Blanc SVSE 4 - Sciences de la vie, de la santé et des écosystèmes : Neurosciences

Why are all spinal motor neurons not affected by neurodegeneration? Testing the hyperexcitability hypothesis – HYPER-MND

Why do only certain types of motoneurons degenerate in Amyotrophic Lateral Sclerosis?

Is Amyotrophic Lateral Sclerosis (ALS) due to an hyperexcitability of motoneurons? The often proposed hypothesis according to which the selective degeneration of motoneurons is due to hyperexcitability has never been demonstrated.

Study of the excitability of the different types of motoneurons.

ALS is a fatal adult-onset neurodegenerative disease for which no cure exist today. Understanding why some motoneuron degenerate in ALS while other survive would constitute a major breakthrough in our understanding of the pathophysiological mechanisms responsible for this disease. One of the hypothesis put forward to explain the selective degeneration of motoneurons propose that some of them become too excitable which would induce their death. In SOD1 mice, an animal model of ALS, the motoneurons innervating the fast contracting but highly fatigable muscle fibers degenerate first, followed by the motoneurons innervating fast contracting but fatigue resistant muscle fibers, while the motoneurons innervating slow contracting fibers do not degenerate. Our project rests in part on a new electrophysiological technique that we’ve developed and that allows, in a deeply anesthetized mouse, recording spinal motoneurons and classifying them based on the contractile properties of the muscle fibers they innervate (Figure). By combining electrophysiology and molecular biology, we are studying 1) the excitability of motoneurons during ALS in adults as well as neonatal animals age 6-10 days old, i.e. the age at which muscle fiber innervation stabilizes itself in control animals; 2) the alterations of the proteins in the part of the axon that governs motoneuron excitability; 3) the deregulation of the genes coding for the proteins controlling the excitability.

We have developed new electrophysiological techniques allowing differentiating the different types of spinal motoneurons. The first technique enable recording adult spinal motoneurons in deeply anesthetized mice to study simultaneously the electrical properties of motoneurons and the force developed by the muscle fibers they innervate (Figure). One can distinguish three types of motoneurons in adult animals based on the contractile properties of the muscle fibers. The second technique allows recording motoneurons on spinal cord slices at an early stage of post-natal development. In this context, one can distinguish two types of motoneurons based on their discharge properties. Once the type of a motoneuron has been established and its excitability properties recorded, we associate our electrophysiological studies with innovative molecular biological techniques to explain the alterations of excitability that we observed (immunohistochemistry of the proteins responsible for setting the excitability, single cell RT-PCR to study the expression of the genes coding for those proteins).

In adults, we have observed that SOD1 mouse motoneurons have a larger input conductance than controls. Yet, their excitability tend to stay constant, which suggests that there is a mechanism, called homeostasis, that tries to maintain the excitability despite the increase in conductance. Our results invalidate the hyperexcitability hypothesis, even more so since we observed that, in SOD1 mice, a fraction of motoneuron (which increases with age) loses its capacity to fire in response to a stationary input (loss of function), as if the homeostasis started to fail. We have shown that the region of the axon in which are concentrated the channels responsible for the action potentials tend to increase in size in SOD1 mice. Yet, the density (number of channels per surface unit) of these channels stays constant, which suggest that the total number of channels increases. Electrophysiological investigations in neonatal mice revealed two types of motoneurons that can be told apart from their firing patterns (« immediate firing » et « delayed firing »). In SOD1 mice, we found the same two types of motoneurons, but the input conductance increases only in the « immediate firing » types, which makes them less excitable. Molecular biology experiments allowing the identification of the motoneurons mRNAs are underway to work out which ion channels are expressed in each types of motoneurons in SOD1 and control mice.

Our results indicate that SOD1 mouse adult motoneurons are not hyperexcitable, but on the opposite, they tend to become less excitable as the animal gets older. We will study if this loss of function depends on the physiological type of the motoneuron. We will check whether the changes in input conductance are due to changes in morphology in specific types of motoneurons.
Our experiments in neonatal mice revealed that, at this stage of development, motoneurons do not exhibit the same sensitivity to the disease. In SOD1 mice, input conductance increases in one type of motoneuron but not in others. We are characterizing the motoneurons through their ion currents and the proteins they express and will try to establish if their differences are linked to their adult fate.

Our first results have already been presented at major international conferences (SFN, International Symposium on ALS/MND, International motoneuron meeting). They will be published in two papers currently being written.

Amyotrophic lateral sclerosis (ALS) is a frequent neurodegenerative disease with an adult onset that is characterized by the degeneration of both corticospinal neurons and motor neurons. No curative treatment is available so far. Transgenic mice over-expressing a mutation of the human SOD1 gene (G93A) develop an ALS phenotype.

One of the striking features observed in ALS mice is that all motor neurons are not equally vulnerable: only those innervating the fast contracting motor units die after their neuromuscular junctions have degenerated. A major hypothesis is that degeneration results from an excitotoxic process due to an hyperexcitability of motor neurons. Impairment in the expression of sodium and potassium channels at the spike-triggering compartment, the axon initial segment (AIS), during post-natal development may well be the cause of this hyperexcitability. The hyperexcitability combined with the low calcium-buffering capacity of spinal motor neurons could result in an excessively high calcium concentration in the cells and lead to the deregulation of numerous cellular pathways. Our project aims at unraveling whether the hyperexcitability is indeed responsible for the specific vulnerability of « fast » motor neurons.

Our project combines complementary approaches. In vivo electrophysiological recordings of motor neurons in SLA mice will allow us to investigate their excitability properties and to relate them to the impairment of the neuromuscular junctions and to the physiological type of the motor unit. Daniel Zytnicki and his collaborators (Team 1) succeeded in performing stable intracellular recordings of spinal motor neurons in anesthetized adult mice, which is the key to the present study. An immunohistochemical study of recorded motor neurons will allow us to compare the expression of proteins involved in excitability (ionic channels within the AIS and their regulating subunits) in affected and unaffected motor neurons (François Couraud and Marc Davenne, Team 2). This immunolabelling data, together with the electrophysiological data, will be incorporated in models of motor neurons to understand which conductance changes account for the switch towards an hyperexcitable state.

Alterations of electrical properties have been reported in motor neurons of an ALS mouse model at an earlier age (P6-P10). It is important to also study motor neurons at this age. Indeed, the molecular composition of the AIS exhibits a drastic change in terms of sodium and potassium channels in the second and third post-natal weeks. Boris Lamotte d’Incamps (Team 1) will perform whole-cell patch-clamp recordings of motor neurons in spinal slices of ALS mice at P6-P10. He will investigate whether the hyperexcitable motor neurons are specifically those that will later innervate a fast contracting motor unit. In vitro recordings will be tightly coupled with RT-PCR studies and the analysis of more global gene expression profiles, thanks to the collaboration already initiated with Judith Melki (Team 3). The cytoplasm will be harvested during the electrophysiological experiments, and the RNA repertoire will be analyzed. We will focus on gene expression and splicing changes that might be responsible for the excitability modifications of the affected motor neurons.

The selective vulnerability of motor neurons also occurs in spinal muscular atrophy (SMA), another severe motor neuron disease. As part of this proposal, we will test the hypothesis that membrane hyperexcitability is also the cause of the selective vulnerability of motor neurons in SMA, we will determine which motor neurons are affected and analyze their gene expression profile.

Project coordinator

Monsieur Daniel Zytnicki (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS A) – daniel.zytnicki@parisdescartes.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LNP CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS A
PMSNC UNIVERSITE PARIS VI [PIERRE ET MARIE CURIE]
INSERM U788 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PARIS XI

Help of the ANR 510,000 euros
Beginning and duration of the scientific project: - 36 Months

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