We have previously reported that OLE reduces Aβ aggregation in vitro
and hinders the appearance of oligomeric pre-fibrillar aggregates, thus protecting the exposed cells against Aβ injury 
. Here we checked whether OLE was also effective in vivo
by investigating whether it protected the CL2006 strain against Aβ3–42
toxicity. The worms, synchronized and placed on E. coli
at 16 °C, were fed with either vehicle or OLE (12.5–500 µM, 100 µl/plate), starting at L1, L2 or L3 larval stages. Worm paralysis was rated 96, 48 or 24 h later, when the worms were at L4 (see for treatment schedule). N2 wild type strain and CL802 worms, in which the Aβ transgene was not expressed and the paralysis did not occur 
, were used as controls. We found that OLE protected CL2006 worms from the paralysis induced by Aβ expression and aggregation in a dose-dependent manner upon administration either at L2 or L3, supporting a specific effect induced by OLE (). The IC50
value, i.e. the drug concentration that inhibited paralysis by 50%, calculated after OLE administration at L2 larval stage, was 14.5±1.0 µM (n
100 worms/group, mean±SE). This value was significantly lower (p
<0.01, Student’s t test) than that determined after OLE administration at L3 (242±8.5 µM, n
100 worms/group, mean±SE).
Effect of OLE on Aβ-induced paralysis in CL2006 transgenic C. elegans strain.
Feeding CL2006 worms at L1 larval stage with 50 µM OLE produced a protective effect significantly lower than that occurring when the same dose of drug was administered at L2 (45.8% vs. 65% reduction of paralysis for L1 and L2 administration, respectively, p<0.01, Student’s t test) () The protective effect at L1 did not increase by feeding worms with 100 µM OLE (46% protection of paralysis) and higher doses resulted to be toxic, causing the death of worms.
The protective effect of 50 µM OLE at L2 larval stage was similar to that occurring when 500 µM of drug was administered at L3; in fact, worm paralysis was reduced by 65.0% and 62.6% respectively (). The OLE effect was comparable to that obtained feeding CL2006 worms with 50 µM tetracycline at L3 (), which resulted in a reduction of nematode paralysis of 66%. The effect of tetracycline when administered at L2 was not here considered due to the toxicity of the drug at this larval stage 
. The double administration of 50 µM OLE both at L2 and L3 larval stage did not further improve drug protection; the reduction of the paralysis was comparable to that observed with the single administration at L2 (data not shown).
The feeding of CL802 worms and wild type N2 nematodes with OLE starting at L1 (50–100 µM), L2 or L3 larval stages (12.5–500 µM), did not modify their viability () indicating that the drug did not cause any aspecific and/or toxic effects in control strains.
We also investigated whether OLE protected CL4176 worms from the paralysis caused by the inducible Aβ1–42
. According to the treatment schedule set out in Figure S1A
, worms were fed either vehicle or OLE (50–500 µM, 100 µl/plate) 30 h before the temperature rise (at L1 larval stage), 6 h before the temperature rise (at L2) or 18 h after the rise of temperature (at L3). A slight reduction of the paralysis of CL4176 worms was observed only when nematodes fed 50–100 µM OLE 30 h before the induction of the Aβ expression (reduction of paralysis of 18%) (Fig. S1
) whereas the other treatment schedules were ineffective (data not shown). Feeding CL4176 at L1 larval stage with OLE at doses higher than 100 µM, as well as the repeated administration of 50–100 µM OLE starting from L1, resulted to be toxic.
These findings indicated that the OLE protective effect was lower in CL4176 strain than in CL2006 worms: this could be ascribed to the fully penetrant paralysis phenotype of the inducible strain. The optimal protective effect of OLE against Aβ-induced paralysis was obtained in CL2006, when drug feeding started from L2 larval stage, and in CL4176 when the drug was administered from L1. These observations indicated that OLE protective effect was maximal when worms were treated from the beginning of the aggregation process.
We then investigated whether OLE reduced the degree of Aβ amyloidosis in CL2006 transgenic worms by evaluating the amount of aggregate deposits in their head region 
. The worms, synchronized and placed at 16 °C for 48 h at L2, were fed either vehicle or OLE (50 µM, 100 µl/plate) for 48 h and then stained with the X-34 dye which recognizes β-amyloid aggregates 
. OLE strongly reduced X-34 positive spots (), indicating that the drug inhibited Aβ fibril deposition.
OLE reduces amyloid deposition and extends the life-span of CL2006 transgenic C. elegans strain.
It is known that compounds able to interact with protein fibrils and aggregates affect age-related changes in protein homeostasis and extend worm lifespan 
. Accordingly, we analyzed the effect of OLE administration on overall CL2006 and CL802 nematode survival. Feeding CL2006 worms at L2 larval stage with 50 µM OLE significantly increased their median survival by 47% () (median survival: 7 and 15 days for worms fed with vehicle or with OLE, respectively. p
0.004, Log-rank test). The same effect on survival was observed after administration of a higher dose of OLE (100 µM) (median survival: 7 and 15 days for worms fed with vehicle or with OLE, respectively. p
0.003, Log-rank test). In addition, the survival of CL2006 worms exposed to a double administration of 50 µM OLE, at L2 and 8 days after, was not different from that of nematodes receiving a single dose of the drug (median survival: 15 days for either worms fed one or two OLE doses. p
0.790, Log-rank test).
The administration of 50 µM OLE at L2 larval stage to CL802 nematodes did not modify their survival () (median survival: 12 and 13 days for worms fed vehicle or OLE, respectively. p
1.000 Log-rank test) supporting the absence of aspecific effects.
Such beneficial effects of OLE were not related to its ability to modify the steady-state of Aβ by interfering with its production and/or degradation. In fact no significant difference in the Aβ immunoreactive signal was observed between worms fed with vehicle or with 50 µM OLE at L2 larval stage, as indicated by the dot blot analysis performed on CL2006 worm lysates using an antibody specific for total Aβ (WO2) (). It is known that the expression of Aβ in paralyzed CL2006 worms is accompanied by an increase of the A11-positive oligomer production 
. We therefore performed dot blot analysis to determine whether the protective effect of OLE could be related to its ability to counteract Aβ oligomer formation. Actually, it transpired that, in nematodes fed with 50 µM OLE at L2 larval stage, the A11-immunoreactivity was reduced by 40% compared with that found in CL2006 worms treated with vehicle (p
9, Student’s t-test) ().
Effect of OLE on Aβ oligomer production.
The increased production of superoxide precedes the paralysis in the transgenic C. elegans
CL2006 strain constitutively expressing the human Aβ3–42
; this was observed under experimental conditions (+64% of superoxide production in CL2006 worms as compared to the CL802 control strain. p
<0.001, Student’s t-test, n
100 worms/group). In the same strain, the protective effect of tetracyclines and of a Ginko biloba
extract against worm paralysis was found to be associated with a reduced superoxide production 
. An in vitro
and in vivo
ROS-scavenging effect of chronic OLE treatment has been reported as well 
and this is a likely explanation for its ability to reduce lipid peroxidation. We then investigated whether, besides the previously described anti-fibrillogenic effect, an antioxidant action contributed to OLE protection against Aβ toxicity. To this end, CL2006 worms were fed with vehicle or with 50 µM OLE at L2 larval stage, followed after 48 h by superoxide measurement. We found that OLE treatment did not reduce the level of intracellular superoxide, whereas a significant reduction by 37% and 31% with respect to vehicle-fed worms, was observed in nematodes treated for 24 h with antioxidant compounds such as 5.0 mM NAC or 50 µM tetracycline, respectively (). We conclude that the protective effect of OLE in Aβ-expressing worms is not related to its antioxidant activity. This matches the conclusion reached when we investigated OLE protection against Aβ and amylin aggregates in cultured cells 
Effect of OLE on oxidative stress in CL2006 transgenic C. elegans strain.
Overall, our data show that OLE treatment of the C. elegans CL2006 strain at early larval stage is significantly effective against Aβ oligomerization, fibrillization and aggregate deposition, without a significant reduction in Aβ levels. The strongly reduced extent of plaque deposition and oligomer level results in a strong protection against the pathological phenotype, such that the worms recover good motility and viability and improve their survival with respect to those fed with vehicle.