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Deficient glutamate tranport is associated with neurodegeneration in Alzheimer's disease.

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Deficient Glutamate Transport Is Associated
E. Masliah, MD,*t M. Alford, BA,* R. DeTeresa, BS,* M. Mallory, BS,* and L. Hansen, MD*t
The mechanisms of synapse damage in Alzheimer’s disease (AD) are not fully understood. Deficient functioning of
glutamate transporters might be involved in synaptic pathology and neurodegeneration by failing to clear excess glutabinding is decreased;
mate at the synaptic cleft. In AD, glutamate transporter activity as assessed by ~-[~H]aspartate
however, it is not clear to what extent it is associated with the neurodegenerative process and cognitive alterations. For
this purpose, levels of D- and L-[3H]aspartate binding in midfrontal cortex were correlated with synaptophysin levels,
brain spectrin degradation product levels, and clinical and neuropathological indicators of AD. Compared to control
t a t e a 30% decrease in ~-[~H]aspartate
brains, AD brains displayed a 34% decrease in levels of ~ - [ ~ H ] a ~ p a ~binding,
binding, and a 48% loss of synaptophysin immunoreactivity. Increased levels of brain spectrin degradation products
binding, and decreased levels of synaptophysin immunocorrelated with a decrease in levels of D-[~H]and ~-[~H]aspartate
reactivity. Levels of L- [3H]aspartate binding correlated with levels of synaptophysin immunoreactivity. These results
suggest that decreased glutamate transporter activity in AD is associated with increased excitotoxicity and neurodegeneration, supporting the possibility that abnormal functioning of this system might be involved in the pathogenesis of
synaptic damage in AD.
Masliah E, Alford M, DeTeresa R, Mallory M, Hansen L. Deficient glutamate transport is associated with
neurodegeneration in Alzheimer’s disease. Ann Neurol 1996;40:759-766
The cognitive alterations in Alzheimer’s disease (AD)
are associated with widespread neurodegeneration
throughout the association cortex and limbic system
[ 1-71. This neurodegenerative process is characterized
by synaptic and neuronal loss [8-121, plaque and tangle formation [13, 141, concomitant decrease in specific
neurotransmitters [I 5-17], and gliosis. Synaptic loss is
significantly correlated with cognitive performance [8,
10, 181 and occurs early in the development of AD
[3, 1I], suggesting that synapse pathology is a primary
rather than a secondary event. The mechanisms of synaptic damage and neurodegeneration in AD are not
completely understood. Recent studies suggested that
abnormal expression or processing of growth-associated
proteins in the central nervous system (CNS) may play
a role in the process leading to synaptic damage and
neurodegeneration in AD [2, 19, 201. Prominent
among these is amyloid precursor protein (APP), a
molecule centrally involved in AD pathogenesis [21,
221, because mutations within the gene that encodes
for this molecule are associated with familial AD [23261 and overexpression of mutated APP in transgenic
mice results in AD-like pathology [27].Recent studies
showed that APP is found primarily in neurons [28301 with a preferential localization at central and pe-
ripheral synaptic sites [31-33], suggesting a possible
role in neuroplasticity [34, 351. Furthermore, studies
also showed that secreted APP (sAPP) fulfills synaptotrophic [35-371 and neuroprotective functions within
the CNS in response to excitotoxicity [38, 391 and
ischemia [39-4 11. Therefore, abnormal functioning of
sAPP may be involved in the mechanisms of synaptic
damage by failing to promote or maintain normal
synaptic populations after excitotoxic challenge [2, 1 1,
From the Departments of *Neurosciences and t Pathology, University of California San Diego, La Jolla, CA.
Address correspondence to Dr Masliah, Department of Neurosciences, University of California San Diego, La Jolla, CA 92093OG2*.
Received Jan 12, 1996, and in revised form Mar 18 and May 15.
Accepted for publication May 15, 1996.
38, 391.
It has been postulated that sAPP might prevent the
toxicity associated with stimulation of glutamate receptors by stabilizing intracellular CaLt levels [39, 42, 431.
Furthermore, neuroprotection might also be achieved
by modulating the levels of glutamate receptors or activity of glutamate-aspartate uptake systems. With respect to the latter, studies showed that potentially neurotoxic neurotransmitters like glutamate are cleared
from the synaptic cleft by high-affinity, Na’-dependent uptake carriers located in both neurons and glia
[44-471. Recently, at least four high-affinity Nafdependent uptake carriers of aspartatdglutamate (also
known as glutamate transporters) were cloned: GLT- 1
(or EAAT2) [48],EAAC1 (or EAAT3) [47],GLAST
[50], (or EAAT1) and the cerebellar transporter (or
Copyright 0 1996 by the American Neurological Association 759
EAAT4) [51]. 'These transporters exhibit 39 to 55%
sequence homology with each other [51]. GLT-1 is
specifically located in astrocytes, EAAC1 has a neuronal
localization that includes nonglutaminergic neurons,
and G U S T is largely localized to astroglia [52]. Thus,
deficient glutamate transporter activity might result in
neurodegeneration by accumulation of excitotoxins at
the synaptic site. Supporting a role for decreased glutamate transporter activity in neurodegeneration, recent
studies showed that (1) in AD the high-affinity
glutamate/aspartate uptake system is 40 to 50% decreased in the neocortex [15, 53, 541, (2) in amyotrophic lateral sclerosis (ALS) there is selective loss of the
glial glutamate transporter (type GLT-1) [ 5 5 ] , and (3)
chronic inhibition of glutamate transporter activity
with ~(-)-threo-3-hydroxy aspartate (THA) in experimental models results in degeneration [56].
However, it is not clear to what extent the deficient
glutamate transporter activity is associated with the
neurodegenerative process and the cognitive alterations
observed in AD. Previous studies used u-['Hlaspartate
binding to assess glutamate transporter activity (for review, see [47]).However, more recent studies suggested
that D- [ 'Hlaspartate binding reflects glial cells, rather
than neuronal activity, and that L-[ 'Hlaspartate binding should be further investigated as a possible marker
for the neuronal transporter (for review, see [47]).For
this purpose, levels of D- and ~-[~H]aspartate
were correlated with synaptophysin (SYN) (general
presynaptic terminal marker), brain spectrin degradation products (marker of calpain I activation and excitotoxicity), and neuropathological (plaques and tangles) and clinical (Blessed score) indicators of AD.
Materials and Methods
ously described [62]. From each brain, frozen tissue blocks
from the MF cortex were homogenized and separated into
cytosoiic and particulate fractions, as previously described
[62, 631. T o ensure equal loading, the protein content of all
samples was determined by Lowry assay [64] and adjusted
with homogenization buffer to 1.25 mg/ml. Forty rnicrograms of cytosolic fraction was loaded per lane onto 6%
sodium dodecyl sulfate-polyacrylamide gels, electrophoresed,
and blotted onto nitrocellulose paper. Blots were then incubated with blocking solution (phosphate-buffered saline
[PBS]/0.1% Tween 20) followed by the anti-brain spectrin
rabbit polyclonal antibody (Chemicon International, Temecula CA) and radioiodinated protein A (30 pCi/pg) (ICN,
Irvine, CA). Autoradiographic imaging of the blots was done
with storage phosphor screens and scanning was done with a
PhosphorImager SF (Molecular Dynamics, Sunnyvale, CA).
Bands were quantified by integrating pixel intensities over
defined volumes using the ImageQuant software.
SYN is an abundant small synaptic vesicle protein [65,
661, the levels of which have been used as an indicator of
synaptic content in the nervous system [63, 67, 681. Levels
of SYN immunoreactivity (SYN-IR) were determined by dot
blot, as previously described [63]. Briefly, for each assay, five
serial dilutions of a normalization standard were run at three
protein concentrations ( 2 , 1, and 0.5 pg) and spotted to
nitrocellulose membrane with a dot blotting manifold
(Schleicher & Shuell, Keene, NH). The blot was blocked in
0.1% Tween 20 in PBS, pH 7.4, for 2 hours at 4°C and
incubated overnight with the mouse monoclonal antibody
against SYN (0.1 pg/ml) (Boehringer Mannheim, Indianapolis, IN), followed by rabbit anti-mouse IgG and "'I-labeled
protein A (0.1 pCi/ml). Autoradiographic images (from storage phosphor screens) of the blots were analyzed with the
PhosphorImager and determinations of the average SYN-IR
were done with the ImageQuant software and expressed as
arbitrary units per 1 pg of the protein [63].All experiments
were repeated at least once to ensure the reproducibility of
the results.
Subjects aizd Neuropathological Examination
A total of 23 autopsy cases from the Alzheimer Disease
Research Center at the University of California San Diego
were included for the present analysis. All patients were extensively assessed before death with a comprehensive set of
neurological and psychometric tests including the Blessed
score. For 16, the diagnosis of AD was confirmed by clinical
and neuropathological analysis. The other 7 were agematched control subjects who were clinically and histopathologically free of neurological disease. Quantitative assessment
of neuritic plaques and tangles in the midfrontal (MF), inferoparietal (IP), and superotemporal (ST) cortex and hippocampus was performed with thioflavine S-stained, paraffinembedded sections [ 121.
lmmunochemical Studies
Brain spectrin is a cytoskeletal protein that contributes to
neuronal integrity [57].Calpain I-mediated proteolysis of
spectrin [58, 591 has been proposed as an indicator of the
Ca2+-activated, excitatory amino acid-induced neuronal
death [60, 611. T o estimate levels of brain spectrin degradation products, Western blot analysis was performed as previ-
760 Annals of Neurology
Vol 40
No 5
November 1996
and L-(-'H]Aspartate Binding Assay
T o estimate glutamate transporter activity, for subsequent
correlative studies with SYN,brain spectrin, and other clinical and neuropathological alterations observed in AD, 11-and
~ - [ ~ H ] a s p a r t a binding
assays were performed on washed
membrane preparations by a modified method of Cross and
colleagues [69]. The 13- and ~-['H]aspartate binding assays
were performed on frozen samples (approximately 100 mg
wet weight) from the M F cortex of control and AD brains
that were sonicated in 1 nil of 50 mM Tris-hydrochloric
acid (HC1)/300 mM sodium chloride (NaCI) buffer, p H
7.4. Nine additimal milliliters of buffer was added followed
by centrifugation a t 21,780g for 30 minutes at 4°C. The
supernatant was decanted and the pellet resuspended in 10
ml of buffer. Total protein was determined by the method
of Lowry and colleagues [64].Samples were diluted to 0.4
mg/ml of total protein. One-milliliter incubation volumes
in triplicate tubes containing 40 pg (100 pl) of washed membranes and a final concentration of 50 nM D- or ~-['H]asparrate (Dupont N E N Research Products, Boston, MA) and
4,000 nM unlabeled (cold) aspartare were incubated at room
Cliiiicopat/~ologirtlChrzrncteristics of the Autopsy Cizses
Age (yr)
72 % 4
Delay (hr)
Blessed Score
Blessed Score
Neuritic Plaques (MF)
( X 1.6 mni')
Tangles (MF)
( X 0.16 nini')
5 f2
O f 0
22 f 2
21 2 4
2.2 ? 0.8
6 5 2
0 2 0
MF = midfrontnl cortex
temperature for 30 minutes. This incubation time was selected based on previous studies that showed optimal results
at this time point [53, 701. However, since ~-['H]aspartate
could be rapidly metabolized, additional binding experiments
were performed by incubating the membrane preparations
for 2 to 4 minutes and comparing with the results at 30
minutes. Nonspecific binding was determined by adding 10fold excess (40,000 nM) cold aspartate to otherwise identical
incubation tubes. Membranes and unbound ligand were separated by rapid filtration through 0.45-p glass-fiber filters on
a disposable filtration manifold (V&P Scientific, San Diego,
CA), followed by 2 X 200-pl washes of 50 tnM Tris HCI/
300 m M NaCl buffer. Filter disks were counted in 7-mI vials
containing 5 ml of EcoLume scintillation cocktail (ICN) on
a TM Analytic 688 1 Mark I11 scintillation specrrophotometer. Results were expressed as piconioles bound per milligram
of total protein.
To further confirm the specificity of D- and ~-['H]asparrate for the glutamate transport under the conditions used
for our assay, displacement curves with unlabeled 1.-gluramate and T H A were performed. Previous studies showed
that 'I'HA [56] specifically inhibits glutamate uptake and induces neurodegeneration in experimental animal models.
Briefly, as previously described by Scott and coauthors 1531,
1-nil incubation volwnes in triplicate tubes containing 20
pg of synaptosonial membranes (by Lowry assay), 20 nM I)['Hlaspartate, and displacers ranging from 1 n M to 30 pM
were incubated 30 minutes at room temperature. Nonspecific binding was defined as binding in the presence of 0.1
m M cold r)-aspartate added to otherwise identical incubation
tubes. Unbound ligand was separated by rapid filtration
through 0.45-p filter disks on disposable microfold filtration
manifolds. Filter disks were washed with 2 X 200 p1 of
buffer and counted in a scintillation spectrophotonieter.
Data were processed by the EBDA and LIGAND programs
to produce a fitted curve 171, 721.
Statistical Am!ysis
All samples were 'issigned a blind code before the experiments were initiated. The code was broken after all the results were downloaded into the database. Statistical analysis
was performed utilizing the StatView program running in a
Macintosh 6100/66. Differences between control and A D
findings were tested using the unpaired, two-tailed Student's
t test. The relationship between two specific variables was
tested using the Pearson product-moment correlation, the I
value, calculated with simple linear regression analysis. In
order to avoid artificial skewing of the regression line by
control cases, only AD cases were included for this analysis.
All results were expressed as mean ? standard error of mean.
Fig 1. Leuels of 1)- and L-['H]aspartnte rrptnkr and synnptophyjiii iinn?unoreactivity are decreased i n the fiorital c0rte.x
of A D brains ( ) I = IG), when rowipnred to c o n t ~ o brrrins
(n = 7).Conlpniisons u m r dnne using the two-tcriled
iinpaired Student; t test.
T h e characteristics of the 23 autopsy cases are presented in the Table. Compared to control brains, AD
brains displayed a 48% loss in the levels of SYN-IR
(df= 21, t = 5.1, p < 0.001) in the MF cortex (Fig
IA), a 34% decrease in the levels of ~-['H]aspartate
binding (df= 21, t = 2.2, p < 0.05) (Fig IB), and
a 300/0 decrease in the levels of I.-[ 'Hlaspartate binding
(df= 19, t = 2 . 1 , ~< 0.05) (Fig IC). Levels of D- and
L-['Hlaspartate binding were correlated ( r = 0.728, p
= 0.0001, n = 16) (not shown). Levels of ~ - [ ' H ] a s p a r late binding at 2 to 4 minutes (n = 3 controls, 152
5 21 prnol/mg; n = 3 AD cases, 68 2 1 prnol/rng)
were relatively lower compared to results at 30 minutes
(n = 3 controls, 216 -+ 26 prnol/mg; n = 3 AD cases,
120 +_ 17 prnol/mg). However, the values on a caseto-case basis were proportionate and strongly correlated
( Y = 0.95, p < 0.01, n = 6), indicating that the binding had not yet reached an equilibrium at 2 to 4 rnin-
Masliah et al: Glutamate Transport and Neurodegeneration i n AD
utes compared to 30 minutes. Further confirmation of
D-[ 3H]~ partate
specificity to bind glutamate transporters was achieved by displacement of the radioligand
binding with THA and L-glutamate. Consistent with
previous studies [53], THA displaced ~-[~H]aspartate
with a I& of 3.1 X
M and a B,,, of 1.16 X lo-'
M, while L-glutamate displaced D-[ %]aspartate with a
& of 2.5 X lo-' M and B,, of 1.3 X lo-' M (Fig 2).
The antibody against brain spectrin recognized a native band at 240 kd and degradation products at approximately 150 kd (Fig 3A). Consistent with previous
studies [62],brain spectrin degradation products were
significantly increased in the AD MF cortex (see Fig
3A), when compared to control cortex. This increased
level in AD brains was correlated with lower levels of
~-['H]aspartate( r = -0.587, p = 0.01, n = 16) (see
Fig 3B) and ~-[~H]aspartate
binding ( Y = -0.57, p =
0.02, n = 16) (see Fig 3C). The extent of the synaptic
damage, as assessed by levels of SYN-IR, was inversely
correlated with the progressive accumulation of brain
spectrin degradation products (Y = -0.55, p = 0.02,
40 20
20 (
1 OE-09
1 OE-08
1 OE-07
1 OE-05
1 OE-06
Log Displace1 Concentration IM1
* L-glutamate
Fig 2. Displacement of ~-pH]aspartatewith ~(-)-threo-3hydroq aspartate (THA) and r2-glutamate. THA displaced D['Hlapartate with a Kd of 3.I X IO-' A4 and a B,, of
1.16 X lo-' M, while L-glutamate displaced ~ [ ' H J a s p a r M and B,, of 1.3 X
tate with a & of 2.5 X
M, conjrming the spec@city of the radioligand f i r the glutamate transporter.
1 :
r=-0.587,p0.016, nS16
r--0.570, p.0.021. 11-16
I a0
1 00
D-[gHJ.aspartate binding (pmollmg)
L-[3H]-asparlate binding (pmollmg)
~ 0 . 5 5p=0.02,
r- 3.488. p-0.045, n-16
1 2 3 4
Synaptophysin immunoreactivity
L-[3H]-aspartate binding
Fig 3. Correlations between markers of neurodegeneration and aspartate uptake in the fiontal cortex. (A) We.stern blot analysis
showed increased levels of brain spectrin degradation products i B A D brains (lanes I -3), when compared to control brains (lanes
4, 5). (8 C) Levels ofbrain spectrin degradation were inversely correlated with D- and i.-(-'H]aspartate binding in AD brains.
(D, E) Levels of synaptophysin immunoreactiviq were inversely correlated with levels of brain spectrin degradation products and
directly correlated with [.-rH]aspartate binding in AD.
762 Annals of Neurology
Vol 40
No 5
November 1996
Synaptophysin immunoreactivity
L-[3H]-aspartate uptake
500 7
r=-0.495, p=0.04, n = l 6
p=O.OOl, n=l6
Blessed score
Blessed score
Fig 4. Covrelations between behavioral and synaptic alterations. The Blessed score, as an indicator of global cognition, was
inversely correlated with synaptophysin immunoreactivity (A) and with levels of L-i3H]aspartate uptake in the fiontal cortex
AD brains (B).
n = 16) (see Fig 3D) and with the severity of cognitive
alterations as determined by the Blessed score ( r =
-0.742, p = 0.001, n = 16) (Fig 4A). The levels
of L-[ 'HI aspartate uptake were directly correlated with
SYN-IR ( r = 0.488, p = 0.04, n = 16) (see Fig 3E)
and inversely correlated with the Blessed score ( r =
-0.495, p = 0.04, n = 16) (see Fig 4 B ) . However,
D-[ 'Hlaspartate uptake only displayed an inverse trend
toward a correlation with the levels of SYN-IR ( r =
0.391, p = 0.13, n = 16), and with the Blessed score
( r = -0.274, p = 0.3, n = 16) (not shown). As to
the neuropathological indicators of AD, neither the
numbers of neuritic plaques ( r = -0.198, p = 0.4, n
= 16) nor neurofibrillary tangles ( r = -0.01, p =
0.96, n = 16) were significantly correlated with the
levels of D- and ~-[jH]aspartatebinding or brain spectrin degradation products (not shown).
Previous studies showed that glial and neuronal transporters are involved in the uptake of aspartate and glutamate [50, 731 and that their abnormal functioning
might result in neurodegeneration by accumulation of
excitotoxins at the synaptic cleft [56]. While in ALS
the glutamate transporter affected is glial [ 5 5 ] , in AD,
the high-affinity Na+-dependent glutamate transporter
affected might be neuronal. Supporting this possibility,
the present study showed that ~-[~H]aspartate
correlated with SYN-IR. This is consistent with recent
studies suggesting that Nat-dependent binding of
~-['H]aspartate rather than ~ - [ ~ H ] a s p a r t a t should
be further investigated as a possible marker for
glutaminergic/aspartergic synapses [47]. The present
study showed that ~-[~H]aspartate
uptake correlated
not only with levels of SYN-IR in the frontal cortex,
but also with the Blessed score, indicating that damage
to glutaminergic terminals plays an important role in
cognitive dysfunction in AD [15, 171. As to the identity of the glutamate transporter responsible for the decrease n- and ~-[~H]aspartate
uptake, future studies
with antibodies that recognize specific glutamate transporters will be necessary to help clarify this issue.
Consistent with previous studies [53, 54, 69, 74,
751, the present study showed a 40% decrease in D['Hlaspartate binding in AD. Furthermore, r.-['H]
aspartate binding was significantly decreased in AD.
Decreased glutamate transporter activity in AD could
result from a reduction in the number of glutamatel
aspartate carriers (BmZeffect) or decreased affinity of
the transporter (& effect), or both. Previous studies
using synaptic membranes and displacement curves
with glutamate and T H A found that a decrease in D['Hlaspartate binding site density might be the most
likely cause for the deficient glutamate transporter activity [53]. These studies [53, 541 showed that differences in B,,,, values were present both in brain regions
displaying AD pathology as well as in the spared areas
of the AD brain. However, additional studies with
['Hlglutamate binding to synaptosomal preparations
will be necessary to further characterize the alterations
in transporter site density in AD.
The deficit in glutamate transporter activity in AD
Masliah et al: Glutamate Transport and Neurodegeneration in AD
could be secondary to the overall loss of synaptic terrninals and neurons or related to abnormal APP metabolism and p-amyloid deposition. T h e present study
showed that progressive accumulation of brain spectrin
degradation products in AD was inversely correlated
with decreased u- and L-[ ’Hlaspartate binding and decreased levels of SYN-IR, but not with the amyloid
load. These results suggest that decreased activity of
glutamate transporters in A11 might be associated with
increased excitotoxicity and neurodegeneration, since
proteolysis of brain spectrin has been proposed as an
indicator of Ca’+-activated, excitatory amino acidinduced neuronal death [60, 611. Delayed glutamate
receptor-mediated injury is mainly the result of abnormally high influx of Ca” into the neurons [76].
Ca2+-activated pathways are rhought to mediate the
toxic effects of elevated intracellular Ca’+, including
Ca’+-activated lipases, oxygen radicals, generation of
nitric oxide, and activation of proteases [61]. In regard
to this latter mechanism, it has been shown that in
fact, calpain inhibition can block the Ca’+-dependent
neuronal death induced by excitotoxins [61]. Then,
Ca”-mediated excitotoxicity may be rriggered by disruption in protease functioning, abnormal functioning
of molecules that regulate Ca” homeostasis, increased
glutamate receptor sensitivity, and/or decreased functioning of glutamate transporters. In AD, deficient
functioning of the glutamate transporters could lead to
increased glutamate levels at the synaptic cleft, which
in turn might trigger excessive stimulation of glutamate
receptors and increased intracellular Ca’+ levels, which
eventually could lead to synaptic damage and neuronal
death. Since APP is centrally involved in the pathogenesis of AD [22], in Ca” homeostasis [39], and in neuroprotection against excitotoxicity [38], it might then
be possible that abnormal processing of APP might be
associated with the deficient functioning of the glutamate transporter system in AD.
This work was supported by National lnsritutes of Healrh/Narional
Insrirure on Aging grants AGIO689 and AGO5131 and by a grant
from the Alzheimer’s Association.
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