Lysophosphatidic Acid and Several Neurotransmitters Converge on Rho-Kinase 2 Signaling to Manage Motoneuron Excitability

Intrinsic membrane excitability (IME) sets up neuronal responsiveness to synaptic drive. Several neurotransmitters and neuromodulators, acting through G-protein-coupled receptors (GPCRs), fine-tune motoneuron (MN) IME by modulating background K+ channels TASK1. However, intracellular partners linking GPCRs to TASK1 modulation are not yet well-known. We hypothesized that isoform 2 of rho-kinase (ROCK2), acting as downstream GPCRs, mediates adjustment of MN IME via TASK1. Electrophysiological recordings were performed in hypoglossal MNs (HMNs) obtained from adult and neonatal rats, neonatal knockout mice for TASK1 (task1(-/-)) and TASK3 (task3(-/-), the another highly expressed TASK subunit in MNs), and primary cultures of embryonic spinal cord MNs (SMNs). Small-interfering RNA (siRNA) technology was also used to knockdown either ROCK1 or ROCK2. Furthermore, ROCK activity assays were performed to evaluate the ability of various physiological GPCR ligands to stimulate ROCK. Microiontophoretically applied H1152, a ROCK inhibitor, and siRNA-induced ROCK2 knockdown both depressed AMPAergic, inspiratory-related discharge activity of adult HMNs in vivo, which mainly express the ROCK2 isoform. In brainstem slices, intracellular constitutively active ROCK2 (aROCK2) led to H1152-sensitive HMN hyper-excitability. The aROCK2 inhibited pH-sensitive and TASK1-mediated currents in SMNs. Conclusively, aROCK2 increased IME in task3(-/-), but not in task1(-/-) HMNs. MN IME was also augmented by the physiological neuromodulator lysophosphatidic acid (LPA) through a mechanism entailing G(alpha i/o)-protein stimulation, ROCK2, but not ROCK1, activity and TASK1 inhibition. Finally, two neurotransmitters, TRH, and 5-HT, which are both known to increase MN IME by TASK1 inhibition, stimulated ROCK2, and depressed background resting currents via G(alpha q)/ROCK2 signaling. These outcomes suggest that LPA and several neurotransmitters impact MN IME via G(alpha i/o)/G(alpha q)-protein-coupled receptors, downstream ROCK2 activation, and subsequent inhibition of TASK1 channels

Targeting autotaxin impacts disease advance in the SOD1-G93A mouse model of amyotrophic lateral sclerosis

A preclinical strategy to broaden the search of potentially effective treatments in amyotrophic lateral sclerosis (ALS) relies on identifying factors controlling motor neuron (MN) excitability. These partners might be part of still unknown pathogenic pathways and/or useful for the design of new interventions to affect disease progression. In this framework, the bioactive membrane-derived phospholipid lysophosphatidic acid (LPA) affects MN excitability through LPA receptor 1 (LPA(1)). Furthermore, LPA(1) knockdown is neuroprotective in transgenic ALS SOD1-G93A mice. On this basis, we raised the hypothesis that the major LPA-synthesizing ectoenzyme, autotaxin (ATX), regulates MN excitability and is a potential target to modulate disease development in ALS mice. We show here that PF-8380, a specific ATX inhibitor, reduced intrinsic membrane excitability (IME) of hypoglossal MNs in brainstem slices, supporting that baseline ATX activity regulates MN IME. PF-8380-induced alterations were prevented by a small-interfering RNA directed against mRNA for lpa(1). These outcomes support that impact of ATX-originated lysophospholipids on MN IME engages, at least, the G-protein-coupled receptor LPA(1). Interestingly, mRNA(atx) levels increased in the spinal cord of pre-symptomatic (1-2 months old) SOD1-G93A mice, thus preceding MN loss. The rise in transcripts levels also occurred in cultured spinal cord MNs from SOD1-G93A embryos, suggesting that mRNA(atx) upregulation in MNs is an etiopathogenic event in the ALS cell model. Remarkably, chronic administration in the drinking water of the orally bioavailable ATX inhibitor PF-8380 delayed MN loss, motor deterioration and prolonged life span in ALS mice. Treatment also led to a reduction in LPA(1)-immunoreactive patches in transgenic animals mostly in MNs. These outcomes support that neuroprotective effects of interfering with ATX in SOD1-G93A mice rely, at least in part, on LPA(1) knockdown in MNs. Therefore, we propose ATX as a potential target and/or a biomarker in ALS and highlight ATX inhibitors as reasonable tools with therapeutic usefulness for this lethal pathology.BFU2015-71422-R (MINECO/FEDER) from Spain's Government; FEDER-UCA18-108475 from the 2014-2020 ERDF Operational Programme and the Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia; LI19/10IN-CO21 from INiBICA to BML and PID2019-110960GB-I00 (MICINN) from Spain's Government to BML and DG