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Keywords:

  • Alzheimer's disease;
  • amyotrophic lateral sclerosis;
  • excitotoxicity;
  • glutamate transporter;
  • ischemia

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

Glutamate uptake by high affinity glutamate transporters is essential for preventing excitotoxicity and maintaining normal synaptic function. We have discovered a novel role for presenilin-1 (PS1) as a regulator of glutamate transport. PS1-deficient neurons showed a decrease in glutamate uptake of approximately 50% compared to wild-type neurons. Gamma-secretase inhibitor treatment mimicked the effects of PS1 deficiency on glutamate uptake. PS1 loss-of-function, accomplished by PS1 deficiency or γ-secretase inhibitor treatment, caused a corresponding decrease in cell surface expression of the neuronal glutamate transporter, EAAC1. PS1 deficiency is known to reduce intracellular calcium stores. To explore the possibility that PS1 influences glutamate uptake via regulation of intracellular calcium stores, we examined the effects of treating neurons with caffeine, thapsigargin, and SKF-96365. These compounds depleted intracellular calcium stores by distinct means. Nonetheless, each treatment mimicked PS1 loss-of-function by impairing glutamate uptake and reducing EAAC1 expression at the cell surface. Blockade of voltage-gated calcium channels, activation and inhibition of protein kinase C (PKC), and protein kinase A (PKA) all had no effect on glutamate uptake in neurons. Taken together, these findings indicate that PS1 and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore may be important in limiting glutamate toxicity in the brain.

Abbreviations used

A-beta peptide

AD

Alzheimer's disease

ALS

amyotrophic lateral sclerosis

APP

amyloid precursor protein

BisII

bisindolylmaleimide II

DHK

dihydrokainate

dpm

disintegrations per minute

DMSO

dimethylsulfoxide

EAAC1

rabbit glutamate transporter

ER

endoplasmic reticulum

ERK

externally regulated kinase

FAD

familial Alzheimer's disease

GLAST

rat glutamate transporter

GLT-1

rat glutamate transporter

KRPH

Krebs–Ringer phosphate HEPES buffer

LDH

lactate dehydrogenase

MAPK

mitogen-activated protein kinase

PDC

l - trans -2,4-pyrrolidine dicarboxylate

PKA

protein kinase A

PKC

protein kinase C

PS1

presenilin-1

PS2

presenilin-2

PMA

phorbol-12-myristate-13-acetate

SOCC

store-operated calcium entry channels

VDCC

voltage-dependent calcium channels

Presenilin 1 and 2 (PS1 and PS2) are ubiquitously expressed polytopic transmembrane proteins that are best understood for their roles in regulating amyloid precursor protein (APP) and A-beta (Aβ) metabolism (Haass 1997). Currently, more than one hundred presenilin mutations have been associated with early onset familial Alzheimer's Disease (FAD) (http://molgen-http://www.uia.ac.be/ADMutations). Although FAD-linked presenilin mutations provoke well-established changes in APP cleavage, these molecules mediate other metabolic and signaling processes as well. For example, PS1 regulates cleavage of several transmembrane proteins (Levitan et al. 1996; Baumeister et al. 1997; Struhl and Greenwald 1999; Ni et al. 2001; Marambaud et al. 2002; May et al. 2002; Ikeuchi and Sisodia 2003). It also plays a role in maintaining intracellular calcium homeostasis (Ito et al. 1994; Begley et al. 1999; Guo et al. 1999; Leissring et al. 1999a, b; Yoo et al. 2000), protein trafficking (Naruse et al. 1998; Cai et al. 2002; Leem et al. 2002a), and apoptosis (Mattson et al. 1998; Wolozin et al. 1998). Thus, PS1 is important in regulating intracellular signaling, neuronal plasticity, and neurotransmission.

These findings demonstrate the broad biological significance of PS1. However, we still have limited insight into the mechanisms whereby PS1 carries out its biological functions, particularly those functions that influence neuronal activity. In an attempt to enlarge our understanding of how PS1 regulates neuronal activity we have studied the effects of PS1 on glutamate uptake. We chose to address this question because glutamate is the major excitatory neurotransmitter in the brain (Fonnum 1984). Removal of extracellular glutamate by uptake transporters is important in regulating synaptic plasticity, maintaining synaptic transmission, and limiting excitotoxicity (Danbolt 1994; Seal and Amara 1999). Moreover, disturbances in the glutamate uptake system are associated with a number of neurological disorders including Alzheimer's disease (AD), CNS ischemic disease, epilepsy, and amyotrophic lateral sclerosis (ALS) (Rothstein et al. 1992, 1995; Bristol and Rothstein 1996; Miller et al. 1997; Masliah et al. 1998; Rossi et al. 2000; Lauderback et al. 2001; Howland et al. 2002).

We have discovered a novel function for PS1 as a significant regulator of neuronal glutamate uptake. PS1-deficient neurons display a marked impairment in glutamate uptake. PS1 loss-of-function appears to mediate its effects on glutamate uptake by reducing cell surface expression of the neuronal glutamate transporter, EAAC1, without altering total EAAC1 expression levels. In an effort to better understand the subcellular mechanisms by which PS1 influences glutamate uptake we noted that PS1 also regulates intracellular calcium homeostasis (Leissring et al. 2000; Yoo et al. 2000; LaFerla 2002). Thus we have explored the role of intracellular calcium signaling in regulating glutamate transport. Using three independent approaches we have found that reducing intracellular calcium in the endoplasmic reticulum (ER) significantly reduced glutamate uptake. Importantly, in each instance the reduced uptake was accompanied by a marked decrease in EAAC1 expressed at the cell surface. These data are the first to link ER-mediated intracellular calcium signaling directly to regulation of glutamate transporter trafficking and indicate that PS1 controls glutamate uptake via regulation of intracellular calcium signaling.

Genotyping and cell culture

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

Embryonic mice derived from PS1 doubly heterozygous (+/–) crosses were genotyped using three PCR primers (primer 1: 5′-ACCTCAGCTGTTTGTCCCGG-3′, primer 2: 5′-GCACGAGACTAGTGAGACGTG-3′ and primer 3: 5′-CTGGAAGTAG-GACAAAGGTG-3′). Primer 1 and 3 generated a 345 base pair (bp) PCR product diagnostic of a wild-type genotype, whereas primer 1 and 2 yielded a 300 bp band indicating a knock-out genotype. Cultured cortical neurons from embryonic day 16–17 mice were prepared as described elsewhere (Yang et al. 1999). Lactate dehydrogenase (LDH) assays were performed using a commercially available kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) as per manufacturer's procedures.

[14C]Glutamate uptake

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

To measure [14C]glutamate uptake, neurons grown on 24-well tissue culture plates (200 000 cells/well) were washed twice with piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES)-buffered balance salt solution (BSS) (135 mm NaCl, 3.1 mm KCl, 2.2 mm CaCl2, 2.2 mm MgSO4, 0.5 mm KHPO4, 6 mm PIPES and 2 mm glucose, pH 7.2) at room temperature immediately before experimentation. Glutamate uptake was initiated by adding 0.167 µCi/mL [14C]glutamate (American Radiochemicals, St. Louis, MO, USA) in the BSS buffer lacking non-labeled, cold glutamate to the cells in a final volume of 300 µL per well. Unless otherwise indicated uptake was carried out at 37°C for 9 min. Uptake was terminated by two ice-cold washes with 500 µL BSS followed by immediate cell lysis in ice-cold 0.1 N NaOH/0.01% lauryl sulfate. [14C]Glutamate uptake was measured in a scintillation counter. Disintegrations per minute (dpm) values were normalized to total protein content in the assay and expressed as dpm/µg total protein. Protein concentrations in the lysates were determined using the bicinchoninic acid method (Pierce, Rockford, IL, USA). Results were presented as the percentage of wild-type or control dpm in each condition. Glutamate uptake inhibitors, l-trans-2,4-pyrrolidine dicarboxylate (PDC), or dihydrokainate (DHK) were obtained from Tocris (Ellisville, MO, USA). Gamma-secretase inhibitor was obtained from Calbiochem (γ-secretase inhibitor II, Cat # 565755; San Diego, CA, USA). Sodium-independent uptake was performed in sodium-free BSS where the sodium (135 mm) had been replaced with choline chloride (135 mm). For each experiment individual samples were assayed in triplicate.

Cell surface biotinylation and western blot procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

Cell surface proteins were biotinylated by placing neurons (400 000 cells/well in 12-well plates) on ice for 10 min followed by two ice-cold washes with Krebs–Ringer phosphate HEPES buffer (KRPH: 128 mm NaCl, 4.7 mm KCl, 1.25 mm CaCl2, 5 mm Na2HPO4, 20 mm HEPES and 1.25 mm MgSO4, pH 7.4). Cultures were then incubated in Sulfo-NHS-Biotin solution (Pierce) (0.5 mg/mL in KRPH, 1 mL/well) on ice for 30 min. The biotinylation reaction was terminated by quenching for 15 min in a solution with 20 mm glycine in KRPH. The neurons were rinsed twice in ice-cold KRPH and then lysed in 1 mL immunoprecipitation (IP) lysis buffer (50 mm Tris, 150 mm saline, 2% Triton X-100, 0.5 mm EDTA, pH 8.0) supplemented with protease inhibitor cocktail (Pierce) (2 µL/mL lysis buffer). Portions of the total cellular lysates (6.5%) were loaded directly, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and western blotted with anti-EAAC1, GLAST (Alpha Diagnostic International Inc., San Antonio, TX, USA), anti-GLT-1 (Novus, Littleton, CO, USA), or anti-APP (carboxyl terminus) (Zymed, San Francisco, CA, USA). To isolate surface expressed proteins the lysates were immunoprecipitated with streptavidin agarose beads (Pierce). Following immunoprecipitation, biotinylated cell surface proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotted with anti-EAAC1 or anti-βAPP as described above. Anti-active MAPK (phosphorylated form) and anti-ERK1/2 (pan-specific form) antibodies were obtained from Promega (Madison, WI, USA). The antiβ-actin antibody was purchased from ICN (Costa Mesa, CA, USA). Protein bands were detected using horseradish peroxidase-conjugated secondary antibodies (Sigma, St. Louis, MO, USA) in conjunction with chemiluminescence (Pierce). Densitometry was performed using the Kodak I.D. 3.0.2 Scientific Imaging System. EAAC1 resolves by sodium dodecyl sulfate–polyacrylamide gel electrophoresis into monomers (65 kDa) and multimers (140–250 kDa) (Haugeto et al. 1996). The proportion of total EAAC1 resolving into monomeric versus multimeric form can vary slightly from experiment to experiment, presumably due to differences in efficiency with which boiling, sodium dodecyl sulfate, and reducing agents denature the multimeric EAAC1 complexes. Thus, band intensity values for EAAC1 monomers and multimers were added together (all bands in the range of 65–250 kDa) to yield total EAAC1 intensity per lane. The results were normalized to total protein content and analyzed as the percentage of EAAC1 intensity in appropriate wild-type or vehicle treated controls. Phorbol-12-myristate-13-acetate (PMA), bisindolylmaleimide II (BisII), forskolin, H-89, thapsigargin, SKF-96563 were obtained from Calbiochem (La Jolla, CA, USA).

Calcium measurements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

Intracellular free calcium [Ca2+]i was quantified by fluorescence ratiometric imaging of the calcium probe fura-2/AM. Briefly, mouse cortical neurons were cultured on 10-cm2 glass coverslips in neurobasal medium at a density of 50 000 cells/cm2, and loaded with 10 µm fura-2/AM (30 min incubation at 37°C) dissolved in dimethylsulfoxide (final [DMSO] < 0.25%). Cells were then washed twice with fresh medium and incubated for another 30 min before experiments. Calcium measurement was carried out using a Nikon inverted microscope (20 × objective) connected to a computerized software, MetaFluor, for calcium ratiometric imaging. The average [Ca2+]i of individual neuron was determined from the ratio of fluorescence emissions resulted from two different excitation wavelengths (340 and 380 nm). The system was calibrated using a fura-2 calcium imaging calibration kit (Molecular Probes, Eugene, OR, USA) according to the formula: [Ca2+]i = Kd[(R − Rmin)/(Rmax − R)](F380 max/F380 min). For each treatment, 50–100 cells were chosen for statistical analysis.

Glutamate uptake is impaired in PS1 knock-out neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

To determine whether PS1 expression influences glutamate transport we measured [14C]glutamate uptake in cultured cortical neurons from PS1 wild-type (+/+), heterozygous (+/–) and knock-out (–/–) mice (Shen et al. 1997). We found that PS1 knock-out neurons showed a mean 53% decrease in glutamate uptake compared to PS1 wild-type neurons (p < 0.001) (Fig. 1a). In addition, PS1 heterozygous neurons showed a statistically significant 18% reduction compared to PS1 wild-type neurons (p < 0.002). For all cells glutamate uptake was dependent on extracellular Na+ (135 mm) and for all experiments incubation buffers lacking non-labeled, cold glutamate were used (see Experimental procedures). We noted no differences in viability between PS1 wild-type, heterozygous, and knock-out neurons as measured by release of LDH in three independent experiments [F(2,9) = 0.344; NS].

image

Figure 1. Glutamate uptake reduced in PS1 knock-out neurons. (a)  [ 14 C]Glutamate uptake was measured in WT, HET and KO neurons with (+ Na) or without (– Na) extracellular sodium. Results are shown as the means of five independent experiments normalized as the percentage of wild-type uptake (WT = 100%). (b)  [ 14 C]Glutamate uptake in WT (▪), HET (▴) and KO (□) neurons after 3, 9, and 15 min of [ 14 C]glutamate uptake. ‘+’ and ‘–’ indicate presence or absence of extracellular Na + . Results are shown as averages from two independent experiments. (c)  Sub-saturating dose of PDC (10 µ m ) partially blocked [ 14 C]glutamate uptake in WT, HET and KO neurons. (d)  Effect of γ-secretase inhibitor treatment on glutamate uptake in PS1 WT (filled bars), HET (shaded bars) and KO (open bars) neurons. Neurons were treated with γ-secretase inhibitor (100 µ m ) or DMSO vehicle control (V) for 4 h prior to [ 14 C]glutamate uptake. The uptake assay was performed at 37°C for 9 min. Results in (d) are presented as the means of three independent experiments and expressed as percentages of uptake in vehicle treated WT neurons (WT Vehicle = 100%). Error bars indicate ± SEM.

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We next examined the kinetics of glutamate uptake in cortical neurons by incubating them with [14C]glutamate for 3, 9, or 15 min. Figure 1(b) shows a consistent pattern of impaired glutamate uptake in PS1 knock-out neurons over a range of uptake durations in the presence of extracellular sodium (+). Consistent with the data in Fig. 1(a), heterozygous neurons displayed intermediate levels of uptake at all time points compared to PS1 knock-out and wild-type cells. In the absence of sodium (–), glutamate uptake was blocked in all cells.

Loss of PS1 did not completely abolish glutamate uptake. This finding indicates that approximately 50% of the total cellular glutamate uptake capacity is dependent on normal PS1 function. The addition of one PS1 allele partially restored the wild-type phenotype (Figs 1a and b). Thus, glutamate uptake in neurons is regulated by PS1 in a gene dose-dependent manner.

To further characterize the specificity of glutamate uptake we treated the cells with a highly selective glutamate transport competitor, PDC (Bridges et al. 1991). This compound has been shown to compete selectively with glutamate for uptake via glutamate transporters both in vivo and in vitro (Zuiderwijk et al. 1996; Wang et al. 1998). Figure 1(c) shows that a subsaturating dose of PDC competed with [14C]glutamate for uptake into neurons from all three PS1 genotypes. This result provides additional evidence that our uptake measures specifically reflected the activity of glutamate uptake transporters.

Gamma-secretase inhibitor impairs glutamate uptake

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

PS1 deficiency causes a marked decrease in Aβ production, a phenotype that is mimicked by treatment with γ-secretase inhibitors (De Strooper et al. 1998; Wolfe 2001). For this reason we asked if γ-secretase inhibitor treatment would, like PS1 deficiency, inhibit glutamate uptake. We therefore treated PS1 wild-type, heterozygous, and knock-out neurons with a γ-secretase inhibitor (γ-secretase inhibitor II, Calbiochem; Wolfe et al. 1999) for 4 h prior to measuring [14C]glutamate uptake. We observed a significant decrease (p < 0.01) in glutamate uptake in wild-type neurons after treatment with γ-secretase inhibitor compared to a vehicle control (Fig. 1d). By contrast, the γ-secretase inhibitor had no effect on uptake in the PS1 knock-out cells compared to the corresponding vehicle control. The effect of the γ-secretase inhibitor in PS1 heterozygous neurons was less pronounced than in wild-type cells, but greater than in knock-out neurons. In a separate experiment, we observed an orderly dose-dependent inhibition of glutamate uptake in wild-type neurons by the γ-secretase inhibitor (data not shown).

These findings strengthen the link between PS1 loss of function and impaired glutamate uptake in neurons. The lack of effect with the γ-secretase inhibitor in PS1 knock-out neurons strongly suggests that the γ-secretase inhibitor affected glutamate uptake via a PS1-dependent regulatory pathway.

PS1 deficiency impairs neuronal glutamate transport in presence of DHK

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

In forebrain and cortical regions the three predominant glutamate transporter isoforms are GLAST (EAAT1) and GLT-1 (EAAT2), both of which are expressed primarily in astrocytes (Rothstein et al. 1994), and EAAC1 (EAAT3), considered a neuronal isoform (Kanai and Hediger 1992; Rothstein et al. 1994). We performed western blots on total cellular lysates from cultured primary cortical neurons. Lysates from whole brain served as a positive control. We observed abundant expression of EAAC1 compared to GLT-1 and GLAST (Fig. 2a). These results were consistent with earlier data obtained from highly purified neuronal cultures (Wang et al. 1998; Guillet et al. 2002) and suggested the possibility that EAAC1 played a predominant role in mediating glutamate uptake in these cultures. However, recent findings indicate that neurons also express the glial transporter, GLT-1 (Chen et al. 2002). To determine the relative contribution of GLT-1 to glutamate uptake in wild-type and PS1 knock-out neurons we treated the cells with DHK, a specific inhibitor of GLT-1 (Fig. 2b) (Arriza et al. 1994). DHK (50–1000 µm) produced modest levels of inhibition (approximately 20–25%) in both wild-type and PS1 knock-out neurons compared to untreated cells of the same genotype. Even when GLT-1 mediated uptake was abolished by DHK, PS1 knock-out neurons still displayed significantly reduced uptake compared to wild-type cells [F(1,35) = 184.88, p < 0.001). Pharmacological agents that distinguish between uptake mediated by EAAC1 and GLAST are not available. Therefore, it is not possible to rule out that GLAST also contributed to glutamate uptake in the wild type and knockout neuronal cultures.

image

Figure 2. Differential expression of glutamate transporters in neuronal cultures. (a)  Western blots compare EAAC1, GLT-1, and GLAST expression in primary cortical neurons. ‘N’ and ‘Ctrl’ indicate total cellular lysates from primary neuronal cultures and control lysates from whole brain, respectively. (b)  [ 14 C]Glutamate uptake was measured in wild-type (WT) and PS1 knock-out (KO) neurons in the presence or absence of DHK (50 µ m thru 1000 µ m ) using the same procedures as in Fig. 1 . DHK-treated (1000 µ m ) PS1 KO neurons exhibited significantly less [ 14 C]glutamate uptake compared to DHK-treated WT cells ( p <  0.007). Histogram shows mean uptake values from three independent experiments. Error bars indicate ± SEM.

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EAAC1 surface expression is reduced in PS1 knock-out neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

To explore the mechanisms by which PS1 regulates glutamate transport we examined cell surface expression of the neuronal glutamate transporter, EAAC1 on PS1 wild-type and knock-out neurons. We labeled cell surface proteins with biotin, immunoprecipitated all biotinylated surface proteins with streptavidin-conjugated agarose beads, and then performed western blots of the immunoprecipitates with antibodies recognizing EAAC1 (see Experimental procedures). As previously reported (Haugeto et al. 1996), western blots showed that EAAC1 appears both in monomeric (65 kDa) and multimeric (145–250 kDa) form (Fig. 3). Blots of whole-cell lysates showed that overall transporter expression levels were comparable in wild-type and knock-out neurons. Equal amounts of total protein were loaded and confirmed by immunoblotting for β-actin (Fig. 3a). A significant 66% decrease (p < 0.002) in cell surface expressed EAAC1 was observed in PS1 knock-out neurons compared to wild-type neurons (Fig. 3b). We next tested the effect of γ-secretase inhibitor treatment on EAAC1 cell surface expression in wild-type neurons (Fig. 3c). Following a 4-h treatment with γ-secretase inhibitor, EAAC1 expression on the cell surface was reduced to approximately 68% of untreated cells (Fig. 3d). To show that PS1 deficiency did not cause a generalized, non-specific decrease in proteins expressed at the cell surface, we also measured APP cell surface expression in PS1 wild-type and knock-out neurons. The knock-out neurons displayed a modest increase in APP expression at the cell surface (data not shown). These findings are consistent with previous studies showing that PS1 affects protein trafficking at the cell surface in a protein specific manner (Naruse et al. 1998; Cai et al. 2002; Goutte et al. 2002; Leem et al. 2002a,b).

image

Figure 3. PS1 loss-of-function reduced EAAC1 cell surface expression. (a)  Cell surface expressed EAAC1 from wild-type (WT) and knock-out (KO) neurons was biotinylated and then immunoprecipitated with streptavidin–agarose beads. Immunoprecipitates were then western blotted for EAAC1. EAAC1 was detected in monomeric (65 kDa; lower bracket) and multimeric forms (140–250 kDa; upper bracket). A portion (6.5%) of total cell lysate was immunoblotted directly to compare total EAAC1 expression levels in WT and KO neurons. Equal amounts of total protein were loaded in WT and KO lanes and confirmed by immunoblotting for β-actin. (b)  Graph shows relative EAAC1 densitometric band intensities for cell surface and total whole cell EAAC1. Data are expressed as means from three independent experiments normalized with reference to WT intensity values (WT = 100%). Band intensities for monomers and multimers were summed. (c)  Western blot of surface expressed EAAC1 on WT neurons treated with γ-secretase inhibitor or DMSO vehicle control. Neurons were exposed to γ-secretase inhibitor for 4 h prior to cell lysis and immunoprecipitate/western blot analyses were performed as in (a). (d)  Histogram shows relative EAAC1 band intensity (performed as in panel b) for γ-secretase inhibitor and vehicle control treated WT neurons. Values are mean of four independent experiments. Error bars indicate ± SEM.

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Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

A number of intracellular second messenger systems have been shown to regulate glutamate transporter trafficking (Zerangue et al. 1995; Dowd and Robinson 1996; Davis et al. 1998; Sims et al. 2000). In addition, PS1 deficiency reduces intracellular calcium stores (Yoo et al. 2000; LaFerla 2002). If reduced intracellular calcium stores were responsible for the effects of PS1 deficiency on glutamate uptake, we therefore reasoned that other experimental manipulations that directly reduce ER calcium stores should recapitulate the PS1-deficient glutamate transport phenotype. Caffeine has been shown to deplete ER calcium by activating ryanodine receptors, thus leading to calcium release (Bhat et al. 1997; Pan et al. 2000). We found that pre-treatment with caffeine for 2 h significantly reduced glutamate uptake in a dose-dependent fashion (Fig. 4a). Importantly, this reduction in uptake was accompanied by a marked loss of EAAC1 expressed at the cell surface (Fig. 4b). Total cellular levels of EAAC1 were unchanged by caffeine treatment. Furthermore, the magnitude of the loss of surface EAAC1 mirrored the reduction in uptake (Fig. 4c).

image

Figure 4. Caffeine reduced glutamate uptake and cell surface expression of EAAC1. (a)  Glutamate uptake was measured in neurons pre-treated with 0.8, 4 and 20 m m caffeine for 2 h. Uptake measurements were carried out at 37°C for 9 min. Results are average of three independent experiments, presented as means ± SEM. (b)  Reduction of cell surface, but not whole-cell EAAC1 levels under caffeine treatments. EAAC1 was measured in a portion of the whole-cell lysate (6.5% of total lysate) and streptavidin immunoprecipitates of cell surface-biotinylated proteins. To ensure equal protein amounts were loaded, an equal portion of lysate (6.5%) was blotted for β-actin. (c)  Band density was calculated as sum of multimer and monomer, and plotted against the control density set as 100%. Results are average of three independent experiments and presented as percentage of control. Error bars indicate ± SEM.

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Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

To further test the hypothesis that ER calcium stores regulates EAAC1 uptake, we treated neurons with thapsigargin, a well-established compound that depletes ER calcium by blocking the ER calcium pump, Ca2+-ATPase (Thastrup et al. 1990). Figure 5(a) showed that thapsigargin treatment produced a dose-dependent impairment of glutamate uptake in neurons. Surface expression of EAAC1 was also reduced by thapsigargin (Fig. 5b), closely matching the reduction of glutamate uptake (Fig. 5c). Total EAAC1 levels in control and thapsigargin-treated cells were equivalent (Figs 5b and c).

image

Figure 5. Thapsigargin impaired glutamate uptake and EAAC2 cell surface expression. (a)  Glutamate uptake was measured in neurons pre-treated with 1, 5 or 10 µ m thapsigargin (TG) for 30 min. Results are average of three independent experiments. (b)  Thapsigargin treatment reduced cell-surface, but not whole-cell EAAC1 expression in a dose-dependent manner. EAAC1 was measured in a portion of the whole-cell lysate (6.5% of total lysate) and in streptavidin immunoprecipitates of cell surface-biotinylated proteins. To ensure equal protein amounts were loaded, an equal portion of lysate (6.5%) was blotted for β-actin. (c)  Densitometric determination of band intensities from EAAC1 expression. Band density was calculated as a sum of both multimer and monomer, and plotted against the control density set as 100%. The results are the average of three independent measurements and presented as percentage of the control. Error bars indicate ± SEM.

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SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

Store-operated calcium entry channels (SOCC) have been proposed to mediate replenishment of ER calcium stores (Patterson et al. 1999). To further test the idea that intracellular calcium stores regulate glutamate uptake, we tested the effect of SKF-96365, a SOCC blocker (Merritt et al. 1990; Mason et al. 1993) on glutamate uptake in neurons. We found that SKF-96365 significantly inhibited glutamate uptake in a dose-dependent manner (Fig. 6a). Importantly, nifedipine and ω-conotoxin GVIA, which inhibit L- and N-type voltage-dependent calcium channels (VDCC), respectively, had no effect on glutamate uptake (Fig. 6b). In addition, cadmium, which blocks all VDCC on the plasma membrane, was also ineffective at reducing glutamate uptake. These data strongly argue that calcium entry from voltage-gated calcium channels did not influence glutamate uptake. SKF-96365 induced a dose-dependent reduction of cell surface EAAC1 expression without altering total EAAC1 expression (Figs 6c and d).

image

Figure 6. SKF-96365, but not nifedipine, ω-conotoxin, or cadmium reduced glutamate uptake. (a)  Dose-dependent reduction of glutamate uptake by SKF-96365 (SKF) treatment. (b)  Nifedipine (Nif), ω-conotoxin (ω-Con), and cadmium (Cad) had no effects on glutamate uptake. Neurons were treated with SKF-96365 (100 µ m ), nifedipine (1 µ m ), ω-conotoxin (2 µ m ), or cadmium (100 µ m ) for 1 h prior to and during uptake. Results were from three independent experiments for both (a) and (b), and data are means ± SEM. (c)  EAAC1 was measured in both a portion of the whole-cell lysate (6.5% of total lysate) and streptavidin immunoprecipitates of cell surface-biotinylated proteins. To ensure equal protein amounts were assayed, an equal portion of lysate (6.5%) was blotted for β-actin. (d) Band density was calculated as sum of multimer and monomer, and plotted against the control density set as 100%. The results are average of three independent experiments and presented as percentage of the control. Error bars indicate ± SEM.

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Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

In order to show that intracellular calcium stores regulate glutamate transporter activity, it was important to prove that our manipulations reduced ER calcium levels as does PS1 deficiency. To accomplish this we pre-treated neurons with caffeine, thapsigargin, or SKF-96365 and then released any calcium remaining in the ER by the addition of thapsigargin (1 µm) in the absence of extracellular calcium. Calcium released into the cytosol was detected by fura-2 ratiometric (R340/R380) imaging. Calcium content in the ER was significantly reduced in caffeine (20 mm, 2 h), thapsigargin (5 µm, 30 min) and SKF-96365 (100 µm 1 h) treated neurons (Fig. 7a). For each of the drugs tested the peak calcium values were significantly lower in the treated cells as compared to controls (Fig. 7b).

image

Figure 7. Calcium store depletion by caffeine, thapsigargin and SKF-96365 treatment. (a)  Neurons were pre-treated with caffeine (20 m m , 2 h) (red, n  = 8 cells), thapsigargin (5 µ m , 30 min) (green, n  = 5 cells), SKF-96365 (100 µ m , 1 h) (blue, n  = 10 cells), and HBSS buffer as a pre-treatment control (black, n  = 7 cells). Cytosolic [Ca 2+ ] i was determined by fluorescence ratiometric imaging using Fura-2 (see Experimental procedures ). Following treatments, release of remaining calcium from intracellular stores was induced by 1 µ m thapsigargin in calcium-free HBSS buffer. (b)  Histogram represents the average of baseline (B) and peak (P) calcium levels before and after bulk calcium release from intracellular calcium stores. There was no statistically significant difference between baselines of treated vs. untreated cells. Compared to control cells (Ctrl), peak calcium levels were significantly reduced in neurons pre-treated with caffeine (Caff, p <  0.05), thapsigargin (TG, p <  0.02), and SKF-96365 (SKF, p <  0.01). Error bars indicate ± SEM.

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Taken together, four approaches that reduce ER calcium levels: caffeine, thapsigargin, SKF-96365, and PS1 deficiency (Yang and Cook, 2004) all decreased glutamate uptake. Decreased glutamate uptake was in all cases accompanied by a decrease in EAAC1 expressed at the cell surface that was proportional to the reduction in uptake.

Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

In order to address whether other intracellular second messenger systems regulate glutamate uptake in neurons, we also examined the effects of modulating protein kinase C (PKC) and protein kinase A (PKA) activity on glutamate transport in primary neurons. Neurons were treated with the PKC or PKA activators PMA (100 nm, 30 min) and forskolin (25 µm, 30 min), respectively. Cells were also treated with the PKC and PKA inhibitors, BisII (2 µm, 15 min) and H-89 (5 µm, 1 h), respectively. None of these reagents induced significant changes in glutamate uptake (Fig. 8a). To insure that these treatments were effective in modulating PKC and PKA activation under our experimental conditions we examined PMA and forskolin-induced activation of MAPK (ERK1/2) using antibodies recognizing either the active, phosphorylated form of ERK1/2 or the inactive, pan-specific form of ERK1/2 (Seger and Krebs 1995). We observed significant phosphorylation of ERK1/2 in response to PMA treatment (Fig. 7b, left panel, lane 2) compared to the control (lane 1). ERK1/2 phosphorylation induced by PMA was attenuated by BisII pre-treatment (lane 3). Similar results were obtained using forskolin and H-89 (Fig. 7b, right panel). Thus, activation or inhibition of either PKC or PKA activity did not exert a significant impact on glutamate uptake in neurons under our experimental conditions.

image

Figure 8. Modulation of PKC or PKA failed to alter glutamate uptake in neurons. (a)  Glutamate uptake in response to PKC or PKA activation or inhibition. Neurons were treated with PMA (100 n m , 30 min), forskolin (FSK, 25 µ m , 30 min), Bisindolylmaleimide II (BisII, 1 µ m , 15 min), and H-89 (5 µ m , 1 h) prior to measuring glutamate uptake. Uptake was conducted at 37°C for 9 min. No significant changes in glutamate uptake were detected under these stimulatory or inhibiting conditions compared to control. The results are averages of three independent experiments presented as means ± SEM. (b) To confirm that PKC and PKA activity was activated or inhibited by the treatments employed in (a), western blot analyses were performed using an antibody specifically recognizing phosphorylated ERK1/2 (upper panel). Total ERK1/2 levels were examined with a pan-specific ERK1/2 antibody (lower panel). PMA treatment (100 n m , 30 min) induced ERK phosphorylation in neurons, which was attenuated by BisII pre-treatment (1 µ m , 15 min), whereas total ERK1/2 levels remained unchanged. Forskolin treatment (25 µ m , 30 min) also increased ERK1/2 phosphorylation which was attenuated by H-89 (right panel).

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Role of PS1 in regulating neuronal glutamate transport

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

We found that PS1 knock-out neurons displayed a marked impairment in sodium-dependent glutamate uptake. The degree of this impairment was related to PS1 gene dose. The effects of knocking out PS1 were mimicked pharmacologically by γ-secretase inhibition. In addition, we found that both PS1 deficiency and γ-secretase inhibitor treatment reduced EAAC1 cell surface expression. This function of PS1 appears to be mediated by the effects of PS1 on intracellular calcium homeostasis because three distinct means of reducing intracellular calcium stores mimicked PS1 deficiency by reducing glutamate uptake and decreasing EAAC1 cell surface expression.

A family of five sodium-dependent glutamate transporter subtypes have been identified (Kanai and Hediger 1992; Pines et al. 1992; Storck et al. 1992; Fairman et al. 1995; Arriza et al. 1997). These transporters are localized at many sites on neurons (including pre- and post-synaptic sites), extra-synaptically on astrocytes, and on other cell types (Danbolt 1994; Kugler and Schmitt 1999). Glutamate transporters are important for protecting neurons against glutamate toxicity (Mangano and Schwarcz 1983; Rosenberg et al. 1992), modulating synaptic transmission by limiting glutamate diffusion (spillover) to surrounding synapses (Robinson et al. 1993; Asztely et al. 1997; Rusakov and Kullmann 1998), and influencing synaptic plasticity (Levenson et al. 2002). To examine the mechanisms by which PS1 loss-of-function influenced glutamate uptake, we focused on EAAC1 because it was expressed abundantly in our cortical neuronal cultures compared to GLT-1 and GLAST, as assayed by western blotting. This pattern of glutamate transporter expression agrees with earlier reports examining neuron-enriched cultures (Wang et al. 1998; Guillet et al. 2002). However, neurons can also express the glial-type transporter, GLT-1 (Chen et al. 2002). Thus it was important to determine how much transporter activity was attributable to GLT-1 in the neuronal cultures. Under our experimental conditions approximately 25% of total glutamate uptake was mediated by DHK-sensitive transporters. Compared to wild-type cells, PS1-deficient neurons still exhibited a significant decrease in glutamate uptake in the presence of DHK, thus suggesting that EAAC1 and possibly GLAST are regulated by PS1.

Influence of γ-secretase inhibitor treatment on glutamate transport

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

The effects of PS1 deficiency on glutamate transport were closely mimicked in wild-type neurons by brief (4 h) γ-secretase inhibitor treatment. The difluoro ketone peptidomimetic compound used in this study has been shown to block production of Aβ40 and Aβ42, but also affects other proteolytic activities (Wolfe et al. 1999). In order to insure that the effects of the γ-secretase inhibitor were specifically related to PS1 activity, we also tested PS1 heterozygous and knock-out neurons. We found that the γ-secretase inhibitor failed to block glutamate transport in the PS1 knock-out neurons. This finding did not reflect a ‘floor’ effect because PS1 knock-out cells retained roughly 50% of their total glutamate transport activity. Thus, these results strongly suggest that the γ-secretase inhibitor effects were specifically mediated by a PS1-dependent mechanism. This conclusion raises intriguing questions as to whether PS1 itself or other molecules directly regulated by PS1 (e.g. Aβ or APP C-terminal fragments) are important in regulating glutamate uptake. Future studies addressing this issue could reveal additional biological effects of APP/PS1 interactions beyond those already well-established for Aβ production.

The development of γ-secretase inhibitors to block Aβ production has emerged as an important candidate approach to the treatment of AD. We found that γ-secretase inhibition dramatically suppressed glutamate uptake in a PS1-dependent fashion. This finding extends and strengthens the conclusion that PS1 is an important modulator of glutamate uptake. However, our findings also raise the disquieting possibility that use of γ-secretase inhibitors could suppress glutamate uptake, the primary means by which the brain clears extracellular glutamate.

Role of PS1 in regulating EAAC1 cell surface expression

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

To investigate the mechanisms by which PS1 regulates glutamate uptake, we examined the effects of PS1 deficiency on the cell surface expression of EAAC1. EAAC1 exists in a large intracellular pool that is available for transport the cell surface (Rothstein et al. 1994; Kugler and Schmitt 1999). We found that PS1 loss-of-function caused a marked decrease in EAAC1 cell surface expression. Total EAAC1 levels (the combined intracellular and surface pools) were comparable in PS1 wild-type and knock-out neurons. Consistent with this, the intensity of EAAC1 immunoreactivity in permeablized PS1 wild-type and knock-out neurons was indistinguishable (data not shown). Cell surface EAAC1 expression levels in neurons were too low to examine by immunofluorescence. The magnitude of the reduction in cell surface EAAC1 expression (approximately 60%) was quite similar to the reduction in glutamate transport activity caused by PS1 deficiency (approximately 50%). Thus, it is plausible that altered transporter trafficking could account for the effects of PS1 deficiency on glutamate uptake.

The selectivity of PS1 in regulating protein trafficking is protein-specific. PS1 loss-of-function mutations cause increased cell surface expression of nicotinic acetylcholine receptors (Leem et al. 2002b) and APP (Cai et al. 2002; data not shown). In contrast to acetylcholine receptors and APP, transport of nicastrin to the cell surface is retarded in PS1 knock-out cells (Goutte et al. 2002; Leem et al. 2002a,b). These data show that PS1 deficiency can either up- or down-regulate specific proteins expressed on the cell surface. However, the majority of the proteins transported to the cell surface are not influenced by the loss of PS1 (Naruse et al. 1998). Thus, the effects of PS1 on EAAC1 cell surface expression appear to be highly specific.

Role of intracellular calcium signaling in regulating glutamate transport

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

A number of mechanisms have been proposed whereby glutamate transporter function may be regulated, including protein phosphorylation (Casado et al. 1993), oxidative modification (Trotti et al. 1997a,b; Lauderback et al. 2001), protein–protein interactions (Jackson et al. 2001; Lin et al. 2001), and membrane trafficking (Dowd and Robinson 1996; Davis et al. 1998). Intracellular second messenger pathways appear to play a particularly influential role in regulating glutamate transporter activity and trafficking in specific cell types (Zerangue et al. 1995; Dowd and Robinson 1996; Davis et al. 1998; Sims et al. 2000). In addition, there is evidence suggesting that glutamate release is associated with intracellular calcium stores (Parpura et al. 1994; Bezzi et al. 1998). Importantly, PS1 deficiency reduces ER calcium levels as evidenced by reduced cytosolic calcium responses evoked by thapsigargin in the absence of extracellular calcium (Leissring et al. 2002) and by increased capacitive calcium entry responses (Yoo et al. 2000). These findings prompted us to examine whether directly reducing intracellular calcium stores would also alter glutamate transporter function. We used three different approaches to reduce intracellular ER calcium levels. Caffeine releases calcium from intracellular stores by activating ryanodine receptors on the ER membrane (Bhat et al. 1997; Pan et al. 2000), whereas thapsigargin depletes ER calcium stores by inhibiting the ER calcium pump, Ca2+-ATPase (Thastrup et al. 1990). Nevertheless, both caffeine and thapsigargin reduced glutamate uptake and decreased cell surface expression of EAAC1. SKF-96365 blocks SOCC (Merritt et al. 1990). SOCC have been proposed to couple the ER and plasma membrane in order to mediate calcium re-entry into the ER (Berridge 1995; Patterson et al. 1999). Consistent with this idea we detected a significant decrease of ER calcium levels in SKF-95365-treated neurons. We found that glutamate uptake was impaired by SKF-95365, but not nifedipine, ω-conotoxin GVIA, or cadmium. These results are in keeping with data showing that calcium influx through store-operated channels is unaffected by VDCC antagonists (Usachev and Thayer 1999). Interestingly, unlike PS1, PS2 deficiency has been shown to have a minimal effect on ER calcium stores (Leissring et al. 2002), thus it is unlikely that PS2 plays a significant role in regulating glutamate.

We have shown that PS1 loss-of-function, caffeine, thapsigargin, and SKF-95365 all reduce glutamate uptake and reduce cell surface expression of EAAC1. Although each of these perturbations affect distinct molecular processes, they all reduce ER calcium levels. This strongly suggests that intracellular calcium stores constitute a key site where the processes affected by these perturbations converge to affect a novel subcellular pathway that regulates glutamate transporter function. In contrast to γ-secretase inhibitors, which depend on the presence of PS1, caffeine, thapsigargin, and SKF-95365 appear to affect glutamate uptake in parallel with PS1 by directly reducing ER calcium levels. Thus, it would not be expected that these agents would strictly depend on PS1 to affect glutamate uptake. Consistent with this idea thapsigargin (10 µm) and SKF-95365 (100 µm) produce comparable reductions in glutamate uptake in both PS1-KO neurons compared to wild-type neurons (data not shown). However, we have observed that caffeine (20 mm) is less effective in reducing glutamate uptake in PS1-KO cells compared to wild-type cells (data not shown). It is likely that these results are attributable to differences in the efficiency with which these compounds deplete intracellular calcium.

Taken collectively, our findings strongly suggest that intracellular calcium stores play an important role in regulating glutamate transporter activity and trafficking. This intracellular signaling pathway appears to modulate glutamate uptake in neurons with specificity because calcium entry mediated by voltage-gated calcium channels, activation/inhibition of PKC, and PKA did not affect glutamate uptake. Interestingly, these data further suggest that glutamate transporter function is regulated in a cell type-specific fashion since PKC is known to be an important regulator of EAAC1 function in C6 glioma cells (Dowd and Robinson 1996; Davis et al. 1998; Gonzalez et al. 2002).

Role of glutamate transporters in human disease

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

Glutamate transporter abnormalities have been associated with a number of neurological disorders, including epilepsy and CNS ischemic disease (Drejer et al. 1985; Otori et al. 1994; Szatkowski and Attwell 1994; Roettger and Lipton 1996; Martin et al. 1997; Rossi et al. 2000), seizures (Meldrum 1994; Miller et al. 1997), AD (Masliah et al. 1998, 2000; Lauderback et al. 2001; Scott et al. 2002), and ALS (Rothstein et al. 1992, 1995; Bristol and Rothstein 1996; Howland et al. 2002). The manifestations of disturbed glutamate transporter function in these diseases are diverse. For example, recent data suggest that glial glutamate transporters are aberrantly expressed in pyramidal neurons in AD patients (Scott et al. 2002). In other circumstances chronic inhibition of glutamate transport may contribute to the pathology associated with ALS (Rothstein et al. 1992, 1995). Thus, the central nervous system appears to be broadly vulnerable to dysregulation of glutamate uptake, resulting in wide ranging pathophysiological consequences. The roles of PS1 and intracellular calcium stores in regulating this important neurotransmitter uptake system may have numerous biomedical implications and require further investigation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References

We gratefully acknowledge the helpful comments and encouragement of Drs Zhu-Chen Ye, Gerard Schellenberg, and Ian D'Souza in preparing this manuscript. This work was supported by NIA AG05136 Alzheimer's Disease Research Center/Project (DGC) and a VA Merit Review Award (DGC).

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  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Genotyping and cell culture
  5. [14C]Glutamate uptake
  6. Cell surface biotinylation and western blot procedures
  7. Calcium measurements
  8. Statistical analysis
  9. Results
  10. Glutamate uptake is impaired in PS1 knock-out neurons
  11. Gamma-secretase inhibitor impairs glutamate uptake
  12. PS1 deficiency impairs neuronal glutamate transport in presence of DHK
  13. EAAC1 surface expression is reduced in PS1 knock-out neurons
  14. Caffeine impairs glutamate uptake in a dose-dependent manner through reducing surface expression of EAAC1 in neurons
  15. Thapsigargin reduces glutamate uptake and cell surface expression of EAAC1 in neurons in a dose-dependent manner
  16. SKF-96365, but not nifedipin, ω-conotoxin, or cadmium impairs glutamate uptake and surface EAAC1 trafficking in neurons
  17. Intracellular calcium is depleted by caffeine, thapsigargin and SKF-96365 in neurons
  18. Modulation of protein kinase C and protein kinase A activity does not influence glutamate transport in neurons
  19. Discussion
  20. Role of PS1 in regulating neuronal glutamate transport
  21. Influence of γ-secretase inhibitor treatment on glutamate transport
  22. Role of PS1 in regulating EAAC1 cell surface expression
  23. Role of intracellular calcium signaling in regulating glutamate transport
  24. Role of glutamate transporters in human disease
  25. Acknowledgements
  26. References
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