Lipid- and receptor-binding regions of apolipoprotein
E4 fragments act in concert to cause mitochondrial
dysfunction and neurotoxicity
Shengjun Chang*, Tian ran Ma*, R. Dennis Miranda*†, Maureen E. Balestra*, Robert W. Mahley*†‡§¶,
and Yadong Huang*†‡¶?
*Gladstone Institute of Neurological Disease and†Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158;
and Departments of‡Pathology,§Medicine, and?Neurology, University of California, San Francisco, CA 94143
Contributed by Robert W. Mahley, September 23, 2005
Apolipoprotein (apo) E4, a 299-aa protein and a major risk factor
for Alzheimer’s disease, can be cleaved to generate C-terminal-
truncated fragments that cause neurotoxicity in vitro and neuro-
degeneration and behavioral deficits in transgenic mice. To inves-
truncations or mutations in transfected Neuro-2a cells. ApoE4
(1–272) was neurotoxic, but full-length apoE4(1–299) and apoE4(1–
240) were not, suggesting that the lipid-binding region (amino
acids 241–272) mediates the neurotoxicity and that amino acids
273–299 are protective. A quadruple mutation in the lipid-binding
region (I250A, F257A, W264R, and V269A) abolished the neurotox-
icity of apoE4(1–272), and single mutations in the region of amino
acids 273–299 (L279Q, K282A, or Q284A) made full-length apoE4
neurotoxic. Immunofluorescence staining showed that apoE4(1–
272) formed filamentous inclusions containing phosphorylated tau
in some cells and interacted with mitochondria in others, leading
to mitochondrial dysfunction as determined by MitoTracker stain-
ing and flow cytometry. ApoE4(241–272) did not cause mitochon-
drial dysfunction or neurotoxicity, suggesting that the lipid-bind-
ing region alone is insufficient for neurotoxicity. Truncation of
N-terminal sequences (amino acids 1–170) containing the receptor-
binding region (amino acids 135–150) and triple mutations within
that region (R142A, K146A, and R147A) abolished the mitochon-
drial interaction and neurotoxicity of apoE4(1–272). Further anal-
from the secretory pathway and that the lipid-binding region
mediates mitochondrial interaction. Thus, the lipid- and receptor-
binding regions in apoE4 fragments act together to cause mito-
chondrial dysfunction and neurotoxicity, which may be important
in Alzheimer’s disease pathogenesis.
Alzheimer’s disease ? mitochondria ? proteolysis
ApoE4 is a major risk factor for Alzheimer’s disease (AD) (5–7).
The apoE4 allele, which is found in 40–65% of cases of sporadic
and familial AD, increases the occurrence and lowers the age of
onset of the disease (7, 8).
Biochemical, cell biological, transgenic animal, and human
studies have suggested several potential mechanisms to explain
the contribution of apoE4 to the pathogenesis of AD. These
mechanisms include modulation of the deposition and clear-
ance of amyloid ? (A?) peptides and the formation of plaques
(9–15), modulation of A?-caused synaptic and cholinergic
deficits (16), acceleration of age- and excitotoxicity-related
neurodegeneration (17), impairment of the antioxidative de-
fense system and mitochondrial function (18–21), dysregula-
tion of neuronal signaling pathways (22), altered phosphory-
lation of tau and neurofibrillary tangle formation (23–28),
depletion of cytosolic androgen receptor levels in the brain
(29, 30), potentiation of A?-induced lysosomal leakage and
apoptosis in neuronal cells (31), and promotion of endosomal
uman apolipoprotein (apo) E, a 34-kDa protein with 299 aa,
has three major isoforms, apoE2, apoE3, and apoE4 (1–4).
abnormalities linked to A? overproduction (32–34). The
mechanisms of these apoE4-mediated detrimental effects are
We have shown that apoE can be cleaved by a neuron-
specific chymotrypsin-like serine protease that generates bio-
active C-terminal-truncated forms of apoE (25, 27, 28). The
fragments are found at higher levels in the brains of AD
patients than in age- and sex-matched controls (27), and apoE4
is more susceptible to cleavage than apoE3. When expressed
in cultured neuronal cells or added exogenously to the cultures,
apoE4 fragments are neurotoxic, leading to cell death (25).
When expressed in transgenic mice, they cause AD-like neu-
rodegeneration and behavioral deficits (27). Because apoE is
synthesized by neurons under diverse pathophysiological con-
ditions (35–49), we hypothesize that apoE4 produced in
neurons in response to stress or injury (e.g., A? toxicity, brain
trauma, or oxidative stress) is uniquely susceptible to proteo-
lytic cleavage and that the resulting bioactive C-terminal-
truncated fragments induce neuropathology and associated
behavioral deficits. ApoE3 also undergoes proteolytic cleavage
but to a lesser extent.
In this study, we investigated the cellular and molecular
mechanisms of the neurotoxicity caused by apoE4 fragments in
cultured neuronal cells. We also evaluated the roles of various
regions [specifically, the receptor-binding region (amino acids
135–150) and the lipid-binding region (amino acids 241–272)] of
apoE (1–4, 50).
Reagents. MEM, Opti-MEM, and FBS were obtained from Life
Technologies (Rockville, MD). Polyclonal goat anti-human
apoE was obtained from Calbiochem. Monoclonal antibodies
that specifically recognize the lipid-binding region of apoE
(3H1) were obtained from Karl H. Weisgraber (Gladstone
IgGs coupled to fluorescein or Texas red were obtained from
Vector Laboratories. MitoTracker Deep Red 633 was obtained
from Invitrogen. A cDNA construct encoding red fluorescent
protein fused with a mitochondrial localization signal peptide
(DsRed2-Mito) was obtained from BD Biosciences.
cDNA Constructs. PCR products encoding WT or N-terminal-
truncated apoE4 with its signal peptide were subcloned into a
pcDNA 3.1(?) vector (Invitrogen) containing the cytomegalo-
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: A?, amyloid ?; AD, Alzheimer’s disease; apo, apolipoprotein; STP-O, Strep-
¶To whom correspondence may be sent at the*address. E-mail: yhuang@gladstone.
ucsf.edu or email@example.com.
© 2005 by The National Academy of Sciences of the USA
December 20, 2005 ?
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no. 51 www.pnas.org?cgi?doi?10.1073?pnas.0508254102
apoE4 fusion protein was also subcloned into the vector. cDNA
truncations were made from the pcDNA–apoE4 or pcDNA–
GFP–apoE4 construct with a QuikChange kit (Stratagene). All
constructs were confirmed by sequence analysis.
Cell Culture and Transfection. Mouse neuroblastoma Neuro-2a
cells (American Type Culture Collection) maintained at 37°C in
MEM containing 10% FBS were transiently transfected with the
apoE4 cDNA constructs by using Lipofectamine 2000 (Invitro-
gen) (25). ApoE4 expression levels were determined by anti-
apoE Western blotting of cell lysates and media. The truncated
and mutated forms of apoE4 that are neurotoxic were expressed
at ?15–30% lower levels than full-length apoE4. To exclude
their potential weaker antibody responses, those forms of apoE4
were tagged with GFP, and their expression levels were deter-
mined by flow cytometry. Again, their expression levels were
?15–30% lower than those of full-length apoE4. Thus, the
results were not due to overexpression.
Immunocytochemistry and Confocal Microscopy. Neuro-2a cells
transiently transfected with various apoE4 cDNA constructs
were grown in serum-free MEM for 18–24 h, fixed in 3%
paraformaldehyde, permeabilized for 45 min at room tempera-
ture with 500 units of Streptolysin-O (STP-O, Sigma) in BBII
buffer (75 mM potassium acetate?25 mM Hepes, pH 7.2) (for
plasma membranes) or 0.5% Tween 20 in PBS (for plasma and
intracellular organelle membranes) (51), and stained with poly-
clonal anti-apoE (1:4,000 dilution) or monoclonal anti-apoE
(3H1, 1:200 dilution) and a fluorescein-coupled secondary an-
tibody (Vector Laboratories) (25). Labeled cells were mounted
in VECTASHIELD (Vector Laboratories) and viewed with a
Radiance 2000 laser-scanning confocal system (Bio-Rad) that
was mounted on an Optiphot-2 microscope (Nikon). Neuro-2a
cells transiently transfected with cDNA constructs encoding
GFP–apoE4 with mutations or truncations were analyzed di-
rectly by confocal microscopy. Some Neuro-2a cells were co-
transfected with various apoE cDNA constructs and a construct
encoding red fluorescent protein fused with a mitochondrial
localization signal peptide (DsRed2-Mito, BD Biosciences),
stained with immunofluorescent polyclonal or monoclonal anti-
apoE antibody, and analyzed by confocal microscopy.
Cell Survival. Neuro-2a cells grown in 24-well plates were
transiently transfected with various apoE4 or GFP–apoE4
cDNA constructs in serum-free Opti-MEM. Cell survival was
estimated with an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-
yltetrazolium bromide (MTT) colorimetric assay (52) at 48 h
Flow Cytometry Analysis of Mitochondrial Function and Integrity.
Neuro-2a cells grown in six-well plates were transiently trans-
fected with various GFP–apoE4 cDNA constructs. The culture
medium was aspirated 48 h after transfection, and MitoTracker
Deep Red 633 (100 nM in MEM containing 10% FBS) was
added for 15 min at 37°C. After a wash with serum-free MEM,
cells were trypsinized and suspended in 1 ml of PBS, washed
twice with PBS by centrifugation (300 ? g for 5 min), resus-
pended in 1 ml of PBS, and filtered through a mesh cap into a
5-ml tube. The fluorescence intensity of GFP, which represents
apoE4 expression levels, and of MitoTracker Deep Red 633,
which represents the levels of mitochondrial function and integ-
rity (53), were analyzed by flow cytometry. Untransfected
Neuro-2a cells served as a negative control.
2,5-diphenyltetrazolium bromide (MTT) assay. (a) Survival of cells transfected with WT apoE4, apoE4(1–272), apoE4(1–240), or apoE4(1–191). (b) Survival of cells
transfected with WT apoE4, apoE4(1–272), or apoE4(1–272) with four mutations (I250A, F257A, W264R, and V269A). (c) Survival of cells transfected with WT
(e) Survival of cells transfected with WT apoE4, apoE4(1–272), apoE4(87–272), apoE(127–272), or apoE(171–272). (f) Survival of cells transfected with WT apoE4,
apoE4(1–272), or apoE4(1–272) with double (K146A and R147A) or triple (R142A, K146A, and R147A) mutations. Values are given as mean ? SD of three to six
assays at 48 h after transfection.*, P ? 0.05 vs. WT apoE4.
The lipid- and receptor-binding regions in apoE4 fragments act in concert to cause neurotoxicity, as determined with a 3-(4,5-dimethylthiazol-2-yl)-
Chang et al. PNAS ?
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vol. 102 ?
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Statistical Analysis. Results are reported as mean ? SD. Differ-
ences were evaluated by t test or analysis of variance.
The Lipid-Binding Region Is Required for ApoE4 Fragment-Related
Neurotoxicity. To assess the neurotoxicity of various apoE4
fragments in Neuro-2a cells, we used a 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression
of apoE4(1–272) caused 35% greater cell death than full-length
apoE4; further C-terminal truncation to amino acids 240 or 191
to remove the lipid-binding region (amino acids 241–272) abol-
ished the neurotoxicity (Fig. 1a). Four mutations of this region
(I250A, F257A, W264R, and V269A) that are conserved across
different species (54) also abolished the neurotoxicity (Fig. 1b).
Single C-Terminal Mutations Make Full-Length ApoE4 Neurotoxic.
ApoE4(1–272) was more neurotoxic than full-length apoE4,
suggesting that the 27 C-terminal amino acids protect against
fragment-related neurotoxicity. Three amino acids in this region
(L279, K282, and Q284) are highly conserved in 10 species (54).
To assess their importance in this neuroprotective effect, we
introduced mutations at each site (L279Q, K282A, or Q284A)
into WT apoE4. Each mutation made full-length apoE4 as
neurotoxic as apoE4(1–272) (Fig. 1c).
Neurotoxicity Requires Both Lipid- and Receptor-Binding Regions. To
determine whether the lipid-binding region alone was neuro-
toxic, we analyzed Neuro-2a cells expressing only amino acids
241–272 of apoE4. No neurotoxicity was observed (Fig. 1d). To
determine which region of the N terminus was also required for
neurotoxicity, we transfected cells with cDNA constructs en-
coding apoE4(1–272) with progressively longer N-terminal trun-
cations. Neurotoxicity was abolished only by a truncation that
removed the receptor-binding region (amino acids 135–150)
Positively Charged Amino Acids in the Receptor-Binding Region Are
Critical for Neurotoxicity. The receptor-binding region contains a
cluster of positively charged amino acids (arginine and lysine)
(1–4). To test their importance in apoE4 fragment-related
neurotoxicity, we introduced double (K146A and R147A) and
triple (R142A, K146A, and R147A) mutations into apoE4(1–
272). The triple mutation abolished the neurotoxic effect of
apoE4(1–272), and the double mutation reduced it (Fig. 1f).
ApoE4 Fragments Escape the Secretory Pathway and Interact with
Cytoskeletal Components and Mitochondria. To investigate the
mechanisms of neurotoxicity, we assessed the intracellular
localization of full-length or truncated apoE4 in Neuro-2a cells
by immunofluorescence staining. Full-length apoE4 was typ-
ically located in the endoplasmic reticulum and Golgi appa-
ratus (Fig. 2a), whereas apoE4(1–272) formed intracellular
filamentous inclusions in some cells and had a granular
distribution in others (Fig. 2b), suggesting mislocalization of
the truncated apoE4 in Neuro-2a cells. Because intracellular
filamentous inclusions contain phosphorylated tau and phos-
phorylated neurofilament proteins, as reported (25, 26), some
of the fragments must have escaped the secretory pathway and
interacted with cytoskeletal components. In cells expressing
both apoE4(1–272) and DsRed2-Mito, the granule-associated
apoE4 fragments were in the mitochondria (Fig. 2c).
Mitochondrial Mislocalization Requires the Lipid- and Receptor-
Binding Regions. Next, we investigated the intracellular location
of apoE(171–272), containing only the lipid-binding region,
and apoE4(1–240), containing only the receptor-binding re-
gion. Neither form was located in the mitochondria, and their
immunocytochemistry and confocal microscopy. (a) Cells transfected with WT
apoE4, permeabilized with Tween 20, and stained with anti-apoE (green). (b)
Cells transfected with apoE4(1–272), permeabilized with Tween 20, and
stained with anti-apoE (green). (c) Cells cotransfected with apoE4(1–272) and
DsRed2-Mito (red), permeabilized with STP-O, and stained with anti-apoE
Intracellular distribution of various forms of apoE4 as determined by
apoE(171–272) (c), apoE4(1–240) (e), apoE4(1–272)-AARA with four mutations (I250A, F257A, W264R, and V269A) in the lipid-binding region (g), or apoE4(1–
with anti-apoE (green). Cells cotransfected with DsRed2-Mito (red) and various apoE4 constructs mentioned above were permeabilized with 500 units of STP-O
(b, d, f, h, and j) and stained with anti-apoE (green). The cells were then analyzed by confocal microscopy for only green (a, c, e, g, and i) or both red and green
(b, d, f, h, and j).
The lipid and receptor-binding regions act in concert to cause mitochondrial mislocalization of apoE4 fragments. Cells transfected with WT apoE4 (a),
www.pnas.org?cgi?doi?10.1073?pnas.0508254102Chang et al.
intracellular distributions were similar to that of full-length
apoE4 (Fig. 3 a–f). The mitochondrial mislocalization was also
abolished by the quadruple mutation in the lipid-binding
region [E4(1–272)-AARA] and the triple mutations in the
receptor-binding region [E4(1–272)-3A] (Fig. 3 g–j).
The Receptor-Binding Region Is Required to Escape the Secretory
Pathway, and the Lipid-Binding Region Mediates Mitochondrial Inter-
action. To dissect the functions of the lipid- and receptor-binding
regions, we assessed the effect of removing the N-terminal
secretion signal peptide from fragments containing only one of
the two regions. When expressed directly in the cytosol,
apoE(171–272), containing only the lipid-binding region, inter-
acted with the mitochondria (Fig. 4 a and b), although the same
fragment with the signal peptide was retained in the secretory
pathway and did not interact with the mitochondria (Fig. 3 c and
d). Also, triple mutation of the receptor-binding region caused
apoE4(1–272) with the signal peptide to be retained in the
secretory pathway and, thus, no interaction with the mitochon-
dria (Fig. 3 i and j). ApoE4(1–191), containing only the receptor-
binding region, did not interact with the mitochondria, even
when expressed directly in the cytosol (Fig. 4 c and d).
Lipid- and Receptor-Binding Regions Together Impair Mitochondrial
Function and Integrity. To investigate the effect of apoE4 frag-
ments on mitochondria, Neuro-2a cells transfected with vari-
ous apoE4 constructs were incubated with MitoTracker Deep
Red 633, and fluorescence intensity was analyzed by flow
cytometry as a measure of mitochondrial function and integ-
rity (53) (Fig. 5). Fluorescence intensity was 25% lower in cells
expressing apoE4(1–272) or apoE(127–272) than in cells ex-
pressing full-length apoE4 (Fig. 5a). Because only functional
mitochondria with a normal membrane potential can effec-
tively take up and store MitoTracker Deep Red 633, this
finding suggests that only apoE4 fragments with both the lipid-
and receptor-binding regions can impair mitochondrial func-
tion and integrity. Importantly, this effect depended on the
level of expression (Fig. 5b). Consistent with the immunocy-
tochemical data, apoE4 fragments containing only one of the
two regions and those fragments with the quadruple mutation
in the lipid-binding region or the triple mutation in the
receptor-binding region had no significant effect on mitochon-
drial function and integrity (Fig. 5).
ApoE4 fragments found in cultured neuronal cells and in AD
brains induce neurofibrillary tangle-like structures and cause
neurotoxicity in vitro (25, 26, 55, 56) and neurodegeneration and
behavioral deficits in transgenic mice (27, 28). This study dem-
onstrates that both the lipid- and receptor-binding regions are
required for neurotoxicity of apoE4 fragments in Neuro-2a cells
and that, in addition to disrupting cytoskeletal structure and
function (25, 27), the apoE4 fragments also interact with mito-
chondria and impair their function and integrity.
Our working model is that positively charged amino acids in
the receptor-binding region enable apoE4 fragments to escape
the secretory pathway and enter the cytosol, whereas the lipid-
binding region mediates interactions with the mitochondria.
When the secretion signal peptide was present, apoE(171–272),
which contains only the lipid-binding region, could not escape
the secretory pathway and did not interact with the mitochon-
dria. However, when the signal peptide was removed and
apoE(171–272) was directly expressed in the cytosol, it did
interact with the mitochondria. Also, the triple mutation in the
receptor-binding region caused apoE4(1–272) with the signal
peptide to be retained in the secretory pathway, where it could
not interact with the mitochondria. ApoE4(1–191), which con-
tains only the receptor-binding region, did not interact with the
mitochondria, even when expressed directly in the cytosol.
The receptor-binding region of apoE shares a feature [the
enrichment of positively charged amino acids (arginine and
lysine)] with the protein-translocation domain (PTD) of many
viral proteins. PTD-containing proteins, such as HIV-1 Tat,
penetrate the plasma membrane of cells in a receptor-
pathway, and the lipid-binding region mediates mitochondrial interaction.
Cells transfected with apoE(171–272) without signal peptide (a) or apoE4(1–
191) without signal peptide (c) were permeabilized with 0.5% Tween 20 and
stained with anti-apoE (green). Cells cotransfected with DsRed2-Mito (red)
and either of those two apoE4 constructs were permeabilized with 500 units
as in Fig. 3.
The receptor-binding region is required to escape the secretory
concert to cause mitochondrial dysfunction, as determined by MitoTracker
Deep Red 633 staining and flow cytometry. (a) Effects of various forms of
given as mean ? SD of three to six assays.*, P ? 0.05 vs. WT apoE4, E4(171–
272), and E4(1–272)-3A. E4(1–272)-3A, apoE4(1–272) with a triple mutation in
the receptor-binding region.
The lipid- and receptor-binding regions in apoE4 fragments act in
Chang et al. PNAS ?
December 20, 2005 ?
vol. 102 ?
no. 51 ?
independent, concentration-dependent fashion (57, 58). The Tat
PTD, a short basic region of 10 aa, has been used as a carrier to
deliver many peptides, proteins, and antisense oligodeoxynucle-
otides into cells (59–61). Likewise, the receptor-binding region
of apoE has also been used to deliver antisense oligodeoxynucle-
otides into cells (62, 63), consistent with the membrane-
penetrating ability that we observed in this study.
In vitro, the lipid-binding region is responsible for the inter-
action of apoE with A? peptides (9, 64), whereas the receptor-
binding region is responsible for binding with tau (23). Thus,
apoE4 fragments might also interact with A? or tau or both via
two different regions and act synergistically to cause dysfunction
of both the cytoskeleton and the mitochondria, leading to
neuronal and behavioral deficits.
Mitochondrial dysfunction in AD (21, 65–67) varies with apoE
genotype, being greater in apoE4 than in apoE3 carriers (19).
ApoE4 is also associated with decreased cerebral glucose me-
tabolism in both AD patients and nondemented subjects (68–
71). Because normal cerebral glucose metabolism requires nor-
mal mitochondrial function, and because apoE4 fragments are
found in AD brains (27), it is tempting to speculate that the
impairment of mitochondrial function and integrity elicited by
the expression of the truncated apoE4 in Neuro-2a cells relates
to the mitochondrial dysfunction or damage observed in AD
brains. Consequently, blocking the interaction of apoE4 frag-
ments with the mitochondria is a potential strategy for inhibiting
the detrimental effects of apoE4 in AD and other neurological
We thank Aubrey Bernardo for assistance in some cell-culture experi-
ments; Drs. Karl Weisgraber, Lennart Mucke, and Luke Esposito for
critical reading of the manuscript; Karina Fantillo and Sylvia Richmond
for manuscript preparation; Stephen Ordway and Gary Howard for
editorial assistance; John C. W. Carroll and Jack Hull for graphics; and
Chris Goodfellow for photography. This work was supported in part by
Program Project Grant P01 AG022074, National Institutes of Health
Grant R01HL37063, and a research grant from GlaxoSmithKline.
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