Jessica Joyce, Heidi Smith, and Jacqueline Curley. Page 2 of 8. In non-AD
elderly, the products of APP cleavage are returned to the plasma membrane,
IN SCHOOL ARTICLE
The Effects of Brain-Derived Neurotrophic Factor (BDNF), Local Cholesterol, and Celastrol on BetaAmyloid Neurotoxicity in an Alzheimer’s Disease Model
Jessica Joyce1*+, Heidi Smith1*, and Jacqueline Curley2 Student1, Teacher2: The Loudoun County Academy of Science, 21326 Augusta Drive Sterling, VA 20164 *These authors contributed equally +Corresponding author: [email protected]
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the loss of neural tissue and the accumulation of extracellular beta-amyloid (Aβ) plaques, leading to the degeneration of neurons via apoptosis. Increased local cholesterol in neuronal systems has been shown to result in the formation of neurotoxic beta-amyloid plaques. Brain-derived neurotrophic factor (BDNF) is essential for neuronal growth and regulation of synaptic plasticity and has been shown to promote cell survival despite the presence of Aβ plaques. Celastrol is a drug that affects beta-amyloid production in AD models. The effects of BDNF and celastrol on Aβ neurotoxicity after an increase in local cholesterol were investigated in this study. An IMR32 cell line was cultured with cholesterol treatment, celastrol treatment, BDNF treatment, and combination treatments. Beta-amyloid formation and cell survival was measured over a 12 day period. Celastrol treatment decreased cell survival and did not affect Aβ production compared to treatment groups, but celastrol treatment did decrease Aβ production when combined with cholesterol treatment compared to cholesterol treatment groups. A combined treatment of cholesterol, celastrol, and BDNF did not affect Aβ production but did increase cell survival compared to untreated groups. Cholesterol treatment resulted in increased production of Aβ and decreased cell survival. BDNF treatment resulted in decreased Aβ production and increased cell survival. Interestingly, BDNF and cholesterol combined treatment increased cell survival and decreased Aβ production beyond levels observed in untreated groups. The observed effects of cholesterol and BDNF suggests a synergistic mechanism through which cholesterol and BDNF decreases Aβ production and increases cell survival, warranting further investigation.
Neurodegenerative diseases are characterized by the loss of neural tissue. Among these diseases is Alzheimer’s disease (AD), which is characterized by dementia that impairs memory, thought, and behavior. Thus, victims of AD experience problems with language, decision-making ability, judgment, and personality. The cause of AD is not known, but factors increasing risk include old age, family history, chronic high blood pressure and cholesterol, as well as a history of head trauma1. Females are also
at a greater risk of contracting the disease1. The symptoms of AD are progressive and are categorized into early symptoms and late symptoms1. Early symptoms include short-term memory loss, lack of interest, and difficulty performing simple tasks requiring concentration1. As the disease progresses, gaps in memory become larger, and victims may become violent, depressed, or agitated1. They may also experience delusions and have difficulty performing everyday activities1. Symptoms of severe AD (late symptoms) include an inability to comprehend language, perform basic daily tasks and lack of recognition of family members1. Closely tied to the onset of Alzheimer’s disease is the accumulation of extracellular beta- amyloid (Aβ) plaques and neurofibrillary tangles2. These plaques and tangles lead to the degeneration of neurons via a process of programmed cell death called apoptosis2. This apoptosis is preceded by an inflammatory response of neurons to oxidative damage, at least partially caused by the accumulation of Aβ plaques3. Aβ proteins are formed by cleavage of the amyloid precursor protein (APP)3. Cleavage of APP differs from other secretase-dependent cellular processes in that it takes place in lipid rafts4. The APP is regularly processed at the cell surface of neurons and is also processed intracellularly after being trafficked into the cell by lipid rafts—specialized microdomains within plasma membranes that are rich in cholesterol and sphingolipid5. The role of lipid rafts is to regulate signal transduction and trafficking. Due to their small size (~20 nm), protein distribution, such as the processing of Aβ, in lipid rafts is observed biochemically (e.g. detergent-resistant membrane (DRM) isolation)5. After APP is brought into a cell by lipid rafts, it is cleaved by the enzymes β-secretase (Bace1) and γsecretase5. This cleavage produces a soluble N-terminal fragment (sAPPβ) and an amino acid amyloid β-peptide (Aβ)5. Normally, the amyloid precursor protein is trafficked into a neuron, where it is cleaved in one of two distinct pathways, one amyloidogenic and one nonamyloidogenic. In the amyloidogenic pathway, APP is trafficked into a neuron and cleaved by β-secretase 99 amino acids from the C-terminus4. This 99 amino acid peptide includes a hydrophobic region, coined the Aβ region4. Following this is further cleavage mediated by γ-secretase into a peptide usually 40 amino acids in length4. The enzyme, however, can alternately cleave the peptide of 99 amino acids into a peptide of 42 amino acids (Aβ42)4. Aβ42 is more hydrophobic than other isoforms of Aβ, and has a greater tendency to amass into fibrils and plaques4.
Jessica Joyce, Heidi Smith, and Jacqueline Curley In non-AD elderly, the products of APP cleavage are returned to the plasma membrane, where they are secreted6. In victims of AD, however, an increase in intracellular APP leads to a greater rate of production of the Aβ peptide, which then accumulates extracellularly to form insoluble, neurotoxic plaques5. The Aβ isoform most prevalent in these plaques is Aβ424. The hydrophobicity of these Aβ plaques contributes to their neurotoxicity; masses of extracellular Aβ interact with the neuronal membrane and disrupt lipid bilayer permeability4. Recent research discovered that an increase in cholesterol resulted in rapid endocytosis of APP, as well as the clustering of β-secretase enzymes and APP in lipid rafts4. This indicated that increased cholesterol levels may not directly cause an increase in the production of Aβ, but be an indirect cause of Aβ production by bringing the β-secretase enzyme and amyloid precursor protein in close proximity5. Further research has also shown that increased cholesterol binds Aβ to the lipid rafts, inducing membrane disturbances through oxidative damage4. In addition, the interaction of cholesterol and Aβ decreases membrane fluidity, resulting in greater processing of APP4. Celastrol has been the subject of recent investigations for its potential uses in treatments for AD3. Celastrol is a triterpene derived from Tripterygium wilfordii (also called the “Thunder of God Vine”), a plant indigenous to southern China. Celastrol is used in the treatment of various neurodegenerative diseases, and has also been found to possess anticancerous properties3. The use of celastrol in the treatment of AD is due to its phenol moiety and anionic carboxyl group3. Together, these groups reduce oxidative damage to cell membranes3. Celastrol is also an NFκB inhibitor3. NFκB proteins are located in the nucleus, where they regulate DNA transcription7. NFκB proteins have been found to regulate genes controlling the expression of Bace17. In this way, they are related to the production of Aβ7. Inhibition of NFκB results in the inhibition of Bace1 expression, which decreases the cleavage of APP into Aβ7. This decrease in APP cleavage and Aβ production prevents the formation of Aβ plaques, and thus decreases cell death due to Aβ toxicity7. Previous research investigating the use of celastrol as a possible treatment for AD has been conducted3. One study found that the anti-inflammatory properties of celastrol resulted in the decreased inflammation of cells and a reduction in cell death. It also found that the anti- inflammatory properties of celastrol were dosage dependent—oxidative damage and cell death decreased as celastrol concentration increased. Another investigation found that celastrol reduced the formation of Aβ in vivo7. The effects of celastrol were dosage dependent7. Another protein implicated in Alzheimer’s disease is the brain-derived neurotrophic factor (BDNF)8. The brain-derived neurotrophic factor monitors synaptic function and plasticity, and is essential for neuronal growth and survival8. Aβ accumulation can inhibit neurotrophic signaling, and BDNF signaling levels are significantly lower in AD transgenic mice models8. It has recently been discovered that Aβ also inhibits BDNF transport8. In a study on BDNF regulation in transgenic AD mouse models, it was seen that the cognitive decline observed after a reduction of BDNF was reversed by the addition of exogenous BDNF9.
Page 2 of 8 A study in 2009 conducted by Blurton-Jones et al., shows the effects of BDNF on AD transgenic mice models10. In this study, the introduction of neural stem cells (NSC) lessened the symptoms of AD. The presence of NSC increased BDNF levels, which resulted in an increase in synaptic density10. The increase in synaptic density observed in this study resulted in regained memory function10. Another recent study investigated the effect of BDNF against Aβ neurotoxicity both in vitro and in vivo in rats11. This study found that the addition of exogenous BDNF did not decrease Aβ plaque levels, but instead reduced the toxicity of Aβ plaques, completely removing neuronal death due to Aβ1-42 and reducing the toxicity of Aβ25-35 by eighty percent. Increased levels of BDNF also prevented natural neuronal death11. In addition, the protein was identified with possible antioxidative properties11. Previous research has investigated the method of Aβ plaque formation, its effect on neuron development6, the relationship between cholesterol levels and lipid raft endocytosis of APP5, and the relationship between Aβ, BDNF levels, and neuron health8. Current research includes investigations of the effect of BDNF on primates and in other neurodegenerative diseases8, as well as the effectiveness of overexpressing the CYP46A1 gene to modify cholesterol levels in order to reduce Aβ plaque formation12. This research addresses the problematic rapid endocytosis of the amyloid precursor protein (APP) and subsequent cleaving by β- and γ- secretase enzymes in nerve cells following an increase in local cholesterol, resulting in increased production of the beta-amyloid protein and formation of neurotoxic beta-amyloid plaques. The brain-derived neurotrophic factor (BDNF) has been shown to decrease cell death despite the presence of beta-amyloid plaques. Celastrol is a drug with antioxidative properties. Our research will investigate the suggested effects of BDNF, celastrol, and cholesterol levels on Aβ plaque formation and neurotoxicity to assess the effect of the introduction of BDNF and celastrol into an IMR-32 neuroblastoma model after an increase in cholesterol levels. IMR-32 neuroblastoma cells can be differentiated into nerve cells, and contain the TrkB receptor protein necessary for the function of BDNF13. IMR-32 also produces the Aβ plaques associated with AD. These characteristics make IMR-32 neuroblastoma cell lines an appropriate model for the study of the effects of neurotrophic factors and cholesterol on human neuronal processes14. One of the hypotheses tested in this study was if BDNF concentration is increased, then cell death will decrease due to decreased beta-amyloid toxicity. If local cholesterol is increased, cell death will increase due to an increase in beta-amyloid production. If celastrol concentration is increased, cell survival will increase due to a decrease in oxidative stress. A combined cholesterol and celastrol treatment should result in no change when compared to untreated groups, due to the reducing effect of celastrol on Aβ and the increase seen in Aβ after an increase in cholesterol. A combined treatment of celastrol and BDNF will result in decreased Aβ production and an increase in cell survival compared to untreated groups due to the effects of both celastrol and BDNF resulting in decreased Aβ production and BDNF’s effect increasing cell survival. A combined cholesterol and BDNF treatment will result in no change when compared to untreated
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groups, due to cholesterol’s effect to increase Aβ production and decrease cell survival and BDNF’s effect to decrease Aβ production and increase cell survival. This study tested the combined effects of BDNF, celastrol, and increased cholesterol on beta-amyloid production and neuronal survival. This research may prove useful in developing treatments for Alzheimer’s disease in the near future that would help to prevent Aβ plaque toxicity causing neuron degeneration and the subsequent dementia seen in the victims of Alzheimer’s disease.
Materials and Methods
An IMR-32 neuroblastoma cell line, obtained from the Coriell Institute for Medical research (Catalog ID GM03320), was used in all trials. An IMR-32 group with no treatment was used as a control group. Beta-amyloid production and cell survival values from this control group were used to determine optimal concentrations and treatment times of cholesterol (Sigma Catalog ID 69068-97-9), celastrol (Sigma Catalog ID 34157-83-0), and BDNF (Prospec (Protein Specialists) Catalog ID CYT-207). These optimized concentrations and treatment times were then used to conduct a combined treatment trials of BDNF and cholesterol, and celastrol and cholesterol. Six trials were conducted for each group. Optimization Trials: Three concentrations were tested for each treatment. Cholesterol was tested at 0.05 mM, 0.10 mM, and 0.15 mM concentrations. BDNF was tested at 50 ng/mL, 75 ng/mL, and 100 ng/mL concentrations. Celastrol was tested at 1.0 μM, 2.0 μM, and 3.0 μM concentrations. The optimal cholesterol concentration, resulting in the greatest Aβ production, was determined to be 0.10 mM. The optimal BDNF concentration, resulting in the greatest cell survival, was determined to be 75 ng/mL. The optimal celastrol concentration, resulting in the least Aβ production, was determined to be 2.0 μM. The data collected during optimization trials is provided in the Supplementary Data Tables, denoted by “Cholesterol Optimization Trials”, “BDNF Optimization Trials”, or “Celastrol Optimization Trials”. Cell culture: IMR-32 cell lines were cultured in 24-well plates using standard protocol according to ATCC15 [number: CCL-127] in order to establish a standard curve of untreated IMR-32 cells based on beta-amyloid productions per cell and cell survival. Once three well plates were seeded, cells were then cultured in a 5% CO2 incubator at 37°C. Measuring beta-amyloid production and cell survival:
Beta-amyloid formation was first measured in each well. Media was removed from each well in the well plate and filled with 1.0 mL of 1.0 μM Congo red, obtained from Sigma Aldrich [number: C6277], in phosphate buffered saline (PBS). After 20 minutes, the 1.0 μM Congo red solution was removed from each well, and a plate reader was used to measure absorbance of Congo red in each well. After absorbance values had been recorded, each well was rinsed with PBS and then digested with trypsin (Cellgro, Mediatech, Inc [number: 25053-CL]), using a standard protocol according to ATCC15 [number: CCL-127]. A hemacytometer and phase- inverted microscope were then used to conduct a cell count with trypan blue for each well in the plate. Aβ production and cell survival were measured in each group. These quantities were then used to determine “Aβ production” per cell values. These values can be found in Supplementary Data Tables 17-24. Aβ production per cell values and cell survival values were compared using ANOVA, Kruskall-Wallis, and Mann-Whitney tests with a 95% confidence interval. Analyzing values: Optimal concentrations and treatment times for cholesterol, celastrol, and BDNF were determined after analysis of optimization trial data with ANOVA tests. These optimal concentrations and treatment times were used during combined treatment trials. Cell counts and beta-amyloid production values were analyzed using parametric t-tests and nonparametric Kruskall-Wallis and MannWhitney tests. Results were analyzed using Minitab software16. Kruskall-Wallis and Mann-Whitney nonparametric tests were used due to the small sample size of treatment groups. Both Kruskall-Wallis and Mann-Whitney tests were used to provide nonparametric corollaries to both ANOVA and t-tests, respectively. The nonparametric tests, in every case, supported our conclusions made based on the results of similar parametric statistics. Control groups were values obtained from untreated IMR-32 groups, as each compound was diluted in untreated media.
Optimization trials were conducted to determine the cholesterol concentration that resulted in the greatest Aβ production, the BDNF concentration that resulted in the greatest cell survival, and the celastrol concentration that resulted in the least Aβ production. These optimal concentrations were determined to be 0.10 mM cholesterol, 75 ng/mL BDNF, and 2.0 μM celastrol. These concentrations were then used in combination treatments of cholesterol and celastrol treatment, cholesterol and BDNF treatment, celastrol and BDNF treatment, and cholesterol, celastrol, and BDNF treatment. Statistical analysis showed a significant increase in Aβ production (t-test: p = 0.021, Mann-Whitney: p = 0.014, Kruskall-Wallis: p = 0.011) (Table 1) and a significant decrease in cell survival (t-test: p = 0.010, MannWhitney: p = 0.020, Kruskall-Wallis: p = 0.016) following cholesterol treatment (Table 2). A significant change in
Figure 1. Cell morphology of IMR-32 with no treatment (control group).
Figure 2. Cell morphology of cholesterol treatment groups shows cells attaching but not spreading. The axons are not visible and cell growth is clearly stunted.
Jessica Joyce, Heidi Smith, and Jacqueline Curley
Table 1: Aβ production - cholesterol
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Table 2 Cell survival - cholesterol.
cell morphology was also noted following cholesterol treatment. As compared to untreated control groups of the same age (Figure 1), IMR-32 treated with cholesterol showed little evidence of cell spreading (Figure 2). Analysis of Aβ production and cell survival for BDNF treatment trials showed no significant change in Aβ production (t-test: p = 0.140, Mann-Whitney: p = 0.128, Kruskall-Wallis: p = 0.109) (Table 3) but exhibited a dramatic increase in cell survival (t-test: p < 0.001, Mann-Whitney: p = 0.005, Kruskall-Wallis: p = 0.004) (Table 4) and when compared to values for untreated control groups. As seen in Figure 3, IMR-32 treated with BDNF showed advanced signs of spreading compared to control treatment groups. Celastrol treatment of IMR-32 resulted in no change in Aβ production (t-test: p = 0.238, MannWhitney: p = 0.298, Kruskall-Wallis: p = 0.262) (Table 5) and decreased cell survival (t-test: p < Figure 3. Cell morphology 0.001, Mann-Whitney: p = 0.005, Kruskall-Wallis: p = 0.004) (Table 6) when compared to values for of IMR-32 BDNF treatment untreated control groups. As observed in Figure 4, cells treated with celastrol exhibited decreased groups shows that cell growth spreading, though did spread at rates greater than those observed in cholesterol treatment trials. The is significantly greater than decrease in cell survival was unexpected. Recent research indicates this negative effect of celastrol on cholesterol treatment groups. cell survival can be due to an alteration in the proteasome pathway17. After a combined treatment of IMR-32 with BDNF and cholesterol, a decrease in Aβ production (t-test: p = 0.026, MannWhitney: p = 0.013, Kruskall-Wallis: p = 0.010) (Table 7) and an increase in cell survival (t-test: p = 0.001, Mann-Whitney: p = 0.005, Kruskall-Wallis: p = 0.004) (Table 8) were observed when compared to untreated control groups. Cells treated with BDNF and cholesterol demonstrated a decreased rate of spreading when compared to Table 3. Aβ production – BDNF. morphology for untreated control groups (see Figure 5). Analysis of cholesterol and celastrol combined treatment data did not demonstrate a significant change in Aβ production (t-test: p = 0.301, Mann-Whitney: p = 0.471, Kruskall-Wallis: p = 0.423) (Table 9) or cell survival (t-test: p = 0.200, Mann-Whitney: p = 0.230, KruskallWallis: p = 0.200) (Table 10) when compared to values for control groups. As seen in Figure 6, cells treated with both cholesterol and celastrol exhibited decreased spreading patterns compared to those seen for untreated control groups. Table 4. Cell survival – BDNF. BDNF and celastrol combined treatment trials resulted in no significant change in Aβ production values (t-test: p = 0.757, MannWhitney: p = 0.936, Kruskall-Wallis: p = 0.873) (Table 11) or cell survival (t-test: p = 0.069, MannWhitney: p = 0.230, Kruskall-Wallis: p = 0.200) (Table 12) compared to those of untreated control trials. A decreased degree of spreading was also observed in BDNF and celastrol combined treatment groups when compared to control groups (see Figure 7). These observed effects were also supported by previous research exhibiting the individual effects of BDNF11 and celastrol18 on cell survival and Aβ neurotoxicity. Treatment of IMR-32 with cholesterol, BDNF, and celastrol resulted in no significant change in Aβ production values when compared to Aβ production values for untreated control groups (t-test: p = 0.456, Mann-Whitney: p = 0.689, Kruskall-Wallis: p = 0.631) (Table 13) and an increase in cell survival when compared to values for untreated control groups (t-test: p < 0.001, Mann-Whitney: p Figure 4. Cell morphology of = 0.005, Kruskall-Wallis: p = 0.004) (Table 14). Cells treated with cholesterol, BDNF, and celastrol IMR-32 celastrol treatment exhibited decreased spreading when compared to morphology for untreated control groups, as seen depicts cells that are not attaching and spreading out. in Figure 8.
Jessica Joyce, Heidi Smith, and Jacqueline Curley
Table 6. Cell survival – celastrol.
Table 5. Aβ production – celastrol.
Figure 5. Cell morphology of IMR-32 combined treatment groups of BDNF and cholesterol shows cells are attaching and spreading out.
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Figure 6. Cell morphology of IMR-32 combined treatment groups of cholesterol and celastrol shows cells are not spreading out.
Figure 7. Cell morphology of IMR-32 combined treatment groups of BDNF and celastrol shows cells are not spreading out.
Table 7. Aβ production – cholesterol & BDNF.
Table 8. Cell survival – cholesterol & BDNF.
Table 9. Aβ production – cholesterol & celastrol.
Table 10. Cell survival – cholesterol & celastrol.
Table 11. Aβ production – BDNF & celastrol.
Table 12. Cell survival – BDNF & celastrol.
Jessica Joyce, Heidi Smith, and Jacqueline Curley Discussion
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This research offers insight into the effects of BDNF on neuronal survival and Aβ production after an increase in cholesterol (typical in victims of AD)19, as well as the effects of celastrol after an increase in local cholesterol. Cholesterol treatment resulted in increased Aβ production and decreased cell survival when compared to untreated groups. These results supported known research and our hypothesis, which predicted that cholesterol treatment would result in increased Aβ production per cell and decreased cell survival due to an increase in endocytosis of the amyloid precursor protein5. BDNF treatment resulted in decreased Aβ production and increased cell survival when compared to untreated groups. The predicted increase in cell survival for BDNF treatment groups Figure 8. Cell morphology of was supported by both parametric and nonparametric statistical tests, as well as previous research11. IMR-32 combined treatment However, an unexpected decrease in Aβ production was observed. This decrease may have been groups of BDNF, celastrol, caused by inhibition of the Aβ production pathway by BDNF-triggered responses, and further and cholesterol shows cells are attached, but not spreading out. research is needed to elucidate this response. Celastrol treatment resulted in no change in Aβ production and decreased cell survival when compared to untreated groups. Previous research suggested that celastrol treatment would result in decreased Aβ production7. Our results, however, did not display a significant difference between Aβ production values for celastrol treatment groups and control groups. An unexpected decrease in cell survival was also observed. Recent research indicates this negative effect of celastrol on cell survival can be due to an alteration in the proteasome pathway17. Celastrol has been shown to critically alter p23 and HSP70 (heat shock protein 70)17, Table 13. Aβ production – cholesterol, BDNF, & celastrol. which assist in the degradation of proteins necessary for cell function. Alteration of these proteins after celastrol treatment may explain the observed cell death for celastrol treatment trials. Our methodology, however, does not elucidate either of these mechanisms. Cholesterol and BDNF combined treatment resulted in decreased Aβ production and increased cell survival when compared to untreated groups. These results were in concurrence with single treatment (cholesterol alone and BDNF alone) trials. Unexpectedly, Table 14. Cell survival – cholesterol, BDNF, & celastrol. however, was the statistical equality of cell survival values for BDNF and cholesterol treatment groups and BDNF single treatment groups. These results may be explained by the neutralization of the neurotoxic effects of cholesterol by BDNF. Synergistic effects of BDNF and cholesterol may explain the decrease in Aβ production compared to control groups despite the presence of increased cholesterol, though the mechanisms behind these effects warrant further investigation. Cholesterol and celastrol combined treatment resulted in no change in Aβ production and no change in cell survival when compared to untreated groups. These results supported the hypothesized effects of cholesterol and celastrol and Aβ production and cell survival, but were not supported by results for cholesterol single treatment (showing an increase in Aβ production and a decrease in cell survival) or celastrol single treatment groups (showing no change in Aβ production and a decrease in cell survival). These results suggest that cholesterol and celastrol may work synergistically to negate the neurotoxic effects of each substance individually. Our research, however, did not provide evidence for the mechanisms through which this negation may occur. Thus, the synergistic effects of celastrol and cholesterol on Aβ production and cell survival warrants further investigation. BDNF and celastrol combined treatment resulted in no change in Aβ production and no change in cell survival when compared to untreated groups. These results are in concurrence with results of both BDNF single treatment and celastrol single treatment groups, which demonstrated that celastrol decreased cell survival and did not affect Aβ production and that BDNF increased cell survival and decreased Aβ production. These observed effects were also supported by previous research exhibiting the individual effects of BDNF11 and celastrol18 on cell survival and Aβ neurotoxicity. Cholesterol, BDNF, and celastrol combined treatment resulted in no change in Aβ production and increased cell survival when compared to untreated groups. The observed increase in cell survival seen after cholesterol, BDNF, and celastrol treatment coincides with cell survival results of BDNF and cholesterol combined treatment trials. The return of Aβ production values to values statistically equivalent to those for untreated control groups is also supported by the observed effects of cholesterol single treatment, BDNF single treatment, and celastrol single treatment groups. The decrease in cell survival observed following celastrol treatment demonstrates that oxidative damage may not be the only mechanism through which Aβ plaque accumulation leads to neuronal apoptosis. Another possible reason for this decrease in cell survival may have been the alteration of p23 and HSP70 (heat shock protein 70), which assist in the degradation of proteins necessary for cell function, demonstrated in recent research17. Alteration of these proteins after celastrol treatment may explain the observed cell death for celastrol
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treatment trials. Our methodology, however, does not elucidate either of these mechanisms. Trials of celastrol and BDNF combined treatment and BDNF, cholesterol, and celastrol combined treatment further demonstrated the beneficial effects of BDNF on cell survival, even in the presence of celastrol (which decreased cell survival). This suggests the complexity of mechanisms behind Alzheimer’s disease pathology, and indicates that treatment of the disease must consider these interactions. The decrease in Aβ in cholesterol and BDNF treatment groups and increase in cell survival in cholesterol and BDNF treatment groups when compared to untreated control groups suggests that there are other factors contributing to the dramatic increase in cell survival that was observed, especially because cholesterol is known to increase Aβ production. A literature review of the effects of BDNF and cholesterol on neuronal processes led to formation of the hypothesis that adenosine receptors may have some role in the activation of BDNF20 and the reversal of Aβ neurotoxicity21. These beneficial effects of combined cholesterol and BDNF treatment certainly warrant further investigation. Continuation of this research could effectively be utilized by conducting repeated trials for each treatment group. Daily measurements of cell survival and beta-amyloid production in each group over a longer duration of time could also offer further insight into the effects of cholesterol, celastrol, and BDNF by determining the time at which these substances begin to affect IMR-32. Observing the effect of varying the time at which each treatment is first introduced and the number of times treatment is introduced over the period of time for which the cells are observed may determine the optimal time and frequency at which treatments may be introduced to most greatly increase cell survival. Future research regarding celastrol may be conducted investigating mechanisms other than oxidative damage via which Aβ plaques cause cell death. Other possible topics of future research include investigating the factors affecting the endocytosis of APP into neuronal cells, mechanisms through which BDNF increased cell survival, factors involved in the mediation of BDNF release, pathways through which celastrol affects beta-amyloid production, and effects of celastrol on specific proteins that may lead to cell death. Current experimentation by these researchers investigates the mechanisms resulting in the effects observed for BDNF and cholesterol combined treatment groups. The study will specifically examine the role of A2A and TrkB receptor proteins in response to increased cholesterol and BDNF concentrations, and the effect of these responses on Aβ neurotoxicity and neuronal survival. Recent research indicates that BDNF interacts with both of these transmembrane proteins20. These responses will be observed via measurement of intracellular and extracellular glutamate concentrations and extracellular acetylcholine concentrations. Previous research has not addressed the effects of A2A and TrkB inhibition and activation after an increase in cholesterol and BDNF. In summary, cholesterol treatment increased Aβ production and decreased cell survival. BDNF treatment decreased Aβ production and increased cell survival. Celastrol treatment did not change Aβ production but decreased cell survival, suggesting negative effects of celastrol that have been observed in other research17. Combined cholesterol and celastrol treatment resulted in no change in Aβ production and no change in cell survival compared to untreated groups, suggesting that cholesterol and celastrol may work synergistically to negate the neurotoxic effects of each substance individually. Combined celastrol and BDNF resulted in both Aβ production and cell survival returning to levels statistically equivalent to those observed in untreated groups, in concurrence with results from the individual BDNF treatment and celastrol treatment groups. Combined cholesterol, BDNF, and celastrol treatment returned Aβ production levels to those observed in untreated groups, but did result in increased cell survival, coinciding with the observed effects of BDNF and cholesterol combined treatment trials. The return of Aβ production values to values statistically equivalent to those for untreated control groups is also supported by the observed effects of cholesterol single treatment, BDNF single treatment, and celastrol single treatment groups. Interestingly, combined cholesterol and BDNF treatment decreased Aβ production and increased cell survival when compared to untreated groups, suggesting beneficial synergistic effects of the two compounds that warrant further investigation.
1. Amieva, H., Le Goff, M., Millet, X., Orgogozo, J. M., Pérès, K., Barberger-Gateau, P., … Dartigues, J. F. (2008). Prodromal Alzheimer’s disease: Successive emergence of the clinical symptoms. Annals of Neurology, 64, 492–498. doi: 10.1002/ ana.21509 2. Lefort, R. (2011). Investigating the role of the amyloid precursor protein in the pathogenesis of Alzheimer’s disease. New York: Columbia University. 3. Allison, A.C., Cacabelos, R., Lombardi, V. R. M., Alvarez, X. A., & Vigo, C. (2001). Celastrol, a potent antioxidant and antiinflammatory drug, as a possible treatment for Alzheimer’s disease. Progressive Neuro-Psychopharmacological & Biological Psychiatry, 25, 1341-1357. 4. Lai, A.Y., & McLaurin, J. (2011). Mechanisms of amyloid-beta
peptide uptake by neurons: the role of lipid rafts and lipid raftassociated proteins. International Journal of Alzheimer’s Disease, 2011, 11. 5. Marquer, C., Devauges, V., Cossec, J.-C., Liot, G., Lecart, S., Saudou, F., … Potier, M. (2011). Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis. The FASEB Journal, 1295-1305. 6. Groemer, T. W., Thiel, C. S., Holt, M., Riedel, D., Yunfeng, H., Huve, J., … Klingauf, J. (2011). Amyloid precursor protein is trafficked and secreted via synaptic vesicles. PLoS ONE, 6(4): e18754. doi:10.1371/ journal.pone.0018754 7. Paris, D, Ganey, N. J., Laporte, V., Patel, N. S., Beaulieu-Abdelahad, D., Bachmeier, C., March, A., Ait-Ghezala, G., & Mullan, M. J. (2010). Reduction of β-amyloid pathology by celastrol in a transgenic mouse
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