Astaxanthin Protects PC12 Cells against Homocysteine- and Glutamate-Induced Neurotoxicity

Memory impairment has been shown to be associated with glutamate (Glu) excitotoxicity, homocysteine (Hcy) accumulation, and oxidative stress. We hypothesize that Glu and Hcy could damage neuronal cells, while astaxanthin (ATX) could be beneficial to alleviate the adverse effects. Using PC12 cell model, we showed that Glu and Hcy provoked a huge amount of reactive oxygen species (ROS) production, causing mitochondrial damage at EC50 20 and 10 mm, respectively. The mechanisms of action include: (1) increasing calcium influx; (2) producing ROS; (3) initiating lipid peroxidation; (4) causing imbalance of the Bcl-2/Bax homeostasis; and (5) activating cascade of caspases involving caspases 12 and 3. Conclusively, the damages caused by Glu and Hcy to PC12 cells can be alleviated by the potent antioxidant ATX.


Introduction
Glutamate is the most well-known excitatory neurotransmitter, occurring in over 50% of nervous tissues [1]; it is involved in the process of learning, cognition, and neurodegeneration [2]. Two major classes of glutamate receptors are recognized, the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs) [3]. All the iGluRs function as nonselective cation channels, allowing the passage of Na + , K + , and small amounts of Ca 2+ in some cases [2]. The mGluRs modify neuronal and glial excitability through G protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and cAMP [2]. Excess extracellular glutamate level could induce brain lesions, neuronal death, and other pathological changes in several organs associated with endocrine function; this pathway is called excitotoxicity, which was coined by   [4]. During the development process for Alzheimer's disease (AD), excessive activated extracellular glutamate receptors prompt changes in ion channels and signaling systems [5][6][7]. The intracellular calcium produces an avalanche of reactive oxygen species (ROS), leading to mitochondria-mediated apoptotic process, in particular, during such a pathological condition [5][6][7]. ROS and the oxidative-stress-induced A concentration of Hcy up to 10 μM has been measured in brain [25]. Elevated concentration of total Hcy in plasma (>12 μmol/L) is a risk factor for several diseases of the central nervous system [10]. However, more modest levels (15-50 mM) are found very commonly in the general population (a condition known as hyper-homocysteinemia) [26,27]. The cell viability could be affected by several factors including the kind of cell line and medium for incubation [28]. Froissard and Duval (1994) reported that glutamate (1-10 mM) led to a dose-dependent cell damage (70% of cell lysis at 10 mM) [29]. In contrast, according to Wang et al. (2016), 50% of severe suppression of Y79 cell viability by glutamate occurred at a dose of 20 mM [30]. Literature elsewhere also often used 25 mM of glutamate for conducting similar studies [5,[31][32][33], consequently, a dose of 10 mM Hcy and 20 mM Glu has been adopted in this article.

Potentiation of Glutamate on the Cytotoxicity of Homocysteine
Both Glu and Hcy are cytotoxic, and we questioned whether these two compounds may exert any additive or synergistic effect on the cell viability. Glu at 5 mM seemed to be totally ineffective to potentiate the cytotoxicity of 10 mM Hcy. At a dose >10 mM (up to 20 mM), Glu significantly potentiated the Hcy cytotoxicity in a dose-and time-dependent manner (Figure 2a). Much of literature has evidenced that Hcy not only induces direct neurotoxicity, but also potentiates both amyloid-β and glutamate neurotoxicity [34]. Similarly, it has been revealed that activation of group III metabotropic glutamate receptors stimulates the excitotoxic action of Hcy and homocysteic acid [2]. Previously, Lecleric et al. evidenced the occurrence of NMDA receptor in PC12 cells and concluded that PC12 cells express predominantly the splice variant NMDAR1-4a and smaller amounts of NMDAR1-1a, NMDAR1-2a, and NMDAR1-3a [35]. More recently, Sibarov et al. implicated that GluN2A subunit-containing NMDA receptors as the preferential neural targets of Hcy [36]. Moreover, Hcy has been confirmed not only to be Ca 2+ -and NMDA receptor-dependent, but also Ca 2+ -independent, mainly mediated by the "synaptic type" GluN1/2 NMDAR [36]. Suggestively, the degree of binding by different ligands like Glu and Hcy for these receptor subunits and the outcome responses may be synergistically additive on one hand, but competitively expelling on the other hand. The significance of the difference was judged by confidence levels of * p < 0.05; # p < 0.05; 〒 p < 0.05; ⊗ p < 0.05;  p < 0.05; ** p < 0.01; ## p < 0.01; ⊗⊗ p < 0.01;  p < 0.01; *** p < 0.005.

Protective Effect of Astaxanthin against The Insult Exerted by Homocysteine and Glutamate
Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41].
Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca 2+ -overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate
The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated  Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41].
Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca 2+ -overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate
The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated   Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via a mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vit cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect vi signaling pathways including NFκB and MAPK pathways, and implicated ATX to be prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glu
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the model in reality has pertinently reflected such a possibility [40]. The calcium ion influx w raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu slightly less effective against that by Hcy (10 mM), and was much less effective against the therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions betw and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined In addition, Hcy has been implicated to indirectly increase intracellular calcium activating ionotropic and metabotropic receptors [41], which, compared with glutamate, m the reasons to elicit different outcomes by such a combination of pharmacological actio [2,36,41].
Two subcellular organelles, namely the mitochondria and endoplasmic reticulum involved in the cellular pathogenesis of AD; an increased oxidative stress and dysreg calcium homeostasis also have been reported [42]. Owing to the important role of ER in the r of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tightly controlled in the phy conditions. When the distance is decreased, Ca 2+ -overload occurs both in the cy mitochondria [40]. The reduction in the distance between ER and mitochondria may be imp Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indi ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulate (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate
The level of reactive oxygen species (ROS) was highly induced by treating with Glu Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were e suppressed by ATX at 5 μM ( Figure 3b). The mitochondrial Ca 2+ -overload can lead to increased generation of reactive oxyge inducing the opening of the mitochondrial permeability transition pore and ultimatel neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway rela hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient wi cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxi molecule neutralizes free radicals or other oxidants by either accepting or donating electron being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX- Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41].
Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca 2+ -overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate
The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated p < 0.05; ** p < 0.01; ## p < 0.01; ⊗⊗ p < 0.01; s implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating sfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac reviously, Zhang et al. found that ATX exerted neuroprotective effect via multiple ys including NFκB and MAPK pathways, and implicated ATX to be used as a mediation agent against neuronal disorder [39].

nthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
ysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell as pertinently reflected such a possibility [40]. The calcium ion influx was highly lt of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment ). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was ve against that by Hcy (10 mM), and was much less effective against the combined igure 3a), implicating insufficient mole number for the interactions between ATX uggestively, a higher dose of ATX may be required for such a combined insult. Hcy has been implicated to indirectly increase intracellular calcium levels by pic and metabotropic receptors [41], which, compared with glutamate, may create it different outcomes by such a combination of pharmacological actions of Hcy lar organelles, namely the mitochondria and endoplasmic reticulum (ER), are ellular pathogenesis of AD; an increased oxidative stress and dysregulation of sis also have been reported [42]. Owing to the important role of ER in the regulation ER-mitochondrial distance in the neurons is tightly controlled in the physiological the distance is decreased, Ca 2+ -overload occurs both in the cytosol and . The reduction in the distance between ER and mitochondria may be implicated in se (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that lutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein g glutamate-induced apoptosis through rescuing the redox balance and inhibiting d calcium influx [24].

nthin on the ROS Production Caused by Homocysteine and Glutamate
eactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), the combined treatment for 24 h (Figure 3b). The ROS produced were effectively X at 5 μM (Figure 3b). drial Ca 2+ -overload can lead to increased generation of reactive oxygen species, ning of the mitochondrial permeability transition pore and ultimately causing ic and necrotic cell death [40]. In addition, the NO pathway relating with s induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique tions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This es free radicals or other oxidants by either accepting or donating electrons, without r becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage ctive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated as implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating ysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac . Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple ays including NFκB and MAPK pathways, and implicated ATX to be used as a emediation agent against neuronal disorder [39].

xanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell has pertinently reflected such a possibility [40]. The calcium ion influx was highly ult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was tive against that by Hcy (10 mM), and was much less effective against the combined Figure 3a), implicating insufficient mole number for the interactions between ATX suggestively, a higher dose of ATX may be required for such a combined insult. , Hcy has been implicated to indirectly increase intracellular calcium levels by opic and metabotropic receptors [41], which, compared with glutamate, may create licit different outcomes by such a combination of pharmacological actions of Hcy lular organelles, namely the mitochondria and endoplasmic reticulum (ER), are cellular pathogenesis of AD; an increased oxidative stress and dysregulation of tasis also have been reported [42]. Owing to the important role of ER in the regulation , ER-mitochondrial distance in the neurons is tightly controlled in the physiological en the distance is decreased, Ca 2+ -overload occurs both in the cytosol and ]. The reduction in the distance between ER and mitochondria may be implicated in ase (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein ing glutamate-induced apoptosis through rescuing the redox balance and inhibiting ed calcium influx [24].

xanthin on the ROS Production Caused by Homocysteine and Glutamate
reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), d the combined treatment for 24 h (Figure 3b). The ROS produced were effectively TX at 5 μM (Figure 3b). ondrial Ca 2+ -overload can lead to increased generation of reactive oxygen species, ening of the mitochondrial permeability transition pore and ultimately causing otic and necrotic cell death [40]. In addition, the NO pathway relating with is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique ctions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This izes free radicals or other oxidants by either accepting or donating electrons, without or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage eactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 µM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41].
Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca 2+ -overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate
The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 µM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated neuroprotection in experimental models of neurological disorders involves antioxidant, anti-inflammatory, and antiapoptotic mechanisms [45][46][47].
2.6. Effect of Astaxanthin on the MDA Production Resulting from Insult of Homocysteine and Glutamate MDA was highly stimulated when treated with Glu (20 mM), Hcy (10 mM), and the combined therapy to levels of 1.48, 1.80, and 2.20 µmol/µg of protein, respectively, which were suppressed by treatment with 5 µM ATX (Figure 3c), similar to the outcomes for Ca 2+ influx ( Figure 3a) and ROS induction (Figure 3b). ATX was evidenced to be a strong peroxyl radical scavenger, exerting a strong protective effect on the human brain [19,48].

Effect of Astaxanthin on the MDA Production Resulting from Insult of Homocysteine and Glutamate
MDA was highly stimulated when treated with Glu (20 mM), Hcy (10 mM), and the combined therapy to levels of 1.48, 1.80, and 2.20 μmol/μg of protein, respectively, which were suppressed by treatment with 5 μM ATX (Figure 3c), similar to the outcomes for Ca 2+ influx ( Figure 3a) and ROS induction (Figure 3b). ATX was evidenced to be a strong peroxyl radical scavenger, exerting a strong protective effect on the human brain [19,48].  Literature has implicated that ATX inhibits homocysteine-ind mitochondrial dysfunction and signal crosstalk [37]. A similar res cell cultures [38]. Previously, Zhang et al. found that ATX exerted n signaling pathways including NFκB and MAPK pathways, and prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected
Calcium ion dysregulation is relevant to the initiation of Alzhe model in reality has pertinently reflected such a possibility [40]. T raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153% (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influ slightly less effective against that by Hcy (10 mM), and was much le therapy (133%) (Figure 3a), implicating insufficient mole number and (Glu + Hcy); suggestively, a higher dose of ATX may be requir In addition, Hcy has been implicated to indirectly increase activating ionotropic and metabotropic receptors [41], which, comp the reasons to elicit different outcomes by such a combination of [2,36,41].
Two subcellular organelles, namely the mitochondria and e involved in the cellular pathogenesis of AD; an increased oxida calcium homeostasis also have been reported [42]. Owing to the imp of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tight conditions. When the distance is decreased, Ca 2+ -overload o mitochondria [40]. The reduction in the distance between ER and m Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40 ATX attenuated glutamate-induced elevation of CHOP and ER cha (GRP78), inhibiting glutamate-induced apoptosis through rescuing glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homocystei
The level of reactive oxygen species (ROS) was highly induce Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). Th suppressed by ATX at 5 μM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased gener inducing the opening of the mitochondrial permeability transiti neuronal apoptotic and necrotic cell death [40]. In addition, hyperexcitability is induced by homocysteine thiolactone (HcyT) [4 cell membrane actions and diverse clinical benefits [44]. ATX is th molecule neutralizes free radicals or other oxidants by either accepti being destroyed or becoming a pro-oxidant in the process [44]. AT and lowers C-reactive protein (CRP) and other inflammation Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41].
Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca 2+ -overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate
The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated p < 0.05; # p < 0.05; ** p < 0.01; ⊗⊗ p < 0.01; ## p < 0.01; Molecules 2020, 25, x Literature has implicated that ATX inhibits homocysteine mitochondrial dysfunction and signal crosstalk [37]. A simila cell cultures [38]. Previously, Zhang et al. found that ATX exert signaling pathways including NFκB and MAPK pathways, prophylactic or remediation agent against neuronal disorder [3

Effect of Astaxanthin on the Intracellular Calcium Ion Level Aff
Calcium ion dysregulation is relevant to the initiation of A model in reality has pertinently reflected such a possibility [4 raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (1 (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium slightly less effective against that by Hcy (10 mM), and was mu therapy (133%) (Figure 3a), implicating insufficient mole num and (Glu + Hcy); suggestively, a higher dose of ATX may be re In addition, Hcy has been implicated to indirectly inc activating ionotropic and metabotropic receptors [41], which, the reasons to elicit different outcomes by such a combinatio [2,36,41].
Two subcellular organelles, namely the mitochondria a involved in the cellular pathogenesis of AD; an increased o calcium homeostasis also have been reported [42]. Owing to the of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is conditions. When the distance is decreased, Ca 2+ -overloa mitochondria [40]. The reduction in the distance between ER an Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signalin ATX attenuated glutamate-induced elevation of CHOP and ER (GRP78), inhibiting glutamate-induced apoptosis through resc glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Homoc
The level of reactive oxygen species (ROS) was highly in Hcy (10 mM), and the combined treatment for 24 h (Figure 3b suppressed by ATX at 5 μM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased g inducing the opening of the mitochondrial permeability tra neuronal apoptotic and necrotic cell death [40]. In additi hyperexcitability is induced by homocysteine thiolactone (Hcy cell membrane actions and diverse clinical benefits [44]. ATX molecule neutralizes free radicals or other oxidants by either ac being destroyed or becoming a pro-oxidant in the process [44] and lowers C-reactive protein (CRP) and other inflammat  Literature has implicated that ATX inhibits homocystei mitochondrial dysfunction and signal crosstalk [37]. A simi cell cultures [38]. Previously, Zhang et al. found that ATX ex signaling pathways including NFκB and MAPK pathways prophylactic or remediation agent against neuronal disorder

Effect of Astaxanthin on the Intracellular Calcium Ion Level A
Calcium ion dysregulation is relevant to the initiation of model in reality has pertinently reflected such a possibility raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium slightly less effective against that by Hcy (10 mM), and was m therapy (133%) (Figure 3a), implicating insufficient mole nu and (Glu + Hcy); suggestively, a higher dose of ATX may be In addition, Hcy has been implicated to indirectly i activating ionotropic and metabotropic receptors [41], which the reasons to elicit different outcomes by such a combina [2,36,41].
Two subcellular organelles, namely the mitochondria involved in the cellular pathogenesis of AD; an increased calcium homeostasis also have been reported [42]. Owing to t of Ca 2+ -signaling, ER-mitochondrial distance in the neurons conditions. When the distance is decreased, Ca 2+ -overl mitochondria [40]. The reduction in the distance between ER Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signa ATX attenuated glutamate-induced elevation of CHOP and (GRP78), inhibiting glutamate-induced apoptosis through re glutamate-induced calcium influx [24].

Effect of Astaxanthin on the ROS Production Caused by Hom
The level of reactive oxygen species (ROS) was highly Hcy (10 mM), and the combined treatment for 24 h (Figure suppressed by ATX at 5 μM (Figure 3b).
The mitochondrial Ca 2+ -overload can lead to increased inducing the opening of the mitochondrial permeability neuronal apoptotic and necrotic cell death [40]. In add hyperexcitability is induced by homocysteine thiolactone (H cell membrane actions and diverse clinical benefits [44]. AT molecule neutralizes free radicals or other oxidants by either being destroyed or becoming a pro-oxidant in the process [4 and lowers C-reactive protein (CRP) and other inflamm p < 0.01; *** p < 0.005; ### p < 0.005.

Western Blot Analysis Indicated Astaxanthin Restored Bax and Bcl-2 Homeostasis
Bcl-2 family members either promote or repress programmed cell death. Bax, a proapoptotic member of the Bcl-2 family of proteins, is a pore-forming, mitochondria-associated protein [49] as well as an activator for the mitochondrial permeability transition pore (mPTP) [50].
Western analysis revealed that the Bax (proapoptotic protein) level was highly upregulated to 128%, 134%, and 170% by Glu (20 mM), Hcy (10 mM), and the combined treatments, respectively ( Figure 4a). Interestingly, results clearly implicated the synergistic effect of Glu plus Hcy, i.e., 70% > (28% + 34%). ATX at 5 µM completely attenuated the suppression caused by glutamate or homocysteine alone and also ameliorated significantly that caused by the combined treatments of glutamate with homocysteine ( Figure 4a). Conversely, Bcl-2 was severely down-regulated by the insult of Glu (20 mM), Hcy (10 mM), and the combined treatment to 78%, 76%, and 56%, respectively ( Figure 4b). Hcy induced neurotoxicity via causing mitochondrial dysfunction to regulate Bcl-2 family and opening of mitochondrial permeability transition pores [37]. Our results have revealed that ATX was unable to have completely attenuated such inhibitions. Although ATX at 5 µM alone was able to increase the Bcl-2 level (antiapoptotic protein) to 116%, compared to 100% of the control (Figure 4b), the % recovery only attained 83% and 84% by the single insult exerted by Glu and homocysteine, respectively, and 70% by the combined insults. As the counterbalance of Bcl-2 and Bax is responsible for the homeostasis of the intrinsic pathway, we further examined the variation of Bcl-2/Bax ratio (Figure 4c). More apparent results were seen when insulted by Glu, Hcy and the combined therapy; the Bcl-2/Bax ratios reached 60%, 58%, and 34%, respectively, compared with 100% of the control (Figure 4c), for which ATX at 5 µM was found to have attenuated the levels to only 75%, 72%, and 52%, respectively, only. ATX (5 µM) alone increased the ratio to 108% (Figure 4c). In contrast, Wang et al.'s study showed the complete reversal of Hcy (8 mM)-induced neurotoxicity after treatment with ATX (5 µM) [37]; suggestively, such a deviation could be due to the difference in the cell model and dose of Hcy used.
In cultured cells, astaxanthin protected the mitochondria against endogenous oxygen radicals, conserved their redox (antioxidant) capacity, and enhanced their energy production efficiency [44]. Two distinct mechanisms leading to cytochrome C release were described by Eskes et al. (1998): one being stimulated by calcium and inhibited by cyclosporine A; the other being Bax-dependent, Mg 2+ sensitive, but cyclosporine insensitive [49]. Apparently, the treatment of PC12 cells with Glu, Hcy and/or the combined therapy could suppress the cell viability ( Figure 1) by up-regulating apoptosis ( Figure 4) [51].    Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via allev mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro ca cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via mu signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used  iterature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating hondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac

Effect of Astaxanthin on the Expression of Caspase-12 and Caspase-3 in PC12 Cells Treated with Homocysteine and Glutamate
Results from Western blot analysis showed the levels of caspase-12 (42 kDa) and caspase-3 (17 kDa) were highly upregulated due to the insult of Glu (20 mM), Hcy (10 mM), and the combined treatment ( Figures 5 and 6). This again confirms that Glu, Hcy and/or the combined insult could suppress the cell viability (Figure 1), up-regulating apoptosis (Figure 4) and the activities of caspase-12 ( Figure 5) and caspase-3 ( Figure 6) and indicating the neurotoxic role of glutamate and Hcy to PC12 cells [51].  Literature has implicated that ATX inhibits homocy mitochondrial dysfunction and signal crosstalk [37]. A cell cultures [38]. Previously, Zhang et al. found that AT signaling pathways including NFκB and MAPK path prophylactic or remediation agent against neuronal diso

Effect of Astaxanthin on the Intracellular Calcium Ion Le
Calcium ion dysregulation is relevant to the initiati model in reality has pertinently reflected such a possib raised by the insult of 20 mM Glu (144%), and/or 10 mM (176%) (Figure 3a). ATX at 5 μM fully alleviated the ca slightly less effective against that by Hcy (10 mM), and w therapy (133%) (Figure 3a), implicating insufficient mo and (Glu + Hcy); suggestively, a higher dose of ATX ma In addition, Hcy has been implicated to indirec activating ionotropic and metabotropic receptors [41], w the reasons to elicit different outcomes by such a com [2,36,41]. Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create The level of caspase-12 was stimulated to 120%, 121%, and 148% due to the insult of Glu, Hcy, and combined treatment, respectively, comparing to 100% of the control ( Figure 5). ATX at 5 μM was able to almost completely alleviate the adverse effects caused by Glu and Hcy (105% and 106%) but was only slightly effective for the combined treatment (138%) ( Figure 5).
Caspase-12, expressed in mouse and human, is classified as an inflammatory caspase [52] that mediates ER-specific apoptosis pathway and contributes to Aβ neurotoxicity [52,53]. Moreover, is was found to mediate carbon tetrachloride-induced hepatocyte apoptosis in mice through the activation of the downstream effector caspase-3 directly and/or indirectly via caspase-9 activation [54]. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. Thus, caspase-12 plays an important role in stretch-induced apoptosis that is associated to endoplasmic reticulum stress by activating caspase-3 [55].
In summary, both Hcy and Glu provoked the intrinsic pathways, resulting in cell apoptosis. In the intrinsic pathway, ROS and calcium ion influx triggered imbalanced Bcl-2/Bax homeostasis and the release of cytochrome c, which in turn upregulated caspase-3 and provoked cell apoptosis. The role of ATX was seen to have dose-dependently alleviated all these adverse effects. Literature  Literature has implicated that ATX inhibits homocy mitochondrial dysfunction and signal crosstalk [37]. A cell cultures [38]. Previously, Zhang et al. found that AT signaling pathways including NFκB and MAPK pathw prophylactic or remediation agent against neuronal diso

Effect of Astaxanthin on the Intracellular Calcium Ion Le
Calcium ion dysregulation is relevant to the initiati model in reality has pertinently reflected such a possib raised by the insult of 20 mM Glu (144%), and/or 10 mM (176%) (Figure 3a). ATX at 5 μM fully alleviated the ca slightly less effective against that by Hcy (10 mM), and w therapy (133%) (Figure 3a), implicating insufficient mo and (Glu + Hcy); suggestively, a higher dose of ATX ma In addition, Hcy has been implicated to indirec activating ionotropic and metabotropic receptors [41], w the reasons to elicit different outcomes by such a com [2,36,41].
Two subcellular organelles, namely the mitochon involved in the cellular pathogenesis of AD; an incre calcium homeostasis also have been reported [42]. Owin of Ca 2+ -signaling, ER-mitochondrial distance in the neur conditions. When the distance is decreased, Ca 2+ -o mitochondria [40]. The reduction in the distance betwee Alzheimer's disease (AD) pathology by enhanced Ca 2+ -s ATX attenuated glutamate-induced elevation of CHOP (GRP78), inhibiting glutamate-induced apoptosis throug glutamate-induced calcium influx [24].  Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41].
Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca 2+ -signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca 2+ -overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer's disease (AD) pathology by enhanced Ca 2+ -signaling [40]. Lin et al.'s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein p < 0.05; # p < 0.05; ** p < 0.01; *** p < 0.005.
The level of caspase-12 was stimulated to 120%, 121%, and 148% due to the insult of Glu, Hcy, and combined treatment, respectively, comparing to 100% of the control ( Figure 5). ATX at 5 µM was able to almost completely alleviate the adverse effects caused by Glu and Hcy (105% and 106%) but was only slightly effective for the combined treatment (138%) ( Figure 5).
Caspase-12, expressed in mouse and human, is classified as an inflammatory caspase [52] that mediates ER-specific apoptosis pathway and contributes to Aβ neurotoxicity [52,53]. Moreover, is was found to mediate carbon tetrachloride-induced hepatocyte apoptosis in mice through the activation of the downstream effector caspase-3 directly and/or indirectly via caspase-9 activation [54]. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. Thus, caspase-12 plays an important role in stretch-induced apoptosis that is associated to endoplasmic reticulum stress by activating caspase-3 [55].
In summary, both Hcy and Glu provoked the intrinsic pathways, resulting in cell apoptosis. In the intrinsic pathway, ROS and calcium ion influx triggered imbalanced Bcl-2/Bax homeostasis and the release of cytochrome c, which in turn upregulated caspase-3 and provoked cell apoptosis. The role of ATX was seen to have dose-dependently alleviated all these adverse effects. Literature elsewhere has indicated that Aβ induced GRP78/Bip expression and activated caspases 4 and 12 [56], implicating the possible alternate target of ATX.

Source of Cell Line
The cell line PC12 cells (BCRC 6008), originating from the pheochromocytoma on the rat adrenal gland, was purchased from the Bioresource Collection and Research Center (Hsin-Chu, Taiwan).

Cultivation of PC12 Cell Line
The method for cultivation of PC12 cell line was conducted as previously reported [14].

Preparation of Differentiating Medium
Serum-free RPMI 1640 medium stored at 4 • C was used. The PC12 cells were dislodged from stock culture dishes and triturated well using a glass Pasteur pipette to break up cell clumps. The cells were seeded onto 150-mm, 100-mm and 35-mm-dishes at densities of 5 × 10 6 , (1-2) × 10 6 , and (2-5) × 10 6 cells, respectively, on poly-l-lysine-coated dishes and cultured in medium supplemented with a final concentration of NGF at 50 ng/mL. The cells were incubated at 37 • C under saturated water vapor and 7.5% CO 2 atmosphere. The medium was changed three times per week. The culture was observed for 72 h. According to the instruction, by 7-10 days of treatment, at least 90% of the cells can generate neurites. (https://biocyclopedia.com/index/cell_biology_methods/cultured_for_neuroanal_ pc12_cells.php) [57]. The differentiated cells were roughly counted by a hematocytometer.

Preparation of Astaxanthin Solution
The work of Bolin et al. (2010) was followed for preparation of astaxanthin DMSO stock solution, which was stored at −20 • C in a brown-colored Eppendorf tube. Appropriate dilution was performed before use [58].

Preparation of Homocysteine and Glutamate Solutions
The work of Zhou et al. (2005) was followed to prepare homocysteine DMSO solution [59], while the method of Kawakami et al. (2009) was conducted to prepare glutamic acid solution (pH 7.0) (denoted as Glu) [60]. With a slight modification, an appropriate amount of Hcy was weighed, dissolved in DMSO, and stored at −20 • C in a brown-colored Eppendorf tube. Appropriate dilution was performed before use.

MTT Assay Cell Viability Test
The cell viability test with MTT assay was carried out as previous reported [14]. The doses of Glu and Hcy were applied as indicated. The absorbance was read with ELISA Reader (ClarioStar, BMG Labtech Japan Ltd., Saitama, Japan) at 570 nm. Cell viability was calculated according to Equation (1): where As is the absorbance of the sample and Ac is the absorbance of the control.

The Cytotoxicity of Astaxanthin
Test for ATX cytotoxicity was conducted as previously reported [45]. The dose of ATX was applied to 24-well culture plates as indicated. The cell viability was calculated according to Equation (1).

Protective Effect of Astaxanthin against the Cytotoxicity of Homocysteine, Glutamate, or Homocysteine Plus Glutamate
The PC12 cells were seeded at a density of 7 × 10 4 cells/well and incubated for 24 h until adhesion. The medium was changed to the differentiating media containing ATX at 0.0, 1.0, 2.0, 5.0, 10.0, and 100.0 µM and incubated for 24 h The medium was replaced fresh with differentiating medium. Hcy (10 mM), Glu (20 mM), or Hcy (10 mM) plus Glu (20 mM) were added and the incubation was continued for 24 and 48 h. MTT assay was carried out as mentioned above. The cell viability was calculated according to Equation (1).

Determination of Intracellular Calcium Ion Concentration
The work of Sul et al. (2009) was followed to determine the intracellular calcium ion concentration [61]. In brief, PC12 cells were seeded onto 24-well plates at a density 7 × 10 4 cells/well and incubated for 24 h until entirely adhered. The following protocol was carried out as cited. The doses of ATX, Hcy, and Glu were 5 µM, 10 mM, and 20 mM respectively. The final supernatant was discarded, and the pellets were rinsed with PBS. Then, 1× trypsin was added and incubated at 37 • C for 5 min. The cell density was counted with the hematocytometer, the cells were seeded onto 96-well plates at a density 2 × 10 4 cells/well, and the medium was replaced fresh with the incomplete medium containing 1 µM Fluo-3/AM (C 51 H 50 C l2 N 2 O 23 ) and incubated at 37 • C for 30 min avoiding direct sunlight. The fluorescence produced after the reaction of Fluo-3/AM with calcium ions was measured at excitation wavelength E x = 488 nm, and emitted wavelength E m = 532 nm. The untreated sample was used as the blank and set at 100% to estimate the change of intracellular calcium ion concentration within different groups.

Determination of Intracellular Reactive Oxygen Species (ROS)
According to Zhang et al. (2008), the intracellular ROS level was determined [62]. The cultivation of PC12 cells and the doses of ATX, Hcy, and Glu were similarly conducted as mentioned in the above section. The final culture was centrifuged at 12,500× rpm for 20 min. The supernatant was discarded, and the pellets were rinsed with PBS. Then, 1× trypsin was added and incubated at 37 • C for 5 min. The cell density was counted with the hematocytometer, the cells were seeded onto 96-well plates at a density 2 × 10 4 cells/well, and the medium was replaced fresh with the incomplete medium containing 10 µM 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) and incubated at 37 • C for 30 min avoiding direct sunlight. The fluorescence of the product dichlorofluorescein (DCF) produced after the reaction of H 2 DCFDA with ROS was measured at excitation wavelength E excitation = 488 nm, and emitted wavelength E emission = 532 nm using a fluorescence ELISA Reader (Bio-Rad, Hercules, CA, USA). The untreated sample was used as the blank and set at 100% to calculate the amount of ROS produced within different groups.

Protein Extraction
Method of protein extraction was carried out as previously cited [63]. The cultivation of PC12 cells and the doses of ATX (5 µM), Hcy (10 mM), and Glu (20 mM) were similarly conducted as mentioned in the above section. The protein content of the final supernatant was determined using the authentic bovine serum protein to establish the calibration curve, and the results were expressed in µg/µL. The remaining supernatant was transferred to a new 5-mL Eppendorf tube and stored at -80 • C in a freezer to keep the integrity of the cells for further use.

Assay for the Malondialdehyde (MDA) Concentration
The determination of MDA was carried out using the protocol reported by Chang et al. (2011) [64]. The final OD was read at 532 nm using an ELISA Reader (Bio-Rad, CA, USA). Authentic 1,1,3,3-tetramethoxy propane (TMP) was used to establish the calibration curve, from which the concentration of MDA in samples was calculated and expressed in µM/µg protein.

Western Blot Analysis
The SDS PAGE (7.5%-12%) electrophoresis was conducted at 100 V as instructed by the manufacturer (Life Science, CA, USA). The 100 µg of sample protein was loaded in each well. The primary antibodies of monoclonal anti-βactin, polyclonal anti-Bax, polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-cleaved caspase-12, and polyclonal anti-BcL-2 (BioVision Inc., Milpitas, CA, USA) were applied as 1/1000 of dilution and left in a 4 • C ice box overnight. The PVDF membranes were rinsed with TBST buffer once for 30 min. The secondary antibodies Gt × Ms IgG(H + L) HRP and goat anti-rabbit IgG pAb HRP (Stressgen Biotechnologies Corporation Corporate, San Diego, CA, USA) were applied as 1/5000 of dilution and left in 4 • C ice box for 30 min. while shaken. The membranes were rinsed with TBST buffer twice, each time for 30 min. Enhanced chemiluminescence (ECL) was added and mixed well for 5 min to facilitate the reaction. The emitted chemiluminescence was taken by the Hansor LIS02 photo system, and the intensity was quantified with Image J (NIH Image, Bethesda, MD, USA).

Statistical Analysis
Data obtained in the same group were analyzed using the SPSS 10.0 statistical software (SPSS, Chicago, IL, USA). Analysis of variance (ANOVA) and Tukey's test were used to analyze the variance. Data are expressed as means ± SD from triplicate experiments. Data with significant differences between groups were identified with statistical level of * ,⊗,#, Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41]. Literature has implicated that ATX inhibits homocysteine-induced neurotoxi mitochondrial dysfunction and signal crosstalk [37]. A similar result was found cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective signaling pathways including NFκB and MAPK pathways, and implicated AT prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocystein
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease ( model in reality has pertinently reflected such a possibility [40]. The calcium ion raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the co (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 slightly less effective against that by Hcy (10 mM), and was much less effective aga therapy (133%) (Figure 3a), implicating insufficient mole number for the interact and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a co In addition, Hcy has been implicated to indirectly increase intracellular activating ionotropic and metabotropic receptors [41], which, compared with glut the reasons to elicit different outcomes by such a combination of pharmacologi [2,36,41].  Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via allevi mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro ca cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via mul signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutama
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC1 model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was hi raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatm (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but slightly less effective against that by Hcy (10 mM), and was much less effective against the comb therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insul In addition, Hcy has been implicated to indirectly increase intracellular calcium level activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may c the reasons to elicit different outcomes by such a combination of pharmacological actions of [2,36,41].   Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via allev mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro c cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via m signaling pathways including NFκB and MAPK pathways, and implicated ATX to be use prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutam
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined trea (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, bu slightly less effective against that by Hcy (10 mM), and was much less effective against the com therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions betwee and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined ins In addition, Hcy has been implicated to indirectly increase intracellular calcium lev activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may the reasons to elicit different outcomes by such a combination of pharmacological actions o [2,36,41].   (20 mM). Da compared to the control; 〒: vs. Hcy 10 mM at the same tim 20 mM at the same time; #: vs. Hcy 10 mM + Glu 20 mM a difference was judged by confidence levels of * p < 0.05; # p < < 0.01; ## p < 0.01; ⊗⊗ p < 0.01;  p < 0.01; *** p < 0.005.
Literature has implicated that ATX inhibits homocyste mitochondrial dysfunction and signal crosstalk [37]. A sim cell cultures [38]. Previously, Zhang et al. found that ATX e signaling pathways including NFκB and MAPK pathway prophylactic or remediation agent against neuronal disord

Effect of Astaxanthin on the Intracellular Calcium Ion Level
Calcium ion dysregulation is relevant to the initiation model in reality has pertinently reflected such a possibilit raised by the insult of 20 mM Glu (144%), and/or 10 mM Hc (176%) (Figure 3a). ATX at 5 μM fully alleviated the calciu slightly less effective against that by Hcy (10 mM), and was therapy (133%) (Figure 3a), implicating insufficient mole n and (Glu + Hcy); suggestively, a higher dose of ATX may b In addition, Hcy has been implicated to indirectly activating ionotropic and metabotropic receptors [41], whi the reasons to elicit different outcomes by such a combin [2,36,41].   Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate
Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult.
In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create p < 0.005.

Conclusions
Glutamate and homocysteine can cooperatively, but not additively or synergistically, damage neuronal cells as evidenced in the PC12 cell model. Also, homocysteine and glutamate compromise neuronal homeostasis by multiple and divergent routes. Both of them can provoke cell apoptosis via oxidative stress and the intrinsic pathway. The mechanisms of action may involve (1) increasing calcium influx; (2) producing ROS; (3) initiating lipid peroxidation (as evidenced by the huge production of MDA); (4) causing imbalance of the Bcl-2/Bax homeostasis; and (5) activating a cascade of caspases involving caspase-12 and caspase-3 ( Figure 7). Meanwhile, ATX can protect the neuron-mimic PC12 cell viability via alleviating these adverse effects. Suggestively, drugs that exhibit potent antioxidative capability can be considered beneficial for treatment of certain neuro-excitatory diseases.