The role of BMP/Smad signalling in motor neuron connectivity
Lead Research Organisation:
Imperial College London
Department Name: Dept of Medicine
Abstract
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.
Technical Summary
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.
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.
Planned Impact
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.
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.
People |
ORCID iD |
Vasso Episkopou (Principal Investigator) |
Publications
Redshaw N
(2013)
TGF-ß/Smad2/3 signaling directly regulates several miRNAs in mouse ES cells and early embryos.
in PloS one
Martin N
(2013)
Interplay between Homeobox proteins and Polycomb repressive complexes in p16INK4a regulation.
in The EMBO journal
Kicheva A
(2014)
Coordination of progenitor specification and growth in mouse and chick spinal cord.
in Science (New York, N.Y.)
Kelly CE
(2013)
Rnf165/Ark2C enhances BMP-Smad signaling to mediate motor axon extension.
in PLoS biology
Delegkou GN
(2023)
E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase.
in Cancers
Chasapis C
(2012)
NMR-based insights into the conformational and interaction properties of Arkadia RING-H2 E3 Ub ligase
in Proteins: Structure, Function, and Bioinformatics
Birkou M
(2022)
Unveiling the Essential Role of Arkadia's Non-RING Elements in the Ubiquitination Process.
in International journal of molecular sciences
Birkou M
(2017)
A Residue Specific Insight into the Arkadia E3 Ubiquitin Ligase Activity and Conformational Plasticity.
in Journal of molecular biology
Birkou M
(2022)
Impact of a Single Nucleotide Polymorphism on the 3D Protein Structure and Ubiquitination Activity of E3 Ubiquitin Ligase Arkadia.
in Frontiers in molecular biosciences
Description | Motor neurons control movement via long axons that extend from the spinal cord to muscles as far as in distant limbs. Little is known about factors that regulate this extensive axonal growth in the periphery. Here we report that the ubiquitin ligase Ark2C (Arkadia2) is expressed in neurons and can serve to amplify neuronal responses to specific signals. We find that these signals belong to the Bone Morphogenetic Protein (BMP) family of secreted factors, which are highly expressed in the periphery and known to regulate the development of the limbs. Loss of Ark2C gene function in mice results in inefficient growth of motor axons to distant muscles, and we show that this process is regulated by BMP signaling. Ark2C targets BMP inhibitors for destruction, and therefore the presence of Ark2C helps to enhance BMP signaling, which in turn is necessary for the innervation of distal muscles. Our experiments reveal a previously unknown function of BMP in motor axon growth and describe a molecular mechanism for how axons and limbs coordinate their growth. |
Exploitation Route | will form the basis of further research and knowledge |
Sectors | Education Healthcare |
URL | http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001538 |
Description | Spyroulias, G. Univ. of Patras NMR structural studies of Arkadia |
Organisation | University of Patras |
Department | Pharmacology Department Patras |
Country | Greece |
Sector | Academic/University |
PI Contribution | The collaborator prof. Spyroulias, paid to send his postdoctoral fellow Dr Birkou in my lab for 2 months to perform revisions for our paper under review in the journal of AAAS "Science Signaling". January-February 2020. |
Collaborator Contribution | The collaborator prof. Spyroulias, paid to send his postdoctoral fellow Dr Birkou in my lab for 2 months to perform experiments (revisions) for our paper under review in the journal of AAAS "Science Signaling". January-February 2020. |
Impact | Delegkou GNN, Birkou M, Fragkaki N, Toro T, Marousis KDD, Episkopou V, Spyroulias GAAet al., 2023, E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase, CANCERS, Vol: 15 Birkou M, Delegkou GN, Marousis KD, Fragkaki N, Toro T, Episkopou V, Spyroulias GAet al., 2022, Unveiling the Essential Role of Arkadia's Non-RING Elements in the Ubiquitination Process, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, Vol: 23 Birkou M, Raptis V, Marousis KD, Tsevis A, Bourikas K, Bentrop D, Episkopou V, Spyroulias GAet al., 2022, Impact of a single nucleotide polymorphism on the 3D protein structure and ubiquitination activity of E3 ubiquitin ligase arkadia, Frontiers in Molecular Biosciences, Vol: 9, ISSN: 2296-889X Birkou M, Chasapis CT, Marousis KD, Loutsidou AK, Bentrop D, Lelli M, Herrmann T, Carthy JM, Episkopou V, Spyroulias GAet al., 2017, A Residue Specific Insight into the Arkadia E3 Ubiquitin Ligase Activity and Conformational Plasticity, JOURNAL OF MOLECULAR BIOLOGY, Vol: 429, Pages: 2373-2386, ISSN: 0022-2836 Birkou M, Tsapardoni M, Marousis K, Chasapis CT, Bentrop D, Episkopou V, Spyroulias GAet al., 2013, CONFORMATIONAL DYNAMICS AND DRUG DESIGN APPROACHES THROUGH NMR, EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES, Vol: 50, Pages: E37-E37, ISSN: 0928-0987 Chasapis CT, Kandias NG, Episkopou V, Bentrop D, Spyroulias GAet al., 2012, NMR-based insights into the conformational and interaction properties of Arkadia RING-H2 E3 Ub ligase, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, Vol: 80, Pages: 1484-1489, ISSN: 0887-3585 Kandias NG, Chasapis CT, Bentrop D, Episkopou V, Spyroulias GAet al., 2009, High yield expression and NMR characterization of Arkadia E3 ubiquitin ligase RING-H2 finger domain, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 378, Pages: 498-502, ISSN: 0006-291X |
Start Year | 2007 |
Description | Thymakou, E. Univ. of Crete Analysis of the central brain phenotype in Arkadia2C mutant mice |
Organisation | University of Crete |
Department | Department of Psychiatry |
Country | Greece |
Sector | Academic/University |
PI Contribution | We generated and provide the animal model for these studies. |
Collaborator Contribution | The collaborators perform behavioral tests, 2 photon imaging on our mutant mice |
Impact | We have preliminary results that can be used to raise funding to complete this study |
Start Year | 2009 |
Description | Zuckerman Institute Columbia Univ NY: The role of BMP-Smads in the spinal cord |
Organisation | Columbia University Medical Center |
Country | United States |
Sector | Academic/University |
PI Contribution | we generated combination of conditional knock out Smad1/5/8 mouse strains |
Collaborator Contribution | generating antibodies against Smad8 and Arkadia2C mouse strains Arkadia2C conditional mouse strains Smad8-ConditionalCre mouse strain Smad8 conditional KO |
Impact | not yet |
Start Year | 2013 |