Beyond the neuromuscular junction: dysfunction of spinal synaptic targets of motoneurons in Amyotrophic Lateral Sclerosis

Lead Research Organisation: University College London
Department Name: Neuroscience Physiology and Pharmacology

Abstract

Every year more than 5000 people are diagnosed with Amyotrophic Lateral Sclerosis (ALS). Its outcome is always fatal, with a median survival time from diagnosis of only 4-5 years. The initial symptoms include muscle weakness, cramps, and twitching. ALS is a disease of the central nervous system that mostly targets motoneurons. Motoneurons are located in the spinal cord and send their long processes throughout the body making contacts with distinct muscle groups. Through these contacts motoneuron activation leads to muscle contraction and to execution of all motor tasks, from walking to grasping, chewing, and even breathing. During the early symptomatic stages of the disease, contacts made by motoneurons onto muscles become ineffective, hampering successful execution of motor tasks. This is followed by progressive motoneuron death, leading to more severe motor deficits, inability to walk, bear weight, or handle objects. The degeneration progresses along the spinal cord and extends to motoneurons responsible for the contraction of the diaphragm necessary for respiration. This is normally the final stage of the disease, in which the patient maintains full awareness, but becomes progressively incapable of unassisted breathing.
Currently there is no cure and the only approved treatments (such as riluzole, a compound that reduces the excitability of motoneurons) only marginally prolong the life expectancy.
The causes of the disease are unknown, but in at least 20% of patients there is a family history that proves a hereditary trait. Indeed, familial studies led to the identification of several mutations in proteins that affect the health of motoneurons. A major breakthrough resulted from this knowledge: these mutations were used to develop animal models of the disease.
We are proposing to use two of these animal models that are quite different from each other, but whose symptoms closely mirror the symptoms and time course of ALS in the corresponding affected human patients. Previous research has focussed on the analysis of cellular events in motoneurons that could lead to degeneration and death. While a number of key events have been identified, the cause-effect relationships proved hard to pinpoint, thus making it difficult to develop therapeutic strategies.
In our proposal we hypothesize that motoneuron degeneration is not only due to events occurring at the molecular level within single cells, but also due to events within circuits regulating motor control. In particular, we will focus on local circuits between motoneurons and a special class of inhibitory neurons called Renshaw cells that have the role of limiting and fine tuning the excitation of motoneurons. Disruption of these circuits may cause a vicious cycle in which motoneurons become over excitable and degenerate as a consequence of the excessive, unchecked amount of excitation they receive.
There is some previous evidence of disruption of this circuit in both affected humans and mutated animals, and our research will quantify the extent and time course of this disruption.
Our investigation has a dual purpose: first, by understanding how and when the local circuits are affected, we may be able to devise a diagnostic test that could reveal early stages of the disease, well before clear symptoms appear. This would potentially open a longer therapeutic time window. Second, the demonstration of the importance of the disruption of local circuits in the development of the disease could open the way to the development of new therapies aimed at preserving and enhancing normal local circuit function.

Technical Summary

Amyotrophic Lateral Sclerosis (ALS) is a fatal motoneuron disease that leads to impairment of motor control, paralysis, and death. While its causes are unknown, a number of different mutations accounts for approximately 20% of cases. This has enabled the development of mouse models that mimic the human disease. We propose to use two different animal models of ALS to study the specificity of spinal circuit dysfunction in ALS: the well-established SOD1G93A and the recently developed aggressive TauONhFUSP525L. While much past research has focused on molecular and electrophysiological changes occurring in motoneurons (Mns), there is evidence that synaptic contacts made by Mns within the spinal cord are also affected. These synapses are the basis for a negative feedback loop mediated by Renshaw cells that inhibit the same Mns they are excited from and a positive feedback loop mediated by contacts of Mns on each other. We will compare the strength of recurrent excitation and inhibition in Mns from wild type and ALS affected animals in order to quantify impairments in synaptic transmission, with each experiment being performed at different stages (beginning pre-symptomatically) of disease. First, we will establish the time course of the impairment of recurrent inhibition using EMG recordings. We will then use electrophysiological (patch clamp) recordings of Mns and Renshaw cells. We will perform quantal analysis of synaptic currents evoked by antidromic stimulation of motoneurons or during paired recordings of connected cells in order to measure features of individual synapses. Furthermore, we will use holographic two-photon stimulation to map the pattern and strength of connectivity between Mns and Renshaw cells, to verify whether ALS alters the architecture of recurrent circuitry. The identification of synaptic and circuit abnormalities in an ALS model could potentially identify a new therapeutic target for controlling the symptoms and slowing progression of disease.

Planned Impact

Both applicants have a long history of spinal cord research including basic circuits (MB and RB), animal model of spinal cord injury (RB), and clinical research and activity (RB) .
This project will make use of our knowledge of the field and our recent specific findings about the recurrent circuitry in the spinal cord to assess the extent of its impairment in two animal models of ALS.
Clinical beneficiaries
Our proposed plan of investigation aims at quantifying early impairment of local spinal circuits. Thus, the prospective target of beneficiaries includes people affected by ALS or other motor neuron diseases, as well as workers in the health sector. The use of acute, minimally invasive, EMG recordings to measure the extent of impairments in the recurrent circuitry has high potential for translation, as these types of measurements can be readily performed in humans. If our data indicate an early impairment in the animal model, we are planning to extend these measurements to human subjects, in the framework of a new collaboration with Prof. Iannetti at UCL, who is a leading expert in motor recordings from human subjects. Such recordings could potentially form the basis of a diagnostic test aimed at either detecting early stages of the disease or monitoring disease progress. If, as available data seem to indicate, the recurrent circuit is affected by ALS and its impairment leads to a vicious cycle of over-excitation that is not balanced by recurrent inhibition, then in the long term, recurrent circuitry may constitute a novel pharmacological target for treating symptoms and possibly slowing its time course. Both the excitatory (mainly cholinergic) and inhibitory (GABAergic and glycinergic) components can be enhanced by available drugs that can increase the activation of Renshaw cells by motoneurons (by inhibiting acetylcholinesterase) or prolong the activation of inhibitory receptors (benzodiazepine) thus dampening the excess excitation. Future development of more targeted therapies would be ideal. (Note that one aspect of RB's clinical practice is insertion of pumps for intrathecal therapies; these techniques could be readily extended to new drugs.)
General public
The details of our outreach and engagement activity will be informed by the progress of our research. The surprising and widespread popularity of the 'ice bucket challenge' two years ago has sharply increased the general public awareness of ALS and the importance of related research. We plan to contribute to this awareness by engaging with the public in the following settings:
Local schools: Both applicants have already given talks on their achievements in basic motor research to high school pupils in the UK (MB) and Canada (RMB). We expect to maintain this activity, including a focus on neurodegenerative motor diseases. By the end of the grant tenure, we aim to incorporate our findings into lectures targeted at high school students, as well as providing didactic material to science teaching staff.
Public exhibitions: during the second year of the project we will submit an application to take part in the Royal Society Summer Science Exhibition. This is an exceptionally popular event that reaches a wide audience and is a chance to highlight the importance of research into motor disorders.
Training of young pupils: during summer we will host one or more young student coming form an unprivileged background through the long established In2Science UK scheme, that was initiated by a former UCL student and now runs in many London research institutions.

Publications

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