Characterization of neuroprotective and immunoprotective signaling pathways in CNS demyelinating disease
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Multiple Sclerosis is a chronic inflammatory disease that affects the central nervous system (CNS) in approximately 400,000 people in the United States and around 2.4 million people worldwide. MS prevalence and incidence is highest in northern climates and in the last several decades has increased in certain areas. Current medications used to treat MS are almost entirely immunomodulatory. No treatments exist to prevent or repair CNS damage. Therefore, understanding signaling molecules and pathways affected in MS is of critical importance to the development of novel therapeutic modalities to repair CNS damage and modulating immune responses.;The Tyro3, Axl, and MerTK (TAM) family of receptor tyrosine kinases, and their ligands Gas6 and ProS1, are involved in innate immune responses and CNS homeostasis. In established chronic MS lesions, there is an inverse correlation between Gas6 and cleaved soluble Axl likely caused by the upregulation of proteases involved in receptor cleavage thereby limiting protective effects afforded by Gas6 signaling. Gas6 is the sole ligand for Axl. To better understand the role of this signaling axis, double knockout mice (DKO) lacking both Gas6 and Axl were generated. The cuprizone model of toxicity is an in vivo model used to study demyelination and remyelination and was employed to compare DKO and wild-type (WT) mice.;We found that naive young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Ibal+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS 1 /Tyro3/MerTK signaling.;Akt is a serine/threonine kinase involved in a wide variety of cellular functions including survival, apoptosis, proliferation, metabolism, activation, differentiation, and growth. There are three Akt isoform (Akt 1, Akt2, and Akt3) that have both distinct and overlapping functions. Akt3 is highly expressed in the CNS and represents ~50% of total Akt in brain and ~30% in spinal cord. Previous studies from our lab show that deletion of Akt3 results in worse clinical outcomes during MOG-induced EAE including increased neuroinflammation and more axonal damage. To further explore the role of Akt3 signaling during MOG-induced EAE, we utilized several mouse strains with different manipulations to Akt3 including mice with enhanced Akt3 kinase activity (NMF350 mice), mice with conditional deletion of Akt3 in CD4+ T cells (CD4 CKO mice), and mice with conditional deletion of Akt3 in neurons (Synl CKO mice). During EAE, NMF350 mice had lower clinical scores with a lag in disease onset, delayed influx of inflammatory cells into the CNS, decreased CNS inflammation, and less axonal damage relative to WT controls. CD4 CKO mice had a delay in disease onset relative to controls, but showed no difference in clinical or pathological outcomes during acute EAE disease. Examination of CD4+ T cell activation and Thl differentiation showed that Akt3 likely does not function in these settings. Additionally, Synl CKO mice showed no differences in EAE clinical outcomes or pathology relative to controls indicating that Akt3 signaling in neurons does not regulated axonal integrity in a CNS autoimmune setting. Lastly, Akt3 is known to regulate post-natal brain development as Akt3-/- mice have ~20% reduction in brain size. No differences were seen in brain sizes of adult Synl CKO mice relative to controls indicating that Akt3 signaling in neurons alone does not regulate brain size. Further characterization of Akt3 signaling in these settings is needed to better understand the contribution of Akt3 signaling during CNS autoimmune inflammation.
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