| 1. Backgroud and significanceAlzheimer’s disease(AD) is the most common form of dementia among the elderly, and the incidence increases with the aging population worldwide, causing a huge social and economic burden for families and societies. Accumulating evidence indicates that amyloid-beta(Aβ) and its oligomers play central roles in the pathogenesis of AD. Despite significant progress has been made towards understanding the pathogenesis of AD in recent years, no efficient disease-modifying therapeutics is available for the management of AD. In recent years, a number of drug candidates targeting Aβ through immunotherapy or using secretase inhibitors have proceeded to clinical trials but all failed to improve cognitive functions in patients. Clearly, lessons have been learnt through failed clinical trials indicating that a drug targeting a single target or pathway does not work on this complex disease. Aβ, over produced and accumulated in AD brains, triggers subsequent pathological events such as synaptic degeneration, Tau-hyperphosphorylation, oxidative stress, neuroinflammation, neurite degeneration and neuronal loss. These secondary pathological events can form vicious cycles themselves and accelerate the disease progression. Therefore, we proposed that it is critical to discover novel drugs, which target multiple key pathways in the pathogenesis of AD, in order to improve or halt the progression of the disease.As a serial of new drugs for AD failed in clinical trials, it is necessary to choose drugs with both an established safety profile and a mechanism-based rationale for futureclinical trials.One approach is to screen current drugs approved by regulatory bodies for other indications and reposition them for AD. In the present study we have taken such an approach and investigated potential therapeutic effect of Edaravone, an oxygen radical scavenger which is currently used for the treatment of acute ischemic stroke. Oxidative imbalance is a manifestation of AD even preceding Aβ deposition and neurofibrillary tangle(NFT). Aβ is a highly redox active peptide that generates reactive oxygen species(ROS). ROS is one of the key factors, which promote several Aβ-driven vicious cycles and propagate the pathogenesis of AD. Previous study found that Edaravone was able to attenuate Aβ-induced oxidative stress and neurotoxicity. Aβ accumulation and aggregation into amyloid plaques in the brain are considered to trigger the AD pathogenesis. In the present study, we found that Edaravone can interact with Aβ and is competent in inhibiting Aβ aggregation and disaggregating preformed Aβ fibrils, suggesting that Edaravone is a scavenger for both ROS and Aβ. In animal models, we found that Edaravone, given before or after the onset of Aβ deposition, reduced Aβ burden in the brain and cerebral arterioles by inhibiting Aβ deposition and reducing BACE1 processing of the amyloid-β precursor protein(APP), attenuated oxidative stressand neuroinflammation, inhibited Tau hyperphosphorylation, protected brain neurons from loss and synaptic degeneration, and finally rescued the cognitive deficits of aged APPswe/PS1dE9(APP/PS1) mice.2. Materials and methodsAβ aggregation and disaggregation assays. The Aβ aggregation and disaggregation assays using ThT, Western blot and TEM methods were performed as described. Edaravone binding epitope mapping was performed by the competition assay using the ThT activated fluorescence method.Neurotoxicity and neurite growth assays.SH-SY5 Y cells were applied for MTT and neurite growth assays as described previously, as well as for examining the effect of Edaravone on Tau phosphorylation and GSK3β activation, in the presence or absence of Aβ. Neonatal primary cortical neurons were used for neurite growth assay, PI staining and ROS determination. SH-SY5Y-APP695 cells were used for examining the effect of Edaravone on BACE1 expression and APP processing.Pharmacokinetic experiment.Adult rats aged 4 months were given with Edaravone at the dose of 10mg/kg intravenously or orally. The concentrations of drug per administration route were examined by LC-MS/MS.Animals and sample collections. Animal studies on APP/PS1 transgenic mice were performed in two parts. In part one, Edaravone was administered intraperitoneally twice per week at the dose of 44.1 mg/kg, which is converted from the dose administered intravenously in stroke patients. In the prevention experiments, 3-month old APP/PS1 mice were treated with Edaravone for 6 months(n=11), and blank controls(age- and sex-matched APP/PS1 mice, n=8) and wild type controls(age- and sex-matched wild type littermates, n=7) were administered with same volume of normal saline(NS). In the treatment experiments, 9-month old female APP/PS1 mice were treated with Edaravone for 3 months(n=12), and age- and sex-matched APP/PS1 controls(n=11) and wild type controls(n=8) were treated with the same volume of NS. Additional group of 9 month old female APP/PS1 mice without any treatment(n=8) were also set as baseline control for the treatment experiment. In part two, Edaravone was administered orally. Accordingly, 3-month old female APP/PS1 mice were administered with or without daily oral Edaravone at the dose of 33.2 mg/kg in drinking water for 9 months(n=8 per group), and wild type littermates were used as control without oral Edaravoneadministration(n=7). After the set dosing, the mice underwent behavioral testing as described below. A subset of 3 mice in peritoneal injection treatment experiment underwent Golgi staining(below).Behavioral tests. Mice in prevention experiment underwent multiple behavioral tests including Morris water maze, Y-maze and open field, and mice in treatment experiment and oral medication experiment only underwent Morris water maze as described. All behavioral tests were performed in a double blind manner.ELISA assays. Frozen brain was homogenized in liquid nitrogen and part of resultant powder was successively extracted with TBS, 2%SDS and 70% formic acid(FA) solutions. Concentrations of Aβ40, Aβ42, IL-6, IL-1β, INF-γ, TNF-α in brain extracts were quantitatively measured by ELISA. α- and β-secretases activities in fresh brain tissues was measured by relevant ELISA kits. The brain homogenates were also subjected to assays for superoxide dismutase(SOD) activity, glutathione peroxidase(GSH-Px) activity, hydroxyl radicals scavenging ability and malondialdehyde(MDA) concentration. Histological staining.For Aβ plaques in parenchyma, a series of five equally spaced tissue sections(~1.3 mm apart) spanning the entire brain were stained with Congo red and 6E10(Aβ antibody) as described previously. The sections were also stained for NeuN, ChAT, MAP-2, CD45, GFAP, pSer396-Tau, 3-NT and Caspase-3 antibodies in 1:100-1:200 dilution, visualized by DAB for ChAT, CD45, GFAP, 3-NT and pSer396-Tau, or by Alexa Fluor fluorescent dyes for NeuN and MAP-2 and Caspase-3.The sagittal sections of brain tissue were subjected to Thioflavin S staining. Each vessel was scored on a four-grade scale for the severity of CAA. Prussian blue staining was also conducted and microhemorrhage profiles were counted as described previously. TUNEL staining was used to detect apoptotic cells. The sections were labeled by in situ Death Detection Kit, POD(Roche) according to the manufacturer’s instructions. Animals in treatment experiment underwent Golgi staining using the manufacturer’s protocols(FD rapid GolgiStainTM kit). Images per staining were collected and quantified by ImageJ, yielding the area fraction of positive staining against the area of tissue analyzed.Western blot.The levels of molecules or enzymes involving Aβproduction and degradation, phosphorylated Tau, oxygen stress, and synapse-related proteins were analyzed using western blot. Proteins in the animal brain homogenate were extracted with RIPA buffer and loaded on SDS-PAGE(4–10% or 4-10-15-18% acrylamide) gels, transferred to nitrocellulose membranes and probed with different antibodies. The membranes were incubated with IRDye 800 CW secondary antibodies(Li-COR) and scanned using the Odyssey fluorescent scanner. The band density was all normalized to β-actin or GAPDH when analyzing.Participant enrollment and blood collection in a pilot clinical study. A total of 30 patients with moderate to severe AD, who admitted to the Department of Neurology of Daping Hospitalfrom July 2013 through July 2014, were recruited.The neuropsychological evaluation was performed using the Chinese version of the Mini-Mental State Examination(MMSE), Clinical Dementia Rating(CDR) and instrumental Activities of Daily Living(ADL). Clinical data were also collected, including age, education and sex. Patients were randomly divided into two groups, Edaravone group and control group. Fasting blood was collected on day 1 and day 8. Serum was separated and measurements of Aβ40 and Aβ42 were conducted according to the manufacturer’s instructions. The protocols of those studies were reviewed by the local ethics committees and registered in the Chinese Clinical Trial Registry. An informed consent was acquired from all subjects and, if needed, from authorized family members.Statistical analysis. Unless otherwise stated, the results were presented as mean ± s.e.m. Statistical comparisons between two groups were tested using Student t-test, or Man-Witney U test, as applicable. The comparisons among groups were tested using one-way ANOVA or two-way ANOVA, and the trend analysis was performed when necessary. P values<0.05 were considered significant.3. ResultsEdaravone inhibits Aβ aggregation and antagonizes Aβ neurotoxicity in vitro. Previous studies suggest that several natural antioxidants such as curcumin and grape-derived polyphenolics can inhibit aggregation of Aβ. Based on these findings, we speculate that Edaravone, an oxygen radical scavenger, might also be able to interfere with Aβ aggregation. Using Thioflavin T(ThT) fluorescence assay, we found that Edaravone, when incubated with Aβ monomers or preformed Aβ fibrils, dose-dependently reduced Aβ fibrillation-induced fluorescence intensity. Western blot assays further showed that Edaravone inhibited the formation of Aβ fibrils during incubation, which was revealed by the bands on the bottom of the loading wells without moving to the gel. Edaravone pre-incubated with Aβ dose-dependently increased the soluble Aβ oligomer species. Moreover, transmission electron microscopy(TEM) assays visually confirmed that Edaravone suppressed the fibrillation of Aβ and disaggregated the preformed Aβ fibrils, as reflected by the twisted long fibrils, short fibrils, and some small and relatively amorphous aggregates mixed with fibrils after treatment with Edaravone. To elucidate the Edaravone binding epitope in Aβ42 we did Aβ42 fragment competition assays using ThT fluorescence measurement. We found that only the peptide of Aβ42 fragment amino acid(aa) 13-18, among 7 peptides which cover the entire Aβ aa sequence, had an ability to increase the Aβ fibril fluorescence which was otherwise suppressed by Edaravone, indicating this peptide may compete for the binding site of Edaravone in Aβ42 and suggesting Edaravone may bind on the Aβ42 sequence aa 13-18. All peptides did not interfere with the formation of Aβ42 fibrils, or formed any fibrils themselves. The putative Edaravone binding epitope aa 13-18 is within the beta strand region of Aβ42.Based on above findings, we examined whether Edaravone can protect neurons from Aβ-induced neurotoxicity. In human neuroblastoma SH-SY5 Y cells, we found that Edaravone dose-dependently protected neurons from cell death and neurite collapse triggered by Aβ. Furthermore, using neonatal primary cortical neurons as another cell model, we also found the protective effects of Edaravone against neurite collapse, cell death and reactive oxygen species(ROS) production triggered by Aβ. These results suggest that Edaravone has a potent capacity of inhibiting Aβ aggregation and neutralizing the toxicity of Aβ in vitro.Edaravone improves cognitive deficits prior to and after onset of Aβ deposition. Based on its properties on oxidative stress and Aβ aggregation, we next investigated the preventive and treatment effects of Edaravone on AD-type pathologies and cognitive deficits in APP/PS1 mice. Compared with the normal saline control of APP/PS1 mice, the mice in prevention and treatment groups performed better in the Morris water maze. This was reflected by significant reductions in the escape latency time and distance to platform in progressive platform learning trials, greater numbers of annulus crossing and more time spent in target quadrant in the probe trial in Edaravone-treated mice. The learning deficit in APP/PS1 mice at 9 months of age was rescued by preventive medication of Edaravone and the escaping time in Edaravone-treated mice was similar to wild type controls. By 12 months of age, while the escaping latency in the treatment group was longer than that in wild type mice, it was similar to the baseline control of 9 months old APP/PS1 mice. There was no difference in swimming speed among control and experimental groups. The mice in the prevention group also performed better in Y-maze tests than the saline-treated control and the baseline controls. In spontaneous alteration tests, the mice treated with Edaravone showed higher spontaneous alteration percentage and greater total entries into three arms. Additionally, in novel arm exploration tests, Edaravone-treated mice showed more entries into and more time spent in the novel arm and greater total entries. We also found a higher number of rearing, and a longer distance travelled in the prevention group in the open field test. No difference was found in the number of the grooming behavior among different groups.Edaravone reduces Aβ burden of APP/PS1 mice. To investigate whether Edaravone affects Aβ deposition in APP/PS1 mice, we performed Congo red staining for compact amyloid plaques and Aβ immunostaining(6E10) for total amyloid plaques. Compared with APP/PS1 controls, the mice treated with Edaravone in the prevention and treatment groups showed significantly lower amyloid plaque burden in the brain than the control group. We further examined cerebral amyloid angiopathy(CAA). Based on the extent to which Aβ is deposited on the blood vessels, we categorized the severity of CAA into four grades from 0(no CAA) to 3(severe CAA). We found that Edaravone significantly reduced proportion of severe CAA by both prevention and treatment. The number of microhemorrhage profiles in the treatment group was also significantly reduced compared to the control. ELISA tests also showed a significant decline in the levels of total Aβ, Aβ40 and Aβ42 in TBS, SDS and formic acid(FA) fractions of brain homogenates in both prevention and treatment groups compared with their respective controls. These data indicate that Edaravone can reduce both parenchymal and vascular Aβ burden in brain.Edaravone inhibits amyloidogenic processing of APP in APP/PS1 mice. Edaravone had no effect on the expression of total APP, however, it significantly reduced β-cleavage and increased α-cleavage of APP in the brains of APP/PS1 mice. The mice treated with Edaravone showed significantly declined levels of β-cleavage products and Aβ production, and increased levels of CTFα and sAPPα in the treatment experiment. Consistent with the in vivo data, the treatment of SH-SY5Y-APP695 cells with Edaravone dose-dependently increased the levels of CTFα and sAPPα and decreased the levels of CTFβ and sAPPβ. Moveover, Edaravone treatment increased the expression of disintegrin and metalloprotease 10(ADAM10) and α-secretase activity, and decreased beta-site APP cleaving enzyme 1(BACE1) expression and activity, but had no effect on the expression of presenlin 1(PS1). Consistent with the results in vivo, Edaravone dose-dependently suppressed BACE1 expression in SH-SY5Y-APP695 cells in vitro. Edaravone had no effect on the expression of Aβ-degrading enzymes neprilysin(NEP) and insulin-degrading enzyme(IDE), and Aβ blood-brain barrier(BBB)-transporting molecules low density lipoprotein receptor-related protein(LRP) and receptor for advanced glycation end products(RAGE).Edaravone rescues neuronal and dendritic loss and attenuates inflammation in the brains of APP/PS1 mice. Compared to saline-treated APP/PS1 controls, APP/PS1 mice in both prevention and treatment groups showed markedly increased positive-staining area fractions of NeuN(neurons), MAP-2(dendrites) and ChAT(cholinergic neurons) immunostainings, and significantly reduced apoptosis detected by caspase-3 immunofluore- scence and TUNEL staining(Fig. 5D) in the hippocampus. Neuroinflammation, microgliosis(detected by CD45 antibody) and astrocytosis(detected by GFAP antibody) in the treatment group were significantly declined. Additionally, the levels of pro-inflammatory cytokines, including TNF-α, IFN-γ, IL-1β and IL-6 in brain homogenates in the treatment group were also lower than APP/PS1 controls. Importantly, we found that synapse-associated protein expressions were significantly increased in brain homogenates of Edaravone-treated APP/PS1 mice in both prevention and treatment groups, and the number of dendritic spines detected by the Golgi staining in the hippocampus was also significantly increased by Edaravone.Edaravone attenuates Tau-phosphorylation in APP/PS1 mice. Edaravone significantly improved Tau pathology in prevention and treatment expeirments. The area fractions of Tau-phospho-Ser396 positive neurons in the subregions of hippocampus and neocortex of Edaravone-treated APP/PS1 mice were significantly lower than those of the controls. Western blots further showed that Tau-phosphorylation at multiple sites, including serine 396, 262, 199 and threonine 231, was consistently and significantly diminished in the brain of APP/PS1 mice in both prevention and treatment groups. In addition, we found that the phosphorylation at Ser9 of GSK3β, an enzyme well known for its role in the phosphorylation of Tau and the pathogenesis of AD, was significantly increased in the treatment group. To examine the underlying mechanism, we treated SH-SY5 Y cells with Aβ in the presence and absence of Edaravone. Indeed, Edaravone dose-dependently inhibited the elevation of phosphorylation of Tau at Ser396 site induced by Aβ, and increased the ratio of pSer9-GSK3β to total GSK3β in vitro. These findings indicate that the suppression of Tau-hyperphosphorylation of Edaravone is via its effect on Aβ.Effect of Edaravone on oxidative stress in the brains of APP/PS1 mice. We performed experiments on oxidative stress markers using brain samples from APP/PS1 mice treated with Edaravone by biochemical and immunohistological methods. Compared with the controls, the activities of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px) and hydroxyl radical scavenging were significantly increased, and the levels of lipid peroxidation products malondialdehyde(MDA), 4-hydroxynonenal(4-HNE), protein peroxidation products 2,4-dinitrophenylhydrazine(DNPH) and 3-nitral tyrosine(3-NT) were significantly reduced in the brain of Edaravone treated mice. Furthermore, we found this decrease in 3-NT levels was more significant in the hippocampus than in the whole brain and cortex. As expected, oxidative stress levels in the brains of Edaravone-treated APP/PS1 mice were significantly reduced.Effect of oral Edaravone treatment in APP/PS1 mice. Based on the pharmacokinetic result the bioavailability of oral Edaravone was 38% of the intravenous delivery, we fed APP/PS1 mice with Edaravone at 33.2mg/kg/day between the ages of 3 and 12 months. Morris water maze test by 12 months of age showed that oral Edaravone prominently attenuated the cognitive deficits, as reflected by reduced escaping latency in platform testing and increased annulus crossing during probing test. Oral intake of Edaravone also markedly alleviated Aβ plaque burden in the hippocampus and neocortex. The Aβ levels in the brain were also significantly reduced by the oral intake.Serum Aβchanges after Edaravone treatment in two pilot clinical studies. The in vitro and in vivo studies described above indicate that Edaravone is able to affect the aggregation state and metabolism of Aβ, we investigated whether Edaravone can alter Aβ levels in the bloods of patients with moderate to severe AD. The concentration changes(the concentration on day 8 minus the concentration on day 1) of Aβ40 and Aβ42 in the Edaravone group were significantly higher than those in the control group, indicating that, similar to Aβ antibodies, Edaravone can also change the steady status of serum Aβ levels in human subjects. Taking the results of two pilot studies together, we speculates that Edaravone has potent bidirectional regulation effects on Aβ to balance Aβ concentration to a beneficial side for our bodies.4. Discussion and ConclusionIn the present study, we found that Edaravone can interact with Aβ via binding to Aβ42 sequence aa 13-18, and is competent in inhibiting Aβ aggregation and disaggregating preformed Aβ fibrils, suggesting that Aβ is a scavenger for both free oxygen radicals and Aβ. Furthermore, we found that Edaravone, given before or after the onset of AD, reduced Aβ burden in the brain and cerebral arterioles by blockingAβ/ BACE1 vicious cycle, attenuated oxidative stress and neuroinflammation, inhibited Tau hyperphosphorylation, protected brain neurons from loss and synaptic degeneration, and finally rescued the cognitive deficits of aged APPswe/PS1dE9 mice.According to the findings in the present study, we propose that multiple target or pathway therapy may become a future direction of drug discovery for AD. Frankly speaking, so far we have not found direct evidence supporting the binding of Edaravone to Aβ, i.e., the putative binding site of Edaravone in Aβ peptide showed in the present study needs further validation. Additionally, the mechanisms underlying the effects of Edaravone on Aβ, Aβ-induced neurotoxicity and α/β secretases are not fully elucidated. The verification of the therapeutic effect of Edaravone on AD requires a mulitiple-centered prospective clinical study. |
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