β-amyloid cytotoxicity is prevented by natural achillolide A

Alzheimer’s disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are desperately needed. Key contributors to AD are amyloid beta (Aβ)- and reactive oxygen species (ROS)-induced cytotoxicities. Plant-derived substances have been shown to affect various potential targets in various diseases including AD. Therefore, phytochemicals which can protect neuronal cells against these insults might help in preventing and treating this disease. In the following research, we have isolated the sesquiterpene lactone achillolide A from the plant Achillea fragrantissima and, for the first time, characterized its effects on Aβ-treated neuroblastoma cells. Aβ is a peptide derived from the sequential cleavage of amyloid precursor protein, and is part of the pathogenesis of AD. Our current study aimed to determine whether achillolide A can interfere with Aβ-induced processes in Neuro2a cells, and protect them from its toxicity. Our results show that achillolide A decreased Aβ-induced death and enhanced the viability of Neuro2a cells. In addition, achillolide A reduced the accumulation of Aβ-induced ROS and inhibited the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase and p44/42 mitogen-activated protein kinase in these cells. We therefore suggest that achillolide A may have therapeutic potential for the treatment of AD.


Introduction
Alzheimer's disease (AD) is the most prevalent cause of dementia in adults. Progressive neuronal loss takes place in specific brain areas and causes memory loss, learning difficulty, diminished recall accuracy, impaired problem-solving ability and cognitive deterioration. One of the hallmarks of the disease is the formation of amyloid plaques formed by aggregated β-amyloid (Aβ) peptides. Aβ is a 4-kDa peptide derived from the sequential cleavage of amyloid precursor protein [1] and its oligomeric form is part of the pathogenesis of AD. It is thought to exert its action through different mechanisms, including the induction of reactive oxygen species (ROS) accumulation, microglial activation, and neuronal death [2,3]. Therefore, phytochemicals that can protect neuronal cells from Aβ toxicity and oxidative stress may assist in coping with AD. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are desperately needed. Plant extracts and purified phytochemicals have been shown to affect various potential targets in AD such as hyperphosphorylated tau and acetyl cholinesterase activity [4][5][6][7].
Sesquiterpene lactones isolated from plants have been shown to possess diverse biological activities and to exhibit several effects, such as immunomodulation, anti-inflammatory, antitumor and antimicrobial activities [8][9][10][11][12]. Sesquiterpene lactones have also been reported to have neuroprotective effects in cultured neurons as well as in vivo, using animal stroke models and cocaine consumption studies. Thus, it is reasonable to assume that these compounds or their active metabolites can cross the blood−brain barrier [8,[13][14][15][16]. Based on these activities, sesquiterpene lactones could be promising candidates for the development of drugs for the treatment of neurodegenerative diseases [17][18][19].
We have previously shown that achillolide A, a sesquiterpene lactone that we isolated from Achillea fragrantissima (Forssk) Sch. Bip, downregulated microglial activation [20], prevented hydrogen peroxide (H 2 O 2 )-induced death of astrocytes [21], and protected neuroblastoma cells from glutamate toxicity [22]. Our current study aimed to determine whether achillolide A can interfere with Aβ-induced processes in Neuro2a (N2a) cells and protect them from its toxicity.

Plant material
The aerial parts of Achillea fragrantissima (Forssk) Sch. Bip (Af) were collected in the Arava Valley. Achillolide A (98% pure) was isolated as previously described from the aerial parts of Af [20].

Determination of cell viability
N2a cells were grown and treated as in the cytotoxicity assay. Cell viability was determined by a modification of the crystal violet assay [23], and the optical density was measured at 540 nm with a 690-nm reference filter in a Syn-ergy2 Multi-Detection Microplate Reader (BioTek Instruments, Inc.).

Determination of intracellular ROS levels
N2a cells were grown and re-plated as in the cytotoxicity assay, using 1% instead of 5% FBS. After 24 h the cells were treated with 20 µM DCF-DA for 30 min at 37 °C. Following incubation, the cultures were rinsed with phosphate-buffered saline, and fresh medium was added to the cells. The ROS levels before and after treatment with achillolide A and Aβ were determined according to fluorescence (excitation at 485 nm and emission at 520 nm) in a Synergy2 Multi-Detection Microplate Reader (BioTek Instruments, Inc.).

Statistical analyses
The results were analyzed by one-way ANOVA followed by Tukey-Kramer multiple comparison tests, using the Graph Pad InStat 3 for Windows (GraphPad Software, San Diego, CA, USA).

Results and discussion
Achillolide A (Fig. 1) was previously shown by us to inhibit microglial activation, protect astrocytes from oxidative stress, and protect neuroblastoma cells from glutamate toxicity [22,24]. Aβ 25-35 is a neurotoxic peptide commonly used in cellular models of AD [25,26]. We have previously shown that exposure of N2a neuroblastoma cells to Aβ 25-35 resulted in their death 20 h later [22]. To examine the effect of achillolide A on the toxicity of Aβ, these cells were treated with 25 μM of the Aβ 25-35 peptide together with different concentrations of achillolide A. Viability and cytotoxicity were determined 20 h later. Our results show that achillolide A reduced Aβ 25-35 -induced cell death by 71% at a concentration of 16 nM (Fig. 2a), as observed by the LDH method, and completely rescued viability, as observed by the crystal violet method (Fig. 2b). It should be noted that achillolide A by itself is not cytotoxic to N2a cells that were exposed to different concentrations (up to 1566 nM) of this molecule, as determined using the crystal violet assay (Fig. 2c).  LDH or (b, c) the crystal violet method. Cytotoxicity (a) was significantly reduced in cells treated with Aβ + achillolide A compared to Aβ-treated cells. Likewise, viability (b, c) was significantly increased in cells treated with Aβ + achillolide A compared to Aβ-treated cells. The results are the mean ± SEM of two experiments (n = 16). The maximal LDH release after disruption of the cells by Triton x-100 was A492 = 0.61 ± 0.04 as measured in two experiments (n = 5). ***P < 0.001 One mechanism by which Aβ may exert cell toxicity is the generation of ROS, leading to neuronal death [3]. Treatment of N2a cells with Aβ 25-35 for 20 h results in a two-fold increase in intracellular ROS levels, as we have previously shown [22]. We therefore tested whether achillolide A could inhibit the elevated production of ROS following treatment with Aβ 25-35 and, as a result, protect the cells from Aβ 25-35induced cytotoxicity. To test this possibility, cells were treated with various concentrations of achillolide A at the time of Aβ 25-35 application, and ROS formation was determined 20 h later. Our results show that treatment with achillolide A inhibits 78% of the intracellular levels of Aβ 25-35induced ROS. We observed that 16 nM achillolide A is the lowest effective dose at both attenuating neuronal cell death and reducing the level of ROS production following Aβ 25-35 treatment (Figs. 2, 3).
The antioxidant characteristics of achillolide A demonstrated in this study in Aβ-treated N2a neuroblastoma cells support our previous observations, showing similar effects in glutamate-treated neuroblastoma N2a cells [22], H 2 O 2 -treated astrocytes and LPS-activated microglial cells [20,24]. Although part of Aβ toxicity is mediated by glutamate [27], the mechanism underlying Aβ cytotoxicity is complex and involves many downstream targets (for review see [28]). As neuronal vulnerability in AD originates in the hippocampal formation, future experiments should examine the effect of achillolide A on these neuronal populations in vitro and in vivo.

Conclusions
In this study, we have shown for the first time that achillolide A, a natural sesquiterpene lactone we isolated from A. fragrantissima, can protect N2a cells from Aβ 25-35 -induced cell death. In addition, achillolide A reduced the accumulation of Aβ-induced ROS in N2a cells and inhibited the phosphorylation of SAPK/JNK and p44/42 MAPK in these cells. Based on our results in astrocytes, microglial cells and N2a neuroblastoma cells, it is proposed that achillolide A has neuroprotective therapeutic characteristics. Further studies are warranted in order to substantiate the therapeutic potential of achillolide A for the treatment of AD.