Calcium Ions Aggravate Alzheimer’s Disease Through the Aberrant Activation of Neuronal Networks, Leading to Synaptic and Cognitive Deficits

Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by the production and deposition of β-amyloid protein (Aβ) and hyperphosphorylated tau, leading to the formation of β-amyloid plaques (APs) and neurofibrillary tangles (NFTs). Although calcium ions (Ca2+) promote the formation of APs and NFTs, no systematic review of the mechanisms by which Ca2+ affects the development and progression of AD has been published. Therefore, the current review aimed to fill the gaps between elevated Ca2+ levels and the pathogenesis of AD. Specifically, we mainly focus on the molecular mechanisms by which Ca2+ affects the neuronal networks of neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, and autophagy. Furthermore, the roles of Ca2+ transporters located in the cell membrane, endoplasmic reticulum (ER), mitochondria and lysosome in mediating the effects of Ca2+ on activating neuronal networks that ultimately contribute to the development and progression of AD are discussed. Finally, the drug candidates derived from herbs used as food or seasoning in Chinese daily life are summarized to provide a theoretical basis for improving the clinical treatment of AD.


INTRODUCTION
Alzheimer's disease (AD) is a neurodegenerative disease with cognitive deficit as the main characteristic (Elgh et al., 2006). During the course of AD development and progression, calcium ion (Ca 2+ ) concentrations are obviously increased in the brains of patients with AD and APP/PS1 Tg mice . One report has shown that β-amyloid protein (Aβ)   has the ability to increase Ca 2+ influx in rat cortical synaptosomes and cultured cortical neurons (MacManus et al., 2000). Similar to  ,  induce the Ca 2+ influx via RyRs in primary cultured hippocampal neurons (Marcantoni et al., 2020). Furthermore, the Aβ 25-35 peptide promotes Ca 2+ influx by activating L-and T-type Ca 2+ channels in rat hippocampal slices (Li et al., 2010). The APP intracellular domain (AICD), a APP cleavage fragment, may act as a transcription factor to activate the Ca 2+ signaling system (Cao and Südhof, 2001;Leissring et al., 2002). Because of the self-aggregating characteristics of Aβ, Aβ oligomers can promote Ca 2+ influx through N-methyl-D-aspartic acid receptor (NMDAR) channels in a short period of time (Kelly and Ferreira, 2006). More directly, Arispe et al. (2010) found that the aggregates of  and  form a cation channel on the surface of an artificial lipid membrane that allows the passage of Ca 2+ . The pore formation ability of Aβ was confirmed and corroborated by atomic force microscopy (Lin et al., 2001), electron microscopy (Lashuel et al., 2002(Lashuel et al., , 2003, and a theoretical model (Durell et al., 1994;Jang et al., 2008).
Reciprocally, Ca 2+ is not a passive contributor to the development and progression of AD. In PS-mutant AD brain tissue, a Ca 2+ metabolic disorder was evident before the formation of APs or NFTs (Etcheberrigaray et al., 1998), which indicated that the metabolic disorder caused by Ca 2+ located in the cytoplasm might be the cause of AD. Based on this hypothesis, previous studies have shown that Ca 2+ influx increases the production and aggregation of Aβ and the phosphorylated tau protein, which affects the learning and memory of patients with AD (Etcheberrigaray et al., 1998;Zempel et al., 2010;Tong et al., 2018). Moreover, Ca 2+ imbalance leads to dysregulated metabolism that affects many neurophysiological functions related to AD, including the regulation of neuroinflammation, response to neuronal injury, neuronal regeneration, neurotoxicity and autophagy (Wahlestedt et al., 1993;Liu and Zukin, 2007;Decuypere et al., 2011a;Sama and Norris, 2013;Song et al., 2019). These actions of Ca 2+ may finally contribute to neuronal death, which results in cognitive decline during the course of AD development and progression.
Given the multiple functions of Ca 2+ in AD, its transporters in the cell membrane, endoplasmic reticulum (ER), mitochondria and lysosomes must be involved in regulating the development and progression of AD. As an antagonist of NMDAR, a Ca 2+ transporter on the surface of the nerve cell membrane, memantine significantly inhibits Ca 2+ influx and was the first Food and Drug Administration (FDA)-approved drug for the treatment of moderate to severe AD in patients (Bullock, 2006). Regarding the important reservoir of Ca 2+ in neurons, the ER has been reported to release Ca 2+ to the cytosol, which contributes to the development and progression of AD (Guan et al., 2021). Although direct evidence showing the relationship between Ca 2+ transport from mitochondria and lysosomes and the learning ability of patients with AD is unavailable, voltage-dependent anion channel protein 1 (VDAC1) is a hub protein that interacts with phosphorylated tau, Aβ, and γ-secretase, and it contributes to their toxic effects on triggering cell death and potentially leading to the dementia that is a characteristic of AD (Shoshan- Barmatz et al., 2018). All this evidence prompted us to summarize the roles of Ca 2+ transporters located in different organelles in regulating the development and progression of AD.
Therefore, this review mainly summarizes the molecular mechanisms by which a Ca 2+ imbalance in individuals with AD affects the regulation of neuroinflammation, neuronal injury, neuronal regeneration, neurotoxicity, neuroprotection, and autophagy, specifically from the perspective of Ca 2+ transporters in the cell, mitochondria, endoplasmic reticulum and lysosomal membranes. By addressing these mechanisms, we will fill the gaps between increased Ca 2+ concentrations and the fate of neurons, which results in dementia.

CROSSTALK BETWEEN FACTORS RESPONSIBLE FOR Ca 2+ DYSHOMEOSTASIS AND NEUROINFLAMMATION Ca 2+ Increases the Production of Proinflammatory Cytokines
Neuroinflammation is widely accepted to be mediated by Ca 2+ dyshomeostasis and induces the cognitive decline associated with AD. This process is studied to understand the inherent mechanisms by which Ca 2+ exerts an effect. For example, Ca 2+ increases the production of interleukin (IL)-1β and tumor necrosis factor α (TNF-α) via calcineurin (CaN) in glial cells (Sama and Norris, 2013). Consistently, an indirect blockade of Ca 2+ entry into lipopolysaccharide (LPS)-activated microglia stimulates the production of proinflammatory cytokines, such as TNF-α and IL-6 (Dolga et al., 2012). These observations revealed critical roles for Ca 2+ in inducing neuroinflammation by concurrently increasing the production of proinflammatory cytokines and decreasing the levels of anti-inflammatory cytokines.

Transporters on the Cell Membrane Mediate the Effects of Ca 2+ on the Secretion of Proinflammatory Cytokines
Based on these observations, Ca 2+ transporters were found to be involved in regulating neuroinflammation. More specifically, NMDAR is critical for mediating the effects of Ca 2+ on stimulating the production of proinflammatory cytokines, such as IL-1β and TNF-α, in primary mouse hippocampal neurons and lamina II neurons of isolated spinal cord slices (Kawasaki et al., 2008;Huang et al., 2011). By deactivating NMDAR, sevoflurane, an NMDAR antagonist, inhibits the production of IL-1β, TNF-α, IL-6, and IL-8, whereas the addition of the NMDAR agonist D-cycloserine restores the suppression of ageing phenotype acquisition in rats (Yang Z. Y. et al., 2020). NMDAR overexpression in primary cultured microglial cells was induced to synthesize nitric oxide (NO) by activating the NF-κB signaling pathway and to exclude the nonspecific action of these pharmacological interventions (Murugan et al., 2011). In the context of inflammation, NMDAR blockade attenuates the clinical symptoms of glutamate excitotoxicity, suggesting that NMDAR exerts potential neuroprotective effects (Wallström et al., 1996). Similar to this observation, blocking the AMPA/kainate receptor also results in the neuroprotection of encephalomyelitis-sensitized mice (Pitt et al., 2000;Smith et al., 2000). Based on this observation, researchers have readily deduced that α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) might also be involved in regulating neuroinflammation. In SG neurons and lamina II neurons isolated from spinal cord slices, AMPAR was reported to mediate Ca 2+ -stimulated secretion of proinflammatory cytokines, such as IL-1β and TNF-α . Perampanel, an AMPAR antagonist, concurrently suppressed the expression of proinflammatory cytokines, including IL-1β and TNF-α, and upregulates the expression of anti-inflammatory cytokines, including IL-10 and Transforming Growth Factor Beta 1 (TGF β1), in a rat model of traumatic brain injury (TBI; . In addition to glutamate receptors serving as transporters of Ca 2+ , some Ca 2+ transporters in the cell membrane are reported to be involved in regulating neuroinflammation. For example, the blockade of L-type voltage-gated calcium channels (L-VGCC) by bepridil, nitrendipine or nimodipine attenuates neuroinflammation by deactivating astrocytes and microglial cells in LPS-stimulated or artificial cerebrospinal fluid (aCSF)injected (i.c.v.) rats and astrocytes from the CA1 region of the hippocampus (Brand-Schieber and Werner, 2004;Daschil et al., 2013;Espinosa-Parrilla et al., 2015;Hopp et al., 2015). These observations were corroborated by the ability of Ca 2+ to induce TNF-α production in cultured rat hippocampal neurons through an L-VGCC-dependent mechanism (Furukawa and Mattson, 1998). In addition, transient receptor potential channels (TRPs) have been identified in mammals and are grouped into six families associated with the onset of neurodegenerative diseases of the central nervous system (CNS): vanilloid TRP (TRPV), melastatin TRP (TRPM), ankyrin TRP (TRPA), polycystin TRP (TRPP), and canonical or classical TRP (TRPC) channels (Morelli et al., 2013). Among these channels, TRPM2 deletion suppresses cytokine production by deactivating microglial cells in TRPM2-knockout mice (Miyanohara et al., 2018;Kakae et al., 2019). Activation of the TRPV1 channel increases the production of proinflammatory cytokines, such as IL-6, in microglial cells (Sappington and Calkins, 2008). The roles of TRPV4 in inflammation are still being debated. By blocking TRPV4 channels, the release of IL-1β and TNFα is inhibited because of the reduced Ca 2+ influx, leading to the attenuation of glial cell-mediated inflammation (Shi et al., 2013). In contrast, the opening of TRPV4 channels by a selective TRPV4 agonist, 4α-phorbol 12, 13-didecanoate (4α-PDD), prevents microglial activation and TNF-α release after LPS treatment, and TRPV4 knockdown eliminates the inhibitory effect of agonists on the release of TNF-α from cultured microglial cells (Konno et al., 2012). According to these findings, TRPV4 activation may be induced by microglial cell swelling after activation with LPS. Channel activation may thus serve as an autoregulator to avoid excess microglial activation. In addition, TRPC1-mediated negative regulation may exert an immunosuppressive effect by blocking the initiation of inflammatory pathways in primary microglial cells ; Figure 1). Although Apolipoprotein E4 (APOE4) is not regarded as a canonical Ca 2+ transporter, human APOE4 increases the activity of microglial cells by inducing the expression of IL-1β in E4F AD mice (Rodriguez et al., 2014). In contrast to APOE4, other isoforms of APOEs inhibit the synthesis of inflammatory mediators, including COX-2, PGE 2 , and IL-1β, in primary cultured microglia obtained from the adult rat brain cortex (Chen et al., 2005).
With opposite effects, proinflammatory cytokines have the ability to modulate the Ca 2+ balance via their transporters. For example, TNF-α, IL-1β, and IFNγ increase the influx of Ca 2+ into microglial cells, which indicates crosstalk between Ca 2+ and neuroinflammatory factors in cultured hippocampal neurons (Goghari et al., 2000;McLarnon et al., 2001;Franciosi et al., 2002). IL-1β increases the expression of AMPAR on the cell surface, which potentially contributes to the entry of Ca 2+ into hippocampal neurons (Viviani et al., 2003;Simões et al., 2012). In contrast to AMPAR, IL-1β inhibits L-VGCC activity by suppressing the protein expression of Ca 2+ channels in primary cultured neurons (Zhou et al., 2006;Zhou, 2010). In addition, IL-1β is responsible for increasing the expression of TRPM2, leading to the influx of Ca 2+ to microglial cells (Fonfria et al., 2006). Similar to IL-1β, IL-6 potentiates Ca 2+ entry through NMDARs in hippocampal neurons (Orellana et al., 2005). Although IL-6 is not expressed in neuronal cells, it downregulates the expression of SERCA2, which blocks Ca 2+ entry into the ER, thus maintaining high levels of cytosolic Ca 2+ in cardiac myocytes (Villegas et al., 2000). Similar to other cytokines, TNF-α increases Ca 2+ currents through NMDARs in cultured rat hippocampal neurons (Furukawa and Mattson, 1998). In addition, TNF-α induces the rapid insertion of AMPAR into the membranes of hippocampal pyramidal neurons (Ogoshi et al., 2005). In addition, the colocalization of GluA1, GluA2 and GluA4 and synaptophysin on the neural crest also indicates the transportation of AMPAR to synapses (Wigerblad et al., 2017). In contrast, TNF-α decreases Ca 2+ influx by inhibiting the activity of L-VGCCs in cultured rat hippocampal neurons and hippocampal CA1 neurons (Furukawa and Mattson, 1998;Sama et al., 2012). Regarding the regulation of intracellular stores, impaired TNF-α signaling disrupts the FIGURE 1 | Ca 2+ participates in regulating neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, autophagy and apoptosis via its transporters located on the cell membrane. Aβ activates Ca 2+ transporters, including NMDAR, AMPAR, LTCC, Na + /K + -ATPase, CALHM1, TRPV1, and Cav1.2, which promote Ca 2+ entry into the cytoplasm and increase the concentration of Ca 2+ in neuronal cells. More importantly, these Ca 2+ transporters mediate the effects of Ca 2+ on neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, autophagy, and apoptosis through different mechanisms. Aβ activates NMDAR, LTCC, CALHM1, and TRPV1, which result in apoptosis induction, leading to cell death. Regarding neuroinflammation, NMDARs mediate the effects of Aβ on activating NF-κB through a Ca 2+ -dependent mechanism, which results in transcriptional regulation of the secretion of IL-1β, IL-6, NO, and TNF-α. Moreover, NMDARs induce LC3 II production, leading to autophagy. effects of InsP3R on mediating Ca 2+ release from the ER to the cytosol in 3xTg mice (Park et al., 2010). Moreover, calcineurin (CaN) is activated by the proinflammatory cytokine TNF-α in astrocytes (Fernandez et al., 2007;Sama et al., 2008;Furman et al., 2012). TNF-α activates a more complicated mechanism to regulate Ca 2+ currents. In addition to TNF-α itself, the TNF-α receptor mobilizes Ca 2+ through an RyR-dependent mechanism in cultured neonatal rat dorsal root ganglion (DRG) neurons (Pollock et al., 2002). In addition to proinflammatory cytokines, most investigations have focused on the roles of anti-inflammatory cytokines on Ca 2+ transporters. Based on this information, researchers also found that anti-inflammatory cytokines, such as IL-10, reduced the intracellular Ca 2+ levels in microglial cells by decreasing Ca 2+ release from the ER through the deactivation of the InsP3R-dependent mechanism in cultured hippocampal neurons (Turovskaya et al., 2012). Therefore, the existence of crosstalk between Ca 2+ and neuroinflammation will result in the aggravation of AD (Figure 2).

Proinflammatory Cytokines Reciprocally Regulate the Activities of Transporters Expressed on Lysosomes to Regulate the Basal Ca 2+ Levels in Glial Cells
In SH-SY5Y cells, IFNγ also induces Ca 2+ influx by activating TRPM2, leading to the apoptosis of cultured neurons . Furthermore, IFNγ reduces the activity of ATPase Sarcoplasmic/Endoplasmic Reticulum Ca 2+ Transporting 2b (SERCA2b) in IL-1β-stimulated OSCC cells (Cardozo et al., 2005;Gkouveris et al., 2018). In addition to these cytokines, inflammatory factors, such as H 2 O 2 , increase TRPM2 activity, which might lead to increased basal Ca 2+ levels in cultured rat microglial cells (Kraft et al., 2004). Poly ADP-ribose polymerase-1 (PARP-1) induces Ca 2+ influx by activating TRPM2 in PARP-2 knockout mice (Kraft et al., 2004). All this evidence revealed crosstalk between Ca 2+ and neuroinflammatory factors, which aggravates AD via the actions of different transporters ( Table 1).

Ca 2+ SIGNALING IMPAIRS NEURONAL FUNCTION
The Effects of Ca 2+ on Impairing Neuronal Functions Given the crosstalk between Ca 2+ and neuroinflammatory factors, we continued to elucidate the roles of Ca 2+ in impairing neuronal functions and its effects on the relationship between neuroinflammation and neuronal apoptosis and death ( Table 2). For example, accumulating evidence has revealed that appropriate activation of microglial cells may exert beneficial effects by attenuating neuronal apoptosis, increasing FIGURE 2 | Ca 2+ channels in the ER are involved in regulating neuroinflammation, apoptosis, tau phosphorylation and Aβ deposition, leading to cognitive impairment. The accumulation of Aβ in neuronal cells induces Ca 2+ influx from the intracellular Ca 2+ store, namely, the ER. In addition, Ca 2+ depletion from the ER triggers sustained extracellular Ca 2+ influx to the cytosol via a SOCE pathway, including TRPC1 and Orai1, by activating the Stim. During these processes, InsP3R and RyR2 play important roles in inducing Ca 2+ influx from the ER to the cytosol, regulating apoptosis, neurogenesis, tau phosphorylation and Aβ deposition and subsequently leading to cognitive impairment. ER, endoplasmic reticulum; SOCE, store-operated calcium entry. neurogenesis, and promoting functional recovery after cerebral ischaemia (Neumann et al., 2008). In contrast, overactivation of microglial cells may result in the apoptosis or death of neurons (Brown and Neher, 2014). Based on these findings, excessive release of Ca 2+ initially protects neuronal cells from death by inducing the expression of Bcl-2 through the activated transcription factor NF-κB (Pahl and Baeuerle, 1996;Mattson and Furukawa, 1997), whereas sustained increases in cytosolic Ca 2+ concentrations induced by neuronal depolarization result in Aβ 1-42 production and subsequent neuronal death (Pierrot et al., 2004). Moreover, a series of studies reviewed in our previous work described the effects of Ca 2+ on cell apoptosis via multiple signaling pathways, and this information is not repeated in the present review .

Proinflammatory Cytokines Induce
Neuronal Apoptosis or Death via Ca 2+ Transporters Located on the Cell Membranes However, transporters have not been considered critical for mediating the effects of Ca 2+ on the apoptosis or death of neurons. Therefore, we further addressed the roles of different types of Ca 2+ transporters in regulating the apoptosis or death of neuronal cells, especially during the course of AD development and progression. Due to its close association with neuroinflammation, neuronal apoptosis in the rat hippocampus is induced by IL-1β through an NMDAR-mediated Ca 2+ influx mechanism (Dong et al., 2017). By coculturing glial cells with primary hippocampal neurons, IL-1β secreted from glial cells triggers neuronal death via tyrosine phosphorylation and NMDAR trafficking mechanisms (Viviani et al., 2006;Dong et al., 2017). In contrast to the action of IL-1β, IL-6 reduces Ca 2+ overload by deactivating NMDARs, which resulted in the death of cultured cerebellar granule neurons (CGNs) via the JAK/CaN pathways . As another type of glutamate receptor involved in Ca 2+ transport, AMPAR, which is trafficked to the plasma membrane, mediates the effects of TNF-α on exacerbating the effects of spinal cord injury on cell death (Ferguson et al., 2008;Beattie et al., 2010). By inhibiting the activities of L-VGCC, Gas6 or nimodipine suppresses Aβinduced neuronal apoptosis by attenuating Ca 2+ influx into primary cultured cortical and hippocampal neurons (Ueda et al., 1997;Yagami et al., 2002). In addition, NMDARs and L-VGCCs mediate the effects of perfluorohexanesulfonate (PFHxS) on activating the AMPK and ERK pathways, leading to the apoptosis FIGURE 3 | Ca 2+ efflux from mitochondria regulates the apoptosis of neuronal cells, which results in cognitive dysfunction. Ca 2+ is transported to the mitochondria via MCU. Under physiological or pathological conditions, Ca 2+ is continuously shuffled between the ER and mitochondria via VDAC. Moreover, Ca 2+ in mitochondria induces the formation of the mPTP, which transports Ca 2+ and small molecules, such as ROS and cytochrome C, from the mitochondria to the cytosol, leading to neuronal apoptosis. The loss of neurons will cause cognitive dysfunction. of P12 cells . Among the Ca 2+ transporters located in the cell membrane, TRPV1 overexpression disrupts mitochondrial function and induces cytochrome c release, which results in the death of a human microglial cell line (HMO6; Kim et al., 2006;Zhang and Liao, 2015). Similarly, ectopically expressed TRPV4 in glial cells induces neuronal damage via an apoptotic mechanism (Shi et al., 2013). Consistent with these findings, pharmacological or genetic interventions targeting TRPV4 suppress neuronal cell death by decreasing the expression of proinflammatory cytokines, such as IL-1β and TNF-α (Konno et al., 2012;Shi et al., 2013). As another type of TRP family protein, TRPM2 is activated to induce Ca 2+ influx, resulting in the death of RIN-5F rat insulinoma cells and rat cortical neurons (Kaneko et al., 2006). TRPM2 knockdown reduces the toxicity of Aβ and subsequent death of primary rat neuron cultures (Fonfria et al., 2005;Li and Jiang, 2018; Figure 1).

Ca 2+ Transporters Located on the ER Membrane Are Responsible for Regulating Neuronal Apoptosis
Although APOE4 is not a canonical Ca 2+ transporter, APOE4 overexpression induces Ca 2+ influx, resulting in neuronal apoptosis (Veinbergs et al., 2002;Jiang et al., 2015).
Through a more complicated mechanism, APOE4 induces neuronal apoptosis in APOE4 knockout mice by activating NMDAR-mediated Calcium/Calmodulin dependent protein kinase II (CaMKII) pathways (Qiao et al., 2017). Moreover, TBI induces apoptosis in the cortex and hippocampus of Tg mice overexpressing human APOE4 by activating APOE4 (Giarratana et al., 2020). In addition, ER stress also mediates the effects of the unfolded protein response (UPR) and misfolded proteins on inducing apoptosis through mechanisms related to Ca 2+ influx (Nishitoh et al., 2009;Moreno et al., 2013). Specifically, Ca 2+ transporters located on the ER membrane, including InsP3R and RyR, are reported to be involved in regulating neuronal apoptosis. For example, type 3 InsP3R regulates cell death by modulating Ca 2+ release from the ER to the cytosol in postnatal cerebellar granule cells (Blackshaw et al., 2000;Wang and Zheng, 2019). Isoflurane treatment induces Ca 2+ influx, leading to caspase-3 activation by cleavage in DT40 cells (Joseph et al., 2014). Upon the stimulation of P2X7R by isoflurane and sulforaphane, InsP3R mediates the effects of Ca 2+ on inducing apoptosis or cell death of NG108-15 and PC12 neuronal cells and cells in nude mice (Wei et al., 2008;Chao et al., 2012;Hudecova et al., 2016). Specifically, Aβ 25-35 induces the apoptosis of murine astrocytes via InsP3R-and Ca 2+ -activating pathways  (Oseki et al., 2014). In addition to InsP3R, the posttranslational modification of RyR2 by S-glutathionylation increases channel activity, resulting in the death of rat cortical neurons (Bull et al., 2008). In contrast, the suppression of RyR3 expression in TgCRND8 neurons increases the neuronal death rate, which suggests a protective role for RyR in the late stages of AD pathogenesis (Supnet et al., 2010). Based on these observations, ethanol dose-dependently increases the intracellular Ca 2+ concentration, which damages HepG2 hepatocytes by upregulating the expression of the Orai1 and Stromal interaction molecule 1 (Stim1) mRNAs and proteins (Liu et al., 2012). Although the pathophysiological effects of decreased Store-operated calcium entry (SOCE) levels in AD remain unclear, several lines of evidence have shown that SOCE deficits lead to neuronal cell death and decreased synaptic plasticity (Soboloff and Berger, 2002;Calvo-Rodriguez et al., 2020). As expected, Stim1 silencing alleviates the apoptosis of H 2 O 2 -treated endothelial progenitor cells . Moreover, the downregulation of Stim1 by an siRNA concurrently increases neuronal viability and inhibits apoptotic cell death by decreasing the intracellular Ca 2+ levels (Selvaraj et al., 2016). In PC3 and DU145 cells, both Stim1 and Orai1 separately mediate the effects of resveratrol (RSV), a natural polyphenol, on activating autophagic cell death (Selvaraj et al., 2016). In addition, resveratrol can mediate the release of Ca 2+ from intracellular stores (Santoro et al., 2020). As a method to exclude nonspecific effects of pharmacological interventions, silencing the expression of Stim1 and Orai1 reduces the apoptosis rate of LPS-treated pulmonary microvascular endothelial cells by blocking SOCE in pulmonary microvascular endothelial cells . Researchers excluded the effects of Stim1 on cell apoptosis by transfecting Orai1 mutants and observed decreases in both SOCE and the rate of thapsigargin-induced apoptosis in human prostate cancer (PCa) cells (Flourakis et al., 2010; Figure 2).

Mitochondrial Dysfunction Is Also Involved in Mediating the Effects of Ca 2+ on Neuronal Apoptosis
However, ER stress is not the only mechanism by which the effects of Ca 2+ on neuronal apoptosis are mediated: mitochondrial dysfunction is also reported to be involved in this process (Yoon et al., 2011). Consistently, Stim1 or Orai1 knockdown attenuates the intracellular Ca 2+ overload, restores the mitochondrial membrane potential, decreases the release of cytochrome c and inhibits ethanol-induced apoptosis (Cui et al., 2015). Without affecting ER stress, curcumin protects mitochondria from oxidative damage by attenuating the apoptosis of cortical neurons (Zhu et al., 2004). In primary cultured spinal neurons, salidroside (Sal) treatment decreases apoptosis by activating PINK-Parkin pathways, leading to mitophagy of mitochondria (Gu et al., 2020). Similar to its effects on AD, Aβ 1-42 induces neuronal apoptosis by concurrently upregulating mitochondrial fission protein dynamin-related protein 1 (Drp1) and downregulating mitofusin 1/2 (Mfn1/2) and dynamin-like GTPase (OPA-1) in primary cultures of mouse cerebral cortical neurons . In addition, Aβ 25-35 induces cytochrome c-mediated apoptosis of NT2 cells through a functional mitochondria-dependent mechanism (Morais Cardoso et al., 2002). In this mechanism, Ca 2+ transport by InsP3R to mitochondria induced by opening  the mPTP induces the release of cytochrome c, which results in the apoptosis of cells (Szalai et al., 1999). In fact, mPTP opening induces matrix swelling, the subsequent rupture of the outer membrane, and nonspecific release of proteins in the intermembrane space into the cytosol upon cannabidiol (CBD) induction of human monocyte apoptosis . By inhibiting the opening of the mPTP in mitochondria, mortalin overexpression blocks Aβ-induced SH-SY5Y cell apoptosis (Qu et al., 2012). In AD mice, CyPD knockout decreases the cell death rate by attenuating the opening of the mPTP in mitochondria (Du et al., 2008;Pahrudin Arrozi et al., 2020). VDAC1 expression induces cell death and apoptosis by activating the Ca 2+ signaling cascade in A549 cells (Weisthal et al., 2014). VDAC is involved in the apoptosis of lymphoblastoid cells carrying a mitochondrial DNA mutation (Yuqi et al., 2009). Through a direct interaction with Bax, VDAC induces the transport of cytochrome c through membranes (Shimizu et al., 1999). Moreover, the cleavage of the pro-apoptotic protein Bid by caspase-8 induces the closure of VDAC, which leads to protein release from mitochondria and apoptosis (Rostovtseva et al., 2004). In contrast, Bcl-xL promotes the opening of the VDAC, which results in a reduced apoptosis rate of cultured FL5.12 cells (Vander Heiden et al., 2001;Bessou et al., 2020). Fatty acid binding protein 5 (FABP5), which is expressed in oligodendrocytes, induces mitochondrial macropore formation through VDAC-1 and Bax, thus accelerating mitochondria-induced glial cell death. These two proteins mediate mitochondrial outer membrane FIGURE 4 | Ca 2+ transporters are responsible for activating neuronal networks. Ca 2+ transporters located in the cell membrane, ER, mitochondria, and lysosome are responsible for regulating neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, autophagy, and apoptosis. permeability, resulting in the release of mitochondrial DNA and cytochrome c into the cytoplasm and activation of apoptotic caspases . More interestingly, BAPTA-AM, a Ca 2+ -chelating reagent, inhibits mitochondria-mediated apoptosis by decreasing the oligomerization of VDAC1 in HeLa and T-REx-293 cells (Keinan et al., 2013). Consistent with this observation, anion transport inhibitors, including 4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), SITS, H 2 DIDS, DNDS, and DPC, inhibit apoptosis-associated VDAC1 oligomerization (Ben-Hail and Shoshan-Barmatz, 2016). In addition, blockade of plasmalemmal VDAC1 with a specific antibody suppresses Aβ-induced neuronal apoptosis (Thinnes, 2011;Lim et al., 2021a). In THP-1 macrophages, DIDS disodium salt, an inhibitor of VDAC1, attenuates the apoptosis of THP-1 macrophages by decreasing intracellular Ca 2+ levels (Chen et al., 2014). Similarly, Ca 2+ transporters generally mediate the regulatory effects of Ca 2+ on neuronal apoptosis, especially in the context of AD (Figure 3).

Ca 2+ INHIBITS THE REGULATION OF NEURONAL STEM CELLS
Ca 2+ Modulates Neuronal Differentiation, Migration and Self-renewal During the Course of Neurogenesis During the course of AD development and progression, neurogenesis is markedly inhibited in the brains of patients with AD and mouse models (Rash et al., 2016). Given the potential roles of Ca 2+ in AD, we summarize the effects of Ca 2+ on neurogenesis during the course of AD development and progression. Indeed, higher frequencies of Ca 2+ oscillations increase the differentiation of hippocampus-derived neural stem cells (NSCs) into neurons in adult rats . Moreover, Epac2 mediates PACAP-induced differentiation of neural progenitor cells (NPCs) into astrocytes along with an increase in intracellular Ca 2+ levels, which also activated the signaling pathway for astrocytogenesis in Epac2-knockout (KO) mice (Seo and Lee, 2016). NSC differentiation is closely related to the expression of VGCC, especially Caveolin 1 (Cav1) through regulating Ca 2+ influx (D'Ascenzo et al., 2006). Moreover, exposure in extremely low-frequency electromagnetic fields (ELFEF) promotes the differentiation of NSCs by upregulating the expression and function of Cav1 (Piacentini et al., 2008c). Furthermore, bidirectional radial Ca 2+ activity elongates the fiber of radial glial cells (RGCs) and simultaneously induces neurogenesis during early cortical column development (Rash et al., 2016). By upregulating the Notch signaling pathway after brain injury, Ca 2+ waves generated in neighboring astrocytes propagate to NPCs, inducing neurogenic behavior, including the self-renewal and migration of progenitor cells (Kraft et al., 2017). Based on these observations, Ca 2+ induces neuronal differentiation, migration and self-renewal during the course of neurogenesis.

Ca 2+ Transporters Located on the Cell Membranes Are Required for Neurogenesis
Given the key roles of Ca 2+ in neurogenesis, its transporters are also required for neurogenesis. In the developing cerebellum, granule cell precursors differentiate upon activation of a homodimeric G protein-coupled receptor that is sensitive to Ca 2+ levels called calcium-sensing receptor (CaSR). CaSR activation in vivo induces the homing of granule cell precursors during differentiation, mainly through CaSR interactions with integrin complexes (Tharmalingam et al., 2016). Among these CaSRs, the lower activity of NMDARs in NR1 +/mice contributes to increased cell proliferation and neurogenesis compared to the activity in the brains of adult NR1 +/+ mice (Bursztajn et al., 2007). In contrast, intraperitoneal injection of the NMDAR agonist NMDA (2 mg/kg/day) promotes cell proliferation in the subventricular zone (SVZ) of rats (Fan et al., 2012). Unfortunately, the researchers did not extend their investigations to Ca 2+ , although NMDAR affects neurogenesis. Compared to NMDARs, the roles of AMPARs in neurogenesis are relatively simple. In rats administered chronic corticosterone (CORT), S47445, a novel AMPARpositive allosteric modulator (AMPA-PAM), exerted significant neurogenic effects on the proliferation, survival and maturation of new hippocampal neurons (Mendez-David et al., 2017). Moreover, AMPAR mediates kainate-induced radial glia-like stem cell proliferation (Shtaya et al., 2018). Human NPCs contain Ca 2+ -permeable AMPARs; however, AMPARs were engineered to become Ca 2+ -impermeable receptors during the course of differentiation from NPCs to neurons or astrocytes through RNA editing of the AMPA receptor subunit GluR2 at the Q/R site (Whitney et al., 2008). Then, the NMDAR subunits NR1 and NR2B and the AMPAR subunit GluR2 in Ca 2+impermeable AMPARs were upregulated at the mRNA level in differentiated neuroepithelial precursors, indicating their likely contribution to neurotransmission after first establishing neuronal networks (Muth-Köhne et al., 2010;Wang et al., 2018).
In addition to NMDARs and AMPARs, different types of VGCCs and TRPs in cell membranes are also involved in regulating neurogenesis. For example, the differentiation of dental pulp stem cells (DPSCs) into neural cells is markedly inhibited by regulating the levels of the distal C-terminus (DCT) upon treatment with nimodipine and knock down of Cav1.2 expression (Ju et al., 2015). In the dentate gyrus (DG) region, deletion of Cav1.2 decreases the numbers of doublecortin-positive adult-born neurons, suggesting important roles for Cav1.2 in adult neurogenesis (Temme et al., 2016). Consistent with these findings, Cav1.3 knockout impairs hippocampal neurogenesis and inhibits neuronal differentiation (Marschallinger et al., 2015). More importantly, Ca 2+ mediates the effects of L-VGCC on the neurogenesis of interneurons in nifedipine-treated NPCs (Brustein et al., 2013). Similar to L-VGCCs, blockade of other types of VGCCs, such as N-and T-VGCCs, decreases the migration and neurite extension of developing neurons (Komuro and Rakic, 1992;Louhivuori et al., 2013). On the other hand, TRPs are also reported to be involved in regulating neurogenesis. For instance, TRPM2 deficiency results in impaired embryonic neurogenesis because it regulates neural progenitor self-renewal through an SP5-dependent mechanism (Li and Jiao, 2020). In addition, the antisense oligonucleotide-mediated knockdown of TRPC1 expression reduces the effects of bFGF on the proliferation of embryonic rat NSCs (Fiorio Pla et al., 2005;Toth et al., 2016). Blocking SOCE activity with YM-58483 (BPT2) decreases the proliferation of SVZ and neural stem cells (Domenichini et al., 2018). By stereotactically injecting a recombinant adeno-associated virus expressing TRPC1 into the DG of the bilateral hippocampus, we observed that neurogenesis, LTP induction, and cognitive enhancement related to environmental enrichment (EE) were effectively rescued in TRPC1 knockout mice (Du et al., 2017). Consistent with this observation, TRPC3 knockout reduces the effect of Ca 2+ on mGluR5-mediated radial glial processes, reducing the neuronal migration rate (Louhivuori et al., 2015;Toth et al., 2016). In addition to these classical Ca 2+ transporters in the cell membrane, both APOE1-3 knockout and APOE4 overexpression suppress neurogenic responses in vivo (Hong et al., 2013;Rijpma et al., 2013;Geffin et al., 2017). Based on this evidence, transporters are involved in mediating the effects of Ca 2+ on the neurogenesis of NPCs and NSCs (Figure 1).

Intracellular Ca 2+ Stores Mediate the Effects of Ca 2+ on Neurogenesis
The ER and mitochondria are major intracellular Ca 2+ stores and thus mediate the regulatory effects of Ca 2+ on neurogenesis. In PC12 cells, ER stress and BDNF-TrkB signaling pathways are involved in the induction of neurogenesis by 3β, 23, 28trihydroxy-12-oleanene 3β-caffeate from Desmodium sambuense (Cheng et al., 2019). In addition, ER stress mediates the effects of tunicamycin and HRD1 deletion on the aberrant induction of neuronal differentiation and inhibition of dendrite outgrowth in retinoic acid-treated P19 mouse embryonic carcinoma cells (Kawada et al., 2014). More interestingly, transcripts encoding the three main isoforms of the two families of intracellular calcium release channels, namely, InsP3R and RyR, were detected during early neurogenesis in the mouse cerebral cortex (Faure et al., 2001). In particular, an antagonist of the InsP3 pathway, wortmannin, prevents neurogenesis in neural crest cells (Evrard et al., 2004). In addition, Ca 2+ waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ) and require connexin hemichannels, P2Y1 ATP receptors, and intracellular InsP3-mediated Ca 2+ release, suggesting critical roles for radial glial signaling mechanisms in cortical neuronal production (Weissman et al., 2004;Lim et al., 2021b). In this process, the G protein-coupled receptor GPR157, an orphan G protein-coupled receptor, is involved in regulating the neuronal differentiation of radial glial progenitors through Gq-InsP3-mediated Ca 2+ cascades (Takeo et al., 2016). In mesenchymal stem cells, caffeine, an RyR agonist, induces an intracellular Ca 2+ response that increases throughout neuronal differentiation (Resende et al., 2010). Specifically, RyR2 knockout decreases the neurogenesis of embryonic stem cells (Yu et al., 2008). Associated with the aforementioned mechanisms, the proliferation of embryonic and adult NPCs cultured as neurospheres and progenitors in the subventricular zone (SVZ) of adult mice in vivo was attenuated by depleting the expression of Stim1 and Orai1, suggesting pivotal roles for SOCE channel-mediated Ca 2+ entry in mammalian neurogenesis (Somasundaram et al., 2014). In addition to Orai1, single knock down of Stim1, a Ca 2+ sensor that mediates SOCE, impairs early and late embryonic stem cell differentiation into neural progenitors, neurons or astrocytes, increasing the cell death rate and suppressing the proliferation of neural progenitors (Hao et al., 2014;Deb et al., 2020). Similarly, pharmacological blockade of SOCE decreases the proliferation and self-renewal of NSCs, driving asymmetric division to the detriment of symmetric proliferative division, reducing the population of stem cells in the adult brain, and impairing the ability of SVZ cells to form neurospheres in culture (Domenichini et al., 2018). CRAC channels serve as a major route of Ca 2+ entry in NSCs/NPCs and regulate key effector functions, including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis (Somasundaram et al., 2014). Similar to the ER, mitochondria are intracellular Ca 2+ stores involved in regulating the neurogenesis of NPCs. For example, the inhibition of mPTPs and a selective reduction in mitochondrial superoxide spikes significantly ameliorates the negative effects of Aβ 1-42 on NPC proliferation and survival (Hou et al., 2014). Moreover, cyclosporin A inhibits neuronal differentiation by suppressing mPTP opening (Hou et al., 2013;Namba et al., 2020). All these observations confirm the involvement of Ca 2+ and its transporters in regulating neurogenesis ( Table 3).

THE EFFECTS OF Ca 2+ ON NEUROTOXICITY Ca 2+ Induces Excitotoxicity via Its Transporters Located on Cell Membranes
Neurotoxicity might be the inherent cause of the Ca 2+mediated impairment of neuronal functions. In primary cultured cerebral cortical neurons, increased levels of Ca 2+ induce excitotoxicity, whereas reduced Ca 2+ release exerts neuroprotective effects (Frandsen and Schousboe, 1991). As the natural ligand of NMDAR, NMDA induces neurotoxicity by activating NMDAR in cerebellar granule cells (Xia et al., 1995). In addition to its natural ligand, the exposure of neurons to ethanol and glutamate also induces neurotoxicity by activating NMDARs (Thomas and Morrisett, 2000;Miao et al., 2012). Similar to its effect on the AD pathway, Aβ 25-35 induces neurotoxicity by deactivating the pCRMP2 and NMDAR2B signaling pathways in SH-SY5Y cells (Ji et al., 2019). However, the researchers did not extend their observations to the involvement of Ca 2+ in neurotoxicity. In cultured cerebellar granule neurons, domoic acid induces neurotoxicity through NMDAR-mediated Ca 2+ influx (Berman et al., 2002). By blocking NMDAR-mediated Ca 2+ influx, dantrolene and ionomycin prevent neurotoxicity in cultured rat cortical and retinal ganglion cell neurons (Lei et al., 1992). Drug-induced inhibition of Glutamate ionotropic receptor NMDA type subunit 2A (GluN2A) NMDAR or deletion of the GluN2A subunit gene attenuates the effects of homocysteine on increasing intracellular Ca 2+ concentrations, leading to neurotoxicity (Deep et al., 2019). In hippocampal neurons, Aβ-induced Ca 2+ influx mediated by NMDARs leads to calpain-dependent neurotoxicity (Kelly and Ferreira, 2006;Deep et al., 2019). Based on these observations, NMDARs have the ability to mediate Aβ-induced neurotoxicity via Ca 2+ -dependent mechanisms. In addition, AMPAR was also reported to be involved in regulating neurotoxicity as another glutamate receptor type functioning as a Ca 2+ transporter. For example, cannabinoid receptor activation attenuates the effects of TNF-α on the surface localization of AMPAR, which resulted in excitotoxicity in cultured hippocampal neurons (Zhao et al., 2010;Ganguly et al., 2019). AMPAR trafficking to the cell membrane of CNS neurons regulates excitotoxicity induced by TNF-α (Ferguson et al., 2008). TNF-α induces a rapid reduction in AMPARmediated Ca 2+ entry by increasing the expression of the GluR2 subunit on the cell surface, which results in excitotoxicity during the progression of neurodegeneration (Rainey- . Moreover, AMPAR mediated AMPA-and kainite-induced neurotoxicity via Ca 2+ influx mechanisms in cultured rat hippocampal neurons (Ambrósio et al., 2000). In addition, ethanol induces neurotoxicity in hippocampal slices by activating AMPAR (Gerace et al., 2021). Of note, either Aβ or trimethyltin has the ability to induce neuronal death via activating L-VGCC, leading to the Ca 2+ overload (Piacentini et al., 2008a,b). Therefore, NMDARs and AMPARs are critical for inducing neurotoxicity by triggering Ca 2+ influx.
In the cell membrane, L-VGCC is also involved in mediating AMPA/Zn 2+ -induced neurotoxicity in primary cultured rat cortical neurons (Ambrósio et al., 2000;Lee et al., 2016). In these cells, L-VGCCs were further reported to be critical for iron-induced neurotoxicity . In cerebral cortical cells, CXCL12 induces neurotoxicity via NMDAR and L-VGCC-dependent p38 MAPK activation (Sanchez et al., 2016). By blocking the L/N-type Ca 2+ channel, cilnidipine protects the retina from neurotoxicity in ischaemia-reperfusion-treated rats (Sakamoto et al., 2009).
Another family of Ca 2+ transporters, TRPs, was also reported to be involved in regulating neurotoxicity. In primary cultures of mouse DRG neurons, the inhibition of TRPV1 with specific blockers, such as capsaicin or resiniferatoxin, reduces the prooxidant capacity of microglial neurotoxicity (Ma et al., 2009). In addition, TRPV1 mediates vanilloid-and low pH-induced neurotoxicity in cultured rat cortical neurons (Shirakawa et al., 2007;Ertilav et al., 2021). In contrast, the inhibition of TRPV1 by the antagonist capsazepine attenuates its neuroprotective effects, indicating that TRPV1 activation contributes to the survival of rat nigral neurons (Park et al., 2012). To the best of our knowledge, no report has reconciled these conflicting results. With respect to TRPC1, neurotoxicity in SH-SY5Y cells is markedly induced by treatment with 1-methyl-4-phenylpyridinium ion (MPP + ) through TRPC1-deactivating Ca 2+ -dependent mechanisms (Bollimuntha et al., 2005). TRPC1 overexpression inhibits neurotoxicity by inhibiting the release of cytochrome c and the  expression of the Bax and Apaf-1 proteins in SH-SY5Y cells (Morelli et al., 2013). In contrast to TRPC1, TRPC6 deletion attenuates the effects of NMDAR-mediated Ca 2+ entry, resulting in a disruption of the effect of Ca 2+ on neurotoxicity in primary cultured neurons . Blocking TRPV4-mediated Ca 2+ influx reduces the neurotoxicity of paclitaxel to small and medium dorsal root ganglion neurons (Boehmerle et al., 2018). Regarding TRPM2, cisplatin-induced neurotoxicity in primary DRG cells is attenuated by treatment with its antagonist, 2-aminoethoxydiphenyl borate . TRPM2 knockout blocks Aβ oligomerinduced neurotoxicity, which results in impaired memory in APP/PS1 mice (Ostapchenko et al., 2015). In hippocampal neurons,  induces neurotoxicity by activating TRPM2 . In addition to these canonical Ca 2+ transporters, decreasing the expression of CALHM1 exerts neuroprotective effects on oxygen and glucose deprivation in hippocampal slices (Garrosa et al., 2020). On the other hand, APOE has been reported to be involved in regulating neurotoxicity. For example, APOE4 promotes the neurotoxicity induced by Aβ aggregation in AD (Ma et al., 1996). Extracellular APOE4 is cytotoxic to human neuroblastoma SK-N-SH cells, and Aβ 1-42 enhances the cytotoxicity of APOE4. The carboxyl terminal mutation of L279Q, K282A or Q284A decreases the ability of APOE4 to form SDS-stable oligomers and decreases its cytotoxicity. Structural and thermodynamic analyses showed that all three APOE4 mutants contain significantly increased α-helical and β-sheet structures, which resulted in reduced exposure of the hydrophobic surface to the solvent and reduced conformational stability during chemical denaturation (Dafnis et al., 2018). In N2a-APP 695 cells, APOE4 exacerbates the effects of ethanol on inducing neurotoxicity by increasing oxidative stress and apoptosis (Ji et al., 2019). In contrast, APOE1-3 has been shown to protect primary cultures of rat cortical neurons from the neurotoxic effects of the nonfibrillar C-terminal domain of Aβ (Drouet et al., 2001;Brookhouser et al., 2021). APOE isoforms play different roles in neurotoxicity by modulating Aβ deposition in the mouse brain (Drouet et al., 2001). Ca 2+ mediates the effects of truncated APOE on neurotoxicity in cultured embryonic rat hippocampal neurons (Tolar et al., 1999).
Through these mechanisms, APOE-related neurotoxicity might be a therapeutic target for AD (Marques and Crutcher, 2003; Figure 1).

The ER Mediates the Effects of Ca 2+ on Inducing Neurotoxicity as an Intracellular Store
Since Ca 2+ regulates neurotoxicity via transporters located in the cell membrane, the roles of Ca 2+ derived from intracellular stores in neurotoxicity are further addressed in Table 4. For example, Aβ induces neurotoxicity in cortical neurons via an ER-mediated apoptotic pathway (Ferreiro et al., 2006;Goswami et al., 2020). In the spinal cord, Ca 2+ mediates the effects of ER stress on neurotoxicity (Li et al., 2014). By alleviating ER stress, nicotine suppresses the activity of MPP + /MPTP associated with neurotoxicity in PC12 cells (Cai et al., 2017). Similar to its role in AD, Aβ induces neurotoxicity in cortical neurons by promoting ER stress (Song et al., 2008).
As Ca 2+ mediates the effects of ER stress on neurotoxicity, Ca 2+ transporters in ER membranes must be associated with neurotoxicity. For example, The generation of InsP3 by activated M3 muscarinic receptors contributes to increased Ca 2+ influx and subsequent cytotoxicity in rat cerebellar granule cells (Limke et al., 2004). Furthermore, cyanide induces the formation of InsP3, which triggers intracellular neurotoxic signaling events in PC12 cells (Yang et al., 1996). In hippocampal neurons, Ca 2+ was also found to be the critical cause of microcystin-LR-induced neurotoxicity through PLC-and InsP3-dependent pathways (Cai et al., 2015). Regarding the receptors of InsP3, InsP3R triggers Ca 2+ influx to mediate isoflurane-induced neurotoxicity, which is facilitated by an APP mutant in SH-SY5Y cells (Liu et al., 2016). In primary cultures of cortical cells, Aβ induces neurotoxic effects by inducing Ca 2+ release from the ER via InsP3R-and RyR-dependent mechanisms (Ferreiro et al., 2004). After inhibiting the activity of InsP3R and RyR, the cytotoxicity and increased Ca 2+ levels are attenuated. More interestingly, the combined inhibition of both receptors paradoxically increases the amount of cytosolic Ca 2+ entering PC12 cells from the extracellular space, increasing cytotoxicity Frontiers in Molecular Neuroscience | www.frontiersin.org  . In addition to InsP3R, RyR alone might be critical for modulating neurotoxicity in human microglia and THP-1 cells (Klegeris et al., 2007;Holland and Pessah, 2021). In cultured mammalian neurons, Xbpls ameliorates Aβ-induced neurotoxicity through an RyR-dependent mechanism (Fernandez-Funez et al., 2010). Thus, the ER is an important intracellular Ca 2+ store for regulating neurotoxicity in neurons (Figure 2).

Mitochondria Are Critical for Regulating Neurotoxicity Through a Ca 2+ -Dependent Mechanism
In addition to the ER, mitochondria are reported to be critical for regulating neurotoxicity through a Ca 2+ -dependent mechanism. In particular, VDAC1, a transporter located in mitochondria, mediates Aβ-induced neurotoxicity in PC12 and SH-SY5Y cells and thus represents a potential target for AD treatment (Smilansky et al., 2015). In addition, the dephosphorylation of VDAC1 by hesperidin blocks Aβ-induced neurotoxicity in PC12 cells through a mitochondria-dependent mechanism (Wang et al., 2013). Aβ directly induces neurotoxicity via the dephosphorylation of VDAC1 in murine septal SN56, SH-SY5Y and hippocampal HT22 cells (Fernandez-Echevarria et al., 2014;Shoshan-Barmatz et al., 2018). In these cells, the interaction between VDAC and mERα at the plasma membrane may lead to the modulation of Aβ-induced neurotoxicity (Marin et al., 2007). In addition to VDAC1, an anti-VDAC2 antibody reduces neurotoxicity by decreasing intracellular Ca 2+ levels in SH-SY5Y cells (Marin et al., 2007;Nagakannan et al., 2019). By inhibiting the opening of the mPTP, cyclosporin A protects SH-SY5Y and PC12 cells from neurotoxicity (Ye et al., 2016). In primary cultures of rat cortical neurons, 4hydroxy-2(E)-nonenal facilitates NMDA-induced neurotoxicity by opening the mPTP, which results in Ca 2+ influx (Choi et al., 2013). This observation is further supported by a report showing that NMDA induced neurotoxicity via the mPTP in cultured murine cortical neurons (Kinjo et al., 2018). Based on this evidence, intracellular Ca 2+ stores are involved in mediating the effects of Ca 2+ on neurotoxicity, which potentially contributes to neuronal apoptosis or death ( As a protein clearing function, autophagy deficiency might be the cause of the aggregation and deposition of Aβ or hyperphosphorylation of tau in APs and NFTs (Pickford et al., 2008;Heckmann et al., 2019). Ca 2+ signaling plays a crucial role in autophagy in various experimental models (Shaikh et al., 2016;Zhang et al., 2016). Logically, Ca 2+ transporters are proposed to be involved in regulating autophagy. According to preliminary evidence, NMDARs on the cell membrane contribute to autophagy and the membrane potential in leukaemic megakaryoblasts (Nursalim, 2016). Specifically, exposure to low-dosage NMDA increases LC3 II production, which results in the degradation of GluR1, a subunit of AMPAR, in cultured rat hippocampal neurons (Shehata et al., 2012). Treatment with an antagonist of NMDAR, memantine, induces the NMDAR1-mediated autophagic cell death of T-98G cells (Yoon et al., 2017). In cultured hippocampal neurons, the NR2B antagonist Ro25-6981 markedly attenuates NMDA-and global ischaemia-induced activation of the autophagy pathway by disrupting the association of NR2B and Beclin1, resulting in cell death (Borsello et al., 2003;Liu and Zhao, 2013). In contrast, autophagy upregulates the expression of AMPAR subunits, including GluR1, GluR2, and GluR3, in oxygenand glucose-deprived and reoxygenated injured neurons (Bao et al., 2017). These observations indicate the involvement of Ca 2+ transporters located in the cell membranes in regulating autophagy. Similarly, VGCC induces Ca 2+ influx to inhibit autophagy by activating calpains that cleave ATG5, an important factor for elongating autophagosomes, in H4 cells (Williams et al., 2008). As an atypical Ca 2+ transporter in the cell membrane, APOE4 potentiates the effects of Aβ on the destabilization and permeabilization of lysosomal membranes, which results in impaired autophagy and the degradation of lysosomes in N2a cells (Ji et al., 2006;Nasiri-Ansari et al., 2021). In addition, rapamycin, an autophagy inducer, enhances mitochondrial autophagy and restores mitochondrial function in APOE4-expressing astrocytes (Schmukler et al., 2020). In astrocytes, APOE4 also impairs autophagy, resulting in attenuated clearance of Aβ (Simonovitch et al., 2016; Figure 1).

ER Stress Induces Autophagy by Modulating the Dyshomeostasis of Ca 2+
In terms of intracellular Ca 2+ stores, ER stress induces autophagy in propofol-stimulated C2C12 myoblast cells (Chen et al., 2018). In SK-N-SH cells, ER stress activates autophagy in UPR-stimulated SK-N-SH cells, which indicates its roles in AD (Nijholt et al., 2011). Specifically, polyglutamine induces LC3 conversion via ER stress, which initiates the onset of autophagy in C2C5 myoblast cells (Kouroku et al., 2007). Similarly, inducers of ER stress, including tunicamycin, DTT and MG132, concurrently decrease the activity of mTOR and increase the conversion of LC3 I to LC3 II in MEFs (Qin et al., 2010). Lithium induces autophagy by suppressing inositol monophosphatase, leading to the depletion of free inositol and InsP3 in SK-N-SH and COS-7 cells (Sarkar et al., 2005). This observation was also confirmed in lithium-treated IMPA1 knockout mice (Sade et al., 2016). In another study, Ca 2+ was reported to be located downstream of InsP3R and mediated 2-aminoethoxydiphenyl borate (2-APB)-induced autophagy flux in neonatal rat ventricular myocytes (NRVMs) and HeLa cells (Wong et al., 2013). In addition, by inhibiting InsP3mediated Ca 2+ signaling, glucocorticoids induce autophagy in T lymphocytes (Harr et al., 2010). Blockade of InsP3R, the receptor of InsP3, restores autophagy and mitochondrial function in muscle fibers from WT and MDX mice (Valladares et al., 2018). InsP3R knockout upregulates the expression of autophagy markers compared to the WT controls (Cárdenas et al., 2010;Khan and Joseph, 2010). Researchers further emphasized the involvement of Ca 2+ in autophagy by inducing autophagy through starvation and the activation of the InsP3R-mediated Ca 2+ signaling pathway, as evidenced by the abolishment of LC3 lipidation and the formation of GFP-LC3 puncta in HeLa cells; these changes were blocked by the Ca 2+ chelator BAPTA-AM and the InsP3R inhibitor xestospongin B (Cárdenas et al., 2010). In PC12 cells, isoflurane induced autophagydependent cell death via InsP3R-Ca 2+ -dependent mechanisms (Peng et al., 2011). Moreover, InsP3R-mediated transfer of Ca 2+ from the ER to mitochondria is required to maintain the proper production of ATP, and Ca 2+ blockade inhibits AMPK activity, leading to the suppression of autophagy in DT40 cells (Cárdenas et al., 2010;Lim et al., 2021a). Regarding the other Ca 2+ transporters in ER membranes, RyR mediates the effects of propofol on inducing autophagy in cortical neuronal progenitor cells (Qiao et al., 2017). In primary cultured cortical neurons, RyR1 and RyR3 upregulation induced by insulin deprivation increase Ca 2+ release from the ER, which increases the production of LC3II, an important autophagy marker (Edinger and Thompson, 2004;Chung et al., 2016). As an antagonist of RyRs, ryanodine stimulates autophagy by decreasing the cytosolic levels of Ca 2+ , leading to neuroprotection in CBE-N2a cells (Liou et al., 2016). By blocking RyR activity, dantrolene and an inhibitory dose of ryanodine reduce the conversion of LC3I to LC3II in HEK293 and C2C12 cells (Vervliet et al., 2017). Similarly, the downregulation of RyR2-mediated Ca 2+ release decreases ATP production by suppressing mitochondrial metabolism, resulting in an increase in the autophagy-dependent death of rat neonatal cardiomyocytes (Pedrozo et al., 2013;McDaid et al., 2020). By depleting Ca 2+ from the ER, SOCE exerts a biological effect on Ca 2+ influx. In PC3 and DU145 cells, autophagic cell death was induced by resveratrol, which downregulated the expression of Stim1 and disrupted its association with TRPC1 and Orai1 (Selvaraj et al., 2016). The overexpression of Stim1 and Orai1 inhibits the effects of starvation-and rapamycin-induced autophagy on A7R5 rat arterial smooth muscle cells (Michiels et al., 2015). Moreover, caerulein promotes the interaction between Stim1 and Orai1, which activates CaN by inducing Ca 2+ overload, leading to the expression of autophagy-related genes in mice with acute pancreatitis . These observations revealed the involvement of ER Ca 2+ stores in regulating autophagy (Figure 2). Based on the aforementioned observations, InsP3R was found to connect mitochondria, potentially contributing to apoptosis and autophagy (Decuypere et al., 2011b). In Aβtreated PC12 cells, moderate activation of autophagy regulates intracellular Ca 2+ levels and the mitochondrial membrane potential (Xue et al., 2016). Reciprocally, mitochondrial fission-mediated Ca 2+ signaling induces the expression of Stim1 and subsequent SOCE, which promoted autophagy through Ca 2+ /CAMKK/AMPK signaling cascades (Huang et al., 2017). Regarding Ca 2+ transporters in mitochondria, VDAC recruits Parkin to defective mitochondria, resulting in the induction of mitochondrial autophagy in HEK293 cells (Sun et al., 2012). In addition, p53 is actively recruited to the outer membrane of mitochondria during nutrient deprivation, resulting in opening of the mPTP, an increase in the conversion of LC3BII to LC3BI, and the formation of LC3-GFP puncta in ventricular myocytes (Eydelnant et al., 2009;Xu H. X. et al., 2020).

Ca 2+ Transporters on the Lysosomal Membranes Are Responsible for Regulating the Degradation of Aggregated Proteins
As the lysosome is the organelle responsible for degrading proteins, studies aiming to elucidate the roles of Ca 2+ transporters located in lysosomes in regulating autophagy would be interesting. For example, Ca 2+ stimulates lysosomal v-ATPase and mTORC1 pathways, which potentially contribute to the effects of orexin and hypocretin on autophagy in HEK293T cells . Rapamycin treatment inhibits mTOR activity by decreasing phosphorylation at two serine residues, leading to the induction of autophagy via a Ca 2+ -dependent mechanism (Onyenwoke et al., 2015). Furthermore, v-ATPase deficiency in Presenilin 1 (PS1) loss-of-function states causes deficits in lysosomes and autophagy, which contributes to abnormal cellular Ca 2+ homeostasis (Lee et al., 2015). In addition, accumulating evidence is showing that the functional regulation of TRP channels contributes to Ca 2+ signaling and subsequent autophagy initiation . Transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) is a lysosomal Ca 2+ channel, which can mediate the release of Ca 2+ from lysosomes to cytoplasm. TRPML1 mutation increases the formation of autophagosomes, disrupts the fusion of autophagosomes and lysosomes, and induces the accumulation of p62 and insufficient removal of ubiquitinated proteins and/or defective mitochondria in fibroblasts from patients with mucolipidosis type IV (MLIV; Vergarajauregui et al., 2008;Nakamura et al., 2020). Under nutrient starvation conditions, TRPML1 upregulation is critical for increasing lysosomal proteolytic activity in COS-1 cells (Wang W. et al., 2015). Moreover, the overexpression of TRPML3/MCOLN3 induces autophagy in HeLa cells via a Ca 2+ -dependent mechanism (Kim et al., 2009). Similarly, both exogenous and endogenous Ca 2+ modulate autophagy via different transporters (Table 5).

THE HERBS USED AS FOOD AND SEASONINGS IN CHINESE DAILY LIFE POTENTIALLY CONTRIBUTE TO AD TREATMENT BY RESTORING THE Ca 2+ CONCENTRATION THROUGH EFFECTS ON ITS TRANSPORTERS
As discussed above, Ca 2+ overload plays important roles in aggravating AD via its transporters. In particular, Ca 2+ overload perturbs the activities of the brain network, which increases the risk of AD and contributes causally to synaptic and cognitive deficits in hAPP mice. Since Ca 2+ homeostasis is regulated by different transporters, transporters might be potential therapeutic targets for treating AD by modulating Ca 2+ homeostasis. However, the outcome is not always consistent with our expectation. For instance, memantine, a noncompetitive NMDA antagonist, is an effective drug approved by the FDA   Kim et al. (2009) for the treatment of AD. The VGCC inhibitor levetiracetam, an antiepileptic drug, exerts positive effects on patients with AD (Cumbo and Ligori, 2010;Vogl et al., 2012), whereas no beneficial therapeutic effect on AD was observed for the VGCC antagonist nilvadipine (Lawlor et al., 2018).
Moreover, active compounds, including homoharringtonine, magnolol, polydatin (PD), and Ginkgo biloba extracts (EGb), derived from herbs, such as Cephalotaxus fortunei, magnolia tree, Polygonum cuspidatum, and Ginkgo biloba, respectively, modulate Ca 2+ homeostasis by regulating the activities of transporters located in the ER through mechanism partially dependent on SOCE or mitochondria (Matsubara et al., 2005;Yang et al., 2013;Guo et al., 2014;Hsieh et al., 2018;Li et al., 2019). Although these herbs have not been used in clinical trials, all this evidence suggests that the herbs used as food and seasonings in Chinese daily life potentially contribute to treating AD by targeting Ca 2+ transporters to restore Ca 2+ concentrations ( Table 6).

CONCLUSIONS
During the development and progression of AD, Ca 2+ concentrations are increased in the cytosol of neuronal cells via transportation from the extracellular space and intracellular stores through transporter-dependent mechanisms. Ca 2+ accumulation in neuronal cells induces the production and deposition of Aβ and hyperphosphorylated tau in APs and NFTs, leading to impaired learning ability in patients with AD. Moreover, transporters in the cell membrane, endoplasmic reticulum, mitochondria, and lysosomal membranes are critical for mediating the effects of Ca 2+ on neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, autophagy, and synaptic plasticity, which contribute to the cognitive decline associated with AD (Figure 4). Based on these theoretical investigations, some bioactive components from Chinese herbal medicines have the potential to treat AD by targeting Ca 2+ transporters. Moreover, Ca 2+ transporters are progressively becoming new therapeutic targets for treating AD.

AUTHOR CONTRIBUTIONS
P-PG and L-LC contributed to conceptualizing and drafting the manuscript. YY contributed to summarizing the data presented in Table 6. PW contributed to conceptualizing, writing, reviewing, and editing the manuscript. All authors have agreed to publish the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported in part or in whole by the National Natural Science Foundation of China (CN; 81771167 and 81870840).