Riluzole But Not Melatonin Ameliorates Acute Motor Neuron Degeneration and Moderately Inhibits SOD1-Mediated Excitotoxicity Induced Disrupted Mitochondrial Ca2+ Signaling in Amyotrophic Lateral Sclerosis

Abstract

Selective motoneurons (MNs) degeneration in the brain stem, hypoglossal motoneurons (HMNs), and the spinal cord resulting in patients paralysis and eventual death are prominent features of amyotrophic lateral sclerosis (ALS). Previous studies have suggested that mitochondrial respiratory impairment, low Ca2+ buffering and homeostasis and excitotoxicity are the pathological phenotypes found in mice, and cell culture models of familial ALS (fALS) linked with Cu/Zn-superoxide dismutase 1 (SOD1) mutation. In our study, we aimed to understand the impact of riluzole and melatonin on excitotoxicity, neuronal protection and Ca2+ signaling in individual HMNs ex vivo in symptomatic adult ALS mouse brain stem slice preparations and in WT and SOD1-G93A transfected SH-SY5Y neuroblastoma cell line using fluorescence microscopy, calcium imaging with high speed charged coupled device camera, together with immunohistochemistry, cell survival assay and histology. In our experiments, riluzole but not melatonin ameliorates MNs degeneration and moderately inhibit excitotoxicity and cell death in SH-SY5YWT or SH-SY5YG93A cell lines induced by complex IV blocker sodium azide. In brain stem slice preparations, riluzole significantly inhibit HMNs cell death induced by inhibiting the mitochondrial electron transport chain by Na-azide. In the HMNs of brainstem slice prepared from adult (14–15 weeks) WT, and corresponding symptomatic SOD1G93A mice, we measured the effect of riluzole and melatonin on [Ca2+]i using fura-2 AM ratiometric calcium imaging in individual MNs. Riluzole caused a significant decrease in [Ca2+]i transients and reversibly inhibited [Ca2+]i transients in Fura-2 AM loaded HMNs exposed to Na-azide in adult symptomatic SOD1G93A mice. On the contrary, melatonin failed to show similar effects in the HMNs of WT and SOD1G93A mice. Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence, an indicator of mitochondrial metabolism and health in MNs, showed enhanced intrinsic NADH fluorescence in HMNs in presence of riluzole when respiratory chain activity was inhibited by Na-azide. Riluzole’s inhibition of excitability and Ca2+ signaling may be due to its multiple effects on cellular function of mitochondria. Therefore formulating a drug therapy to stabilize mitochondria-related signaling pathways using riluzole might be a valuable approach for cell death protection in ALS. Taken together, the pharmacological profiles of the riluzole and melatonin strengthen the case that riluzole indeed can be used as a therapeutic agent in ALS whereas claims of the efficacy of melatonin alone need further investigation as it fail to show significant neuroprotection efficacy.

Riluzole and edaravone: A tale of two amyotrophic lateral sclerosis drugs

“…Riluzole belongs to the benzothiazole class, a glutamate antagonist, and it appears to block the excessive release of glutamate from MNs. 30,31 Unrestrained secretion of glutamate at synaptic junction overstimulates the MNs receiving the signals, which leads to abnormally high levels of calcium in MNs soma and glial cells.…”

Discrete mitochondrial aberrations in the spinal cord of sporadic ALS patients

“…Increased Ca 21 levels may also stimulate nitric oxide synthase leading to nitric oxide-mediated inhibition of complex IV (Beal, 1999). Consistent with this possibility is evidence linking deficits in motor neuron mitochondrial Ca 21 buffering capacity and ALS (Jaiswal, 2014(Jaiswal, , 2017Tadic, Prell, Lautenschlaeger, & Grosskreutz, 2014).…”

Ionic Homeostasis Maintenance in ALS: Focus on New Therapeutic Targets

“…Recently, an additional possible mechanism of action has been reported for riluzole. This involves a considerable reduction of [Ca 2+ ] i transient currents and a reversible inhibition of [Ca 2+ ] i inward currents in MNs of adult symptomatic SOD1 G93A mice (Jaiswal, 2017 ); thus confirming the importance of Ca 2+ homeostasis in ALS pathophysiology.…”

“…In these patients, riluzole, a drug able to increase survival rate, has shown to prevent MNs deterioration and moderately reduce excitotoxicity and cell loss by blocking tetrodotoxin-sensitive sodium channels which are associated with damaged neurons. Deregulation of intracellular calcium homeostasis has also been described in the terminal motor axons of subjects affected by ALS, as well as in the spinal MNs of ALS animals, both in the sporadic and familial forms (Jaiswal, 2017 ). Recently, an additional possible mechanism of action has been reported for riluzole.…”