Albert Sun⁽¹⁾, Bozena Draczynska-Lusiak⁽²⁾, Grace Sun^{(3)
(1)(2)(3)}Department of Pharmacology. University of Missouri - Columbia. United States

INTRODUCTION

Recent studies have demonstrated the involvement of oxidative stress in the pathogenesis of Alzheimer’s disease and Ab and apoE have been implicated as the key factors contributing to these oxidative events (Lyras et al. 1997; Sayre et al. 1997; Mattson, 1998; Mattson and Pedersen, 1998; Behl 1999a). ApoE are found in amyloid plaques, neurofibrillary tangles and vasculatures of autopsied AD brain. Lipoproteins (LP) in the central nervous system (CNS), particularly those associated with apolipoprotein E (apoE), are known to play important roles in support of many brain functions. For example, they are known for their ability to mediate intercellular lipid transport, promote neurite outgrowth, maintenance of cholesterol homeostasis and repair of membrane during injury (Ignatius et al. 1986; 1987; Holtzman et al., 1995; Fagan-Niven et al., 1996; Poirier et al., 1993; Beffert et al, 1998). Lipoproteins in cerebral spinal fluid (CSF) are of the high-density type and are different in lipid composition as compared to those in the circulatory system (Pitas et al., 1987a,b). Lipoproteins released from astrocytes contain high levels of phospholipids and cholesterol but low levels of triglycerides and cholesterylesters (LaDu et al., 1998). There is evidence that LP released from astrocytes are taken up by neurons through a receptor-mediated endocytotic process (Fagan-Niven et al., 1996).

We contemplate the possibility that in AD brain, the oxidative environment results in LP oxidation and exacerbates the progression of the disease. In our recent studies, we observed the ability of oxidized low-density lipoproteins (LDL) in serum to enhance oxidative stress and apoptotic cell death in PC12 cells (Draczynska-Lusiak et al. 1998 a,b). Oxidized LDL has also been implicated to cause cell differentiation, inflammation and cytotoxicity in embryonic neuronal cells (Keller et al, 1999a). Due to the high content of polyunsaturated fatty acids (PUFA) in brain membranes (Sun and Sun, 1976), it is possible that the phospholipids in brain LPs also contain high PUFA and are more susceptible to oxidative stress. In agreement with the increased oxidative stress in AD brain, there is evidence that LPs in cerebral spinal fluid of AD patients are more vulnerable to oxidation (Bassett et al., 1999). Therefore, an objective for this study is to test the hypothesis that oxidized LPs are more readily taken up and internalized by neurons and that oxidized LPs are cytotoxic and can induce neuron cell death.

Accumulation of neuritic plaques and amyloid beta peptides (Aß) are important pathological landmarks of AD (Strittmatter et al., 1993 a,b; Price et al., 1999). Amyloid beta peptides with 39 to 43 amino acid residues are derived from the amyloid precursor protein (APP) through cleavage by secretases. APP is a transmembrane protein present in both neurons and glial cells in the brain. While the cellular function of APP is still unknown, accumulation of Aß, especially in their aggregated form, is known to cause a number of cytotoxic events and exacerbate oxidative stress in the brain (Mattson and Pedersen, 1998). Under normal conditions, soluble Aß can be detected in the CSF and plasma at levels between 10^-8 to 10^-10 M. However, when Aß peptides (1-40 and 1-42) are converted to their fibrillar form, these peptides can enhance the production of reactive oxygen species (ROS), resulting in protein carbonyl formation and lipid peroxidation, and subsequent alteration of cellular functions (Yatin et al, 1999; Huang et al, 1999ab). Despite the pro-oxidative properties of aggregated Aß, whether this form of Ab can exacerbate the cytotoxic effect of oxidized LP on neurons has not been examined in detail. In this study, LPs were prepared using lipids extracted from the brain and subsequently enriched with apoE3 or apoE4. The brain LPs (BLP) were then subjected to oxidation and both native and oxidized BLPs were used to test their uptake by primary neurons in culture in the presence and absence of Ab ^(1-42) and their ability to induce cell death. These results provide evidence supporting the notion that aggregated Ab can exacerbate the cytotoxic effect of oxidized BLP.

MATERIALS AND METHODS

Primary cortical neuronal culture

Mixed neocortical neuronal cell cultures, containing mainly neurons and a small amount of glial cells, were prepared from fetal rats at 18-19 days of gestation using protocols as described by Cheng and Sun (1994). Dissociated cortical cells were plated in Eagle’s minimal essential medium (MEM) supplemented with 20 mM glucose, 2 mM glutamine and 10% fetal bovine serum. When the culture cells reached confluency, non-neuronal cell division was arrested by exposing the culture to 10 mM cytosine arabinofuranoside for 1-3 days. Cultures were maintained at 37^oC in a humidified incubator containing 5% CO₂ and atmospheric oxygen. In most instances, cells were used for experiments after maintaining in culture for 10-14 days.

Preparation of aggregated beta amyloid peptide (Ab)

Preparation and aggregation of Ab was similar to that descrebed by Hu et al. (1998). Briefly, 1 mg of Ab ( 1-42 ) (BACHEM, King of Prussia, PA) was first dissolved in 100 ml dimethysulfoxide (DMSO) and then diluted 10 fold with sterile distilled water. Ab suspension was then aged at room temperature for 4 days. To verify aggregation, an aliquot of the aged Abwas examined under an inverted microscope (Nikon Diaphot 300, Nikon Corp. Melville, NY). Aggregated Abs formed a turbid solution and when examined under the microscope, aggregates of beta sheet conformation could be observed.

Preparation of lipoproteins with brain lipids (BLPs).

For extraction of lipids, rat brain was homogenized with phosphate-buffered saline (PBS) and partitioned with 4 vol of chloroform-methanol (2:1, v/v). After evaporating the solvents under nitrogen, the lipids were hydrated in a medium composed of 0.1 M KCl, 0.01 M Tris-HCl (pH 8.0) and bovine serum albumin (BSA, 1mg/ml). This mixture was sonicated and purified as described by Rensen et al. (1997). Briefly, BLPs were concentrated by density gradient ultracentrifugation using NaBr/EDTA density solution at 43,500 rpm for 18-22 hr at 4^oC. The lipoprotein band was isolated by aspiration. BLPs were then divided into two fractions, one was subjected to overnight oxidation with 0.1 M FeCl₃ (Evans et al. 1997). Both native and oxidized BLP were subjected to dialysis overnight against a phosphate buffered saline-EDTA solution. ApoE e3 and apoE e4 were initially gifts from Dr. Holtzman (Washington University, St. Louis, MO) and later purchased from PanVern Corp, Madison, WI). Native and oxidized BLPs (0.5 mg/ml) were incubated with apoE (10 mg/ml) at 37^o for 30 min to obtain the apoE3- or apoE4-enriched BLPs.

Methods to assess uptake of BLPs by primary cortical neurons

1. Confocal microscopy using the fluorescent probe 3,3-dioctadecylindocarbocyanine (Di-I): Native and oxidized BLPs were labeled with 50 ml of Di-I (3mg/ml) at 37^oC for 15 hr and dialyzed with saline solution containing 0.01% EDTA according to procedures described by Pitas et al. (1981). Neuronal cells were plated on the coverslips and treated with the Di-I labeled native or oxidized BLPs for 3-4 hr. After incubation, the cells were washed five times with PBS containing 2 mg/ml BSA and once with PBS, and then fixed for 30 minutes with 3% paraformaldehyde in PBS (pH 7.4). Confocal and fluorescence microscopy was performed with epifluorescent illumination using a rhodamine filter package.
2. Labeling with [¹⁴C]cholesterol: In this preparation, a known amount of [¹⁴C] cholesterol was added to the lipid extract and the preparation of BLP was performed as described above. Both native and oxidized BLPs were enriched with apoE e3 or apoE e4, and were incubated with primary neurons for various time periods. After incubation, neuronal cells were washed two times with PBS, suspended in 0.5 ml of water and the amount of radioactivity incorporated into the cells was counted using a Beckman’s 5800 liquid scintillation counter (Beckman, Sunnyvale, CA).

Lactate dehydrogenase (LDH) release assay.

Neuronal cell death was determined by assessing the release of lactate dehydrogenase (LDH) into the medium using the spectrophotometric method described by (Cheng et al, 1994). Briefly, 0.2 ml of the culture medium was added to a phosphate-buffered pyruvate (0.6 mM, pH 7.5) (Sigma Chem Corp., St. Louis, MO) and followed by addition of NADH to initiate the reaction. The assays were carried out at 25^oC and changes of absorbency at 340 nm were measured using a Beckman spectrophotometer equipped with a kinetic program.

Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Neuronal cell survival was also assessed by the MTT reduction assay protocol. This method is based on the reduction of the yellow MTT (Sigma Chem Corp., St. Louis, MO) to its blue formazan product by dehydrogenases in the mitochondria. The amount of formazan produced is proportional to the number of living cells (Li and Sun, 1999). Cells were treated with native and oxidized BLPs and after an appropriate time (24-48 hr), MTT was added (0.5mg/ml) and incubated for 3 hr at 37oC. Upon removal of the media, blue formazan produced was solubilized in DMSO and measured with a spectrophotometer at 540 nm. Results were expressed as percent of control.

RESULTS

Uptake of native and oxidized BLP by primary cortical neurons

Using native and oxidized BLPs enriched with apoE3 or apoE4 and labeled with the fluorescent probe Di-I, we examined uptake of these BLP by primary cortical neurons. Micrographs in Fig 1 show a small uptake of native BLPs into neurons, regardless whether they are enrichment with apoE3 (A) or apoE4 (C) (see pink color). However, when oxidized BLP were presented to the neurons, large amounts of the BLPs were taken up regardless whether they are enriched with apoE3 or apoE4.

Uptake of BPLs by neurons was also examined using BLPs constructed with brain lipids containing [¹⁴C]cholesterol. Data in Fig 2 showed a time-dependent increase in uptake of labeled BLPs by neurons. In agreement with the uptake of Di-I BPL, oxidized BLPs were taken up more readily by the neurons than native BLPs.

Neuronal cell death induced by oxidized BLPs

Experiments were carried out to determine whether increased uptake of oxidized BLPs by neurons was associated with enhanced neuronal cell death. Data in Fig 3 show the results using the MTT reduction assay. In this experiment, native BLPs (enriched with apoE4) exposed to neurons for 48 hrs showed no decrease in neuronal cell survivability as compared with controls. However, when oxidized BLPs were added to neurons for the same period of time, neuronal survivability was significantly reduced by 40% (Fig 3). We also tested the effect of resveratrol, a polyphenolic compound known to ameliorate oxidant stress in cells (Chanvitayapongs et al., 1997). When neurons were pretreated with resveratrol (0.5 mM) prior to exposure to oxidized BLPs, resveratrol was able to protect the neurons completely from cell death induced by oxidized BLP.

Similar results were obtained using lactate dehydrogenase (LDH) release was use as an index of cell death. As shown in Fig 4, exposure of neurons to oxidized but not native BLP resulted in an increase in cell death. Furthermore, pretreatment of neurons with resveratrol could similarly reduce cell death due to oxidized BLPs (Fig. 4).

Neuronal cell death mediated by Aß and BLPs

In this study, we examined the effects of Aband oxidized BLPs on neuron cell death. When aggregated Ab were added to the neurons, honeycomb-like structures could be seen in the medium as well as attached to the neurons. Based on results of MTT assay, exposure of neurons to aggregated Aß (1-42, 2 mM) for 24 hr resulted in a decrease in MTT by 23% (Fig 5). Native BLP did not alter cell viability and did not decrease cell viability when incubated with Ab. However, addition of Fe²⁺/DTP (50 mM) to Ab together with native BLP could give slightly greater reduction of MTT as compared to that mediated by Ab alone. However, cells exposed to oxidized BLP showed MTT reduction (24%) similar to that mediated by Ab alone. Nevertheless, when neurons were exposed to Ab together with oxidized BLPs, MTT reduction (40%) was greater than those due to Ab and oxidized BLP alone. These results indicate that Aß and Fe²⁺ may cause additional oxidative stress to the cells, and Aß may potentiate the damaging effects of oxidized BLP.

Representative micrographs of primary cortical neurons exposed to Ab and/or oxidized BLP are shown in Fig 6. In panel A, control neurons are bright and show strong connections. When neurons were exposed to Ab (Panel B) or oxidized BLP (Panel D) for 24 hr, cells became dull and neurites became thin and started to disintegrate (see arrows). The combined treatment of Aß and oxidized BLP (Panel C) resulted in complete disappearance of neurites and extensive cell death.

DISCUSSION

It is recognized that oxidative stressors such as ionizing radiation, inflammatory agents, excitotoxic insults, stroke and ischemia, or release of transition metal ions (Fe³⁺, Cu²⁺), may cause production of reactive oxygen species (ROS) in the brain (Sun and Chen, 1998). Lipoproteins are likely targets of the oxidative insult. For example, increase in oxidized LPs was found associated with traumatic brain injury (Borovic et al., 1995). Our previous studies further demonstrated that oxidized low-density lipoproteins (LDL) from human plasma could damage these cells and cause apoptotic cell death (Draczynska-Lusiak et al. 1998ab; Sun and Chen, 1998). In this study, we tested LPs prepared with lipids extracted from the brain and enriched with human recombinant apoE (Resen et al. 1997). Ultracentrifugation of the BLP preparation showed that these LP had the characteristic high density similar to those isolated from CSF (Pitas et al., 1987a,b). Our results show that regardless of enrichment with apoE3 or apoE4, BLP could be taken up by primary neurons. However, oxidized BLPs were more effectively taken up by neurons that the native BLPs. Furthermore, the greater uptake of oxidized BLP is associated with their ability to induce neuron cell death. Taken together, studies by our laboratory (Draczynska-Lusiak et al. 1998ab) as well as those from others (Keller et al., 1999a, b, c) well indicate the ability of oxidized LPs to be taken up by neurons and subsequently cause cytotoxicity to these cells.

An important pathological landmark of Alzheimer’s disease is the accumulation of neuritic plaques surrounded with activated glial cells and degenerating neurons (Price et al. 1998). There is immense interest to understand mechanisms that can regulate the production of Ab and its interaction with apoE. Aßs have been shown to disrupt neuronal ion homeostasis (Yankner 1996), increase ROS production and cause oxidative damage to neurons (Butterfield 1997; Mark et al., 1999). Aß peptides (39 to 43 amino acids) are derived from the amyloid precursor protein (APP) present in both neurons and glial cells in the brain. While the cellular function of APP is yet to be elucidated, this protein is comprised of a hydrophobic membrane-spanning domain, N-glycosylation sites, and sites for binding Zn²⁺ and Cu²⁺ with high affinity (Hesse et al. 1994). Copper is present at substantial levels in the brain and its release as a result of synaptic activation can reach as high as 100 mM in the synaptic cleft (Kardos et al. 1989). The interaction between APP and Cu²⁺ may result in the reduction of Cu²⁺ to Cu⁺ and the formation of an intra-molecular disulfide bond (Mucke et al., 1994). In the presence of oxidant stressors such as H₂O₂, the APP-Cu interaction may cause APP fragmentation and increase in the production of Ab (Smith et al. 1997). Under normal conditions, Abs appear to be normal products of APP metabolism and are present in CSF and plasma. Although the exact molecular mechanism underlying neurotoxicity of Aß is still unknown, Abin their fibrillar form have been shown as a direct source for ROS production (Huang et al. 1997; 1999). Studies by these investigators gave evidence for a direct interaction between Aß and Fe³⁺/Cu²⁺ to create a strong positive formal reduction potential, which can rapidly reduce Fe³⁺ and Cu²⁺ ions and trap molecular oxygen to generate H₂O₂. These results suggest that Aß, in the order of Aß 1-42> Aß 1-40, may directly contribute to the oxidative stress in the AD brain. In this study, we examined the effects of aggregated Aß ^(1-42) on the survivability of neuronal cells. Our data are in agreement with the notion that aggregated Aßs are cytotoxic and can cause neuron cell death (Fig 5). Furthermore, more extensive neuronal damage can be observed when aggregated Aßs were added to neurons together with oxidized BLPs. Lipoprotein oxidation is known to result in the generation of a number of products such as 17 oxycholesterol, acylhydroperoxide, malonyldialdehyde, and 4-hydroxynonenal. In our study, oxidation of BLP resulted in the decrease in phospholipids, particularly, ethanolamine plasmalogen, as well as the PUFA content of these phospholipids (data not shown). These lipid peroxidation products are likely the major factors underlying neuronal cell death induced by oxidized LP (Chanvitayapongs et al 1997; Sun and Chen 1998; Neely et al., 1999; Keller et al., 1999b).

Antioxidants such as Vitamin E have been used to ameliorate oxidative damage in AD brain (Behl 1999; Mattson and Goodman, 1995). Our recent studies have focused on the polyphenolic flavonoid compound, resveratrol, which is an amphipathic molecule capable of ameliorating oxidative stress in both cytosol and membrane compartments in the cells. This molecule is effective in scavenging free radicals due to its ability to form a stable resonance structure. Resveratrol has been the active ingredient in red wine that provides protective effects on cardiovascular diseases (Fremont, 2000). In the peripheral system, resveratrol has been shown to inhibit LDL oxidation and to prevent the cytotoxicity of modified LDL (Frankel et al., 1993). In our recent studies with PC12 cells, resveratrol was shown to protect oxidative injury due to t-butyl hydroperoxide (Chanvitayapongs et al., 1997). Resveratrol can also inhibit NF-kB/AP-1 activation and apoptotic cell death induced by oxidized lipoproteins (Draczynska-Lusiak et al., 1998b; Sun et al., 1998). Thus, it is not surprising that resveratrol can rescue neurons from cell death due to oxidized BLP. Based on these data, it is reasonable to consider resveratrol as a possible therapeutic agent to ameliorate progression of neurodegenerative diseases in general and Alzheimer’s disease in particular.

ACKNOWLEDGEMENT

We thank Dr. David Holtzman (Neurology Department, Washington University, St Louis, MO) for the generous gift on human recombinant apoE. This study was supported by grants from Missouri Alzheimer’s Disease and Related Disorder Research Program and the Research Board of the University of Missouri.

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Discussion Board

Any Comment to this presentation?

[ABSTRACT] [INTRODUCTION] [MATERIALS AND METHODS] [RESULTS] [IMAGES] [DISCUSSION] [ACKNOWLEDGEMENT] [REFERENCES] [Discussion Board]

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Albert Sun, Bozena Draczynska-Lusiak, Grace Sun
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Last update: 31/1/2000