Multiple Sclerosis (MS) is a debilitating neurologic disease affecting young adults. A central issue for the treatment of MS is how cell signaling pathways regulate oligodendrocyte cell survival and remyelination after the inflammatory response. Oligodendrocytes are the myelinating cells of the central nervous system; their fine-tuning and survival is paramount for normal neurological function. Toward this goal, we have used a molecular approach to identify several genes implicated in signaling pathways that regulate oligodendrocyte and neuronal survival.  One group of genes encodes proteins belonging to the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases.  TAM are located at the cell surface and bind the growth factor growth arrest-specific protein 6 (Gas6).  Gas6 binding serves as a signal that subsequently activates downstream signaling proteins that have multiple functions in the oligodendrocyte including protection of oligodendrocytes from cell death via the PI3 kinase/AKT pathway. Gas6 signaling is involved in clearance of apoptotic cells and myelin debris following an immune attack.  In established MS lesions, increased protease activity upregulates Axl and Mer and negatively correlates with Gas6 reducing the interaction of Gas6 with surface-bound receptors.  We have initiated in vivo studies to determine whether Gas6 is therapeutic following an inflammatory attack in the CNS in mouse models of neuroimmune demyelination.  Another ongoing project is examining the role of AKT3 in the CNS and in T cell cell function.  AKT3 is the major AKT isoform expressed in brain (50%), AKT3^-/-mice, transgenic and conditional knockout mice will be examined for their role in neuronal survival resulting from inflammatory attacks to the CNS.  Immunohistochemical analysis revealed decreased numbers of Foxp3+ regulatory T cells in the spinal cord of AKT3 null mice compared with WT mice during MOG-induced EAE, whereas in vitro suppression assays showed that AKT3-deficient Th cells were less susceptible to regulatory T cell–mediated suppression than their WT counterparts. These results indicate that AKT3 signaling contributes to the protection of mice against EAE.

Ongoing projects include applying techniques of molecular & cell biology, biochemistry, immunocytochemistry, confocal and electron microscopy to address questions concerning the structure, function and regulation of myelination in the normal CNS and remyelination following neuroimmune injury.

John GR, Shankar SL, Shafit-Zagardo B, Massimi A, Lee SC, Raine CS, Brosnan C.  2002.  Multiple Sclerosis: Re-expression of a developmental pathway that restricts remyelination. Nature Med 8:1115-1121.

Shankar SL, O’Guin K, McMorris FA, Varnum B, Basch RS, Shafit-Zagardo B.  2003. The growth arrest-specific gene product Gas6 promotes the survival of human oligodendrocytes via a phosphatidylinositol 3-kinase-dependent pathway.  J Neurosci 23(10):4208-4218.

Shankar SL, O’Guin K, Kim, M, Varnum B, Lemke G, Brosnan CF, Shafit-Zagardo B.  2006.  Gas6/Axl signaling activates the phosphatidylinositol 3-kinase/Akt1 survival pathway to protect oligodendrocytes from tumor necrosis factor alpha-induced apoptosis. J Neurosci 26:5638-5648.

Weinger JG, Gohari P, Yan Y, Backer JM, Varnum B, Shafit-Zagardo B. 2008.  In brain, Axl recruits Grb2 and the p85 regulatory subunit of PI3 kinase; in vitro mutagenesis defines the requisite binding sites for downstream Akt activation. J Neurochem. 106:134-146.

Weinger JG, Omari KM, Marsden K, Raine CS, Shafit-Zagardo B.  2009. Upregulation of soluble Axl and Mer receptor tyrosine kinases negatively correlates with Gas6 in established multiple sclerosis lesions.  Am. J. Pathol. 175:283-293.

Tsiperson V, Li X, Schwartz G, Raine CS, Shafit-Zagardo B.  2010. Gas6 enhances clearance of myelin debris, remyelination and axonal survival after cuprizone toxicity.  PlosOne.  Dec 23;5(12):e15748.

Weinger JG, Brosnan CF, Loudig O, Goldberg MF, Macian F, Arnett H, Prieto AL, Tsiperson V, Shafit-Zagardo B.  2011.  Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis.  J Neuroinflammation.  8:49-56.

Weinger JG, Davies P, Acker CM, Brosnan CF, Tsiperson V, Bayewitz A, Shafit-Zagardo B. 2012. Mice devoid of Tau have increased susceptibility to neuronal damage in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. J Neuropathol Exp Neurol. 71:422-433.

Tsiperson V, Gruber R, Goldberg M, Jordan A, Weinger JG, Macian F, Shafit-Zagardo B. 2013. Suppression of Inflammatory Responses during Myelin Oligodendrocyte Glycoprotein–Induced Experimental Autoimmune Encephalomyelitis Is Regulated by AKT3 Signaling.  J.  Immunol. 190:1528-1539. 

 O’Guin KN, Raine CR, Gruber RC, Poulus BK, Shafit-ZagardoB. 2014. Gas6 enhances axonal ensheathment by MBP⁺ membranous processes in human DRG/OL promyelinating co-cultures,  ASN Neuro.  ASN NEURO 6(1):art:e00135.doi:10.1042/AN20130022. 

Gruber RC, Ray A, Guzik HM, Burek D, García de Frutos P, Kaspar B, Shafit-Zagardo B.  2014. Targeted growth-arrest specific protein 6 (Gas6) delivery to the CNS protects axons from damage during experimental autoimmune encephalomyelitis (EAE).  J. Neurosci. 34:16320-16335.

Ray A, DuBois JC, Gruber RC, Guzik HM, Gulinello ME, Perumal G, Raine C, Kozakiewicz L, Williamson J, Shafit-Zagardo B. Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation and less remyelination following cuprizone exposure. Glia, 65(12):2051-2069, 2017. PMID: 28925029

Shafit-Zagardo B, Gruber RC, DuBois JC. The role of TAM family receptors and ligands in the nervous system: From development to pathobiology. Pharmacol Ther. 188:97-117, 2018. doi: 10.1016/j.pharmthera.2018.03.002.

Mocholi E, Dowling SD, Botbol Y, Gruber RC, Ray AK, Vastert S, Shafit-Zagardo B, Coffer PJ, Macian F. Autophagy Is a Tolerance-Avoidance Mechanism that Modulates TCR-Mediated Signaling and Cell Metabolism to Prevent Induction of T Cell Anergy.
Cell Rep. 24(5):1136-1150, 2018. PMID: 30067971

DuBois JC, Ray AK, Gruber RC, Zhang Y, Aflakpui R, Macian-Juan F, Shafit-Zagardo B. Akt3-Mediated Protection Against Inflammatory Demyelinating Disease. Front Immunol. 10:1738, 2019. PMID: 31404142

DuBois JC, Ray AK, Davies P, Shafit-Zagardo B. Anti-Axl antibody treatment reduces the severity of experimental autoimmune encephalomyelitis. J Neuroinflammation. 17(1):324, 2020. PMID: 33121494