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NRF2 and CREB as therapeutic targets in LM‐031‐treated ΔK280 TauRD‐DsRed SH‐SY5Y cells. (a) Western blot analysis of CREB, pCREB, cleaved CASP3, and TauRD‐DsRed levels in compound‐treated cells infected with CREB‐specific or scrambled shRNA‐expressing lentivirus. GAPDH was used as a loading control. To normalize, the relative CREB, pCREB, or CASP3 of uninduced cells, or relative TauRD‐DsRed of untreated cells was set at 100%. (b) Western blot analysis of NRF2 and TauRD‐DsRed levels (loading control: GAPDH) and caspase 3 activity assay in compound‐treated cells infected with NRF2‐specific or scrambled shRNA‐expressing lentivirus. To normalize, the relative NRF2 level or caspase 3 activity of uninduced cells, or relative TauRD‐DsRed of untreated cells was set at 100%. (c) Neurite outgrowth quantification. p values: comparisons between induced and uninduced cells (#: p < .05 and ##: p < .01), or between compound‐treated and untreated cells (*: p < .05 and **: p < .01) (n = 3). (one‐way ANOVA with a post hoc Tukey test)
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Microtubule‐associated protein Tau, abundant in the central nervous system (CNS), plays crucial roles in microtubule assembly and stabilization. Abnormal Tau phosphorylation and aggregation are a common pathogenic hallmark in Alzheimer's disease (AD). Hyperphosphorylation of Tau could change its conformation and result in self‐aggregation, increase...
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Assembly of tau protein into paired helical filaments (PHFs) and straight filaments is a key feature of Alzheimer's disease (AD). Aggregation of tau has been implicated in neurodegeneration, cellular toxicity and the propagation which accompanies disease progression. We have reported previously that a region of tau (297–391), referred to as dGAE, a...
Traumatic brain injury confers a significant and growing public health burden. It is a major environmental risk factor for dementia. Nonetheless, the mechanism by which primary mechanical injury leads to neurodegeneration and an increased risk of dementia-related diseases is unclear. Thus, we aimed to investigate the effect of stretching on SH-SY5Y...
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The physical properties of cytoskeletal microtubules have a multifaceted effect on the expression of their cellular functions. A superfamily of microtubule-associated proteins, MAP2, MAP4, and tau, promote the polymerization of microtubules, stabilize the formed microtubules, and affect the physical properties of microtubules. Here, we show differe...
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Background
Microtubules (MTs) are critical for cell structure, function, and survival. MT instability may contribute to Alzheimer’s disease (AD) pathogenesis as evidenced by persistent negative regulation (phosphorylation) of the neuronal microtubule-associated protein tau. Hyperphosphorylated tau, not bound to MTs, forms intraneuronal pathology th...
Citations
... The anti-aggregation activity of congo red (positive control) and ZN/VB compounds (5-20 µM) was assessed using E. coli-expressed ∆K280 Tau RD protein [33] and thioflavin T (Sigma-Aldrich) fluorescence assay. The trend of ∆K280 Tau RD amyloid aggregate formation depicted with enhanced and red-shifted fluorescence was recorded by a FLx800 microplate fluorescence reader (BioTek Instruments), where the procedure was previously reported in detail [34]. ...
... Next, we examined the effects of congo red and ZN/VB compounds on Tau misfolding using His-tagged ∆K280 Tau RD proteins purified from E. coli cells [33] in thioflavin T spectroscopic assay. As shown in Figure 3A, after 48 h incubation at 37 • C, markedly increased thioflavin T fluorescence was observed with ∆K280 Tau RD protein (from 193 to 12,215 arbitrary unit (AU); p < 0.001), and the increase was significant compared to wild-type Tau RD protein (12,215 versus 3202 AU; p < 0.001). ...
... We next examined the therapeutic targets in TRKB signaling for neuroprotection of ZN/VB compounds. LM-031, a coumarin derivative displaying neuroprotective potential by up-regulating CREB-dependent BDNF/BCL2 pathway in pro-aggregatory Tau SH-SY5Y cells [33], was included for comparison. As shown in Figure 5, auto-phosphorylation of TRKB at residues Y516 and Y817 decreased upon the induction of ∆K280 Tau ...
Misfolded aggregation of the hyperphosphorylated microtubule binding protein Tau in the brain is a pathological hallmark of Alzheimer’s disease (AD). Tau aggregation downregulates brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB) signaling and leads to neurotoxicity. Therefore, enhancement of BDNF/TRKB signaling could be a strategy to alleviate Tau neurotoxicity. In this study, eight compounds were evaluated for the potential of inhibiting Tau misfolding in human neuroblastoma SH-SY5Y cells expressing the pro-aggregator Tau folding reporter (ΔK280 TauRD-DsRed). Among them, coumarin derivative ZN-015 and quinoline derivatives VB-030 and VB-037 displayed chemical chaperone activity to reduce ΔK280 TauRD aggregation and promote neurite outgrowth. Studies of TRKB signaling revealed that ZN-015, VB-030 and VB-037 treatments significantly increased phosphorylation of TRKB and downstream Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase 1/2 (ERK) and AKT serine/threonine kinase (AKT), to activate ribosomal S6 kinase (RSK) and cAMP response element-binding protein (CREB). Subsequently, p-CREB enhanced the transcription of pro-survival BDNF and BCL2 apoptosis regulator (BCL2), accompanied with reduced expression of anti-survival BCL2-associated X protein (BAX) in ΔK280 TauRD-DsRed-expressing cells. The neurite outgrowth promotion effect of ZN-015, VB-030 and VB-037 was counteracted by a RNA interference-mediated knockdown of TRKB, suggesting the role of these compounds acting as TRKB agonists. Tryptophan fluorescence quenching analysis showed that ZN-015, VB-030 and VB-037 interacted directly with a Pichia pastoris-expressed TRKB extracellular domain, indirectly supporting the role through TRKB signaling. The results of up-regulation in TRKB signaling open up the therapeutic potentials of ZN-015, VB-030 and VB-037 for AD.
... Previously, we found that a novel coumarin derivative, LM-031, displayed neuroprotective potential by up-regulating nuclear factor erythroid 2-related factor 2 (NRF2) and CREB expression in pro-aggregatory tau SH-SY5Y cells and hyperglycemic triple-transgenic AD mice [28]. In this study, we evaluated the potential of LM-031 and its analogues LMDS-1 to -4 in treating tauopathies by examining if these compounds exert neuroprotective effects through enhancing TRKB signaling in ΔK280 tauRD-DsRed folding reporter SH-SY5Y cells. ...
... Thioflavin T is widely used to visualize and quantify the presence of misfolded amyloid protein aggregates in vitro [74]. The tau aggregation inhibiting potential of Congo red, LM-031 and LMDS-1 to -4 was assessed by using E. coli-expressed ΔK280 tauRD protein [28]. Briefly, purified ΔK280 tauRD protein (20 μM) was incubated with Congo red or coumarin compounds (1-10 μM) in 150 mM NaCl and 20 mM Tris-HCl pH8.0, at 37 °C with shaking for 48 h to form aggregates. ...
Hyperphosphorylation and aggregation of the microtubule binding protein tau is a neuropathological hallmark of Alzheimer’s disease/tauopathies. Tau neurotoxicity provokes alterations in brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB)/cAMP-response-element binding protein (CREB) signaling to contribute to neurodegeneration. Compounds activating TRKB may therefore provide beneficial effects in tauopathies. LM-031, a coumarin derivative, has demonstrated the potential to improve BDNF signaling in neuronal cells expressing pro-aggregated ΔK280 tau mutant. In this study, we investigated if LM-031 analogous compounds provide neuroprotection effects through interaction with TRKB in SH-SY5Y cells expressing ΔK280 tauRD-DsRed folding reporter. All four LMDS compounds reduced tau aggregation and reactive oxygen species. Among them, LMDS-1 and -2 reduced caspase-1, caspase-6 and caspase-3 activities and promoted neurite outgrowth, and the effect was significantly reversed by knockdown of TRKB. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in these cells, implying that the neuroprotective effects of LMDS-1/2 are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. Furthermore, LMDS-1/2 demonstrated their ability to quench the intrinsic fluorescence of tryptophan residues within the extracellular domain of TRKB, thereby consolidating their interaction with TRKB. Our results suggest that LMDS-1/2 exert neuroprotection through activating TRKB signaling, and shed light on their potential application in therapeutics of Alzheimer’s disease/tauopathies.
... The cognitive function of presenilin 1 (PS1)/APP transgenic mice is significantly improved by increasing CREB phosphorylation with phosphodiesterase inhibitors [18,19]. In our previous study, a synthetic coumarin-chalcone hybrid LM-031 upregulates CREB and demonstrates the neuroprotective potential to reduce Aβ, tau, expanded polyglutamine (polyQ) aggregation, and oxidative stress [20][21][22]. Here, we investigate three synthetic coumarin-chalcone derivatives made in our laboratory to determine their effects on CREB expression. ...
... The potential of Congo red and coumarin-chalcone derivatives in inhibiting tau misfolding was assessed using the E. coli-derived ΔK280 tau RD protein [21]. In brief, ΔK280 tau RD protein (20 μM) was incubated with the tested 2 ...
... Aβ/Tau Aggregation Inhibition. Coumarin-chalcone derivatives have been shown to upregulate the CREB pathway in 4 Oxidative Medicine and Cellular Longevity AD and polyQ cell models [20][21][22]. Here, we examined three synthetic coumarin-chalcone derivatives LM-016, LM-021, and LM-022 ( Figure 1(a)), all of which fulfilled Lipinski's criteria for oral bioavailability on the basis of molecular weight, hydrogen bond donors, hydrogen bond acceptors, and calculated octanol/water partition coefficient [29] ( Figure S1A). ...
Abnormal accumulations of misfolded Aβ and tau proteins are major components of the hallmark plaques and neurofibrillary tangles in the brains of Alzheimer’s disease (AD) patients. These abnormal protein deposits cause neurodegeneration through a number of proposed mechanisms, including downregulation of the cAMP-response-element (CRE) binding protein 1 (CREB) signaling pathway. Using CRE-GFP reporter cells, we investigated the effects of three coumarin-chalcone derivatives synthesized in our lab on CREB-mediated gene expression. Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells were used to evaluate these agents for possible antiaggregative, antioxidative, and neuroprotective effects. Blood-brain barrier (BBB) penetration was assessed by pharmacokinetic studies in mice. Of the three tested compounds, (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-4-hydroxy-2H-chromen-2-one (LM-021) was observed to increase CREB-mediated gene expression through protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) in CRE-GFP reporter cells. LM-021 exhibited antiaggregative, antioxidative, and neuroprotective effects mediated by the upregulation of CREB phosphorylation and its downstream brain-derived neurotrophic factor and BCL2 apoptosis regulator genes in Aβ-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells. Blockage of the PKA, CaMKII, or ERK pathway counteracted the beneficial effects of LM-021. LM-021 also exhibited good BBB penetration ability, with brain to plasma ratio of 5.3%, in in vivo pharmacokinetic assessment. Our results indicate that LM-021 works as a CREB enhancer to reduce Aβ and tau aggregation and provide neuroprotection. These findings suggest the therapeutic potential of LM-021 in treating AD.
... It has been reported that derivatives of coumarin could prevent misfolded Aβ aggregation [21]. In AD cell and mouse models, synthetic coumarin-chalcone hybrid LM-031 demonstrates neuroprotective potential by regulating CREB and anti-oxidative pathways [22,23]. Coumarin derivative imperatorin also activates BDNF and CREB signaling to improve learning and memory deficits in prenatally stressed rats [24]. ...
... Analogs of coumarins showing pharmacological activities have been described [49]. Coumarin and its derivatives demonstrate their potential in treating AD through several mechanisms such as inhibiting AChE [50] and β-secretase [51], preventing misfolded Aβ aggregation [21], upregulating CREB and anti-oxidative pathways [22,23], and promoting BDNF-TRKB and CREB signaling [24,25]. Here, we found the potential of new coumarin derivatives ZN014 and ZN015 for AD treatment by reducing Aβ aggregation, ROS, caspase-1, and AChE as well as promoting neurite outgrowth (Figures 2 and 3) and TRKB signaling (Figure 4). ...
... AChE-inhibitory activities of coumarin derivatives have been reported [50]. Resembling coumarin and LM-031 [23], ZN015 exhibited inhibitory activity on both AChE ( Figure 3B) and Aβ aggregation ( Figure 1B). ...
Alzheimer’s disease (AD) is a common neurodegenerative disease presenting with progressive memory and cognitive impairments. One of the pathogenic mechanisms of AD is attributed to the aggregation of misfolded amyloid β (Aβ), which induces neurotoxicity by reducing the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TRKB) and increasing oxidative stress, caspase-1, and acetylcholinesterase (AChE) activities. Here, we have found the potential of two novel synthetic coumarin derivatives, ZN014 and ZN015, for the inhibition of Aβ and neuroprotection in SH-SY5Y neuroblastoma cell models for AD. In SH-SY5Y cells expressing the GFP-tagged Aβ-folding reporter, both ZN compounds reduced Aβ aggregation, oxidative stress, activities of caspase-1 and AChE, as well as increased neurite outgrowth. By activating TRKB-mediated extracellular signal-regulated kinase (ERK) and AKT serine/threonine kinase 1 (AKT) signaling, these two ZN compounds also upregulated the cAMP-response-element binding protein (CREB) and its downstream BDNF and anti-apoptotic B-cell lymphoma 2 (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of ZN014 and ZN015. A parallel artificial membrane permeability assay showed that ZN014 and ZN015 could be characterized as blood–brain barrier permeable. Our results suggest ZN014 and ZN015 as novel therapeutic candidates for AD and demonstrate that ZN014 and ZN015 reduce Aβ neurotoxicity via pleiotropic mechanisms.
... Also, long-term exposure to oxidative stress conditions is harmful. It has been shown that chronic oxidative modification of proteins leads to protein aggregation, diminished cellular function, and finally to apoptotic cell death in elderly animal [35]. Glutathione-SH (GSH) antioxidants system is also indispensable for the cellular detoxification of ROS in cells. ...
Excessive reactive oxygen species (ROS) may cause oxidative stress which is involved in aging and in the pathogenesis of various human diseases. Whereas unregulated levels of the ROS may be harmful, regulated basal level of ROS are even necessary to support cellular functions as a second messenger for homeostasis under physiological conditions. Therefore, redox medicine could develop as a new therapeutic concept for human health-benefits. Here, we introduce the involvement of ROS on the crossroads of stemness, senescence, and carcinogenesis in a stem cell and cancer cell biology. Amazingly, the anti-proliferative (APRO) family anti-proliferative proteins characterized by immediate early growth responsive genes may also be involved in the crossroads machinery. The biological functions of APRO proteins (APROs) seem to be quite intricate, however, which might be a key modulator of microRNAs (miRNAs). Given the crucial roles of ROS and APROs for pathophysiological functions, upcoming novel therapeutics should include vigilant modulation of the redox state. Next generation of medicine including regenerative medicine and/or cancer therapy will likely comprise strategies for altering the redox environment with the APROs via the modulation of miRNAs as well as with the regulation of ROS of cells in a sustainable manner.
... Notably, the identified framework is present in some of the already reported Taubased drug candidates currently undergoing clinical trials (e.g., xalsalate and curcumin) ( Figure S3) [69,70], as well as in the natural compounds xanthohumol and licochalcone A that act as Tau aggregation inhibitors [70,71] (Figure S3). The latter chemotype is currently under evaluation on several targets related to neurodegenerative diseases, including monoaminoxidase B, αβ-amyloid and α-sinuclein [72][73][74]. ...
... Notably, the identified framework is present in some of the already reported Tau-based drug candidates currently undergoing clinical trials (e.g., xalsalate and curcumin) ( Figure S3) [69,70], as well as in the natural compounds xanthohumol and licochalcone A that act as Tau aggregation inhibitors [70,71] ( Figure S3). The latter chemotype is currently under evaluation on several targets related to neurodegenerative diseases, including monoaminoxidase B, αβ-amyloid and α-sinuclein [72][73][74]. ...
Tau is a highly soluble protein mainly localized at a cytoplasmic level in the neuronal cells, which plays a crucial role in the regulation of microtubule dynamic stability. Recent studies have demonstrated that several factors, such as hyperphosphorylation or alterations of Tau metabolism, may contribute to the pathological accumulation of protein aggregates, which can result in neuronal death and the onset of a number of neurological disorders called Tauopathies. At present, there are no available therapeutic remedies able to reduce Tau aggregation, nor are there any structural clues or guidelines for the rational identification of compounds preventing the accumulation of protein aggregates. To help identify the structural properties required for anti-Tau aggregation activity, we performed extensive chemoinformatics analyses on a dataset of Tau ligands reported in ChEMBL. The performed analyses allowed us to identify a set of molecular properties that are in common between known active ligands. Moreover, extensive analyses of the fragment composition of reported ligands led to the identification of chemical moieties and fragment combinations prevalent in the more active compounds. Interestingly, many of these fragments were arranged in recurring frameworks, some of which were clearly present in compounds currently under clinical investigation. This work represents the first in-depth chemoinformatics study of the molecular properties, constituting fragments and similarity profiles, of known Tau aggregation inhibitors. The datasets of compounds employed for the analyses, the identified molecular fragments and their combinations are made publicly available as supplementary material.
... Lin et al. [159] studied licochalcone A and five derivatives for their ability to prevent tau misfolding, ROS scavenging and neuroprotection in human cells expressing proaggregant ΔK280 TauRD-DsRed. Among tested compounds, licochalcone A and compound 61 showed reduced tau misfolding and associated ROS. ...
Chalcone [(E)-1,3-diphenyl-2-propene-1-one], a small molecule with α, β unsaturated carbonyl group is a precursor or component of many natural flavonoids and isoflavonoids. It is one of the privileged structures in medicinal chemistry. It possesses a wide range of biological activities encouraging many medicinal chemists to study this scaffold for its usefulness to oncology, infectious diseases, virology and neurodegenerative diseases including Alzheimer’s disease (AD). Small molecular size, convenient and cost-effective synthesis, and flexibility for modifications to modulate lipophilicity suitable for blood brain barrier (BBB) permeability make chalcones a preferred candidate for their therapeutic and diagnostic potential in AD. This review summarizes and highlights the importance of chalcone and its analogs as single target small therapeutic agents, multi-target directed ligands (MTDLs) as well as molecular imaging agents for AD. The information summarized here will guide many medicinal chemist and researchers involved in drug discovery to consider chalcone as a potential scaffold for the development of anti-AD agents including theranostics.
... In the study of Sobolova et al., berberine core was substituted at position 9-O of its aromatic ring region, and these derivatives, especially the top-ranked compound (90, Fig. 18), showed multi-targeted profile inhibiting tau/Ab aggregation, prolyl oligopeptidase, AChE and butyrylcholinesterase [282]. In addition, Lin et al., examined the ability of licochalcone A and its five derivatives in cell assays for inhibiting tau aggregation, oxidation, and providing neuroprotection [283]. Among them, LM-031 (91, Fig. 18) showed high potency in cell assays, reduced tau and Ab levels in the hippocampus and cortex, and rescued cognitive deficits in streptozocininduced hyperglycemic 3 Â Tg-AD mice. ...
... Among them, LM-031 (91, Fig. 18) showed high potency in cell assays, reduced tau and Ab levels in the hippocampus and cortex, and rescued cognitive deficits in streptozocininduced hyperglycemic 3 Â Tg-AD mice. In parallel artificial membrane permeability assay, LM-031 was categorized as the high BBB permeable compound [283]. Now the development of MTDLs is in preclinical stage. ...
Alzheimer's disease (AD) is the most common form of dementia characterized by presence of extra-cellular amyloid plaques and intracellular neurofibrillary tangles composed of tau protein. Currently there are close to 50 million people living with dementia and this figure is expected to increase to 75 million by 2030 putting a huge burden on the economy due to the health care cost. Considering the effects on quality of life of patients and the increasing burden on the economy, there is an enormous need of new disease modifying therapies to tackle this disease. The current therapies are dominated by only symptomatic treatments including cholinesterase inhibitors and N-methyl-D-aspartate receptor blockers but no disease modifying treatments exist so far. After several failed attempts to develop drugs against amyloidopathy, tau targeting approaches have been in the main focus of drug development against AD. After an overview of the tauopathy in AD, this review summarizes recent findings on the development of small molecules as therapeutics targeting tau modification, aggregation, and degradation , and tau-oriented multi-target directed ligands. Overall, this work aims to provide a comprehensive and critical overview of small molecules which are being explored as a lead candidate for discovering drugs against tauopathy in AD.
Licochalcone A (Lico-A) is a flavonoid compound derived from the root of the Glycyrrhiza species, a plant commonly used in traditional Chinese medicine. While the Glycyrrhiza species has shown promise in treating various diseases such as cancer, obesity, and skin diseases due to its active compounds, the investigation of Licochalcone A’s effects on the central nervous system and its potential application in Alzheimer’s disease (AD) treatment have garnered significant interest. Studies have reported the neuroprotective effects of Lico-A, suggesting its potential as a multitarget compound. Lico-A acts as a PTP1B inhibitor, enhancing cognitive activity through the BDNF-TrkB pathway and exhibiting inhibitory effects on microglia activation, which enables mitigation of neuroinflammation. Moreover, Lico-A inhibits c-Jun N-terminal kinase 1, a key enzyme involved in tau phosphorylation, and modulates the brain insulin receptor, which plays a role in cognitive processes. Lico-A also acts as an acetylcholinesterase inhibitor, leading to increased levels of the neurotransmitter acetylcholine (Ach) in the brain. This mechanism enhances cognitive capacity in individuals with AD. Finally, Lico-A has shown the ability to reduce amyloid plaques, a hallmark of AD, and exhibits antioxidant properties by activating the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of antioxidant defense mechanisms. In the present review, we discuss the available findings analyzing the potential of Lico-A as a neuroprotective agent. Continued research on Lico-A holds promise for the development of novel treatments for cognitive disorders and neurodegenerative diseases, including AD. Further investigations into its multitarget action and elucidation of underlying mechanisms will contribute to our understanding of its therapeutic potential.
Decreased BDNF and impaired TRKB signaling contribute to neurodegeneration in Alzheimer's disease (AD). We have shown previously that coumarin derivative LM-031 enhanced CREB/BDNF/BCL2 pathway. In this study we explored if LM-031 analogs LMDS-1 to -4 may act as TRKB agonists to protect SH-SY5Y cells against Aβ toxicity. By docking computation for binding with TRKB using 7,8-DHF as a control, all four LMDS compounds displayed potential of binding to domain d5 of TRKB. In addition, all four LMDS compounds exhibited anti-aggregation and neuroprotective efficacy on SH-SY5Y cells with induced Aβ-GFP expression. Knock-down of TRKB significantly attenuated TRKB downstream signaling and the neurite outgrowth-promoting effects of these LMDS compounds. Among them, LMDS-1 and -2 were further examined for TRKB signaling. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in Aβ-GFP cells, implicating the neuroprotective effects are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. LMDS-1 and -2 are blood-brain barrier permeable as shown by parallel artificial membrane permeability assay. Our results demonstrate how LMDS-1 and -2 are likely to work as TRKB agonists to exert neuroprotection in Aβ cells, which may shed light on the potential application in therapeutics of AD.
