Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Molecular Biology and Genetics

First Advisor

Jeffrey A. Loeb

Abstract

During peripheral nervous system development, successful communication between axons and glial cells including Schwann cells in peripheral nervous system and oligodendrocytes in central nervous system, is required for the proper functions of both neurons and glia. Three types of alternatively-spliced proteins belonging to the neuregulin1 (NRG1) gene family of growth and differentiation factors are essential for Schwann cell survival and peripheral nerve development. While membrane-bound NRG1 forms (type III) has been strongly implicated in the regulation of myelination process at late stage of Schwann cell development, little is known about the role of soluble, heparin-binding forms of NRG1 (type I/II) in regulating early Schwann cell development in vivo. These forms are rapidly released from axons in vitro by Schwann cell-secreted neurotrophic factors, and, unlike membrane-bound forms, have a unique ability to diffuse and adhere to heparan sulfate-rich cell surfaces. We harness this natural targeting ability of soluble NRG1 to develop a novel antagonist by fusing its heparing-binding domain (HBD) to the soluble human epidermal growth factor receptor 4 (HER4). This fusion protein retains high affinity for heparin binding and to specific cell surface that express heparan sulfates resulting in a much more potent NRG1 antagonist than any other inhibitors for this molecule. In vivo, it is targeted to peripheral nerve segments where endogenous soluble NRG1 binds to and efficiently blocks the activity of NRG1 as a Schwann cell survival factor, leading to significant cell apoptosis in both motor and sensory axon area dose-dependently.

In this thesis work, we also show that axon-derived soluble NRG1 translocates from axonal to Schwann cell surfaces in the embryonic chick between days 5-7, corresponding to the critical period of Schwann cell survival during the normal development of peripheral nervous system. Down-regulating endogenous soluble NRG1 signaling with the targeted antagonist or shRNA via chick in ovo electroporation, blocks their differentiation from precursors into immature Schwann cells and increases programmed cell death, while up-regulating NRG1 rescues Schwann cells from normal-occurring apoptosis. Furthermore, exogenous BDNF also promotes Schwann cell survival through promoting the local release of axonal NRG1 by binding axonal trkB or p75 receptor. Consistently, increased Schwann cell death occurs both in trkB knock-out mice and after knocking-down axonal trkB in chick embryos, which can then be rescued with soluble NRG1. These findings suggest a localized, axoglial feedback loop through soluble NRG1 and BDNF critical for early Schwann cell survival and differentiation in vivo, which may not only be important for the axoglial communication, but may also be helpful in understanding nervous system diseases that involve the axoglial interface and the providing better therapeutic strategies for their treatments.