Date of Award

January 2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

John A. Watt

Abstract

Ciliary neurotrophic factor (CNTF) is believed to promote neuronal sprouting and survival within the magnocellular neurosecretory system (MNS) by means of astrocyte activation following injury. When CNTF binds to its receptor complex, CNTF receptor alpha, located on the extracellular surface of the astrocyte, it initiates the Jak/STAT3 pathway. The activation of this pathway and the translocation of phosphorylated STAT3 (pSTAT3) to the nucleus is believed to lead to the release of multiple factors that act in a paracrine manner to influence survival and sprouting of MNS neurons. To confirm the ability of CNTF to activate the astrocyte, the three receptor components were confirmed to be present by means of immunocytochemistry and/or Western Blotting on in-vitro rat primary astrocyte cell cultures. Next activation of the astrocyte, translocation of pSTAT3 to the nuclease, was measured using Nuclear Extractions of cell cultures that had been incubated in rat recombinant CNTF (rrCNTF) and compared to non-treated cell cultures. Western blotting of the nuclear extractions indicates that rrCNTF activation of the Jak/STAT3 pathway occurs within 30 minutes of application. Using Rat Cytokine Antibody Arrays from RayBio, the comparison of 72 hour rrCNTF treated astrocyte cell cultures supernatants to non-treated, showed an increase release of roughly 20% in several factors; Fractalkine (66.67%), IL-6 (22.22%), LIX (18.42), and VEGF (36.54%). Further

analysis of Fractalkine, IL-6, LIX and VEGF by means an Elisa on the cytosolic portions of nuclear extractions and the corresponding supernatants did not indicate any significant changes in protein production or release. To test levels of RNA multiple real time reverse transcriptase polymerase chain reaction (RT² PCR) profiler arrays were performed on RNA collected from both control and rrCNTF treated astrocyte cell cultures after 6, 12 or 24 hour incubations. The gene analysis indicates numerous changes falling beyond a threefold cut off. The inhibition of the Jak/STAT3 pathway within the cultured astrocytes was tested by the application of AG490 to inhibit Jak2 and cucurbitacin to inhibit STAT3. Within the cultured astrocytes, AG490 did not successfully inhibit the CNTF activated pathway, although the inhibition of STAT3 by cucurbitacin arrested the activation process. To test if astrocytes do indeed release neuronal promoting factors, astrocyte conditioned media from a non-treated control, rrCNTF treated, AG490, AG490 plus rrCNTF, cucurbitacin and cucurbitacin plus rrCNTF treatments was applied to hypothalamic organotypic explant cultures and the MNS neurons were counted and compared. The comparison showed a significant increase in neuronal survival for explants exposed to CNTF as well as AG490 plus CNTF. This indicates that AG490 does not inhibit the CNTF activation of cultured astrocytes. The exposure to cucurbitacin does effectively prevent the astrocytes from becoming activated by CNTF, preventing the release of pro-survival neuronal factors. In summary, CNTF potentiates the survival of axotomized magnocellular neurons by means of the resident astrocytes.

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