STIM1 Deficiency Leads to Specific Down-Regulation of ITPR3 in SH-SY5Y Cells

Abstract

STIM1 is an endoplasmic reticulum (ER) protein that modulates the activity of a number of Ca2+ transport systems. By direct physical interaction with ORAI1, a plasma membrane Ca2+ channel, STIM1 activates the ICRAC current, whereas the binding with the voltage-operated Ca2+ channel CaV1.2 inhibits the current through this latter channel. In this way, STIM1 is a key regulator of Ca2+ signaling in excitable and non-excitable cells, and altered STIM1 levels have been reported to underlie several pathologies, including immunodeficiency, neurodegenerative diseases, and cancer. In both sporadic and familial Alzheimer’s disease, a decrease of STIM1 protein levels accounts for the alteration of Ca2+ handling that compromises neuronal cell viability. Using SH-SY5Y cells edited by CRISPR/Cas9 to knockout STIM1 gene expression, this work evaluated the molecular mechanisms underlying the cell death triggered by the deficiency of STIM1, demonstrating that STIM1 is a positive regulator of ITPR3 gene expression. ITPR3 (or IP3R3) is a Ca2+ channel enriched at ER-mitochondria contact sites where it provides Ca2+ for transport into the mitochondria. Thus, STIM1 deficiency leads to a strong reduction of ITPR3 transcript and ITPR3 protein levels, a consequent decrease of the mitochondria free Ca2+ concentration ([Ca2+]mit), reduction of mitochondrial oxygen consumption rate, and decrease in ATP synthesis rate. All these values were normalized by ectopic expression of ITPR3 in STIM1-KO cells, providing strong evidence for a new mode of regulation of [Ca2+]mit mediated by the STIM1-ITPR3 axis.

Application of CRISPR/Cas9 in Alzheimer’s Disease

“…In 2020, Pascual-Caro et al ( 2020) found a remarkable reduction of ITPR3 following the knock-out of STIM1 gene, which caused the mitochondrial function abnormality. This further confirmed the potential role of STIM1-ITPR3 axis in the pathogenesis of AD (Pascual-Caro et al, 2020). Similarly, Knupp et al (2020) found that the depletion of SORLA in hiPSCs by using CRISPR/Cas9 can cause early endosome enlargement in hiPSCs-derived neurons rather than hiPSC microglia, and inhibition of BACE is not able to rescue the endosome enlargement phenotype in hiPSC neurons, indicating the independent function of SORLA in AD pathogenesis.…”

Plasma Membrane and Organellar Targets of STIM1 for Intracellular Calcium Handling in Health and Neurodegenerative Diseases

“…This includes regulation of ER refilling through mGluR1 and 5 [155,156] and of synaptic plasticity [157], as well as Ca 2+ overload through upregulation of L-type VGCCs and mitochondrial dysfunction [152]. A recent work has demonstrated that STIM1-deficient SH-SY5Y cells display reduced expression of the IP 3 receptors type 3 (IP 3 R3) that is, in turn, responsible for the low mitochondrial Ca 2+ concentration ([Ca 2+ ]) and for the reduced efficiency of this organelle [158], which are common features in AD patients [159].…”

Target Molecules of STIM Proteins in the Central Nervous System

“…However, in some mammalian cells, IP 3 Rs have been shown to co-localize with Orai1 (Lur et al, 2011 ) and interact with STIM1, Orai1, and TRPCs (Hong et al, 2011 ). The expression of IP 3 R isoform (IP 3 R 3 ) was shown to be significantly lower in STIM1-deficient SH-SY5Y cells, meaning that STIM1 is a positive regulator of ITPR3 gene expression in these cells (Pascual-Caro et al, 2020 ). IP 3 R 3 is a Ca 2+ channel that is localized mainly at the ER-mitochondrion junction, which transfers Ca 2+ from the ER to mitochondria (Ivanova et al, 2014 ).…”

“…Thus, STIM1 deficiency leads to a decrease in mitochondrial Ca 2+ concentrations, leading to cell death. The overexpression of IP 3 R 3 restores mitochondrial Ca 2+ homeostasis and bioenergetics, ATP production, and cell survival in STIM1-KO neuronal-like cells (Pascual-Caro et al, 2020 ). These results provide evidence of a novel STIM1-IP 3 R 3 -mediated pathway of mitochondrial Ca 2+ levels, the dysregulation of which contributes to neurodegeneration.…”

“…These results provide evidence of a novel STIM1-IP 3 R 3 -mediated pathway of mitochondrial Ca 2+ levels, the dysregulation of which contributes to neurodegeneration. Mitochondria from AD patients have lower Ca 2+ uptake (Kumar et al, 1994 ), which is attributed to lower IP 3 R 3 and STIM1 levels (Pascual-Caro et al, 2020 ).…”