Detergent‐Insoluble EAAC1/EAAT3 Aberrantly Accumulates in Hippocampal Neurons of Alzheimer's Disease Patients

Abstract

Disturbed glutamate homeostasis may contribute to the pathological processes involved in Alzheimer's disease (AD). Once glutamate is released from synapses or from other intracellular sources, it is rapidly cleared by glutamate transporters. EAAC1 (also called EAAT3 or SLC1A1) is the primary glutamate transporter in forebrain neurons. In addition to transporting glutamate, EAAC1 plays other roles in regulating GABA synthesis, reducing oxidative stress in neurons, and is important in supporting neuron viability. Currently, little is known about EAAC1 in AD. To address whether EAAC1 is disturbed in AD, immunohistochemistry was performed on tissue from hippocampus and frontal cortex of AD and normal control subjects matched for age and gender. While EAAC1 immunostaining in cortex appeared comparable to controls, in the hippocampus, EAAC1 aberrantly accumulated in the cell bodies and proximal neuritic processes of CA2‐CA3 pyramidal neurons in AD patients. Biochemical analyses showed that Triton X‐100‐insoluble EAAC1 was significantly increased in the hippocampus of AD patients compared to both controls and Parkinson's disease patients. These findings suggest that aberrant glutamate transporter expression is associated with AD‐related neuropathology and that intracellular accumulation of detergent‐insoluble EAAC1 is a feature of the complex biochemical lesions in AD that include altered protein solubility.

INTRODUCTION

The task of clearing extracellular glutamate is accomplished by a family of five sodium‐dependent glutamate transporter subtypes referred to as GLAST, GLT‐1, EAAC1, EAAT4 and EAAT5 (also known as EAAT1‐5, respectively) 3, 16, 27, 47, 60. Glutamate transporters are localized at many sites on neurons, astrocytes and other cell types. GLAST, GLT‐1 and EAAC1 are the primary glutamate transporters in the hippocampus and cortex. GLAST is expressed principally by astrocytes. GLT‐1 is highly expressed in astrocytes and more recently, it has become clear it is also expressed by some neurons (7). In the central nervous system (CNS), EAAC1/EAAT3 (hereafter in this report referred to as EAAC1) is expressed primarily by neurons (50).

A growing number of reports indicate that glutamate transporters are disturbed in Alzheimer's disease (AD). GLT‐1 and GLAST levels are reduced in AD 13, 26, 34, 39. GLAST is aberrantly expressed in cortical neurons of AD patients (56). AD patients exhibit increased oxidative damage to glutamate transporters, which is also seen in astrocytes treated with amyloid‐β (Aβ) peptide 8, 19, 20, 28, 31. GLT‐1 and GLAST, but not EAAC1, are reduced in transgenic mice overexpressing the amyloid precursor protein (APP) harboring a familial AD mutation (40). Finally, it has recently been shown that GLT‐1 levels are reduced in APPswe/PS1M146V double transgenic mice (38). In addition to clearing glutamate, EAAC1 plays a number of other complementary roles in regulating neuronal activity and viability. These roles include facilitating Gamma‐aminobutyric acid (GABA) synthesis 41, 57 and protecting neurons from oxidative stress by mediating glutathione synthesis 2, 11, 22. Reducing EAAC1 expression via antisense RNA approaches increases the susceptibility of neurons to excitotoxic injury (10) and causes dendritic swelling in the hippocampus (52). Importantly, it has also recently been shown that EAAC1 deficiency causes age‐related neuron loss (2), thus demonstrating that EAAC1 is critical for supporting neuron viability. Taken together, these findings raise the question of whether EAAC1 is disturbed in AD. Most of the studies addressing the role of glutamate transporters in AD pathogenesis have examined GLAST and GLT‐1. By comparison, less attention has been focused on EAAC1. Thus, the aim of the studies reported herein was to address whether the expression patterns and biochemical structure of EAAC1 are disturbed in AD.

METHODS

Patient tissue

Tissue from the hippocampus and frontal cortex was obtained from the brain banks of the Alzheimer's Disease Research Center (ADRC) at the University of Washington and the Alzheimer's Disease Center (ADC) at Oregon Health & Science University (OHSU). Appropriate informed consent was obtained for use of all patient tissues using procedures approved by the human subjects' Institutional Review Boards of the University of Washington and OHSU. All patients had clinical and neuropathological diagnoses made according to established consensus criteria (1). AD patients selected for study were free of coexisting Lewy body disease or vascular damage. A total of 34 AD, 20 normal controls, 4 Huntington's disease (HD) and 4 Parkinson's disease (PD) patients were studied. The Braak system was used for staging neurofibrillary tangles (NFT) pathology in AD (9), and Consortium to Establish a Registry for Alzheimer's Disease (CERAD) plaque scores (43) were accomplished with modified Bielschowsky‐stained sections. For each experiment, the characteristics (neuropathological assessment, age and gender) of the specific groups being examined are summarized in the results section.

Immunohistochemistry

Standard immunohistochemical staining methods were used. Briefly, paraffin‐embedded sections were dewaxed with xylenes and were partially hydrated through washes of 100%, 95% and 70% ethanol. Endogenous peroxidase activity was quenched by a 20‐minute pretreatment with 3% hydrogen peroxide. Tissue was rehydrated and treated with Revealit antigen recovery solution at 85°C for 10 minutes (ImmunoSolution, Newcastle, NSW, Australia). At room temperature, sections were washed in H₂O, phosphate buffered saline (PBS), and incubated in blocking solution [0.5% bovine serum albumin (BSA), 0.01% Triton X‐100, 0.05% sodium azide] for 1 h at 37°C. Slides were incubated with affinity‐purified rabbit polyclonal EAAC1/EAAT3 antisera (Alpha Diagnostics, San Antonio, TX, USA) overnight at 37°C, rinsed and then incubated with biotinylated goat antirabbit secondary antibody (Vector Laboratories, Burligame, CA, USA). Positive immunostaining was evidenced by the insoluble 3, 3′‐diaminobenzidine (DAB) reaction product developed using an ABC biotinylated‐avidin‐horseradish peroxidase kit (Vector Laboratories). Lesions containing a‐synclein in PD tissue were detected with the monoclonal antibody LB509 (4). In some experiments, double immunostaining was performed to detect EAAC1 using DAB as a brown chromogen, and either tau (Tau‐2, Sigma Chemical Co., St. Louis, MO, USA) or Aß (4G8, Covance/Signet Laboratories, Deadham, MA, USA) using a Vector Red chromogen (Vector Laboratories). Monoclonal antibodies were detected with a biotinylated horse antimouse IgG secondary antibody (Vector Laboratories) and the ABC alkaline phosphatase kit (Vector Laboratories). Alpha‐synuclein immunostained tissue was counterstained with hemotoxylin to stain neuron cell bodies. Counterstains were omitted in double‐label experiments and in all single‐label experiments where images were presented as monochromatic gray‐scale images. Microscopy was carried out using a Nikon Optiphot‐2 microscope equipped with an Insight QE digital camera (Diagnostics Instruments, Sterling Heights, MI, USA). Double‐label confocal microscopy was performed with a Leica confocal microscope (Leica microsystems, Bannockburn, IL). Primary antibodies were stained with goat antirabbit or antimouse Alexa‐488 or Alexa‐555 conjugated secondary antibodies (Molecular Probes/Invitrogen, Carlsbad, CA, USA). Image acquisition and processing were performed using Spot imaging software (Diagnostics Instruments) and were formatted with Photoshop (Adobe Systems, San Jose, CA, USA). For each experiment, microscopic images were acquired and digitally processed under identical conditions. Digital image processing was limited to linear brightness and contrast adjustments that were performed identically on experimental and control images. Gray‐scale images were converted from red, green, blue (RGB) color to gray scale.

Detergent‐insoluble EAAC1 measurements

Snap‐frozen hippocampus was dissected, homogenized and sequentially extracted in buffer A (10 mM Tris, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM Dithiothreitol (DTT) and 10% sucrose, pH 7.5) and buffer B (buffer A with 1% Triton X‐100) using previously described methods (68). Phosphatase inhibitors (20 mM NaF and 1 mM sodium orthovanadate) and protease inhibitor cocktail (Sigma Chemical Co.) were added to buffers A and B immediately before use. Detergent‐insoluble material in buffer B was extracted with 70% formic acid (FA) as previously described. (69) FA extracts of detergent‐insoluble proteins were dried by vacuum centrifugation and resolubilized by sonication in 20 volumes of 5 M guanidine HCl, 100 mM Tris, pH 7.4, with 0.002% bromphenol blue added to confirm elimination of FA. These were further diluted 128‐fold in 100 mM Tris, pH 7.4, with 0.05% sodium azide and 0.002% bromphenol blue, and 100 µL was spotted onto 96‐well plates and incubated overnight at room temperature in a humidified chamber. The plates were subsequently washed twice with PBS, blocked with 1% BSA in PBS with 0.05% sodium azide and washed again with PBS. Anti‐EAAC1 (Alpha Diagnostics) and anti‐Presenilin 1 (PS1) (polyclonal rabbit antibody provided by Dr. G.Schellenberg, Seattle, WA, USA) antibodies were used to detect detergent‐insoluble EAAC1 and PS1 by standard enzyme‐linked immunosorbence assay (ELISA) methods using tetramethylbenzidine as a substrate and absorbances determined at 450 nm.

Detergent‐soluble EAAC1 measurements

Detergent‐soluble protein lysates from hippocampus were prepared for Western blot analyses as described above except that extracts were solubilized in 10 volumes of 1 × Laemmli sample buffer (Bio‐Rad, Hercules, CA, USA), without vacuum centrifugation. Total protein concentrations for each lysate were determined by the BCA method (Pierce, Rockford, IL, USA). Proteins were resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Resolved proteins were transferred to nitrocellulose and Western blotted with anti‐EAAC1. Protein bands were detected using horseradish peroxidase‐conjugated secondary antibodies (Sigma Chemical Co.) in conjunction with chemiluminescence (Pierce). Densitometry was performed using NIH image software (National Institutes of Health, Bethesda, MD). Brain tissue from EAAC1 knockout mice was the generous gift of Dr. Raymond Swanson (University of California, San Francisco, CA, USA).

Glutamate transporter expression vectors

The lentiviral transfer vector (pRRL‐cPPT‐CMV‐X‐PRE‐SIN) used for these studies was the generous gift of Dr. William Osborne (Department of Pediatrics, University of Washington, Seattle, WA, USA). This transfer vector directs transgene expression with the cytomegalovirus (CMV) promoter and includes an incorporated central polypurine tract (cPPT) element from the pol region of HIV‐1 to increase transduction efficiency. Transgene expression is further enhanced by inclusion of a posttranscriptional regulatory element (PRE) element from human hepatitis B virus. (6) Cloning of the cDNAs encoding the glutamate transporters GLT‐1a, GLT‐1b, GLAST and EAAC1 is described in detail elsewhere 6, 61. Each of the glutamate transporter cDNAs was cloned into the pRRL‐cPPT‐CMV‐X‐PRE‐SIN multiple cloning site to generate the required expression vector. All constructs were purified using Qiagen Plasmid Maxiprep kits (Qiagen, Valencia, CA) according to the manufacturer's protocol. DNA sequencing analysis confirmed the accuracy of the constructs. The resulting lentiviral glutamate transporter transfer vectors were expressed in HEK 293T cells using standard calcium phosphate transfections procedures to transduce the cells according to previously published procedures (6).

RESULTS

EAAC1 is aberrantly elevated in AD hippocampus

In order to investigate EAAC1 expression in AD patients, the specificity of our Western blot and immunohistochemical approaches were first tested in cell lines expressing different glutamate transporter subtypes and in brain tissue from wild‐type and EAAC1 knockout mice. This was particularly important as it has been shown that some antibodies raised against unique EAAC1 domains can display unexpected affinity for other proteins lacking any amino acid sequence homology to EAAC1, even though such antibodies can appear specific as judged by peptide preadsorption assays (24). EAAC1 forms functional homotrimers (71) that, depending on the state of the tissue and the processing conditions, resolve by SDS‐PAGE as monomers (approximately 66–69 kDa) and higher‐order multimers. These multimers can become more prominent when EAAC1 undergoes oxidative modification (21). Figure 1A shows that EAAC1 was detected in 293T cells expressing lentiviral cDNA constructs directing EAAC1 expression, but not in cells similarly transduced to express the glutamate transporters GLT‐1a, GLT‐1b and GLAST. More importantly, positive EAAC1 immunoreactivity in Western blots of wild‐type mouse brain tissue (Figure 1B) and in immunostained wild‐type hippocampus (Figure 1C) was absent in EAAC1 knockout mice, thus satisfying the most stringent criterion for antibody specificity (53).

Figure 1.

Specificity of EAAC1 immunoreactivity. 293T cells were transduced with lentiviral transfer vectors directing expression of GLT‐1a, GLT‐1b, EAAC1 or GLAST (A). Non‐transduced cells served as negative control. EAAC1 was detected only in EAAC1‐transduced cells. EAAC1 resolved in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis both as a monomer (approximately 66 kDa) and higher molecular weight multimers (140–250 kDa). EAAC1 was detected by Western blot in brain tissue from wild‐type (WT), but not EAAC1 knockout (KO) mice (B). EAAC1 immunoreactivity was detected in the hippocampus of wild‐type, but not EAAC1 knockout mice (C). Scale bar: 200 µm.

To examine whether EAAC1 expression is disturbed in AD, immunohistochemical analyses were carried out on a panel of seven AD (four and three Braak stages V and VI patients, respectively) and seven controls (four and three Braak stages I and II patients, respectively) that were comparable on the basis of age (AD ranging from 72 to 88 years of age, mean = 81.7 ± 2 years; controls ranging from 73 to 85 years of age, mean = 79.7 ± 2 years) and gender (AD: three males and four females, controls: four males and three females). In the brain, EAAC1 expression is most pronounced in neurons of the hippocampus, cortex and caudate‐putamen (50). In this study, the hippocampus and frontal cortex were examined because these brain regions display AD pathologic changes at the Braak stages investigated. In six of the seven AD cases, hippocampal EAAC1 immunoreactivity appeared more intense in pyramidal neurons of the CA2‐CA3 compared with age‐matched controls as judged by an experimenter blind to the diagnosis associated with the tissue (Figure 2A–D). In contrast to the CA2‐CA3, the pattern of EAAC1 immunoreactivity in CA1 and the dentate gyrus of AD subjects were comparable to controls (Figure 2E–H). In addition, consistent with previously published results (34), no differences in the intensity of neuronal EAAC1 immunoreactivity were apparent in AD cortex compared to controls (Figure 2I,J). Thus, the aberrant neuronal EAAC1 immunoreactivity in CA2‐CA3 was anatomically discrete both within the hippocampus and with respect to the frontal cortex. This data also show that aberrant elevated EAAC1 immunoreactivity is not simply a feature of neuronal degeneration because the cortex, which undergoes neuronal degeneration in AD, appeared comparable to the cortex from normal controls.

Figure 2.

EAAC1 is aberrantly expressed in Alzheimer's disease (AD) hippocampal CA2‐CA3 neurons. Representative EAAC1 immunostaining of AD hippocampus (panels A,C,E,G,I) and age‐matched normal control (panels B,D,F,H,J) are shown. Aberrant EAAC1 immunoreactivity in AD pyramidal neurons of the CA3 and CA2 (panels A,C) was more pronounced compared with control (panels B,D). In contrast to this, the neuronal EAAC1 immunostaining in CA1, dentate gyrus (DG) and frontal cortex of AD (panels E,G,I) appeared comparable to control (F,H,J) Scale bars: 50 µm.

Elevated EAAC1 is associated with degenerating neurons

Inspection at higher magnification revealed further distinctions in EAAC1 expression between AD and control CA2‐CA3 pyramidal neurons (Figure 3). In contrast to the relatively uniform pattern of cytoplasmic staining that was more characteristic of neurons from normal subjects (Figure 3C), in AD hippocampus (Figure 3A,B), neurons with aberrantly elevated EAAC1 levels frequently had an uneven, punctate expression pattern in the cell bodies. It was not uncommon to observe this aberrant staining pattern in cells also displaying granulovacuolar degeneration (GVD). GVD lesions are histologically defined as distinct cytoplasmic vacuolar bodies containing dense granules of aggregated protein that are associated with AD and a number of other neurodegenerative diseases 30, 45, 55. In the CA2 of AD hippocampus, we estimated that approximately 28% ± 6% (n = 4) of the neurons displaying aberrant EAAC1 immunoreactivity also possessed structures consistent with GVD pathology.

Figure 3.

Aberrant EAAC1 immunoreactivity is associated with granulovacuolar degeneration (GVD). EAAC1 immunostaining in hippocampal pyramidal neurons from CA2‐CA3 of an Alzheimer's disease (AD) patient (A,B) was manifested by intense patchy intracellular staining in the cell bodies and proximal neuritic processes. This was in contrast to the uniform cytoplasmic staining that was more characteristic of hippocampal neurons in normal subjects (C). Evidence of GVD in AD neurons (arrowheads in B) suggested aberrant EAAC1 immunoreactivity was associated with degenerating neurons. Scale bars: 20 µm.

Neurofibrillary tangles constitute the most prominent intracellular histopathological lesion associated with AD (32). Double‐label immunostaining of EAAC1 and tau (stained with the monoclonal antibody Tau‐2) was performed to determine whether aberrant EAAC1 expression was associated with tau‐bearing neurofibrillary tangles. Pyramidal neurons in AD CA2‐CA3 displaying aberrant EAAC1 immunoreactivity were frequently positive for tau, both in the cell bodies (Figure 4A) and in dendritic processes (Figure 4B). In the CA2 of AD patients (n = 7), 42% ± 7% [standard error of the mean (SEM)] of the morphologically intact neurons with aberrant EAAC1 immunostaining also displayed elevated tau immunoreactivity. This degree of co‐expression may be an underestimate as the intense tau‐immunoreactivity associated with neurofibrillary tangles may have obscured EAAC1 staining in some neurons. Using double‐label confocal microscopy, examples of aberrant EAAC1 (green) and tau (red) immunoreactivity in AD hippocampal pyramidal neuronal cell bodies are more easily visualized (Figure 4E,F).

Figure 4.

Aberrant EAAC1 expression in Alzheimer's disease (AD) is associated with neurofibrillary tangles but not amyloid plaques. In AD hippocampus, degenerating neurons were often immunopositive for both EAAC1 (stained brown) and tau (stained red with monoclonal antibody, Tau‐2) (A). Open arrowheads indicate examples of hippocampal CA2‐CA3 pyramidal neurons with aberrant EAAC1 immunoreactivity in the cell bodies and also in neuritic processes (closed arrow head). Panel B is a higher magnification of panel A showing co‐staining of EAAC1 and tau in a neuritic process. EAAC1 immunoreactivity (stained brown) was not associated with senile plaques (stained red with monoclonal 4G8 antibody recognizing Aβ) in AD hippocampus (C) and in AD cortex (D). Panels E and F show examples of aberrant perisomal EAAC1 (green) and tau (red) immunoreactivity localized in the same neurons in the hippocampus of an AD patient. Scale bars: panels A, C, D (20 µm); panel B (10 µm).

Extracellular senile plaques are comprised chiefly of amyloid β peptides (Aβ), but these plaques can also be sites where other proteins accumulate in AD brain tissue (35). To determine whether EAAC1 accumulates in association with senile plaques, double‐label immunohistochemistry was performed by staining for EAAC1 and Aβ (labeled with the monoclonal antibody, 4G8). In the hippocampus and cortex, EAAC1 immunoreactivity was not associated with senile plaques (Figure 4C,D). Collectively, these data suggest that aberrant elevated intracellular EAAC1 expression is associated with neuronal degeneration in AD.

Aberrant EAAC1 accumulation is specifically associated with AD

If EAAC1 accumulation were a feature specifically associated with degenerating neurons in AD, then other neurodegenerative diseases that tend to spare the hippocampus might be expected to display less aberrant neuronal EAAC1 expression. To address this issue, HD and PD cases were examined because both are pathologically well‐defined neurodegenerative disorders with less severe hippocampal pathology compared with AD. EAAC1 immunostaining was compared in the hippocampus from a panel of four AD patients (two Braak III and two Braak III/IV stage cases composed of two males and two females ranging from 76 to 84 years of age, mean = 80 ± 1.8 years), four HD patients (four males ranging from 72 to 81 years, mean = 73.3 ± 3.1 years) and seven controls (four Braak stage I and three Braak stage II cases composed of four males and three females ranging from 73 to 85 years of age, mean = 79.7 ± 2.0 years). This control group was the same as examined in Figure 2. We also examined four PD cases (one Braak stage I and three Braak stage II, three males and one female ranging from 74 to 93 years, mean = 85.5 ± 4.0 years) that were compared to four controls (two Braak stage I and two Braak stage II cases composed of four males ranging from 56 to 85 years, mean = 68.8 ± 8.3 years controls). EAAC1 immunoreactivity again appeared aberrantly elevated in AD hippocampal CA2 pyramidal neurons compared to controls (Figure 5A,C). In contrast to this, EAAC1 immunostaining in the HD cases appeared comparable to controls (Figures 5B,C). EAAC1 immunoreactivity in CA2 hippocampal neurons of PD patients also appeared comparable to controls (Figure 5D,E). Although hippocampal damage associated with PD is considered less severe than AD, consistent with other reports 12, 25, in each of the PD patients, moderate to severe neuritic CA2 hippocampal pathology was evidenced by the accumulation of α‐synuclein‐positive Lewy neurites (Figure 5F) that were absent in the control cases (not shown). These results suggest that aberrant intracellular EAAC1 accumulation is associated with degenerating neurons in AD, but not with features of α‐synucleinopathy in PD hippocampus.

Figure 5.

EAAC1 expression in CA2 hippocampus of Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson's disease (PD). EAAC1 immunoreactivity in CA2 hippocampal neurons of a representative AD case (A) revealed greater aberrant intracellular EAAC1 accumulation compared with CA2 hippocampus in HD (B) and control (C). EAAC1 immunoreactivity in CA2 hippocampal pyramidal neurons of a representative PD case (D) appeared comparable to neurons in the control CA2 (E). Representative PD Lewy neurite patholology in the CA2 region of the hippocampus (evident by brown 3, 3'‐diaminobenzidine labeled α‐synuclein immunoreactivity) (F) was absent in controls (not shown). In panel F only, neuron cell bodies were counterstained with hemotoxylin. Scale bars: 20 µm.

Detergent‐Insoluble EAAC1 accumulates in AD hippocampus

To further examine EAAC1 expression in AD, Western blots were performed on hippocampal protein Triton X‐100‐soluble extracts obtained from a cohort of 23 AD (1 and 22 Braak stages V and VI, respectively) and 9 controls (3, 3, 1 and 2 Braak stages 0 and II, III and IV, respectively) that were comparable on the basis of age (AD ranging from 58 to 92 years of age, mean = 76.3 ± 1.8 years, SEM; controls ranging from 41 to 95 years of age, mean = 70.1 ± 7.6 years, SEM) and gender (AD: 12 males and 11 females, controls: 6 males and 3 females).

As with other glutamate transporters, EAAC1 monomers form functional non‐covalently associated homotrimers (71) that can resolve in SDS‐PAGE as monomers and multimers (21). Consistent with previous studies in humans 5, 51, under our experimental conditions, EAAC1 in hippocampal Triton X‐100‐soluble protein extracts resolved as a monomer of approximately 69 kDa (Figure 6A). Quantification of hippocampal EAAC1 levels in Western blots from 23 AD and 9 controls (Figure 6B) revealed that soluble EAAC1 levels in the AD patients were significantly lower than controls (P < 0.01).

Figure 6.

Detergent‐insoluble EAAC1 increased in Alzheimer's disease (AD) hippocampus. A. Western blot of Triton X‐100‐soluble EAAC1 levels in dissected hippocampal tissue shows EAAC1 resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis as an approximately 69‐kDa EAAC1 monomer. B. Quantification of EAAC1 Western blot immunoreactivity revealed that Triton X‐100‐soluble EAAC1 levels in hippocampal AD tissue (n = 23) were statistically significantly lower (P < 0.01) than controls (n = 9). C. Hippocampal tissue was multiply extracted with Triton X‐100 to remove detergent‐soluble proteins in AD (n = 23), Parkinson's disease (PD) (n = 4) and controls (n = 9). Remaining Triton X‐100‐insoluble protein was solublized in formic acid and then analyzed by enzyme‐linked immunosorbence assay to determine detergent‐insoluble EAAC1 levels, which were statistically significantly higher in AD cases compared with PD (P < 0.05) and controls (P < 0.05). Triton X‐100‐insoluble EAAC1 levels in PD hippocampus were moderately lower, but were not significantly different than controls. (D) In contrast to detergent‐insoluble EAAC1, there was a statistically nonsignificant difference in detergent‐insoluble Presenilin‐1 (PS1) levels in the same group of hippocampal tissue samples [AD (n = 23), PD (n = 4) and controls (n = 9)].

This finding raised the question why detergent‐soluble EAAC1 levels were reduced in AD hippocampus while EAAC1 immunohistochemistry suggested that EAAC1 levels were elevated. One possibility is that because Western blots were performed on total hippocampal tissue, the increased EAAC1 levels in CA2‐CA3 may have been diluted by inclusion of other hippocampal regions in the protein lysates where distinctions between AD and control EAAC1 immunostaining were not evident and where AD‐related neuron loss is prominent (eg, CA1). However, another possibility is that the aberrant EAAC1 expression pattern reflected an accumulation of EAAC1 in a detergent‐insoluble or aggregated state. Under such circumstances, the EAAC1 may have been excluded from detergent‐soluble lysates or may have failed to enter and resolve in the gels. To address this possibility, the same hippocampal tissue analyzed above (Figure 6B) was multiply extracted with Triton X‐100 to remove detergent‐soluble proteins. The remaining pelleted detergent‐insoluble proteins were subsequently solublized in formic acid (FA) and then analyzed by ELISA to determine the levels of Triton X‐100‐insoluble but FA‐soluble EAAC1. We and others have used this method previously to obtain abnormal tau, Aβ and other proteins from AD cerebrum 17, 18, 29, 44, 54, 58, 68. Detergent‐insoluble hippocampal EAAC1 levels were also measured in the same panel of four PD patients examined above (Figure 5). HD patients were not tested because frozen hippocampal tissue was not available.

The results in Figure 6C show that Triton X‐100‐insoluble EAAC1 levels in the hippocampus were significantly greater in AD patients compared with controls and PD patients [ANOVA: F 2, 33 = 5.275, P < 0.01; Newman–Kuels test for multiple comparisons: AD vs. PD, P < 0.05, AD vs. controls, P < 0.05 and PD vs. Controls, nonsignificant]. The lack of co‐localization between EAAC1 and amyloid plaques (Figure 4) indicates that the increased levels of detergent‐insoluble EAAC1 in AD hippocampus were not caused by potentially nonspecific associations between insoluble Aβ and EAAC1 occurring in vivo. In addition, the increased levels of EAAC1 in the Triton X‐100‐insoluble protein fractions could not be explained by nonspecific interactions between EAAC1 and detergent‐insoluble Aβ or tau during the extraction procedure because the correlations between levels of detergent‐insoluble EAAC1 vs. insoluble Aβ and insoluble EAAC1 vs. insoluble tau in AD hippocampus were nonsignificant (P < 0.22 and P < 0.12, respectively). Also, as detergent‐soluble EAAC1 levels in AD hippocampus were significantly lower than controls, it is quite unlikely that the observed increase in insoluble EAAC1 could represent nonspecific contamination or incomplete extraction of the soluble protein fraction. As a further control for the potentially nonspecific effects of the extraction procedure, we also examined the levels of detergent‐insoluble PS1 in the same AD, PD and control hippocampal samples (Figure 6D). Like EAAC1, PS1 is a protein with multiple hydrophobic transmembrane domains that are, in principle, capable of forming nonspecific protein–protein complexes upon detergent solubilization. Nonetheless, we found a nonsignificant difference in the levels of detergent‐insoluble PS1 among AD, PD and control groups [ANOVA: F 2, 33 = 1.463, nonsignificant]. Because the overall ANOVA was nonsignificant, no further post hoc analyses were appropriate. Taken together, these data strongly suggest that AD pathogenesis and the development of aberrant EAAC1‐positive intraneuronal structures in the hippocampus is specifically associated with increased detergent‐insoluble EAAC1 in AD.

DISCUSSION

Glutamate transporters govern glutamate exposure in the CNS

The crucial role glutamate transporters play in buffering extracellular glutamate is underscored by the dramatic consequences of injecting potent glutamate transport inhibitors into mice, which die within minutes (59). Despite the importance of glutamate transporters in preventing excitotoxic cell death, their subtler role in regulating the spread and exposure duration of even normal glutamate levels has a significant impact on the long‐term health and functionality of the CNS. Glutamate is rapidly cleared from peri‐synaptic and extra‐synaptic spaces in roughly 1–2 ms (15) and is maintained at levels recently shown to be in the range of 25 nM (23). Even small changes in the time it takes to restore basal extracellular glutamate levels can have a significant impact on how far glutamate spreads from the site of release. By limiting its spread from one synaptic domain to another (often referred to as spillover), glutamate transporters have been shown to modulate the fidelity of spatial and temporal information processing 14, 48, 65. Glutamate transporters have also been shown to regulate long‐term potentiation and contextual fear conditioning (33).

The significance of EAAC1 dysfunction

A growing body of data suggests that AD is associated with disturbed glutamate transporter function 8, 13, 19, 20, 26, 28, 31, 34, 38, 39, 40. Most of this work has focused on GLT‐1 and GLAST with comparatively little attention paid to EAAC1, in part because EAAC1 appears to be expressed at lower levels than either GLT‐1 or GLAST (21). From this point of view, EAAC1 has been considered to play a less significant role in protecting neurons from excitotoxicity. This perspective has been reinforced by the phenotypes of GLT‐1, GLAST and EAAC1 knockout mice. While GLT‐1 deficiency is lethal (63) and GLAST deficiency produces mice with motor disturbances and increased susceptibility to neurotoxic brain damage (66), initial studies of EAAC1 knockout mice showed comparatively mild phenotypes that did not involve neurological or cognitive abnormalities (46). However, more recent studies of EAAC1 have refined the more limited view of its role in regulating neuronal activity and viability. Recent data now show that EAAC1 deficiency causes age‐related neuronal loss, increased oxidative stress and profound impairment in performing the Morris water maze task (2). In addition to transporting glutamate, EAAC1 also transports cysteine. In mature neurons, EAAC1‐mediated cysteine uptake is a rate‐limiting factor in the synthesis of the major antioxidant molecule, glutathione 11, 22. It is also now appreciated that EAAC1 can modulate GABA synthesis 41, 57.

Such data support the idea that EAAC1 dysfunction could give rise to a compounding array of damaging neuropathological consequences that could include decreased GABA synthesis, increased susceptibility to oxidative neuronal injury, reduced glutamate clearance rates and eventual neuron loss. This report presents evidence that intracellular EAAC1 aberrantly accumulates in pyramidal neurons of the AD hippocampus. Those neurons displaying aberrant elevated EAAC1 immunoreactivity also tended to exhibit increased levels of tau immunoreactivity and GVD pathology, thus strongly suggesting that intracellular EAAC1 accumulation is a process associated with AD‐related neurodegeneration in the hippocampus.

In order for EAAC1 to function, it must be transported from regulated intracellular pools to the cell surface. Thus, the EAAC1 accumulating in degenerating AD neurons is not properly localized to transport glutamate or cysteine. With the current data, one cannot determine whether this expression pattern arises from damage to EAAC1 that is caused by independent pathological processes (discussed below) or whether it reveals an apparent mislocalization of EAAC1 that could facilitate the ongoing neuronal dysfunction associated with AD pathogenesis. Interestingly, EAAC1 expression at the cell surface is regulated by calcium signaling (70), and calcium signals evoked by activating N‐methyl‐D‐aspartic acid (NMDA) receptors induce EAAC1 internalization (67). Thus, it is reasonable to wonder if excessive NMDA receptor activation in AD could drive aberrant EAAC1 internalization. The consequences of this would be diminishing EAAC1 activity that could further undermine the ability of neurons to regulate excitability and/or protect against oxidative stress. Much further work will be required to address this possibility. However, the efficacy of the uncompetitive NMDA receptor antagonist, memantine, in treating AD hints at the plausibility of such an idea (36).

Detergent‐insoluble EAAC1 is a novel biochemical lesion in AD

A number of neurodegenerative disorders are associated with intracellular accumulation of specific detergent‐insoluble proteins including prion proteins in Creutzfeld–Jakob disease, α‐synuclein containing Lewy bodies in PD and Huntingtin in HD (42). Intracellular neurofibrillary tangles composed of aberrantly phosphorylated tau, along with extracellular amyloid plaques, are defining neuropathological lesions in AD. In addition to tau and Aβ, proteomic surveys indicate that a subset of proteins expressed in the brain exhibits altered detergent insolubility in AD (68). We have found that detergent‐insoluble EAAC1 is significantly increased in the hippocampus of AD patients, but not PD patients, even though the PD hippocampus exhibited α‐synuclein pathology. Our immunohistochemical findings are consistent with the idea that this insoluble EAAC1 is most likely localized in neuron cell bodies and proximal neuritic processes. Thus, these data constitute the first evidence that aberrant detergent‐insoluble EAAC1 accumulation is specifically associated with AD in the hippocampus. Moreover, the lack of aberrant EAAC1 immunoreactivity in the AD cortex argues that this pattern of EAAC1 expression is specific to the hippocampus and is not simply a sign of generalized neurodegeneration.

In contrast to insoluble EAAC1, we found that detergent‐soluble EAAC1 levels declined significantly in AD hippocampus. This could be caused by a nonspecific overall loss of neurons. It may be significant that neurons with aberrant EAAC1 expression patterns were more readily observed in CA2 because this region of the hippocampus is relatively spared in AD.

It is conceivable that this aberrant pattern of EAAC1 expression reflects an attempted compensatory response by neurons reacting to ongoing neurological insults. However, this seems unlikely as neurons normally express more EAAC1 than they use at the cell surface. Unlike GLT‐1 and GLAST, which are localized primarily on the cell surface, EAAC1 is prominently localized in regulated intracellular pools from which it is trafficked on and off the cell surface in response to the dynamic needs of the cell (49). Alternatively, it is also possible that EAAC1 accumulates in AD neurons because it is in a biochemically compromised state and cannot traffic or function properly. For example, the uptake activity of EAAC1 is very sensitive to oxidative modification. In addition, oxidatively modified EAAC1 readily forms higher‐order detergent‐insensitive oligomers 21, 64. There is evidence that oxidative damage begins to accumulate early in AD pathogenesis 37, 62. It is plausible that a variety of pathologic processes, including oxidative stress, could inhibit EAAC1 activity or its ability to localize properly by facilitating oxidative modifications that induce EAAC1 to form large detergent‐insoluble oligomers. Under such circumstances, EAAC1 failure could augment the progressive cycle of increasing neuronal dysfunction that characterizes AD.

In summary, detergent‐insoluble intracellular EAAC1 accumulates specifically in the hippocampus of AD patients. This may be significant because EAAC1 plays multiple important roles in controlling neuronal activity and viability. The findings in this report cannot address whether EAAC1 dysfunction has a direct role in contributing to the complex manifestations of AD pathogenesis. However, considering that there is strong evidence EAAC1 loss of function, itself can cause progressive neurodegeneration (2), these findings provide insights into a novel potentially pathological pathway in AD that requires further attention and may ultimately suggest new approaches designed to forestall the disease process.