using tissue clearance techniques that identified columnar structures in the cord"s long axis, particularly in the periphery. Astrocytes were the predominantly activated cell-type. The extent of activation correlated with disease severity as demonstrated by flow cytometry. Fingolimod administration or astrocyte targeted S1P1 removal reduced the number of activated astrocytes. GFP-positive EAE-activated astrocytes were isolated by FACS and processed for RNAseq, revealing differential transcriptional changes.
Conclusions: Astrocyte activation correlates with clinical severity; both are reduced by genetic or pharmacological S1P1 signaling loss in murine EAE. Examination of the lower spinal cord by using tissue clearance technologies during the course of EAE development identified increases in activated astrocytes within lesions that produced discrete 3-dimensional geometries in the spinal cord. Distinct transcriptional changes in isolated astrocytes processed by FACS and RNAseq during EAE were identified and will be discussed.
Disclosure:
JC has received compensation for serving as a consultant or speaker, or having received research funds from AbbVie, Amira Pharmaceuticals, Arena, Biogen Idec, Cellular Bioengineering Inc., GlaxoSmithKline, Johnson & Johnson, Novartis, Pfizer, Ono Pharmaceuticals, and Taisho Pharmaceutical Corporation.