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J Neuropathol Exp Neurol
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Vol. 68, No. 5
May 2009
pp. 503Y514
ORIGINAL ARTICLE
Increased Association Between Rough Endoplasmic
Reticulum Membranes and Mitochondria in Transgenic Mice
That Express P301L Tau
Sébastien Perreault, MD, Olivier Bousquet, PhD, Michel Lauzon, Jacques Paiement, PhD,
and Nicole Leclerc, PhD
Abstract
INTRODUCTION
From the Département de Pathologie et Biologie Cellulaire, Université de
Montréal, Montréal, Québec, Canada.
Send correspondence and reprint requests to Nicole Leclerc, PhD, Département
de Pathologie et Biologie Cellulaire, Université de Montréal, C.P.6128,
Succ. Centre-ville, Montréal, Québec, Canada H3C 3J7; E-mail: nicole.
[email protected]
Sébastien Perreault and Olivier Bousquet equally contributed to the
experimental work of this study.
Sébastien Perreault had a studentship from Groupe de Recherche sur le
Système Nerveux Central.
This study was supported by Grant MOP-81092 from the Canadian Institute
of Health Research and by Grant ASC-0659 from the Alzheimer Society
of Canada.
The neuropathologic hallmarks of Alzheimer disease
(AD) are plaques that contain amyloid deposits and neurofibrillary tangles (NFTs) formed by paired helical filaments
(PHFs) that are composed of insoluble tau protein (1). The
numbers of NFTs more consistently correlate with the degree
of dementia than amyloid plaques in AD (2Y6). Tau is
preferentially associated with the microtubules of the axonal
compartment in normal brain (7). During the process of
neurodegeneration in AD, tau becomes hyperphosphorylated,
has a decreased binding affinity to microtubules, accumulates
in the somatodendritic compartment, and aggregates into
insoluble intraneuronal filaments called PHFs (8Y10).
Insoluble tau filaments are also characteristics of
neurodegenerative diseases called tauopathies. These diseases
include frontotemporal lobar degeneration with Pick bodies,
corticobasal degeneration, progressive supranuclear palsy,
sporadic multiple system tauopathy with dementia, argyrophilic grain disease, neurofibrillary tangle dementia, and
frontotemporal dementia (FTD) with microtubule-associated
protein tau gene mutation, also called FTD with parkinsonism linked to chromosome 17; all these belong to a group of
diseases termed frontotemporal lobar degeneration (9, 11).
The importance of tau dysfunction in neurodegeneration was
recently supported by the identification of mutations in its
gene in patients who have FTD with parkinsonism linked
to chromosome 17 (12, 13). The precise role of tau in neurodegeneration, however, remains obscure.
The contribution of tau to neurodegeneration was
confirmed in mouse models in which wild-type human tau
is overexpressed. Although these models do not entirely
recapitulate the landmarks of tauopathies (i.e. they did not
develop NFTs), they indicate that too much tau is detrimental
to neurons (14Y17). In the spinal cord, inclusions of hyperphosphorylated tau in the axonal proximal segment were
associated with axonal degeneration, fewer microtubules, and
reduced axonal transport. Since the discovery of tau
mutations, several transgenic mice have been produced that
express mutated forms of human tau. For example, the
overexpression of the R406W mutation induced formation of
tau inclusions that shared some properties with NFTs (18).
Most notably, these inclusions were associated with memory
deficits. In the transgenic mice JNPL3 that express the mutant
J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
503
In several neurodegenerative diseases, including Alzheimer
disease, the neuronal microtubule-associated protein tau becomes
hyperphosphorylated, accumulates in the somatodendritic compartment, and aggregates into insoluble filaments. The consequences of
the accumulation of hyperphosphorylated tau in the somatodendritic
compartment remain poorly characterized at the early stage of
disease before the formation of tau insoluble filaments. We
investigated the ultrastructural changes induced by this accumulation in the neuronal soma of motor neurons in asymptomatic
JNPL3 mice that overexpress mutant tau, P301L. More numerous
contacts between rough endoplasmic reticulum (RER) membranes
and mitochondria were observed in JNLP3 mice compared with
wild-type mice. This correlated with a preferential increase of the
amount of tau at the surface of RER membranes but not at the
surface of mitochondria, as revealed by tau immunogold labeling.
Using a subcellular fractionation procedure, an increased amount of
phosphorylated tau was identified in the rough microsome subfraction, wherein the RER marker, ribophorin, was enriched. A
similar increase was noted in the rough microsome subfraction
isolated from Alzheimer disease brains. The association of hyperphosphorylated tau with ER membranes was confirmed by double
immunogold labeling of the subfraction enriched in ER membranes
isolated from Alzheimer disease brains. These results suggest that
more contacts between RER membranes and mitochondria resulting
from the accumulation of tau at the surface of RER membranes
might contribute to tau-induced neurodegeneration.
Key Words: Alzheimer disease, Microtubule-associated protein tau,
Mitochondria, Neurodegeneration, P301L, Rough endoplasmic
reticulum.
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Perreault et al
J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
human form of tau P301L, true NFTs have been observed
and correlated with neurodegeneration (19). This mutation is
located in the second repeat of tau microtubule-binding
domain and reduces its binding to microtubules (20). In
JNPL3 mice, hyperphosphorylated tau accumulates in the
somatodendritic compartment and forms NFTs in the motor
neurons of the spinal cord, whereas neurons at the preYtangle
stage predominate in the brain (19).
The accumulation of tau protein in the soma of neurons
is associated with neurodegeneration, but a pool of tau is
detected in the soma in normal conditions. This population of
tau has a level of phosphorylation higher than the axonal tau
(21, 22). In a recent study, we showed that somatic tau was
associated with Golgi membranes (23). The somatic pool of
tau can increase under certain physiological conditions. A
somatodendritic accumulation of phosphorylated tau in CA3
pyramidal cells was found in the hippocampus of hibernating
squirrels (24). This accumulation was correlated with the
regression of the synaptic contacts of the mossy fibers on the
apical dendrites of the pyramidal cells and disappeared when
synaptic contacts were reformed at the end of hibernation,
indicating that the somatodendritic accumulation of hyperphosphorylated tau is a transitory and reversible event.
In tauopathies, the somatodendritic accumulation of
hyperphosphorylated tau becomes irreversible. The consequences of this permanent accumulation on the integrity of
the somatodendritic compartment remain poorly characterized. Alterations of the morphology of the endoplasmic
reticulum (ER) and the Golgi apparatus have been reported in
AD brain (25, 26), but it is unclear whether these changes are
caused by the accumulation of hyperphosphorylated tau in
the soma. The subcellular distribution of tau filaments was
examined in neuronal soma in AD brain using immunogold
labeling. Tau filaments were found to arise from the nuclear
membrane (which is formed of rough ER [RER] membranes)
and smooth ER membranes (27).
We recently reported that the overexpression of tau
induced a fragmentation of the neuronal Golgi both in
neuronal cultures and in JNPL3 mice (28). This event was
more frequent when a mutated form of tau associated with
frontotemporal lobar degeneration was overexpressed in
neuronal cultures. The fragmentation of the Golgi induced
by tau was not related to apoptosis in neurons. In JNPL3
mice, the fragmentation of the Golgi coincided with the
somatodendritic accumulation of hyperphosphorylated tau
before tangle formed, indicating that this reorganization of
the Golgi occurred early in the process of neurodegeneration
(28). In transgenic mice that overexpress the P301S mutant, a
decrease of mitochondria and RER membranes was observed
(29). Ultrastructural analysis of tau transgenic mice revealed
that tau filaments were often observed in the vicinity of
membranes. In JNPL3 mice, tau filaments were located in the
vicinity of the nuclear membrane (30). The overexpression of
human tau in lamprey also resulted in the formation of tau
filaments associated with membranous elements including
ER membranes (31).
In previous studies, the consequences of tau accumulation on the ultrastructural integrity of the soma were
examined at the end stage of the disease when tau filaments
were present within a neuron. Several studies have demonstrated, however, that the accumulation of hyperphosphorylated tau in the somatodendritic compartment can be
detrimental to a neuron without the formation of tau
filaments. For example, in Drosophila, the accumulation of
hyperphosphorylated tau after the overexpression of either
human wild-type or mutant tau was sufficient to induce
neurodegeneration (32). In a mouse model expressing the
human mutant form of tau P301L that could be repressed by
doxycycline, memory improvement was noted when human
tau expression was suppressed although NFTs continued to
form (33). In vitro studies have provided some insights on
how hyperphosphorylated tau exerts its toxicity. Hyperphosphorylated tau but not PHFs isolated from AD brain
inhibited microtubule assembly and promoted the disruption
of preformed microtubules by the sequestration of normal tau
and MAP2 (34, 35).
In the present study, we examined the ultrastructural
changes induced by the accumulation of hyperphosphorylated tau in the soma occurring before the formation of NFTs
to understand how this accumulation can be detrimental to a
neuron. This point was investigated in motor neurons of the
spinal cord of JNPL3 mice in which NFTs are observed.
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MATERIALS AND METHODS
Electron Microscope Immunocytochemistry
on Spinal Cord Sections
JNPL3 and age-matched wild-type mice were purchased from Taconic Farms (Germantown, NY). The use of
animals and all surgical procedures described in this article
were carried out according to The Guide to the Care and Use
of Experimental Animals of the Canadian Council on Animal
Care. Adult mice were deeply anesthetized with intraperitoneal sodium pentobarbital (Somnotol, MTC Pharmaceuticals, Cambridge, Ontario, Canada, 65 mg/kg). They were
perfused through the ascending aorta with 20 mL of 0.1 M
cacodylate buffer ([CB] pH 7.4, room temperature), followed
by 100 mL of freshly prepared fixative containing 2.5%
glutaraldehyde (J. B. Em Services, Pointe Claire, Quebec,
Canada) and 1% paraformaldehyde (Fisher, Ottawa, Ontario,
Canada) in CB. The spinal cords were removed and kept in
the same fixative for 1 hour at room temperature after which
they were washed in CB. The spinal cords were cut in slices
of 100 Km with a vibratome. The slices were then cut in half,
with one half containing the posterior horns and the other half
containing the anterior horns. The anterior horn half slices
containing motor neurons were postfixed in 1% osmium
tetroxide (J. B. Em Services) and 1.5% potassium ferrocyanide (Sigma, Oakville, Ontario, Canada) in CB for 30
minutes at 4-C. After an extensive wash in buffer, the slices
were dehydrated in a graded series of alcohols at 4-C, then
washed twice for 10 minutes in 100% ethanol at room
temperature and embedded in LR White resin (J. B. Em
Services). The resin was polymerized under anaerobic
conditions for 48 hours at 56-C. Ultrathin sections (approximately 60 nm) were cut with an ultramicrotome and placed
on single-slot nickel grids coated with Formvar (Mecalab
Ltd, Pointe-aux-Trembles, Montréal, Quebec, Canada). Each
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J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
RER/Mitochondria Association in Mutant Tau Mice
grid contained 2 sections of 1 mm (width) 1.5 mm
(height). These sections were then processed for postembedding immunocytochemistry, as previously described
(36). A polyclonal antibody directed against the human tau
346Y352 (kindly provided by Virginia M.-Y. Lee, University
of Pennsylvania, Philadelphia, PA) was used for tau
immunogold labeling (37) at a dilution of 1:100. Sections
from 1 transgenic and 1 wild-type mouse were simultaneously processed in each immunogold labeling experiment.
Four grids per animal were processed at a time for a total of
2 times to make sure that the pattern of the immunogold
staining was reproducible.
were again washed and then revealed by chemiluminescence
(Amersham Pharmacia Biotech, Québec, Quebec, Canada).
The following primary antibodies were used: the monoclonal
antibody directed against tau not phosphorylated at S198,
S199, or S202, Tau-1 (1:1000; Oncogene Research Products,
San Diego, CA), a polyclonal phosphorylation-insensitive
anti-tau antibody directed against human tau 346-352
(1:1000; kindly provided by Virginia M.-Y. Lee, University
of Pennsylvania) (37), the monoclonal antibody HT7 directed
against human tau (1:100; Pierce Endogen, Rockford, IL), a
polyclonal antibody against ribophorin (1:500; kindly provided by Dr G. Kreibich, New York University School of
Medicine, New York, NY), a polyclonal antibody against
calnexin (1:3000; Stressgen Biotechnologies, Victoria, British Columbia, Canada), the monoclonal anti-KDEL receptor
(KDELR) antibody (1:250), the monoclonal antiYsyntaxin-6
antibody (1:1000; Stressgen Biotechnologies), and the monoclonal antibody anti-MAP2 (1:1000; clone HM2, Sigma).
The following primary antibodies directed against phosphorylated tau were used: the monoclonal antibody AT8
(1:200; Pierce Endogen), CP9 (1:100), CP13 (1:100), PG-5
(1:100), and PHF-1 (1:100; kindly provided by Dr Peter
Davies, Albert Einstein, Bronx, NY) directed against tau
phosphorylated at S199/S202/T205, T231, S202, S409, and
S396/S404, respectively.
Quantitative Morphometric Analysis of Number
of Contacts Between RER and Mitochondria
The initial goal of the study was to investigate the
subcellular localization of tau in JNPL3 mice before the
formation of NFTs. Only motor neurons with well-defined and
preserved Golgi, RER, and nuclear membranes were chosen
for the analysis. One micrograph that included the Golgi and a
portion of the nucleus was taken per motor neuron at a
magnification of 28K. During the quantitative analysis of tau
immunogold on membranes, we noted that the number of
contacts between RER membranes and mitochondria was
increased in JNPL3 mice. To quantify this observation, the
number of RER/mitochondria contacts per micrometer of RER
membranes was calculated for each micrograph representing
1 motor neuron. In wild-type and JNPL3 mice, 52 and
126 motor neurons, respectively, were analyzed.
Subcellular Fractionation
Brains were dissected from wild-type and JNPL3 mice,
and subcellular fractions were generated using the protocol
previously described by Lavoie et al (38) in 1996 and
modified as described in our recent studies (23, 39). Briefly,
total microsomes were isolated by differential centrifugation,
and ER and Golgi elements were subsequently separated by
ultracentrifugation in a sucrose step-gradient. The same
protocol of subcellular fractionation was applied to frozen
human tissues from the frontal cortex. Human tissues from
4 AD patients and 4 control patients presenting no sign of
cognitive impairment were obtained at the Brain Bank from
the Douglas Hospital Research Center affiliated to McGill
University, Montréal, Quebec, Canada. A neuropathologic
analysis was performed blinded to the clinical diagnosis in
accordance with established criteria. The use of human tissue
was approved by the institutional ethical review committee of
the University of Montreal.
Immunoblot Analysis
Protein assay was performed on the subfractions
isolated from mouse brain and human brain (Bio-Rad kit,
Bio-Rad Laboratories, Mississauga, Ontario, Canada). Equal
amounts of proteins were loaded in each lane and electrophoresed in a 7.5% polyacrylamide gel. After separation,
proteins were electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose strips were incubated with
the primary antibodies for 90 minutes at room temperature.
They were then washed with PBS and incubated with the
peroxidase-conjugated secondary antibodies. Membranes
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Electron Microscopy on the Rough
Microsome Subfraction
The rough microsome (RM) subfraction isolated from
control and AD brain was fixed using 2.5% glutaraldehyde
(J. B. Em Services), recovered onto Millipore membranes
(Millipore, Bedford, MA) by the random filtration technique
of Baudhuin et al (40) and processed for electron microscopy
as previously described (38). The PHF-1 antibody was
diluted 1:10, and the anti-calnexin antibody, 1:100. An antimouse antibody conjugated to 10-nm gold particles (1:10)
and an anti-rabbit antibody conjugated to 5-nm gold particles
(1:10) were used to reveal the localization of PHF-1 and the
anti-calnexin antibody, respectively (Sigma). Immunogold
labeling was performed on the RM subfractions prepared
from 2 control and 2 AD brains.
Statistical Analysis
The counts of the number of gold particles on the
membranous elements in motor neurons of the spinal cord of
wild-type and JNPL3 mice were subjected to a 2-way
analysis of variance test to detect any interaction between
the sets of experiments. Because no interaction was found
between the sets of experiments, they were combined. The
differences between the types of mice (wild-type and JNPL3
mice) were analyzed by a 1-way analysis of variance.
Statistical significance was accepted if p G 0.05.
RESULTS
The Association of RER Membranes With
Mitochondria Was Increased in JNPL3 Mice
The ultrastructural changes induced by the accumulation of hyperphosphorylated tau in the somatodendritic
505
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Perreault et al
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FIGURE 1. Electron microscopy of spinal cord motor neurons of wild-type (Wt) and JNPL3 transgenic (Tg) mice. There are
contacts between mitochondria and rough endoplasmic reticulum (RER) membranes in both Wt and Tg mice. The RER
membranes are identified by the presence of ribosomes at their surface. These contacts were twice as frequent in Tg as in Wt mice.
Immunogold particles label tau protein. Scale bar = 100 nm.
compartment are not well characterized at the initial stage of
AD. We examined these changes in motor neurons of the
spinal cord in JNPL3 mice that overexpress P301L tau. In
hemizygous JNPL3 mice, P301L is expressed at levels
approximately equivalent to those of endogenous tau (50%
of total tau) (19). This mutant tau becomes hyperphosphorylated, accumulates in the somatodendritic compartment, and
forms tangles in the motor neurons of the spinal cord in
JNPL3 mice. The ultrastructural analysis was carried out in
asymptomatic 5- to 10-month-old mice before tangle formation. Neurons that expressed P301L were identified by tau
immunogold labeling. No gold particles were found when the
primary antibody was omitted as well as when this primary
antibody was preabsorbed with pure tau protein. The most
noticeable ultrastructural difference between JNPL3 and
wild-type mice was an increased number of contacts between
RER membranes and mitochondria (Fig. 1). At the regions of
contacts, the RER membranes were deprived of ribosomes
and were in close apposition to the outer membrane of mitochondria, as previously noted in neuronal and nonneuronal
cells (41, 42). The numbers of contacts between mitochondria
and RER membranes in motor neurons were evaluated for
each mouse type in 2 sets of experiments, and the mean T SD
was calculated for each animal. Because no difference was
noted between the 2 sets of experiments, the results of each
mouse type were combined. In wild-type mice, the number of
contacts between mitochondria and RER membranes was
0.58 T 0.72 for each micrograph taken from each motor
neuron; in JNPL3 mice, the number almost doubled to 1.14 T
1.08 (Table 1).
The increased number of contacts between mitochondria and RER membranes could be caused by an increase in
the number of mitochondria in the soma. Indeed, over-
506
expression of tau in neuronal cultures was shown to block the
transport of mitochondria to the neurites resulting in their
accumulation in the soma (43). Therefore, the numbers of
mitochondria were determined. The number of mitochondria
per micrograph (each corresponding to a single motor
neuron) was 3.09 T 1.26 in wild-type and 3.02 T 1.21 in
JNPL3 mice. These numbers were not statistically different
(Table 1). The numbers of mitochondria per micrometer of
RER membranes for each micrograph taken from each motor
neuron were also determined, and no statistical difference
TABLE 1. Quantitative Analysis of the Number of Contacts
Between RER and Mitochondria
No. contacts between
RER membranes and
mitochondria in each
micrograph
No. mitochondria per
micrometer of RER
membrane in each
micrograph
No. mitochondria in
each micrograph
Total no. contacts
between RER
membranes and
mitochondria
Wild-Type Mice
JNPL3 Mice
p
0.59 T 0.72
n = 52
1.14 T 1.08
n = 126
G0.001
0.87 T 0.72
n = 52
0.85 T 0.58
n = 126
G0.996
3.09 T 1.26
n = 52
31
3.02 T 1.21
n = 126
143
n = number of motor neurons used for the quantitative analysis of the number of
rough endoplasmic reticulum (RER)/mitochondria contacts for each type of mice. Each
number represents the mean T SD.
The total number of contacts between mitochondria and RER membranes results
from the addition of the number of contacts in each type of mouse.
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J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
RER/Mitochondria Association in Mutant Tau Mice
was identified for this parameter (Table 1). These observations are consistent with a previous study in which the
numbers of mitochondria in the soma and neurites were not
found to be modified in another transgenic mouse model with
P301L expression under the neuron-specific promoter,
mThy1.2 (44). The total number of RER/mitochondria
contacts for the 2 wild-type and 2 JNPL3 mice was evaluated
(Table 1), with 31 and 143 contacts between RER membranes and mitochondria observed in wild-type and JNPL3
mice, respectively.
procedure generates a subfraction called RM enriched in
RER membranes and 3 subfractions, I1, I2, and I3, which
predominantly contain Golgi membranes. In adult rat brain,
tau was concentrated in the I2 subfraction (23). We
previously confirmed the enrichment of RER membranes by
Western blotting analysis using ER markers such as
ribophorin and calnexin as well as by ultrastructural analysis
(23, 39). As for the ultrastructural analysis, asymptomatic
mice were used to reveal the membranous association of tau
at the early stage of its accumulation in the somatodendritic
compartment. The fractionation procedure was applied to the
brains of wild-type and JNPL3 mice and not to the spinal
cord because of the relatively limited amount of tissue. Wildtype and JNPL3 mice (n = 5 each) were used for this
analysis. Ribophorin, a marker of RER, was enriched in the
RM subfraction isolated from wild-type and JNPL3 mice
(Fig. 2). Moreover, the dendritic microtubule-associated
protein MAP2 was enriched in the RM subfraction, as noted
in adult rat brain (39). Syntaxin-6, a marker of the Golgi
membranes, was found in both the RM and I2 subfractions.
In wild-type mice, tau was predominantly found in the I2
subfraction using a polyclonal phosphorylation-insensitive
The Association of P301L With RER Membranes
Was Preferentially Increased in JNPL3 Mice
To investigate how the accumulation of tau in the soma
could induce an increased association between RER membranes and mitochondria, tau distribution on membranous
elements was examined using a subcellular fractionation
procedure and immunogold labeling. Subfractions enriched
in RER and Golgi membranes were isolated from wild-type
and JNPL3 mice using the fractionation protocol that we
previously used to show that tau was associated with the
Golgi membranes in normal adult rat brain (23). This
FIGURE 2. Immunoblot analysis of subfractions isolated from a wild-type (Wt1) and 3 JNPL3 (Tg1, Tg2, and Tg3) mice. Upper
portions: An antibody directed against the endoplasmic reticulum marker ribophorin (Ribo) demonstrates enrichment of rough
endoplasmic reticulum membranes in the rough microsome (RM) subfractions. The dendritic microtubule-associated protein,
MAP2, was preferentially found in the RM subfractions as revealed by the anti-MAP2 antibody (HM2). An antibody directed
against syntaxin-6 shows that Golgi membranes were found in the I2 and RM subfractions. Lower portions: The distribution of
total tau was visualized using the polyclonal anti-tau antibody and the distribution of phosphorylated tau using the paired helical
filament 1 (PHF-1) antibody. An arrowhead indicates an additional higher molecular weight band observed in the RM subfraction
of the Tg mice by the PHF-1 antibody. The HT7 antibody specific to human tau was used to confirm the presence of human tau in
the RM subfraction. E, cytoplasmic extract; I, interface; P, total membrane extract; S, cytosolic fraction.
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Perreault et al
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anti-tau antibody recognizing human and rodent tau, as noted
in adult rat brain (Fig. 2). In JNPL3 mice, tau was also
enriched in the I2 subfraction. The distribution of phosphorylated tau was also examined in the subfractions generated
from wild-type and JNPL3 mice using the antibody PHF-1
recognizing tau phosphorylated at S396/S404 (Fig. 2). Tau in
the I2 subfraction was reactive to PHF-1 in both wild-type
and JNPL3 mice. Interestingly, in JNPL3 mice, an additional
PHF-1Ypositive band of higher molecular weight was
detected in the RM subfraction, indicating that tau found in
this subfraction was more phosphorylated in JNPL3 than in
wild-type mice. No additional band was detected by the
phospho-dependent tau antibodies AT8 and T231 (data not
shown).
The HT7 antibody that specifically recognizes human
tau was used to confirm the presence of human tau in the RM
subfraction (Fig. 2). A similar pattern of staining was
generated by the HT7 antibody and the polyclonal antibody
reactive to both rodent and human tau, indicating that human
tau was present in all subfractions.
The above results suggested that hyperphosphorylated
tau could be preferentially associated with RER membranes
in the soma in motor neurons in JNPL3 mice. Because other
membranous markers such as the Golgi markers syntaxin-6,
GM130, and TGN38, and mitochondrial markers such as
VDAC were found in the RM subfraction, however, we
could not conclude that tau was preferentially found at the
surface of RER membranes (23, 39). To directly address this
point, we quantified tau immunogold labeling at the surface
of the membranous elements in the soma of motor neurons in
asymptomatic mice. Quantitative analysis of the immunogold
labeling was carried out for the Golgi membrane surfaces,
RER membranes, nuclear membrane, and the mitochondria in
wild-type and transgenic animals. To verify whether the
mutant P301L had a distribution similar to that of endogenous tau, the polyclonal phosphorylation-insensitive anti-tau
FIGURE 3. Immunogold labeling of tau on spinal cord sections of wild-type (Wt1 and Wt2) and transgenic (Tg1 and Tg2) mice.
, rough endoplasmic reticulum (RER) membranes decorated
Tau immunogold labeling was observed on the Golgi membranes
, and the nuclear membrane
. Panels (a’) and (b’) represent enlarged views of RER membrane portions
with ribosomes
identified by (a) and (b) in the micrograph above them. In panels (a’) and (b’), ribosomes and gold particles are indicated by
arrowheads and arrows, respectively. Scale bar = 250 nm.
508
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TABLE 2. Quantitative Analysis of tau Immunogold Labeling
Membranous Organelles
No. particles per square
micrometer of
Golgi membranes
No. gold particles per
micrometer of
RER membranes
No. gold particles per
micrometer of
nuclear membrane
Wild-Type Mice
JNPL3 Mice
p
8.22 T 3.03
n = 52
13.13 T 4.47
n = 126
G0.001
1.39 T 1.12
n = 52
2.75 T 1.41
n = 126
G0.001
1.65 T 1.43
n = 16
1.93 T 1.25
n = 72
G0.049
n = number of motor neurons used for the quantitative analysis of tau immunogold
labeling for each type of mouse.
Mean T SD are indicated.
RER, rough endoplasmic reticulum.
antibody recognizing human and rodent tau was used to
compare the subcellular localization of total tau in wild-type
and JNPL3 mice by immunogold labeling (Fig. 3). The motor
neurons of the spinal cord have a large Golgi with multiple
stacks of saccules that seem discontinuous around the
nucleus (45). Fenestration was used to identify the cis side
of the Golgi, and clathrin-coated vesicles were used to
identify the trans Golgi side (45Y47). To evaluate the amount
of tau on Golgi membranes, the surface of the Golgi was
directly measured on enlarged electron negatives using the
Northern Eclipse image analysis system. The numbers of
gold particles were then counted per square micrometer of
Golgi membrane (gold particles per square micrometer) for
each micrograph taken from 1 motor neuron. The mean T SD
was calculated for each animal. Because no difference was
noted between the 2 sets of experiments, the results for each
type of mice were combined. The quantitative analysis
revealed 8.22 T 3.03 and 13.13 T 4.47 gold particles per
square micrometer on Golgi membranes of wild-type and
JNPL3 mice, respectively (Table 2). Tau immunolabeling
was also noted on the RER membranes in the vicinity of the
Golgi. These membranes were identified by the presence of
ribosomes at their surface. The number of gold particles per
micrometer of RER membrane length present in each micrograph from 1 motor neuron was determined. In wild-type
mice, the number of gold particles per micrometer of RER
membrane was 1.39 T 1.12, whereas in JNPL3 mice, 2.75 T
1.41 gold particles per micrometer of membrane were found
(Table 2). Because the nuclear membrane is composed of
RER membranes, the number of gold particles per micrometer of this membrane was also evaluated for each micrograph taken from 1 motor neuron. There were 1.65 T 1.43
gold particles per micrometer and 1.93 T 1.25 per micrometer
found in wild-type and JNPL3 mice, respectively. The
amount of tau found on the nuclear membrane, Golgi, and
RER membranes was 1.1, 1.6, and 1.9 times higher in JNPL3
mice than in wild-type mice. These differences were statistically significant (Table 2). Finally, the number of gold
particles per micrometer of mitochondrial outer membrane
was analyzed. In the first set of experiments, the number of
gold particles was significantly higher in JNPL3 (1.39 T 0.84)
than in wild-type (0.73 T 0.63) mice (p e 0.002) (Table 2);
this difference was not observed in the second set of
Ó 2009 American Association of Neuropathologists, Inc.
RER/Mitochondria Association in Mutant Tau Mice
experiments (p e 0.375). The above data indicate that the
association of the mutant P301L with the RER membranes
was preferentially increased in JNPL3 mice and suggest that
the increased association between RER membranes and
mitochondria could be caused by the accumulation of tau at
the surface of RER membranes.
Tau Filaments Were Associated With RER
Membranes in AD Brain
We next investigated the membranous distribution of
tau in AD brain. The RM, I1, I2, and I3 subfractions were
isolated from control and AD brains (Table 3). The antibody
directed against the RER marker ribophorin did not react
with the human protein on Western blots. An antibody
directed against calnexin, a protein present in both smooth
and rough ER membranes, was used to show that ER
membranes were concentrated in the RM subfraction in
control and AD brains (Fig. 4). Calnexin immunolabeling
was also noted in the I3 subfraction in the AD brain. The
distribution of the Golgi membranes was investigated using
an anti-KDELR antibody. This receptor mediates the recycling of ER resident proteins that have escaped to the Golgi
by binding to their KDEL sequence. At steady state, this
receptor is concentrated at the Golgi (48). In both control and
AD brains, KDELR was predominantly found in the I1
subfraction (Fig. 4). We used the HT7 antibody to reveal the
membranous distribution of total tau. In control brains, tau
was found in the I2 subfraction as observed in mouse and rat
brains; the highest amount of tau was in the I1 subfraction, as
noted for the Golgi marker, KDELR (Fig. 4). The distribution
of tau dramatically changed in AD brains. Most of the tau
was redistributed from the I1 and I2 subfractions to the RM
subfraction. This corroborated a previous study reporting the
enrichment of hyperphosphorylated tau in a microsomal
fraction isolated from the AD brain (49). The signal of tau
immunoreactivity detected by the HT-7 antibody in the I1
and RM subfractions was quantified by densitometry in 4
control and 4 AD brains. The mean of the ratio I1/RM was
3.30 and 0.07 in control and AD brains, respectively. These
results suggested that hyperphosphorylated tau presented a
membranous distribution distinct from that of normal tau.
We then examined the state of tau phosphorylation in
the I1, I2, and RM subfractions (Fig. 4). In control brains,
unphosphorylated tau detected by the Tau-1 antibody was
TABLE 3. Characteristics of Human Tissues
Clinical Diagnosis
AD1
AD2
AD3
AD4
NCI1
NCI2
NCI3
NC4
Sex
Age at Death,
years
Postmortem Delay,
hours
F
F
F
M
F
F
M
F
80
85
79
71
72
67
79
75
8.25
11.25
10.75
10
13.5
17
14.79
6.5
AD, Alzheimer disease; F, female; M, male; NCI, no cognitive impairment.
509
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Perreault et al
J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
FIGURE 4. Immunoblot analysis of subfractions isolated from control and Alzheimer disease (AD) brains. An antibody directed
against the endoplasmic reticulum (ER) marker, calnexin, reveals the enrichment of ER membranes in the rough microsome (RM)
subfraction. The Golgi proteins are found in the interface I1, I2, and I3 subfractions as revealed by an antibody directed against
the KDEL receptor (KDELR). The distribution of total tau is visualized using the monoclonal anti-tau antibody HT7. The distribution
of hyperphosphorylated tau is revealed using the antibodies PHF-1, AT-8, CP13, CP9, and PG-5; the distribution of
unphosphorylated tau is shown using the antibody Tau-1. In control brain, tau is concentrated in the I1 and I2 subfractions,
whereas in AD brains, tau is enriched in the RM subfraction. E, cytoplasmic extract; P, total membrane extract; S, cytosolic
fraction.
concentrated in the I1 subfraction. A similar distribution was
noted in the AD brains. Several antibodies directed against
phosphorylated tau were used to examine the distribution of
this population of tau protein (PHF-1, AT8, CP-9, CP-13,
and PG-5). All of these antibodies revealed that hyperphosphorylated tau was predominantly found in the RM
subfraction in AD brain. No staining was detected with the
phospho-dependent anti-tau antibodies in the subfractions
prepared from control brain in these experiments.
The previous observations indicated that hyperphosphorylated tau could be associated with RER membranes in
the RM subfraction. To examine this point, preembedding
immunogold labeling of the RM subfraction was performed
using the anti-tau antibody PHF-1 (Fig. 5). In the RM
subfraction isolated from control brain, weak staining was
present on vesicles (Figs. 5AYC). In the RM subfraction
510
prepared from the AD brain, PHF-1Ypositive filaments were
attached to vesicles (Figs. 5DYF). To confirm the presence of
tau on ER membranes, the RM subfraction isolated from AD
brain was double labeled with an anti-calnexin antibody and
the PHF-1 antibody (Fig. 6). The PHF-1Ypositive tau
filaments associated with calnexin-positive vesicles were
found in this RM subfraction. This observation corroborated
a previous observation reporting the association of tau
filaments with ER-like membranes in intact AD tissue (25).
DISCUSSION
We examined the ultrastructural alterations at the early
stage of the somatodendritic accumulation of tau in motor
neurons of the spinal cord in JNPL3 mice that overexpress
the mutant human tau P301L. The most intriguing initial
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J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
RER/Mitochondria Association in Mutant Tau Mice
FIGURE 5. Immunogold labeling of tau with 10-nm gold particles in the rough microsome (RM) subfraction isolated from control
(AYC) and Alzheimer disease (AD) (DYF) brains. The antibody recognizing tau hyperphosphorylated at S396/S404, PHF-1 was
used. In the RM subfraction isolated from control brain, some tau immunogold labeling was found at the surface of vesicles
(AYC). In the RM subfraction prepared from AD brains, PHF-1Ypositive filaments were associated with vesicles (DYF). Scale bar =
100 nm.
observation was that there were more numerous contacts
between RER membranes and mitochondria in JNPL3 mice
than in wild-type mice. Tau immunogold labeling indicated
that this increased number of contacts might result from the
preferential association of tau with RER membranes. The
association of tau with ER membranes was also demonstrated
in AD brains. Hyperphosphorylation of tau seemed to favor
this association in both JNPL3 mice and AD brains.
In normal conditions, approximately 20% of the mitochondrial surface is in direct contact with the ER membranes
(50), and mitochondria have been shown to interact with both
smooth and rough ER membranes (51, 52). The molecular
link between ER and mitochondria remains poorly characterized, but a recent study reported that mitofusin 2, a protein
known to stabilize the interactions between mitochondria, is
also found at the surface of ER membrane where it is
responsible for the tethering of ER and mitochondria by
establishing homotypic and heterotypic interactions with
mitofusin 1 or 2 located at the surface of mitochondria (53).
Interestingly, mutations of mitofusin 2 in humans induce
Charcot-Marie-Tooth Type IIa, a peripheral sensorimotor
neuropathy (54).
The interaction between mitochondria and RER membranes has important physiological roles, such as homeostasis
FIGURE 6. Double immunogold labeling of the rough microsome subfraction isolated from Alzheimer disease brain with the antitau antibody PHF-1 and an anti-calnexin antibody. (AYC) The PHF-1 antibody and the anti-calnexin antibody were revealed using
a secondary anti-mouse antibody conjugated to 10-nm colloidal gold particles and a secondary anti-rabbit conjugated to 5-nm
colloidal gold particles, respectively. Tau immunogold labeling was present on calnexin-positive vesicles either as individual gold
particles (B) or as several gold particles attached to tau filaments (A, C). Scale bar = 100 nm.
Ó 2009 American Association of Neuropathologists, Inc.
511
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Perreault et al
J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
of intracellular calcium, metabolic flow, protein transport,
and apoptosis (51, 52). Contacts between mitochondria and
ER membranes are also necessary for the biosynthesis of
mitochondrial lipids. This biosynthesis occurs at mitochondria-associated membranes, which correspond to patches of
ER membranes bound to mitochondria that contain several
phospholipids and glycosphingolipid biosynthetic enzymes
(55). Furthermore, Ca2+-sensing ER chaperones are enriched
at mitochondria-associated membranes where they stabilize
Ca2+-handling proteins and thereby regulate signaling of
mitochondrial Ca2+ (56). Mitochondria and ER communicate
by transfer of Ca2+ from 1 organelle to the other (52, 57).
Mitochondria play an important role in regulating intracellular Ca2+ levels by rapidly sequestering Ca2+ released
from the ER (52), and a stable interaction between these
organelles is necessary for Ca2+ uptake by mitochondria from
ER (58). This uptake regulates diverse processes such as
aerobic metabolism, release of caspase cofactors, and feedback control of neighboring ER and plasma membrane Ca2+
channels (52). In stress conditions, an excessive uptake of
Ca2+ by mitochondria can trigger apoptosis by leading to a
decrease of adenosine triphosphate production, an increase in
reactive oxygen species formation, induction of mitochondrial permeability transition pore, and a release of small
proteins such as cytochrome c and apoptosis-inducing factor
from the mitochondria to the cytosol (52). In neuronal
cultures, phosphorylation of tau on T231 by CDK5 led to
its release from microtubules and a concomitant increase of
the retrograde transport of mitochondria and ER membranes,
resulting in their closer apposition around the centrosome,
transfer of Ca2+ from the ER to mitochondria, release of
cytochrome c, caspase activation, and apoptosis (59). In
JNPL3 mice, tau becomes hyperphosphorylated at several
sites including T231 in motor neurons of the spinal cord (19).
We did not observe any difference between the numbers of
mitochondria per micrometer of RER membrane in the soma
of motor neurons in JNPL3 mice. Moreover, although a
decrease of adenosine triphosphate and an increase of
reactive oxygen species production were observed in mice
that overexpress P301L, no apoptotic cell death has been
detected in these mice (28, 44). Interestingly, in a recent
proteomic study in transgenic mice and Drosophila that
overexpress P301L, it was reported that the protein levels of
Bcl-2, an antiapoptotic protein, was increased in regions
such as the cerebellum, which is not affected by tau
pathology (60).
The mechanisms that regulate the interaction between
mitochondria and ER are poorly characterized. High levels of
free cytosolic Ca2+ facilitate the interactions between mitochondria and ER membranes (42), and overexpression of
either normal or mutated tau was accompanied by an
elevation of intracellular Ca2+ levels in neuronal and nonneuronal cells. SH-SY5Y human neuroblastoma cells transfected with the tau mutants, N279K and V337M, display
higher levels of intracellular Ca2+ induced by serum withdrawal than nontransfected cells (61). In the same cells, a
depolarization-induced increase in intracellular Ca2+ was
significantly enhanced by the expression of V337M (62).
Coexpression of AA42 and tau cooperatively elevated basal
levels of myoplasmic-free Ca2+, an effect that was accompanied by depolarization of the plasma membrane (63).
These observations suggest that the overexpression of P301L
in transgenic mice could lead to an increase of contacts
between mitochondria and RER membranes by increasing
intracellular Ca2+ levels.
The amount of tau on RER membranes was significantly higher in JNPL3 mice than in control mice. As
previously reported in neuronal and nonneuronal cells, at the
contact region between RER and mitochondria, no ribosome
was found at the surface of RER membranes in motor
neurons (41, 42). This results in a close apposition of the
RER membrane with the outer mitochondrial membrane, an
event that seems to be necessary for efficient communication
between these membranes. We speculate that accumulation
of tau at the surface of RER could prevent the binding of
ribosomes to RER membranes and thereby favor the
formation of additional contacts between RER and mitochondria. Indeed, ribosomes are located in the cytosol at the initial
stage of protein synthesis before binding to ER membranes
for the completion of the translation process. With respect to
mitochondria, the amount of tau was only increased in 1
JNPL3 mouse, suggesting that the association of tau with
mitochondria is not necessarily involved in an enhanced
interaction between RER and mitochondria. The inconsistency of the amount of tau found at the surface of
mitochondria might indicate that tau has a higher affinity
for binding to RER, Golgi, and the nuclear membrane than to
mitochondria. Tau would then only begin to accumulate at
the surface of mitochondria when its expression reaches a
certain level. This level was not attained in the other
transgenic mouse examined. Taken together, these observations suggest that the increased association of tau with RER
membranes might be responsible for the formation of higher
numbers of contacts between RER and mitochondria in
JNPL3 mice.
The accumulation of tau on RER membranes could
contribute to the alteration of their function leading to
neurodegeneration. The ER is involved in protein synthesis,
protein folding and trafficking, cellular responses to stress,
and Ca2+ homeostasis (57). The ER presents stress-signaling
pathways that enable it to adapt to a panoply of stress factors.
For example, the accumulation of misfolded proteins in the
ER lumen induces an unfolded protein response, which
switches off protein synthesis and upregulates the production
of new chaperones to reestablish ER homeostasis (57). If ER
stress is maintained for too long and ER homeostasis is not
reestablished, the unfolded protein response triggers the
activation of caspases and ultimately apoptosis. Indeed, ER
stressYmediated apoptotic signal pathways may contribute to
the pathogenesis of AD (64). It remains to be determined,
however, whether the accumulation of tau on ER membranes
does contribute to this pathway in AD. The fragmentation of
the Golgi observed in transgenic mice and in primary
neuronal cultures overexpressing either normal or mutated
tau could indicate that the trafficking of protein from the ER
to Golgi is compromised (28). This could result in an
accumulation of proteins in the ER. Another possibility is
that the attachment of tau to ER membranes could modify
512
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J Neuropathol Exp Neurol Volume 68, Number 5, May 2009
RER/Mitochondria Association in Mutant Tau Mice
their biophysical properties and thereby hinder protein
trafficking and exit from these membranes.
The association of tau with the nuclear membranes
composed of RER membranes was increased before the
formation of tau filaments in JNPL3 mice. This is consistent
with previous observations indicating that tau filaments are in
close proximity to the nuclear membranes and pores in AD
brains (27). These membranes could serve as nucleating
structures for tau filaments. It remains unclear, however,
whether the association of tau with the nuclear membrane can
compromise the communication between the nucleus and the
cytoplasm. In a recent study, nuclear transport factor 2 was
found to accumulate in the cytoplasm in a subset of hippocampal neurons with and without NFTs in AD brains (65).
The overexpression of P301L tau results in an increase
of its phosphorylation state in JNPL3 mice (19). We found
that phosphorylated tau at S396/S404 was increased in the
RM subfraction, where the RER marker ribophorin was
enriched. In AD brains, hyperphosphorylated tau was
exclusively found in the RM subfraction enriched in ER
membranes. These results corroborate previous observations
indicating that hyperphosphorylated tau was present on RER
membranes in AD brain (66, 67). Although we cannot exclude
the possibility that hyperphosphorylated tau was associated
with more than 1 membranous element in this subfraction, its
binding to ER membranes was confirmed by double
immunogold labeling. Tau phosphorylated at T231 was also
found to be associated with RER membranes in AD brain by
immunocytochemistry (68), but we observed no signal in the
RM subfraction isolated from the brain of JNPL3 mice using
an antibody directed against tau phosphorylated at T231. The
absence of signal could be explained by the fact that the RM
subfraction was isolated at a very early stage of tau pathology
when tau is moderately phosphorylated. Taken together,
these observations suggest that hyperphosphorylation of tau
might favor its association with RER membranes.
It was recently reported that annonacin, a natural mitochondrial complex I inhibitor, induced a depletion of adenosine triphosphate, an increased retrograde transport of
mitochondria to the cell soma, and the accumulation of
hyperphosphorylated tau in the soma in rat striatal primary
cultures (69). These observations indicate that oxidative
damage and mitochondrial dysfunction observed at an early
stage of AD could trigger the somatodendritic accumulation
of hyperphosphorylated tau (70). In the light of our present
results, this accumulation could in turn amplify the dysfunction of mitochondria by favoring their interaction with RER
membranes. Mitochondrial dysfunction and the somatodendritic accumulation of tau could cooperate to engage a neuron
on an irreversible pathway of cell death in AD brain.
the anti-ribophorin antibody. The authors also thank Dr
Moise Bendayan for helpful discussions.
ACKNOWLEDGMENTS
The authors thank Dr Virginia Lee (University of
Pennsylvania, Philadelphia, PA) for providing the polyclonal
anti-tau antibody, Dr Peter Davies (Albert Einstein University, Bronx, NY) for the phospho-dependent anti-tau antibodies, CP9, CP13, PG-5, and PHF-1, and Dr Gert Kreibich
(New York University School of Medicine, New York, NY) for
Ó 2009 American Association of Neuropathologists, Inc.
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