Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I

An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (gamma 1, kappa), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The construct (VL-linker-VH) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography. The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). The affinity constant was determined by a biosensor method using the BIAcore system. The results showed that the yield of correctly refolded scFv was more than 20 mg l-1 of E. coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74 x 10(-8) M (Kd). A notable thing is that guanidine-HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was. This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies.     

Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100

We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.     

Identification of sequence homology between human plasma apolipoprotein B-100 and apolipoprotein B-48

The structural relationship between apolipoprotein B-100 (apo-B-100) and apolipoprotein B-48 (apo-B-48) has not been elucidated. A peptide fragment (MDB-18) of approximately 6 kDa was isolated from a tryptic digest of apo-B-100. The sequence of the first 22 amino acids of MDB-18 was determined by Edman degradation. A 15-residue peptide corresponding to this sequence was synthesized by the solid-phase method and was utilized to develop a sequence-specific polyclonal antibody. On immunoblot analysis, the antibody recognized both intact apo-B-100 and apo-B-48. In addition, preincubating the antibody with the synthetic peptide abolished the recognition of both apo-B-100 and apo-B-48. These data are interpreted as indicating that there is an amino acid sequence homology between apo-B-100 and apo-B-48. Since the MDB-18 peptide is located in the carboxyl region of the B-100 molecule, we propose apo-B-100 and apo-B-48 share a common carboxyl region sequence.     

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody (MabB23) specific for human plasma apolipoprotein B-100

We have determined the nucleotide sequences encoding the heavy and light chains of the Fab fragment of murine monoclonal antibody MabB23 (γ2b, λ), which is specific for human plasma apolipoprotein B-100 of low-density lipoproteins. The sequence analyses revealed that the variable regions of the heavy and light chains are members of mouse heavy-chain subgroup I(B) and λ light-chain, respectively. A few unusual amino acids in the framework and constant regions of the heavy-chain were also noticed     

Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100.

A monoclonal antibody to apolipoprotein (apo) B-100 (JI-H) with unique binding properties has been used to separate a population of triglyceride-rich lipoproteins from blood plasma of normotriglyceridemic individuals and patients with various forms of hypertriglyceridemia. This antibody fails to recognize an apoE-rich population of very low density lipoproteins (VLDL) containing apoB-100 as well as all triglyceride-rich lipoproteins containing apoB-48, but it binds other VLDL that contain apoE and almost all lipoproteins that contain apoB-100, but no apoE. The unbound triglyceride-rich lipoproteins separated by ultracentrifugation after separation from plasma by immunoaffinity chromatography contained 10-13% of the apoB of triglyceride-rich lipoproteins from three normotriglyceridemic individuals, 10-29% of that from five patients with endogenous hypertriglyceridemia, 40-48% of that from three patients with familial dysbetablipoproteinemia, and 65% of that from a patient with lipoprotein lipase deficiency. In all cases, the unbound triglyceride-rich lipoproteins contained more molecules of apoE and cholesteryl esters per particle than those that were bound to monoclonal antibody JI-H, and they were generally depleted of C apolipoproteins. These properties resemble those described for partially catabolized remnants of chylomicrons and VLDL. The affinity of the unbound lipoproteins for the low density lipoprotein (LDL) receptor varied widely, and closely resembled that of the total triglyceride-rich lipoproteins from individual subjects. Our results demonstrate that remnant-like chylomicrons and a population of remnant-like VLDL can be isolated and quantified in blood plasma obtained in the postabsorptive state from normotriglyceridemic and hypertriglyceridemic individuals alike.     

Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.     

Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed inEscherichia coli as periplasmic inclusion bodies. Their variable heavy (V_H) and light (V_L) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II ofDrosophila melanogaster and rat MAb Yol1/34, specific for pig brain α-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv′)₂ was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv′)₂ have binding constants quite close to those of the parental MAbs and fourfold higher than scFv′ monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.     

Degradation of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins

Human plasma low density lipoproteins (LDL) contain one major apoprotein of apparent Mr = 550,000 designated apolipoprotein B-100 (apo-B-100) and in some LDL preparations, minor components termed apo-B-74 (Mr = 410,000) and apo-B-26 (Mr = 145,000). The structural and metabolic relationships among these LDL apoproteins remain obscure. In the present study, we show that the mixing of proteolytic inhibitors with blood at the moment of collection prevents the appearance of apo-B-74 and -26 in plasma LDL indicating that these peptides are derived by proteolytic degradation of apo-B-100. In order to simulate the degradation in vitro, LDL were digested with plasmin, trypsin, chymotrypsin, thrombin, and tissue and plasma kallikreins and the degradation products analyzed by polyacrylamide gradient gel electrophoresis. While plasmin, trypsin, and chymotrypsin caused extensive degradation of apo-B-100, thrombin, and tissue and plasma kallikreins generated limited cleavage patterns. LDL digested with thrombin contained stoichiometric amounts of two peptides with apparent Mr = 385,000 and 170,000. Mixing experiments showed that the thrombin-derived peptides of apo-B-100 did not co-migrate with apo-B-74 and B-26 during electrophoresis indicating that these peptides were different. In contrast, LDL digested with kallikrein contained stoichiometric amounts of two peptides with apparent molecular weights identical to apo-B-74 and -26. Together, the above results indicate that apo-B-74 and -26 are degradation products of apo-B-100 and are not produced by the action of thrombin. Whether the expression of a kallikrein-like activity in vivo accounts for the specific degradation of LDL B-100 to yield LDL B-74 and -26 remains to be determined.     

Distribution of lipid-binding regions in human apolipoprotein B-100

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.     

Bacterial expression and refolding of human trypsinogen

The expression of recombinant trypsinogens from different mammalian origins in Escherichia coli typically leads to the formation of insoluble aggregates. This work describes the high level expression of human trypsinogen 1 in E. coli using the T7 expression system. Direct expression of trypsinogen was not possible, but the N-terminal fusion of the first 11 amino acids of the T7 protein 10 resulted in an expression level of 200 mg g(-1) bacterial dry mass. A refolding procedure was optimized, and a method using continuous feed of denatured product was developed. Thus the working concentration of trypsinogen could be raised four-fold, while the yield of active protein could be maintained at 20-35%. The refolded trypsinogen was converted to trypsin by autocatalytic activation, and the utility for the detachment of mammalian cells in culture was proven.     

Bacterial cytoplasm production of an EGFP-labeled single-chain Fv antibody specific for the HER2 human receptor

The human epidermal growth factor receptor 2 (HER2) is the main diagnostic marker of breast and ovary cancers. Here, to obtain a rapid and sensitive immunodiagnostic tool a single-chain antibody (scFv800E6) specific for the HER2 was fused to the N-terminus of the enhanced green fluorescent protein (EGFP) by a flexible linker. The soluble production of the novel scFv800E6-EGFP protein in the cytoplasm of Escherichia coli was investigated at different induction temperatures (25, 30 and 37°C); the intrinsic fluorescent properties and the binding activity to HER2 positive tumour cells of the fusion protein were analysed. Western blotting and fluorescence analysis of SDS-PAGE revealed the presence of two scFv800E6-EGFP forms, with different mobility and optical properties, their ratio depending on the induction temperature. The fluorescent form maintained the optical fluorescence properties of EGFP and exhibited a binding activity to the HER2-expressing cells comparable to that of the non-fused scFv800E6. In addition, to provide an insight into the effect of the induction temperature on the molecular structure, the folding of the fusion protein was assessed at atomic level by performing molecular dynamics simulations of the homology-derived model of scFv800E6-EGFP at 300 K and 310 K. The comparison of the data collected at these two temperatures revealed that the higher temperature affects specific structural elements. To improve the production of the soluble and functional scFv800E6-EGFP protein, "in silico" results could be utilised for ad hoc design of the molecular structure.     

Functional expression of a single-chain antibody specific for the HER2 human oncogene in a bacterial reducing environment

Recombinant antibody fragments represent useful tools for cancer diagnosis and therapy. Aberrant expression of the HER2 receptor is implicated in metastatic breast and ovary cancers, two malignancies with a high prevalence in young women. In this study, we focussed on a single-chain fragment of variable antibody regions specific for HER2 (scFv800E6) that can be expressed in a functional form in the cytoplasm of Escherichia coli. ScFv800E6 was extracted from bacterial cultures following induction at different temperatures and purified. The yield of both soluble and insoluble forms was measured. We found that scFv800E6 was functional when expressed in the soluble fraction in the bacteria cytosol. In addition, scFv800E6 extracted from inclusion bodies was easily refolded and largely recovered its functionality. Thus, scFv800E6 is intrinsically capable of efficient and functional folding in a reducing environment and represents one of the few described antibody fragments with a framework well adapted for cytoplasmic expression.     

Unusually high reactivity of apolipoprotein B-100 among proteins to radical reactions induced in human plasma

Relative reactivities of proteins to radical reactions caused in human plasma were studied for the first time utilizing an immunoblotting assay. When radical reactions were caused by Cu(2+), apolipoprotein B-100 (apoB) underwent extensive fragmentation concurrently with the decrease in alpha-tocopherol, while human serum albumin (HSA) and transferrin (TF) were not decreased at all. When radical reactions were initiated by Cu(2+) with hydrogen peroxide or 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), alpha-tocopherol and apoB were also decreased steadily but HSA and TF were not decreased. These observations indicate that apoB is extremely reactive, even comparable to alpha-tocopherol, towards radical reactions. These results also suggest that the radical reaction of apoB is a possible process in vivo and it is involved in atherogenesis along with low density lipoprotein lipid peroxidation, which has been studied extensively.     

Bacterial expression and purification of the Fab fragment of a monoclonal antibody specific for the low-density lipoprotein receptor-binding site of human apolipoprotein E.

We report the bacterial expression and the purification of a monoclonal antibody (mAb) specific for an epitope that coincides with the LDL receptor (LDLr)-binding domain of human apolipoprotein E (apoE). This antibody resembles the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire light chain and the Fd portion of the heavy chain, was expressed in Escherichia coli and purified to homogeneity. Purification was facilitated by including a five-histidine carboxyl-terminal extension on the Fd chain. A 5- to 10-fold difference in yield of the antibody was observed when the plasmid was expressed in two different strains of E. coli. Typically 2-6 mg of rFab per liter of culture medium was recovered in the periplasm of the TG1 strain and less than 1 mg was recovered in the periplasm of the XL1-Blue strain. Culture temperatures above 35 degrees C or inclusion of sucrose in the medium reduced rFab yields. The 2E8 rFab was indistinguishable from Fab prepared from 2E8 hybridoma-generated IgG with respect to its affinity and fine specificity. We are using this system to express a panel of 2E8 variant Fabs that will be used as probes to establish the structural features responsible for the binding of apoE to the LDLr.     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30°C or 37°C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic \-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30°C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A ₆₆₀ at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.     

Expression of apolipoprotein B-100 in isolated human small intestine epithelium.

Apolipoprotein B-48 (apoB48) is synthesized in the small intestine and becomes a component of chylomicrons (CM). Apolipoprotein B-100 (apoB100) is synthesized in liver and becomes a component of both very low density lipoprotein (VLDL) and low density lipoprotein (LDL). To evaluate whether apoB100 is present in the human small intestine, we performed immunohistochemical staining using anti-apoB100 monoclonal antibody (mAb). Jejunal samples stained positive and the granular staining was noted in the supranuclear region of epithelial cells. We also identified apoB100 expression in the epithelial cells by immunoblotting and dot-blotting of PCR-amplified cDNA. In order to exclude submucosal stroma contaminated with blood, we used isolated epithelium from human small intestine obtained by a crypt isolation technique. The results indicate that not only apoB48, but also apoB100 are expressed in human small intestine epithelium. The expression of apoB100 suggests that the dietary VLDL may be synthesized in human small intestine epithelium and converted into LDL, which might play an important role in atherosclerosis.     

Overexpression of human apolipoprotein B-100 induces severe neurodegeneration in transgenic mice

Recent studies showed correlation between increased serum apolipoprotein B-100 (apoB-100) level and Alzheimer's disease. To reveal the possible role of apoB-100 in neurodegeneration, we analyzed the serum lipoprotein and cerebral protein profiles, amyloid plaque formation, apoptosis and brain morphology of transgenic mice overexpressing the human apoB-100 protein. Serum lipoprotein profile showed significant increase of the plasma triglyceride level, while no alteration in total cholesterol was detected. The antibody microarray experiment revealed upregulation of several cytoskeletal, neuronal proteins and proteins that belong to the mitogen activated protein kinase pathway, indicating active apoptosis in the brain. Histochemical experiments showed formation of amyloid plaques and extensive neuronal death. Biochemical changes severely affected brain morphology; a dramatic genotype-dependent enlargement of the third and lateral ventricles in the brain was detected. On the basis of earlier and present results, we conclude that overexpressed human apoB-100 protein significantly increases the level of serum lipids (triglyceride upon normal chow diet and cholesterol on cholesterol-rich diet) which leads to cerebrovascular lesions and subsequently induces apoptosis and neurodegeneration.