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Keywords:

  • Alzheimer's disease;
  • glutamate;
  • microglia;
  • nitric oxide;
  • synapse;
  • xc exchange

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Microglial activation as part of a chronic inflammatory response is a prominent component of Alzheimer's disease. Secreted forms of the β-amyloid precursor protein (sAPP) previously were found to activate microglia, elevating their neurotoxic potential. To explore neurotoxic mechanisms, we analyzed microglia-conditioned medium for agents that could activate glutamate receptors. Conditioned medium from primary rat microglia activated by sAPP caused a calcium elevation in hippocampal neurons, whereas medium from untreated microglia did not. This response was sensitive to the NMDA receptor antagonist, aminophosphonovaleric acid. Analysis of microglia-conditioned by HPLC revealed dramatically higher concentrations of glutamate in cultures exposed to sAPP. Indeed, the glutamate levels in sAPP-treated cultures were substantially higher than those in cultures treated with amyloid β-peptide. This sAPP-evoked glutamate release was completely blocked by inhibition of the cystine–glutamate antiporter by α-aminoadipate or use of cystine-free medium. Furthermore, a sublethal concentration of sAPP compromised synaptic density in microglia–neuron cocultures, as evidenced by neuronal connectivity assay. Finally, the neurotoxicity evoked by sAPP in microglia-neuron cocultures was attenuated by inhibitors of either the neuronal nitric oxide synthase (NG-propyl-l-arginine) or inducible nitric oxide synthase (1400 W). Together, these data indicate a scenario by which microglia activated by sAPP release excitotoxic levels of glutamate, probably as a consequence of autoprotective antioxidant glutathione production within the microglia, ultimately causing synaptic degeneration and neuronal death.

Abbreviations used
AAA

α-aminoadipic acid

amyloid β-peptide

AD

Alzheimer's disease

APV

2-amino-5-phosphonovaleric acid

βAPP

β-amyloid precursor protein

[Ca2+]i

intracellular free calcium concentration

FBS

fetal bovine serum

HBH

HEPES-buffered Hank's balanced salt solution

IFN

interferon

IL

interleukin

iNOS

inducible nitric oxide synthase

MEM

minimal essential medium

MTT

methyltetrazolium

nNOS

neuronal nitric oxide synthase

ROS

reactive oxygen species

sAPP

secreted β-amyloid precursor protein

Accumulating evidence indicates that chronic neuroinflammatory processes play a significant role in the pathogenesis of Alzheimer's disease (AD). Non-steroidal anti-inflammatory drugs decrease the risk of incidence and slow the progression of AD (Breitner 1996). Moreover, the neuritic plaques pathognomic for AD contain activated microglia (reviewed in Griffin et al. 1998), as do those plaques that accumulate in β-amyloid precursor protein (βAPP) transgenic mice (Frautschy et al. 1998; Huang et al. 1999; Stalder et al. 1999). Several substances are produced by these activated microglia that are implicated in models of neurotoxicity: pro-inflammatory, neurotoxic cytokines (Shohami et al. 1994; Chao et al. 1995; Yeung et al. 1995); reactive oxygen species (ROS), such as those formed from nitric oxide (NO) (Beckman 1991; Lipton et al. 1993); and glutamate (Patrizio and Levi 1994; Piani and Fontana 1994; Klegeris and McGeer 1997). A role for glutamate is consistent with indications of calcium-mediated excitotoxic damage in Alzheimer's disease. Specifically, cells expressing ionotropic glutamate receptors appear to be preferentially affected (Jansen et al. 1990) and the products of calcium-mediated toxicity are elevated in Alzheimer-afflicted brains (Saito et al. 1993). Even more compelling therapeutically is the attenuation of clinical dementia in patients treated with memantine, a non-competitive NMDA receptor antagonist (Winblad and Poritis 1999).

Genetics, neuropathology, biochemistry and cell biology all point towards the β-amyloid precursor protein (βAPP) as a pathogenic instigator in AD, but the relative inflammatory capacities of its various isoforms and proteolytic fragments remain unclear. Mechanistic hypotheses seeking to combine inflammatory indications with βAPP biology have focused on the ability of amyloid β-peptide (Aβ) to activate microglia (Meda et al. 1995), but microglia are also activated by secreted βAPP (sAPP) (Barger and Harmon 1997). Although CSF sAPP levels reportedly are depressed in some AD patient populations (Van Nostrand et al. 1992; Wagner et al. 1994), the striking abundance of βAPP in plaque-associated dystrophic neurites (Shoji et al. 1990; Cras et al. 1991; Joachim et al. 1991) suggests that sAPP could be elevated locally. Indeed, a recently described mutation associated with familial AD results in elevated sAPP levels without an alteration of Aβ production (Ancolio et al. 1999). Secreted APP activates microglia independently of costimuli and is effective even as a β-secretase product. Moreover, sAPP is approximately three orders of magnitude more potent and over five-fold more efficacious than Aβ1-42 in activating microglia (Barger et al. 1999).

The activation of microglia by sAPP results in elevated neurotoxicity in microglia-neuron cocultures where the two cell types are not in contact (Barger and Harmon 1997), suggesting the involvement of a diffusible toxin. Potential neurotoxins identified include NO, as evidenced by elevated medium nitrite levels and inducible nitric oxide synthase (iNOS) expression. However, the possibility that excitotoxins are released by activated microglia suggests that NO could be produced by neuronal nitric oxide synthase (nNOS) within the neurons themselves. The present study identifies glutamate as an excitotoxin elaborated in response to sAPP treatment and demonstrates the resulting effects on neuronal survival, as well as functionally important consequences for synaptic integrity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

Protein preparations of sAPPα, sAPPβ and the sAPPα304-612 deletion mutant were performed as described (Barger and Harmon 1997; Barger 1998). Proteins were expressed from a prokaryotic expression vector with a polyhistidine tag for uniformity of purification. The sAPPα304-612 construct was created by removal of an XhoI fragment from the sAPPα expression vector (Barger and Harmon 1997). Aminoadipic acid (AAA) and 2-amino-5-phosphonovaleric acid (APV) were obtained from Sigma (St Louis, MO, USA). Aβ1-42 was from AnaSpec (San Jose, CA, USA); no ‘aging’ protocol was utilized, as this sequence aggregates very rapidly in aqueous solutions (confirmed in direct neurotoxicity assays). Rat interferon-γ was from PeproTech (Rocky Hill, NJ, USA). Fura-2/acetomethoxyester (/AM) was from Molecular Probes (Eugene, OR, USA). NG-propyl-l-arginine was from Calbiochem (La Jolla, CA, USA) and 1400 W was from Alexis Biochemicals (San Diego, CA, USA).

Cell cultures

Primary microglia were acquired through differential adherence from mixed glial cultures (Barger and Harmon 1997). Initial cultures were established from the cerebral cortices of 3-day-old rats. After 10–14 days in minimal essential medium (MEM) containing 10% fetal bovine serum (FBS) and 10 µg/mL gentamycin-sulfate, astrocytes had reached confluency and microglia were sufficiently numerous and detached to be removed by gentle stream flow of the medium aspirated and dispensed from a serological pipette three times. The suspended cells were collected by centrifugation of the medium, resuspended in MEM with 10% FBS, and plated at 16 000 cells per well into 96-well plates (nitrite assays) or 200 000 per well into 24-well plates (glutamate assays) or 35-mm Millipore culture-well inserts (neurotoxicity assays). Treatments were administered 18–24 h after replating.

Primary neuronal cultures were established from embryonic rats at 18 days of gestation as described (Mattson et al. 1995). Hippocampal neurons were plated into 6-well plates at ∼1 × 105 cells per well (neurotoxicity assays) or into glass-bottomed 35-mm plates (calcium imaging). Hippocampal neurons were maintained in Neurobasal medium supplemented with B27 (Gibco/Life Technologies Inc., Rockville, MD, USA) and 10 µg/mL gentamycin-sulfate. Neocortical cultures were plated into glass-bottomed 35-mm plates at ∼3 × 105 cells per plate and were maintained in MEM containing 10% FBS, 10 mm additional glucose, 14.7 mm additional KCl and 10 µg/mL gentamycin-sulfate. All culture surfaces utilized for neuronal cultures were coated with polyethyleneimine as described by Mattson et al. (1995).

Calcium measurements

For conventional measurements of responses to glutamate or conditioned medium, primary hippocampal neurons were loaded with fura-2/acetomethoxyester (AM) as described (Barger 1999). Immediately before imaging, the culture medium was replaced with a HEPES-buffered Hank's balanced salt solution (HBH) (Barger 1999). Fluorescent images were acquired at ∼5-s intervals from 15–25 neuronal somata at a time, and intracellular free calcium concentration ([Ca2+]i) was calculated by an InCa dual-wavelength imaging system (Intracellular Imaging Inc., Cincinnati, OH, USA). Traces represent the mean [Ca2+]i measured in all of the neurons from three cultures. For synchronous oscillations, primary neocortical cultures were loaded with fura-2AM and placed in HBH. Fluorescent images were acquired at ∼2/s from ∼20 neuronal somata. During data acquisition, the HBH was replaced (by three complete buffer changes) with a similar buffer that was nominally free of magnesium, as indicated. Traces represent the mean of all neurons from a single plate that is representative of three replicates. Frequency of oscillation is represented as the number of spikes > 50 nm occuring per min, as calculated during the first 400 s after magnesium removal, for three cultures per condition. Following imaging, these cultures were assessed for viability via the methyltetrazolium (MTT) reduction assay as described (Mao and Barger 1998).

Nitrite measurements

Primary microglia plated in 96-well plates were changed to serum-free MEM one day after plating, and treatments were applied. After 18 h, 100 µL of medium was transferred to a new plate. An equal volume was added of the indicator reagent: 0.5% sulfanilamide and 0.05% naphthylethyleneamine dihydrochloride in 0.25% phosphoric acid. After 10 min, the absorbance was measured at 540 nm. Absolute concentrations of nitrite were calculated by interpolation in a standard curve generated with known concentrations of sodium nitrite. Values represent the mean ± SEM of quadruplicate determinations.

Glutamate measurements

Primary microglia plated in 24-well plates were changed to serum-free medium and treated as indicated. For cystine-free medium, Gibco Select-AMINE kit was prepared with cystine omitted. After 20 h, the culture medium was transferred to microfuge tubes and cleared by centrifugation at 350 g for 5 min. The supernatant was transferred to new tubes and stored at − 20°C. For quantification, glutamate was derivatized with o-pthaldialdehyde, followed by HPLC separation and fluorescence measurement (Espey et al. 1998).

Neurotoxicity assays

After 8–9 days in culture, hippocampal neuronal cultures were exposed to Millipore culture-well inserts containing microglia that had been pre-treated 24 h with 10 nm sAPP. At the time of introduction of the microglial inserts and after an additional 24 h, random fields were marked and photographed. The percentage of initial cells surviving at 24 h was determined by visual comparison of established morphological criteria by an observer blinded to the treatment conditions (Mattson et al. 1995).

Statistics

Data sets were analyzed by anova, followed by Scheffe's post hoc test for pair-wise comparison between groups. A p-value less than or equal to 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

As observed in several inflammatory paradigms, the activation of microglia by sAPP involves the exhibition of enhanced neurotoxicity (Barger and Harmon 1997). Because of the intimate involvement of calcium regulation in various aspects of neuronal survival, we assayed the effects of microglial products on intracellular free calcium concentrations ([Ca2+]i) in primary hippocampal neurons. Primary microglia obtained from the cerebral cortices of neonatal rats either were left untreated or were treated for 20 h with sAPPβ. The calcium-modulating activity of sAPP is dependent upon the unique carboxyterminal residues of sAPPα (Furukawa et al. 1996); therefore, sAPPβ was used to avoid confounding effects in the subsequent assay. The conditioned medium from these microglial cultures was then diluted into the buffer covering primary hippocampal neurons during measurement of [Ca2+]i through fura-2 microfluorescent imaging. Upon the addition of conditioned medium from untreated microglia, a small, gradual increase in [Ca2+]i was observed (Fig. 1). In contrast, the addition of conditioned medium from sAPP-activated microglia was followed immediately by a much larger and more rapid elevation of [Ca2+]i. This response of the hippocampal neurons to the conditioned medium was reversed by the addition of 2-amino-5-phosphonovaleric acid (APV), an inhibitor of ionotropic glutamate receptors.

image

Figure 1. Release of a glutamate receptor agonist by sAPP-treated microglia. Primary hippocampal neurons were assayed for [Ca2+]i by fura-2 fluorometric microscopy. The bar on the x-axis indicates the presence (at a 1 : 40 dilution) of conditioned medium from primary microglial cultures. The microglia from which the conditioned medium was collected were either untreated (‘Con-CM’) or treated with 10 nm sAPPβ for 24 h. As a positive control, glutamate (200 µm) was added to the neurons treated with Con-CM at the time indicated. Likewise, APV (100 µm) was added to the neurons treated with sAPPβ-CM at the time indicated.

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The APV-sensitive elevation of [Ca2+]i by conditioned medium from sAPP-treated microglia suggested that a glutamatergic agonist was released by these microglia. To test directly the possibility that glutamate itself was being released, levels of the amino acid in conditioned medium were assayed by HPLC. sAPPα evoked a significant, dose-dependent release of glutamate from primary microglia (Fig. 2). We compared the effect of sAPP to that of Aβ1-42, which often requires a priming costimulus such as interferon gamma (IFNγ) to exert significant pro-inflammatory stimulation. The response to sAPP was both more efficacious and more potent than that to Aβ, even in the presence of IFNγ. The effect of sAPP on microglial glutamate release also showed specific structural requirements: namely, sAPPα and sAPPβ had similar potency and efficacy, while an aminoterminal deletion mutant was approximately four-fold less potent (Fig. 3).

image

Figure 2. Release of glutamate by sAPP-treated microglia. Primary microglia were treated for 20 h with the indicated concentrations of sAPPα (nm) or Aβ1-42m). These treatments also were tested in the presence of 10 ng/mL rat interferon-γ. The medium was assayed for glutamate by fluorescence HPLC. Values reflect the mean (+ SEM) for triplicate cultures.

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image

Figure 3. Structural requirements for sAPP stimulation of microglial glutamate release. Primary microglia were treated with the indicated concentrations of sAPPα, sAPPβ or sAPPα304-612. After 20 h, the medium was assayed for glutamate. Values reflect the mean (+ SEM) for triplicate cultures. (p < 0.01, sAPPα304-612 versus each other group)

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One mechanism for glutamate transport across cell membranes is via a cystine–glutamate antiporter. At high extracellular glutamate concentrations, this exchanger can deprive the cytosol of the cystine needed for glutathione synthesis, leading to oxidative stress (Murphy et al. 1989). Under conditions where glutathione (and, thus, cystine) consumption exceeds extracellular glutamate levels, the antiporter discharges substantial amounts of glutamate from the cell. To test whether this mechanism was involved in the release of glutamate triggered by sAPP, we applied two approaches. First, we assayed the effects of sAPP on glutamate release in a medium nominally devoid of cystine. While stimulation of primary microglial cultures in cystine-free medium with sAPP resulted in increased release of glutamate into the medium, the absolute concentrations of glutamate released were greatly diminished (Fig. 4). The second approach was to treat microglia with α-aminoadipate (AAA), an inhibitor of the cystine–glutamate antiporter. Exposure to sAPP did not stimulate glutamate release from microglia in the presence of AAA (Fig. 4).

image

Figure 4. Dependency of microglial glutamate release on the cystine antiporter. Primary microglia were treated for 20 h with 10 nm sAPPα in the absence or presence of 2.5 mmα-aminoadipate (AAA). A set of cultures was also tested in a culture medium lacking cystine. Values reflect the mean (+ SEM) for triplicate cultures (*p < 0.01).

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Activated microglia can evoke neuronal cell death, but equally harmful effects on function could result from the loss of synaptic connectivity. Furthermore, glutamate has been documented to prune dendrites dosimetrically and chronologically prior to causing outright cell death (Mattson and Barger 1993). Therefore, we tested whether microglia activated by sublethal concentrations of sAPP could have this kind of subtle effect. As an index of synaptic integrity, we measured the frequency of synchronous [Ca2+]i oscillations. These oscillations occur spontaneously upon dramatic reductions of buffer Mg2+ concentration, and their frequency has been shown to be a reliable index of synaptic density (Muramoto et al. 1993). Primary microglia were placed in coculture with high-density cultures of neocortical neurons grown on glass-bottomed plates; microglia were introduced in basket-type culture-well inserts (Barger and Harmon 1997). Such cocultures were left untreated or were exposed to sAPPβ at a concentration 10-fold lower than that shown to evoke significant neurotoxicity. (These high-density cortical cultures were more resistant to toxicity than the lower density hippocampal cultures used in other experiments.) After 24 h, the culture-well inserts containing the microglia were removed, and the neurons were subjected to high-frequency fluorometric imaging for measurement of [Ca2+]i. As illustrated in Fig. 5, the reduction of extracellular Mg2+ initiated a series of synchronous [Ca2+]i oscillations with a period of approximately 12 s in neuronal cultures exposed only to microglia. However, in cocultures treated with sAPPβ, the period was increased. Analysis of oscillation frequency across multiple cultures yielded mean values (spikes per min ± SEM) of 5.6 + 0.6 for control cultures and 2.2 + 0.2 for sAPP-treated cultures (p < 0.02, Student's t-test). Following the fluorometric imaging, each neuronal culture was assayed for cell viability by MTT reduction assay, and the results indicated no decline of viability in sAPPβ-treated cultures relative to controls. As an additional control for the direct effects of sAPPβ on synaptic function, we treated cortical neurons with sAPPβ at the same dose and duration in the absence of microglia; no effect was observed (data not shown).

image

Figure 5. Alteration of synaptic function by sAPP-activated microglia. Primary microglia were placed in basket-type insert cocultures with primary neocortical neurons. Some cultures were treated with 3 nm sAPPβ. After 24 h, the neurons were loaded with fura-2 and subjected to fluorometric microscopy, measuring [Ca2+]i at 1.5-s intervals before and after the removal of Mg2+ from the imaging buffer. The traces represent the mean [Ca2+]i (nm) in 28 (Con) to 31 (sAPP-treated) cells. The bar indicates the period when Mg2+ was present.

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The damage evoked by an excitotoxin such as glutamate can be limited to dendrites dosimetrically; higher concentrations evoke whole-cell killing (Mattson and Barger 1993). Similarly, concentrations of sAPP can be reached that will cause neuronal death in neuron/microglia cocultures (Barger and Harmon 1997). It has been speculated that NO produced by glial iNOS under similar paradigms of activation could be responsible for such glia-mediated forms of neurotoxicity, but excitotoxins also generate NO from nNOS. To test the relative contributions to neurotoxicity of NO from these distinct sources, basket-type cocultures of microglia and hippocampal neurons were treated with sAPPβ in the presence of NOS inhibitors relatively selective for nNOS or iNOS. NG-propyl-l-arginine is more potent against nNOS than iNOS (published Ki = 57 nm versus 180 µm), whereas 1400 W is a more specific inhibitor of iNOS (published Ki = 7 nm versus 1.4 µm). After a 24-h treatment with sAPPβ, ∼60% of neurons were killed (in contrasted with ∼30% in the absence of sAPPβ). NOS inhibitors significantly ameliorated this neurotoxicity (Fig. 6). In simple dosimetry, NG-propyl-l-arginine and 1400 W had similar neuroprotective potencies.

image

Figure 6. Role of NO in the neurotoxicity of sAPP-activated microglia. Primary microglia were placed in basket-type insert cocultures with primary hippocampal neurons. Cultures were treated with 10 nm sAPPβ in the presence of various concentrations of (▪) NG-propyl-l-arginine or (○) 1400 W (doses were selected to bracket the reported Ki of each drug.) Neurons surviving 24 h later are reflected as the percentage of the original cell number. Values reflect the mean + SEM of triplicate cultures.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Inflammatory activation of microglia appears to contribute to neuronal cell death in a variety of conditions, and considerable evidence suggests that such processes may occur in Alzheimer's disease. The βAPP gene is connected to Alzheimer's both genetically and biochemically, and microglia can be activated by two of its proteolytic products: Aβ and sAPP. We have explored the mechanism behind microglial neurotoxicity in the context of activation by sAPP. The data indicate that sAPP promotes the release of glutamate from microglia, which can dramatically elevate neuronal calcium levels. This glutamate release is dependent on extracellular cystine and appears to involve recruitment of cystine–glutamate antiporter activity to support antioxidant pathways. Notably, sAPP was much more potent and efficacious than Aβ in triggering this response from microglia. The glutamate release can evoke neurotoxicity, which may be restricted to the loss of neurites and synapses. These data suggest a sequence of events that begins with a stress-related elevation in βAPP expression and culminates in a subcellular excitotoxic event, resulting in loss of neurotransmission with or without neuronal cell death.

The cystine–glutamate antiporter has been implicated in other neurotoxic paradigms. The cystine–glutamate antiporter expressed in macrophages has been implicated in transcellular neuronal killing similar to that described here (Piani and Fontana 1994). Working in the opposite direction, the antiporter also can contribute to glutamate toxicity in its own cell of expression (Miyamoto et al. 1989; Murphy et al. 1989; Sagara et al. 1993; Pereira and Oliveira 1997; Tan et al. 1998). Thus, immature neurons or other cell types can succumb to glutamate toxicity via a mechanism that does not involve ionotropic glutamate receptors. As cystine is a precursor to glutathione, it seems likely that the cystine–glutamate exchange responsible for the exudation of glutamate in activated phagocytes results from the consumption of glutathione by the reactive oxygen species they produce. Therefore, it would be interesting to determine whether supplanting this mechanism by protecting against cellular oxidation in other ways would alleviate neurotoxicity. Perhaps this is one mechanism through which antioxidants suppress inflammation-related neurodegeneration (e.g., al-Shabanah et al. 1996).

The present data suggest that the indirect neurotoxicity of sAPP mediated by microglia involves both NO derived from activated microglia and that which is mobilized in the neurons themselves by calcium-mediated activation of nNOS. NO is produced at a high rate under both inflammatory and excitotoxic conditions and has been associated with several detrimental mechanisms (Dawson and Dawson 1998). Much of the NO produced in inflammation is generated by iNOS, expressed in activated macrophages and microglia. However, nNOS is present in the neurons themselves; it responds to calcium elevation and appears to be responsible for a large fraction of calcium-dependent excitotoxicity (Dawson et al. 1991). Notably, iNOS can potentiate glutamate toxicity mediated by the NMDA class of receptors (Hewett et al. 1994). In the MPTP model of Parkinson's disease, animals devoid of iNOS show better neuronal survival in the substantia nigra but not in the striatum (Liberatore et al. 1999), suggesting that iNOS has a differential role in different regions or situations. In the model presented here, the amelioration of microglial toxicity by NG-propyl-l-arginine, a relatively specific inhibitor of nNOS, was similar to that observed with 1400 W, which has specificity for iNOS (and a lower Ki for its respective target). However, the Ki of each drug for its preferred target differs by more than eight-fold; therefore, one might expect 1400 W to be more potent than NG-propyl-l-arginine if nNOS and iNOS were equally involved in toxicity. In preliminary studies, a distinction was observed between nitro-l-arginine, a NOS inhibitor somewhat selective for nNOS, and N-methyl-l-arginine, a non-selective NOS inhibitor. Therefore, it appears that NO from both sources contributed to neurotoxicity in our model, but the relative contributions may not be equal. Additional data from cells bearing targeted deletion of each NOS isoform might be useful in resolving this issue.

Microglia appear to be capable of releasing sufficient levels of glutamate to contribute to neurodegeneration. Extracellular levels of glutamate generally are controlled tightly by the combined activities of glutamate transporters and glutamine synthase in astrocytes (Schousboe et al., 1998). However, oxidative stress or energy restriction can compromise these functions (Butterfield et al. 1997; Trotti et al. 1997), a reasonable concern considering the indications that oxidation and/or mitochondrial abnormalities contribute to Alzheimer's disease (Butterfield et al. 1999; Beal 2000). It is also possible that there are microenvironments in which the apposition of microglia and neurons makes astrocyte actions less significant. Indeed, glutamate release by microglia could even play a diffuse signal-modulating role. However, empirical evidence also pertains: we have reported previously that an inflammatory state induced in vivo by viral infection broadly elevates the levels of glutamate in CSF and parenchymal microdialysates by a mechanism sensitive to α-aminoadipate and independent of astrocytic transport or catabolism (Espey et al. 1998).

Regarding dose and structural requirements, the results reported here are consistent with other indices of microglial activation by sAPP, including the apparent involvement of an ApoE-binding domain in the aminoterminal 411 amino acids (Barger and Harmon 1997). Despite some caveats, the potential pro-inflammatory role of sAPP is intriguing as an explanation of Alzheimer pathogenesis or injury responses in general. Overexpression of βAPP in transgenic mice results in Alzheimer-like pathology and symptoms, including strong evidence of inflammatory events (Frautschy et al. 1998; Stalder et al. 1999). Nishimura et al. (1998) delivered a βAPP expression vector to hippocampal neurons in vivo and observed neurodegeneration with a microglial involvement. While some of these conditions may elevate Aβ production, it does not appear to be required. For instance, the in vivo delivery of βAPP by Nishimura et al. did not lead to production of Aβ or the carboxyterminal product generated by β-secretase, a requisite intermediate in Aβ production. Furthermore, a recently described mutation associated with familial AD resulted in elevated sAPPα production while reducing Aβ levels (Ancolio et al. 1999). Finally, pathology and behavioral deficits can occur in βAPP transgenic models without Aβ accumulation (Hsiao et al. 1995). Confirmed neuronal loss is rare in βAPP transgenic mouse lines. However, the synaptic effects detected here indicate that sublethal aberrations can result from microglial activation, which could explain synaptic abnormalities apparent in βAPP transgenic mice (Games et al. 1995; Chapman et al. 1999). Synapse integrity is an early functional and pathological parameter in AD, correlating better with cognitive function than do other neuropathological indices (DeKosky and Scheff 1990; Terry et al. 1991).

In accordance with the strong support for a role of Aβ in AD, many studies have focused on the effects of Aβ on glial activation. The data indicate that Aβ can stimulate pro-inflammatory responses in microglia, including elevated cytokine release, NOS expression and neurotoxicity (Meda et al. 1995). The vast majority of studies have found a requirement for a costimulus such as IFNγ. The required doses are quite high, equivalent with those that evoke direct neurotoxicity in culture. In contrast, sAPP appears competent to activate similar reactions in microglia at much lower concentrations with no costimulus (Barger et al. 1999; hic, Fig. 2). Furthermore, the magnitude of the response to sAPP in multiple assays appears to be several times greater than the response to Aβ (Barger et al. 1999; hic, Fig. 2). Nevertheless, the enhanced release of glutamate from Aβ-activated phagocytes has been documented (Klegeris and McGeer 1997; Noda et al. 1999). In addition, an excitotoxin distinct from glutamate has been reported to accumulate in the medium of microglia exposed to material from amyloid plaques (Giulian et al. 1995). It would be of interest to determine the relative contribution of this or other excitotoxins to the microglia-mediated neurotoxicity of Aβ, as well as the potential involvement of the xc-transporter in Aβ effects.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Technical assistance from Mandy M. Lucas is appreciated. Funds from the National Institute of Aging (2P01AG12411-04A10003) and the Alzheimer's Association contributed to the execution of these studies.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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