Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer's disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer's disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.

Curcumin is a curry spice with multiple biological activities that is also effective in transgenic AD mouse models [5] , [6] . To improve the potency and pharmacokinetic properties of curcumin, we synthesized a series of hybrid molecules between curcumin and cyclohexyl-bisphenol A (CBA), a compound that has neurotrophic activity which curcumin lacks [7] . The best compound in our initial library was CNB-001, a molecule that has improved stability over curcumin and that is neuroprotective in multiple neurotoxicity assays in which curcumin is inactive [8] . We then generated a large number of derivatives of CNB-001 and selected the best compound on the basis of activity in our multiple toxicity assays. The result was a much more potent molecule called J147. It was then asked if J147 is effective in two paradigms of age-associated pathology, AD and memory. It is shown here that this broadly neuroprotective compound has the ability to enhance memory in normal animals as well as to prevent memory deficits in AD transgenic mice. The neurotrophic and memory-enhancing activities of J147 are associated with an increase in brain derived neurotrophic factor (BDNF) levels and the expression of BDNF responsive proteins, the enhancement of LTP, synaptic protein preservation, the reduction of markers for oxidative stress and inflammation, the reduction of amyloid plaques, and lower levels of soluble Aβ 1–42 and Aβ 1–40 . These pleiotrophic effects of a single molecule suggest that J147 has potential for the treatment of AD.

Since age is the prime risk factor for AD as well as many other neurodegenerative conditions, we developed a drug screening procedure that is based upon old age-associated pathologies without requiring pre-selected molecular targets. A series of cell culture assays were brought together that mimic multiple pathways of CNS nerve cell damage and we required that a drug candidate show efficacy in all of these assays before it could be moved forward into AD animals. The assays include the loss of trophic support, oxidative stress, the reduction of energy metabolism (in vitro ischemia and glucose starvation) and amyloid toxicity. As potential lead drug candidates to screen in these assays, we chose a representative from the original pharmacopeia, plant-based polyphenolics. Natural products have a wide range of molecular targets because they resemble biosynthetic intermediates to a greater extent than synthetic drugs and are therefore able to compete with substrates for multiple enzymes [4] .

At present, there are few drugs that improve the memory deficits associated with normal aging and none that prevent cognitive decline in chronic neurodegenerative conditions such as Alzheimer's disease (AD). Except for the rare cases of familial AD, the cause of AD is not known, but the disease is highly correlated with aging, a process involving a wide variety of physiological changes. Therefore, it is likely that the cells in the aging brain are compromised not from a single cause but from the convergence of multiple insults. However, the currently most widely used approach to drug discovery is to identify a single molecular target and then make a drug that alters this target [1] . Unfortunately, drugs for AD that were developed through this approach have all failed in clinical trials, perhaps because one target is not sufficient or because the targets are also critical for normal brain function so that their inactivation results in toxicity. Therefore, a different approach to drug discovery for AD is needed [2] , [3] .

Results

Because many of the pathological consequences of AD are enhanced features of the stresses that occur with old age [9], we devised a drug discovery scheme that uses multiple assays to test efficacy against these distinct stresses. We started with a multi-target natural product, curcumin [3], and required that synthetic derivatives have both improved medicinal chemical properties and lower EC 50 s in all of our assays of age-related stresses, not just one. Figure 1 summarizes this approach. Briefly, a pyrazole derivative of curcumin called CNB-001 (Figure 1A) is neuroprotective in multiple cell culture assays designed to reflect the various cellular toxicities associated with AD [8]. Figure 2 shows the assays that were used to select the best compounds and presents a comparison between curcumin and CNB-001. Because aromatic hydroxyl groups may be covalently modified in vivo by sulfation and carbohydrate addition thereby limiting brain penetration [10], derivatives of CNB-001 were synthesized that did not contain hydroxyl groups but still maintained its biological activities. A limited structure-activity relationship (SAR) analysis of CNB-001 showed that the hydroxyl groups are not necessary for activity and that activity was maintained following the addition of two methyl groups to the nitrogen-associated aromatic ring of CNB-001 (Fig. 1B, CNB-023. Table 1). To improve the pharmacological properties of CNB-023 and explore the possibility that two aromatic rings connected by a nitrogen containing bridge are sufficient for activity, a large number of molecules were synthesized in a single reaction, followed by the selection of the most active compound in the mix and the determination of its structure. To generate diversity, we carried out a reaction between 2,4 dimethylphenyl hydrazine and m-anisaldehyde under conditions which generate a wide variety of reaction products. The reaction products were then separated initially by thin layer chromatography (TLC) followed by reverse phase chromatography. Biological activity was followed by three primary rat cortical neuron culture assays that are detailed below; an assay for trophic factor activity [11]; [8], a glutamate-induced oxidative stress assay called oxytosis [12], and amyloid toxicity toward hippocampal neurons [13]. When the most active TLC fraction was separated on a HPLC column, only one peak was effective in all three assays (Fig. 1D). The structure of this compound was determined by NMR spectroscopy and confirmed by synthesis and crystal structure (Figs. 1C and D). It was designated J147 (Fig. 1C). J147 has the medicinal chemical properties of a good CNS drug candidate with respect to size (351 MW), cLogP (4.5), total polar surface area (41.9) and pharmacokinetics (not shown) [14]. Also included in the legend of Fig. 1 is an abbreviated list of assays used to identify both potential drug targets and screens to identify potential toxicities. To date there is no evidence for potential side effects, nor has a molecular target been identified. As detailed below, J147 also has a low EC 50 in multiple neurotoxicity assays.

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larger image TIFF original image Download: Figure 1. Selection of J147. CNB-001 (A) is a broadly neuroprotective and neurotrophic derivative of curcumin [8]. CNB-023 (B) is a derivative of CNB-001 lacking hydroxyl groups but with similar activity that was identified by SAR analysis of CNB-001. The boxed area shows the hypothesized biologically active fragment of CNB-023. A large collection of molecules around this chemical space was generated by the reaction of m-anisaldehyde with 2.4 dimethylphenyl hydrazine (see Materials and Methods) and J147 (C) was selected from the reaction products on the basis of its activity in trophic factor withdrawal (TFW), oxidative stress (oxytosis) and Aβ toxicity assays (see Figure 2). (D) Final HPLC run of reaction mixture that generated J147. The structure of the most active product (fraction 10) was determined by NMR. J147 was then synthesized, and its crystal structure (insert) determined, confirming its biological activity and structural identity. The following are de-risking and target identification screens, with no significant reproducible hits. J147 was used at 10 micromolar unless indicated. (1) LeadProfiling+P450 screen. Over 60 CNS receptors and transporters (work done by MDS Pharma). (2) hERG (work done by MDS and Absorption Systems). (3) Acute toxicity in rats. Negative at 2 grams/kilogram (work done by Absorption systems). (4) CeeTox “Safe” up to 90 micromolar plasma concentration (work done by CEETOX, INC). (5) 352 protein kinases (done by Ambit). (6) MDRI-MDCK brain penetration classification “High” (work done by Absorption Systems). (7) Enzyme Assays. Cox 1; Cox 2; Lox 5,12,15; Sirt 1,2,3; LT4 hydrolase; LTC4 synthase; cathepsin B; matrix metalloprotease 1; phosphodiesterases 10A1, 11A1, 1A, 2A, 3A, 4A1A, 4B1, 5A, 6, 7A, 7B, 8A1, 9A2; proteasome (done by MDS). (8) Enzyme assays. PPARα, γ; deubiquitinases, BAP1, UCH-L1, L3, USP5; acetylcholine esterase; MAOA; MAOB; phosphatases (19 of them), proteases and caspases (8) (done by Caliper). https://doi.org/10.1371/journal.pone.0027865.g001

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larger image TIFF original image Download: Figure 2. Biological Activities of J147. J147 is active with EC 50 s between 10 and 200 nM in six different assays for neurotrophic activity and neurotoxicity. o-o, J147; x-x, CNB-001; Δ-Δ, curcumin. (A) Trophic Factor Withdrawal. Primary cortical neurons were prepared from 18-day-old rat embryos and cultured at low cell density with or without the three compounds. Cell viability was assayed 2 days later. (B) BDNF-like Activity. HT22 cells expressing the TrkB (BDNF) (open circles, J147) receptor or no TrkB (black circles, J147) were placed in serum-free medium in the presence of 50 ng/ml BDNF or the indicated amounts of compounds. Cell viability was determined 2 days later. Curcumin had no activity in this assay up to one micromolar. BDNF was used at 50 ng/ml and active only in cells expressing TrkB (open bar), not in its absence (black bar). (C) Oxidative Stress. E18 rat cortical neurons were treated with 5 mM glutamate and different concentrations of compounds one day after plating when no ionotropic glutamate receptors are expressed. Cell viability was measured 24 hr later. (D) Glucose Starvation. PC12 cells were starved for glucose plus or minus 20 nM J147, 0.2 µM CNB-001 or 10 µM curcumin and cell viability determined 48 hr later. J147 and NGF increase cell viability in the absence of glucose, *P<0.001 vs. control. CNB-001 and curcumin are inactive at 0.2 and 10 µM respectively (curcumin not shown). (E) Chemical ischemia. HT22 cells were treated with 20 µM iodoacetic acid for 2 hr alone or in the presence of varying concentrations of J147, CNB-001 or curcumin. Percent survival was measured after 24 hr. (F) Amyloid toxicity. Primary hippocampal cells were exposed to 5 µM Aβ 1–42 in the presence of increasing amounts of compounds and cell viability determined 48 hr later. All data shown are mean ± SEM, n = 3 or 4. The curcumin and CNB-001 data which were included for comparison with J147 have been presented, in part, previously [8], [84]. https://doi.org/10.1371/journal.pone.0027865.g002

J147 is Broadly Neuroprotective Since the original lead compound, CNB-001, was selected on the basis of its broad neuroprotective activity [8] and J147 on the basis of a subset of these activities, it was first asked if J147 maintained all of the activities of CNB-001. Figure 2 shows that J147 is biologically active in six assays that represent distinct neurotoxicity pathways related to aging and neurodegenerative disease. In one assay of trophic factor withdrawal (TFW), primary embryonic cortical cells are plated at low density in serum-free medium (Fig. 2A). At low density, the cells die within two days but can be rescued by combinations of neurotrophic growth factors, but not by one alone [[11] and unpublished]. In contrast, cell death is prevented by J147 alone with an EC 50 of 25 nM. In a second assay, J147 is able to rescue a clone of the hippocampal nerve cell line HT22 expressing the BDNF receptor, transmembrane receptor kinase B (TrkB), from serum starvation under conditions where the cells can be protected by BDNF (Fig. 2B) [15]. J147 is equally neuroprotective using cells lacking TrkB, showing that J147 does not require the BDNF receptor for activity. Figure 2C shows that J147 also rescues primary cortical neurons from oxytosis, an oxidative stress-induced programmed cell death pathway caused by glutathione (GSH) depletion [12]. A reduction in GSH is a common denominator of old age and essentially all chronic CNS diseases [16]. J147 is also neuroprotective against glucose starvation (Fig. 2D) and the loss of energy metabolism in an in vitro ischemia model (Fig. 2E) [17], [18]. Finally, J147 is able to block extracellular amyloid toxicity using rat hippocampal neurons with an EC 50 of about 200 nM (Fig. 2F). J147 does not, however, bind to and directly inhibit Aβ 1–42 aggregation using the thioflavin S assay (not shown), as do its precursors curcumin and CNB-001 [8], [6]. Curcumin is only marginally active in two neuroprotection assays [8], the trophic factor withdrawal and Aβ toxicity assays, at the indicated concentrations (Fig. 2 A and F), while CNB-001 is significantly less effective than J147 in all assays. These data show that our drug discovery scheme is effective at identifying a highly potent neuroprotective molecule. J147 has neurotrophic and BDNF-like activities that are independent of the BDNF receptor, TrkB (Fig. 2B). BDNF is a molecule that is also involved in promoting memory, is reduced in the brain with age, and in AD as well as in other neurological disorders [19], [20], [21], and it has long been considered a drug target for AD [22].

J147 Enhances Long-Term Potentiation and Memory Given the association between memory, BDNF and AD, we first asked whether or not J147 could affect long-term potentiation (LTP) in hippocampal slices. LTP is considered a good model of how memory is formed at the cellular level. Although J147 had no direct effect on basal synaptic transmission in the CA1 area of rat hippocampal slices (Fig. 3A), it induced LTP in slices exposed to a weak titanic stimulation (15 pulses at 100 Hz), which by itself failed to induce LTP (Fig. 3B). The LTP facilitating effect of J147 was concentration-dependent in the range of 0.01–1 µM (Fig. 3C). An inactive analog of J147 (−187–88) in which the two nitrogens were replaced by carbons had no activity in this assay (Fig. 3D). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. J147 Facilitates the Induction of LTP in Schaffer Collateral CA1 Pyramidal Cell Synapses in Rat Hippocampal Slices. (A) Effect of J147 (1 µM) on basal synaptic transmission. Hippocampal slices were exposed to J147 during the time indicated by the black bar. The fEPSP slope is expressed as the percentage of the value immediately before the addition of J147. J147 does not affect basal synaptic transmission. (B) J147 facilitates the induction of LTP after a weak tetanic stimulation (15 pulses at 100 Hz) which alone does not induce LTP in control slices. The effect of J147 is concentration-dependent. Time course of changes in the fEPSP slope. The hippocampal slices were untreated (o, n = 9) or exposed to J147 (▾, 0.01 µM, n = 6; ▴, 0.1 µM, n = 5; •, 1 µM, n = 7) for the time indicated by the black bar and weak tetanic stimulation was applied at time 0. The fEPSP slope is expressed as the percentage of the value immediately before the application of weak tetanic stimulation. (C) Concentration-dependency. To compare the data among the groups, the averages of the fEPSP slopes 30–60 min after tetanic stimulation were calculated as an index of LTP magnitude. J147 demonstrated a concentration dependent effect with 1000 nM having the greatest effect on the fEPSP slope (n = 7 slices per rat: one-way ANOVA F(3,23) = 4.4, **P = 0.01). (D) Negative Control. −187–88, the alkene form of J147 in which nitrogens are replaced by carbons (o, n = 9; ▴, 0.1 µM, n = 5; •, 1 µM, n = 5) showed no effect. All data shown are means ± SEM. https://doi.org/10.1371/journal.pone.0027865.g003 It has recently become possible to identify early presymptomatic stages of AD in which there is minimal cognitive impairment, the best time to initiate treatment [23]. Current measures of outcome in AD clinical trials require a significant improvement in cognition for FDA approval, but none of the recently developed drugs showed such an effect in clinical trials and may require very long treatment durations to achieve significance. Therefore, we asked if J147 is active in promoting learning and memory following a short-term treatment using multiple assays in normal animals. These assays include the novel object recognition (NOR) test in rats, and the novel object location (NOL) test, Barnes and Y mazes in mice; they all measure various aspects of spatial learning and memory. The innate exploratory nature of rodents can be used to assess memory using the NOR and NOL tests [24], for both require habituation where the rodent becomes familiar with objects in certain locations. The habituation period is followed by the testing period where the animal is exposed to a novel object in a known location or a known object in a novel location. In the NOR test, during the habituation period rats were presented with two identical objects, which they explored for a fixed time period. Memory is assessed one day later when two objects are presented, one of which was presented previously during the training and is thus familiar; while the other object is new to them. It is hypothesized that memory for the familiar object results in more time exploring the novel object. J147 was administered orally to the rats before the start of the habituation period to assess its effects on memory, specifically consolidation of the familiar object. Figure 4A shows that oral administration of J147 only one hour prior to the habituation period enhanced memory of the familiar object in the rat NOR test. There was a statistically significant increase in the recognition index following all 3 doses of J147. A derivative of J147 that was inactive in the LTP assay, −187–88 (Fig. 3D), was also inactive in the NOR test (not shown). The positive control for this assay was the acetylcholinesterase inhibitor, galantamine, injected IP at 3 mg/kg. J147 is not, however, an acetylcholinesterase inhibitor or a phosphodiesterase inhibitor (PDI) as assayed against acetylcholinesterase and 12 PDIs at 10 µM, (MDS Pharma Services, not shown). The inhibition of both enzymes promotes memory and both are drug targets for memory enhancement [25], [26]. Therefore, the mode of action of J147 on memory may be distinct from the traditional memory enhancing drugs. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Behavioral Assays. (A) The novel object recognition test (NOR) was employed for compound J147. Doses of 1, 2 and 5 mg/kg were administered by oral gavage to 7 wk old Sprague-Dawley rats 1 hr prior to training. Treatment with J147 increased the recognition index with all three concentrations. Galantamine, injected IP at 3 mg/kg, served as a positive control, one-way ANOVA F (3, 40) = 12.9. *P<0.05 n = 12 per group (Done by Behavioral Pharma, La Jolla, CA). In panels B-J normal male C57BL/6J mice were fed J147 at 200 ppm for 2 wks and the Barnes maze assay was then done, which required 8 weeks to complete. This was followed by the novel object location (NOL) and Y-maze assays. (B) Novel object location (NOL) assay; the assay was performed similarly to (A) except that one object was used and it was moved to a new location instead of being replaced by another. J147 treated mice had significantly greater recognition indexes than control mice in this assay (n = 12 per group: one-way ANOVA F(1,22) = 2.5, *P<0.05). (C) Y-maze. The tendency of rodents to sequentially explore 3 arms of the Y-maze is a measure of short-term memory. While the groups did not differ in total arm entries, mice fed J147 exhibited a higher percentage sequential entries into all 3 arms (spontaneous alterations). F(1,22) = 3.3 p = 0.0032. (D) The error rate in the acquisition phase of the Barnes maze, 9 consecutive days, pooled data from 3 days per block. A lower error rate indicates better learning. The difference between control (white bar) and J147-fed mice (black bar) in Blocks 1 and 3 are almost significant (n = 12 per group: one-way ANOVA F(1,22) = 2 and F(1,22) = 1.8, respectively, P>0.05 for both). Six weeks after the final acquisition phase, mice were re-assayed and J147 significantly improved memory (retention test) (n = 12 per group: one-way ANOVA F(1,22) = 2.6, *P<0.01) but had no effect when the escape tunnel was relocated (reversal test). (E–J) The strategy used by the mice to find the escape tunnel was determined from videotapes and mice fed J147 tended to use a spatial strategy rather than sequential or random ones (z = 2.33, P = 0.019 for Block 3; z = 1.73, P = 0.80 for retention). All data shown are means ± SEM. https://doi.org/10.1371/journal.pone.0027865.g004 To explore the ability of chronic J147 treatment to stimulate various aspects of memory in young mice, they were fed J147 at 200 ppm (10–20 mg/kg/day) in their chow. After 2 wks on J147 chow, they were sequentially assayed in the Barnes maze, Y-maze and NOL. The NOL test relates to spatial short-term recognition memory where the exploration of a novel object over repeated trials and its subsequent change in location assays the ability of mice to build up spatial representations of their environment. J147 treated mice demonstrated a greater difference between the final trial with the old location and the trial with the new location (P = 0.0006 vs. P = 0.017) yielding a significant difference in the recognition index (F(1,22) = 2.5, P<0.05; Fig. 4B). This result demonstrates that J147 treatment resulted in an improvement in spatial memory in these mice. To assay the ability of J147 to alter another aspect of memory, we used the Y maze. The spontaneous tendency to alternate free choices in entering the 3 arms of the Y maze is a measure of short-term memory [27], [28]. The groups did not differ in overall numbers of arm entries, suggesting that activity levels were not altered by J147 treatment. However, J147 treated mice made a higher percentage of spontaneous alternations (defined as consecutive entries into the 3 different arms) in this test F(1,22) = 3.3, P = 0.0032) (Fig. 4C). These data indicate that J147 improves short-term memory. The Barnes maze assay is also a spatial learning and memory test [29]. It is less stressful and physically taxing than the Morris water maze and suitable for studies in all ages and abilities of mice. In addition, the strategies used by the animals to perform the task are readily revealed. Mice were trained to find an escape tunnel among 20 choices in a circular platform for 3 blocks of 3 days each for the acquisition phase. The retention test for long-term memory was done 6 weeks later with the escape tunnel in the same position. The escape tunnel was then moved to a new location and the behavior of the mouse recorded (the reversal test). Although there was no statistically significant differences between groups (F(1,22) = 2.9, P>0.05), there were trends toward decreased errors in the J147 group in block 1 (F(1,22) = 2.0, P = 0.057) and block 3 (F(1,22) = 1.8, P = 0.086) trials in the acquisition phase (Fig. 4D). Importantly, a greater proportion of J147-treated mice used a spatial strategy to locate the escape chamber in block 3 (z = 2.33, P = 0.019) (Fig. 4E and H). This difference appeared to hold during the retention (memory) test (Fig. 4F, I), where the J147 treated mice made significantly fewer errors than control mice (F(1,22) = 2.6, P = 0.01) (Fig. 4D). There was no group difference in errors made when the escape chamber was moved in the reversal test (Fig. 4D). However, a greater number of J147 mice utilized a sequential strategy during this test (Fig. 4G, J), suggesting that they adopted a working memory strategy in the face of change. J147-treated mice used more optimal strategies in the Barnes maze, resulting in superior spatial memory as evidenced in the retention test. Therefore, J147 meets the initial criterion of a drug for AD in that it has the ability to enhance several different aspects of memory with short-term treatment in multiple animal models.

J147 Prevents Memory Deficits in an Alzheimer's Disease Animal Model Since J147 is orally active in memory assays in normal animals, we asked if it could prevent the behavioral deficits in a transgenic mouse model of AD. The line 85 APPswe/PS1/ΔE9 transgenic AD mouse (huAPP/PS1) was used because there is an extensive literature on their pathology and behavior [30], [31], [32], [33]. There is good pathology (plaques and loss of synaptic markers) at 9 months along with clear deficits in the Morris water maze (MWM) assay. The transgenic mice were fed J147 in their chow at 200 ppm starting at 4 mo of age. This dose was chosen based upon the potency of J147 in the memory assays (Fig. 4). Since we already had extensive behavioral data on J147 in wild type rodents, control animals plus J147 were not included in these experiments. Mice were assayed for memory deficits in the MWM and for pathological and biochemical changes after 5 additional months on J147 or control diet. In the acquisition task portion of the MWM, an assay for learning [34], the transgenic mice showed deficits that were corrected by treatment with J147 (Fig. 5A). Since these were older animals, even the non-transgenic littermate control mice were relatively slow learners [35]; [36]. However, by day 5 the mice with J147 diet had learned the task, while the untreated AD mice did not. In the probe trial, an assay for spatial memory, there was also significant improvement, with the J147 diet restoring memory of the platform location to control levels (Fig. 5B). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. J147 Improves Learning and Short-Term Spatial Memory. Line 85 mice were fed J147 at 200 ppm between 3 and 10 months of age and then assayed for learning and memory in the Morris water maze. (A) Task acquisition phase. Data are expressed as standard deviation ± SEM and analyzed by ANOVA of repeated measures. F(2,14) = 8.3. **P<0.01 AD vs AD+J147; P<0.05 AD vs. control. (B) Probe test. One-way ANOVA F(2,54) = 16.6. **P<0.001 AD vs AD+J147; P<0.05 AD vs. control. These data show that both wild type and line 85 mice are slow learners, but that J147 corrects the deficit in both learning (day 5) and their spatial memory (probe test). (C) Aβ oligomers between 30 and 50 kD in soluble fraction from AD (left 2 lanes) and AD+J147 (right 2 lanes) animals. J147 treatment decreased these oligomers. Two-tailed P value, *P<0.05. Aβ 1–40 and Aβ 1–42 levels were measured by ELISA in control AD animals (open bars) and AD animals fed J147 (hatched bars) in the insoluble (100,000×g pellet) (D) and RIPA soluble fractions (E and F). J147 treatment decreased both soluble Aβ 1–40 and Aβ 1–42 levels. Two-tailed P values *P<0.05; **P<0.01; *** P<0.001. All data shown are means ± SEM. n = 6 to 9 mice per group. https://doi.org/10.1371/journal.pone.0027865.g005

J147 Reduces Soluble Aβ Levels in the Hippocampus Since some compounds that reduce memory loss in AD mice reduce Aβ plaque burden, we next examined plaque size and density as well as Aβ levels in the RIPA insoluble (100,000×g pellet) and soluble (RIPA supernatant) fractions of the hippocampi of J147 treated and control huAPP/PS1 mice. Neither Aβ 1–42 nor Aβ 1–40 levels as measured by ELISA, were altered in the RIPA insoluble fraction in animals fed J147 relative to untreated AD transgenic animals (Fig. 5D). Although there was no difference in plaque size between control and J147 treated animals, the average area occupied by plaques and the total number of plaques in each group was significantly different (Fig. 6). Therefore, while treatment with J147 enhanced the cognitive ability of the huAPP/PS1 mice, it had no significant effect on insoluble Aβ levels but a small but significant reduction of plaque load. However, it is now thought that soluble Aβ polymers are major contributors to the toxicity associated with the peptide [37]. Figure 5E and F show that the J147 diet reduces the amount of RIPA soluble Aβ 1–40 and Aβ 1–42 in the hippocampus and Figure 5C demonstrates a reduction in Aβ polymers, detected by the 6E10 antibody, in the 40–60,000 molecular weight range in the transgenic mice fed J147 relative to control animals on a drug-free diet. It follows that J147 has a small but significant effect on Aβ metabolism by reducing Aβ 1–42 in the soluble fraction of the hippocampus. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. Immunohistochemical analysis of condensed Aβ deposits. Immunohistochemical analysis was done using brain coronal sections of 13-month old AD mice on control diet (A) or J147 diet (B) using antibody 6E10. In comparison to AD control mice (A), condensed Aβ deposits were significantly reduced in the hippocampus region of AD mice on J147 diet (B). 30 micron thick sections of similar regions from each mouse, (n = 6) were examined and plaque counts in the hippocampus were quantified. All images were quantified using Image J Software. (C) The average of plaque counts for each mouse group is expressed as a number of plaques ± the SD (n = 6 per group: unpaired t-test, *P<0.05). (D) Reduced Aβ plaque load in AD mice on J147 diet. The Aβ plaque load was determined by measuring the area occupied by Aβ-immunoreactive condensed deposits, (n = 6 per group: unpaired t-test, *P<0.05). (E) The plaque size did not change between AD mice on control and J147 diets (n = 6 per group: unpaired t-test, *P>0.05). https://doi.org/10.1371/journal.pone.0027865.g006

J147 Reduces Oxidative Stress and the Inflammatory Response As outlined in the Introduction, the biochemical profile of the brain changes with age and many pathological aspects of the aged brain are enhanced in AD. Specifically, there is an increase in pro-oxidants and markers of oxidative stress as well as inflammation, and these have been associated with a decline in cognitive skills, both in AD and normal aging [38] [39]. At the same time, cells in the AD brain are trying to survive the pathological insults associated with the disease by increasing their neuroprotective mechanisms. It is the relative balance of the two processes that determines the fate of the individual cell. A successful AD drug should alter this balance in favor of survival, returning expression levels of the molecules involved to control values. To determine if J147 is able to alter any markers for oxidative stress in huAPP/PS1 transgenic mice, Western blots of hippocampal tissue were carried out to determine the expression levels of several stress-related proteins. Figure 7A shows that there is a large increase in heme oxygenase 1 (HO-1) in huAPP/PS1 mice that was decreased to control levels by J147. HO-1 is frequently thought of as an antioxidant enzyme [40], but it is elevated in AD brain and can act as a pro-oxidant under some conditions [41]. Microglia mediate a significant part of the inflammatory immune response in the CNS and are activated and increased in number in AD [39]. Biochemical markers for microglia include Iba-1 and inducible nitric oxide synthase (iNOS). Figure 7B shows that there is a significant reduction in the amount of Iba-1 in the hippocampus of J147-treated huAPP/PS1 mice relative to control huAPP/PS1 mice while the reduction of iNOS is not quite statistically significant due to the large mouse to mouse variability with this marker (Fig. 7C). Finally, 5-lipoxygenase (5-LOX) is responsible for the synthesis of leukotrienes that are potent mediators of inflammation and chemotaxis of immune cells [42], and are overexpressed in AD [43]. 5-LOX is highly upregulated in the huAPP/PS1 mice and brought down to control levels by J147 (Fig. 7D). Together these data suggest that J147 is able to modulate the level of oxidative stress and the inflammatory response in huAPP/PS1 mice. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. J147 Reduces Pro-oxidant Enzymes in the Brains of huAPP/PS1 mice. Cell lysates of hippocampal tissue from huAPP/PS1 and control mice were analyzed by western blotting and the images quantified. C = control; A = AD transgenic; A+ = AD transgenic fed J147. (A) Heme oxygenase-1 is increased in AD transgenic mice compared to control and these levels are decreased in mice fed J147. (B) Iba-1 levels are decreased in AD transgenic mice fed J147. (C) iNOS levels are not quite significantly decreased in AD transgenic mice fed J147. P = 0.053. (D) 5-LOX is significantly decreased in AD transgenic mice fed J147. *P<0.05, **P<0.01, and ***P<0.001. All data shown are means ± SEM, n = 5 per group. https://doi.org/10.1371/journal.pone.0027865.g007

J147 Reduces Heat-Shock Proteins and Increases Synaptic Protein Expression Oxidative stress damages proteins and degrades their ability to fold properly. To compensate, cells increase the synthesis of proteins that promote proper folding, including members of the large heat shock family. In AD, there is a large increase in the expression of the heat shock family of chaperones and the co-chaperone HOP [44]. Figure 8A–C shows that the levels of HSP70, 90 and the co-chaperone HOP are increased in huAPP/PS1 mice and decreased to control levels by J147. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 8. J147 Reduces Heat Shock Protein Stress Response and Increases Markers for Synaptic Function. Cell lysates from hippocampal tissue of line 85 mice fed J147 for 7 months were analyzed by Western blotting. C = control; A = AD transgenic; A+ = AD transgenic fed J147. (A) HSP90 levels are decreased in AD transgenic mice fed J147. (B) HOP levels are decreased in AD transgenic mice fed J147. (C) HSP70 levels are increased in AD transgenic mice and these levels are decreased with treatment of J147. (D) Drebrin levels are decreased in AD transgenic mice and J147 treatment restores levels to above control levels. (E) Synapsin-1 levels are decreased in AD transgenic mice and are restored significantly beyond control by J147. (F) Synaptophysin levels are decreased in AD transgenic mice and levels are restored beyond control levels with J147. *P<0.05, **P<0.01), and ***P<0.001. All data shown are means ± SEM, n = 5 per group. https://doi.org/10.1371/journal.pone.0027865.g008 To obtain some insight into the structural basis of the protection from cognitive loss afforded by J147, the expression of three major synaptic proteins was examined. Drebrin, synapsin 1 and synaptophysin were chosen because their loss has been correlated with both human AD and behavioral deficits in transgenic mouse lines [45]; [46]; [47]. Figure 8D–F shows that all three proteins are reduced in huAPP/PS1 mice and their levels brought to control levels or higher by J147.