Amyloid Protein Precursor Stimulates Excitatory Amino Acid Transport
IMPLICATIONS FOR ROLES IN NEUROPROTECTION AND PATHOGENESIS*

Eliezer MasliahDagger §, Jacob Raber§, Michael AlfordDagger , Margaret MalloryDagger , Mark P. Mattsonparallel , Daseng Yang, Derek Wong, and Lennart Mucke**Dagger Dagger

From the Dagger  Departments of Neurosciences and Pathology, University of California San Diego, La Jolla, California 92093-0624, the  Gladstone Institute of Neurological Disease and Department of Neurology, University of California San Francisco, San Francisco, California 94141-9100 the parallel  Sanders-Brown Research Center on Aging and Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536 and ** Neuroscience Program, University of California, San Francisco, California 94141-9100

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Excitatory neurotransmitters such as glutamate are required for the normal functioning of the central nervous system but can trigger excitotoxic neuronal injury if allowed to accumulate to abnormally high levels. Their extracellular levels are controlled primarily by transmitter uptake into astrocytes. Here, we demonstrate that the amyloid protein precursor may participate in the regulation of this important process. The amyloid protein precursor has been well conserved through evolution, and a number of studies indicate that it may function as an endogenous excitoprotectant. However, the mechanisms underlying this neuroprotective capacity remain largely unknown. At moderate levels of expression, human amyloid protein precursors increased glutamate/aspartate uptake in brains of transgenic mice, with the 751-amino acid isoform showing greater potency than the 695-amino acid isoform. Cerebral glutamate/aspartate transporter protein levels were higher in transgenic mice than in non-transgenic controls, whereas transporter mRNA levels were unchanged. Amyloid protein precursor-dependent stimulation of aspartate uptake by cultured primary astrocytes was associated with increases in protein kinase A and C activity and could be blocked by inhibitors of these kinases. The stimulation of astroglial excitatory amino acid transport by amyloid protein precursors could protect the brain against excitotoxicity and may play an important role in neurotransmission.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The amino acids L-glutamate and L-aspartate are the main excitatory neurotransmitters in the central nervous system (1) and, if allowed to accumulate to abnormally high levels, can derange the neuronal calcium homeostasis and trigger cell death, a process referred to as "excitotoxicity" (2). Excitotoxicity appears to play an important role in a number of neurodegenerative diseases (3).

In vitro and in vivo studies indicate that the amyloid beta  protein precursor (APP)1 may function as an important endogenous excitoprotectant (4, 5). APP is expressed at relatively high levels in the central nervous system of diverse species (6, 7). Multiple APP isoforms are derived from a single gene by alternative splicing (see Ref. 8 for review). The most prevalent forms of APP are 695-770-amino acid glycoproteins with a large extracellular region, a hydrophobic membrane-spanning domain, and a short cytoplasmic segment (9). Proteolytic processing of APP by a presumed "alpha -secretase" results in the secretion of a large N-terminal ectodomain (s-APP). An alternative APP processing pathway gives rise to the Abeta peptide, which appears to play a central role in Alzheimer's disease (10). Cell types differ with respect to the predominant APP isoform they produce and in how much of the APP they synthesize is secreted or retained within the cell. APP695 is produced primarily by neurons, whereas APP751, which contains a Kunitz-type protease inhibitor domain, is produced by many different cell types (11). Neurons are the primary source of APP in the brain and, in humans, express both hAPP695 and hAPP751 (11). In vitro studies indicate that brain cells secrete roughly 10-40% of their APP (12, 13). Expressing full-length APPs in cultured cells or adding s-APP to their media promotes neurite outgrowth, enhances cell survival, and protects cells from a variety of cytotoxic agents (14, 15).

A number of studies suggest that APP may play an important role in neuroplasticity. APP is localized preferentially at central and peripheral synaptic sites (16-18), has been identified in growing neurites of immature rat brain (19), and reaches its highest level of central nervous system expression during postnatal brain maturation and completion of synaptic connections (20, 21).

To elucidate the effects of hAPPs on the central nervous system in vivo, we previously placed cDNAs encoding full-length hAPP695 or hAPP751 downstream of the neuron-specific enolase (NSE) promoter and used the resulting fusion genes to generate multiple lines of NSE-hAPP transgenic mice (22). The levels of neuronal hAPP expression directed by the NSE promoter resulted in only moderate elevations of total APP expression above levels found in nontransgenic controls and, hence, were substantially lower than those found in hAPP models that develop Alzheimer's disease-type neuropathology (8, 22-24). NSE-driven neuronal overexpression of hAPP in transgenic mice or intracerebroventricular infusion of hAPP fragments in nontransgenic rodents increased the number of presynaptic terminals in the neocortex and protected neurons against ischemia and excitotoxins (5, 22, 25-27), suggesting that APP may fulfill important neurotrophic and neuroprotective functions in vivo. In vitro studies suggest that s-APP may prevent excitotoxicity by stabilizing intraneuronal calcium levels (4). However, the mechanisms by which APP exerts excitoprotective effects in vivo have not yet been identified.

Recent studies indicate that high affinity, Na+-dependent uptake of excitatory amino acid transmitters from the extracellular space of the brain is necessary for the prevention of excitotoxicity and neurodegeneration (28, 29). This uptake is mediated by a family of membrane proteins, the excitatory amino acid transporters (EAATs) (30-33). Five EAATs have been cloned: EAAT1 (or GLAST), EAAT2 (or GLT-1), EAAT3 (or EAAC1), EAAT4, and EAAT5 (34-37). EAAT1 is localized in subsets of neurons and astrocytes, whereas other EAATs are expressed primarily in astrocytes (EAAT2) or neurons (EAAT3) (38). Here, we report that hAPP stimulates aspartate uptake in cultures of primary murine astrocytes and in brains of NSE-hAPP transgenic mice. We also provide evidence that hAPP increases the availability of EAATs and that this process involves the activity of protein kinase A and C. This newly identified APP function could play an important role in the prevention of excitotoxic neuronal injury and in neurotransmission.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Transgenic Mice and Kainic Acid Injections---Male and female C57BL/6 × SJL mice, 9-10 months old, were used in this study. Transgenic mice were from previously described NSE-hAPP lines (22): NSE-hAPP751-28 (n = 4), NSE-hAPP751m-57 (n = 14), NSE-hAPP695-60 (n = 4), and NSE-hAPP695m-25 (n = 12). Nontransgenic littermates (n = 17) served as controls. The levels and distribution of hAPP expression in brains of mice from different NSE-hAPP lines have been described in detail elsewhere (5, 8, 22). All transgenic mice included in this study were heterozygous with respect to the transgene. Some mice were challenged with the excitotoxin kainic acid essentially as described previously (5). Kainic acid (Sigma) was diluted in 0.9% NaCl and injected intraperitoneally at a single dose of 25 mg/kg body weight. Mice were analyzed 4 days after the injection. Controls were either untreated or sham-injected with vehicle, as specified under "Results," and analyzed in parallel.

Tissue Processing-- Mice were deeply anesthetized with chloral hydrate and perfused transcardially with cold 0.9% NaCl. Brains were divided sagittally, and individual hemibrains were snap frozen in isopentane cooled in a Histobath (Shandon Lipshaw, Pittsburgh, PA). Subsequently, frontal and parietal neocortices were isolated and processed for Western blot analysis or aspartate uptake assay as described further below.

RNA Analysis-- Total RNA was isolated from snap frozen hemibrains with TRI reagent (Molecular Research Center, Cincinnati, OH). Levels of EAAT1, EAAT2, and EAAT3 mRNAs were determined by RNase protection assay (RPA), essentially as described (8), using the following 32P-labeled antisense riboprobes (protected sequences in parentheses): EAAT1 probe (nucleotides 1586-1706 of murine EAAT1; GenBank accession no. L19565), EAAT2 probe (nucleotides 1686-1914 of murine EAAT2; GenBank accession no. U11763), EAAT3 probe (nucleotides 1488-1651 of murine EAAT3; GenBank accession no. D43797), actin probe (nucleotides 480-559 of murine beta -actin cDNA; GenBank accession no. M18194). RNA (10 µg) hybridized to 32P-labeled antisense riboprobes was digested with 43 units/ml RNase T1 (Life Technologies, Inc.) and 20 µg/ml RNase A (Sigma) in 100 µl of digestion buffer followed by protein digestion with 3.5 mg/ml proteinase K (Sigma) in 6.5% laurylsarcosine. Subsequently, RNA was isolated with 4 M guanidine thiocyanate and precipitated in isopropanol. Samples were separated on 5% acrylamide, 8 M urea Tris-borate-EDTA gels, and dried gels were exposed to Biomax® film (Eastman Kodak Corp.).

Western Blot Analysis-- Rabbit polyclonal antibodies against different EAATs (0.25 µg/ml) were raised at Research Genetics Inc. (Huntsville, AL) with synthetic peptides corresponding to the C-terminal region of the EAAT1, EAAT2, and EAAT3 proteins, as described (38). The particulate fraction was isolated from neocortical homogenates and subjected to Western blot analysis essentially as described (22, 39). To ensure equal loading, the protein content of all samples was determined by the Lowry method (40) and adjusted with homogenization buffer to 1.25 mg/ml; 40 µg of brain protein were loaded per lane. Samples from the particulate fractions were electrophoretically separated on sodium dodecyl sulfate-polyacrylamide gels and blotted onto nitrocellulose membranes. Blots were incubated sequentially with blocking solution, primary antibody (diluted 1:1000, incubated at 4 °C overnight), and radioiodinated protein A (ICN, Irvine, CA) (0.1 µCi/ml, incubated at room temperature for 2 h). Signals were quantitated with a Phosphor-Imager SF (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software and expressed as integrated pixel intensities over defined volumes (22). As controls, similar blots were incubated with preimmune sera or with the antibody preadsorbed with a 20-fold excess of the corresponding specific peptide.

Astroglial Cultures and s-hAPP Treatments-- Primary astrocytes were obtained from whole brains of nontransgenic neonatal mice, essentially as described previously (41). Briefly, brains were disrupted in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum by passing gently through an 18-gauge needle. The cell suspension was transferred into T25 flasks coated with poly-L-lysine (Sigma) (one brain per flask). On day 2, the flasks were shaken at 100 rotations/min for 2 h at 37 °C to remove cell debris and nonastrocytic cells. Primary astrocytes (>95% pure by immunolabeling with antibodies to cell-specific markers) from different mice were then combined and maintained in culture for 21 days. Two days before the aspartate uptake assay (culture day 19), the medium was replaced with Neurobasal medium (Life Technologies, Inc.) containing N2 supplement (Life Technologies, Inc.).

Recombinant s-hAPP695 and s-hAPP751 were produced and purified as described (42). The integrity of these preparations was verified after HPLC purification by comparing immunoreactive bands obtained on Western blot analysis with antibodies directed against the N terminus or the C terminus of alpha -secreted hAPP. s-hAPP695 and s-hAPP751 were dissolved separately in 1 M NaCl and 20 mM Tris (pH 7.2). Astrocyte cultures were treated (without exchange of media) for 30 min with different concentrations of these s-hAPP isoforms (1 pM, 10 pM, 0.1 nM, and 1 nM) or with vehicle alone.

Aspartate Binding and Aspartate Uptake Assays-- Binding of D-[3H]aspartate (NEN Life Science Products) to membrane preparations from neocortex was determined by a method modified from Cross et al. (43) and used to estimate neocortical glutamate/aspartate uptake, essentially as described previously (44). Before analysis, all samples were assigned codes, and the codes were broken only after all results had been downloaded into the data base. Frozen samples of neocortex (50-70 mg, wet weight) were sonicated in 1 ml of buffer (50 mM Tris-HCl/300 mM NaCl, pH 7.4); 9 ml of buffer were added, and the samples were centrifuged at 21,780 × g for 30 min at 4 °C. The supernatant was decanted and the pellet resuspended in 6 ml of buffer. Total protein was determined by the Lowry method (40). Samples were diluted to 0.4 mg/ml total protein. Triplicate tubes (1 ml total volume per tube) each containing 40 µg (100 µl) of washed membranes, D-[3H]aspartic acid (50 nM final concentration), and cold D-aspartic acid (4,000 nM final concentration) were incubated at room temperature for 30 min. Nonspecific binding was determined by adding cold aspartic acid at 10-fold excess to otherwise identical incubation tubes. Bound ligand was separated from free ligand by filtration through 0.45-µm glass fiber filters on a disposable filtration manifold (V&P Scientific, San Diego, CA). After addition of 5 ml of EcoLume scintillation mixture (ICN BioMedicals, Costa Mesa CA), filter disks were counted with a TM Analytic 6881 Mark III scintillation spectrophotometer. Results were expressed as picomoles of D-aspartate bound/mg of total protein. To determine the kinetics of D-aspartate binding, neocortical membrane preparations (20 µg of total protein) were incubated for 30 min at room temperature with D-[3H]aspartic acid (0.05 µM) in the presence of increasing concentrations of cold D-aspartate (0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 4.8, and 6.4 µM). For each mouse and dose of cold D-aspartate, D-[3H]aspartate binding was then determined in quadruplicate, essentially as described above.

To confirm the specificity of D-[3H]aspartate binding to glutamate/aspartate transporters under the conditions used here, displacement curves were obtained with unlabeled L-glutamate or L-beta -threo-3-hydroxyaspartate (THA) (both from Sigma). THA has been shown previously to specifically inhibit glutamate/aspartate uptake (45). 20 µg of neocortical membrane proteins (by Lowry assay), 20 nM D-[3H]aspartate, and displacers ranging in concentration from 1 nM to 30 µM were incubated in 1-ml incubation volumes in triplicate tubes for 30 min at room temperature, as described previously (44). Nonspecific binding was defined as binding in the presence of 0.1 mM cold D-aspartate added to otherwise identical incubation tubes. Unbound ligand was separated by rapid filtration through 0.45-µm filter disks on disposable microfold filtration manifolds. Filter disks were washed twice with 200 µl of buffer and counted in a scintillation spectrophotometer.

For aspartate uptake measurements in primary astrocyte cultures, cells were seeded in 96-well plates at 104 cells/well. D-[3H]Aspartic acid (NEN Life Science Products) was used at 0.6 µCi/well, and the final concentration was adjusted to 4,000 nM with cold D-aspartic acid. After an additional 30 min of incubation at 37 °C the cultures were washed twice with phosphate-buffered saline, followed by cell lysis in 2 N KOH, scintillation counting, and protein determination (Micro BCA protein assay reagent kit; Pierce). Exposure of cultures to L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC; 20 µM final concentration) (Research Biochemicals International, Natick, MA) was used to specifically inhibit glutamate/aspartate transporters (46). Two independent batches of primary astrocytes were used to determine the kinetics of astroglial aspartate uptake. In each experiment, cells were incubated for 30 min with vehicle (control) or s-hAPPs (1 nM) in the presence of D-[3H]aspartate (570 nM) and increasing concentrations of cold aspartate (0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 µM). Four wells were analyzed per treatment and dose of cold aspartate.

Aspartate binding and aspartate uptake data were processed with the EBDA and LIGAND programs (Biosoft, Cambridge, UK) to produce fitted curves and to determine Km and Vmax values as described (47).

Assessments of Protein Kinase A and C Activities-- To investigate the involvement of protein kinase C (PKC) and protein kinase A (PKA) in s-hAPP effects on astroglial aspartate uptake, cultured astrocytes (see above) were exposed to GF 109203X (GFX, an inhibitor of PKC (see Ref. 48); Research Biochemicals International, Natick, MA), to adenosine 3', 5'-cyclic monophosphothioate (Rp isomer (Rp), an inhibitor of PKA (49); Calbiochem, La Jolla, CA), or to adenosine 3',5'-cyclic monophosphothioate (Sp isomer (Sp), an activator of PKA (50); Calbiochem). GFX was first dissolved in dimethyl sulfoxide (Me2SO), and Rp and Sp in water; these reagents were then diluted 1:10 in phosphate-buffered saline and added individually (either with or without recombinant s-hAPP) to astrocyte cultures (without exchange of media) resulting in final concentrations of 1 µM (GFX) and 10 µM (Rp, Sp). The activities of PKC and PKA in astroglial cell extracts were measured with assay systems from Life Technologies, Inc. per manufacturer's instructions. In preliminary experiments, different dilutions of astroglial cell extracts were analyzed to ensure that the enzyme activities of test samples were within the linear range of the assays (data not shown). PKC activity was expressed as picomoles/min/106 cells. For PKA activity, we first determined basal and s-hAPP-induced PKA activities. cAMP was then added to each test sample to maximally stimulate PKA. Final values for basal and s-hAPP-induced PKA activities were expressed as percent of total activated PKA.

Statistical Analysis-- Statistical analysis was performed with the StatView and Superanova programs (Abacus, Berkeley, CA). All data are expressed as mean ± S.E. Differences between means were analyzed by unpaired Student's t test, differences among means by one-way analysis of variance. When analysis of variance showed significant differences, pairwise comparisons between means were tested by Dunnett's or Tukey-Kramer post hoc tests. The null hypothesis was rejected at the 0.05 level.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Increased Cortical Aspartate Uptake in NSE-hAPP Mice-- To determine whether hAPP influences the transport of excitatory amino acid transmitters in the central nervous system in vivo, neocortical aspartate uptake was compared in unmanipulated nontransgenic controls (n = 9) and NSE-hAPP mice expressing either hAPP751 (n = 10) or hAPP695 (n = 8) at moderate levels (22). Additional NSE-hAPP695, NSE-hAPP751, and nontransgenic mice were injected intraperitoneally with either saline or kainic acid (n = 4/group and treatment) 4 days before analysis. Systemically injected kainic acid traverses the blood-brain barrier and results in extensive neuronal damage in nontransgenic mice and NSE-hAPP695 mice, whereas NSE-hAPP mice expressing the hAPP751 isoform are relatively protected against this acute excitotoxic challenge (5, 27). The cerebral hAPP expression levels in the stable NSE-hAPP lines used here have previously been shown by PhosphorImager quantitations of RPAs and radiolabeled Western blots to increase total APP levels in the neocortex by roughly 10-20% over levels found in non-transgenic controls (5, 22). The NSE-hAPP751 and NSE-hAPP695 lines selected for comparison of aspartate uptake were matched with respect to cerebral hAPP expression levels (data not shown). The neocortex was chosen for analysis because we previously found that this brain region shows maximal levels of transgene expression and is protected from excitotoxic injury in this model (5, 22, 27).

D-Aspartate is transported by the same EAATs as L-glutamate and L-aspartate but, in contrast to these L-isomers, is not metabolized effectively by cells (30). Therefore, D-[3H]aspartate is widely used to estimate glutamate/aspartate uptake. As documented in previous studies (51), binding of D-[3H]aspartate to membrane preparations can be used to estimate cerebral glutamate/aspartate uptake in vivo. Here, we used this approach to evaluate glutamate/aspartate uptake in the neocortex of transgenic and nontransgenic mice. Saline-injected NSE-hAPP751 mice showed on average twice as much neocortical aspartate binding as nontransgenic controls (Fig. 1). Although there was also a tendency toward increased aspartate binding in NSE-hAPP695 mice (Fig. 1), this increase did not reach statistical significance. Similar differences between transgenic and nontransgenic mice and between transgenic mice expressing hAPP751 versus hAPP695 were observed in independent experiments in which neocortical aspartate binding was measured in unmanipulated transgenic mice from lines NSE-hAPP751m-57 (n = 6), NSE-hAPP751-28 (n = 4), NSE-hAPP695m-25 (n = 4), and NSE-hAPP695-60 (n = 4) and nontransgenic controls (n = 9) (data not shown).


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Fig. 1.   Increased aspartate uptake in the neocortex of NSE-hAPP transgenic mice. A, nontransgenic controls (Non-Tg) and transgenic mice from lines NSE-hAPP751m-57 (hAPP751) and NSE-hAPP695m-25 (hAPP695) were injected intraperitoneally with saline or kainic acid (n = 4/group). Four days later, brains were analyzed for aspartate uptake as described under "Experimental Procedures." Neocortical aspartate uptake was significantly increased in both saline-treated NSE-hAPP751 mice (compared with all other groups) and in kainic acid-treated NSE-hAPP751 mice (compared with nontransgenic controls and kainic acid-treated NSE-hAPP695 mice) (*, p < 0.05 by Tukey-Kramer post hoc test). Although there was a trend toward increased aspartate uptake in NSE-hAPP695 mice, the differences between similarly treated NSE-hAPP695 mice and nontransgenic controls were not statistically significant. B, the specificity of D-[3H]aspartate binding to aspartate/glutamate transporters in neocortical membrane preparations was assessed by displacement of radioligand with L-glutamate or THA. Data points represent the average of assay results from two different membrane preparations. Consistent with previous studies (44, 45), L-glutamate displaced D-[3H]aspartate from membrane preparations with a KD of 35.66 µM and a Bmax of 57.7 µmol/mg of protein, while THA displaced D-[3H]aspartate with a KD of 48.72 µM and a Bmax of 72.9 µmol/mg of protein.

The kinetics of neocortical aspartate binding were compared in transgenic mice from line NSE-hAPP751m-57 and age-matched nontransgenic controls as described under "Experimental Procedures." The expression of hAPP751 significantly increased the Bmax (nontransgenic: 183.4 ± 11.7 pmol/mg of protein, transgenic: 281.2 ± 44.9 pmol/mg of protein; p < 0.04) but not the KD (nontransgenic: 0.366 ± 0.024 µM, transgenic: 0.419 ± 0.027 µM; p > 0.09) of neocortical aspartate binding (samples were assayed in quadruplicate; data represent means ± S.E. from four mice per group; P values were obtained by unpaired one-tailed Student's t test).

Kainic acid treatment decreased neocortical aspartate binding in nontransgenic and transgenic mice (Fig. 1). However, even after kainic acid treatment, neocortical aspartate binding in NSE-hAPP751 mice was significantly higher than in saline-treated nontransgenic controls (Fig. 1).

Effect of hAPP Expression on Cerebral EAAT mRNA and Protein Levels-- To determine whether hAPP expression modulates cerebral EAAT gene expression, brain RNA from unmanipulated NSE-hAPP751 mice (n = 6) and nontransgenic controls (n = 3) was analyzed by RPA using riboprobes specific for EAAT1, EAAT2, or EAAT3. Transgenic and nontransgenic mice showed similar cerebral steady-state levels for all three transporter mRNAs (Fig. 2). EAAT protein levels were assessed by semiquantitative Western blot analysis of brain homogenates (Fig. 3). Compared with nontransgenic controls, transgenic mice showed increases in neocortical EAAT2 and EAAT3 protein levels with differences reaching statistical significance only in NSE-hAPP751 mice but not in NSE-hAPP695 mice (Table I). An analysis of neocortical EAAT2 protein levels in saline- or kainic acid-treated NSE-hAPP mice revealed similar results (Fig. 4).


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Fig. 2.   Cerebral levels of mRNAs encoding different glutamate/aspartate transporters. Total RNA was extracted from entire hemibrains of transgenic mice (lines NSE-hAPP751-28 (lanes 1-3) and NSE-hAPP751m-57 (lanes 4-6)) and nontransgenic controls (lanes 7-9) and analyzed by RPA as described under "Experimental Procedures." U, undigested probes (indicated on left) without sample RNA; D, probes without sample RNA digested with RNases. The mRNA/probe fragments specifically protected in samples 1-9 are indicated on the right. Note the similar levels of glutamate/aspartate transporter mRNA in transgenic and nontransgenic brains.


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Fig. 3.   Determination of EAAT2 levels in neocortex by Western blot analysis. Proteins were extracted from the neocortex of nontransgenic controls (lanes 1) and mice transgenic for NSE-hAPP695 (lanes 2) or NSE-hAPP751 (lanes 3) and subjected to Western blot analysis as described under "Experimental Procedures." Incubation of blots with antibody to EAAT2 revealed an expected band of approximately 75 kDa, whereas no labeling was observed after incubation of blots with preimmune sera or anti-EAAT2 antibody that had been preadsorbed with EAAT2 peptide. Western blot signals for EAAT2 (Fig. 4 and Table I) and for other EAATs (Table I) were compared semiquantitatively by PhosphorImager analysis.

                              
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Table I
Relative levels of glutamate/aspartate transporter proteins in the neocortex of NSE-hAPP mice and nontransgenic controls
The relative protein levels of the glutamate/aspartate transporters EAAT1, EAAT2, and EAAT3 in the neocortex of nontransgenic controls (Non-Tg) and mice transgenic for NSE-hAPP751 (hAPP751) or NSE-hAPP695 (hAPP695) were determined by semiquantitative Western blot analysis as described under "Experimental Procedures." *, p < 0.05 by Dunnett's post hoc test (compared with nontransgenic controls).


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Fig. 4.   Neocortical EAAT2 protein levels in saline-treated and kainic acid-challenged NSE-hAPP mice and nontransgenic controls. Nontransgenic controls (Non-Tg) and mice transgenic for NSE-hAPP751 (hAPP751) or NSE-hAPP695 (hAPP695) were injected intraperitoneally with saline or kainic acid (n = 4/group). Four days later, brains were analyzed for EAAT2 levels as outlined in Fig. 3. Compared with saline-treated nontransgenic controls, only saline-treated NSE-hAPP751 mice showed a significant increase in EAAT2 levels (*, p < 0.05 by Dunnett's post hoc test). Among kainic acid-treated mice, the increase in EAAT2 expression over nontransgenic control levels was statistically significant only in NSE-hAPP751 mice (°, p < 0.05 by Tukey-Kramer post hoc test), but not in NSE-hAPP695 mice.

Effect of Recombinant s-hAPP on Aspartate Uptake by Primary Astrocytes-- Because astroglial transporters appear to be responsible for most of the excitatory amino acid uptake in the central nervous system (29), we next examined the effects of recombinant s-hAPP695 and s-hAPP751 on the uptake of aspartate by cultured primary murine astrocytes. s-hAPP695 and s-hAPP751 represent the large N-terminal hAPP ectodomains that are released from cells after proteolytic cleavage of the respective membrane-anchored precursor molecules by alpha -secretase activity (see Introduction). At concentrations of 0.01, 0.1, and 1 nM, both s-hAPP695 and s-hAPP751 clearly increased astroglial aspartate uptake (Fig. 5A). However, at 0.1 or 1 nM, the increase in astroglial aspartate uptake induced by s-hAPP751 was significantly greater than that induced by s-hAPP695 (Fig. 5A). The s-hAPP-induced increase in astroglial aspartate uptake was blocked completely in the presence of trans-PDC (Fig. 5B), a selective inhibitor of EAATs (46).


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Fig. 5.   Effect of recombinant s-hAPP695 and s-hAPP751 on aspartate uptake by primary mouse astrocytes. Primary astrocytes from nontransgenic neonatal mouse brains were maintained in culture for 21 days as described under "Experimental Procedures." Astrocytes were then exposed for 30 min to s-hAPP695 or s-hAPP751 or to vehicle alone (Control). Astroglial uptake of D-[3H]aspartate during the exposure period was determined as described under "Experimental Procedures." n = 4 wells per treatment. A, to assess whether hAPP affects astroglial aspartate uptake, astrocytes were exposed to s-hAPP695 or s-hAPP751 at the concentrations (nanomolar) indicated. *, p < 0.05 and **, p < 0.01 (versus control) by Dunnett's post hoc test. At 0.1 and 1 nM, s-hAPP751 had a stronger effect on astroglial glutamate/aspartate uptake than s-hAPP695 (°, p < 0.01 by Tukey-Kramer post hoc test). B, to assess the specificity of the hAPP effects observed in this assay, astrocytes were treated with vehicle (Control) or s-hAPP (1 nM) in the presence or absence of the glutamate/aspartate transporter inhibitor, trans-PDC (20 µM). *, p < 0.05 and **, p < 0.01 (versus control without trans-PDC) by Dunnett's post hoc test. s-hAPP751 had a stronger effect on astroglial aspartate uptake than s-hAPP695 (p < 0.05 by Tukey-Kramer post hoc test), and trans-PDC completely blocked the hAPP-dependent uptake stimulation.

To determine if s-hAPP alters the kinetics of astroglial excitatory amino acid transport, D-[3H]aspartate uptake was examined after 30 min incubation of astrocytes with 1 nM s-hAPP695 or 1 nM s-hAPP751 in the presence of increasing concentrations of cold aspartate. Under these conditions, both s-hAPP695 and s-hAPP751 increased the Vmax (picomoles/min/mg of protein): mock-treated control, 56.13 ± 5.46; s-hAPP695-treated, 75.28 ± 9.71; s-hAPP751-treated, 132.78 ± 12.18. The effects of s-hAPP treatments on the KmM] were less consistent: mock-treated control, 0.59 ± 0.18; s-hAPP695-treated, 0.39 ± 0.2; s-hAPP751-treated, 1.06 ± 0.22.

Involvement of PKA and PKC in s-hAPP-induced Aspartate Uptake Stimulation-- To begin to elucidate how s-hAPP stimulates astroglial aspartate uptake, we examined whether PKA and PKC may function as mediators of this process. Inhibition of PKA or PKC activity prevented the hAPP-induced increase in astroglial aspartate uptake (Fig. 6A). While these findings demonstrate that s-hAPP-induced aspartate uptake stimulation is dependent on the activities of PKC and PKA, they do not clarify whether exposure of astrocytes to s-hAPP actually increases the activities of these kinases. Therefore, we next assessed this issue directly using enzyme activity assays specific for PKC or PKA. Both s-hAPP695 and s-hAPP751 up-modulated PKC (Fig. 6B) and PKA (Fig. 6C) activities in astrocytes, indicating that these enzymes may function as critical mediators of the s-hAPP-dependent aspartate uptake stimulation.


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Fig. 6.   Involvement of PKC and PKA in the s-hAPP-dependent stimulation of astroglial aspartate uptake. Primary astrocytes from nontransgenic neonatal mouse brains were maintained in culture for 21 days as described under "Experimental Procedures" and then exposed for 30 min to s-hAPP695 (1 nM), s-hAPP751 (1 nM), or vehicle alone (Control). A, astroglial uptake of D-[3H]aspartate during the exposure period was determined as described under "Experimental Procedures." n = 4 wells per treatment. To determine the dependence of the s-hAPP-induced uptake stimulation on the activity of PKC and PKA, astrocytes were exposed either to s-hAPP alone (-) or to s-hAPP in combination with antagonists of PKC (GFX) or PKA (Rp). The Rp analog Sp, which stimulates PKA, was used as a control. Exposure of astroglial cultures to GFX, Rp, Sp, or Me2SO in the absence of s-hAPP stimulation had no significant effect on astroglial aspartate uptake (data not shown). B and C, to assess the effect of s-hAPP on astroglial PKC (B) and PKA (C) activity, astrocyte cultures were treated with s-hAPP or vehicle alone (control) without exchange of media, and then analyzed for PKC and PKA activity (six wells/treatment/enzyme) as described under "Experimental Procedures." *, p < 0.05 and **, p < 0.01 (versus control) by Dunnett's post hoc test.   

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Five principal findings emerge from this work. First, moderate levels of transgene-mediated hAPP expression in the central nervous system increase cerebral aspartate uptake in untreated mice as well as in mice exposed to an excitotoxic challenge. Second, at roughly similar levels of expression, hAPP751 stimulates aspartate uptake in the brain more strongly than hAPP695. Third, whereas central nervous system expression of hAPP751 does not affect EAAT mRNA levels, it increases the cerebral content of the glutamate/aspartate transporters EAAT2 and EAAT3 at the protein level. Fourth, recombinant s-hAPP increases aspartate uptake by cultured primary astrocytes. Fifth, s-hAPP increases astroglial PKA and PKC activities, and inhibitors of these kinases block the s-hAPP-dependent aspartate uptake stimulation.

EAATs play an important role in glutamate clearance from the synaptic cleft (30, 52). Synapses are frequently ensheathed by astrocytes (53, 54), and D-[3H]aspartate-labeled glial processes are specifically arranged around glutaminergic axons (55). Consistent with these observations, recent studies in which the synthesis of individual EAATs was inhibited by chronic administration of antisense oligonucleotides indicate that astroglial EAATs are essential for maintaining low extracellular glutamate and for preventing chronic excitotoxicity (28, 29). It is therefore of interest to determine which factors regulate the level and activity of EAATs. The current investigation has pinpointed APP as a likely candidate.

The assay we used to estimate neocortical aspartate uptake could reflect the activity of neuronal and glial EAATs. In NSE-hAPP mice, full-length hAPPs were expressed specifically in neurons (22), the main source of APP in the brain (11). However, neurons have been shown to secrete s-APP (12, 13), and release of s-APP from neurons can be stimulated by neuronal depolarization (56, 57). Furthermore, most of the glutamate/aspartate uptake in the brain is mediated by astroglial transporters, particularly EAAT2 (29). It is therefore likely that the increased aspartate uptake in the neocortex of NSE-hAPP mice reflects, at least in large part, effects exerted by neuronally derived s-hAPP on neighboring astrocytes. Consistent with this interpretation, recombinant s-hAPP stimulated aspartate uptake in astrocyte cultures (Figs. 5 and 6).

Based on our findings, it is tempting to speculate that neurons might use APP to regulate the uptake of the excitatory neurotransmitters they release. Release of s-hAPP could prevent excitotoxic neuronal injury by increasing the uptake of excitatory amino acids from the extracellular space in the central nervous system. Indeed, this effect may play an important part in the excitoprotective effects of hAPP observed in experimental models of neurological disease (5, 25, 27). It is interesting in this context that hAPP751, which appears to be expressed at robust levels by neurons of the human brain (11), was more excitoprotective than hAPP695 when different lines of NSE-hAPP mice were challenged with kainic acid (5). The current study indicates that this may be due, at least in part, to the differential effects these two isoforms have on the uptake of excitatory amino acids. Before and after kainic acid challenge, NSE-hAPP751 mice showed significantly higher levels of aspartate uptake in their neocortex than saline-challenged nontransgenic controls, whereas NSE-hAPP695 mice did not. Whether these differences between hAPP751 and hAPP695 somehow relate to the activity of the Kunitz-type protease inhibitor domain, which is present in hAPP751 but absent from hAPP695, or to potential differences in the secretion of these hAPP isoforms remains to be determined (see Refs. 5, 8, and 27 for further discussion).

In principle, hAPP could increase glutamate/aspartate uptake by up-modulating the expression, availability, or activity of EAATs. The lack of hAPP effects on EAAT mRNA levels suggests that hAPP does not regulate glutamate/aspartate uptake by increasing EAAT gene expression. hAPP695 also had no significant effect on EAAT protein level (Table I and Fig. 4). In contrast, hAPP751 increased neocortical EAAT2 and EAAT3 protein levels in transgenic mice (Table I and Fig. 4). Neither hAPP751 nor hAPP695 altered the KD of neocortical aspartate binding, suggesting that they do not significantly increase the affinity of EAATs for their substrate. Because the local concentration of excitatory amino acid transmitters around synaptic clefts can be expected to far exceed the KD, it is the availability of transporter molecules rather than their affinity for substrate that will determine the efficiency of astroglial glutamate/aspartate uptake. It is therefore interesting that hAPP695 and hAPP751 consistently increased the Vmax of aspartate uptake in vivo and in vitro, suggesting that both hAPP isoforms increase the availability of transporter molecules.

Although additional studies are needed to fully characterize the pathways leading from hAPP exposure to increased glutamate/aspartate uptake, our preliminary analysis of potential second messengers revealed that PKA and PKC may play an important role in s-hAPP-dependent EAAT stimulation. EAATs contain phosphorylation sites for PKA and PKC, and PKC activation has previously been shown to increase EAAT activity (58). In our cell culture studies, inhibition of PKA or PKC prevented the s-hAPP-induced aspartate uptake stimulation (Fig. 6A), suggesting a dependence of this s-hAPP effect on the activity of these kinases. In addition, we observed that s-hAPP increased astroglial PKA and PKC activities (Fig. 6, B and C). Taken together, these findings identify PKA and PKC as likely mediators of the s-hAPP-dependent aspartate uptake stimulation. It is possible that phosphorylation of EAATs or other proteins by these kinases results in an increased translocation of intracellular stores of transporters to the plasma membrane or diminishes the turnover of EAATs at the cell surface. The s-hAPP-induced increase in the Vmax of astroglial aspartate uptake and the relatively rapid in vitro effects of s-hAPP would seem consistent with this interpretation.

It has recently been demonstrated that the activity of EAATs is significantly decreased in the neocortex of humans with Alzheimer's disease (44, 59). This disorder is associated with the increased cerebral deposition of the hAPP-derived peptide Abeta (10, 60). The Abeta peptide has been shown to inhibit astroglial glutamate/aspartate transporters in cultured astrocytes and in synaptosomes (61), and this may contribute to the decrease in neocortical aspartate uptake seen in patients with Alzheimer's disease. However, the presumed "beta -secretase" processing pathway, which gives rise to the Abeta peptide, also results in the secretion of an N-terminal hAPP ectodomain that is truncated at its C-terminal end. Interestingly, compared with the longer alpha -secreted hAPP, beta -secreted hAPP appears to be functionally impaired (62). This raises the intriguing questions of whether beta -secreted hAPP may be less effective at regulating astroglial glutamate uptake and whether this, in combination with Abeta effects, contributes to the neurodegenerative process in Alzheimer's disease. Recent evidence suggests that in sporadic Alzheimer's disease the production of neuroprotective hAPP could also become impaired as a result of abnormal processing of hAPP transcripts in vulnerable neuronal subpopulations (63).

In conclusion, our data indicate that APP regulates excitatory amino acid levels in the central nervous system and that this effect may be mediated, at least in part, by PKA and PKC. These observations provide a mechanistic link between hAPP and excitoprotective effects observed in different in vivo models (5, 25-27). Our findings also suggest that alterations of hAPP levels or functions by genetic or environmental processes could impair astroglial glutamate/aspartate uptake and thereby result in excitotoxicity and disturbed neurotransmission.

    ACKNOWLEDGEMENTS

We thank S. Li for providing the EAAT riboprobes, E. Rockenstein and C. E. Westland for excellent technical assistance, S. Ordway for editorial assistance, and R. Haines for help with the preparation of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AG11385 (to L. M.), NS34602 (to L. M.), AG10869 (to E. M.), AG05131 (to E. M.), NS29001 (to M. P. M.), and NS30583 (to M. P. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this study.

Dagger Dagger To whom correspondence should be addressed: Gladstone Institute of Neurological Disease, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-695-3819; Fax: 415-826-6541; E-mail: lennart_mucke{at}quickmail.ucsf.edu.

1 The abbreviations used are: APP, amyloid beta  protein precursor; h, human; hAPP, human APP; EAAT, excitatory amino acid transporter; NSE, neuron-specific enolase; PKC, protein kinase C; PKA, protein kinase A; RPA, RNase protection assay; GFX, GF 109203X; s-hAPP, secreted N-terminal fragment of human APP (resulting from alpha -secretase cleavage); s-APP, secreted APP; THA, L(-)-threo-3-hydroxyaspartate; trans-PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; Abeta , amyloid beta  peptide; Tg, transgenic.

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Abstract
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Results
Discussion
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