Study Of The Correlation Between Cholesterol And Aβ Production

Alzheimer's disease (AD) is a most common neurodegenerative disease in the elderly, which is characterized with deposition ofβ-amyloid peptide (Aβ) outside neurons. The reasons for Aβproduction are required for the prevention and treatment of AD.Aβis produced primarily fromβ-amyloid precursor protein (AβPP) through beta-site amyloid precursor protein cleaving enzyme 1(BACE1) cleavage. Increased cholesterol in brain or neurons has been reported to be one of reasons for Aβproduction. Higher levels of cholesterol promoted BACE1 activity to increase Aβproduction in neuron cells; reversely, lower levels of cholesterol decrease Aβproduction.Serum cholesterol is also one of factors which are related with Aβproduction. High cholesterol diet-mediated hypercholesterolemia promoted Aβproduction and deposition in the brain of transgenic mouce; treatment with statins attenuates brain Aβproduction in transgenic mouse. It is well known that cholesterol can not go through blood-brain barrier, so we are wondering whether brain cholesterol is related with serum cholesterol-mediated Aβproduction.Luteinizing Hormone (LH) is one of the most important hormones in hypothalamic-pituitary-gonadal (HPG) axis. Recently, it is found that LH is significantly increased in AD patients'blood and brain and BACE1 activity and Aβare both increased in neurons after LH treatment. LH can bind to LH receptor to regulate cholesterol analysis in sexual gland. LH receptor also exists in brain. Based on these findings, we are wondering whether cholesterol is related with LH-mediated Aβproduction.To elucidate the role of cholesterol in above-mentioned conditions, in this project, we observed brain or cellular cholesterol levels after treated Wistar rats with high cholesterol diet (PartⅠ) or statins (PartⅡ) altering serum cholesterol and treated neuroblastoma M17 cells with LH (PartⅢ) to result in the change of brain or cellular Aβ. This project was divided into two parts: PartⅠHypercholesterolemia increasing brain Aβlevels does not alter brain cholesterol amountPurpose: to study the role of brain cholesterol in serum cholesterol affecting Aβproduction, brain cholesterol levels were observed after serum cholesterol was elevated in Wistar rats treated with high cholesterol diet and brain Aβlevels were changed.Methods: A total of 18 healthy Wistar male rats used in this study. Rats were randomly assigned into normal-chow group (NC, n=9) and high-cholesterol-chow group (HCC, n=9), chow diet containing 2% cholesterol and 10% fat. After raised for total 13 weeks, rats were killed. Serum was used for serum cholesterol quantification assay; brain tissues were used for brain cholesterol quantification assay, total RNA extract and brain Aβquantification assay.Results:1 High cholesterol diet elevated serum cholesterol concentrationsSerum cholesterol concentrations were detected by automatic biochemistry analyzer. Compared with NC, serum cholesterol levels in HCC was significantly increased (3.15±0.62 mmol/l vs 1.62±0.10 mmol/l,p<0.001), confirming the induction of hypercholesterolemia by high cholesterol diet.2 Elevated serum cholesterol increased endogenous AβlevelsMeasurement of total Aβwas performed by radioimmunoassay. Compared with NC, Aβlevels in HCC were significantly increased (315.23±73.06 pg/mg protein vs 266.01±36.41 pg/mg protein, p<0.05). The levels of brain Aβwere positively correlated with the levels of serum total cholesterol (r=0.89, r2=0.78, p<0.001), suggesting that elevated serum cholesterol increased brain Aβlevels.3 No effects of elevated serum cholesterol on brain cholesterol levelsCholesterol levels were determined by CHOD-PAP method. Compared with NC, brain cholesterol levels were found no changes in HCC (5.96±1.26 mg/10g vs 6.08±0.51 mg/10g, p>0.05), suggesting that elevated serum cholesterol had no effects on brain cholesterol levels.4 No effects of elevated serum cholesterol on mRNA expression of genes involving in brain cholesterol metabolismRT-PCR was performed to detect mRNA expression of genes involving in brain cholesterol metabolism. Brain cholesterol is balanced by de novo synthesis which is catalyzed by HMG CoA reductase (HMGR) and conversion catalyzed by cholesterol 24S-hydroxylase (CYP46) to 24S-hydroxycholesterol. Compared with NC, HMGR (0.58±0.05 vs 0.54±0.03, p>0.05) and CYP46 (0.57±0.01 vs 0.61±0.02, p>0.05) mRNA expression levels were found no changes in HCC, suggesting that elevated serum cholesterol had no effects on mRNA expression of brain cholesterol metabolism-related genes which proved further that serum cholesterol had no effects on brain cholesterol.Conclusions:1 The levels of brain Aβwere positively correlated with the levels of serum total cholesterol.2 Elevated serum cholesterol increasing brain Aβproduction was brain cholesterol-independent.PartⅡReduced serum cholesterol decreasing brain Aβproduction does not alter brain cholesterol levelsPurpose: to study the role of brain cholesterol in serum cholesterol affecting Aβproduction, brain cholesterol levels were observed after serum cholesterol was reduced in Wistar rats treated with fluvastatin and brain Aβlevels were changed.Methods: A total of 20 healthy Wistar male rats used in this study. Rats were randomly assigned into control group (Con, n=10) and fluvastatin group (FV, n=10). Fluvastatin was suspended in 1% carboxymethyl cellulose and given by oral gavage at a final dose of 20 mg/kg/day for 28 d. The control group was given equivalent vehicle. After rats sacrificed, serum was used for serum cholesterol quantification assay; brain tissues were used for brain cholesterol quantification assay, total RNA extract, brain Aβquantification assay and AβPP immunohistochemistry.Results:1 Fluvastatin reduced serum cholesterol concentrationsSerum cholesterol concentrations were detected by automatic biochemistry analyzer. Compared with Con, serum cholesterol was significantly decreased in FV (1.13±0.10 mmol/l vs 1.53±0.13 mmol/l,p<0.05), confirming the induction of hypocholesterolemia by fluvastatin.2 Reduced serum cholesterol decreased endogenous AβlevelsMeasurement of total Aβwas performed by radioimmunoassay. Compared with Con, Aβlevels were significantly decreased in FV (213.36±45.21 pg/mg protein vs 142.53±27.48 pg/mg protein, p<0.05). The levels of brain Aβwere positively correlated with the levels of serum total cholesterol (r=0.54, r2=0.24, p<0.05), suggesting that reduced serum cholesterol levels decreased brain Aβlevels.3 No effects of reduced serum cholesterol on brain cholesterol levelsCholesterol levels were determined by CHOD-PAP method. Compared with Con, brain cholesterol levels were found no changes in FV (6.32±0.97 vs 6.00±0.58, p>0.05), suggesting that reduced serum cholesterol had no effects on brain cholesterol levels.4 No effects of reduced serum cholesterol on mRNA expression of genes involving in brain cholesterol metabolismRT-PCR was performed to detect mRNA expression of genes involving in brain cholesterol metabolism. Compared with Con, HMGR (0.57±0.05 vs 0.53±0.05, p>0.05) and CYP46 (0.88±0.06 vs 0.99±0.12, p>0.05) mRNA expression levels were found no changes in FV, suggesting that reduced serum cholesterol had no effects on mRNA expression of brain cholesterol metabolism-related genes which proved further that serum cholesterol had no effects on brain cholesterol.5 Reduced serum cholesterol affected the expression of Aβ? production-related genes RT-PCR and immunohischemistry were performed to detect the expression of Aβproduction-related genes. Compared with Con, Brain AβPP protein expression and BACE1 mRNA expression were decreased andα-secretase (ADAM10), the enzyme involving in alternative AβPP cleavage which does not product Aβ, mRNA expression was increased, suggesting that reduced serum cholesterol-mediated brain Aβreduction was related with altered expression of AβPP and AβPP processing-related enzymes.Conclusions:1 The reduced brain Aβlevels were positively correlated with reduced serum total cholesterol.2 Serum cholesterol reducing brain Aβproduction was brain cholesterol-independent.PartⅢLH promotes Aβproduction and increases cellular cholesterol in neuron cellsPurpose : to clarify the role of cholesterol in LH regulating Aβproduction.Methods: Neuroblastoma M17 cells were treated with LH for 5d. Medium was collected to perform secreted Aβquantitative analysis; cells were collected to perform cellular Aβquantitative analysis, cholesterol assay and mRNA expression of genes involving in cholesterol synthesis and Aβproduction.Results:1 Secreted and cellular Aβlevels were increased after LH treatmentAβlevels in medium were quantified by radioimmunoassay. Secreted and cellular Aβwas significantly increased in dose-dependent manner, suggesting that LH promoted A? ?production in neuron cells.2 Cellular cholesterol was increased after LH treatmentCellular cholesterol was increased after LH treatment and positively correlated with total A??levels, suggesting that cholesterol might have a role in LH promoting A? p?roduction.3 mRNA expression of HMGR was increased after LH treatment The mRNA expression of HMGR, the rate-limiting enzyme in cholesterol synthesis, was detected by real-time PCR. HMGR mRNA expression was increased after LH treatment, suggesting that increased cellular cholesterol might be related with increased HMGR mRNA expression.4 mRNA expression of SREBP2 was increased after LH treatmentThe mRNA expression of SREBP2, the upstream regulator of HMGR, was detected by real-time PCR. SREBP2 mRNA expression was increased after LH treatment, suggesting that increased HMGR mRNA expression might relate with increased SREBP2 expression.5 LH affected mRNA expression of genes involving in AβproductionThe mRNA expression of Aβproduction-related genes was detected by real-time PCR. The mRNA expression of BACE1, which involves inβ-secretase cleavage of AβPP, was increased, but ADAM10 involving inα-secretase pathway and AβPP mRNA expression had no changes after LH treatment, suggesting that LH-mediated increased Aβlevels might be related with increased BACE1 mRNA expression.Conclusions: LH might promote cholesterol synthesis through SREBP2-HMGR-cholesterol pathway, thereby increasing Aβlevels in neuron cells.Summary:1 Brain Aβlevels change as serum cholesterol, but this kind of change of Aβis brain cholesterol-independent.2 LH increasing Aβlevels in neuron cells is cholesterol-dependent.3 Factors which lead to altered brain Aβare not all related with brain cholesterol. Neuroblastoma (NB) is the most common extracranial solid malignant tumor in childhood. It is composed of undifferentiated sympathetoblast, which make it hard to cure and easy to relapse. Chemotherapy resistance is one of primary reasons for patients'poor prognosis. Thus, better understanding of drug resistance mechanisms has been required.The p53 tumor suppressor induces apoptosis in response to cell stresses as a transcription factor. It is thought that p53 inactivation contributes to sensitivity to chemoradiation therapies in a large number of tumors. Although the molecular mechanisms of p53 inactivation in NB have not been completely elucidated, the role of HDM2 has been paid much attention in the inactivation of NB.HDM2 is a major negative regulator of p53 function. HDM2 functions as an ubiquitin-protein isopeptide ligase and facilitates degradation of p53 by the proteasome and also can block the transactivation ability of p53 by direct protein-protein interaction. It has been reported that excess expressed HDM2 could dramatically inhibit p53-mediated apoptosis in wile-type p53 NB cells and that small molecular inhibitor nutlin-3 disturbing the interaction between HDM2 and p53 recovered p53 activities in p53-wild type tumors. From these data, it can be speculated that higher expression of HDM2 might result in drug resistance through interacting with p53 and inhibiting apoptotic cell death.Noxa is an important pro-apoptotic effector which induces NB cell apoptotic death after doxorubicin (Doxo, a commonly used antitumour drug for the treatment of cancers, including neuroblastoma) treatment in a p53-dependent manner. These results promoted us to wonder whether HDM2 has a role in p53-related apoptotic cell death by regulating Noxa expression or by regulating proteasome-mediated p53 degradation.To clarify the role of HDM2 in drug resistance of NB, we divided this project into two parts and employed several NB cell lines as objects. First, we identified Doxo-resistant NB cells in which HDM2 and p53 expression were observed; then, we studied the correlation between HDM2, apoptotic cell death and drug resistance by knocking down HDM2 expression in Doxo-resistant NB cells and by over-expressing HDM2 in Doxo-sensitive NB cells; Next, we observed the interaction of HDM2 and p53 in resistant cells; finally, we elucidated the detailed mechanism of HDM2 resulting in drug resistance in NB. Our results provided experimental evidence for development of new therapies for NB.PartⅠThe study of correlation between HDM2 and NB drug resistanceObjective: to identify Doxo-resistant NB cells in which HDM2 expression was observed and study the correlation between HDM2, apoptotic cell death and drug resistance by knocking down HDM2 expression in Doxo-resistant NB cells and by over-expressing HDM2 in Doxo-sensitive NB cells; to confirm the interaction of HDM2 and p53 in resistant cells; to clarify whether HDM2-related apoptotic cell death and drug resistance is through promoting p53 degradation by observing p53 levels after treating cells with a proteasome inhibitor MG132.Methods: cell death was detected by WST assay in SK-N-SH, NB-9, IMR32 and NB-19 cells 24h after 0.1μg/ml Doxo treatment; HDM2 and p53 expression were detected by western blot and RT-PCR in above-mentioned cells lines; SK-N-SH cells were infected by retrovirus vector expressing HDM2 and selected by G418 to make HDM2 over-expressing stable cell line and sub-G0/G1 fraction analysis was performed 24h after Doxo treatment; p53 wild-type IMR32 and NB-19 and p53 mutated SK-N-DZ cells were transfected with HDM2 siRNA to knock down HDM2 expression, morphological analysis after DAPI staining was performed 24 hours after Doxo treatment; immunoprecipitation was performed in IMR32 and NB-19 cells; p53 amount was detected by western blot in IMR-32, NB-19, SK-N-SH and HDM2-overexpressing SK1 cells after MG132 treatment.Results: 1 IMR32 and NB-19 cells were Doxo-resistantCell death was obvious in SK-N-SH and NB-9 cells but rare in IMR32 and NB-19 cells, suggesting that IMR32 and NB-19 cells are Doxo-resistant and SK-N-SH and NB-9 are Doxo-sensitive.2 HDM2 had higher expression in Doxo-resistant NB cellsHDM2 mRNA and protein expression were both higher in Doxo-resistant IMR32 and NB-19 cells than Doxo-sensitive SK-N-SH and NB-9 cells, suggesting that HDM2 has higher expression in Doxo-resistant NB cells.3 HDM2 overexpression in Doxo-sensitive cells inhibited apoptotic cell deathApoptosis rate was lowered after HDM2 overexpression in Doxo-sensitive SK-N-SH cells, suggesting that HDM2 overexpression in Doxo-sensitive cells inhibited apoptotic cell death.4 HDM2 knockdown sensitizes resistant NB cells to Doxo treatmentApoptosis rate was elevated after HDM2 knockdown in Doxo-resistant IMR32 and NB-19 cells, suggesting that HDM2 knockdown sensitizes resistant NB cells to Doxo treatment.5 p53 had higher expression in Doxo-resistant NB cellsp53 mRNA and protein expression were both higher in Doxo-resistant IMR32 and NB-19 cells than Doxo-sensitive SK-N-SH and NB-9 cells, suggesting that p53 has higher expression in Doxo-resistant NB cells.6 HDM2-mediated apoptosis inhibition was p53-dependentApoptosis rate was elevated after HDM2 knockdown in Doxo-resistant IMR32 and NB-19 cells but no changes in p53 mutated SK-N-DZ cells, suggesting that HDM2-mediated apoptosis inhibition was p53-dependent.7 HDM2 interacted with p53 in Doxo-resistant NB cellsHDM2 was immunoprecipitated after crosslinking using p53 antibody and p53 was immunoprecipitated after crosslinking using HDM2 antibody in Doxo-resistant IMR32 and NB-19 cells, suggesting HDM2 interacted with p53 in Doxo-resistant cells.8 Proteasome-mediated p53 degradation was inhibited in Doxo-resistant NB cells p53 amount was not increased after MG132 treatment in Doxo-resistant IMR32 and NB-19 cells, suggesting that proteasome-mediated p53 degradation was inhibited in Doxo-resistant cells.9 p53 was degradated through proteasomes in Doxo-sensitive NB cells p53 amount was increased after MG132 treatment in Doxo-sensitive SK-N-SH cells, suggesting that p53 could be degradated through proteasomes in Doxo-sensitive NB cells.10 Proteasome-mediated p53 degradation was inhibited after HDM2 overexpression in Doxo-sensitive cellsp53 amount was not increased HDM2 overexpression in Doxo-sensitive SK-N-SH cells, suggesting that HDM2 overexpression could inhibit proteasome-mediated p53 degradation.Conclusion:1 Higher expressed HDM2 interacts with p53 in p53 wild-type Doxo-resistante NB cells which makes cells resistant to apoptosis and chemotherapy drugs.2 The negative regulation of higher expressed HDM2 in p53 wild-type Doxo-resistant NB cells is not related with proteasome-mediated p53 degradation.PartⅡThe molecular mechanism of HDM2 resulting in drug resistance in NBObjective: To elucidate molecular mechanism of HDM2 resulting in drug resistance in NBMethods: Noxa expression was detected by western blot and RT-PCR 24 hours after Doxo treatment when p53 wild-type IMR32 and NB-19 and p53 mutated SK-N-DZ cells were transfected with HDM2 siRNA to knock down HDM2 expression; after SK-N-SH cells infected by retrovirus vector expressing HDM2 and selected by G418 to make HDM2 over-expressing stable cell line, p53, p53 phosphorylation, p53 target genes p21 and Noxa expression were detected by western blot and p21 and Noxa expression was detected by RT-PCR 24 hours after Doxo treatment; ChIP and luciferase assay were performed in SK-N-SH and HDM2-overexpressing SK1 cells 24 hours after Doxo treatment,Results:1 HDM2 overexpression in Doxo-sensitive NB cells inactivated p53Compared with SK-N-SH cells, the amount of p53 was not increased, p53 phosphorylation was inhibited, the expression of p53 target genes p21 and Noxa were both inhibited in HDM2 overexpressed SK1 cells, suggesting that HDM2 overexpression in Doxo-sensitive NB cells inactivated p53.2 Noxa expression was increased in Doxo-resistant cells after HDM2 knockdownNoxa mRNA and protein levels were both increased in Doxo-resistant cells after HDM2 knockdown. Unlike wild type-p53 NB cells, Noxa expression was not increased in p53 mutated SK-N-DZ cells, suggesting that HDM2-regulated Noxa expression was p53-dependent.3 Noxa expression was inhibited in Doxo-sensitive cells after HDM2 overexpressionCompared with Doxo-sensitive SK-N-SH cells, Noxa mRNA and protein levels were both inhibited in HDM2 overexpression SK1 cells, suggesting that p53 transcriptional activities might be inhibited by higher expressed HDM2.4 HDM2-regulated Noxa expression was p53-dependentUnlike wild type-p53 NB cells, Noxa expression was not increased in p53 mutated SK-N-DZ cells, suggesting that HDM2-regulated Noxa expression was p53-dependent.5 HDM2 overexpression inhibited the binding of p53 to Noxa promoterCompared with non-HDM2 overexpressed SK-N-SH cells, the binding of p53 to Noxa promoter was inhibited in HDM2 overexpressed SK-N-SH cells.Conclusion: Higher expressed HDM2 interacts and inactivates p53, inhibiting p53 transcriptional activity to its target gene Noxa, down-regulating Noxa expression, thereby inhibiting apoptosis and resulting in the resistance of NB to Doxo. Summary: Higher expressed HDM2 inactivates p53 and inhibites p53 transcriptional activity to its target gene Noxa, down-regulating Noxa expression, thereby inhibiting apoptosis and resulting in the resistance of NB to Doxo which is the mechanism of drug resistance in p53 wild-type NB cells.