Rosiglitazone increases dendritic spine density and
rescues spine loss caused by apolipoprotein E4
in primary cortical neurons
Jens Brodbeck*, Maureen E. Balestra*, Ann M. Saunders†, Allen D. Roses†, Robert W. Mahley*‡§¶?**,
and Yadong Huang*‡¶**††
*Gladstone Institute of Neurological Disease and‡Gladstone Institute of Cardiovascular Disease, The J. David Gladstone Institutes, 1650 Owens Street,
San Francisco, CA 94158; Departments of§Medicine,¶Pathology, and††Neurology and?Cardiovascular Research Institute, University of California,
San Francisco, CA 94143; and†GlaxoSmithKline Research and Development, Research Triangle Park, NC 27709
Contributed by Robert W. Mahley, October 18, 2007 (sent for review July 20, 2007)
Convergent evidence has revealed an association between insulin
resistance and Alzheimer’s disease (AD), and the peroxisome pro-
liferator-activated receptor-? (PPAR-?) agonist, rosiglitazone, an
insulin sensitizer and mitochondrial activator, improves cognition
in patients with early or mild-to-moderate AD. Apolipoprotein
(apo) E4, a major genetic risk factor for AD, exerts neuropatho-
dendritic spine structure and mitochondrial function. Here we
show that rosiglitazone significantly increased dendritic spine
density in a dose-dependent manner in cultured primary cortical
rat neurons. This effect was abolished by the PPAR-?-specific
antagonist, GW9662, suggesting that rosiglitazone exerts this
effect by activating the PPAR-? pathway. Furthermore, the C-
terminal-truncated fragment of apoE4 significantly decreased den-
dritic spine density. Rosiglitazone rescued this detrimental effect.
Thus, rosiglitazone might improve cognition in AD patients by
increasing dendritic spine density.
Alzheimer’s disease ? mitochondria ? peroxisome proliferator-activated
receptor-? ? apolipoprotein E fragment ? synaptogenesis
affects millions of people globally (1). Alarmingly, currently
ment of functional deficits. Thus, AD cases will increase dispro-
portionately as the global elderly population increases. A major
risk factor for late-onset AD is apolipoprotein (apo) E4, which
increases the risk and lowers the age of onset in a gene
dose-dependent manner (2, 3). The three isoforms of human
apoE (apoE2, apoE3, and apoE4) have different roles in lipid
metabolism and neurobiology (4–8). ApoE3 promotes neurite
outgrowth, dendritic arborization, and synaptogenesis, whereas
apoE4 inhibits these processes both in vitro (9–11) and in vivo
(12–14). Furthermore, apoE4 transgenic mice have deficits in
synaptic plasticity, synaptic terminal remodeling, synaptogen-
esis, and learning and memory (12–16). Loss of synaptophysin-
immunoreactive presynaptic terminals, indicating synaptic loss
(12, 17), occurs early in AD and is considered the best patho-
logical correlate of cognitive decline (18–20).
Previously, we showed that neurons express apoE in response
to injury (21) and that neuronal apoE is cleaved into C-terminal-
truncated fragments resembling those in AD brains (22). ApoE4
is more susceptible to this cleavage than apoE3 (23, 24). ApoE4
fragments are neurotoxic in vitro and cause neurodegeneration
and behavioral deficits in transgenic mice (22–24). In neurons,
truncated apoE4 escapes the secretory pathway, enters the
cytosol, and interacts with tau, increasing its phosphorylation
and causing preneurofibrillary tangles (22, 23). In the cytosol,
apoE4 fragments also interact with the mitochondria, impairing
their membrane potential and function (25). Mitochondrial
impairment in AD is greater in apoE4 than in apoE3 carriers
lzheimer’s disease (AD), a devastating neurodegenerative
disease that usually develops in the sixth decade of life,
(26). Thus, apoE4 may contribute to AD pathogenesis by causing
mitochondrial dysfunction and synaptic deficits (6).
Type 2 diabetes also increases the risk of developing AD
(27–29), particularly among diabetic patients carrying apoE4
(30). Insulin resistance, the core defect in type 2 diabetes, results
in hyperinsulinemia to compensate for the reduced action of
insulin in peripheral tissues (31). Correspondingly, AD patients
resistance (32, 33). In addition, AD brains from autopsied
patients have markedly lower levels of insulin mRNA and
tyrosine phosphorylation than control brains (34). Indeed, in-
hibition of the neuronal insulin receptor has been proposed as
a model for sporadic AD (35). Thus, type 2 diabetes and AD
might share a common pathogenic feature that can be modified
by apoE genotype.
The thiazolidinediones (e.g., troglitazone, pioglitazone, and
rosiglitazone) are agonists of the nuclear receptor peroxisome
proliferator-activated receptor-? (PPAR-?). Because they in-
crease peripheral insulin sensitivity and stimulate mitochondrial
biogenesis and function (36, 37), thiazolidinediones are widely
used to treat type 2 diabetes (38, 39). In clinical trials, rosigli-
tazone improved cognition in a subset of AD patients (40, 41)
and also reduced learning and memory deficits in a mouse model
of AD (42). However, the mechanisms underlying the potential
beneficial effects of rosiglitazone in AD remain unclear. In this
study, we tested the hypothesis that rosiglitazone increases
dendritic spine density and rescues the dendritic spine loss
caused by apoE4.
Rosiglitazone Increases Dendritic Spine Density. To assess the effect
of the PPAR-? agonist, rosiglitazone, on dendritic spine density,
we incubated primary cortical rat neurons, previously cultured
for 24 h. Four to 7 days before the experiment, the cells were
transfected with EGFP-tagged ?-actin (EGFP–?-actin), a cy-
toskeletal protein that is abundant in dendritic spines (43).
EGFP–?-actin expression does not impair neuronal function or
synaptic morphology (44). Rosiglitazone increased dendritic
spine density, visible at both low (Fig. 1A) and high (Fig. 1B)
magnification of representative neurons and dendrites, respec-
tively, as shown by microscopic analysis. Quantitative analyses
Author contributions: J.B., A.D.R., R.W.M., and Y.H. designed research; J.B. and M.E.B.
A.M.S. contributed new reagents/analytic tools; and J.B., A.D.R., R.W.M., and Y.H. wrote
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
**To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
© 2008 by The National Academy of Sciences of the USA
January 29, 2008 ?
vol. 105 ?
no. 4 ?
showed 58.5 ? 3% greater dendritic spine density in treated
neurons than in controls (Fig. 1C). However, rosiglitazone did
not affect other parameters of neuronal plasticity, including the
area of the dendritic field (estimated by counting the proximal
extensions of the dendritic tree), dendrite length (longest dis-
tance between the soma and the proximal dendritic extension),
and the number of dendritic branch points (extensions originat-
ing from primary dendrites) (Fig. 2).
Rosiglitazone’s Effects on Dendritic Spine Density Are Dose-
efficacy of rosiglitazone in increasing dendritic spine density.
After 14–17 days in culture, primary cortical neurons were
incubated with 0.1, 0.5, 5, and 10 ?M rosiglitazone at 37°C for
24 h. A comparison of the dentritic complexity of neurons
incubated with rosiglitazone versus control suggested no toxic
effects at all concentrations used. Dendritic spine density was
increased by 29.6 ? 5.2% at a dose of 0.5 ?M and by 47.03 ?
3.4% at 5 ?M (Fig. 3). No further increase was seen at 10 ?M.
Rosiglitazone Increases Dendritic Spine Density by Activating the
PPAR-? Pathway. Depending on the concentration, thiazo-
lidinediones exert both PPAR-?-dependent and PPAR-?-
independent effects (45). To assess the specificity of rosiglita-
zone’s effect on dendritic spine density, we cultured primary
cortical neurons for 14–17 days and incubated them with 5 ?M
rosiglitazone and 1 ?M PPAR-?-specific antagonist GW9662 at
37°C for 24 h. GW9662 abolished the rosiglitazone-induced
increase in dendritic spine density (Fig. 3).
Rosiglitazone Rescues the Dendritic Spine Loss Caused by ApoE4.
ApoE4 and C-terminal-truncated fragments of apoE4
structure and mitochondrial function (12, 22, 23, 25). Because
cytoskeletal integrity and mitochondrial function are critical for
normal synaptic morphology and function (46, 47), we tested the
effects of various forms of apoE on dendritic spine density in
primary cortical neurons. ApoE4 significantly reduced dendritic
spine density by 25.5 ? 4%, and the apoE4(1–272) fragment
reduced it by 45.6 ? 3%, compared with apoE3 (Fig. 4). Thus,
apoE4 and, to a greater extent, its fragment appear to impair
synaptogenesis or synaptic maintenance. Rosiglitazone rescued
the dendritic spine loss caused by apoE4(1–272) (Fig. 4). Ros-
iglitazone also abolished the difference in dendritic spine density
between apoE4 and apoE3, although the effect did not reach
statistical significance, compared with apoE4 alone (Fig. 4).
This study shows that the PPAR-? agonist, rosiglitazone, in-
creases the density of dendritic spines on cultured primary rat
neurons in a dose-dependent manner. In addition, rosiglitazone
dendrite length. The cells, transiently transfected with the synaptic marker EGFP–?-actin and cultured for 14–17 days, were incubated with 5 ?M rosiglitazone
for 24 h or DMSO only as a control. (A) Representative images showing the stimulatory effect of rosiglitazone on dendritic spine density. (B) Representative
dendrites from three different neurons. (C) Dendritic spine densities of eight randomly selected cells per condition were quantified. Values are mean ? SEM.
*, P ? 0.01 versus control (two-tailed t test).
Rosiglitazone increases dendritic spine density in rat primary cortical neurons. Spine density is defined as the number of spines per micrometer of
(µm2 x 104)
(µm x 102)
with the synaptic marker EGFP–?-actin and cultured for 14–17 days, were incubated with 5 ?M rosiglitazone for 24 h or DMSO only as a control. The area of
dendritic field (A), dendrite length (B), and the number of dendritic branches (C) from 15 randomly selected cells per condition were quantified. Values are
mean ? SEM.
www.pnas.org?cgi?doi?10.1073?pnas.0709906104 Brodbeck et al.
rescued the loss of dendritic spines caused by the C-terminal-
truncated fragments of apoE4.
In clinical trials, rosiglitazone had beneficial effects in patients
with early or mild-to-moderate AD (40, 41). Rosiglitazone also
reduced learning and memory deficits in a mouse model of AD
(42). Our data suggest that rosiglitazone improves cognition by
increasing dendritic spine density. In one trial, rosiglitazone at
once-daily doses of 2, 4, and 8 mg improved cognition in AD
patients carrying apoE3, but not in those carrying apoE4 (41).
However, rosiglitazone clearly prevented the dendritic spine loss
caused by the apoE4 fragments in primary neuronal cultures.
Because rosiglitazone does not readily cross the blood–brain
barrier at least in rodents (40), this discrepancy may indicate that
the lower doses of rosiglitazone used in AD patients might not
achieve brain levels high enough to overcome the detrimental
effects of apoE4 and its fragments despite a potentially com-
promised blood–brain barrier.
PPAR-? agonists, such as rosiglitazone, have a complex
pharmacology beyond their established peripheral effect in
activating the PPAR-? pathway. For instance, they appear to
have diverse roles in neuroprotection, such as promoting the
expression of the mitochondrial uncoupling protein 2 after
ischemia-induced hippocampal injury (48) and suppressing the
proinflammatory transcription factor NF-?B through PPAR-
?-dependent (49, 50) or PPAR-?-independent pathways (51).
This finding raises the question of whether the effects of
rosiglitazone on dendritic spine density are related to its ability
to activate PPAR-? or other actions. We found that the
PPAR-?-specific antagonist, GW9662, abolished the rosigli-
tazone-induced increase in dendritic spine density, strongly
suggesting that it exerts beneficial effects by activating the
PPAR-? pathway. Further studies are required to elucidate the
How does rosiglitazone increase dendritic spine density and
rescue dendritic spine loss induced by apoE4? One possibility is
that rosiglitazone stimulates mitochondrial biogenesis and func-
tion (37, 52, 53). Normal mitochondrial dynamics and function
are essential for generating and maintaining distinct axonal and
dendritic microdomains in response to local metabolic demand
(47). Moreover, failure of mitochondria to traffic to appropriate
sites causes energy starvation and impairs synaptogenesis and
memory formation (47, 54). Thus, rosiglitazone might increase
mitochondrial biogenesis or function, thereby improving synap-
togenesis or maintenance of dendritic spines. Furthermore,
because apoE4 fragments impair mitochondrial integrity and
function (25), the rescue of dendritic spine loss also might reflect
the beneficial effects of rosiglitazone on mitochondrial biogen-
esis or function.
Materials and Methods
Reagents. Rosiglitazone maleate was provided by GlaxoSmithKline. Recom-
binant apoE3, apoE4, and apoE4(1–272) were provided by Karl Weisgraber
(The J. David Gladstone Institutes). The pPDGF–EGFP–?-actin construct was a
gift of Yukiko Goda (University College London, London, U.K.). All plasmids
were purified with the Plasmid Maxi kit from Qiagen. GW9662, a PPAR-?
antagonist, was from Sigma–Aldrich.
Primary Neuron Culture and Transfection. Cortices from neonatal rat pups
postnatal day 1 were dissected, treated for 30 min with 10 units/ml papain
15 min. After trituration, dissociated neurons were plated on 12-mm glass
centimeter. After 2 h, cells were transferred into neurobasal medium supple-
mented with B27, 1 mM L-glutamine, and 100 ?g/ml penicillin/streptomycin
(Invitrogen). Neurons were routinely transfected after 10 days in culture and
used for experiments 4–7 days after transfection. Cells were maintained in a
2 ?g of pPDGF–EGFP–?-actin construct with 3 ?l of Lipofectamine 2000
Treatment with Rosiglitazone and ApoE. After 14–17 days in culture, primary
cortical rat neurons were incubated with the rosiglitazone concentrations
indicated in Fig. 3 or DMSO as a control with or without 1 ?M GW9662 or 7.5
?g/ml apoE (various forms) at 37°C for 24 h.
Confocal Microscopy. After treatment, the neurons were fixed for 20 min in
ice-cold 4% paraformaldehyde in PBS (pH 7.4) and mounted on microscope
slides with VECTAshield (Vector Laboratories). Digital images of EGFP were
collected on a laser-scanning confocal microscope with a Bio-Rad Radiance
2000 scanhead mounted on an Optiphot-2 microscope (Nikon) with a ?60 oil
images were analyzed with National Institutes of Health ImageJ software
(http://rsb.info.nih.gov/ij). The length of dendrites was measured by tracing
their extension using the segmented line selection. Dendritic spines were
counted manually by using the point picker function of the particle analysis
plug-in. The complexity of the dendritic tree was quantified by using the
ImageJ NeuronJ plug-in to trace dendritic branches. The area of the dendritic
field was estimated by connecting the outmost dendritic extensions and
calculating the area of the resulting polygon.
Statistical Analysis. A two-tailed t test assuming equal variances was used for
statistical analyses. P ? 0.05 was considered statistically significant.
apoE3, apoE4, and apoE4(1–272); Dr. Yukiko Goda for providing the EGFP–
?-actin construct; Karina Fantillo and Sylvia Richmond for manuscript
preparation; John Carroll for graphics; and Stephen Ordway and Gary
Howard for editorial assistance. This work was supported by The J. David
Gladstone Institutes and GlaxoSmithKline Research and Development.
0.10.5 5.0 10.0
+ 1 µM
07.058 A J .Brodbeck
rat primary cortical neurons. The cells, transiently transfected with the syn-
aptic marker EGFP–?-actin and cultured for 14–17 days, were incubated for
24 h with rosiglitazone in the presence or absence of 1 ?M PPAR-? antagonist
GW9662 or DMSO only as a control. Dendritic spine densities of eight ran-
domly selected cells per condition were quantified. Values are mean ? SEM.
*, P ? 0.05;**, P ? 0.01 versus control (two-tailed t test).
Dose-dependent effect of rosiglitazone on dendritic spine density in
Spines (% of Control)
p p < 0.001 < 0.001
p < 0.001
p p < 0.05 < 0.05
p < 0.05
p < 0.01
fragment. Rat primary cortical neurons, transiently transfected with the syn-
aptic marker EGFP–?-actin and cultured for 14–17 days, were incubated with
7.5 ?g/ml apoE (various forms) for 24 h in the presence or absence of 5 ?M
rosiglitazone. Dendritic spine densities of 10 cells per condition were quanti-
fied. Values are mean ? SEM.
Rosiglitazone rescues dendritic spine loss caused by the apoE4
Brodbeck et al. PNAS ?
January 29, 2008 ?
vol. 105 ?
no. 4 ?
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