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Amyotrophic lateral sclerosis is characterized by progressive death of motor neurons (MNs) with glutamate excitotoxicity and mitochondrial Ca2+ overload as critical mechanisms in disease pathophysiology. We used MNs from G93AhSOD1 and nontransgenic embryonic cultures and adult mice to analyze the expression of the main mitochondrial calcium uniporter (MCU). MCU was overexpressed in cultured embryonic G93AhSOD1 MNs compared to nontransgenic MNs but downregulated in MNs from adult G93AhSOD1 mice. Furthermore, cultured embryonic G93AhSOD1 were rescued from kainate-induced excitotoxicity by the Ca2+/calmodulin-dependent protein kinase type II inhibitor; KN-62, which reduced MCU expression in G93AhSOD1 MNs. MCU activation via kaempferol neither altered MCU expression nor influenced MN survival. However, its acute application served as a fine tool to study spontaneous Ca2+ activity in cultured neurons which was significantly altered by the mutated hSOD1. Pharmacological manipulation of MCU expression might open new possibilities to fight excitotoxic damage in amyotrophic lateral sclerosis.

Aberrations in intracellular calcium (Ca2+) have been well established within amyotrophic lateral sclerosis (ALS), a severe motor neuron disease. Intracellular Ca2+ concentration is controlled in part through the endoplasmic reticulum (ER) mitochondria Ca2+ cycle (ERMCC). The ER supplies Ca2+ to the mitochondria at close contacts between the two organelles, i.e. the mitochondria-associated ER membranes (MAMs). The Sigma 1 receptor (Sig1R) is enriched at MAMs, where it acts as an inter-organelle signaling modulator. However, its impact on intracellular Ca2+ at the cellular level remains to be thoroughly investigated. Here, we used cultured embryonic mice spinal neurons to investigate the influence of Sig1R activation on intracellular Ca2+ homeostasis in the presence of G93AhSOD1 (G93A), an established ALS-causing mutation. Sig1R expression was increased in G93A motor neurons relative to non-transgenic (nontg) controls. Furthermore, we demonstrated significantly reduced bradykinin-sensitive intracellular Ca2+ stores in G93A spinal neurons, which were normalized by the Sig1R agonist SA4503. Moreover, SA4503 accelerated cytosolic Ca2+ clearance following a) AMPAR activation by kainate and b) IP3R-mediated ER Ca2+ release following bradykinin stimulation in both genotypes. PRE-084 (another Sig1R agonist) did not exert any significant effects on cytosolic Ca2+. Both Sig1R expression and functionality were altered by the G93A mutation, indicating the centrality of Sig1R in ALS pathology. Here, we showed that intracellular Ca2+ shuttling can be manipulated by Sig1R activation, thus demonstrating the value of using the pharmacological manipulation of Sig1R to understand Ca2+ homeostasis.

Amyotrophic lateral sclerosis (ALS) is a multisystemic neurodegenerative disorder. Given that peripheral blood mononuclear cells (PBMCs) serve as a “window to the central nervous system” we aimed to answer whether endoplasmic reticulum (ER) stress in ALS-PBMCs is related to disease aggressiveness. We studied ER stress in the PBMCs of 49 patients with ALS and 31 age- and sex-matched healthy controls. The expression of a main ER stress marker, activating transcription factor 6 (ATF6), was significantly higher in ALS compared to controls, but did not correlate with age, disease severity, disease duration and disease progression rate. When ATF6 expression levels were plotted against relative D50 (rD50)-derived disease phases derived from the D50 ALS model, two distinct clusters of patients were observed: cluster 1, with progressively increasing ATF6 expression levels and cluster 2, which demonstrated stable ATF6 expression over the disease course. Individuals in the two clusters did not significantly differ in terms of ALS Functional Rating Scale-Revised (ALSFRS-R), disease aggressiveness, disease duration and subtype. However, patients with the increasing ATF6 level were significantly younger, indicating that aging processes might be related to ER stress in ALS. Our data suggest that the reaction to ER stress during disease course may be compromised in older patients with ALS.

Motor neurons are markedly vulnerable to excitotoxicity mostly by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) stimulation and are principal targets in the neurodegenerative disease Amyotrophic Lateral Sclerosis. Interferon-gamma (IFN-γ), a pro-inflammatory cytokine, can independently cause neuronal dysfunction by triggering calcium influx through a calcium-permeable complex of IFN-γ receptor 1(IFNGR1) subunit and AMPAR subunit GluR1. This receptor complex is formed via a non-canonical neuron-specific IFN-γ pathway that involves Jak1/Stat1 and Protein Kinase A. In this study, we explore the expression of the pathway's participants for the first time in the hSOD1G93A Amyotrophic Lateral Sclerosis mouse model. Elevated IFNGR1 and GluR1 are detected in motor neurons of hSOD1G93A symptomatic mice ex vivo, unlike the downstream targets - Jak1, Stat1, and Protein Kinase A. We, also, determine effects of IFN-γ alone or in the presence of an excitotoxic agent, kainate, on motor neuron survival in vitro. IFN-γ induces neuronal damage, but does not influence kainate-mediated excitotoxicity. Increased IFNGR1 can most likely sensitize motor neurons to excitotoxic insults involving GluR1 and/or pathways mediated by IFN-γ, thus, serving as a potential direct link between neurodegeneration and inflammation in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis is characterized by progressive death of motor neurons (MNs) with glutamate excitotoxicity and mitochondrial Ca

Aberrations in intracellular calcium (Ca

Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with intracellular Ca 2+ dysregulation. The P2X receptor family is comprised of ligand-gated ion channels that respond to extracellular adenosine triphosphate (ATP) and increases permeability of calcium into the cell. The underlying mechanisms of purinergic signalling on peripheral blood mononuclear cells (PBMCs) in ALS remain unclear. Herein, we studied the expression of P2X4/P2X7 receptors and calcium homeostasis in blood cells of ALS patients. Methods We used PBMCs from 42 ALS patients and 19 controls. Purinergic receptors P2X4 (P2X4R) and P2X7 (P2X7R) were examined using western blot analysis. The effect of exogenous ATP on intracellular Ca 2+ homeostasis in monocytes was measured using fluorimetry by Fura-2 on a single-cell level. Results Western blot analysis revealed stable P2X4R expression in patients and controls. P2X7R expression was significantly reduced (p = 0.012) in ALS patients. Repetitive long-term ATP stimulation caused a sustained decrease in Ca 2+ levels in the ALS group as measured by the area under the curve, peak amplitude and peak height. Conclusion These results confirm our hypothesis that Ca 2+ abnormalities in ALS are measurable in immune cells. These findings suggest that the reduction of P2X7 receptor expression on PBMCs leads to intracellular calcium dysregulation. Our study improves the understanding of ALS pathophysiology and proposes PBMCs as a non-invasive source to study ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. Although the etiology remains unclear, disturbances in calcium homoeostasis and protein folding are essential features of neurodegeneration in this disorder. Here, we review recent research findings on the interaction between endoplasmic reticulum (ER) and mitochondria, and its effect on calcium signaling and oxidative stress. We further provide insights into studies, providing evidence that structures of the ER mitochondria calcium cycle (ERMCC) serve as a promising targets for therapeutic approaches for treatment of ALS.