Calbindin 28k blocks mutant presenilin 1 apoptosis, reduces ROS and preserves mitochondrial function
Tim
Neurobiology
Calbindin D28k blocks the proapoptotic actions of mutant presenilin 1:
Reduced oxidative stress and preserved mitochondrial function
(Alzheimer's disease / amyloid / calcium binding protein / endoplasmic
reticulum / mitochondrial transmembrane potential)
Qing Guo*, Sylvia Christakos, Nic Robinson*, and Mark P. Mattson*,
* Sanders-Brown Research Center on Aging and Department of Anatomy and
Neurobiology, University of Kentucky, Lexington, KY 40536; and
Department of Biochemistry and Molecular Biology, University of
Medicine and Dentistry of New Jersey-New Jersey Medical School,
Newark, NJ 07103
Edited by Solomon H. Snyder, Johns Hopkins University School of
Medicine, Baltimore, MD, and approved January 6, 1998 (received for
review August 15, 1997)
ABSTRACT
Top
Abstract
Introduction
Materials
Results
Discussion
References
Mutations in the presenilin 1 (PS-1) gene account for many cases of
early-onset autosomal dominant inherited forms of Alzheimer's disease.
Recent findings suggest that PS-1 mutations may sensitize neurons to
apoptosis induced by trophic factor withdrawal and exposure to amyloid
-peptide (A). We now report that overexpression of the calcium-binding
protein calbindin D28k prevents apoptosis in cultured neural cells
expressing mutant PS-1 (L286V and M146V missense mutations).
Elevations of the intracellular Ca2+ concentration and generation of
reactive oxygen species induced by A, and potentiated by mutant PS-1,
were suppressed in calbindin-overexpressing cells. Impairment of
mitochondrial function by A (which preceded apoptosis) was exacerbated
by PS-1 mutations and was largely prevented by calbindin. These
findings suggest that PS-1 mutations render neurons vulnerable to
apoptosis by a mechanism involving destabilization of cellular calcium
homeostasis, which leads to oxidative stress and mitochondrial
dysfunction.
INTRODUCTION
Top
Abstract
Introduction
Materials
Results
Discussion
References
Degeneration and death of neurons in brain regions involved in
learning and memory processes underlie Alzheimer's disease (AD), a
fatal disorder that affects millions of persons worldwide. Although
the cause(s) of the vast majority of cases of AD are unknown, a small
percentage of cases are inherited in an autosomal dominant manner.
Missense mutations in three different genes, -amyloid precursor
protein (APP; chromosome 21), presenilin 1 (PS-1; chromosome 14), and
PS-2 (chromosome 1) have been causally linked to early-onset familial
forms of AD (1). APP is a transmembrane protein that is the source of
the 40- to 42-aa amyloid -peptide (A) that forms fibrillar aggregates
(plaques) associated with degenerating neurons in the brain in AD.
Several lines of evidence suggest a major role for A in the
neurodegenerative process in AD as follows: APP mutations result in
increased production of cytotoxic forms of A (2); transgenic mice
expressing mutated human APP exhibit A deposition and cognitive
impairments (3, 4); and A damages and kills cultured neurons by a
mechanism involving oxidative stress and disruption of cellular
calcium homeostasis (5-10). PS-1 and PS-2 encode integral membrane
proteins with six or eight membrane-spanning domains (11-13) and are
localized in the endoplasmic reticulum (ER; ref. 14). PSs are
expressed in neurons throughout the brain (15-17) and appear to be
present in both degenerating and nondegenerating neurons in AD brain
(18, 19). Mutations in PS-1 account for approximately 50% of all cases
of familial AD (20); understanding how PS-1 mutations promote neuron
degeneration is, therefore, a critical issue in AD research.
Increased oxidative stress and disruption of neuronal calcium
homeostasis appear to be interrelated final common pathways that
mediate the neurodegenerative process in AD (for review, see refs. 21
and 22). There is increased protein, lipid, and DNA oxidation in
association with degenerating neurons in AD (23-27). Exposure of
cultured neurons to A induces membrane lipid peroxidation (6, 7, 28),
which mediates impairment of membrane ion-motive ATPases and glucose
transporters and thereby renders neurons vulnerable to excitotoxicity
and apoptosis (9, 10, 29, 30). Studies of postmortem AD brain revealed
evidence for calcium-mediated proteolysis in degenerating neurons (31)
and suggest an inverse relationship between expression of
calcium-binding proteins in neurons and their vulnerability to death
(32). Cell culture studies have shown that the mechanism of A toxicity
involves excessive accumulation of calcium within neurons (5, 8, 9)
and that calcium influx can elicit cytoskeletal alterations similar to
those seen in neurofibrillary tangles in AD (33). Expression of the
calcium-binding protein calbindin D28k in cultured hippocampal neurons
is correlated with increased resistance to cell death induced by a
variety of insults including exposure to A (34, 35).
Apoptosis is a form of cell death characterized by cell shrinkage,
mitochondrial alterations, and nuclear DNA condensation and
fragmentation (36-38). Neuronal apoptosis is increasingly implicated
in AD based on studies of postmortem brain tissues (39, 40) and cell
culture studies showing that A can induce neuronal apoptosis (30, 41,
42). Recent findings suggest that PS mutations may predispose neurons
to apoptotic death. Overexpression of mutant (and to a lesser extent
wild type) PS-2 increased the vulnerability of pheochromocytoma (PC12)
cells to apoptosis induced by trophic factor withdrawal (43, 44).
Expression of mutant PS-1 in PC12 cells increased their vulnerability
to apoptosis induced by A and trophic factor withdrawal (45, 46). We
now report that overexpression of calbindin in neural cells
counteracts the proapoptotic actions of PS-1 mutations by a mechanism
involving stabilization of intracellular calcium levels, suppression
of oxidative stress, and preservation of mitochondrial function.
MATERIALS AND METHODS
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Abstract
Introduction
Materials
Results
Discussion
References
Generation and Characterization of PC12 Cell Lines. The PS-1 gene was
amplified from a human lymphoblastoid cDNA library by PCR using 30
cycles of Expand high-fidelity PCR (Boehringer Mannheim), and the
product was then cloned into the vector PCR3 (Invitrogen) yielding a
vector designated PCRS182. For in vitro mutatgenesis, the KpnI-XhoI
fragment from PCR182 was subcloned into pAlter-1 (Promega). The L286V
or M146V mutations were generated by using mutagenic oligonucleotides
according to the manufacturer's instructions (Promega). The DNA
sequence of the wild-type and mutated PS-1 genes were verified by
using AmpliTaq to label the DNA with dye terminator before detection
on an ABI373 DNA sequencer (Perkin-Elmer). Full-length human PS-1
cDNAs containing the L286V or M146V mutations were subcloned into
pRcCMV expression vector to generate pCMV-PS1L286V and pCMV-PS1M146V.
The full-length wild-type human PS-1 cDNA was subcloned into pRcCMV
expression vector to generate pCMV-PS1. PC12 cells were transfected by
using Lipofectamine and stable clonal transfectants from each group
were selected with G418 for 4 weeks and screened for the presence of
expression of the transgene by reverse transcription-coupled PCR and
Western blot analysis. Because the L286V mutation creates a new PvuII
restriction site and the M146V mutation destroys a BspHI restriction
site in PS-1, the introduction of the mutation in the transfected
cells was confirmed by reverse transcription-coupled PCR analysis. For
the L286V mutation, the primers used were 5'-GTGGCTGTTTTGTGTCCGAA-3'
and 5'-GCTTCCCATTCCTCACTGAA-3'. For the M146V mutation, the primers
used were 5'-TCACAGAAGATACCGAGACT-3' and 5'-CGTTATAGGTTTTAAACACT-3'
(ref. 45 and data not shown).
PC12 cell lines stably expressing calbindin D28k were established by
transfection with an expression vector containing the rat calbindin
D28k cDNA subcloned into the pREP4 expression vector (Invitrogen) in
which expression is under the control of Rous sarcoma virus long
terminal repeat promoter. To coexpress PS-1, PS-1 L286V, or PS-1 M146V
with calbindin D28k, PC12 cells overexpressing calbindin D286K were
further transfected by using Lipofectamine (GIBCO/BRL) with the
expression construct pCMV-PS1, pCMV-PS1 L286V, or pCMV-PS1M146V, where
the expression of the transgenes were driven by enhancer-promoter
sequences from the immediate-early gene of the human cytomegalovirus.
Control PC12 cells were transfected with pREP4 and pRc/CMV vectors
alone. Stable transfection of PC12 cells with pCMV-PS-1 or pCMV-PS1
L286V construct did not significantly affect the viability of these
cells during G418 selection procedures, and the cells grew well under
basal culture conditions during early passages. Transfected cells were
maintained at 37°C (5% CO2/95% air atmosphere) in RPMI 1640 medium
containing G418 (400 µg/ml, for PS-1- or PS-1 L286V-transfected cells)
or hygromycin (400 µg/ml, for calbindin D28K-transfected cells) or
both in cotransfected cell lines and supplemented 10% with
heat-inactivated horse serum and 5% with heat-inactivated fetal bovine
serum.
Western Blot Analysis. Relative levels of expression of calbindin,
wild-type PS-1, and PS-1L286V in the various cell lines were
characterized by Western blot analysis using methods similar to those
described (45, 47). Briefly, 50 µg of proteins in cell homogenates was
separated by electrophoresis through a SDS/polyacrylamide gel and then
transferred to a nitrocellulose sheet. After blocking with 5% milk and
a 3- to 4-hr incubation in the presence of primary antibody, the
nitrocellulose sheet was further processed by using horseradish
peroxidase-conjugated anti-mouse secondary antibody and a
chemiluminescent system (Amersham). The primary antibodies were rabbit
anti-rat calbindin (1:2,000 dilution; ref. 47), rabbit anti-human PS-1
(1:200 dilution; ref. 46), and mouse mAb against -tubulin (Sigma).
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Fig. 1. Calbindin overexpression protects PC12 cells against the
proapoptotic action of mutant PS-1. (A) Representative Western blots
showing levels of PS-1 and calbindin protein expression in the PC12
cell lines used. Proteins in homogenates from the indicated cell lines
were separated by SDS/PAGE (50 µg of protein per lane), transferred to
a nitrocellulose sheet, and immunoreacted with a polyclonal
anti-calbindin antibody (Top), a polyclonal anti-PS-1 antibody
(Middle), or a monoclonal anti--tubulin antibody (Bottom). UT,
untransfected parent cell line; VA, line transfected with empty
vector; PS-1C1, a line overexpressing wild-type PS-1; L286VC6, a line
overexpressing the PS-1 L286V mutation; M146VC4, a line overexpressing
the PS-1 M146V mutation; CB13, a line overexpressing calbindin;
CB+PS1C7, a line overexpressing both wild-type PS-1 and calbindin;
CB+L286VC1, a line overexpressing PS-1L286V and calbindin; and
CB+M146VC12, a line overexpressing both PS-1M146V and calbindin. (B)
Cultures of the indicated cell lines were exposed for 24 hr to either
vehicle or 50 µM A1-42. Cells were stained with Hoescht 33342, and the
percentage of cells in each culture with apoptotic nuclei (condensed
and fragmented DNA) was determined. Values are the mean ± SD of
determinations made in four cultures (ANOVA with Scheffe's posthoc
tests).
Experimental Treatments and Quantification of Apoptosis. Immediately
before experimental treatment, the medium was replaced with Locke's
solution (154 mM NaCl/5.6 mM KCl/2.3 mM CaCl2/1.0 mM MgCl2/3.6 mM
NaHCO3/5 mM glucose/5 mM Hepes, pH 7.2). Synthetic A1-42 was
synthesized by the University of Kentucky Macromolecular Structure
Facility, and stocks were prepared at a concentration of 1 mM in water
and allowed to incubate for 2 hr at 37°C before addition to cultures;
during this preincubation period, peptide aggregates formed as
described (8, 9, 46). For analysis of apoptosis, cells were stained
with the fluorescent DNA-binding dye Hoescht 33342 as described (30,
46). Cells with apoptotic nuclei (condensed and fragmented DNA) were
counted in four random ×40 fields per culture; counts were made
without knowledge of cell line or treatment history.
Analyses of Intracellular Calcium Levels, Oxidative Stress, and
Mitochondrial Function. Fluorescence ratio imaging of the calcium
indicator dye fura-2 was performed as described (5). Cells were
exposed to A or vehicle for 4 hr, loaded with fura-2, and then imaged
(in the continued presence of A or vehicle). The ratio of the
fluorescence emission at two different excitation wavelengths (334 nm
and 380 nm) was used to determine the intracellular Ca2+ concentration
([Ca2+]i) as described (48). Levels of cellular oxidative stress were
measured by using the fluorescent probe 2,7-dichlorofluorescin
diacetate (DCF; Molecular Probes) as described (7). Briefly, cells
were incubated for 50 min in the presence of 50 µM DCF, followed by
washing in Hanks' balanced saline solution containing 10 mM Hepes
buffer and 10 mM glucose. Cells were imaged by using a confocal laser
scanning microscope (Molecular Dynamics) coupled to an inverted
microscope (Nikon). Cells were located under bright-field optics and
then scanned once with the laser (488-nm excitation and 510-nm
emission). Values of cellular fluorescence (average pixel intensity
per cell) were obtained with the software supplied by the manufacturer
(Molecular Dynamics).
Levels of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) reduction, a measure of mitochondrial
function (49) were quantified as described (30). Mitochondrial
transmembrane potential was assessed by using the dye rhodamine 123
(50). Cultures were incubated for 30 min in medium, containing 5 µM
rhodamine 123, and then washed with Locke's solution. Cellular
fluorescence was imaged by using a confocal laser scanning microscope
with excitation at 488 nm and emission at 510 nm, and the average
pixel intensity per cell was determined by using IMAGESPACE software
(Molecular Dynamics).
RESULTS
Top
Abstract
Introduction
Materials
Results
Discussion
References
PS-1 Mutations Increase Vulnerability of PC12 Cells to A-Induced
Apoptosis: Prevention by Calbindin. The following clonal lines of PC12
cells were used: the untransfected parent cell line, mock transfected
cell lines (values for a line transfected with the empty vector
pRc/CMV used for PS-1 expression, a line transfected with the empty
vector pREP4 used for calbindin D28K expression, and a line
transfected with both vectors were combined), two lines overexpressing
wild-type PS-1 (PS-1C1 and PS1C5), two lines overexpressing the PS-1
L286V mutation (L286VC1 and L286VC6), two lines overexpressing the
PS-1 M146V mutation (M146VC4 and M146VC11), a line overexpressing
calbindin (CBC13), two lines overexpressing calbindin and wild-type
PS-1 (CB+PS1C2 and CB+PS1C7), two lines overexpressing calbindin and
the PS-1 L286V mutation (CB+L286VC1 and CB+L286VC8), and two lines
overexpressing calbindin and the PS-1 M146V mutation (CB+M146VC1 and
CB+M146VC13). Western blot analyses of these lines showed that levels
of full-length PS-1 protein were approximately 5-fold higher in the
lines transfected with wild-type or mutant human PS-1 compared with
untransfected, vector-transfected, and calbindin-transfected cell
lines (Fig. 1A). In addition to a 46-kDa band corresponding to
full-length PS-1, immunoreactive bands of approximately 34 and 17 kDa
were present. From previous findings (14), the 17-kDa band probably
corresponds to a C-terminal endoproteolytic fragment of PS-1; the
34-kDa band may represent either a proteolytic product or a
posttranslationally modified form of PS-1. All three bands were seen
in lines overexpressing either wild-type or mutant PS-1. Levels of
calbindin D28k were at or below the limit of detection in control cell
lines and lines overexpressing wild-type or mutant PS-1; cell lines
transfected with the pREP-CB expressed calbindin at markedly higher
levels (Fig. 1A). Cell lines overexpressing PS-1, PS-1 L286V, or
PS1M146V were selected based on their similar levels of overexpression
of PS-1 protein, as determined by Western blot analysis (Fig. 1A).
To examine the impact of overexpression of wild-type PS-1, mutant
PS-1, and/or calbindin on neural cell apoptosis, each of the cell
lines was exposed for 24 hr to A1-42 (50 µM) and cells with apoptotic
nuclei were counted. Basal levels of apoptosis ranged from 5% to 15%
among the various cell lines (Fig. 1B). In control cell lines and
lines overexpressing wild-type PS-1, A induced apoptosis in 33-44% of
the cells (Fig. 1B). Levels of A-induced apoptosis were significantly
increased to 70-80% in cell lines expressing either of the PS-1
mutations. Overexpression of calbindin significantly attenuated
A-induced apoptosis and largely blocked the proapoptotic action of
mutant PS-1 (Fig. 1B).
A-Induced Elevations of [Ca2+]i and Reactive Oxygen Species Are
Enhanced by Mutant PS and Blocked by Calbindin Overexpression. Levels
of [Ca2+]i were quantified in the different cell lines 4 hr after
exposure to vehicle or A1-42. Basal levels of [Ca2+]i were similar in
all cell lines examined, ranging from 70 to 100 nM (Fig. 2A). In
control cell lines and lines overexpressing wild-type PS-1, A induced
increases of [Ca2+]i to levels of 150-200 nM during a 4-hr exposure
period (Fig. 2). In contrast, the [Ca2+]i was increased to 350-500 nM
in cells expressing either the L286V or the M146V PS-1 mutations.
Overexpression of calbindin significantly attenuated A-induced
elevation of [Ca2+]i and completely blocked the enhanced calcium
response in cells expressing mutant PS-1 (Fig. 2). Because oxidative
stress is increased in cells exposed to A and may play a role in
A-induced apoptosis (6, 7, 10, 30), we used the fluorescent probe DCF
to measure relative levels of reactive oxygen species (ROS) in the
various cell lines 4 hr after exposure to vehicle or A1-42. In control
cell lines and lines overexpressing wild-type PS-1, A induced
significant 3- to 4-fold increases in ROS levels (Fig. 3). The
A-induced increase in levels of ROS was significantly exacerbated in
cells expressing either of the PS-1 mutations. In contrast, levels of
ROS were not increased after exposure to A in cell lines
overexpressing calbindin alone or in combination with wild-type or
mutant PS-1 (Fig. 3).
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Fig. 2. Increases of [Ca2+]i induced by A are enhanced in cells
expressing mutant PS-1 and attenuated by overexpression of calbindin.
(A) Cells were exposed for 4 hr to 50 µM A1-42 and the [Ca2+]i in
individual cells was quantified by fluorescence ratio imaging of the
calcium indicator dye fura-2 (see Fig. 1 for cell lines). Values are
the mean ± SD of determinations made in three cultures (50-80 cells
per culture; ANOVA with Scheffe's posthoc tests). (B) Ratio images of
intracellular calcium levels in PC12 cells expressing mutant PS-1
alone (PS1L286V) or in combination with calbindin (CB+PS1L286V) 4 hr
after exposure to either vehicle (water) or 50 µM A1-42. The [Ca2+]i
is represented on a color scale shown at the right (values are nM).
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Fig. 3. Increases of cellular reactive oxygen species induced by
A are enhanced in cells expressing mutant PS-1 and are prevented by
overexpression of calbindin. Cells were exposed for 4 hr to 50 µM
A1-42 and levels of ROS in individual cells were measured by using the
fluorescent probe DCF (see Fig. 1 for cell lines). Values are the mean
± SD of determinations made in three cultures (40-65 cells per
culture; ANOVA with Scheffe's posthoc tests).
Mutant PS-1 Enhances A-Induced Impairment of Mitochondrial Function:
Prevention by Calbindin. Mitochondrial alterations, including
decreased energy charge/redox state and membrane depolarization, occur
at relatively early stages in cells undergoing apoptosis (51, 52).
Mitochondrial impairment has also been documented in brain tissues
from AD patients (53), in cultured neurons exposed to A (30, 50), and
in fibroblasts from patients harboring PS-1 mutations (54). To
determine the effects of A on mitochondrial transmembrane potential,
we used the fluorescent probe rhodamine 123 (50). Exposure of control
PC12 cell lines, and the lines overexpressing wild-type human PS-1, to
A resulted in 30-40% decreases in levels of rhodamine 123 fluorescence
(Fig. 4). The decrease in rhodamine 123 fluorescence induced by A was
significantly enhanced (greater than 60% decrease) in cells expressing
either the L286V or M146V PS-1 mutations (Fig. 4). The decrease in
rhodamine 123 fluorescence after A exposure was largely prevented by
overexpression of calbindin in the various cell lines, with the effect
of calbindin being greatest in the lines expressing a mutant PS-1
(Fig. 4). Exposure of control PC12 cell lines and lines overexpressing
wild-type human PS-1 to A resulted in a 20-30% decrease in levels of
MTT reduction during a 4-hr exposure period, indicating a decrease in
electron transport activity. In parallel cultures of PC12 cells
expressing mutant PS-1, A caused a greater than 60% decrease in levels
of MTT reduction; mitochondrial function was largely preserved in the
various cell lines overexpressing calbindin alone or in combination
with wild-type or mutant PS-1 (data not shown). Collectively, these
findings suggest that the elevation of [Ca2+]i induced by A and
enhanced by PS-1 mutations played a role in impairment of
mitochondrial function.
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Fig. 4. Decrease in mitochondrial transmembrane potential induced
by A is exacerbated in cells expressing mutant PS-1s and is prevented
by overexpression of calbindin. (A) Cells were exposed for 12 hr to 50
µM A1-42 and levels of rhodamine 123 fluorescence were quantified (see
Fig. 1 for cell lines). Values are the mean ± SD of determinations
made in three cultures (ANOVA with Scheffe's posthoc tests). *, P <
0.01 compared with values for untransfected, vector-transfected, and
PS-1 C1 cell lines exposed to A; **, P < 0.01 and ***, P < 0.001
compared with the value for the corresponding cell line not expressing
calbindin. (B) Confocal laser scanning microscope images of rhodamine
123 fluorescence in PC12 cells expressing mutant PS-1 alone (PS1L286V)
or in combination with calbindin (CB+PS1L286V) 12 hr after exposure to
either vehicle (water) or 50 µM A1-42. Note that A1-42 caused a marked
decrease in rhodamine 123 fluorescence in cells lacking calbindin but
not in cells overexpressing calbindin.
DISCUSSION
Top
Abstract
Introduction
Materials
Results
Discussion
References
Our data suggest that PS-1 mutations promote an apoptotic cascade of
events involving disruption of calcium homeostasis, oxidative stress,
and mitochondrial dysfunction. A primary action of mutant forms of
PS-1 on calcium homeostasis is indicated by the ability of calbindin
overexpression to suppress A-induced oxidative stress and
mitochondrial dysfunction in cells expressing mutant PS-1. Similar
results were obtained with two different PS-1 mutations, suggesting
that a similar pathogenic mechanism may apply to all PS-1 mutations.
Increased [Ca2+]i and oxygen radical production may both contribute to
the proapoptotic action of PS-1 mutations because agents that block
Ca2+ release from ER or influx through plasma membrane channels and
antioxidants, such as vitamin E, can protect PC12 cells overexpressing
mutant PS-1 against cell death induced by A or trophic factor
withdrawal (45, 46). The specific mechanism whereby mutant PS-1
disrupts cellular calcium homeostasis is unknown but may involve an
effect on ER calcium regulation, as suggested by the localization of
PS-1 in the ER (14) and the demonstrations of enhanced calcium
responses to agonists that induce calcium release from ER in PC12
cells expressing mutant PS-1 (45) and in fibroblasts from human
carriers of PS-1 mutations (55). Indeed, calcium release from ER
induced by the ER Ca2+-ATPase inhibitor thapsigargin is sufficient to
trigger apoptosis in several types of cells (56, 57).
Immunohistochemical analyses of postmortem human brain and spinal cord
tissue have suggested that neurons expressing high levels of calbindin
are relatively resistant to death in several prominent
neurodegenerative conditions including AD (58), Parkinson's disease
(59), Down's syndrome (60), Huntington's disease (61), and amyotrophic
lateral sclerosis (62). Calbindin-immunoreactive neurons are also
spared in hippocampus from human patients with severe temporal lobe
epilepsy and in rats subjected to kainate-induced seizures (63).
Interestingly, levels of calbindin expression in neurons in the brain
may decrease with normal aging (64), a change that would be expected
to render the neurons susceptible to a variety of insults and disease
states. Cell culture studies have shown that hippocampal neurons
expressing calbindin are relatively resistant to death induced by
exposure to excitatory amino acids (34) and A (35). The present data
show that overexpression of calbindin in PC12 cells results in a
marked attenuation of the increase of [Ca2+]i induced by A. Further
evidence for a neuroprotective role for calbindin comes from studies
showing that calbindin expression can be induced by brain injury (47,
65) and that trophic factors that induce calbindin expression in
cultured hippocampal neurons (e.g., basic fibroblast growth factor and
tumor necrosis factor) also protect those neurons against excitotoxic
and metabolic insults (66-68). Calbindin, which is localized primarily
in the cytosol and ER (69, 70), is thought to prevent sustained
elevations of [Ca2+]i by acting as a calcium buffer (71).
An increasing amount of data suggest that apoptosis may be the
predominant form of neuronal death in AD. Several laboratories have
provided evidence that DNA damage and morphological changes in neurons
in AD brain that are consistent with apoptosis (39, 40). In addition,
altered expression of apoptosis-related genes such as c-jun and bcl-2
family members have been documented in vulnerable neurons in AD brain
(72). Previous work in our laboratory (45, 46) and other laboratories
(43) has shown that PS mutations can increase vulnerability of
cultured cells to apoptosis induced by trophic factor withdrawal and
exposure to A. Deng et al. (44) and Wolozin and associates (43)
provided evidence that overexpression of wild-type and mutant PS-2 can
induce apoptosis in the absence of an apoptotic insult. We have not
observed spontaneous apoptosis in PC12 cell lines stably
overexpressing either wild-type or mutant PS-1. This indicates that
the effect of overexpression of PS-1 may be different from that of
PS-2 and is consistent with the observation that overexpression of
wild-type or mutant PS-1 does not result in any apparent neuronal
death under basal conditions in transgenic mice (73). However, we
cannot rule out the possibility that lines overexpressing very high
levels of mutant PS-1 died during the selection process. Our data
suggest that mutant PS-1 may promote neuronal apoptosis in AD by
perturbing cellular calcium homeostasis. Additional data support a
proapoptotic role for PS mutations, including recent studies showing
that caspase-mediated cleavage of PSs is enhanced in cells expressing
mutant PSs (74) and that PS mutations alter APP processing in a manner
that increases production of neurotoxic A and decreases levels of
neuroprotective secreted forms of APP (75, 76).
Previous studies have shown that increases of [Ca2+]i can induce
oxidative stress, which may result from an adverse effect of sustained
elevations of [Ca2+]i on mitochondrial electron transport leading to
superoxide anion radical production and its conversion to hydrogen
peroxide and peroxynitrite (77-79). We found that A induced an
increase in DCF fluorescence in PC12 cells that was significantly
enhanced in cells expressing mutant PS-1. It should be noted that
although the increased DCF fluorescence likely reflects hydrogen
peroxide accumulation (7, 50, 77), DCF can be oxidized by other ROS
including peroxynitrite. Calbindin overexpression largely suppressed
the A-induced increase in ROS production and blocked the adverse
effect of mutant PS-1 on ROS accumulation. These findings suggest that
PS-1 mutations may promote cellular oxidative stress by disrupting
calcium homeostasis. Consistent with the latter possibility, we have
found that dantrolene (an agent that blocks calcium release from the
ER) can block A-induced increases in ROS levels in cells expressing
mutant PS-1 (Q.G. and M.P.M., unpublished data). Other studies have
shown that increases in ROS levels occur early in the process of
thapsigargin-induced apoptosis of thymocytes (80). Antioxidants and
Bcl-2 appear to act downstream of elevation of [Ca2+]i in protecting
cells against thapsigargin-induced apoptosis (81). Consistent with a
role for enhanced oxidative stress in the proapoptotic action of
mutant PS-1, Bcl-2 protected PC12 cells expressing mutant PS-1 against
apoptosis induced by trophic factor withdrawal (46).
Mitochondrial impairment after exposure of PC12 cells to A, as
indicated by decreases in levels of MTT reduction and rhodamine 123
fluorescence, was exacerbated in cells expressing mutant PS-1;
calbindin prevented this adverse effect of mutant PS-1. Decreases in
levels of MTT reduction occur early in cells undergoing apoptosis in
response to many different insults including exposure to A (30, 50,
79). Additonal mitochondrial alterations linked to apoptosis include
loss of mitochondrial transmembrane potential, formation of
permeability transition pores, and release of proteins that trigger
nuclear apoptosis such as cytochrome c and an apoptosis-inducing
factor (38). We found that loss of mitochondrial transmembrane
potential after exposure to A was significantly exacerbated in PC12
cells expressing mutant PS-1 and that calbindin counteracted the
adverse effect of mutant PS-1. These findings suggest that elevated
[Ca2+]i mediates apoptotic mitochondrial alterations induced by A and
their potentiation by PS-1 mutations. Our data are consistent with
previous studies suggesting a mechanistic link between ER calcium
release and mitochondrial alterations involved in apoptosis. For
example, thapsigargin can induce mitochondrial permeability transition
(82), and agents that block the permeability transition (e.g.,
cyclosporin A) can prevent thapsigargin-induced apoptosis (83). A more
detailed understanding of the mechanism whereby PS-1 modulates
neuronal calcium homeostasis and cell survival may reveal molecular
targets upon which to aim antiapoptotic therapeutic approaches in AD.
ACKNOWLEDGEMENTS
We thank W. Fu, H. Luo, and J. Xie for technical assistance and B.
Sopher for providing plasmids containing wild-type and mutant PS-1
cDNAs. This work was supported by grants to M.P.M. from the National
Institutes of Health (AG14554, AG05144, NS35253, and AG10836).
FOOTNOTES
To whom reprint requests should be addressed at: 211 Sanders-Brown
Building, University of Kentucky, Lexington, KY 40536-0230. e-mail:
mmat...@aging.coa.uky.edu.
This paper was submitted directly (Track II) to the Proceedings
Office.
Abbreviations: A, amyloid -peptide; AD, Alzheimer's disease; APP,
-amyloid precursor protein; PS, presenilin; [Ca2+]i, intracellular
Ca2+ concentration; ER, endoplasmic reticulum; DCF,
2,7-dichlorofluorescin diacetate; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ROS,
reactive oxygen species.
REFERENCES
Top
Abstract
Introduction
Materials
Results
Discussion
References
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