Neurotrophin Signaling Laboratory

Lab Interests | Techniques | PhD and Honours Projects | Contacts

The Neurotrophin Signaling Laboratory has primary interests in:

• initiation and regulation of neurotrophin signaling
• characterisation of the neurotrophin receptor NRH2
• examining the role the neurotrophins play in regulating central and peripheral nervous system myelination

The focus of this laboratory centres on a family of growth factors essential for normal nervous system function, called the neurotrophins. We are interested in several aspects of neurotrophin biology, particularly neurotrophin signaling and the role that the neurotrophins play in the myelinating process. We use a variety of biochemical, molecular and cellular techniques to investigate these events.

The laboratory is located in the Centre for Neuroscience, on the 7 th floor of the Medical Building at the University of Melbourne . The lab has close collaborative links with the Multiple Sclerosis Research Group at the Howard Florey Institute.

Group Leader

Simon Murray

Research Fellow

Junhua Xiao

Research Assistant

Lab Interests

Mechanism of Neurotrophin Signal Transduction.

The neurotrophins regulate diverse and apparently opposing biological functions in the Central and Peripheral Nervous Systems. Nerve growth factor (NGF), the prototypical neurotrophin, has long been known for its effects on neuronal cell survival and differentiation. The actions of neurotrophins, such as NGF, are mediated by an unusual transduction system that utilizes two distinct classes of transmembrane receptors: the Trk family of receptor tyrosine kinases, and the p75 neurotrophin receptor (p75NTR), a member tumor necrosis factor receptor superfamily. The neurotrophins can signal independently through each of these two receptors, or utilise a receptor complex consisting of both these receptors to initiate a diverse range of signalling events. The Trk receptors are well characterised as initiating survival signals, however the neurotrophins are also capable of inducing a cell death signal through p75NTR. This represents a biological paradox, in which life and death decisions in the nervous system are dependent upon the expression and action of two receptors with distinctive signaling mechanisms.

This paradox can partly be explained on the basis that the neurotrophins are initially synthesized as precursors (pro-neurotrophins) that are post-transationally cleaved to produce ‘mature' proteins. These mature proteins act as ligands for the Trk family of receptor tyrosine kinases. Recent studies indicate that the pro-neurotrophins may serve as signalling molecules that specifically interact with p75NTR, and exert biological effects opposite to that of the mature neurotrophins. Therefore, the proteolytic cleavage of pro-neurotrophins represents a mechanism that controls the diverse function of the neurotrophins.

In addition, neurotrophin receptor co-expression is a critical factor in determining outcome of neurotrophin signalling. The recent identification of the Neurotrophin Receptor Homolog (NRH), a transmembrane protein highly similar to the p75NTR neurotrophin receptor, raises new questions on the role that receptor co-expression plays in regulating neurotrophin signaling and biology. We have recently shown that the affinity of NGF for TrkA is increased when NRH2 is co-expressed, despite the fact that NGF does not bind to NRH2. In addition, we have shown that this high-affinity NGF binding regulates TrkA signaling, selectively enhancing Erk activation. We have identified a highly-conserved region in the juxtamembrane domain of NRH2 and p75NTR that mediates this effect. These data indicate that the co-expression of neurotrophin receptors represents another layer of complexity in regulating neurotrophin biology. We are currently continuing our analysis of NRH2, and post-graduate projects are available in this field.

The Role that the Neurotrophins play in Regulating Myelination.

The neurotrophins have also been recently identified as key modulators of nervous system myelination. In addition to receptor signal transduction, our laboratory is also interested in defining the mechanisms that the neurotrophins utilize to promote myelination by oligodendrocytes and Schwann cells. Specifically, we are interested in identifying the receptor complexes and cell types that the neurotrophins act through to regulate myelination. As a long-term goal, using a combination of molecular and cellular approaches, we are aiming to define the molecular signals that regulate myelination, both the axonally derived signals that regulate the permissiveness of axons to myelinaton and the glial derived signals (from oligodendrocyte and Schwann cells) that drive the myelination process.

We are currently utilising in vitro myelination assays to investigate the mechanisms of neurotrophin signal transduction that regulate myelination by oligodendrocytes and Schwann cells. In addition, in collaboration with the Multiple Sclerosis Research Group at the Howard Florey Institute, we are also analysing the effects other growth factors have on myelination, and investigating the role that multi-potential neural stem cells have in the myelination process. We are currently continuing our investigation into the molecular regulation of the myelination process, and post-graduate projects are available in this field.

Techniques

• Culture and co-culture of primary cells:

Dorsal Root Ganglion neurons, oligodendrocytes and Schwann cells

• Fluorescent immunostaining of cultured primary cells
• Fluorescent and Confocal Microscopy
• Western blot analysis of cultured primary cells
• Culture of cell lines
• Transfection of cloned DNA
• Generation of stable transfectants
• Western blotting and immunoprecipitaiton
• in vitro kinase assays
• PCR, RT-PCR
• cDNA subcloning
• Molecular generation of deletion, mutant and chimeric constructs
• Immunohistochemistry - paraffin and frozen sections

PhD and Honours Projects

The laboratory is currently offering 3 projects for post-graduate study:

1. Generating diversity in Nerve Growth Factor Signaling.

2. The Role of the Neurotrophins in Promoting Myelination.

3. Inhibiting Nerve Growth Factor Signaling.

Project Title: Generating diversity in Nerve Growth Factor Signaling.

Supervisors: Dr. Simon Murray

Project Overview:

Nerve Growth Factor (NGF) was the first identified and is the prototypic neurotrophic factor. It supports the development, survival and differentiation of multiple neuronal cell types and is absolutely required for normal neuronal development and function of the nervous system.

NGF signals through two distinct receptors, the TrkA tyrosine kinase receptor and the structurally unrelated p75 neurotrophin receptor. Recently, a new co-receptor for NGF was identified and termed the Neurotrophin Receptor Homolog (NRH). We have shown that NRH interacts with both receptors for NGF, and significantly alters the manner in which NGF binds to TrkA. Our unpublished data shows that NRH2 modulates specific downstream signaling cascades initiated by NGF.

This project will continue a biochemical and molecular analysis of NRH2, investigating the nature of its interaction with TrkA and p75, further analysing the sub-cellular signalling cascades it modulates, and determining its mechanism of action. This project will identify some of the vitally important cellular events that control the development and growth of the mammalian nervous system, and will potentially provide insight into new approaches that can be utilised to halt the progression of degenerative neurological diseases.

Project Title: The Role of the Neurotrophins in Promoting Myelination.

Supervisors: Dr. Simon Murray and Professor Trevor Kilpatrick

Project Overview:

Schwann cells and oligodendrocytes are the cells in the nervous system that are responsible for ensheathing neurons with myelin that is essential for normal brain function. Multiple sclerosis is a disease where these myelinating cells progressively die, disrupting normal neural function, leading to a progressive and debilitating disease.

The neurotrophins have recently been shown to modulate myelination during development and re-myelination following injury. They have been used to enhance Schwann cell and oligodendrocyte myelination, however the underlying mechanisms mediating these effects remain unknown. This project will examine the profile of neurotrophin receptors expressed in Schwann cells and oligodendrocytes, and determine the receptor complexes that are activated to enhance myelination. In addition, in vitro myelination assays will be generated to analyse the specific role that the neurotrophins play in the myelinating process.

The project will encompass biochemical, molecular and cellular techniques. The successful applicant will learn to:

• derive, manage and culture primary cells in vitro
• analyse neurotrophin receptor expression from tissue samples in vivo and cultured cells in vitro
• use biochemical analyses to determine the receptor complexes formed following neurotrophin activation

This project is designed to analyse the role the neurotrophins play in promoting the myelination process and identify potential new therapies to enhance nervous system myelination following de-myelinating insults such as Multiple Sclerosis.

Project Title: Inhibiting Nerve Growth Factor Signaling.

Supervisors: Dr. Simon Murray and Dr. Chris Hovens

This project involves a close collaboration with the Molecular Neurobiology Laboratory in the Department of Surgery at the Royal Melbourne Hospital .

Project Overview:

Normal development requires the highly co-ordinated and regulated control of cellular growth and differentiation. One family of receptors well known to regulate these events are the receptor tyrosine kinases. These receptors are activated by growth factors that induce positive signals for cellular growth.

Precise control of cellular growth requires not only the regulated activation of such receptors, but also the negative control or inhibition of signaling. Two recently identified protein families, the Spred and Sprouty proteins, act to antagonize these signalling cascades. They negatively regulate receptor tyrosine kinase activation, and have been shown to inhibit signaling through the growth factors EGF, FGF, and VEGF.

Recently, Spred has been identified as an inhibitor of the Nerve Growth Factor receptor TrkA. The TrkA receptor is predominantly expressed in neuronal cells and is essential for normal neuronal development. This project will examine the molecular interaction between Spred and TrkA, investigate the signaling cascades regulated by this interaction and determine the extent of co-expression between these proteins.

The project will encompass biochemical, molecular, and cellular techniques. The successful applicant will learn to:

• manage and passage cell lines in vitro
• examine the interaction between TrkA and Spred, and use molecular techniques to identify the protein domains required for this interaction
• analyse TrkA signalling to determine the signaling cascades regulated by Spred co-expression

determine the extent of in vivo co-expression of these proteins within the nervous system.

Contact the Lab

Neurotrophin Signaling Laboratory

Centre for Neuroscience

Level 7 West, Medical Building,

University of Melbourne VIC 3010 AUSTRALIA

Phone: (+61 3) 8344 7572

Email: ssmurray@unimelb.edu.au

Website: www.cns.unimelb.edu.au