Therapeutic Effects of Natural Compounds and Small Molecule Inhibitors Targeting Endoplasmic Reticulum Stress in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease, characterized by progressive cognitive impairment and memory loss. So far, the pathogenesis of AD has not been fully understood. Research have shown that endoplasmic reticulum (ER) stress and unfolded protein response (UPR) participate in the occurrence and development of AD. Furthermore, various studies, both in vivo and in vitro, have shown that targeting ER stress and ER stress-mediated apoptosis contribute to the recovery of AD. Thus, targeting ER stress and ER stress-mediated apoptosis may be effective for treating AD. In this review, the molecular mechanism of ER stress and ER stress-mediated apoptosis, as well as the therapeutic effects of some natural compounds and small molecule inhibitors targeting ER stress and ER stress-mediated apoptosis in AD will be introduced.


INTRODUCTION
The endoplasmic reticulum (ER) is a crucial organelle of eukaryotic cells, whose functions include protein synthesis and folding, lipid biogenesis and calcium metabolism (Schwarz and Blower, 2016). Numerous cellular stresses, such as disequilibrium of calcium homeostasis, redox imbalance, changes in protein glycosylation, or protein folding defects in the ER, can trigger ER stress response, causing accumulation of unfolded or misfolded proteins in the ER lumen (Senft and Ronai, 2015). In order to maintain the homeostasis of the ER, the unfolded protein response (UPR), an integrated signal transduction pathway, is activated (Walter and Ron, 2011). UPR can inhibit protein transcription and translation, promote the degradation of misfolded proteins and increase the ability of correct protein folding (Hetz, 2012). However, under chronic or excessive ER stress, UPR fails to maintain the homeostasis of ER, then the apoptosis signaling pathway is activated, leading to a variety of diseases, including Alzheimer's disease (AD) (Uddin et al., 2020).
AD is the most common neurodegenerative disease, characterized by progressive cognitive impairment and memory loss. Besides, abnormal accumulation of misfolded proteins in the brain, such as β-amyloid (Aβ) peptide and hyperphosphorylated tau protein, is the main neuropathological hallmark of AD (Villain and Dubois, 2019;Ghemrawi and Khair, 2020). So far, the pathogenesis of AD has not been fully understood, and there is no effective treatment for this disease. The existing treatments can only relieve the symptoms or slow down the progression of the disease, but the patients can't be cured (Morris et al., 2018). Therefore, it is necessary to find a novel and effective treatment for AD.
Recently, research have shown that ER stress and UPR participate in the occurrence and development of AD (Hoozemans et al., 2009;Salminen et al., 2009;Uddin et al., 2020). Furthermore, various studies, both in vivo and in vitro, have shown that targeting ER stress and ER stress-mediated apoptosis contribute to the recovery of AD (Xu et al., 2018;Zhu et al., 2019;Song et al., 2020;Oliveira et al., 2021). Thus, targeting ER stress and ER stress-mediated apoptosis may be effective for treating AD. In this review, the molecular mechanism of ER stress and ER stress-mediated apoptosis, as well as the therapeutic effects of some natural compounds and small molecule inhibitors targeting ER stress and ER stress-mediated apoptosis in AD will be introduced.

ER STRESS, UPR, AND APOPTOSIS ER Stress-Mediated UPR Signaling Pathways
Early ER stress will induce UPR, which might be protective to cells (Walter and Ron, 2011). Three ER transmembrane proteins are involved in UPR signaling pathways: PERK (protein kinase RNA-like ER kinase), IRE1 (inositol-requiring enzyme 1) and ATF6 (activating transcription factor 6) (Chen and Brandizzi, 2013;Hillary and FitzGerald, 2018;Rozpedek et al., 2019). Under physiological conditions, these proteins are combined with ER chaperone BiP (immunoglobulin binding protein, also called glucose-regulated protein 78, GRP78), and located in the ER membrane in an inactive state (Carrara et al., 2015;Ghemrawi and Khair, 2020). However, under ER stress, when a misfolded or unfolded protein binds to BiP, BiP is dissociated from these transmembrane proteins, then downstream signaling pathways of UPR are initiated (Figure 1; Ron and Walter, 2007;Parmar and Schroder, 2012). These three transmembrane proteins play a key role in the three signaling pathways of UPR, promoting the correct folding of unfolded and misfolded proteins, thus maintaining ER homeostasis.
PERK is a type I ER transmembrane protein with a serine/threonine protein kinase domain (Rozpedek et al., 2019). Under mild ER stress, PERK first undergoes autophosphorylation, and then eukaryotic initiation factor 2α (eIF2α) is activated by phosphorylation. Phosphorylated eIF2α can block protein synthesis and reduce the protein load of ER (Cnop et al., 2017). IRE1 is also a type I ER transmembrane protein with endonuclease activities, which can be divided into two isforms, IRE1α and IRE1β (Chen and Brandizzi, 2013). When ER stress occurs, IRE1α is first activated by autophosphorylation and dimerization (Hetz et al., 2011). Then, through unconventional splicing of X-box binding protein 1 (XBP1) mRNA, an active transcription factor, XBP1s, is produced. XBP1s can promote the correct folding of unfolded and misfolded porteins, thereby restore ER homeostasis (Hollien et al., 2009). ATF6 is a type II transmembrane protein, which encodes a basic leucine zipper (bZIP) transcription factor (Hillary and FitzGerald, 2018). In conditions of ER stress, ATF6 moves from the ER to the Golgi, where it undergoes proteolytic cleavage. Subsequently, the active transcription factor, ATF6α, is produced and transferred to the nucleus, where it regulates the expression of UPR related genes, such as CHOP, BiP, and XBP1 (Wu et al., 2007;Ghemrawi and Khair, 2020).

Chronic or Excessive ER Stress-Mediated Apoptosis Signaling Pathways
On the contrary, under chronic or excessive ER stress, UPR fails to maintain the homeostasis of ER, then the apoptosis signaling pathways are activated, even resulting in cell death (Senft and Ronai, 2015;Iurlaro and Munoz-Pinedo, 2016). ER stress can induce apoptosis through three major signaling pathways: CHOP pathway, JNK pathway and caspase-12 pathway (Figure 1; Hu et al., 2018).

PERK-CHOP Pathway
Phosphorylated eIF2α allows the translation of activating transcription factor 4 (ATF4), an activator of apoptosis-related genes (Hu et al., 2018). Subsequently, AFT4 up-regulates the expression of several apoptosis-related genes, for example, C/EBP homologous protein (CHOP) (Oyadomari and Mori, 2004). CHOP can regulate the expression of some BCL-2 family members, including the down-regulation of antiapoptotic proteins like BCL-2 and BCL-XL, and the upregulation of pro-apoptotic proteins like BIM, BAK, and BAX (Hata et al., 2015). Furthermore, the oligomerization of BAK-BAX will lead to the release of cytochrome c, which causes apoptosis through the mitochondrial pathway (Brenner and Mak, 2009).

Caspase-12 Pathway
ER stress can activate caspase-12, and then the caspase cascade is activated (Iurlaro and Munoz-Pinedo, 2016). Activated caspase-12 moves from the ER to the cytosol, where it cleaves procaspase-9 to form caspase-9 (Szegezdi et al., 2003). Caspase-9 in turn activates caspase-3, the main executioner of apoptosis (Lossi et al., 2018). FIGURE 1 | ER stress-mediated UPR signaling pathways and apoptosis signaling pathways. Three ER transmembrane proteins, IRE1, ATF6, and PERK, are involved in UPR signaling pathways. Under normal conditions, these proteins are combined with ER chaperone BiP, and located in the ER membrane in an inactive state. Under ER stress, when a misfolded protein binds to BiP, BiP dissociates from IRE1, ATF6, and PERK. Subsequently, PERK and IRE1 undergo autophosphorylation, and ATF6 undergoes proteolytic cleavage. Downstream signaling pathways of UPR are initiated, thereby maintaining ER homeostasis. However, under chronic or excessive ER stress, UPR fails to maintain the homeostasis of ER, then apoptosis signaling pathways are activated. All the three ER transmembrane proteins can up-regulate CHOP, leading to apoptosis by regulating the expression of some BCL-2 family members. Activated IRE1 can activate JNK, inducing apoptosis by up-regulating the expression of some pro-apoptotic proteins. Caspase-12 can induce apoptosis by activating the caspase cascade reaction.

APPLICATIONS OF NATURAL COMPOUNDS AND SMALL MOLECULE INHIBITORS TARGETING ER STRESS AND ER STRESS-MEDIATED APOPTOSIS IN TREATING ALZHEIMER'S DISEASE
Researches have shown that ER stress and UPR participate in the occurrence and development of AD (Hoozemans et al., 2009;Salminen et al., 2009;Uddin et al., 2020). During AD, the continuous accumulation of Aβ or p-tau leads to unbalanced ER calcium homeostasis, abnormal protein folding and ER stress (Sobow et al., 2004;Mondragon-Rodriguez et al., 2012). Neuronal cells are especially sensitive to protein misfolding, therefore, the excessive accumulation of Aβ or p-tau will result in synaptic dysfunction and apoptosis, even neuronal death (Remondelli and Renna, 2017;Ghemrawi and Khair, 2020). In addition, some UPR-related proteins, such as phosphorylated PERK, eIF2α and IRE1 were found in AD brains (Devi and Ohno, 2014;Duran-Aniotz et al., 2017;Uddin et al., 2020). Meanwhile, ER stress markers, including GRP78, CHOP and caspase-12, are up-regulated in AD patients (Santos and Ferreira, 2018).
Recently, various studies, both in vivo and in vitro, have shown that targeting ER stress and ER stress-mediated apoptosis contribute to the recovery of AD (Xu et al., 2018;Zhu et al., 2019;Song et al., 2020;Oliveira et al., 2021). Several active components extracted from the plants and small molecule inhibitors of UPR signaling pathways and other signaling pathways have displayed good therapeutic effects on AD models. A brief summary of some natural compounds and small molecule inhibitors targeting ER stress in AD studies is shown in Table 1.

Targeting PERK-CHOP Signaling Pathway
The PERK-CHOP signaling pathway plays an essential role in inducing cell apoptosis. In the early stage of ER stress, activated PERK can further activate eIF2α, which can block protein synthesis and exert a protective effect on cells (Rozpedek et al., 2019). However, under chronic or excessive ER stress, PERK-CHOP signaling pathway will be activated, leading to apoptosis (Hu et al., 2018).

Schisandrin (Sch)
Schisandrin (Sch) is an active component extracted from schisandra chinensis Baill (Zhu et al., 2019). In the Streptozotocin (STZ)-induced AD rats, after the treatment of Sch, the expression of ER stress markers, including GRP78, CHOP, and cleaved caspase-12, was obviously decreased (Song et al., 2020). Moreover, Sch also improved the learning and memory capacity of STZ-induced AD rats, by enhancing the activity of Sirtuin 1 (SIRT1, an enzyme contributes to the acquisition and maintenance of memory) (Song et al., 2020).

Resveratrol and Crocin
In the Aβ 25−35 -induced AD rats, both enhanced expression of GRP78, CHOP, caspase-3, BAX and attenuated expression of BCL-2 were observed in the hippocampal CA1 region (Hippo) and prefrontal cortical (PFC), while the situation was reversed by treatment with resveratrol (a polyphenolic stilbenoid, present in grapes, mulberries, peanuts and other plants; Malaguarnera, 2019) and crocin (a non-tetraquinone pigment extracted from Crocus sativus L.; Shafahi et al., 2018). Furthermore, improved learning and memory ability and decreased number of apoptotic neurons were also detected in AD rats (Lin et al., 2019).

Gastrodin (Gas)
Tg2576 transgenic mouse is an AD model with high expression of β-site APP-cleaving enzyme 1 (BACE1), which is a rate-limiting enzyme for Aβ generation and could aggravate the process of AD (O'Connor et al., 2008). Gastrodin (Gas), an active component of Gastrodia elata Blume, was found to improve learning and memory abilities and attenuate intracellular oxidative stress of Tg2576 mice (Zhang et al., 2016). Moreover, Gas down-regulated BACE1 expression via inhibiting activation of PKR and eIF2α (Zhang et al., 2016).

Quercetin
In the APP23 AD model mice, after long-term feeding with quercetin, a polyhydroxylated flavonoid, the expression of growth arrest and DNA damaged-inducible gene 34 (GADD34) was induced, leading to the down-regulation of phosphorylated-eIF2α and ATF4 (Ohta et al., 2011;Hayakawa et al., 2015). This resulted in improvement of memory in aged AD mice, and delayed deterioration in memory of the mice at the early stage of AD (Hayakawa et al., 2015).

LDN-0060609
Compound LDN-0060609 is a small molecule PERK inhibitor (Rozpedek et al., 2019). In rat astrocytic DI TNC1 cell line, LDN-0060609 pretreatment attenuated the pro-apoptotic, PERKdependent signaling pathway induced by thapsigargin (Th) treatment. It significantly inhibited eIF2α phosphorylation and increased cell survival in vitro. Notably, LDN-0060609 has no cytotoxicity to DI TNC1 cells and also has no influence on cell cycle progression (Rozpedek et al., 2019;Rozpedek-Kaminska et al., 2020). Therefore, it may contribute to prevention against apoptosis and neurodegeneration in AD without the cytotoxic effect.

ISRIB
ISRIB is a small molecule integrated stress response (ISR) inhibitor, which can reverse the phosphorylation of eIF2α and inhibit the downstream targets of eIF2α, such as ATF4, CHOP, and GADD34 (Sidrauski et al., 2015). In an AD mouse model induced by intracerebroventricular injection of amyloidβ oligomers (AβOs), ISRIB treatment counteracted the increase in ATF4 protein level, protecting mice from long-term memory impairment (Oliveira et al., 2021). Furthermore, ISRIB was shown to attenuate translational repression, restore synaptic plasticity and memory in transgenic APP SWE /PS1 E9 AD mice (Oliveira et al., 2021).

Xestospongin C (XeC)
Xestospongin C (XeC), a compound isolated from the Xestospongia species, is a reversible IP 3 receptor antagonist (Gafni et al., 1997). It was found that XeC could improve the cognitive behavior of APP/PS1 AD mice. XeC also reduced the number of Aβ plaques and down-regulated the expression of GRP78, CHOP and caspase-12 in APP/PS1 mice (Wang et al., 2019). In addition, XeC significantly ameliorated Aβ 1−42induced neuronal apoptosis and intracellular Ca 2+ overload in primary cultured hippocampal neurons (Wang et al., 2019).

Targeting IRE1-JNK Signaling Pathway
Activated IRE1 can activate JNK, leading to the up-regulation of pro-apoptotic proteins and subsequent apoptosis (Urano et al., 2000;Deng et al., 2001). Activation of IRE1-JNK signaling pathway plays a key role in ER stress-mediated apoptosis.

Bajijiasu
Bajijiasu (also known as bajisu) is a natural active ingredient isolated from Morinda officinalis (Chen et al., 2014). It was shown to play a protective role against Aβ 25−35 -induced neurotoxicity in PC12 cells . In a double transgenic APP/PS1 mouse model of AD, oral administration of bajijiasu improved learning and memory abilities of APP/PS1 mice. Also, Bajijiasu protected neurons from apoptosis by down-regulating the expression of IRE1α, PERK, eIF2α, and CHOP. Moreover, reduced ROS and MDA levels in both the hippocampus and cortex were detected (Xu et al., 2018).

Ginsenoside Rg1
Ginsenoside Rg1 is a steroidal saponin highly abundant in ginseng (Wu et al., 2013). In a APP/PS1 rat model, AD rats were fed with 0.5% Rg1-enriched food to investigate the neuroprotective effects of Rg1. After Rg1 treatment, the accumulation of Aβ plaque and neurofbrillary tangles (NFTs) was significantly decreased in the AD rats, as well as the reduced expression of caspase-3 and the number of apoptotic cells. Furthermore, down-regulation of GRP78, IRE1, and p-JNK was observed in the AD rats, indicating that Rg1 exhibited neuroprotective effects by inhibiting the ER stress-mediated JNK apoptotic pathway (Mu et al., 2015).

Targeting ATF6-AKT Signaling Pathway
Under ER stress, the ATF6 pathway can mediate cell survival by regulating the activation of Akt and the expression of GRP78 and CHOP (Cui et al., 2017).

Taurine
Taurine, a free amino acid, is abundant in the brain and plays an important role in the central nervous system (Seidel et al., 2019). It was found that taurine can protect PC12 cells against H 2 O 2 -induced ER stress, leading to the increase in cell viability, as well as the down-regulation of GRP78, CHOP, and BIM (Pan et al., 2010). Meanwhile, in primary neuronal cultures, taurine can also modulate hypoxia-and glutamate-induced ER stress by down-regulating the expression of caspase-12, CHOP, cleaved ATF6 and p-IRE1 (Pan et al., 2012). Later, in APP/PS1 transgenic AD mouse model, oral administration of taurine significantly ameliorated cognitive deficits of the adult AD mice (Kim et al., 2014). Further study conducted in the oligomeric Aβ-infusion AD mouse model showed that taurine can ameliorate cognitive impairment by directly binding to oligomeric Aβ (Jang et al., 2017). Nevertheless, mechanisms underlying taurine mediated cognitive improvement still need further elucidation.

DA-CH3
Chronic treatment with DA-CH3, a novel dual GLP-1/GIP receptor agonist (Holscher, 2018), could rescue the spatial acquisition and memory impairments of APP SWE /PS1 E9 mice. Additionally, excessive plaque deposition, gliosis and synaptic damage was ameliorated in the APP SWE /PS1 E9 brain. Further research found that the alleviated ER stress and autophagy impairments in the APP SWE /PS1 E9 mouse brain might be attributed to the up-regulation of the Akt activation (Panagaki et al., 2018).

DISCUSSION
It is proved that ER stress and UPR play an essential role in the occurrence and development of AD (Devi and Ohno, 2014;Duran-Aniotz et al., 2017;Uddin et al., 2020). The related findings, both in vivo and in vitro, have shown that many natural compounds and small molecular inhibitors targeting ER stress and ER stress-mediated neuronal apoptosis contribute to the recovery of AD (Xu et al., 2018;Zhu et al., 2019;Song et al., 2020;Oliveira et al., 2021). However, there are still a lot of questions to be considered if we want to convert these agents into a safe and reliable drug for clinical application, such as the safety, efficacy, and applicability.
Nevertheless, due to the species differences between humans and mice, the existing AD mouse models cannot fully simulate the pathologic and clinical features of human AD, which limits their application in preclinical studies of AD (de Bem et al., 2020). Therefore, it is necessary to develop animal models more similar to human AD to test the safety and efficacy of these agents (Medina and Avila, 2014). Recently, with the development of efficient genome editing technology, larger animals, such as rabbits, pigs and non-human primates, can be used to construct models of AD (Bosze et al., 2003;Chan, 2013;Yang and Wu, 2018). By using the CRISPR/Cas9 system and base editing system, these animal models can accurately simulate the gene mutation sites of human AD, and provide an ideal platform for AD pathogenesis research and preclinical evaluation (Tu et al., 2015;Gaudelli et al., 2017). In the near future, more unknown mechanisms between ER stress and AD will be clarified, which will offer more safe and effective therapeutic strategies to AD.

AUTHOR CONTRIBUTIONS
XG and YX initiated the project, wrote, revised, and finalized the manuscript. XG searched the database. Both authors contributed to the article and approved the submitted version.