Ca2+ dysregulation in the pathogenesis of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease without appropriate cure. One of the main reasons for the lack of a proper pharmacotherapy in ALS is the narrow knowledge on the molecular causes of the disease. In this respect, the identification of dysfunctional pathways in ALS is now considered a critical medical need. Among the causative factors involved in ALS, Ca2+ dysregulation is one of the most important pathogenetic mechanisms of the disease. Of note, Ca2+ dysfunction may induce, directly or indirectly, motor neuron degeneration and loss. Interestingly, both familial (fALS) and sporadic ALS (sALS) share the progressive dysregulation of Ca2+ homeostasis as a common noxious mechanism. Mechanicistically, Ca2+ dysfunction involves both plasma membrane and intracellular mechanisms, including AMPA receptor (AMPAR)-mediated excitotoxicity, voltage-gated Ca2+ channels (VGCCs) and Ca2+ transporter dysregulation, endoplasmic reticulum (ER) Ca2+ deregulation, mitochondria-associated ER membranes (MAMs) dysfunction, lysosomal Ca2+ leak, etc. Here, a comprehensive analysis of the main pathways involved in the dysregulation of Ca2+ homeostasis has been reported with the aim to focus the attention on new putative druggable targets.

Lysosomal calcium is modulated by STIM1/TRPML1 interaction which participates to neuronal survival during ischemic preconditioning.

A robust activity of the lysosomal Ca2+ channel TRPML1 is sufficient to correct cellular defects in neurodegeneration. Importantly, lysosomes are refilled by the endoplasmic reticulum (ER). However, it is unclear how TRPML1 function could be modulated by the ER. Here, we deal with this issue in rat primary cortical neurons exposed to different oxygen conditions affecting neuronal survival. Under normoxic conditions, TRPML1: (1) showed a wide distribution within soma and along neuronal processes; (2) was stimulated by the synthetic agonist ML-SA1 and the analog of its endogenous modulator, PI(3,5)P2 diC8; (3) its knockdown by siRNA strategy produced an ER Ca2+ accumulation; (4) co-localized and co-immunoprecipitated with the ER-located Ca2+ sensor stromal interacting molecule 1 (STIM1). In cortical neurons lacking STIM1, ML-SA1 and PI(3,5)P2 diC8 failed to induce Ca2+ release and, more deeply, they induced a negligible Ca2+ passage through the channel in neurons transfected with the genetically encoded Ca2+ indicator GCaMP3-ML1. Moreover, TRPML1/STIM1 interplay changed at low-oxygen conditions: both proteins were downregulated during the ischemic preconditioning (IPC) while during IPC followed by 1 hour of normoxia, at which STIM1 is upregulated, TRPML1 protein was reduced. However, during oxygen and glucose deprivation (OGD) followed by reoxygenation, TRPML1 and STIM1 proteins peaked at 8 hours of reoxygenation, when the proteins were co-immunoprecipitated and reactive oxygen species (ROS) hyperproduction was measured in cortical neurons. This may lead to a persistent TRPML1 Ca2+ release and lysosomal Ca2+ loss. Collectively, we showed a new modulation exerted by STIM1 on TRPML1 activity that may differently intervene during hypoxia to regulate organellar Ca2+ homeostasis.

STIM Protein-NMDA2 Receptor Interaction Decreases NMDA-Dependent Calcium Levels in Cortical Neurons.

Neuronal Store-Operated Ca2+ Entry (nSOCE) plays an essential role in refilling endoplasmic reticulum Ca2+ stores and is critical for Ca2+-dependent neuronal processes. SOCE sensors, STIM1 and STIM2, can activate Orai, TRP channels and AMPA receptors, and inhibit voltage-gated channels in the plasma membrane. However, the link between STIM, SOCE, and NMDA receptors, another key cellular entry point for Ca2+ contributing to synaptic plasticity and excitotoxicity, remains unclear. Using Ca2+ imaging, we demonstrated that thapsigargin-induced nSOCE was inhibited in rat cortical neurons following NMDAR inhibitors. Blocking nSOCE by its inhibitor SKF96365 enhanced NMDA-driven [Ca2+]i. Modulating STIM protein level through overexpression or shRNA inhibited or activated NMDA-evoked [Ca2+]i, respectively. Using proximity ligation assays, immunofluorescence, and co-immunoprecipitation methods, we discovered that thapsigargin-dependent effects required interactions between STIMs and the NMDAR2 subunits. Since STIMs modulate NMDAR-mediated Ca2+ levels, we propose targeting this mechanism as a novel therapeutic strategy against neuropathological conditions that feature NMDA-induced Ca2+ overload as a diagnostic criterion.

Photosensitizer Activation Drives Apoptosis by Interorganellar Ca2+ Transfer and Superoxide Production in Bystander Cancer Cells.

In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca2+) and other second messengers, resulting in cell demise. Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called "bystander effects". To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca2+ transfer and the production of mitochondrial superoxide anion (O2-∙) and hydrogen peroxide (H2O2) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca2+ uptake. Efficient Ca2+ efflux from the ER was required to promote mitochondrial O2-∙ production in these bystander cells. Our results support a key role for ER-mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing.