Contribution of Lysosome and Sigma Receptors to Neuroprotective Effects of Memantine Against Beta-Amyloid in the SH-SY5Y Cells

Purpose: Memantine is an approved drug for the treatment of Alzheimer’s disease (AD). Autophagy, lysosome dysfunction, and sigma receptors have possible roles in the pathophysiology of AD. Therefore, we aimed to investigate the contribution of sigma receptors and lysosome inhibition to the neuroprotective effects of memantine against amyloid-beta (Aβ)-induced neurotoxicity in SH-SY5Y cells. Methods: We determined the neuroprotective effects of memantine (2.5 µM), dizocilpine (MK801, as a selective N-methyl-D-aspartate (NMDA) receptor antagonist) (5 μM) against Aβ25– 35 (2 μg/μL)-induced neurotoxicity. We used chloroquine (10, 20, and 40 μM) as a lysosome inhibitor and BD-1063 (1, 10, and 30 μM) as a selective sigma receptor antagonist. The MTT assay was used to measure the neurotoxicity in the SH-SY5Y cells. Data were analyzed using the one-way ANOVA. Results: Memantine (2.5 µM), dizocilpine (5 µM), chloroquine (10 and 20 µM) and BD-1063 (1, 10 and 30 µM) decreased the neurotoxic effects of Aβ on the SH-SY5Y cells. However, chloroquine (40 µM) increased the neurotoxic effects of Aβ. Cell viability in the cells treated with memantine + Aβ + chloroquine (10, 20, and 40 μM) was significantly lower than the memantine + Aβ-treated group. Moreover, cell viability in the memantine + Aβ group was higher than the memantine + Aβ + BD-1063 (10 and 30 μM) groups. Conclusion: The lysosomal and sigma receptors may contribute to the neuroprotective mechanism of memantine and other NMDA receptor antagonists. Moreover, the restoration of lysosomes function and the modulation of sigma receptors are potential targets in the treatment of AD.

memantine is elusive.
Sigma receptors are widespread in the CNS and they are involved in different brain functions such as learning and memory, neuroprotection and immunomodulation. 16,17 Various experimental models have documented the neuroprotective effects of the sigma receptors in brain injury and neurodegeneration. 18 Sigma receptors regulate several neurotransmitter systems such as glutamatergic, noradrenalinergic, dopaminergic, and cholinergic systems. 19 Although the NMDA receptors can be modulated by the sigma-1 receptors, 19,20 the interaction between these two receptors has remained controversial. 21 Some studies have shown that the activation of the sigma receptor produces neuroprotection by interfering with the intracellular machinery of NDMA receptors. 22 By considering the role of the NMDA receptors in the neurotoxic effects of Aβ peptide 1 , the sigma receptors may have a possible role in the neuroprotective effects of NMDA antagonists like memantine. In this study, we aimed to explore the contribution of sigma receptors and lysosome inhibition to the neuroprotective effects of memantine against Aβ-induced neurotoxicity in the SH-SY5Y cells.

Neuronal cell culture
We used SH-SY5Y neuronal cells to determine the neurotoxic and neuroprotective activities of the administered agents. We seeded the SH-SY5Y cells at the density of 1 x 10 5 cells/well in the 96-well plates. The cell culture medium contained DMEM/F12 (1:1), fetal bovine serum (10 %), penicillin (100 U/mL), and streptomycin (100 µg/mL). The cells were kept in a standard condition of the humidified atmosphere of 95% air/5% CO2 and 37°C.

Study design and treatments
We used the fibril form of Aβ to conduct the neurotoxicity test. Aβ25-35 (2 μg/μL) was incubated in the water bath for four days at 37°C to induce the aggregation process. Memantine (2.5 µM), dizocilpine (MK801, as a selective NMDA antagonist) (5 μM), chloroquine (10, 20, 40 µM), BD-1063 (1, 10, 30 μM), and Aβ (20 μM) were dissolved in the sterile water. We selected the concentrations of each agent according to the previous studies and a pilot study. The cells were treated with Aβ with or without memantine, dizocilpine, chloroquine, and BD1063 for 24h. The procedure was repeated for four samples.

Cell viability assay
The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was used to measure the neuronal viability. In brief, the MTT reagent (5 mg/mL) was poured into the wells containing the treated cells. After 4 hours, the media was removed and the precipitate dissolved in dimethyl sulfoxide (DMSO) (100 µL). The absorbance of each well was determined at 570 nm by a microplate reader (Synergy HT, Biotek ® ) as an index of cell viability.

Statistical analysis
The results were analyzed using the one-way analysis of variance (ANOVA) test, followed by the LSD test as the post hoc test. P values <0.05 was considered statistically significant. We used the SPSS software (version 23) to analyze the data.
The findings of this study showed that the NMDA antagonists such as memantine and dizocilpine inhibited the neurotoxic effects of Aβ. In vitro and in vivo experiments have shown that memantine reduces the neurotoxic effects of Aβ. 23 This effect might be due to the behavioral effects of memantine. 4 Moreover, Harkany et al 23 have shown that dizocilpine diminished the Aβinduced neurotoxicity. The over-activation of NMDA receptors may mediate the neurotoxic effects of Aβ, and the inhibition of NDMA receptors suppresses this neurotoxic effect. Thus, the NMDA receptor antagonists have potential roles in the AD amelioration. 24 However, the cellular process of neuroprotection produced by the NMDA antagonists is relatively unknown.
Our study showed that the lower concentrations of chloroquine (10 and 20 μM) suppressed the neurotoxic effects of Aβ. In contrast, chloroquine (at a higher concentration: 40 μM) increased the neurotoxic effects of Aβ. According to previous studies, chloroquine has produced both neuroprotective or neurotoxic effects. The low concentration of chloroquine had no apparent effect on the lysosomes and produced neuroprotective effects in the different models. 25,26 In contrast, chloroquine at the concentrations higher than 25 μM decreased primary telencephalic neuronal viability. 27 Another in vitro study has also demonstrated the neurotoxic effects of chloroquine at concentrations higher than 25 μM). 28 Accordingly, chloroquine may have detrimental or beneficial effects on neurons.
The inhibition of lysosomal activity may be related to the neurotoxic effects of chloroquine. Lysosome dysfunction leads to Aβ accumulation and enhances neurotoxicity. 29 Simultaneously, Aβ causes lysosomal dysfunction in the SH-SY5Y cells. 30 Moreover, the neurotoxic effects of Aβ are similar to lysosomotropic agents. 30 On the contrary, the pharmacologic induction of autophagy produces neuroprotective effects against proteinopathies. 31 Chloroquine is a weak base that disrupts the lysosome activity in neurons by the inhibition of autophagosomes inhibition to the lysosome. 32 Therefore, the chloroquine inhibitory effects on lysosome may block the Aβ degradation and increase the Aβ-induced neurotoxicity. In this regard, in vivo studies have shown that lysosomal inhibition may lead to the reduction of Aβ degradation and neurodegeneration. 26,33 Therefore, the chloroquine effects on lysosome especially at the higher concentrations overcomes the neuroprotective effects of this agent and potentiates the neurotoxic effects of Aβ.
There are some controversies regarding the memantine effects on the autophagy. In a study conducted by Yoon et al, the authors reported that a high concentration of memantine activated the autophagy and apoptosis in the glioma cell lines. 34 In contrast, memantine has decreased autophagy in a cellular model of the AD. 15 The difference may be due to the dose of memantine and the cells used in these two experiments. However, there are limited data about the memantine effects on the lysosomal activity. According to our study, lysosome and autophagy inhibition decreased the neuroprotective effects of memantine and dizocilpine. Therefore, autophagy activation and the normal lysosomal function may be protective against the Aβ-induced neurotoxicity. In this regard, the autophagy induction has potentiated neuroprotection against Aβinduced neurotoxicity in the SH-SY5Y cells. 35 Accordingly, the NMDA receptor activation may cause lysosomal defect and magnify the Aβ neurotoxic effects. It is possible to assume that the NMDA antagonists may modulate the lysosome activity and autophagy in the process of neurodegeneration.
This study showed that a sigma receptor antagonist suppressed the Aβ-induced neurotoxicity. There are some inconsistencies in the literature about the neuroprotective or neurotoxic effects of sigma receptors. Some reports have shown that the sigma receptor agonists have produced neuroprotective effects in the neurodegenerative disorder models. 18 On the other hand, BD1063, as a selective sigma receptor antagonist, protected against methamphetamineinduced neurotoxicity in the rat. 36 In vitro and in vivo studies have also shown that AC927, another sigma receptor antagonist, prevented the methamphetamineinduced neurotoxicity. 37 Therefore, the inconsistencies may be related to the models and the neurotoxic agent have been used in these experiments.
Our study showed that a sigma receptor antagonist decreased the neuroprotective effects of two NMDA antagonists against Aβ-induced neurotoxicity. Thus, the activation of sigma receptors may potentiate the neuroprotective effects of memantine and dizocilpine. Other studies have shown that the activation of sigma receptors prevented glutamate-mediated neurotoxicity. 38 In contrast to our results, some studies have shown that the sigma receptor agonists have potentiated the glutamate activity by increasing the expression of the NMDA receptors subunits and the plasma membrane level of NDMA receptors. 22 These inconsistencies may be related to the different interactions between NMDA receptors and sigma receptors in the normal and degenerative conditions.
Our study had some limitations. We did not measure the lysosomal function and NMDA receptor trafficking in the treated cells. Moreover, some agents may have nonspecific effects. Thus, future studies may help to discover the cellular interaction between the NMDA antagonists and the lysosome and sigma receptor functions in the process of neurodegeneration.

Conclusion
The lysosomal function and sigma receptors may contribute to the neuroprotective mechanism of memantine and other NMDA receptor antagonists. Moreover, the restoration of lysosomal function and the modulation of sigma receptors may be potential strategies in the treatment of AD. Future studies may help to discover the exact contribution of lysosome and sigma receptor functions to the neuroprotective effects of the NMDA receptors.

Ethical Issues
Not applicable.