The role of BMP/Smad signalling in motor neuron connectivity

Humans have billions of neurons that are all interconnected via axons and synapses. These provide neurons with the ability to communicate information over long distances. Wiring patterns are essential for body functions, movement, coordination cognition, learning and memory. How neurons grow their axons over great distances to reach and connect to other axons from different neurons or to muscles is not well understood. Axon elongation is one of the essential properties in neuronal connectivity and it is believed that in the adult it provides neurons with the ability to remodel their connections. This plasticity of neuronal connections underlies experience, learning and memory. Furthermore, neurons need to maintain some capacity to elongate their axons and regenerate their axonal projections to repair possible damage during life. Little is known about factors that regulate axon elongation particularly that of motor neurons, which possess the longest axons in the body i.e. from the spinal column to the end of the fingertips and toes. Understanding how our nervous system is wired will give us insights into how it develops and functions. Once we master this we can manipulate various aspects of connectivity to correct defects and disabilities or repair damaged neurons and cure neurodegenerative disease. In this research we aim to examine the role of the Bone Morphogenetic Protein factors in motor neuron connectivity in mouse models and provide basic information that would be beneficial for Motor Neuron disease and for peripheral axon regeneration.

We have revealed that BMP-Smad signalling is involved in motor neuron (MN) axon elongation and identified Arkadia2C (Ark2C) as a positive regulator required for this (Kelly et al Nat. Neurosc. resubmitted). Ark2C is an E3 ubiquitin ligase specifically expressed in the nervous system that enhances BMP-Smad signalling by ubiquitin-mediated degradation of negative regulators.
Expression analysis showed that MN harbour activated Smads and treatment of a MN cell line with BMP inhibitor diminished axon growth in culture. Genetic reduction of BMP signalling, by removal of a BMP-receptorII allele or loss of Smad8 BMP effectors in Ark2C+/- mice causes the emergence of innervation defects otherwise observed only in Ark2C-/-.
Ark2C-/- mice are born but progressively thin and die before weaning, suggesting that BMP/Ark2C are involved in maintenance of the neuromuscular synapse. To explore the importance of motor axon elongation at postnatal stages, the involvement of BMP signalling, and mechanisms:
1. We will generate chimaeras with mixtures of Ark2C-/- and wild type ES-cells that will, respectively, express GFP and RFP in MN, allowing side-by-side comparison of pre-synaptic terminals, branching and synapses. The chimaeras should survive but exhibit defects dependent on contribution of mutant cells. If all Ark2C-/- MN are outcompeted by wild type MN we will pursue a conditional knockout strategy. Smad8-/- mice also exhibit MN defects and these will be analysed to explore BMP-Smad requirements independently of Arkadia2C.
2. Using high throughput sequencing we have identified BMP/Ark2C dependent target genes in ES-cell derived MN that are expected to be relevant to axon elongation. Functional studies on candidate targets will be performed by knockdowns in MN axon elongation assays that we have developed in culture, focusing on genes implicated in axon guidance, adhesion, cytoskeleton remodelling, signal reception and transcription.

This is a basic research project and the immediate beneficiaries are likely to be other academics, but the links I have established with clinicians and clinician scientists studying motor neuron diseases, in my local environment, nationally and internationally, will facilitate translation of any results.
Mutations in the BMP pathway underlie several diseases and this has led to the use of recombinant BMPs in clinical practice for the treatment of bone and kidney disorders. Our research is expected to focus future studies on determining whether BMP signalling is involved in MN axon plasticity, degeneration and regeneration, and whether BMP can be used to modulate these events in disease and injury. More long term studies would address the role of BMPs in regeneration of neurons in the spinal cord and the brain where axon regeneration is very limited, bringing hope to patients with devastating spinal cord injury causing severe paralysis.
We would therefore hope that our research will directly influence further research at all levels, ultimately leading to treatments. Prior experience of the use of BMP, their agonists and antagonists in clinical situations, should speed up their application in problems of the nervous system, both for practical and regulatory reasons.