Restoration of excitation/inhibition balance to modulate motoneuron degeneration in ALS – SynaptALS

Paris group performed all the in vivo electrophysiological intracellular recordings of identified motoneurons from specific motor pools in adult anaesthetized mice. This approach allowed us to investigate the electrical intrinsic properties of single MN, the simple neural circuits to MN (monosynaptic Ia pathway, disynaptic Renshaw inhibition…), to inject iontophoretically in the recorded MN (through the intracellular microelectrode) either an agonist of cAMP (cAMP-SP) or a dye (alexa, dextran) in order to label the MN under investigation for further histological processing. After intracardiac PFA-perfusion, histological procedures were performed by the Paris group in order to visualize the recorded MNs and to detect whether they were infected by the mutated-AAV9 virus (intramuscular injections, three weeks before electrophysiology). Paris group also made an extensive screening of retrograde infectious capabilities of a large set of mutated-AAV9 viruses. Imaging was performed using confocal microscopy.

Ulm group performed the techniques (qPCR, quantitative in situ hybridization) used for the transcriptomics investigations of Gs-coupled (PKA) receptors and ion channels; and it also used immunohistochemistry approach to investigate disease markers. Ulm group constructed the viruses used in the project. In particular they made the GFE3 construct (EGFP-FingR-E3 domains). This system was used for the control of the inhibitory synapses: the synaptic scaffold protein gephyrin is targeted for proteasomal degradation by an E3 ubiquitin ligase, which is brought in close proximity to gephyrin itself by a nanobody-like recognition molecule conjugated to the E3 ubiquitin. This system has been proved to effectively degrade gephyrin clusters and to induce a loss in GABAR/GlyR current. Ulm group also used super resolution imaging to determine the content of glycinergic receptor at Renshaw-cells/motoneuron synapses (identified by Calbindin immunolabelling).

Aim 1

- beta2 and beta 3 adrenergic receptors, that are Gs coupled receptors, are minimally or not dysregulated in adult presymptomatic SOD1 mice (P45-P50) offering entering points for pharmacological activation of the pKA signaling (Ulm).

- Acute iv delivery of beta2/3 agonists increases the slope (gain) of the firing frequency/current intensity (F/I) relationship (indicating increased excitability) whereas it has no effect on the input resistance and the recruitment current (Paris). It upregulates the transcription of immediate early genes, indicating increased neuronal activity, and affects transcription of ion channels that set excitability (Ulm).

- Intracellular injections of a cAMP agonist in single motoneurons (MN) also increase F/I gain demonstrating that beta2/3 agonists neuromodulate excitability through cAMP/PKA pathway (Paris).

- A prolonged (10-days) delivery of beta2/3 agonists does not increase anymore motoneuron F/I gain both in WT and SOD1 mice, indicating a homeostatic regulation of excitability (Paris). It downregulates beta2/3 receptors themselves and has no effect on disease markers (Ulm).

- Altogether, the combined Paris and Ulm work demonstrates that MN are subjected to a, so far overlooked, adrenergic neuromodulation through beta2/3 receptors. This type of adrenergic neuromodulation displays a substantial homeostatic control through several feedback loops.

- This homeostatic control is not dysregulated in presymptomatic adult SOD1 mice, contrasting with a recent hypothesis according to which ALS pathophysiology involved dysregulated homeostasis.

Aim 2

- Synaptic conductance elicited by Renshaw inhibition is similar in MN from WT and SOD1 mice (complex in vivo electrophysiology, Paris).

- Small alteration of Glycinergic Receptors on SOD1 MN at P21 but at a lesser extent at P45 (time of electrophysiological experiments), but there is a paradoxical upregulation of GABAR clusters (Ulm)

- No change in the kinetics of Renshaw inhibition suggesting that changes in Glycinergic and GABA receptors are compensating each other (Paris).

- Both GlyR and GABAR clusters are sensitive to changes in excitability (by PSAM/PSEM chemogenetics) and to changes in PKA signaling (by DREADD-Gs), responding toward a restoration of excitation setpoint; Gephyrin degradation (using functionalized anti-Gephyrin intrabody) has no consequences on excitatory synapses while the disease markers are significantly reduced (Ulm in collaboration with Paris).

- Identification of a new AAV9 with mutations in the capside that retrogradely (muscle injection) infects MN in adult mice at a rate higher than 50% (Paris and Ulm).

- This allows us to perform in vivo intracellular electrophysiological recordings in spinal MN infected by these mutated-AAV9s at adulthood, opening the door to in vivo electrophysiological recordings in the MN in which gene expression is manipulated using a mutated-AAV9 vector (Paris).

Our results indicate that a prolonged pharmacological activation of the Gs-coupled receptors may not be the best way to elicit a sustained activation of the PKA pathway. Other pharmacological strategies, acting more directly on the pathway, should be envisioned (for instance drugs acting on the accumulation/degradation of the cAMP could be tested).

Our discovery of a mutated-AAV9 vector endowed with good retrograde infectious capability pave the way to experimentations where the transcription of many membrane receptors, ion channels, synaptic proteins may be manipulated. This will allow to further investigate the mechanisms by which synaptic inputs and MN excitability interfere with ALS pathophysiology and, hopefully, offer a potential tool for gene therapy.

Amyotrophic Lateral Sclerosis (ALS) is an invariably fatal motoneuron (MN) disease causing the progressive degeneration of upper and lower motoneurons severely affects voluntary movements, speech and breathing. Median survival after diagnosis is estimated to be 4–5 years. To date, there is neither preventive nor efficient therapeutic approach that can alter disease progression. Multiple genetic murine models have revealed differential vulnerability of MNs to the disease across motor pools, or within each motor pool. It has recently been demonstrated, and the two laboratories involved in this project have largely contributed to this demonstration, that vulnerable MNs experience a reduced excitation before degeneration, and that restoring excitation reduces some disease markers. Thus, loss of MN excitation is a newly appreciated critical step in the degeneration of MNs in ALS. What is the origin of such phenotype and how can it be addressed therapeutically? Preliminary data from the two applicant laboratories clearly show that synaptic inputs to MN are abnormal very early in disease progression and that the dysfunction originates from the disruption of the post-synaptic structures. Our goal is to investigate whether dysfunction of excitatory synapses can be reversed by selective chemogenetic manipulations and to show that restoration of the excitation/inhibition balance re-instates MN firing capacity, ameliorates disease markers and slows down the disease progression. This would be a new therapeutic strategy with translational applications. This objective will be achieved by complementary and cutting-edge in vivo technologies that our two laboratories master: advanced viral (AAV) vectors and in vivo chemogenetic manipulation of synaptic inputs and intrinsic excitability (Ulm team); in vivo electrophysiological probing of synaptic inputs and MN properties in mice (Paris team). The demonstration of synaptic disturbances as primum movens of the disease would be a significant conceptual advancement, implying that neurodegeneration is actually highly dependent on proper synaptic input, instead of being dependent on biochemical parameters, and can be therefore therapeutically targeted at synaptic level.