The Antioxidant Activity of Limonene Counteracts Neurotoxicity Triggered byAβ1-42 Oligomers in Primary Cortical Neurons

Many natural-derived compounds, including the essential oils from plants, are investigated to find new potential protective agents in several neurodegenerative disorders such as Alzheimer’s disease (AD). In the present study, we tested the neuroprotective effect of limonene, one of the main components of the genus Citrus, against the neurotoxicity elicited by Aβ1-42 oligomers, currently considered a triggering factor in AD. To this aim, we assessed the acetylcholinesterase activity by Ellman’s colorimetric method, the mitochondrial dehydrogenase activity by MTT assay, the nuclear morphology by Hoechst 33258, the generation of reactive oxygen species (ROS) by DCFH-DA fluorescent dye, and the electrophysiological activity of KV3.4 potassium channel subunits by patch-clamp electrophysiology. Interestingly, the monoterpene limonene showed a specific activity against acetylcholinesterase with an IC50 almost comparable to that of galantamine, used as positive control. Moreover, at the concentration of 10 µg/mL, limonene counteracted the increase of ROS production triggered by Aβ1-42 oligomers, thus preventing the upregulation of KV3.4 activity. This, in turn, prevented cell death in primary cortical neurons, showing an interesting neuroprotective profile against Aβ1-42-induced toxicity. Collectively, the present results showed that the antioxidant properties of the main component of the genus Citrus, limonene, may be useful to prevent neuronal suffering induced by Aβ1-42 oligomers preventing the hyperactivity of KV3.4.


Introduction
Alzheimer's disease (AD) is a complex, multifarious syndrome characterized by the progressive loss of episodic memory and cognitive abilities [1]. Intracellular and extracellular deposits of the amyloid-β (Aβ) peptide play a key role in AD pathology [2]. Accumulating evidence supporting the amyloid cascade hypothesis shows that Aβ oligomers intervene in different pathways leading to AD neurodegeneration including synaptic dysfunction and neuronal network disruption [3]. Besides the well-known neurotoxic role of Aβ per se, the amyloidogenic protein transthyretin intervenes in the aggregation of amyloid fibrils, thus modulating its function overall [4]. At the molecular level, Aβ oligomers affect neuronal and glial cell functions by inducing unregulated production of reactive oxygen species (ROS) and subsequent oxidative stress, aberrant Ca 2+ signaling, abnormal neuronal electrical activity, mitochondrial damage, endoplasmic reticulum (ER) stress, and apoptosis [5][6][7][8][9]. neurons exposed to oligomeric species of the neurotoxic Aβ 1-42 peptide. In particular, we assessed the ability of limonene to counteract the effect of Aβ 1-42 oligomers on neuronal viability, ROS production, and K V 3.4-mediated I A currents.

In Vitro Anti-Acetylcholinesterase Activity
AChE inhibitory activity assay was performed according to a previously described spectrophotometric method [42]. Absorbance was measured at 405 nm in a spectrophotometer (Thermo Scientific Multiskan GO, Monza, Italy). Galantamine was used as positive control and bidistillated water as a negative control. The percentage inhibition of AChE activity was calculated by comparison with the negative control using the following equation: AChE inhibition % = [(A 0 − A 1 )/A 0 ]*100, where A 0 is the absorbance of the control without sample and A 1 is the absorbance of the sample.

Primary Cortical Neurons
Cortical neurons were obtained from brains of 14/16-day-old Wistar rat embryos and dissected as reported previously [44]. Neurons were cultured in a humidified 5% CO 2 atmosphere, and the culture medium was changed every 2 days. For microfluorimetric and electrophysiological studies, cells were seeded on glass coverslips (Fisher, Springfield, NJ, USA) coated with poly-D-lysine and used at least 12 h after seeding. Italian Ministry of Health and the local Animal Care Committee of "Federico II" University of Naples (Italy) approved all animal procedures adopted (D. Lgs. 4th March 2014 from Italian Ministry of Health; DIR 210/63 UE; 12/2018-UT7).

MTT Assay
Mitochondrial dehydrogenase activity was assessed by the MTT assay as previously described [45]. Data are expressed as a percentage of cell viability compared to control cultures.

Assessment of Intracellular ROS Production
Cortical neurons plated on glass coverslips were exposed to limonene in the presence or in absence of Aβ 1-42 oligomers for 24 h. At the end of the treatment, cells were incubated with a physiological solution containing DCFH-DA (17.5 µM) [35,46]. Cells were washed with a stopping solution containing EGTA. Each coverslip was rapidly placed into a perfusion chamber (Medical System, Co. Greenvale, NY, USA) and acquired with the Zeiss Axiovert 200 microscope (Carl Zeiss, Germany) equipped with MicroMax 512BFT cooled CCD camera (Princeton Instruments, Trenton, NJ, USA). Using a 40X objective, each coverslip was exposed at 485-nm excitation for 10 s and the emitted light was passed through a 530-nm barrier filter.

Electrophysiology
K + currents were recorded from primary rat cortical neurons using a commercially available amplifier (Axopatch 200B, Axon Instruments, Union City, CA, USA), as previously described [35,36]. Currents were filtered at 5 kHz and digitized using a Digidata 1322A interface (Molecular Devices, CA, USA). Data were acquired and analyzed using pClamp software (version 9.0, Molecular Devices, CA, USA). The pipette solution contained the following (in mM): 100 K-gluconate, 20 NaCl, 1 Mg-ATP, 0.  (50 nM) and nimodipine (10 µM) were added to extracellular solution to inhibit Na + and Ca 2+ currents. The K + current components (inactivating component I A and delayed-rectifier non-inactivating component I DR ) were discriminated using the appropriated electrophysiological protocols as previously described [35,36]. Possible changes in cell size occurring upon specific pharmacological treatments were calculated by monitoring the capacitance of each cell membrane, which is directly related to membrane surface area, and the current amplitude was expressed as current densities (pA/pF) as previously described [35,36].

Assessment of Nuclear Morphology
Nuclear morphology was studied by Hoechst-33258 as previously described [35] Cells were fixed in 4% paraformaldehyde and then incubated with Hoechst 33258 (1 µg/mL/5 min/37 • C). Images were acquired with a CoolSnap camera (Media Cybernetics Inc, Silver Spring, MD, USA) using the Nikon Eclipse E400 microscope (Nikon, Torrance, CA, USA). Image analysis was performed with the Image-Pro Plus 4.5 software (Media Cybernetics Inc, Silver Spring, MD, USA). A set of 330 nm/450 nm filters was used to detect Hoechst-33258. Pathological nuclei are characterized by chromatin condensation, fragmentation, and decrease in size. Values acquired in all conditions were expressed as percentage of total nuclei.

Statistics
GraphPad Prism 6.02 was used for statistical analyses (GraphPad Software, La Jolla, CA, USA). Data are expressed as the mean ± SEM (Figures 1-4) or mean ± SD ( Figure S1) of the values obtained from individual experiments. Statistical comparisons between groups were performed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls' test; p < 0.05 was considered significant.

Effect of Limonene on Acetylcholinesterase Activity
AChE, the enzyme involved in the hydrolysis of acetylcholine, plays an important role in the neurodegeneration occurring in AD. The AChE inhibitors are currently included in AD therapy since they display efficacy in relieving cognitive symptoms in AD patients [10,11]. Interestingly, limonene showed a significant activity against acetylcholinesterase with a calculated IC 50 of 7.7 µg/mL that was measured in vitro by the Ellman's colorimetric method (Table 1). Of note, the reported IC 50 for limonene activity is almost comparable to that of galantamine, which has been used as positive control (Table 1). Before investigating the possible neuroprotective effect of limonene against Aβ 1-42 -induced neurotoxicity, we assessed mitochondrial dehydrogenase activity of NGF-differentiated PC12 cells exposed to different concentrations of limonene (5, 10, and 25 µg/mL/24 h) in order to exclude any putative cytotoxicity of the compound as well as to identify the appropriate concentration for subsequent experiments. Importantly, NGF-differentiated PC12 cells treated for 24 h with limonene did not display any significant reduction but rather a moderate increase in mitochondrial dehydrogenase activity. In fact, at the concentration of 10 µg/mL and 25 µg/mL respectively, it showed a significant ability to increase mitochondrial dehydrogenase activity ( Figure 1A). Of note, the concentration of 10 µg/mL was very similar to the IC 50 calculated for the inhibition of AChE by galantamine used as positive control (see Table 1). Therefore, the putative neuroprotective effect of 10 µg/ml limonene was investigated in NGF-differentiated PC12 cells and in primary cortical neurons exposed to Aβ 1-42 oligomers (5 µM/24 h) ( Figure 1B,C). In particular, NGF-differentiated PC12 cells and neurons were pre-incubated with 10 µg/mL limonene 30 min before the exposure to Aβ 1-42 oligomers. After 24 h of incubation with Aβ 1-42 oligomers, mitochondrial dehydrogenase activity was assessed. Both NGF-differentiated PC12 cells and primary cortical neurons treated with 5 µM Aβ 1-42 oligomers alone displayed a significant reduction in mitochondrial dehydrogenase activity in comparison to untreated cells. By contrast, the reduction of mitochondrial dehydrogenase activity was prevented in NGF-differentiated PC12 cells and primary cortical neurons pre-treated with 10 µg/mL limonene ( Figure 1B,C). From a transductional point of view, the EO of Citrus medica cv. 'rugosa' containing 67% of limonene [15] produced a significant downregulation of pERK and PKA expression in SH-SY5Y cells ( Figure S1). NGF-differentiated PC12 cells and primary cortical neurons treated with 5 µM Aβ1-42 oligomers alone displayed a significant reduction in mitochondrial dehydrogenase activity in comparison to untreated cells. By contrast, the reduction of mitochondrial dehydrogenase activity was prevented in NGF-differentiated PC12 cells and primary cortical neurons pre-treated with 10 µg/mL limonene ( Figure 1B,C). From a transductional point of view, the EO of Citrus medica cv. 'rugosa' containing 67% of limonene [15] produced a significant downregulation of pERK and PKA expression in SH-SY5Y cells ( Figure S1).

Effect of Limonene on Nuclear Morphology Alteration Induced by Aβ1-42 Oligomers in Primary Cortical Neurons
To further study the neuroprotective effect of limonene against Aβ1-42 toxicity, we also performed labeling experiments with the fluorescent DNA binding dye Hoechst-33258 on primary cortical neurons treated with Aβ1-42 oligomers (5 µM/24 h) in the presence and in absence of limonene. In accordance with the reduction of mitochondrial dehydrogenase activity, nuclear morphological assessment revealed a marked pyknosis, fragmentation, and decrease in size in neurons exposed to Aβ1-42 oligomers compared to untreated neurons (Figures 2 and S2). On the other hand, 30 min of pre-treatment with 10 µg/mL limonene was able to significantly counteract the alteration of nuclear morphology induced by Aβ1-42 oligomers (Figures 2 and S2). Effects of limonene on mitochondrial dehydrogenase activity in NGF-differentiated PC12 cells and primary cortical neurons exposed to Aβ 1-42 oligomers. (A) Evaluation of the mitochondrial dehydrogenase activity by MTT assay in NGF-differentiated PC12 cells exposed to limonene at different concentrations (5, 10, and 25 µg/mL) for 24 h. (B,C) Quantification of mitochondrial dehydrogenase activity assessed by MTT assay in NGF-differentiated PC12 cells (B) and primary cortical neurons (C) exposed to Aβ 1-42 oligomers (5 µM/24 h) in the presence and in absence of limonene (10 µg/mL, 30 min pre-treatment). Data are shown as percentage of mitochondrial dehydrogenase activity (compared to control cells) and values are expressed as mean ± SEM of three independent experimental sessions (* p < 0.05 vs control; ** p < 0.05 vs. Aβ 1-42 ).

Effect of Limonene on Nuclear Morphology Alteration Induced by Aβ 1-42 Oligomers in Primary Cortical Neurons
To further study the neuroprotective effect of limonene against Aβ 1-42 toxicity, we also performed labeling experiments with the fluorescent DNA binding dye Hoechst-33258 on primary cortical neurons treated with Aβ 1-42 oligomers (5 µM/24 h) in the presence and in absence of limonene. In accordance with the reduction of mitochondrial dehydrogenase activity, nuclear morphological assessment revealed a marked pyknosis, fragmentation, and decrease in size in neurons exposed to Aβ 1-42 oligomers compared to untreated neurons ( Figure 2 and Figure S2). On the other hand, 30 min of pre-treatment with 10 µg/mL limonene was able to significantly counteract the alteration of nuclear morphology induced by Aβ 1-42 oligomers (Figure 2 and Figure S2). Antioxidants 2021, 10, x 7 of 15

Effect of Limonene on ROS Production Induced by Aβ1-42 Oligomers in Primary Cortical Neurons
A great amount of studies suggested that oxidative stress associated with increased ROS production may constitute an upstream event in AD pathogenesis. Previous studies by our group showed that Aβ1-42 oligomers at the concentration of 5 µM induce an increase of ROS production that peaks at 3 h and lasts for 24 h in both NGF-differentiated PC12 cells and primary hippocampal neurons [35,36]. Therefore, the generation of ROS was detected by DCFH-DA fluorescent dye in primary cortical neurons exposed to Aβ1-42 oligomers (5 µM/24 h) in the presence and in absence of 10 µg/mL limonene. In line with our previous observations, primary cortical neurons treated with Aβ1-42 oligomers displayed increased DCF-monitored fluorescent intensity indicating a significant increase of ROS production compared with untreated neurons (Figures 3 and S3). Importantly, the pre-treatment with limonene at the concentration of 10 µg/mL prevented the significant over-production of ROS induced by Aβ1-42 oligomers, as indicated by a decrease in DCF-monitored fluorescent intensity compared with Aβ1-42-treated neurons (Figures 3  and S3).

Effect of Limonene on ROS Production Induced by Aβ 1-42 Oligomers in Primary Cortical Neurons
A great amount of studies suggested that oxidative stress associated with increased ROS production may constitute an upstream event in AD pathogenesis. Previous studies by our group showed that Aβ 1-42 oligomers at the concentration of 5 µM induce an increase of ROS production that peaks at 3 h and lasts for 24 h in both NGF-differentiated PC12 cells and primary hippocampal neurons [35,36]. Therefore, the generation of ROS was detected by DCFH-DA fluorescent dye in primary cortical neurons exposed to Aβ 1-42 oligomers (5 µM/24 h) in the presence and in absence of 10 µg/mL limonene. In line with our previous observations, primary cortical neurons treated with Aβ 1-42 oligomers displayed increased DCF-monitored fluorescent intensity indicating a significant increase of ROS production compared with untreated neurons (Figure 3 and Figure S3). Importantly, the pre-treatment with limonene at the concentration of 10 µg/mL prevented the significant over-production of ROS induced by Aβ 1-42 oligomers, as indicated by a decrease in DCFmonitored fluorescent intensity compared with Aβ 1-42 -treated neurons (Figure 3 and Figure S3). Antioxidants 2021, 10, x 8 of 15

Effect of Limonene on the Upregulation of Fast-Inactivating IA Currents Triggered by Aβ1-42 Oligomers in Primary Cortical Neurons
Previously, we provided evidence that Aβ1-42 oligomers induced a selective up-regulation of KV3.4 channels through the ROS-dependent activation of the transcription factor NF-kB [36] and that the subsequent increase of K + efflux was involved in neuronal and astrocytic damage [32,36,40]. Since the blockade of KV3.4 appeared to be an effective strategy to counteract Aβ1-42-mediated caspase-3 overactivation [38,39], we here tested the hypothesis that limonene could prevent the ROS-dependent up-regulation of fast-inactivating IA currents mediated by KV3.4 in primary cortical neurons exposed to Aβ1-42 oligomers. First, we performed patch-clamp experiments in primary cortical neurons treated with Aβ1-42 oligomers (5 µM/24 h) to assess fast-inactivating IA current amplitude carried by KV3.4 channels. In line with our previous reports, patch-clamp experiments revealed that Aβ1-42 oligomers were able to markedly enhance IA density. On the other hand, we found that pre-treatment with 10 µg/mL limonene largely prevented the increase of fast-inactivating IA currents induced by Aβ1-42 oligomers (Figure 4).

Effect of Limonene on the Upregulation of Fast-Inactivating I A Currents Triggered by Aβ 1-42 Oligomers in Primary Cortical Neurons
Previously, we provided evidence that Aβ 1-42 oligomers induced a selective upregulation of K V 3.4 channels through the ROS-dependent activation of the transcription factor NF-kB [36] and that the subsequent increase of K + efflux was involved in neuronal and astrocytic damage [32,36,40]. Since the blockade of K V 3.4 appeared to be an effective strategy to counteract Aβ 1-42 -mediated caspase-3 overactivation [38,39], we here tested the hypothesis that limonene could prevent the ROS-dependent up-regulation of fastinactivating I A currents mediated by K V 3.4 in primary cortical neurons exposed to Aβ 1-42 oligomers. First, we performed patch-clamp experiments in primary cortical neurons treated with Aβ 1-42 oligomers (5 µM/24 h) to assess fast-inactivating I A current amplitude carried by K V 3.4 channels. In line with our previous reports, patch-clamp experiments revealed that Aβ 1-42 oligomers were able to markedly enhance I A density. On the other hand, we found that pre-treatment with 10 µg/mL limonene largely prevented the increase of fast-inactivating I A currents induced by Aβ 1-42 oligomers (Figure 4).

Discussion
In the present study, we evaluated the neuroprotective effects of the monoterpene limonene, the main constituent of plants from Citrus genus, against AD in an in vitro model of the disease represented by primary cortical neurons exposed to Aβ1-42 oligomers. The results obtained suggested that limonene was able to protect primary cortical neurons from cell damage induced by Aβ1-42 oligomers by preventing ROS production and KV3.4 channel hyperfunction (Scheme 1).

Discussion
In the present study, we evaluated the neuroprotective effects of the monoterpene limonene, the main constituent of plants from Citrus genus, against AD in an in vitro model of the disease represented by primary cortical neurons exposed to Aβ 1-42 oligomers. The results obtained suggested that limonene was able to protect primary cortical neurons from cell damage induced by Aβ 1-42 oligomers by preventing ROS production and K V 3.4 channel hyperfunction (Scheme 1).
Molecularly, limonene showed a specific activity against acetylcholinesterase almost comparable to galantamine, a well-known drug used in AD therapy. Moreover, limonene was able to prevent Aβ 1-42 oligomer-induced decrease in mitochondrial dehydrogenase activity and increase in ROS production, thus exerting a neuroprotective effect in primary cortical neurons exposed to Aβ 1-42 oligomers. Our findings are in accordance with a previous in vivo study showing that limonene may exert a neuroprotective effect against the toxicity of Aβ 1-42 in a Drosophila AD model through a strong antioxidant action [26].
In the present study we showed that limonene, acting on ROS production, prevented the K V 3.4 current enhancement induced by Aβ 1-42 oligomers. Of note, the increase in ROS level observed in AD is recognized to be an early biochemical event leading to the enhancement of K V 3.4 currents induced by Aβ 1-42 oligomers [32,36,40]. Therefore, consistent with our previous results, we hypothesized that a marked increase in ROS levels observed here may produce the upregulation of K V 3.4 activity also in primary cortical neurons. This result bears striking homology with previous data showing that the antioxidant action of Vitamin E is able to prevent the upregulation of K V 3.4 channel activity induced by Aβ 1-42 oligomers in neurons [35]. Moreover, it has been shown that the increased expression and function of K V 3.4 following Aβ 1-42 oligomers exposure are critically dependent on Ca 2+ -induced increase in ROS production, which in turn prompts K V 3.4 transcriptional activation through a nuclear factor κB-dependent (NF-κB) pathway [35,36]. Remarkably, NF-κB was the first transcription factor shown to be redox-regulated [47,48]. Molecularly, limonene showed a specific activity against acetylcholinesterase almost comparable to galantamine, a well-known drug used in AD therapy. Moreover, limonene was able to prevent Aβ1-42 oligomer-induced decrease in mitochondrial dehydrogenase activity and increase in ROS production, thus exerting a neuroprotective effect in primary cortical neurons exposed to Aβ1-42 oligomers. Our findings are in accordance with a previous in vivo study showing that limonene may exert a neuroprotective effect against the toxicity of Aβ1-42 in a Drosophila AD model through a strong antioxidant action [26].
In the present study we showed that limonene, acting on ROS production, prevented the KV3.4 current enhancement induced by Aβ1-42 oligomers. Of note, the increase in ROS level observed in AD is recognized to be an early biochemical event leading to the enhancement of KV3.4 currents induced by Aβ1-42 oligomers [32,36,40]. Therefore, consistent with our previous results, we hypothesized that a marked increase in ROS levels observed here may produce the upregulation of KV3.4 activity also in primary cortical neurons. This result bears striking homology with previous data showing that the antioxidant action of Vitamin E is able to prevent the upregulation of KV3.4 channel activity induced by Aβ1-42 oligomers in neurons [35]. Moreover, it has been shown that the increased expression and function of KV3.4 following Aβ1-42 oligomers exposure are critically dependent on Ca 2+ -induced increase in ROS production, which in turn prompts KV3.4 transcriptional activation through a nuclear factor κB-dependent (NF-κB) pathway [35,36]. Remarkably, NF-κB was the first transcription factor shown to be redox-regulated [47,48].
Of note, limonene is able to decrease NF-κB nuclear activation via AMP-activated protein kinase phosphorylation [49]. Therefore, the blockade of ROS-induced NF-κB activation could be involved in the neuroprotective mechanism elicited by limonene. However, a direct ROS scavenging action of the natural compound in the present AD model cannot be ruled out. Scheme 1. Scheme of the putative mechanism of limonene action in cortical neurons exposed to Aβ 1-42 oligomers. Of note, limonene is able to decrease NF-κB nuclear activation via AMP-activated protein kinase phosphorylation [49]. Therefore, the blockade of ROS-induced NF-κB activation could be involved in the neuroprotective mechanism elicited by limonene. However, a direct ROS scavenging action of the natural compound in the present AD model cannot be ruled out.
On the other hand, the important involvement of K V 3.4 channels in the Aβ 1-42 neurotoxicity is further supported by the results showing that BDS-I, a K V 3.4 blocker [50], may exert a potent neuroprotective action both in AD neurons and astrocytes exposed to Aβ 1-42 oligomers [36,40].
In respect to the effect of limonene on ROS production, these species seem to play a relevant role in the neurotoxic cascade of Aβ 1-42 . In fact, the transient influx of Ca 2+ ions induced by Aβ 1-42 oligomers may trigger intracellular cascades that lead not only to increased levels of ROS but also to simultaneous mitochondrial functional impairment characterized by activation of the permeability transition pore in the inner mitochondrial membrane, cytochrome c release, and depletion of ATP [35]. In this regard, it has been demonstrated that the blockade of K V 3.4 may inhibit MPP+-induced cytochrome c release from the mitochondrial intermembrane space to the cytosol and mitochondrial membrane potential depolarization [41].
Another important neuroprotective process modulated by limonene is autophagy. Interestingly, limonene stimulates the autophagic flux through a rapid ERK activation [51]. Of note, the pharmacological inhibition of Kv3.4 by BDS-I counteracts intracellular pH regulation and ERK activation in A549 cells [52], thus further supporting the transduction modulation of K V 3.4 channel by limonene. Besides a plethora of functions mediated at cellular and subcellular level, the ERK1/2 transduction element may negatively regulate the expression of β-secretase, the proteolytical enzyme mainly involved in the production of the neurotoxic Aβ 1-42 peptide [53]. On the other hand, several studies provide direct evidence on the possible involvement of MAP kinase pathway in the hyper-phosphorylation of tau underlining the role played by ERK1/2 activation in the Aβ 1-42 deposition during AD [54]. In addition, targeting ERK1/2 activity may slow tau spreading in sporadic AD thus offering a new putative neuroprotective strategy in the major form of AD [55]. In accordance to the latter study, our preliminary data ( Figure S1) suggested that the EO Citrus medica cv rugosa, containing high levels of limonene, reduced ERK1/2 activation.
Another aspect that deserves attention is the putative clinical relevance of the present data. In fact, the use of essential oil containing limonene, or limonene alone, would be desirable in the therapy of AD symptoms, which is in line with our results showing the antioxidant properties of the natural compound and considering its ability to counteract Aβ 1-42 -induced K V 3.4 hyperfunctionality in cortical neurons. Of course, our in vitro results should be reproduced in vivo to prove not only the efficacy of the treatment in a more complex model of the disease but also to define the pharmacokinetic profile of limonene. However, in line with our in vitro data, a recent manuscript shows a significant cognitiveenhancing effect of essential oil containing limonene in a scopolamine-induced amnesia model [56]. Interestingly, the authors correlate this therapeutic effect to the essential oil ability in inhibiting acetyl/butirrylcholinesterase activities [56]. Although our results are in line with this recent manuscript, we additionally demonstrated that limonene alone may exert the same AChE inhibitory activity than the essential oil containing other components. However, it would be desirable to go even further by performing in vivo experiments in AD transgenic mice to set up a therapeutic window of limonene and to study its protective effect in a more complex model.
Considering that ROS-mediated K V 3.4 overexpression may intervene in both neurodegeneration and neuroinflammation underlying AD development [29,32,[35][36][37][38][39][40], limonene may assume a novel neuroprotective meaning. Therefore, limonene, controlling the modulation of K V 3.4 channels in AD brain via ROS production, might represent a novel therapeutic approach for slowing down the progression of the disease. Therefore, after an accurate examination of the molecular pathway involved in its mechanism, the modification of limonene to serve as prominent scaffold in designing novel bioactive compounds should be taken into consideration as a new potential avenue in AD intervention.

Conclusions
In this manuscript we showed that limonene exerts a novel neuroprotective effect in AD. In particular, limonene, controlling the modulation of K V 3.4 channels via ROS level reduction, might represent a novel therapeutic approach for slowing down the progression of the disease. Moreover, limonene displays a specific activity against AChE almost comparable to galantamine, a well-known drug used in AD therapy. In this respect, the involvement of AChE metabolic activity in Aβ fibril formation is considered one of the most interesting future perspectives in AD therapy. For instance, AChE activity has been mainly associated with the amyloid core of senile plaques in the brain of AD patients [57,58]. Moreover, AChE activity increases and accelerates the aggregation of Aβ [59], as detected by thioflavin-T fluorescence assay [60]. Consequently, AChE inhibitors, such as donepezil and tacrine, reduce Aβ aggregation thus showing a certain therapeutic potential in AD [61]. Another important consideration is that limonene may exert a neuroprotective effect against Aβ toxicity through several molecular mechanisms including the inhibition of AChE, the antioxidant activity, the inhibition of K V 3.4 hyperfunction and the downregulation of pERK. These mechanisms are not all simultaneously shared by the other AChE inhibitors. In fact, among the most studied drugs, tacrine and donepezil are the only two therapeutic compounds displaying some of the mechanisms displayed by limonene [61]. Therefore, limonene could represent an interesting multi-target molecule useful to design novel bioactive compounds slowing down AD progression.