AMPA- and GABA(A) receptor signalling and corticospinal motor neuron excitability in mouse models of ALS
Lead Research Organisation:
University College London
Department Name: Neuroscience Physiology and Pharmacology
Abstract
The proper function of our bodies relies on the chemical communication that takes place among nerve cells in the brain and between nerve cells and peripheral tissues. Nerve cells that convey information to muscles are known as motor neurons. The progressive and selective death of these neurons is a defining feature of the debilitating, and ultimately fatal, neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Unable to function without correct input, the muscles weaken and waste, leaving the patient incapable of voluntary movement. Among the mechanisms thought contribute to this motor neuron loss, 'excitotoxic damage' triggered by elevation of intracellular calcium ions is held to be especially important. Like other nerve cells, motor neurons receive excitatory signals mediated by the neurotransmitter glutamate, which acts by binding to receptor proteins at points of contact between the cells. A combination of poor calcium buffering capacity and the expression of one subclass of AMPA-type glutamate receptors that allow the entry of calcium ions, is thought to contribute to the vulnerability of motor neurons in ALS. However, details of calcium-permeable AMPA receptor function and regulation in motor neurons are poorly understood. Transgenic rodents provide valuable models of the disease, but the study of spinal motor neurons using patch-clamp recording (a technique allowing high-resolution examination of a cell's electrical activity) is possible only in immature, asymptomatic, animals. Unlike motor neurons in the spinal cord, 'upper' motor neurons in the cortex, which also undergo degeneration in ALS, are amenable to patch-clamp recording in slices from mature animals. We plan to take advantage of this, and selectively label these corticospinal motor neurons (CSMNs), allowing us to record from identified motor neurons in acute brain slices from 'ALS' mice at different stages of disease progression. We wish to examine the prevalence and regulation of calcium-permeable AMPA receptors in CSMNs and determine how changes in inhibitory input mediated by a different neurotransmitter receptor, the GABA(A) receptor, contribute to motor neuron excitability, and thus excitotoxic damage, in ALS. By performing electrophysiological studies of motor neurons in both control and symptomatic animals, this project will shed light on normal brain function and provide answers to outstanding questions regarding the mechanisms underlying vulnerability of motor neurons in ALS.
Technical Summary
We will determine functional characteristics of excitatory and inhibitory influences on identified CSMNs in brain slices prepared from SOD1(G93A) 'ALS' mice at different stages of disease progression. Cells will be retrogradely labelled with FluoroGold or latex beads applied to the cervical spinal cord. We will determine the active and passive electrical properties of the neurons and establish any general alterations in their properties at different stages of the disease. We will then examine the contribution of calcium-permeable AMPARs and their auxiliary proteins (TARPs and CNIHs) to synaptic and extrasynaptic signalling. We will record pharmacologically isolated AMPAR-mediated macroscopic currents and synaptic currents. The contribution of specific proteins will be determined by comparing the kinetics, voltage-dependence, calcium-permeability, conductance and pharmacology of synaptic and extrasynaptic receptors with those of recombinant receptors expressed with specific auxiliary subunits in tsA201 cells. To determine how GABAergic signalling is altered during disease progression, we will quantify the relative contribution to charge transfer of phasic and tonic GABA(A)R activation. We will distinguish between pre- and postsynaptic changes by examining pharmacologically isolated mIPSCs (amplitude and kinetics, and the conductance of the underlying channels). To determine the origin of changes in tonic GABA(A)R-mediated currents will examine the properties of native receptors in outside-out somatic patches and compare these with those of recombinant GABA(A)Rs expressed in tsA201 cells. We will assess the impact of GABA(A)R activation on cell excitability and potential changes in chloride homeostasis in recordings using the gramicidin perforated-patch technique to preserve intracellular chloride. Our research will contribute to the understanding of normal brain function and highlight potential strategies for limiting excitotoxic damage of motor neurons in ALS.
Planned Impact
Academic impact: this work has the potential to enhance the knowledge economy by providing new knowledge and scientific advancement. The principal beneficiaries of this project will be neuroscience researchers. By providing a clearer understanding of the properties of calcium-permeable AMPARs in cortical motor neurons and the impact of GABA(A)R-mediated inhibition in regulating cortical motor neuron excitability during disease progression in ALS mice, our work may held identify research avenues that will ultimately lead to strategies to limit motor neuron damage. In addition, academic benefit may come from our development of any new methodologies and techniques to achieve our goals. In the broader sense, our work will also be of benefit to neuroscience researchers wishing to understand normal brain function. Immediate benefits (through exchange of scientific knowledge) are likely to accrue in the short to medium term, and certainly within the lifetime of the project. The research will also have capacity-building impact through the training of highly skilled researchers, who will acquire specific competencies and broader transferable skills.
Economic and societal impact: ALS and related neurodegenerative conditions cause an important economic and social burden. In the long term, by informing drug development or otherwise helping to facilitate rational approaches to therapy, our studies may ultimately lead to enhanced health and well-being.
Economic and societal impact: ALS and related neurodegenerative conditions cause an important economic and social burden. In the long term, by informing drug development or otherwise helping to facilitate rational approaches to therapy, our studies may ultimately lead to enhanced health and well-being.
Publications
Bats C
(2013)
A role of TARPs in the expression and plasticity of calcium-permeable AMPARs: evidence from cerebellar neurons and glia.
in Neuropharmacology
Coombs ID
(2019)
Homomeric GluA2(R) AMPA receptors can conduct when desensitized.
in Nature communications
Coombs ID
(2021)
Single-channel mechanisms underlying the function, diversity and plasticity of AMPA receptors.
in Neuropharmacology
Coombs ID
(2017)
Dual Effects of TARP ?-2 on Glutamate Efficacy Can Account for AMPA Receptor Autoinactivation.
in Cell reports
Davenport EC
(2017)
An Essential Role for the Tetraspanin LHFPL4 in the Cell-Type-Specific Targeting and Clustering of Synaptic GABAA Receptors.
in Cell reports
Greger IH
(2017)
Structural and Functional Architecture of AMPA-Type Glutamate Receptors and Their Auxiliary Proteins.
in Neuron
Lalanne T
(2018)
Synapse Type-Dependent Expression of Calcium-Permeable AMPA Receptors.
in Frontiers in synaptic neuroscience
Lalanne T
(2015)
Synapse-specific expression of calcium-permeable AMPA receptors in neocortical layer 5
in The Journal of Physiology
Magno L
(2021)
Transient developmental imbalance of cortical interneuron subtypes presages long-term changes in behavior
in Cell Reports
McGee TP
(2015)
Auxiliary Subunit GSG1L Acts to Suppress Calcium-Permeable AMPA Receptor Function.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Rigby M
(2015)
Transmembrane AMPAR regulatory protein ?-2 is required for the modulation of GABA release by presynaptic AMPARs.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Soto D
(2014)
Molecular mechanisms contributing to TARP regulation of channel conductance and polyamine block of calcium-permeable AMPA receptors.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Sullivan S
(2017)
TARP ?-2 Is Required for Inflammation-Associated AMPA Receptor Plasticity within Lamina II of the Spinal Cord Dorsal Horn
in The Journal of Neuroscience
Zonouzi M
(2015)
GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury.
in Nature neuroscience
Description | A. Acevedo collaboration |
Organisation | MRC Harwell |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Analysis of CST neuron numbers in TDP models of ALS |
Collaborator Contribution | Provision of mutant mice |
Impact | Poster - 2014 UCL Neuroscience symposium. Output listed in Publications. Multidisciplinary - genetics, behaviour, confocal microscopy |
Start Year | 2012 |
Description | L. Greensmith collaboration |
Organisation | University College London |
Department | Institute of Neurology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Electrophysiology expertise / analysis |
Collaborator Contribution | Planed provision of ALS model mice |
Impact | None |
Start Year | 2012 |
Description | P.H. Ozdinler collaboration |
Organisation | Northwestern University |
Department | Ken & Ruth Davee Department of Neurology |
Country | United States |
Sector | Hospitals |
PI Contribution | Electophysiological recording and analysis |
Collaborator Contribution | Provision of UCHL1-eGFP and hSOD1(G93A)-UeGFP mice |
Impact | None |
Start Year | 2013 |