Mimicking a cure

A collaboration of Melbourne scientists who describe themselves as a “happy confluence” is shedding much-needed light on how the body controls nervous system myelination, and how their tiny peptide ‘mimic’ could help patients with neurodegenerative disease.

The team in question comprises University of Melbourne researchers Drs Simon Murray and Junhua Xiao from the Neurotrophin and Myelin Laboratory and Associate Professor Tony Hughes, who heads the Drug Design Laboratory … and they are even happier now, with the recent publication of their combined experimental efforts in the Journal of Neurochemistry. In a nutshell, this paper reports how a novel peptide mimetic (functional mimic) of a key neurotrophic factor promotes the myelination of peripheral neurons both in the laboratory culture dish and in rats.

These findings are important for the field of neuroscience in general because the work is revealing more than the researchers themselves ever expected about some of the basic mechanics of neurotrophin action, which have remained frustratingly elusive up to now. Importantly, the results are welcome news for those working to develop better treatments for neurological diseases and, of course, for people living with these debilitating diseases.

Meeting the players

The neurotrophins are a family of secreted neurotrophic factors that drive and regulate the development, function and survival of vertebrate nervous systems. As soluble molecules with the ability to affect neurological function, neurotrophins quickly became the subject of intense clinical and pharmaceutical interest when first discovered in the 1980s.

Their potential for use as therapeutic agents for treating neurodegenerative disorders and nerve injury looked promising. However, these very large proteins are cleared rapidly by the body before enough can get to the site of action to have the desired effect - and so neurotrophins themselves do not actually make very good drugs.

Neurotrophins exert their action on nerve cells through two biologically distinct classes of cell-surface receptors - the tropomyosin-related kinase (Trk) receptors and the p75 neurotrophin receptor (p75NTR). From there it just gets more complex and at times murky with activation of these different receptors by the same neurotrophin producing functionally different outcomes in different cells - much about the mechanics of neural regulation via these factors and their receptors remains unknown.

Of the four neurotrophins, brain-derived neurotrophic factor (BDNF) is the focus of this work. Although primarily a growth and survival factor, more recent evidence also indicates that BDNF has a crucial influence on nervous system myelination. The importance of myelination to healthy nerve function is evidenced by the large proportion of neurological diseases involving defective myelin formation or demyelination including multiple sclerosis, leukodystrophy and Guillain-Barré syndrome.

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The crucial partner

The myelin sheath around the axon of neurons is an essential structural feature that allows electrical transmission to occur with miraculous speed and, at times, over relatively huge distances. Myelination is accomplished by the outgrowth of adjacent glial cells, which are sort of like the ‘Robin’ of the nervous system to the ‘Batman’ neurons. In the peripheral nervous system (PNS), Schwann cells snuggle up to the neurons to wrap them in myelin, while oligodendrocytes do the business in the central nervous system (CNS).

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Establishing the confluence

According to Hughes, teaming up with Murray and Xiao a few years back came about through a shared interest in the molecular mechanisms of myelination, and in producing a clinically useful outcome based on their collective expertise. Indeed, the original shared goal was to develop potential therapeutic agents to induce new myelin formation or ‘fix’ defective myelination by targeting specific neural pathways.

A pharmacologist with a background in pharmaceutical and medicinal chemistry, Hughes had worked for many years designing and making peptide-based molecules as potential drugs. But it was his postdoc in Germany in the early 1990s that sparked a particular focus on neurotrophins.

“At that time, neurotrophic factors were going into clinical trials for treating neurological disorders, but the issues with their use as drugs quickly became pretty clear. So, when I came back to Australia in 1995, it made sense to start making small-molecule mimetics for these factors.”

Soon after, Murray, who had also trained in one of the big neurotrophin labs as a postdoc, began to help with some functional studies of a few small-molecule peptides. Hughes and his team had designed these peptides to mimic different regions of the BDNF molecule.

According to Murray, “one of our motivating hypotheses was that activating one of the receptors bound by BDNF and not the other using such a molecule might produce a more unified biological response compared to the diverse outcomes incurred with binding of the entire BDNF protein.”

Hughes soon found that one of his mimetics, a tiny circular peptide called cyclo-DPAKKR (CP) designed to mimic the region of BDNF binding the p75 receptor, promoted survival in a cell culture model and was indeed functionally relevant.

Team success

Meanwhile, back in their lab, Murray and Xiao’s interest in the effects of BDNF on myelination was growing. They had begun to see that BDNF could exert contrasting effects on peripheral myelination, depending on which receptor it was binding.

They realised that the structural-based approach of Hughes would be valuable in gaining the functional selectivity needed to demonstrate a therapeutic benefit of myelination in the clinic. Thus, the trio formed to start looking at the CP peptide’s effect, specifically on myelination.

Early experiments with the CP mimetic using a co-culture of peripheral neurons and Schwann cells (to represent an in-vitro model of peripheral nerve myelination) showed a strong and unified pro-myelinating effect, regardless of the type of neuron. This was just the result they were looking for because BDNF itself is only pro-myelinating in some types of peripheral neuron - it has inhibitory effects in others, even within the same population of nerve cells.

“These data were very clear - the peptide mimic was having only the one effect,” said Murray. “We then spent a lot of time confirming this mechanism of CP action in vitro to prove quite clearly that the peptide was acting through p75NTR expressed by the target neurons.”

The icing on the cake then came with Xiao’s experiments in rodents, which provided strong evidence that CP actually promotes the myelination of peripheral neurons in vivo.

“Injection of CP adjacent to the sciatic nerve of newborn rats significantly up-regulated myelin protein expression and increased the proportion of myelinated axons,” added Xiao. “In contrast, injection of BDNF failed to exert a significant effect.

“We are now investigating whether CP also exerts a protective effect upon myelin using rodent models of peripheral demyelinating diseases such as Guillain-Barré syndrome, a step that is taking our research in a new direction.”

Murray also emphasised that not only was this mimetic now a clear potential tool for clinical application, it also provided important information about the biology of the system because of the p75NTR-specific effects they saw on myelination in peripheral neurons.

“And, by design luck more than intent, CP also proved to be very metabolically stable in vivo due to its cyclic structure,” added Hughes. “This is in contrast to other, non-circular peptides that tend to be more susceptible to cleavage by proteases in the body and thus have very short half lives in a patient’s circulation.”

Spreading the peptide wings

Murray and Xiao’s genetics studies clearly show that CP exerts its pro-myelinating influence through the p75NTR receptor, and now they want to know more about that binding.

“One of our main aims now is to look at the interaction kinetics,” Murray explained. “How is CP snuggled up to p75NTR, exactly which residues are involved and where they are - some of the more structural and molecular aspects of the interaction.”

One of the mysteries of the system is the specific signalling mechanism used by this receptor to bring about a cellular effect, which the researchers say is remarkable because p75NTR has been known as an entity for around 30 years.

“It is a source of both fascination and embarrassment that no-one can figure this out. It is not a classic receptor, and people are still waving their hands when they talk about how it works,” Murray described.

Part of the murkiness around p75NTR is because there is no real robust biochemical assay to test its activity. However, Murray thinks the co-culture systems they have developed to test the myelination effects together with the p75NTR-specific signalling might just fill that gap.

“I hope over the next couple of years we will start to tease all these aspects out and thus make a contribution to both myelination and perhaps potential therapies for remyelination, but also get to the bottom of what p75NTR does and how it does it … that is the greater plan.”

Another direction that Xiao and Murray would like to take now is using the same drug-like, peptide-based approach to look at myelination in the central nervous system. Just to make things even more complex, it seems to be regulated differently from the process in the periphery, via the other neurotrophin receptor TRK. The trio are currently using another peptide designed by Hughes, which mimics the region on BDNF that binds to the TRK receptor, and investigating whether it can promote myelination in the CNS.

Hughes is also excited about these next steps as he sees it feeding into further peptide design activities on his side of things (see below).

“I think the project is taking quite a nice leap at the moment, because we brought the project together specifically to do the myelination stuff and it is now spreading out again as we move into other areas,” he said. “The happy bit is that we all like working together and so as well as being successful scientifically, that makes it a lot of fun.”

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Grander designs

The CP (cyclo-DPAKKR) peptide has yielded some interesting drug design offshoots for Hughes’ own group. The structure of this cyclic peptide has intrinsic and useful drug-like properties: it is quite low in molecular weight (~580 daltons), metabolically stable and easily transported across cell membranes. But the structure of this molecule is also quite intriguing, according to Hughes.

“By NMR spectroscopy we see that this tiny pentapeptide of five amino acid residues forms a very tight and strong circle that has just a single conformation in solution. It actually turns out to be quite a nice template for other chemical and functional groups. We could potentially use it as a sort of starting point to make other compounds that are not peptides - and then we could do all sorts of other useful chemistries with it.”

“So, we are now using University of Melbourne Interdisciplinary Seed Funding to see if we can jump from this rather neat peptide and produce some other scaffolds that may give us the same sorts of biological activities, but get even better control over the pharmacokinetic properties of the molecule,” said Hughes.

They have already started by using in silico techniques to nut out how to present the functional groups on the peptide in the same way, but without the peptide backbone as a scaffold, and will then go on to do some binding studies with the peptide target. “If we can replace the peptide scaffold, it opens up that whole area of how this potential therapeutic molecule can be used.”

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Dr Simon Murray (centre) originally trained as a physiotherapist and returned to study, completing his PhD in 2000. He then spent three postdoctoral years at the New York School of Medicine working on neurotrophin signalling, before returning to work at Melbourne’s Florey Neuroscience Institutes. He moved his lab to the University of Melbourne in 2005. Dr Junhua Xiao (left) completed her clinical medical training in China in 2000, before moving to Melbourne to do a PhD in neurobiology at the University of Melbourne in 2002. She took up a postdoc position in Murray’s Neurotrophin Signalling Laboratory in 2005, focusing on mechanisms that control myelination, particularly neurotrophins. She joined the Department of Anatomy and Neuroscience as a lecturer in 2013. After studying pharmacy, Associate Professor Tony Hughes (right) completed a Master’s Degree in molecular modeling and a PhD in peptide chemistry at the University of London. He then undertook a postdoc stint at the Max Planck Institute for Psychiatry in Martinsried, just outside Munich, where he shifted fields to look at the cellular and molecular biology of neurotophic factors. He returned to Australia in 1995 to take up a teaching and research position in the Department of Pharmacology at the University of Melbourne.