Study On The Relation Of Abnormal Lipid Metabolism With Neuron Death And Alzheimer's Disease

AimPhospholipase A₂ (PLA₂) comprises a diverse family of enzymes that cleave the sn - 2 fatty acyl ester bond of glycerophospholipids to yield a free fatty acid and a lysophopholipid. PLA₂ participates in the pathophysiological processes by producing Arachadonic acid and precursors of various types of proinflammatory lipid mediators, such as, prostaglandins (PGs) , leukotriens (LTs) , thrombox-anes (TXs) , and platelet - activating factor. V subtype of secretory phospholipase A₂ (sPLA₂ - V) is abundant in many mammals and have identified biological functions. sPLA₂ - V mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high - affinity binding sites of sPL A₂ - V have been documented. However, it remains obscure whether sPL A₂ - V causes physiologic or pathologic response in the CNS. To this end, we examined effects of sPL A₂ - Von neuronal survival in primary cultures of rat embryonic cortical and rat cotical and hippocampal neurons. sPLA₂ - V induced neuronal cell death in a concentration -dependent manner. sPLA₂ - Vnot only liberated arachidonic acid (AA) and generated prostaglandin E₂ (PGE₂) from neurons, but also increased the expression of the receptor of PGE₂. The inhibitor of sPLA₂ - V significantly prevented neurons from sPLA₂ - V induced neuronal cell death. In conclusion, we demonstrate a novel neurobiological response of sPLA₂ - V in the CNS.In animal model, a direct correlation between cholesterol levels, brain APOE expression and the typical amyloid neuropathology found in AD has been" described. However human studies couldnt show such correlation, suggesting that additional important factors are involved in the regulation of APOE levels inthe human brain and development of the pathological features of brains with Alzheimer's disease. One Single Nucleotide Polymorphisms (491A/T,) in the proximal regulatory region of the human APOE gene promoter have been described to regulate the expression of APOE protein and to have a role in the development of AD. We will study that Apoe promoter polymorphism loci 491 AA frequency among AD patients. Research its correlation with high levels of cholesterol for amyloid accumulation in brain.Materials and methodsAnimal and reagents;Sprague - Dawley male and pregnancy female rat were used weighing from 200 g, all reagents were obtained from Sigma Company if we dont have special illustration. Indoxam were synthesized at the Dennis Research Laboratories. sPLA2 - V was obtained from Boehringer Mannheim. Ara-binosylcytosine C (Ara C) , and poly -1 - lysine were obtained from Sigma Eagle s medium (MEM) , Leibovitz's L - 15 medium, trypsin, fetal bovine serum (FBS) , horse serum ( HS) , penicillin, and streptomycin were obtained from Gibco. PGD21 PGE2, and LTB4 were purchased from Cascade Biochem Ltd. [3 H] AA and [ 3H] PGD2assay system was purchased from Amersham. [' I] PGE2 and [3H]LTB4 RIA kits were purchased from Dupont NEN DNAase were purchased from Promega. RNAeasy were purchased from Qiagen. The iScript First- strand cDNA Synthesis Kit were purchased from Bio - Rad. 10% Tris - HCL ready - made gels were purchased from Bio - Rad and transferred to nitrocellulose membranes were purchased from Amersham Pharmacia. An Odyssey infrared imaging system ( LI - COR) with Alexa Fluor 680 goat anti - rabbit IgG were purchased from Molecular Probes, Inc. or widely -used enhanced chemi-luminescence (ECL) system with ECL kit were purchased from Amersham RPN 2109. Primary antibody and secondary antibody (fluorolinkCy - labeled goat anti— rabbit IgG supplied by Amersham Pharmacia.Subjects and DNA Extraction The 132 AD and 132 other diseases patients autopsy after death. Genomic DNA wasextracted from the liver samples using GENEclean for Acient DNA kit (Q - BlOgene) and samples were stored at -20<€ until use. 132 unrelated consecutive cases of American nationality from 37 to 88 years old with AD were recruited from patients admitted to our Department of Neurology. Diagnosis of AD was made according to the International Classification of Disease. During the same time, by the principle of 1:1 matched case - control study, 132 controls free of AD and matched for age (within 3 years) , race,sex,history of hypertension and diabetes mellitus( yes or no) were enrolled from other departments of the same hospital. A detailed clinical questionnaire including present and past history, physical examination, clinical findings, personal history (BMI, smoking and drinking status) , family history of AD was obtained from every cases The study was approved by the local ethics committee and all participants gave their informed consents.Neuronal cell cultures were prepared from the cerebral cortex and hippocampus of day -4,8,11 Sprague Dawley rat embryos neuron were dissociated in isotonic buffer with 4mg/rnL trypsin and 0.4mg/mL deoxyribonuclease I. Cells were plated at a density of 3. 0 x 105 cells/cm2 on poly - 1 - lysine - coated dishes in conditioning medium, LeibovitzS L - 15 medium supplemented with 5% FBS, and 5% HS at 37T! in 5% CO2 incubator On day 1 after plating, cultures were treated with 0. 1 m Ara C. The present cultures contained at least 80% neurons. sPLA2 - V was sufficiently activated under our experimental conditions.Analysis of neuronal survival Neurons (3.0 x 105 cells/cm2) were treated with sPLA2 - V in the presence or absence of indoxam at 371. Two different methods were employed for assessment of neurotoxicity of sPLA2 - V. First, the LDH assay was employed. Second, residual cells were counted according to morphologic criteria;neurons with intact neurites and a smooth, round soma were considered viable, whereas those with degenerated neurites and an irregular soma were considered non - viable.Measurement of released [ H ] AA and generated eicosanoids Arachadonic acid Neurons (3.0X105 cells/cm2) were incubated for 24 h in culture medium containing 1 Ci/ml [3 H ] AA, washed twice with culture medium and treated with sPLA2 - V in the presence or absence of 10u,m indoxam at 37^. The radioactivity of [3 H ] AA in each supernatant was measured. Eicosanoids were ex-tracted according to the method. Supematants of culture medium (lml) were mixed homogenously with cold ethanol (4ml). The mixture was centrifuged at 1500g at 4^ for 10 min for removal of particulate matter. Supematants were diluted with an appropriate volume of distilled water to yield a final concentration of 10% ethanol, and the pH was adjusted to 3. 5 -4. 0. Samples were loaded onto reversed phase ( C18) Sep -Pack cartridges, which had been prepared by washing with ethanol, followed by distilled water. Samples were washed onto the Sep - Pak with 15mL of 10% aqueous ethanol, followed by 15mL of petroleum ether. Samples were extracted with 5 mL of methyl formate. The methyl formate effluents were pooled, evaporated with a heating module, and dissolved in RIA buffer. The samples were stored frozen until radioimmunoassay (RIA) analysis for PGD2, PGE2 and LTB4.RNA extraction Sprague - Dawley rats (15weeks) were anesthetized with ketamine and decapitated, immediately thereafter total RNA was extracted from hippocampus and cortex with TRI reagent from Molecular Research Center. RNA was treated with DNAase following recommended instructions before further purification with RNAeasy. Samples with ratios of OD 260 to 280 > 2. 0 were used for further experiments. Integrity of RNA was checked on 1% agarose gel.Real - time RT - PCR Samples were stored at - 80^! or processed directly by reverse transcription using the iScript First - strand cDNA Synthesis Kit. Forward and reverse primers were designed using Beacon Designer 2. 0. Each 25jxl PCR synthesis mix contained 12. 5jxl SYBR master mix 50 nM -300 nM primers, and approximately 5 ng templates. A 18s internal control (Taq-man) was used to normalize template variation. PCR conditions were 951 for 3 minutes, 42 cycles of 95^ for 30 seconds, 60^ for 30 seconds, and 72t for 30 seconds, and the melting curve was checked immediately after PCR. PCR products were confirmed by sequencing. PCR product was gel - extracted with a kit and quantified by spectrometer, on the basis of the A^ OD. Standard curves were made with the PCR product. Efficiencies of all standard curves were within the range between 0.97 and 1.02. Copies of DNA were counted with use of the equation 2x6. 023 x 1023 (C/MW, in which MW = base pairs x 6. 3 x 102, and C was the weight of standards in grams.Immunoblot Whole cortical and hippocampal tissues were extracted from deeply anesthetized animals and frozen immediately. Frozen samples were homogenized in a one - to - one volume of lysis buffer (consisting of a number of protease inhibitors, i. e. , trypsin inhibitor, leupeptin, PMSF, aprotinin, and 1% SDS) and sonicated and frozen again until use with no further processing. 1:1 Sample and Laemmli buffer mixture were run on 10% Tris - HCL ready -made gels and transferred to nitrocellulose membranes (Amersham Pharmacia). An Odyssey infrared imaging system (LI - COR) with Alexa Fluor 680 goat anti - rabbit IgG or widely - used enhanced chemiluminescence (ECL) system with ECL kit was applied to visualize the bands.Immunocytochemistry Animals were deeply anesthetized and transcardial-ly perfused with physiologic saline followed by 4% paraformaldehyde solution in 0.1 M phosphate - buffered saline (PBS, pH 7. 4). Brains were kept in the same solution for one day and then transferred to 30% sucrose in PBS. Slices were obtained orthogonal to the septotemporal axis to include subiculum and kept in PBS at 4 C until they were stained (4 to 20 days). Therefore tissue fixations were done only with 4% paraformaldehyde. During the simple staining procedure, slices were blocked with 10-20% horse serum for 1 hour, incubated with primary antibody overnight and with secondary antibody for 1 hour, washed with PBS (2-3% BSA) three times after each step. After final washes, slices were immediately mounted on slides and fixed under coverslips with a droplet of glyc-erol;fluorescence was observed under a microscope. Control staining was performed by the same staining procedure as the antibody staining with the exception of primary antibody incubation. As the primary antibodies were incubated, the control slices were kept in the blocking buffer.DNA AmplificationFirst - round PCR. Genomic DNA was amplified by PCR using primers spanning the promoter region of the human APOE gene. The primers (invitro-gen) had the following sequences: upstream 5' - etc cag aat cct aac ctt aac cca gaa g - 3';downstream 5' - gtc gag tec tec tat ggc tta cat c - 3'. First - round PCR was carried out using the following conditions: 2 min at 95X! (1 cycle, hot start);30 sec at 95^ , 30 sec at 60^ , 2 min at 12X, (40 cycles);5 min at12X, (1 cycle, extension). Run electrophoresed on a 1% agarose/1 xTBE gel to check product for this PCR spans 1274 bp in size relative to the transcription start site of the APOE gene.Second - round PCR Genomic DNA PCR products from first - round amplification were diluted 100 - fold with Primer sequences: upstream 5 ' — tea age gat tct cct gec tea g - 3' and downstream 5' - agt aat aca gac ace etc etc cat tc - 3'. PCR conditions and the check are same as first - round amplification. The expected product for this PCR spans 404 bp in size relative to the transcription start site of the APOE gene Total samples Second - round amplification DNA was sequenced.Third - round PCR. PCR products from Second - round amplification were diluted 100 - fold with a pair of " nested" primers. The primers had the following sequences: upstream ( mismatches underlined ) 5 ' -TCITCGCCAGGCTCGTTTTAA - 3 ';downstream 5 ' - CCTCCTTTCCTCAC-CCTCTCC - 3 '. The mismatched upstream primer, corresponding to bases -512 to -492 of the APOE gene, introduces Dral sites (5' -TTTAAA -3' ) in the PCR product amplified from individuals with one or more -491A alleles. Third - round PCR was carried out using the following conditions: 12 min at 94X (1 cycle, hot start);30 sec at 94X , 30 sec at SOX, 1 min at 72X (35 cycles);10 min at 721 (1 cycle, extension). After the program was complete , tubes were held at 4X until products were analyzed. To check the fidelity of this PCR reaction was electrophoresed on a 1.5% agarose/1 xTBE gel. The expected product was 228 bp in size.-491 and -427 Genotyping To Genotype the regulatory region of the APOE gene, 2. 5 ui of the Third - round PCR product was digested with both Dral and Alul or either Dral or Alul alone. The mixture was electrophoresed at 200 V on a 4 - 20% nondenaturing polyacrylamide gel/1 x TBE ( Bio - Rad) for 45 min. After electrophoresis, the gel was stained with SYRB in 1 x TBE for 30 min before being viewed by ultraviolet transillumination.The APOE [ -491, -427] haplotypes of subjects were determined from the pattern of restriction fragments on the gel: [ A -T], 144 and 65 bp;[T -T] 144 and 84 bp.Statistical analysis Statistical analysis of the data for multiple comparisons was performed by anova followed by T test.ResultsEffects of sPLA2 - Von cell survival Primary cultures of dissociated cortical and hippocampal neurons were exposed to sPLA2 - V, and neuronal cell death was quantified 24h later. There was a close correlation between LDH activity and morphologic criteria. sPLA2 - V caused neuronal cell death in a dose - dependent manner when cell viability was evaluated by LDH - activity. In addition, sPLA2 - V caused neuronal cell death in a developmental time - dependent manner.Effects of a sPLA2 inhibitor on sPLA2 - V induced neuronal cell death Indoxam, an indolizine derivative, was created as a novel sPLA2 inhibitor by the Dennis Research Laboratories. To test this possibility, we examined the effects of indoxam on sPLA2 - V induced neuronal cell death As shown indoxam prevented sPLA2 - V induced neuronal cell death in a concentration - dependent manner, indoxam suppressed neuronal cell death when applied within 6h after sPLA2 — V treatment.light microscopic changes in neurons by sPLA2 - V. Examination of cultures treated with sPLA2 - V by light microscopy showed some disruption of neurites at 24 h. In control cultures, neurons had extended neurites and smooth, round cell bodies. On the other hand, most cell bodies shrank and lost their neurites in cultures treated with sPLA2 - V. Although indoxam had no effect on control neurons, the inhibitor significantly ameliorated the morphologic disruption in neurons treated with sPLA2.sPLA2 - V induced liberation of AA We examined free [3 H ] AA release during sPLA2 - V - induced neuronal cell death. Neurons were incubated with [3H] A A for 24h for equilibration among phospholipids. Free [3H] AA was spontaneously released horn neurons into the medium. On the other hand;sPLA2 — V significantly increased the liberation of [3 H ] AA from neurons prior to neuronal cell death . The level of free [3 H ] AA (% of total incorporated [3H] AA) was dependent on the concentration of sPLA2 - V. Tliese results indicated that there was a close correlation between sPLA2 - V induced AA release and neuronal cell death.sPLA2 - V induced generation of eicosanoids sPLA2 - V hydrolyzes phos-pholipids in membranes to generate eicosanoids from various cells. Therefore, we ascertained whether sPLA2 - V generates eicosanoids from neurons. The basal levels of eicosanoids tested were not altered throughout the culture* When neurons were exposed to sPLA2 — V, generation of PGE2 was observed within 4h, increased transiently at 24h and returned to the basal level thereafter On the other hand, LTB4 increased slightly, but significantly, 6h after sPLA2 - Vtreatment. sPLA2 - V generated these eicosanoids from neurons in a concentration -dependent manner. Thus, the generation of PGD2, PGE2, and LTB4 was induced by sPLA2 - V from neurons, although their generating patterns differed from each other.Effects of eicosanoids on neuronal cell survival To determine how eicosanoids are involved in sPLA2 - V induced cell death, we examined the effects of various eicosanoids on neuronal survival . PGE2 caused neuronal cell death in a concentration - dependent manner.The effect of indoxam on sPLA2 - V inducing liberation of AA and generation of PGE2 Indoxam attenuated the sPLA2 - V - induced AA and PGE2 liberation and neuron death. At 10|xm, the inhibitor suppressed A A release completely by co - treatment with sPLA2 - V.Real - time RT - PCR allowed us to quantify the copies of EPj ₄ mRNA. They were EPj, EP2, EP3, EP4in rat hippocampus and cortex. Students t test was used to test hippocampus and cortex of different EP subtype, significantly meaning (P<0.05).Immunoblot data showed strongly immunoreactive bands at approximately 50 kDa for EP,, 52 kDa, 70 kDa, and 120 kDa for EP2, 45 kDa, 57 kDa, and 105 kDa for EP3, and 46 kDa for EP4. Samples were from rat hippocampus and neocortex as indicated.Immuohistochemical detection of EP receptors As shown in Figure, all four EP subtypes were detected in both hippocampus and cortical Neuron .Amplification results: First - round amplifications of the promoter region of the APOE gene produced several products of varying size, In each case the 1274 - bp product was apparent and the 404 - bp fragment was always amplified in the second round and the 228 - bp fragment was always amplified in the second round with the nested primers. Despite varying annealing temperature, primer concentration, and magnesium ion concentration, we have been unable to eliminate another band in first - round PCR because it is an internal fragment of the APOE promoter. Also, we have purified, cloned, and sequenced 404 bpfrag-ment.Enzyme Digest PCR product and DNA sequence Digestion of the 228 - bp PCR product with Dral and Alul together, alone, and no enzyme was used. Produce a range of different fragment lengths on the polyacrylamide it is matched the DNA sequence result. The polymorphisms in the regulatory region of the apolipoprotein E gene, -491 A/T have been reported to be associated with the risk of Alzheimer's disease (AD).Conclusions1. The different concentration of sPLA2 - V cause neuronal cell death in vitro. The degree of neuronal cell death is dependent dose of sPLA2 - V and aging of embryonic Neuron.2. The mechanism of sPLA2 - V inducing neuron death is AA liberation and the generation of prostaglandin E2 four subtypes receptors in rat hippocampus and cortical Neuron.3. APOE promoter polymorphism loci -491 AA has higher frequency a-mong AD patients. It will be correlated with high levels of cholesterol for amyloid accumulation in brain.