Amyloid β -but not Tau-induced neurotoxicity is suppressed by Manuka honey via HSP-16.2 and SKN-1/Nrf2 pathways in an in vivo model of Alzheimer ’ s disease

Alzheimer ’ s is a chronic degenerative disease of the central nervous system considered the leading cause of dementia in the world. It is characterized by two etiopathological events related to oxidative stress: the aggregation of β -amyloid peptide and the formation of neuro ﬁ brillary tangles of hyperphosphorylated Tau protein in the brain. The incidence of this disease increases with age and has been associated with inadequate lifestyles. Some natural compounds have been shown to improve the hallmarks of the disease. However, despite its potential, there is no scienti ﬁ c evidence about Manuka honey (MH) in this regard. In the present work we evaluated the e ﬀ ect of MH on the toxicity induced by A β aggregation and Tau in a Caenorhabditis elegans model. Our results demonstrated that MH was able to improve indicators of oxidative stress and delayed A β -induced paralysis in the AD model CL4176 through HSP-16.2 and SKN-1/ NRF2 pathways. Nevertheless, its sugar content impaired the indicators of locomotion (an indicator of tau neurotoxicity) in both the transgenic strain BR5706 and in the wild-type


Introduction
][3] These modifications are accompanied by neuronal loss 2,4 along with synaptic and brain tissue damage 2,5 resulting in memory impairment, 1,6 depression, disorientation, poor judgment and, in later stages, speaking and walking difficulties. 5It is estimated that AD causes between 60-80% of all dementia cases, 5 representing an important economic burden worldwide. 4Currently, most of the approved drugs for the treatment of the illness only alleviate symptoms, but do not modify the underlying biology of Alzheimer's or alter the course of the disease.In addition, most of these drugs usually are accompanied by side effects including nausea, headache, 5 insomnia and hepatotoxicity. 3Only one of the FDA-approved drugs, aducanumab, can reduce Aβ plaques in the brain. 5ence, the search for new drug candidates to fight AD is still an attractive research area.In the last years, several studies have shown that bioactive compounds from natural products possess neuroprotective properties and are capable of relieving AD symptoms, indicating that natural source-based drugs could be a valid alternative in this therapeutic area. 3,4,71][12] However, to the best of our knowledge, there is no evidence on its neuroprotective potential.
On the other hand, the nematode Caenorhabditis elegans (C.elegans), due to its short lifespan and its limited number of neurons and tractable genetics, represents an ideal system to study aging and age-related diseases, such as AD. 1,2,13ikewise, there are multiple transgenic strains, specifically designed to examine the Aβ plaque accumulation and tanglecaused neurotoxicity. 2,6,13n the present study, we investigated the effects of MH on Aβ aggregation and tau neurotoxicity in C. elegans AD models.

Honey samples and reagents
MH was bought in a local supermarket in Spain after importation into Europe by the Comptoirs et Compagnies.All reagents were purchased from Sigma-Aldrich (St Louis, Missouri, USA), Merck (Darmstadt, Germany), Thermo Fisher (Waltham, Massachusetts, USA) or Roche (Basel, Switzerland).

MH characterization
2.2.1 Total phenolic compounds (TPC) and total flavonoids content (TFC) determination.TPC of MH were determined by the Folin-Ciocalteu method, 14 while its flavonoid content was evaluated according to the protocol described by Navarro-Hortal et al. 15 2.2.2Total antioxidant capacity (TAC).6][17] In all cases, the absorbance was measured in a Neo2 microplate reader (Biotek, Winooski, Vermont, U.S.A.) and the results were expressed as μM of Trolox equivalents (TE) per gram (g) of honey.
2.2.3 Extraction, identification, and quantification of phenolic compounds.The extraction of phenolic compounds was performed according to Afrin et al. 11 For their identification a SYNAPT G2 HDMS Q-TOF system (Waters, Mildford, USA) equipped with a dual electrospray ionization probe was used, following the protocol reported by Esteban-Muñoz et al. 18 UPLC separation was performed using an ACQUITY UPLC ™ HSS T3 2.1 × 100 mm, 1.8 mm column and the program for chromatography was set with a binary gradient consisting of (A) water with 0.5% acetic acid and (B) acetonitrile, as follows: initial 0.0 min 5% (B), 15.0 min 95% (B); 15.1 min 5% (B); and 18.0 min 5% (B) and the flow rate was 0.4 mL min −1 .Phenolic compounds were identified through the MassLynx V4 software (Waters Laboratory Informatics, Mildford, USA) by comparing the ions and molecular fragments obtained with previous data.
The quantification was carried out by an ACQUITY UPLC I-Class System (Waters, Mississauga, ON, Canada) equipped with a mass spectrometer Waters XEVO TQ-XS with ionization performed by UniSpray (US), according to the method described by Sánchez-Hernández et al. 19 A gradient consisting of (A) water and (B) methanol with 0.1% [v/v] acetic acid for 25 min at a flow rate of 0.4 mL min −1 was set as follows: 0.0 min 5% B, 15-15.10min 95% B and 15.10-25 min 5% B. An ACQUITY UPLC ™ HSS T3 1.8 mm column was used.Quantification of the phenolic compounds was achieved by comparing the retention times of peaks and fragmentation data obtained, with those of the phenolic compound standards.Measurements in Multiple reaction monitoring was used.To obtain age-synchronized nematodes, the bleaching protocol was used.Briefly, gravid hermaphrodites were exposed to an alkaline bleach solution (0.5 N NaOH in 20% NaOCl) and vigorously stirred for six minutes followed by centrifugation at 1280g × 2 minutes.Settled eggs were then resuspended and washed for three times with M9 medium.After that, eggs were seeded on fresh NGM agar plates with a lawn of E. coli OP50 and allowed to hatch.Worms were allowed to mature until the desired larval stage, changing the plates every 2-3 days.

Lethality test
To assess acute lethality, N2 Bristol worms synchronized at L4 larval stage were placed on NGM plates containing 0, 25, 50, 100, 200 or 300 mg mL −1 of MH for 24 h at 20 °C in the absence of E. coli OP50.After that time, nematodes were scored as live or dead by using a Motic dissecting microscope (Motic Inc., Ltd, Hong Kong, China).Worms that did not respond to repeated stimuli inflicted with a platinum wire were considered dead.At least 30 worms were used per group and the experiment was repeated at least three times.Results were expressed as survival rate.For the subsequent experiments a non-lethal submaximal concentration (100 mg mL −1 ) of MH was used.

Pharyngeal pumping assay and growth evaluation
Egg-synchronized N2 worms were placed on NGM plates seeded with E. coli OP50 and containing or not 100 mg mL −1 MH and then incubated at 20 °C.Ninety-six hours later, worms were transferred to fresh plates and the number of contractions per minute of the pharynx terminal bulb was counted using a Motic microscope (Motic Inc.Ltd Hong Kong, China).At the end, worms were collected by washing with M9 medium and analyzed through a MultiRange large particle flow cytometer Biosorter (Union Biometrica.Massachusetts, USA) to determine the time of flight (TOF) which is indicative of the length.At least 10 worms were used per group for pharyngeal pump counting and about 100 nematodes to determine the size.Both experiments were performed in triplicate.

Eggs fertility assay
L4 stage synchronized N2 worms were individually placed in 24 well NGM plates seeded with E. coli OP50 and containing or not 100 mg mL −1 MH.After 24 h at 20 °C, the laid eggs were counted by using a Motic dissecting microscope (Motic Inc., Ltd, Hong Kong, China) and the worms were moved to a fresh plate to distinguish from the offspring.This procedure was repeated every single day until egg-laying stopped.At least 15 worms were used per group and the experiment was repeated at least three times.Results were expressed as the mean of the total number of eggs per group.

Oxidative stress resistance assays
To assess oxidative stress resistance, the intracellular reactive oxygen species (ROS) levels in L4 stage-synchronized N2 worms were measured by using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay, as previously described by Navarro-Hortal et al. 15 Briefly, eggs-synchronized embryos were placed on NGM plates seeded with E. coli OP50 containing or not 100 mg mL −1 MH for 48 h at 20 °C.Next, young-adult worms were collected and washed three times with M9 medium in order to remove bacteria and were exposed (except control group) to 2.5 μM AAPH (2,2′-azobis-2-amidinopropane dihydrochloride) for 15 min to induce oxidative stress.Worms were further washed with M9 medium and directly incubated with the fluorogenic dye (final concentration, 25 μM) for 2 h.The fluorescence was measured by a MultiRange large particle flow cytometer Biosorter (Excitation wavelength 490 nm, emission wavelength 510 nm) (Union Biometrica.Massachusetts, USA).Results were expressed as the mean of the fluorescence intensity normalized by TOF.At least 200 nematodes were used per group and the experiments were repeated in triplicate.

Paralysis test
Transgenic strain CL4176 and the non-paralyzable control strain CL802 were synchronized as described above in the maintenance conditions section and placed on NGM plates seeded with E. coli OP50 containing or not 100 mg mL −1 MH.Worms were incubated for 48 h at 16 °C and then transferred to a 25 °C incubator to stimulate the Aβ-induced paralysis.After 20 h, 25 nematodes of each group were picked to fresh NGM plates (maintaining temperature conditions) and scored for paralysis every 2 h until 32 h after the temperature rise.Nematodes were considered paralyzed when they did not respond to repeated stimuli inflicted with a platinum wire or if they only moved the head without showing a full body wave.Results were expressed as percentage of non-paralyzed worms.The experiment was performed in triplicate.

β-Amyloid aggregates visualization
Worms of the paralyzable strain CL4176 and the negative control strain CL802 were grown as described above for the paralysis assay.After 26 h of incubation at 16 °C, worms were fixed with a 4% paraformaldehyde/M9 medium solution ( pH 7.4) for 24 h and subsequently permeabilized with a solution containing 5% fresh β-mercaptoethanol, 125 mM Tris and 1% Triton X-100 ( pH 7.4) for another 24 h at 37 °C.Aβ-aggregates were stained with 0.125% Thioflavin T (Sigma-Aldrich, St Louis, MI, USA) in 50% ethanol for 30 min and were observed by using a Nikon epi-fluorescence microscope (Eclipse Ni, Nikon, Tokyo, Japan).About 20-30 images per group were acquired at 40× magnification using the GFP filter with a Nikon DS-Ri2 camera (Tokyo, Japan).

RNA Interference (RNAi) Assay
Superoxide dismutase (SOD)-2 and 3, heat shock protein 16.2 (HSP-16.2),transcription factor skinhead-1 (SKN-1) and dauer formation (DAF-16) gene expression was knocked down by feeding CL4176 worms with E. coli strain HT115 carrying the specific dsRNA.Briefly, L3/L4 stage-synchronized CL4176 worms were transferred to RNAi plates containing 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 25 μg mL −1 carbenicillin and incubated for 48-72 h.Once they reached the fertile age, another synchronization was carried out and the eggs/embryos obtained were placed on fresh RNAi plates containing or not 100 mg mL −1 MH and were allowed to mature to L4 young larvae.The resulting adult worms were used for the paralysis assay as previously described.

Green fluorescence protein (GFP)-reporter transgenic strains
Egg-synchronized worms of the transgenic strains CF1553, CL2166, LD1, OS3062, TJ375, and TJ356 expressing the GFP reporter SOD-3, glutathione S-transferase 4 (GST-4), SKN-1, heat-shock transcription factor 1 (HSF-1), HSP-16.2 and DAF-16, respectively, were grown in NGM plates seeded with E. coli OP50 containing or not 100 mg mL −1 MH for 48 h at 20 °C.Next, 20-30 worms per group were immobilized with sodium azide (1 mM) on a slide and subjected to imaging using a Nikon epi-fluorescence microscope (Eclipse Ni, Nikon, Tokyo, Japan).The total GFP fluorescence was analyzed using the NIS-Elements BR software (Nikon, Tokyo, Japan).The subcellular DAF-16 distribution in the TJ356 strain was analyzed by classifying the expression/location patterns into three categories: cytosolic, intermediate, and nuclear; each of which was assigned a value (1, 2, 3) to carry out a semi-quantification.The experiments were repeated at least three times.

Locomotion behavior analysis
Egg-synchronized embryos of wild type-N2 and BR5706 strains were placed on NGM plates seeded with E. coli OP50 contain-ing or not different treatments (100 mg mL −1 MH, 100 mg mL −1 artificial honey (AH) or 232 µg mL −1 of MH hydroalcoholic extract (ME) for 72 h at 20 °C.Next, about 20-30 worms per group were forced to swim by placing them in a drop of M9 medium in the center of a slide.Worms movement was tracked, recorded and analyzed by using a WormLab Imaging System (MBF Bioscience, Williston, Vermont, EE.UU).The wavelength, swimming speed and activity were analyzed as representative parameters of locomotive behavior.The experiment was carried out at least in triplicate.

Statistical analysis
Statistical analysis was performed by SPSS 24.0 software (IBM, Armonk, NY, USA).Data were subjected to one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) post hoc test to compare statistical difference among multiple groups or to t-student test for pairwise data sets.A p < 0.05 was considered statistically significant.Data are expressed as mean ± SEM from at least three independent experiments.

Short term toxicity of MH
The short-term toxicity of MH in C. elegans was evaluated through different assays, including 24 h lethality, pharyngeal pumping, egg-laying (fertility) and growth.As shown in Fig. 1A, no statistically significant differences ( p < 0.05) were found between the control group and worms treated with increasing concentrations (up to 300 mg mL −1 ) of MH in terms of survival percentage.Likewise, MH supplementation did not affect the number of pumps per minute or the worm's fertility, understood as their spawning capacity.However, worms treated with 100 mg mL −1 MH were slightly smaller ( p < 0.05) (Fig. 1B).

Effects of MH against AAPH-induced oxidative stress
An escalation in endogenous ROS levels accelerates the aging process and, in turn, the nematodes ability to deal with harmful stimuli decreases drastically over time.To evaluate the effects of MH against AAPH-induced stress, we measured intracellular ROS levels by DCF-DA method.As shown in Fig. 2, nematodes treated with MH presented a greater resistance against these stress conditions.MH completely prevented the increase in ROS observed in the stressed group as no significant difference (p < 0.05) was found compared to the control.

Effects of MH against Aβ-induced paralysis
Aβ aggregation is considered one of the major pathological causes of AD.To evaluate whether MH ameliorated the Aβ-induced toxicity, we used the transgenic C. elegans strain CL4176, which expresses the human Aβ peptide in body wall muscle cells following temperature upshift from 16 to 25 °C, resulting in a paralysis phenotype.As shown in Fig. 3A, nontreated CL4176 worms exhibited almost complete paralysis after 34 h of the temperature upshift, while 100 mg mL −1 MH treatment significantly ( p < 0.05) delayed this process.In the MH-treated group, no significant differences ( p < 0.05) were observed with respect to the non-paralyzable strain CL802 (negative control) up to 30 h after temperature upshift.At the end point of the analysis, the paralyzed worms represented only 25% of the entire population.In addition, MH significantly reduced Aβ deposits in the tail regions of CL4176 worms as evidenced by thioflavin T staining (Fig. 3B).
To further elucidate the molecular mechanisms underlying the protective effects of MH on Aβ toxicity, we repeated the paralysis test in CL4176 worms silencing some genes by RNAi, including sod-2 and sod-3, hsp-16.2,skn-1, and daf-16.34 h after the temperature upshift, RNAi for sod-3 and daf-16 had no effect on the percentage of non-paralyzed worms compared to the

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Food & Function

Effects of MH on Tau Proteotoxicity
Along with Aβ plaques, the presence of tau protein aggregates is another hallmark of AD.Although the mechanisms of tauassociated neurodegeneration remain unclear, aggregation of this protein is thought to be toxic to neurons.Therefore, the effects of MH on the transgenic strain BR5706 were also evaluated.BR5706 is characterized by accelerated aggregation of insoluble Tau, resulting in severe developmental defects of the nervous system, impaired locomotion, and slowed growth.Worms treated with 100 mg mL −1 MH showed a significant decrease ( p < 0.05) in swimming speed (by 70.76%), wavelength (by 21.68%) and activity (by 39.32%) compared to the control group (Fig. 6A and B).Trying to find an explanation for these results, the contribution of sugars to the assayed parameters was evaluated.For that, two additional experimental groups were included: (i) worms treated with 232 µg mL −1 of a hydroalcoholic extract (ME) only containing the phenolic compounds of MH and (ii) worms treated with 100 mg mL −1 of an artificial honey (AH).AH was prepared by dissolving 22.0 g of maltose, 4.4 g of sucrose, 98.5 g glucose and 119.1 g of fructose in 50 mL of de-ionized water. 9As it can be appreciated in Fig. 6A and B, no significant differences ( p < 0.05) were found between the ME-treated group and the control group, while AH addition showed the same worsening effects as MH, leading to 39.43-, 71.21-and 70.51-fold diminution of the swimming speed, wavelength, and activity, respectively, compared to the control.
To further determine whether the observed effects were directly related to Tau protein aggregation, the mobility assay was performed in the wild type N2 strain (Fig. 7A and B).Also in this case, MH decreased swimming speed, wavelength, and activity by 54.92, 28.96 and 22.32%, respectively, compared to non-treated nematodes.Considering these data, it can be suggested that the sugar content of MH affects the general mobility of C. elegans and not necessarily the aggregation of the Tau protein.However, future analyzes are needed to corroborate this hypothesis.

Discussion
Despite the fact that MH is a natural product with remarkable biological properties both in vitro [8][9][10][11][36][37][38] and in vivo, 39,40 little is known about its neuroprotective potential. Consiering its ability to scavenge free radicals and the implication of oxidative stress in the pathogenesis of age-related diseases such as AD, we decided to investigate the possible effects of MH in alleviating Aβ and Tau-induced toxicity in C. elegans models of AD.
The qualitative profile of MH phenolic compounds determined by UPLC-TOF-MS/MS was similar to that found by other authors. 8,9,11,41,42Nevertheless, we were unable to quantify some of the most representative compounds reported for this honey, such as syringic acid, 11 methyl syringate, 9,41,42 pinocembrin and pinobanksin. 8,42It is well known that the extraction, conservation, and detection methods may influence these results.Regarding the TPC and TFC, the values obtained (0.47 ± 0.03 mg GAE g −1 and 0.05 ± 0.01 mg CAE g −1 , respectively) were slightly lower than those reported by Y.-Z.Zhang et al., 43 Gośliński et al., 44 Marshall et al., 45 and Anand et al. 46 However, the TAC values (2.65 ± 0.10 μM TE g −1 by FRAP; 1.40 ± 0.13 μM TE g −1 by TEAC and 4.58 ± 0.22 μM TE g −1 by DPPH) were quite similar to those reported by Sadia Afrin et al. 10 and Marshall et al. 45 Besides that, 100 mg mL −1 MH did not show short term toxicity in the C. elegans nematodes as demonstrated by the results of the lethality, pharyngeal pumping and egg-laying (fertility) assays.Next, we also evaluated the effects of MH against AAPH-induced oxidative stress, since multiple lines of evidence indicate that ROS overproduction and consequently, an oxidative stress escalation, not only contributes to the aging process 4,47 but is also one of the main factors involved in the initiation and progression of neurodegenerative diseases. 48In the particular case of AD, oxidative stress is related to Aβ aggregation 4,15 and the phosphorylation and polymerization of Tau, two hallmarks of the disease. 4Here, we demonstrated that MH increased resistance to stress conditions as revealed by the DCF-DA method.In similar studies, echinacoside, a phenylethanoid glycoside from Cistanche deserticola, 47 carnosic acid, 3 cannabidiol, 48 gennianchun, a Chinese herbal formula 7 and caffeic acid 4 reduced ROS levels in the wild-type C. elegans strain.
In addition to the ROS theory of AD, there are multiple hypotheses about AD etiology, including the (i) tau protein hyperphosphorylation hypothesis, (ii) mitochondrial function hypothesis, (iii) cholinergic hypothesis and (iv) amyloid cascade hypothesis. 3Although the topic is the subject of debate, the amyloid cascade hypothesis, which suggests that Aβ deposition triggers a series of events (microglial activation, inflammatory response, reactive astrocytosis) that lead to neuronal dysfunction, is the most widely accepted. 3,7,49,50onsequently, alleviating Aβ accumulation has been considered one of the most promising therapeutic approaches for AD. 48To evaluate whether MH was able to delay the Aβ-induced neurotoxicity, we conducted a paralysis test in the transgenic strain CL4176, which has been widely used to study this process because of its ability to express human Aβ peptide in nematode body wall muscle and develop a progressive and reproducible paralysis phenotype. 2,6,13,51Our results showed that MH significantly delayed the paralysis of CL4176 worms caused by Aβ aggregation in response to the temperature upshift and reduced Aβ deposits as evidenced by thioflavin T staining.It would be interesting, for future research, to assess whether MH, in addition to reducing the aggregation of the Aβ peptide, is capable of reducing Aβ oligomers, since it is well known that they are more toxic than Aβ monomers and have been related to memory loss and severity of dementia 13,52 Aβ oligomers may damage neurons by interfering with neuron-toneuron communication at synapses. 5Many other natural compounds, such as betaine, 49 Ginkgo biloba extract, 7 gennianchun, 7 Sideritis scardica extracts, 53 fucoidan, 54 ergothioneine, 55 frondoside A, 56 spermidine, 57 essential Oil of Acorus tatarinowii Schott, 58 allophycocyanin, 50 caffeic acid, 4 strawberry extract, 15 carnosic acid, 3 scutellarein, 59 oleuropein, 17 hydroxytyrosol, 16,59 and red-beet betalain pigments, 60 also protect C. elegans nematodes against Aβ-induced toxicity.
Most of the studies evaluating the mechanism underlying the effect of natural compounds against Aβ-induced paralysis in C. elegans, focus on pathways related to oxidative stress, heat shock response, insulin signaling cascade and redox homeostasis. 51Hence, we knocked down the expression of SOD-2 and SOD-3, HSP-16.2,SKN-1, and DAF-16 genes by RNAi technology and repeated the paralysis test in the CL4176 strain.
DAF-16 is the only homologue of the forkhead box transcription factors class O (FOXO) in C. elegans and the main output of the insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway.Therefore, it plays a fundamental role in the regulation of growth, aging, metabolism, development, reproduction, stress resistance and immunity. 7,52,61,623][64] Moreover, the response against oxidative stress is also mediated by the joint action of other factors such as SKN-1, SMK-1 (a regulatory subunit of the PP4 protein phosphatase complex) and HSF-1. 62Up to the present, it has been reported that several compounds extend lifespan and reduce Aβ-induced toxicity in C. elegans via DAF-16, as for example royal jelly, 65 monascin, 66 dianxianning, 52 gengnianchun, 7 phosphatidylcholine, 64 Zijuan Pu'er tea extract, 67 Hibiscus sabdariffa L. extract, 68 strawberry extract, 15 oleuropein, 17 Moringa oleífera extract, 69 and hydroxytyrosol. 16However, under our experimental conditions, DAF-16 and SOD-3 RNAi failed to delay the Aβ-induced paralysis, indicating that MH attenuation of neurotoxicity is not mediated by these pathways.On the contrary, SKN-1 and HSP-16.2 were required for MH protective effects.Zhu et al. 70 also demonstrated that a rose essential oil was protective in a C. elegans model of Aβ-toxicity through SKN-1 activation but independent of DAF-16.Other compounds that have been shown to ameliorate Aβ-induced toxicity through the SKN-1 pathway are Hibiscus sabdariffa L. extract, 68 Panax Notoginseng saponins, 71 strawberry extract, 15 and Cratoxylum formosum extract. 72KN-1 is a transcription factor that can act in parallel to DAF-16, that can also inhibit the Aβ-induced toxicity by promoting the expression of stress responsive genes. 52This tran-

Food & Function Paper
1][72] For its part, the overexpression of the chaperone HSP-16.2 may improve the Aβ associated toxicity by increasing abnormal protein degradation, sequestration and refolding. 52,71Guarana (Paullinia cupana Mart.) 73 and palmatine 74 also act through this mechanism.In correspondence with the RNAi results, MH did not cause the translocation of DAF-16 from the cytoplasm to nuclei in the transgenic strain TJ356 carrying a GFP-tagged reporter of this gene.In addition, MH did not cause any change in the expression of SKN-1 compared to the control group and it decreased the expression of HSP-16.2 in the transgenic strains LD1 and TJ375, respectively, suggesting that MH effects on Aβ-paralysis mediated by these genes occurs only under stress conditions and not in a basal state.Likewise, MH did not increase the expression of GST-4, SOD-2 and SOD-3, which are target genes of DAF-16 or SKN-1 implicated in ROS elimination, suggesting that the intrinsic antioxidant capacity of MH is essential for its striking effects on the oxidative stress resistance and attenuation of Aβ-toxicity.As mentioned before, neurofibrillary tangles represent another distinctive feature of AD.6][77][78] Thus, we also evaluated the effect of MH against Tau-induced neurotoxicity, observing that 100 mg mL −1 MH was not able to improve the locomotion defects related to Tau protein accumulation in the transgenic strain BR5706 nor in N2 nematodes.In fact, MH-treated worms showed significantly lower swimming speed, wavelength, and activity compared to the control group.
Depending on the growth media, C. elegans can swim, burrow and crawl 79 and according to the Wormlab software used in this study, the swimming speed refers to the distance covered by the worm along its central axis over a two-stroke interval, whereas activity represents the brush stroke normalized by the time taken to the two strokes.For its part, the wavelength denotes the distance between negative and positive inflection points.This parameter is approximately twice the body length, generating alternating C-shapes with each ventral or dorsal muscle contraction. 79Thus, the effects observed in terms of worm mobility are in correspondence with the length diminution observed in the MH-treated groups.Considering that no differences were observed between MH and AH but with the ME (only containing the phenolic fraction of honey), we ascribed the deleterious effects of MH on locomotion to the sugar content of this matrix.Moreover, since the results were similar in both BR5706 and wild-type strain, we assume that the sugar effect is not related to Tau aggregation but directly to mobility.However, further analyzes are needed in this direction.

Conclusions
In this report it has been demonstrated that MH improved tolerance to oxidative stress induced by AAPH in the wild-type N2 C. elegans strain, and alleviated Aβ-induced paralysis in the AD model CL4176 through HSP-16.2 and SKN-1/Nrf2 pathways.Likewise, it has been shown that the sugar content of MH impaired the indicators of locomotion in both, the transgenic strain BR5706 and in the wild-type N2 worms but did not influence the rest of the favorable effects described above.Results presented here indicated that the antioxidant activity of MH may be responsible for the observed neuroprotective effects.

Fig. 1
Fig. 1 Short-term toxicity of Manuka honey in the N2 wild type strain.(A) 24 h lethality.(B) Pharyngeal pumping, egg-laying (fertility) and growth.Worms were treated with the indicated concentration of MH for 24 h.Data are expressed as mean ± SEM of three independent experiments (n = 3).Asterisk marks (*) indicate statistically significant differences ( p < 0.05) compared to control (non-treated worms).CTL: control; MH: Manuka honey.
-3::GFP, GST-4::GFP, SKN-1::GFP, HSP-16.2::GFP and DAF-16::GFP expressionTo deepen the comprehension of the effects of MH on the genes knocked down in the experiments described above, we used several transgenic strains carrying the GFP transcriptional reporters of interest.A marked diminution (p < 0.05) in the expression of HSP-16.2::GFP in TJ375 worms treated with MH (by 34.41%) as well as in the expression of SOD-3::GFP in CF1553 nematodes (by 16.30%) was found.No significant effects from MH addition (p < 0.05) were observed on the expression of GFP from the promoters for GST-4, SKN-1 and HSF-1 compared to control.Also, MH did not induce the translocation of DAF-16 from the cytoplasm to nuclei (Fig.5A and B).

Fig. 6
Fig. 6 Effects of Manuka honey on Tau-induced altered locomotive behavioral phenotype.The BR5706 transgenic strain was used in all groups.(A) Swimming speed, wavelength and activity.Data are expressed as mean ± SEM of three independent experiments (n = 3).Different superscript letters for the same set of data indicate statistically significant differences (P < 0.05) compared to control (non-treated worms).(B) Image sequence illustrating how the nematodes move from left to right in the experimental groups.Images were obtained from frame-by-frame tracking data provided by the Wormlab system/software.CTL: control; MH: Manuka honey, ME: Manuka honey hydroalcoholic extract, AH: artificial honey.

Fig. 7
Fig. 7 Effects of Manuka honey on locomotive behavior in wild-type C. elegans.Wild type-N2 strain was used in all groups.(A) Swimming speed, wavelength, and activity.Data are expressed as mean ± SEM of three independent experiments (n = 3).Different superscript letters for the same set of data indicate statistically significant differences (P < 0.05) compared to control (non-treated worms).(B) Image sequence illustrating how the nematodes move from left to right in the experimental groups.Images were obtained from frame-by-frame tracking data provided by the Wormlab system/software.CTL: control; MH: Manuka honey, ME: Manuka honey hydroalcoholic extract, AH: artificial honey.

Table 1
Phenolic compounds identified in MH

Table 2
Quantification of the phenolic compounds identified in MH LOQ: limit of quantification.Data are means (n = 3).

Table 3
Phytochemical characterization, antioxidant capacity and yield of MH