The Molecular Neurobiology of Breathing

Lead Research Organisation: King's College London
Department Name: MRC Ctr for Developmental Neurobiology

Abstract

Most ?higher? animals that live on land, including humans, breathe air through lungs. The expansion and
contraction of the lungs during breathing(respiration) is driven by a group of muscles that are attached to the rib cage. The breathing rhythm itself is generated in the brain and then conveyed to the muscles through a specialized type of nerve cell called respiratory motor neuron . I propose to investigate the genetic program that instructs some embryonic cells to become respiratory motor neurons prior to birth. Such a program typically consists of two sets of molecules: One set controls when and where genes are active within the body, and it defines the identity of the cells. A second set of molecules, regulated by the first one, allows developing motor neurons to connect to their ?right? target muscle. The genetic elements that constitute the ?respiratory motor neuron? program and their logical relationships to each other are currently
not known. Once I have decoded the program, I may be able to artificially create new nerve cells of this
type from embryonic stem cells and use them to replace dead or disconnected ones. Such a therapeutic
approach could restore the ability to breathe in patients suffering from neuromuscular disease or spinal cord injury.

Technical Summary

All land vertebrates, including humans, use lungs to breathe air. The inspiratory and expiratory movements
of the lungs are driven by a complex neural circuitry that consists of a central network in the brainstem that generates breathing rhythms and an output layer of motor neurons which connect to respiratory muscles.
These respiratory circuits develop prenatally and have to become functional immediately after birth. While significant progress has been made in understanding the central pattern generator itself, very little is known about the formation of neural circuits that turn breathing rhythms into coordinated motor output. Here, I propose several experimental approaches to address how respiratory motor neurons are specified and connect to the appropriate target muscles. Using a combination of in vitro differentiation of motor neurons from mouse ES cells, mouse genetics, imaging and systematic analysis of gene expression, I aim at defining the transcriptional program of respiratory motor neuron identity. More generally, this study should also provide insights into the crucial, but still largely unresolved, question of how neurons acquire their individual identities.

Publications

10 25 50
 
Description BBSRC Research Grant
Amount £750,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description Research Grant
Amount € 152,000 (EUR)
Organisation Thierry Latran Foundation 
Sector Charity/Non Profit
Country France
Start 09/2014 
End 08/2016
 
Description Research and Development Challenge Fund
Amount £83,000 (GBP)
Organisation King’s Health Partners 
Sector Academic/University
Country United Kingdom
Start 06/2013 
End 05/2014
 
Description Stem cell therapeutic approaches
Amount £65,000 (GBP)
Organisation Thierry Latran Foundation 
Sector Charity/Non Profit
Country France
Start 01/2011 
End 12/2011
 
Description Trampoline Grant
Amount € 31,200 (EUR)
Organisation French Muscular Dystrophy Association (AFM) 
Sector Charity/Non Profit
Country France
Start 01/2012 
End 05/2013
 
Title GFAP::CD14 ES cells 
Description The GFAP::CD14 ES cells (clone H6) carry a stable transgene that allows the isolation of astrocytes directly differentiated from mouse ES cells by magnetic cell sorting 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact Together with the Mnx1::CD14-IRES-GFP ES cell lines, this line allows the generation of CNS-like neuron/glia cocultures to simulate neural patterning/neural circuitry in vitro. The system does not require the use of experimental animals, but closely resembles primary neural cultures. 
URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947756/
 
Title Hb9::CD14-IRES-GFP mouse ES cells 
Description This mouse ES cell line allows the magnetic isolation of motor neurons directly differentiated in vitro. The surface tag used, CD14, is superior to those previously available. We will make the reagent available (through UK Stem Cell Bank or other distributors) once it is published. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact Most existing ES cell lines with motor neurons-specific promotors use fluorescent markers, which require flow cytometers for cell sorting (costs: GBP 100K - 300K). Magnetic cells sorting using surface tags works almost as efficiently and allows the purification of much larger numbers of cells (>10^8) at a fraction of the costs (ca. GBP 300 for the magnet + stand). Once this ES cell line is available, many more research groups will be able to afford working with ES cell derived neurons to explore neural development and neurological diseases. 
URL http://dev.biologists.org/content/141/4/784
 
Title Statistical method to identify neural subtype identity determinants 
Description Our collaborator on the Machado et al. paper, Dr. Eric Blanc, developed a method to analyse transcriptional patterns evoked by candidate transcription factors in ES cell-derived tissue. The method allowed us to filter out potential determinants of phrenic neuron identity. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact This method, which, to my knowledge, has not been used in developmental biology before, will allow researchers to study genetic networks of rare cell type that are largely inaccessible to conventional in vivo methods. 
URL http://dev.biologists.org/content/141/4/784
 
Description Analysis of protein networks during neural circuit formation 
Organisation Charité - University of Medicine Berlin
Department Institute of Chemistry and Biochemistry
Country Germany 
Sector Academic/University 
PI Contribution The collaboration with Prof. Britta Eickholt aims at unraveling protein signaling networks active during sucessive step of neural development, such as neurogenesis, axon outgrowth, dendrite patterning and synaptogenesis.
Collaborator Contribution Prof. Eickholt contributes expertise in biochemical and imaging methods, such as capillary protein electrophoresis, immuno-precipitation and FRET. My contributions to the project include in vitro derivation of neural cells from ES cells, cell sorting and genetic modification of ES cells.
Impact While the project is still in an early phase, we have already generated several crucial reagents, such as ES cell lines, and established long-term cultures of neurons on ES cell-derived astrocytes which allow us to study mature neural circuits in vitro.
Start Year 2011
 
Description Assembly of neuromuscular circuitry in vitro 
Organisation National University of Singapore
Department Mechanobiology Institute (MBI)
Country Singapore 
Sector Academic/University 
PI Contribution My group is contributing technology that allows us to differentiated cellular components of such circuits, motor neurons, glia and skeletal muscle, from ES cells and isolate them by magnetic sorting.
Collaborator Contribution My collaborator Prof. Viasnoff will provide us with expertise and manufacturing facilities to produce custom-made compartmentalized tissue culture devices.
Impact The partnership has just started, but we anticipate that the devices we are developing may be used for pharmaceutical screens aimed at identifying potential therapeutics for neuromuscular disease
Start Year 2013
 
Description Developing a hybrid biological-optoelectronic vagus nerve stimulator device: a new stem cell-based therapeutic strategy to minimise arrhythmias & myocardia ischaemia 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution With two colleagues from UCL, Prof Alexander Gourine and Dr. Pier Lambiase, I have recently started to develop an new type of autonomic neural prosthesis. The aim of the collaboration is to develop a neural modulator device which communicates with the heart through contacts formed stem cell derived grafted tissue. The graft in turn will express optogenetic probes so that it can be controlled through an optoelectronic control system. Long-term, the device will be used to minimize the effects of myocardial ischemia and ventricular arrhythmia. My contribution to the project is my expertise in stem cell biology and in vitro derivation of neural cells.
Collaborator Contribution Prof. Gourine will contribute his expertise in cardiac physiology and in vivo optogenetics, Dr. Lambiase, a cardiologist, will advise us on experimental strategies and translation into clinical practice.
Impact The collaboration has started recently. At this point, the only output is a grant application for the GSK Bioelectronic Medicines program, which we will submit in the next 1-2 weeks.
Start Year 2014
 
Description Generation of motor neurons from ground-state iPSCs 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are collaborating with Dr. Pentao Liu on deriving neural subtypes, glia and skeletal muscle from a new type of human induced pluripotent stem cell. Long-term, we intent to use this material to study neuromuscular disease and basic synaptic function in artificial neural circuits assembled form stem cell-derived tissue.
Collaborator Contribution Dr. Liu contributes his technology of generating ground-state iPSCs, which appears to be superior to methods currently used by most researchers in the field.
Impact The collaboration is at an early stage, but we could already confirm several of Dr. Liu's observation on properties of cells derived with his technology.
Start Year 2013
 
Description In vitro modeling axonal pathology in ALS 
Organisation King's College Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution My team is contributing expertise in modeling neuromuscular circuits with stem cells derived neural tissue and muscle.
Collaborator Contribution Prof Shaw contributed iPSC lines derived from ALS patients.
Impact We have produced preliminary data for grant applications and submitted two application on different aspects of ALS pathology so far (MRC, Wellcome Trust).
Start Year 2015
 
Description Restoration of respiratory motor function through optogenetic neural implants 
Organisation Newcastle University
Department School of Civil Engineering and Geosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution My team provides the know-how of embryonic stem cell culture and in vitro differentiation techniques for motor neurons and astrocytes. The long-term aim of this collaboration is to develop a neural implant that restores respiratory function in patients suffering from ALS
Collaborator Contribution Through this collaboration, I gained access to rodent models for motor neuron disease and succeeded in securing a charity grant.
Impact Though the collaboration is still in an early phase, it has already allowed us to do proof-of-principle experiments for the proposed neural implant and helped to secure funding for a pilot study. We have recently obtained data which suggests that the optogenetic neural implants can survive for more than 5 weeks in vivo.
Start Year 2010
 
Description Restoration of respiratory motor function through optogenetic neural implants 
Organisation University College London
Department Institute of Neurology
Country United Kingdom 
Sector Academic/University 
PI Contribution My team provides the know-how of embryonic stem cell culture and in vitro differentiation techniques for motor neurons and astrocytes. The long-term aim of this collaboration is to develop a neural implant that restores respiratory function in patients suffering from ALS
Collaborator Contribution Through this collaboration, I gained access to rodent models for motor neuron disease and succeeded in securing a charity grant.
Impact Though the collaboration is still in an early phase, it has already allowed us to do proof-of-principle experiments for the proposed neural implant and helped to secure funding for a pilot study. We have recently obtained data which suggests that the optogenetic neural implants can survive for more than 5 weeks in vivo.
Start Year 2010
 
Title Optogenetic Stem Cell Prosthesis 
Description The central idea of the patent is to use optogenetic, stem cell-derived motor neurons to restablish neural control of denervated muscle. The stem cell-derived neurons will form synapses on the muscle and will allow its pacing with an artificial control system (pacemaker device) that produces a rhythm encoded in light flashes. 
IP Reference WO2012052727 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact We have recently submitted a manuscript which shows that this new type of neural prosthesis works in vivo in principle, and plan to develop this idea towards a pre-clinical study and, eventually, application in human patients.
 
Title Optogenetic neural implant 
Description Together with two collaborators, Prof. Linda Greensmith (IoN, UCL) and Patrick Degenaar (U. Newcastle), I am in the initial stages of developing a new type of respiratory pacemaker device. The device will consist of ES cell-derived neural tissue (motor neurons and glia), as well as optoelectronic elements. It will be used to drive respiration in patients that have lost control of their diaphragm muscle due to neurodegenerative disease, such as ALS. We have filed a patent application to protect our intellectual property. 
Type Therapeutic Intervention - Medical Devices
Current Stage Of Development Initial development
Year Development Stage Completed 2012
Development Status Under active development/distribution
Impact It will take 10-15 years to develop this novel technology, but once it can be used on ALS patients, it is likely to result in a significant improvement in quality of life. The neural pacemaker devices we are developing will allow them to breathe without the aid of a mechanical respirator. Furthermore, the ES cell-derived, optogenetic neural tissue represents a new type of body/machine interface and could have other applications, such as artificial sensory organs. 
 
Description Internship for GSCE-level students 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact My group participated in a scheme organized by the charity In2Science UK that provides 2-week lab placements to gifted students from underprivileged backgrounds. We were impressed by the interest and enthusiasm of the student who worked with us and plan to host more students from this scheme in the future.

The student who worked with us, Adam, gave an excellent presentation to other In2Science UK students, and, hopefully, will be motivated to pursue a career in Science or Medicine in the future.
Year(s) Of Engagement Activity 2012,2013
 
Description Teaching material for High School Science classes 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact I have assisted Science in the Classroom (http://scienceintheclassroom.org/), an educational resource published by Science/AAAS, in creating an extensively annotated version of the Bryson et al. paper and a set of data analysis tasks for high school students, which will serve as an educational tool for Science teaching in classrooms in the USA and elsewhere.
Year(s) Of Engagement Activity 2014
URL http://scienceintheclassroom.org/research-papers/lights-motor-neurons-and-muscle-action/university