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Keywords:

  • β-amyloid;
  • cortical neurons;
  • mesencephalic neurons;
  • microglia;
  • nicotinamide-adenine dinucleotide phosphate oxidase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

The purpose of this study was to assess and compare the toxicity of β-amyloid (Aβ) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia-mediated Aβ-induced neurotoxicity. Incubation of cortical or mesencephalic neuron-enriched and mixed neuron–glia cultures with Aβ(1–42) over the concentration range 0.1–6.0 μm caused concentration-dependent neurotoxicity. High concentrations of Aβ (6.0 μm for cortex and 1.5–2.0 μm for mesencephalon) directly injured neurons in neuron-enriched cultures. In contrast, lower concentrations of Aβ (1.0–3.0 μm for cortex and 0.25–1.0 μm for mesencephalon) caused significant neurotoxicity in mixed neuron–glia cultures, but not in neuron- enriched cultures. Several lines of evidence indicated that microglia mediated the potentiated neurotoxicity of Aβ, including the observations that low concentrations of Aβ activated microglia morphologically in neuron–glia cultures and that addition of microglia to cortical neuron–glia cultures enhanced Aβ-induced neurotoxicity. To search for the mechanism underlying the microglia-mediated effects, several proinflammatory factors were examined in neuron–glia cultures. Low doses of Aβ significantly increased the production of superoxide anions, but not of tumor necrosis factor-α, interleukin-1β or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Aβ toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Aβ-induced neurotoxicity in neuron–glia mixed cultures. The role of NADPH oxidase-generated superoxide in mediating Aβ-induced neurotoxicity was further substantiated by a study which showed that Aβ caused less of a decrease in dopamine uptake in mesencephalic neuron–glia cultures from NADPH oxidase-deficient mutant mice than in that from wild-type controls. This study demonstrates that one of the mechanisms by which microglia can enhance the neurotoxicity of Aβ is via the production of reactive oxygen species.

Abbreviations used

β-amyloid

AD

Alzheimer's disease

CAT

catalase

DA

dopamine

DPI

diphenyleneiodonium

FBS

fetal bovine serum

GFAP

glial fibrillary acidic protein

HBSS

Hanks' balanced salt solution

IL-1β

interleukin-1β

IR

immunoreactive

MAP-2

microtubule-associated protein-2

MEM

minimum essential medium

Neu-N

neuron-specific nuclear protein

NO

nitric oxide

PBS

phosphate- buffered saline

PD

Parkinson's disease

PMA

phorbol 12-myristate 13-acetate

ROS

reactive oxygen species

SOD

superoxide dismutase

TH

tyrosine hydroxylase

TNF-α

tumor necrosis factor-α

The pathological hallmark of Alzheimer's disease (AD) is the presence of numerous senile plaques, of which β-amyloid peptide (Aβ) is a major component, throughout the hippocampus and cerebral cortex. The excessive deposition of Aβ is linked to neurodegenerative changes in neurons. Several studies indicate that Aβ exerts its neurotoxic effects by diverse mechanisms. One theory indicates that Aβ can directly injure neurons by specifically or non-specifically interacting with molecules on the cell surface (Yankner et al. 1990). Within 1 h after exposure to Aβ there is ultrastructural evidence of damage to the Golgi apparatus, mitochondria and other membrane systems in the cytoplasm, as well as a breakdown of microtubules in neurites, whereas the nucleus remains essentially intact (Behl et al. 1994). These data suggest that cytoplasmic membrane damage is an early event in Aβ toxicity. Direct toxicity of Aβ may occur because Aβ peptide itself, under certain conditions, can acquire a free radical state in vitro (Hensley et al. 1994), or because it can stimulate the production of intracellular reactive oxygen species (ROS) (Klegeris et al. 1994; Meda et al. 1995a; El Khoury et al. 1996; Van Muiswinkel et al. 1996). In contrast to this direct neurotoxicity theory, several reports show that Aβ causes little cytotoxicity by itself, but is indirectly neurotoxic by activating microglia to release various proinflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO) and superoxide (McDonald et al. 1997; Minghetti and Levi 1998), which in turn kill neurons. Akiyama et al. (2000) have shown that Aβ deposition provokes a microglia-mediated inflammatory response. Abundant reactive microglia and astrocytes have been shown to surround the β-amyloid plaques in the AD brain (McGeer and McGeer 1995; Cotman and Su 1996). The interaction of Aβ(1–42) or Aβ(25–35) in vitro with rat cortical neurons (Schubert et al. 1995; Behl et al. 1994) or hippocampal neurons (Goodman et al. 1994) induces the production of ROS and causes neuronal death. Vitamin E, an antioxidant and free radical scavenger, is able to protect PC12 cells from damage by Aβ(1–42) and Aβ(25–35) (Behl et al. 1992). These investigators have suggested that the cells died because of oxidative stress and free radical-generated damage.

Despite numerous studies on Aβ-induced neurotoxicity, it is still unclear whether or not Aβ-induced neurotoxicity can be mediated through the activation of glia. We addressed this question by comparing the neurotoxic effects of Aβ on both neuron-enriched and neuron–glia cultures prepared from the cortex and mesencephalon of rat embryos. We used cell cultures from the cortical region in this study because senile plaques are most abundant in the cortices of patients with AD. Cell cultures from the mesencephalic region were also studied because Aβ precursor protein has been found in the Lewy bodies of patients with Parkinson's disease (PD) (Arai et al. 1992). Here we report that, although Aβ is capable of damaging neurons directly, the presence of microglia greatly potentiated the neurotoxic effects of Aβ. We also found that increased production of superoxide free radicals from activated microglia plays a major role in enhancing the toxic effects of Aβ.

Reagents

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Aβ(1–42) was purchased from the American Peptide Company (Sunnyvale, CA, USA). Cell culture ingredients were obtained from Life Technologies (Grand Island, NY, USA). [3H]Dopamine (DA) (28 Ci/mmol) and [2,3-3H]GABA (81 Ci/mmol) were purchased from NEN Life Science (Boston, MA, USA). Monoclonal antibodies against the CR3 complement receptor (OX-42), neuron-specific nuclear protein (Neu-N) and microtubule-associated protein-2 (MAP-2) were obtained from Pharmingen (San Diego, CA, USA). The polyclonal antibody against glial fibrillary acidic protein (GFAP) came from DAKO (Carpinteria, CA, USA). The polyclonal antibody against tyrosine hydroxylase (TH) was a gift from Dr John Reinhard of Glaxo Wellcome (Research Triangle Park, NC, USA). Rat monoclonal antibody raised against F4/80 antigen was purchased from Serotec (Washington, DC, USA). Biotinylated horse anti-mouse and goat anti-rabbit secondary antibodies were purchased from Vector Laboratories (Burlingame, CA, USA). Dako antibody diluent was from Dako Corporation (Carpinteria, CA, USA). TNF-α and IL-1β ELISA kits were purchased from R & D Systems Inc. (Minneapolis, MN, USA). All other reagents came from Sigma Chemical Co. (St Louis, MO, USA).

Mesencephalic neuron–glia culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Rat or mouse ventral mesencephalic neuron–glia were prepared following a protocol described previously (Liu et al. 2001), with modification. Briefly, ventral mesencephalic tissues were dissected from embryonic day 14/15 Fischer 344 rats or embryonic day 13 C57BL/6 (wild-type controls) and NADPH oxidase-deficient mice (mutation in gp 91 of NADPH oxidase; from Jackson Laboratory, Bar Harbor, ME, USA), and dissociated by mild mechanical trituration in minimum essential medium (MEM). Cells were plated (5 × 105/well) in 24-well culture plates precoated with poly d-lysine (20 µg/mL). The culture medium consisted of MEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10% heat-inactivated horse serum, 1 g/L glucose, 2 mm l-glutamine, 1 mm sodium pyruvate, 100 µm non-essential amino acids, 50 U/mL penicillin and 50 µg/mL streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Three days later, the cultures were replenished with 0.5 mL/well fresh medium. Seven-day cultures were used for treatment. Upon treatment, cultures were switched to fresh medium containing 2% each of heat-inactivated FBS and heat-inactivated horse serum. The composition of the mesencephalic neuron–glia cultures was determined by immunocytochemistry. Rat mesencephalic neuron–glia cultures contained approximately 11% OX-42-immunoreactive (IR) microglia, 48% GFAP-IR astrocytes and 40% Neu-N-IR neurons, of which 2% were dopaminergic neurons. The composition of mouse mesencephalic neuron–glia cultures was as follows: 10–11% F4/80-IR microglia, 40% Neu-N-IR neurons and 1% TH-IR dopaminergic neurons. The remaining cells were presumed to be astroglia.

Cortical neuron–glia culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Rat cortical neuron–glia were prepared from the brains of embryonic day 16/17 Fischer 344 rats. The cortex was dissociated by mild mechanical trituration in MEM. After pelleting by centrifugation, cells were resuspended and plated (5 × 105/well) in 24-well culture plates precoated with poly d-lysine. Cultures were maintained in the same medium as described above for mesencephalic neuron–glia cultures. Seven-day cultures were used for treatment. The composition of cortical neuron–glia cultures was determined by immunostaining with antibodies against Neu-N and CR3 complement receptor (OX-42). Cortical neuron–glia cultures contained 60% Neu-N-IR neurons and 3.1% OX-42-IR microglia. The remaining cells were presumed to be astroglia.

Microglia-enriched cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Primary microglia were prepared from whole brains of 1-day Fisher 344 rat pups following a protocol described previously (Liu et al. 2000). Briefly, brain tissues were triturated after removing the meninges and blood vessels. Cells (5 × 107) were seeded in a 150-cm2 culture flask. After a confluent monolayer of glial cells had been obtained, microglia were shaken off and replated at 1 × 105/well on top of existing cortical neuron–glia cultures. After plating the enriched microglia for 24 h, the cells were treated with vehicle or Aβ.

Astrocyte-enriched cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Primary astrocytes were prepared as described previously (Liu et al. 2001). Briefly, the mixed glial cultures, after the separation of microglia, were detached with trypsin–EDTA and seeded in the same culture medium as that used for microglia. After at least five consecutive passages, cells were seeded (5 × 104/well) into 96-well plates for superoxide measurement. In a separate experiment, cells (1 × 105/well) were added to cortical neuron–glia cultures in 24-well plates for GABA uptake experiments. Immunocytochemical staining of the astrocyte-enriched cultures with either OX-42 or anti-GFAP antibody indicated purity of ≥ 98%.

Immunocytochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Neurons were stained with the antibody against MAP-2, a marker for both cell bodies and neurites, and with the antibody against Neu-N, for neuronal cell bodies but not neurites. Microglia were visualized by staining for the CR3 complement receptor with the monoclonal antibody OX-42, and astrocytes were stained with the antibody against GFAP, an intermediate filament protein whose synthesis is restricted to astrocytes. Dopaminergic neurons were detected with the polyclonal antibody against TH. Briefly, cells were fixed for 20 min at room temperature (22°C) in 3.7% paraformaldehyde in phosphate-buffered saline (PBS). After washing twice with PBS, the cultures were treated with 1% hydrogen peroxide for 10 min. The cultures were washed three times with PBS and then incubated for 40 min with blocking solution (PBS containing 1% bovine serum albumin, 0.4% Triton X-100, and 4% appropriate serum: normal horse serum for MAP-2, Neu-N or OX-42, and normal goat serum for TH or GFAP staining). The cultures were incubated overnight at 4°C with the primary antibody diluted in Dako antibody diluent, and the cells were then washed three times for 10 min each in PBS. The cultures were next incubated for 1 h with PBS containing 0.3% Triton X-100 and the appropriate biotinylated secondary antibody (MAP-2, Neu-N or OX-42: horse anti-mouse antibody, 1 : 227; TH or GFAP: goat anti-rabbit antibody, 1 : 227). After washing three times with PBS, the cultures were incubated for 1 h with Vectastain ABC reagents (Vector Laboratories, Piscataway, NJ, USA) diluted in PBS containing 0.3% Triton X-100. After washing twice with PBS, the bound complex was visualized by incubating cultures with 3,3′-diaminobenzidine and urea–hydrogen peroxide tablets (Sigma) dissolved in water. Color development was terminated by removing the reagents and washing the cultures twice with PBS. For cell counting, nine representative areas per well in the 24-well plate were counted under the microscope at 100 × magnification.

Uptake assays for [3H]DA or [3H]GABA

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Cells were incubated for 15 min at 37°C with 1 μm[3H]DA or 5 μm[3H]GABA in Krebs–Ringer buffer (16 mm NaH2PO4, 16 mm Na2 HPO4, 119 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.3 mm EDTA, pH 7.4). Non-specific uptake was blocked by 10 µm mazindol for DA uptake, or 10 µm NO-711 and 1 mmβ-alanine for GABA uptake. After washing the cells three times with ice-cold Krebs–Ringer buffer (1 mL/well) and lysing with 1 N NaOH (0.5 mL/well), the lysate was mixed with 15 mL scintillation fluid and radioactivity was determined with a liquid scintillation counter. The specific [3H]DA or [3H]GABA uptake was calculated by subtracting the amount of radioactivity obtained in the presence of mazindol or NO-711 and β-alanine from that obtained in the absence of mazindol or NO-711 and β-alanine.

Superoxide assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

The amount of superoxide produced was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c, as described in Chao et al. (1994). Primary mesencephalic or cortical neuron–glia were plated at 5 × 104/well in 150 μl culture medium in 96-well plates, precoated with poly d-lysine (20 μg/mL), and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Three days later, the cultures were replenished with 150 μl fresh medium. Seven-day cultures were treated with vehicle or Aβ. Cells were washed twice with Hanks' balanced salt solution (HBSS). To each well, 70 μl HBSS with or without SOD (600 U/mL), 50 μl Aβ and 80 μl ferricytochrome c (80 μm) in HBSS were added. The cultures were incubated for 90 min at 37°C in 5% CO2 and 95% air. The absorbance at 550 nm then was read with a Spectra Max Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The amount of SOD-inhibitable superoxide was calculated and expressed as percentage of the control value.

Nitrite assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

The accumulation of nitrite in the culture supernatant, an indicator of the production of NO, was determined with a colorimetric assay with Griess reagent (0.1%N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide and 2.5% H3PO4) (Green et al. 1982). Equal volumes of culture supernatant and Griess reagent were mixed, the mixture was incubated for 10 min at room temperature in the dark, and the absorbance at 540 nm was determined with the Spectra Max Plus microplate spectrophotometer. The concentration of nitrite in samples was determined from a sodium nitrite standard curve.

Presence of microglia enhanced Aβ-induced neurotoxicity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

The neurotoxic effects of Aβ were compared in neuron-enriched cultures and neuron–glia cultures prepared from both mesencephalon and cortex. Dose–response studies showed that Aβ reduced the uptake capacity for neurotransmitters in neuron-enriched cultures in a concentration-dependent manner. Aβ exerted a more potent reduction in DA uptake in mesencephalic neuron–glia cultures and neuron-enriched cultures (Fig. 1a) than GABA uptake in cortical neuron–glia cultures and neuron-enriched cultures (Fig. 1b). It is significant that in neuron–glia cultures, the dose–response curves shifted to the left: the ED50 shifted from 2.0 to 0.5 μm for DA uptake and from more than 6.0 to 2.0 μm for GABA uptake. These results indicate that the presence of glia potentiated the toxic effect of Aβ (Figs 1a and b). The glia-enhanced toxicity of Aβ observed in the uptake studies was confirmed in morphological studies. Immunocytochemical analysis showed that Aβ (0.25–1.0 μm for mesencephalon; 1.0–2.0 μm for cortex) caused morphological damage to dopaminergic neurons (TH-IR) in mesencephalic neuron–glia cultures (Fig. 1e) and to MAP-2-IR neurons in cortical neuron–glia cultures (Fig. 1f). In mesencephalic neuron–glia cultures, numerous healthy TH-IR neurons with extensive neurites were observed in the control cultures. Following Aβ treatment, the neurites became fewer and shorter (Fig. 1e). Similar changes were observed in cortical neuron–glial cultures (Fig. 1f). In contrast, the same concentrations of Aβ did not produce any morphological changes in neurons in neuron-enriched cultures (data not shown). Cell count analysis revealed that Aβ treatment of cell cultures from both brain regions caused a much greater loss in the number of TH-IR and Neu-N-IR neurons in neuron–glia cultures than in neuron-enriched cultures (Figs 1c and d). Results from cell count analysis are consistent with the conclusion based on the DA uptake studies; however, the ED50 values for Aβ from the two measurements are different because DA uptake is a more sensitive functional measurement of cell damage than morphological analysis. Addition of enriched microglia obtained from rat whole brains rendered cortical neuron–glia cultures more sensitive to Aβ-induced neurotoxicity (Fig. 2). At 0.5 μm Aβ, GABA uptake was reduced from 90 to 50% of control after adding microglia (1 × 105/well) to cortical neuron–glia cultures (5 × 105/well). In contrast, addition of astrocytes (1 × 105/well) to cortical neuron–glia cultures did not enhance Aβ-induced neurotoxicity (Fig. 2). Taken together, both the biochemical and morphological studies clearly demonstrate the potentiated neurotoxicity of Aβ in the presence of microglia.

image

Figure 1. Microglia enhanced Aβ-induced neurotoxicity. Mesencephalic or cortical neuron–glia cultures and neuron-enriched cultures seeded in 24-well plates at 5 × 105 cells/well were treated with different concentrations of Aβ for 9 days. [3H]DA uptake in mesencephalic neuron–glia cultures and neuron-enriched cultures (a) or [3H]GABA uptake in cortical neuron–glia cultures and neuron-enriched cultures (b) was determined. Cultures were immunostained for TH or Neu-N. The number of TH-IR neurons in mesencephalic cultures (c) and Neu-N-IR neurons in cortical cultures (d) was counted. Immunocytochemical analysis of Aβ-induced neurotoxicity in mesencephalic neuron–glia cultures with anti-TH antibody (e) and in cortical neuron-glia cultures with anti-MAP-2 antibody (f) was performed. The images shown are representative of three experiments. The data are expressed as mean ± SEM from three independent experiments with triplicate samples for each condition. *p < 0.05, **p < 0.01 versus control cultures (anova with Bonferroni's test).

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image

Figure 2. Addition of microglia rendered cortical neuron–glia cultures more sensitive to Aβ-induced neurotoxicity, but addition of astrocytes did not enhance Aβ-induced neurotoxicity. Cortical neuron–glia were cultured alone or supplemented with microglia or astrocytes (1 × 105/well) from whole rat brains. Twenty-four hours later, the cultures were treated with vehicle or Aβ. GABA uptake was determined 9 days after the treatment. Data are expressed as the percentages of control cultures and are the mean ± SEM from three independent experiments in triplicate. **p < 0.01 versus corresponding vehicle control cultures (anova with Bonferroni's test).

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Aβ induced the activation of microglia

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Previous reports from our laboratory indicated that among the various glia in the brain, microglia were the primary source of the proinflammatory factors which mediated lipopolysaccharide-induced neurotoxicity in rat mesencephalic neuron–glia cultures (Kim et al. 2000). To examine the possibility that microglia might mediate Aβ- induced neurotoxicity, we evaluated the activation of microglia by performing immunostaining with the antibody against OX-42, which detects surface expression of the complement CR3 receptor, a marker for microglia and macrophages. Primary mesencephalic or cortical neuron–glia cultures were treated with vehicle or Aβ for 48 h. Cells were fixed and immunostained with the monoclonal antibody against OX-42. As can be seen in Figs 3a and b, microglia were predominantly in a resting state in the control cultures. In contrast, cultures treated with Aβ at 1.0 or 2.0 μm displayed the characteristics of activated microglia: increased cell size, irregular shape and intensified OX-42 staining. Consistent with previous reports (Lawson et al. 1990; Kim et al. 2000), we found that mesencephalic neuron–glia cultures contained a greater number of OX-42-IR cells than did cortical neuron–glia cultures, and these differences were even more prominent after Aβ treatment.

image

Figure 3. Immunocytochemical analysis of microglia. Mesencephalic or cortical neuron–glia cultures were treated with vehicle or Aβ for 48 h and then immunostained with OX-42 antibody. (a) OX-42-IR microglia in mesencephalic neuron–glia cultures. (b) OX-42-IR microglia in cortical neuron–glia cultures. Images presented are representative of three independent experiments.

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Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Activated microglia can release various proinflammatory cytokines, such as TNF-α and IL-1β, and free radicals, such as NO and superoxide, which in turn kill neurons. Superoxide levels were therefore measured to assess the role of ROS in Aβ-induced neurotoxicity. Mesencephalic or cortical neuron–glia cultures were treated with vehicle or Aβ over the concentration range 0.1–2.0 μm, and superoxide levels were measured as the SOD-inhibitable reduction in ferricytochrome c. Significant amounts of superoxide were generated in mesencephalic neuron–glia cultures (Fig. 4a) and in cortical neuron–glia cultures (Fig. 4b) in a concentration-dependent manner after Aβ treatment. In order to explore the cell types that contribute to the generation of superoxide, levels of superoxide were determined in neuron-enriched, microglia-enriched and astrocyte- enriched cultures. Significant amounts of superoxide were detected in microglia-enriched cultures after Aβ treatment; however, the same concentrations of Aβ did not increase levels of superoxide in neuron-enriched and astrocyte-enriched cultures (Fig. 5). In contrast, treatment of mesencephalic or cortical neuron–glia cultures with the same concentrations of Aβ for 72 h did not produce detectable levels of NO, TNF-α and IL-1β (data not shown; detection limit: NO, 0.5 μm; TNF-α, 5.1 pg/mL; IL-1β, 3.0 pg/mL). The selective increase in the production of superoxide by lower concentrations of Aβ suggests an important role of the ROS in mediating Aβ-induced neurotoxicity. However, we cannot rule out the possibility that other factors, such as NO, TNF-α and IL-1β, may participate in Aβ-induced neurotoxicity, despite the fact that they were below the limits of detection in this study.

image

Figure 4. Aβ increased the generation of superoxide in neuron–glia cultures. Mesencephalic or cortical neuron–glia seeded at 5 × 104/well in 96-well plates for 7 days were treated with vehicle or the indicated concentration of Aβ for 90 min. Superoxide production was measured as the SOD-inhibitable reduction of ferricytochrome c. (a) Superoxide production in mesencephalic neuron–glia cultures. (b) Superoxide production in cortical neuron–glia cultures. (c) Effect of DPI on Aβ-stimulated superoxide production in mesencephalic neuron–glia cultures. Data are mean ± SEM of three experiments performed in triplicate. *p < 0.05, **p < 0.01 versus control cultures; #p < 0.01 versus Aβ-treated cultures (anova with Bonferroni's test).

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image

Figure 5. Aβ did not increase the generation of superoxide in neuron-enriched or astrocyte-enriched cultures. Enriched cultured microglia, neurons or astrocytes seeded at 5 × 104 cells/well in 96-well plates were treated with vehicle or Aβ for 90 min. Superoxide production was measured as the SOD-inhibitable reduction of ferricytochrome c. Data are mean ± SEM of three experiments performed in triplicate. *p < 0.05, **p < 0.01 versus control cultures (anova with Bonferroni's test).

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Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

If superoxide is an important mediator in Aβ-induced toxicity, then CAT and SOD, which convert superoxide to hydrogen peroxide and then to water, should reduce Aβ-induced neurotoxicity. To examine this possibility, both CAT and SOD were added to mesencephalic or cortical neuron–glia cultures before and during Aβ treatment. After 9 days, neurotoxicity was determined by [3H]DA uptake and immunostaining with the anti-TH antibody in mesencephalic neuron–glia cultures, and by [3H]GABA uptake and immunostaining with the anti-Neu-N antibody in cortical neuron–glia cultures. CAT and SOD significantly prevented the Aβ-induced decrease in DA (Fig. 6a) or GABA (Fig. 6c) uptake. Immunocytochemical analysis revealed that CAT/SOD significantly reduced Aβ-induced damage and loss of TH- and Neu-N-IR neurons (Figs 6b and d).

image

Figure 6. CAT and SOD protected neurons from Aβ toxicity. CAT and SOD (50 U/mL each) were added to mesencephalic or cortical neuron–glia cultures (5 × 105 cells/well) 30 min before Aβ treatment. Addition of CAT/SOD was repeated on the 3rd and 5th day. Cultures were harvested on day 9 and neurotoxicity was determined by [3H]DA uptake (a) and immunostaining with anti-TH antibody (b) in mesencephalic neuron–glia cultures, or by [3H]GABA uptake (c) and immunostaining with the anti-Neu-N antibody (d) in cortical neuron–glia cultures. Data are mean ± SEM of three experiments performed in triplicate. *p < 0.05, **p < 0.01 versus corresponding vehicle-treated control cultures (anova with Bonferroni's test).

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NADPH oxidase mediates Aβ-induced neurotoxicity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

Activation of NADPH oxidase by Aβ is thought to be one of the sources of superoxide production in Aβ-induced neurotoxicity (Bianca et al. 1999). Diphenyleneiodonium (DPI) has frequently been used to inhibit the production of superoxide by NADPH oxidase (Li and Trush 1998). DPI significantly inhibited the Aβ-induced induced production of superoxide (Fig. 4c). The result demonstrated that the Aβ-induced release of superoxide free radicals was involved in the activation of NADPH oxidase. Treatment of neuron–glia cultures with DPI significantly prevented the Aβ-induced decrease in DA uptake in mesencephalic neuron–glia cultures (Fig. 7a), and GABA uptake in cortical neuron–glia cultures (Fig. 7c). DPI also significantly protected TH- and Neu-N-IR neurons from Aβ-induced damage and loss (Figs 7b and d). As DPI is not a specific inhibitor of NADPH oxidase (Li et al. 1998), mutant mice that are deficient in this enzyme activity were used to confirm the role of superoxide in Aβ-induced neurotoxicity. Incubation of mesencephalic neuron–glia cultures with Aβ over the concentration range of 0.5–1.0 μm caused less of a decrease in DA uptake in cultures from NADPH oxidase-deficient mutant mice than that from wild-type controls (Fig. 8). These results strongly indicate an important role for NADPH oxidase-generated superoxide in Aβ-induced neurotoxicity.

image

Figure 7. Effect of NADPH oxidase inhibitor (DPI) on Aβ-induced neurotoxicity. Mesencephalic or cortical neuron–glia were pretreated with DPI (4 nm) for 30 min before treatment with different concentrations of Aβ. Neurotoxicity was determined 9 days after Aβ treatment by [3H]DA uptake (a) and immunostaining with the anti-TH antibody (b) in mesencephalic neuron–glia cultures, or [3H]GABA uptake (c) and immunostaining with the anti-Neu-N antibody (d) in cortical neuron-glia cultures. Data are mean ± SEM of three experiments performed in triplicate. *p < 0.05, **p < 0.01 versus corresponding vehicle-treated control cultures (anova with Bonferroni's test).

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image

Figure 8. Αβ caused less of a decrease in DA uptake in NADPH oxidase-deficient mutant mice than wild-type controls. To confirm that NADPH oxidase-deficient mutant mouse cells are unable to produce extracellular superoxide free radicals, we measured superoxide production by phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide in microglia-enriched cultures from NADPH oxidase-deficient mutant mice (Cybb) and from wild-type controls (C57BL/6). PMA and lipopolysaccharide caused significant superoxide generation in microglia-enriched cultures from C57BL/6, but not from Cybb mice. Mesencephalic neuron–glia cultures at 5 × 105/well were treated with different concentrations of Aβ. Neurotoxicity was determined 9 days after Aβ treatment by [3H]DA uptake. Data are mean ± SEM of three experiments performed in triplicate. *p < 0.05, **p < 0.01 versus corresponding wild-type control cultures (anova with Bonferroni's test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References

In this paper we have addressed two questions. Does Aβ exert differential neurotoxicity in the presence and absence of glia and, if glia do enhance neurotoxicity, then what are the factors released from glia that mediate this effect? Using neuron-enriched and neuron–glia cultures from both the cortical and mesencephalic regions of the rat brain, we clearly demonstrate that Aβ exerts a much stronger neurotoxic effect in the presence of glia than in neuron-enriched cultures without glia. Our data also indicate that superoxide free radicals released from microglia play a major role in potentiating this Aβ-induced neurotoxicity. In addition, the present results show that Aβ causes neuronal damage by two different mechanisms. At higher concentrations, Aβ is capable of directly causing the death of neurons, as shown in the neuron-enriched cultures. On the other hand, in the presence of microglia, at much lower concentrations, Aβ was non-toxic in neuron-enriched cultures but exerted potent neurotoxic effects in neuron–glia cultures. Although there was a distinct difference in the concentration response of Aβ in the presence or absence of glia, it is important to point out that these two mechanisms are not mutually exclusive. Many laboratories have described intimate interactions between glia and neurons (Meda et al. 1995b; Kim et al. 2000). Not only can overactivation of glial cells damage neurons, but loss of neurons can also cause reactive gliosis (McMillian et al. 1994). Thus, the mode of the neurotoxic effect of Aβ depends on the concentration of Aβ in its microenvironment in the brain. At lower concentrations, Aβ may cause neuronal damage indirectly by activation of glia. At high concentrations, however, Aβ may not only cause directly toxic effects to and loss of neurons, but also could initiate an additional mechanism for reactive gliosis as a result of localized damage to neurons. In addition, our data show that Aβ is more neurotoxic to mesencephalic neuron–glia cultures than cortical neuron–glia cultures. This observation is consistent with our previous finding that the mesencephalic region has a much higher density of microglia than the cortex (Kim et al. 2000). Thus, dopaminergic neurons in mesencephalic neuron–glia cultures may encounter an excessively high level of oxidative stress after Aβ treatment.

Astroglia and microglia are the major immune cells in the brain. To further understand the glia-mediated mechanism of Aβ it is important to determine which type of glial cell is responsible. Several studies from Van Eldik's lab show that Aβ stimulates NO production in astrocytes, and glial-derived proteins such as IL-1β and S100 activate cultured astrocytes and enhance Aβ-induced glial activation (Akama et al. 1998; Hu and Van Eldik 1999). These studies suggest a role for astrocytes in mediating the neurotoxic effect of Aβ. There was also a report indicating the presence of NADPH oxidase in neurons, which contributes to oxidase stress-induced apoptosis in nerve factor-deprived sympathetic neurons (Tammariello et al. 2000). Results from our studies, however, provide convincing evidence that microglia play a much more dominant role than astrocytes in mediating the Aβ-induced neurotoxicity in neuron–glia cultures. First, addition of microglia, but not astroglia, enhanced the Aβ-induced GABA uptake decrease in cortical neuron–glia cultures (Fig. 2). Second, Aβ produced a significant increase in the production of superoxide in enriched microglia, but not in enriched astrocytes or neurons (Fig. 5). These results are consistent with our previous report showing that microglia, rather than astroglia, mediated the lipopolysaccharide-elicited neurotoxicity in mesencephalic neuron–glia cultures (Kim et al. 2000).

Microglia are the resident immune cells in the brain (Kreutzberg 1996; Aloisi 1999). Under normal conditions, microglia serve an important role in immune surveillance. Microglial activation has been associated with neurodegeneration through the production of neurotoxic factors, such as proinflammatory cytokines, NO and superoxide free radicals (McDonald et al. 1997; Griffin et al. 1998; Combs et al. 1999, 2000; Li et al. 2000). Among the factors produced by activated microglia, ROS may play a prominent role in neurodegeneration (Kim et al. 2000; Kang et al. 2001). Aβ produced ROS via the activation of NADPH oxidase in microglia, monocytes and neutrophils (Bianca et al. 1999), and so ROS could function as an intracellular signaling molecule to further mediate the Aβ-induced production of proinflammatory factors such as TNF-α or NO, which in turn damage neurons (Combs et al. 2001; Kang et al. 2001). In this study, Aβ caused microglial activation, with dramatic changes in the morphological appearance of cortical and mesencephalic neuron–glia cultures (Figa 3a and b), and also caused a significant increase in the production of superoxide anions (Figs 4a, 4b and 5). Treatment of mesencephalic or cortical neuron–glia cultures with the same concentrations of Aβ for 72 h did not produce detectable levels of NO, TNF-α and IL-1β. The lack of effect of Aβ on the production of NO, TNF-α and IL-1β in our study is different from published reports (Ii et al. 1996; Viel et al. 2001). The difference is mainly attributed to the much lower concentrations of Aβ used in our study (0.1–2.0 μm) compared with most of the other reports (10–30 μm). With higher concentrations of Aβ (10 μm), significant amounts of NO and TNF-α were detected in our neuron–glia cultures (data not shown) as described in other reports (Ii et al. 1996; Viel et al. 2001). Taken together, the inhibition of microglia-generated superoxide might be the most efficient way to protect neurons from damage by Aβ. Indeed, we have recently demonstrated that inhibition of superoxide release from Aβ-activated microglia by naloxone affords significant protection both in cortical and mesencephalic neurons (Liu et al. 2002).

Our study indicated a major role of superoxide free radicals released from activated microglia, but it is interesting to note that Giulian et al. (1995) reported a low molecular weight neurotoxin released from microglia found in Alzheimer brain which was toxic to pyramidal neurons in vivo when infused into rat hippocampus. Their study showed that this neurotoxin acts on NMDA-containing neurons. In an effort to examine whether excitatory amino acid transmitters could participate in inflammation-related neurotoxicity, we determined the levels of various excitatory amino acids in the supernatants of neuron–glia cultures treated with one of the most potent inflammagens, lipopolysaccharide. HPLC analysis showed that there was no appreciable increase in the release of glutamate and aspartate from microglia. In addition, several NMDA antagonists were found ineffective in preventing lipopolysaccharide-induced neuronal death in our neuron–glia cultures (W. Kim and J. Hong, unpublished results).

Superoxide scavengers such as CAT and SOD, which remove superoxide and H2O2, significantly protected neurons from Aβ-induced neurotoxicity. This observation suggests that superoxide mediates Aβ-elicited toxicity. This hypothesis was further supported by the increase in superoxide production by Aβ (Figs 4a, 4b and 5). The ROS produced by Aβ could come from different sites in the microglia. The most common cellular sources of ROS include the mitochondrial respiratory chain, microsomal enzymes, xanthine/xanthine oxidase, NADPH oxidase, and perhaps some unidentified NADH–NADPH-dependent enzymes (Cross and Jones 1991). Activation of NADPH oxidase by Aβ is a major source of superoxide production in Aβ-induced neurotoxicity (Bianca et al. 1999). NADPH oxidase is a multicomponent complex. Upon activation, cytosolic subunits are translocated to the membrane and associated with membrane-bound subunits to form the active enzyme and to catalyze the transfer of one electron from NADPH to oxygen, giving rise to superoxide. DPI has frequently been used to inhibit superoxide production mediated by NADPH oxidase (Li et al. 1998). The present study shows that inhibition of NADPH oxidase activity by DPI significantly prevented the Aβ-induced decrease in DA and GABA uptake (Figs 7a and c), and the loss of neurons (Figs 7b and d). Furthermore, Aβ caused less of a decrease in DA uptake in mecensephalic neuron–glia cultures from NADPH oxidase-deficient mutant mice than that from wild-type controls (Fig. 8). In addition, our superoxide analysis demonstrates that the generation of superoxide by Aβ is significantly inhibited by DPI (Fig. 4c). These data provide strong evidence that superoxide plays a critical role in Aβ-induced neuronal death. Therefore, we propose that one of the possible mechanisms by which microglia enhance the neurotoxicity of Aβ is by the production of ROS.

Brain cells are at particular risk from damage caused by free radicals. The brain has an extremely high rate of oxygen consumption and neuronal membranes have a high content of polyunsaturated fatty acids that are susceptible to lipid peroxidation. In healthy cells, there is a well balanced equilibrium between free radical generation and various enzymatic and non-enzymatic antioxidant defense systems (Gutteridge and Halliwell 1989; Ames et al. 1993). In neuronal diseases, an imbalance leading to free radical accumulation occurs. Free radicals have been implicated in several neurodegenerative diseases including PD and AD. It has been reported that free radicals are generated in dopaminergic neurons by the oxidation of DA via monoamine oxidase (Olanow 1992). The explanations for AD should take into account the fact that age is the primary risk factor for the disease (Viel et al. 2001). One important factor associated with aging is the accumulation of oxidative damage caused by free radicals (Ames et al. 1993; Lees 1993). Free radicals are also involved in the neurodegeneration that occurs in AD. Our data suggest that superoxide, one of many free radicals, is an important mediator of Aβ-induced loss of cortical neurons, a hallmark for AD, and the loss of dopaminergic neurons responsible for PD. The data presented here provide the first evidence that Aβ causes significant damage to and loss of dopaminergic neurons, and that superoxide is a major contributor to Aβ-induced dopaminergic neurodegeneration. The similar mechanisms of toxicity by Aβ in the cortex and mesencephalon may partly explain the overlap in pathological features and clinical symptoms observed in AD and PD. Most importantly, the results suggest that therapeutic modulation of microglia may provide hope for prevention and treatment of AD, PD and other related neurodegenerative diseases.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Reagents
  5. Mesencephalic neuron–glia culture
  6. Cortical neuron–glia culture
  7. Mesencephalic and cortical neuron-enriched cultures
  8. Microglia-enriched cultures
  9. Astrocyte-enriched cultures
  10. Immunocytochemistry
  11. Uptake assays for [3H]DA or [3H]GABA
  12. Superoxide assay
  13. Nitrite assay
  14. TNF-α and IL-1β assays
  15. Preparation of Aβ
  16. Statistical analysis
  17. Results
  18. Presence of microglia enhanced Aβ-induced neurotoxicity
  19. Aβ induced the activation of microglia
  20. Aβ caused production of superoxide anion but not NO, TNF-α or IL-1β
  21. Catalase (CAT) and SOD protected neurons from Aβ-induced neurotoxicity
  22. NADPH oxidase mediates Aβ-induced neurotoxicity
  23. Discussion
  24. References
  • Akama K. T., Albanese C., Pestell R. G. and Van Eldik L. J. (1998) Amyloid β-peptide stimulates nitric oxide production in astrocytes through an NFκB-dependent mechanism. Proc. Natl Acad. Sci. USA 95, 57955800.
  • Akiyama H., Barger S., Barnum S., Brandt B., Bauer J., Cole G. M., Cooper N. R., Eikelenboom P., Emmerling M., Fiebich B. L., Finch C. E., Frautschy S., Griffin W. S., Hampel H., Hull M., Landreth G., Lue L., Mrak R., Mackenzie I. R., McGeer P. L., O'Banion M. K., Pachter J., Pasinetti G., Plata-Salaman C., Rogers J., Rydel R., Shen Y., Streit W., Strohmeyer R., Tooyoma I., Van Muiswinkel F. L., Veerhuis R., Walker D., Webster S., Wegrzyniak B., Wenk G. and Wyss-Coray T. (2000) Inflammation and Alzheimer's disease. Neurobiol. Aging 21, 383421.
  • Aloisi F. (1999) The role of microglia and astrocytes in CNS immune surveillance and immunopathology. Adv. Exp. Med. Biol. 468, 123133.
  • Ames B. N., Shigenaga M. K. and Hagen T. M. (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl Acad. Sci. USA 90, 79157922.
  • Arai H., Lee V. M., Hill W. D., Greenberg B. D. and Trojanowski J. Q. (1992) Lewy bodies contain beta-amyloid precursor proteins of Alzheimer's disease. Brain Res. 585, 386390.
  • Behl C., Davis J., Cole G. M. and Schubert D. (1992) Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem. Biophys. Res. Commun. 186, 944950.
  • Behl C., Davis J. B., Lesley R. and Schubert D. (1994) Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77, 817827.
  • Bianca V. D., Dusi S., Bianchini E., Dal Pra I. and Rossi F. (1999) Beta-amyloid activates the inline image forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease. J. Biol. Chem. 274, 1549315499.
  • Chao C. C., Gekker G., Sheng W. S., Hu S., Tsang M. and Peterson P. K. (1994) Priming effect of morphine on the production of tumor necrosis factor-alpha by microglia implications in respiratory burst activity and human immunodeficiency virus-1 expression. J. Pharmacol. Exp. Ther. 269, 198203.
  • Combs C. K., Johnson D. E., Cannady S. B., Lehman T. M. and Landreth G. E. (1999) Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins. J. Neurosci. 19, 928939.
  • Combs C. K., Johnson D. E., Karlo J. C., Cannady S. B. and Landreth G. E. (2000) Inflammatory mechanisms in Alzheimer's disease: inhibition of beta-amyloid-stimulated proinflammatory responses and neurotoxicity by PPARgamma agonists. J. Neurosci. 20, 558567.
  • Combs C. K., Karlo J. C., Kao S. C. and Landreth G. E. (2001) β-Amyloid stimulation of microglia and monocytes results in INFα-dependent expression of inducible nitric oxide synthase and neuronal apoptosis. J. Neurosci. 21, 1791188.
  • Cotman C. W. and Su J. H. (1996) Mechanisms of neuronal death in Alzheimer's disease. Brain Pathol. 6, 493506.
  • Cross A. R. and Jones O. T. (1991) Enzymic mechanisms of superoxide production. Biochim. Biophys. Acta 1057, 281298.
  • El Khoury J., Hickman S. E., Thomas C. A., Cao L., Silverstein S. C. and Loike J. D. (1996) Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils. Nature 382, 716719.
  • Giulian D., Haverkamp L. J., Li J., Karshin W. L., Yu, J., Tom D., Li X. and Kirkpatrick J. B. (1995) Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain. Neurochem. Int. 27, 119137.
  • Goodman Y., Steiner M. R., Steiner S. M. and Mattson M. P. (1994) Nordihydroguaiaretic acid protects hippocampal neurons against amyloid beta-peptide toxicity, and attenuates free radical and calcium accumulation. Brain Res. 654, 171176.
  • Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J. S. and Tannenbaum S. R. (1982) Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131138.
  • Griffin W. S., Sheng J. G., Royston M. C., Gentleman S. M., Mckenzie J. E., Graham D. I., Roberts G. W. and Mrak R. E. (1998) Glial–neuronal interactions in Alzheimer's disease: the potential role of a ‘cytokine cycle’ in disease progression. Brain Pathol. 8, 6572.
  • Gutteridge J. M. and Halliwell B. (1989) Iron toxicity and oxygen radicals. Baillieres Clin. Haematol. 2, 195256.
  • Hensley K., Carney J. M., Mattson M. P., Aksenova M., Harris M., Wu J. F., Floyd R. A. and Butterfield D. A. (1994) A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease. Proc. Natl Acad. Sci. USA 91, 32703274.
  • Hu J. and Van Eldik L. J. (1999) Glial-derived proteins activate cultured astrocytes and enhance beta amyloid-induced glial activation. Brain Res. 842, 4654.
  • Ii M., Sunamoto M., Ohnishi K. and Ichimori Y. (1996) β-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity. Brain Res. 720, 93100.
  • Kang J., Park E. J., Jou I., Kim J. H. and Joe E. H. (2001) Reactive oxygen species mediate Aβ(25–35)-induced activation of BV-2 microglia. Neuroreport 12, 14491452.
  • Kim W. G., Mohney R. P., Wilson B., Jeohn G. H., Liu B. and Hong J. S. (2000) Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J. Neurosci. 20, 63096316.
  • Klegeris A., Walker D. G. and McGeer P. L. (1994) Activation of macrophages by Alzheimer beta amyloid peptide. Biochem. Biophys. Res. Commun. 199, 984991.
  • Kreutzberg G. W. (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312318.
  • Lawson L. J., Perry V. H., Dri P. and Gordon S. (1990) Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience 39, 151170.
  • Lees G. J. (1993) Contributory mechanisms in the causation of neurodegenerative disorders. Neuroscience 54, 287322.
  • Li Y. and Trush M. A. (1998) Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem. Biophys. Res. Commun. 253, 295299.
  • Li Y., Liu L., Kang J., Sheng J. G., Barger S. W., Mrak R. E. and Griffin W. S. (2000) Neuronal–glial interactions mediated by interleukin-1 enhance neuronal acetylcholinesterase activity and mRNA expression. J. Neurosci. 20, 149155.
  • Liu B., Du L. and Hong J. S. (2000) Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J. Pharmacol. Exp. Ther. 293, 607617.
  • Liu B., Wang K., Gao H. M., Mandavilli B., Wang J. and Hong J. S. (2001) Molecular consequences of activated microglia in the brain: overactivation induces apoptosis. J. Neurochem. 77, 182189.
  • Liu Y. X., Qin L. Y., Wilson B. C., An L. J., Hong J. S. and Liu B. (2002) Inhibition by naloxone stereoisomers of beta-Amyloid peptide (1–42)-induced superoxide production in microglia and degeneration of cortical and mesencephalic neurons. J. Pharmacol. Exp. Ther. 302, 12121219.
  • McDonald D. R., Brunden K. R. and Landreth G. E. (1997) Amyloid fibrils activate tyrosine kinase-dependent signaling and superoxide production in microglia. J. Neurosci. 17, 22842294.
  • McGeer P. L. and McGeer E. G. (1995) The inflammatory response system of the brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res. Rev. 21, 195218.
  • McMillian M. K., Thai L., Hong J. S., O'Callaghan J. P. and Pennypacher K. R. (1994) Brain injury in a dish: a model for reactive gliosis. Trends Neurosci. 17, 138142.
  • Meda L., Bonaiuto C., Szendrei G. I., Ceska M., Rossi F. and Cassatella M. A. (1995a) Beta-Amyloid(25–35) induces the production of interleukin-8 from human monocytes. J. Neuroimmunol. 59, 2933.
  • Meda L., Cassatella M. A., Szendrei G. I., Otvos L. Jr, Baron P., Villalba M., Ferrari D. and Rossi F. (1995b) Activation of microglial cells by beta-amyloid protein and interferon-gamma. Nature 374, 647650.
  • Minghetti L. and Levi G. (1998) Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide. Prog. Neurobiol. 54, 99125.
  • Olanow C. W. (1992) An introduction to the free radical hypothesis in Parkinson's disease. Ann. Neurol. 32, S2S9.
  • Schubert D., Behl C., Lesley R., Brack A., Dargusch R., Sagara Y. and Kimura H. (1995) Amyloid peptides are toxic via a common oxidative mechanism. Proc. Natl Acad. Sci. USA 92, 19891993.
  • Tammariello S. P., Quinn M. T. and Estus S. (2000) NADPH oxidase contributes directly to oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neurons. J. Neurosci. 20, RC53.
  • Van Muiswinkel F. L., Veerhuis R. and Eikelenboom P. (1996) Amyloid beta protein primes cultured rat microglial cells for an enhanced phorbol 12-myristate 13-acetate-induced respiratory burst activity. J. Neurochem. 66, 24682476.
  • Viel J. J., McManus D. Q., Smith S. S. and Brewer G. J. (2001) Age- and concentration-dependent neuroprotection and toxicity by TNF in cortical neurons from β-amyloid. J. Neurosci. Res. 64, 454465.
  • Yankner B. A., Duffy L. K. and Kirschner D. A. (1990) Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science 250, 279282.