Neuron

Articlep73 Regulates Neurodegeneration and Phospho-Tau
Accumulation during Aging and Alzheimer’s Disease
Monica K. Wetzel,1,2 Sibel Naska,1,2 Christine L. Laliberté,1 Vladimir V. Rymar,5 Masashi Fujitani,1,2Jeffrey A. Biernaskie,2 Christy J. Cole,3,6 Jason P. Lerch,4 Shoshana Spring,4 S.-H. Wang,3 Paul W. Frankland,3,6
R. Mark Henkelman,4,7 Sheena A. Josselyn,3,6 Abbas F. Sadikot,5 Freda D. Miller,2,6,8,* and David R. Kaplan1,8
1Cell Biology
2Developmental and Stem Cell Biology
3Neuroscience and Mental Health
4Mouse Imaging Groups, Hospital for Sick Children
5Division of Neurosurgery
Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4
6Department of Physiology
7Department of Medical Biophysics
8Department of Molecular Genetics
University of Toronto, Toronto, Canada M5G 1X5*Correspondence: fredam@sickkids.ca
DOI 10.1016/j.neuron.2008.07.021

SUMMARY

The genetic mechanisms that regulate neurodegeneration are only poorly understood. We show thatthe loss of one allele of the p53 family member,p73, makes mice susceptible to neurodegeneration
as a consequence of aging or Alzheimer’s disease(AD). Behavioral analyses demonstrated that old,but not young, p73+/ mice displayed reduced motorand cognitive function, CNS atrophy, and neuronaldegeneration. Unexpectedly, brains of aged p73+/
mice demonstrated dramatic accumulations ofphospho-tau (P-tau)-positive filaments. Moreover,when crossed to a mouse model of AD expressing
a mutant amyloid precursor protein, brains of thesemice showed neuronal degeneration and early androbust formation of tangle-like structures containing
P-tau. The increase in P-tau was likely mediated by
JNK; in p73+/ neurons, the activity of the p73 target
JNK was enhanced, and JNK regulated P-tau levels.
Thus, p73 is essential for preventing neurodegeneration, and haploinsufficiency for p73 may be a susceptibility factor for AD and other neurodegenerative
disorders.
INTRODUCTION
The cellular mechanisms that regulate nervous system degeneration during aging or in neurodegenerative disorders are only
poorly understood, in spite of their clinical importance. For example, much of what we know regarding neural cell death comesfrom studies of naturally occurring developmental death or injury-induced death, but the relevance of these relatively well-understood apoptotic mechanisms to aging or neurodegeneration isstill an open question (for example, see Dusart et al., 2006; Zhu

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et al., 2006). Moreover, the pathology observed in many neurodegenerative disorders indicates that neurons accumulate intracellular protein aggregates and/or cellular damage that leads to theirslow degeneration and death, as opposed to a rapid and controlled apoptotic suicide. Instead, increasing evidence indicates
that, in the aged or degenerating nervous system, prolonged
exposure to relatively minor insults leads to cumulative neuronaloxidative stress and/or DNA damage, which in turn causes
neurons to senesce, reenter the cell cycle and/or atrophy, andultimately degenerate (Lin and Beal, 2006; Herrup and Yang,2007). Thus, both the cause and the enactment of neuronal cell
death differ during development versus aging or degeneration.
While many neurodegenerative disorders appear to be much
more than just accelerated aging, several lines of evidence suggest some similarities. For example, overexpression in Drosophila motor neurons of SOD1, one gene that is mutated in familial
amyotrophic lateral sclerosis (ALS), significantly extends fly
lifespan (Parkes et al., 1998). Moreover, brains of some aged,
apparently normal individuals display neurodegenerative hallmarks such as P-PHF-tau neurofibrillary tangles (Imhof et al.,2007). Thus, perturbations that accelerate neural aging may contribute to and/or predispose individuals to neurodegenerative
disorders.
In this regard, the tumor suppressor p53 and its family member, p63, have been implicated in aging outside of the nervous
system; mouse models with enhanced p53 activity display
accelerated aging and decreased lifespan (Tyner et al., 2002;
Lavigueur et al., 1989; Maier et al., 2004), and aging is enhanced
in p63+/ mice (Keyes et al., 2005). These phenotypes arethought to occur as a consequence of disruptions in p53 family-mediated regulation of genomic stability and cellular senescence, which in turn impact on cellular/organismic aging (reviewed in Papazoglu and Mills, 2007). While aging phenotypes
in these mice were nonneural, they raise the possibility that thethird p53 family member, p73, which is enriched in the PNS
and CNS (Yang et al., 2000; Pozniak et al., 2000), might play
an analogous role in the nervous system. However, althoughNeuronp73 in Neurodegeneration

Figure 1. Aged p73+/ Mice Display Behavioral Changes Indicative of Neurodegeneration(A) Photographs showing the normal clasping behavior of an aged, wild-type, p73+/+ mouse and the abnormal clasping behavior of an aged, p73+/ mouse. Notethat the p73+/ mouse draws its limbs into its body rather than splaying them away from its trunk.
(B) Quantification of the percentage of young or aged p73+/+ versus p73+/ mice displaying the abnormal clasping behavior shown in (A).
(C–F) Young (3-month-old) and aged (17-month-old) male p73+/+ and p73+/ mice were tested for behavioral changes associated with neurodegeneration (n = 13
young p73+/+, 15 young p73+/ , 16 aged p73+/+, and 12 aged p73+/ mice). (C) The mean time mice spent hanging from a suspended grid. Mice were tested threetimes, and the maximum time was recorded. (D–F) Results of the open-field test, showing representative traces of open-field exploration during a 30 min trial (D),the mean percent time spent in the interior versus exterior of the open field (E), and the mean total distance traveled during the trial (F).
(G–I) Results of the Morris water maze test, showing the mean percent time spent in the target versus nontarget zone during the probe test (G), the mean time toreach the platform (latency) (H), and the mean swim speed (J). (n = 16 aged p73+/+ and n = 12 aged p73+/ mice).
All values are mean ± SEM. *p < 0.05, ***p < 0.001, NS = p > 0.05. In all panels, WT = p73+/+ and HET = p73+/ .

we previously showed that p73 is predominantly expressed asa truncated, dominant-inhibitory isoform, DNp73, that preventsneuronal apoptosis during development and following injury(Pozniak et al., 2000, 2002; Walsh et al., 2004), there is noevidence to suggest that p73 is important for cellular necrosis
or degeneration. Nonetheless, two recent reports indicate that
decreased p73 expression occurs in some cases of AD (Liet al., 2004) and that some individuals have lost one copy of the
genomic locus encompassing the p73 gene (Wong et al., 2007).
Here, we have asked whether p73 might play a previously
unsuspected role in neurodegeneration and show that haploinsufficiency for p73 causes age-related neurodegeneration.
Moreover, we demonstrate that decreased p73 causes formation of P-PHF-tau tangle-like structures in the aged nervous system, and coincident with the formation of amyloid plaques in an
amyloid-based mouse model for AD. Thus, p73 is essential toprevent neurodegeneration during aging, and heterozygosity

for p73 may be a susceptibility factor for AD or other neurodegenerative disorders.
RESULTS
Normal Levels of p73 Are Essential to Prevent
Behavioral Changes Indicative of Age-Related
Neurodegeneration
To ask whether changes in p73 copy number would sensitize
mice to neurodegeneration, we compared the behavior of young(3- to 4-month-old) and aged (16- to 18-month-old) p73+/ versus p73+/+ mice. Initially, we examined an abnormal clasping
behavior seen in several mouse neurodegeneration models(Liou et al., 2003; Lewis et al., 2000), where mice clasp their limbs
into their bodies when picked up by their tails. Aged p73+/ but
not p73+/+ adults displayed this abnormal clasping behavior(Figure 1A and Movie S1 available online). Young adult p73+/

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Figure 2. p73 Haploinsufficiency Causes Age-Related Neuronal Degeneration and CNS Atrophy(A) Combined representative MR images of 5 p73+/ aged mouse brains where blue indicates regions that are significantly smaller and red regions that are significantly larger with over 95% certainty in aged p73+/ versus p73+/+ mouse brains. The reconstruction of the entire combined brain is shown in Movie S1. The redregions encompass the space between the skull and brain, which is larger in aged p73+/ mice due to brain atrophy.
(B and C) The mean relative voxel size in aged (B) and young (C) brain regions identified as significantly different between p73+/+ versus p73+/ mice in the MR
image atlas created in (A). (n = 5 per group). All values are mean ± SEM. *p < 0.05. MC, motor cortex; CC, corpus callosum; DG, dentate gyrus; PC, posterior
cerebellum.
(D) Photomicrograph of the 325 mm strip of motor cortex (boxed) that was assessed for quantification. LV, lateral ventricle.
(E) Representative photomicrographs of Nissl-stained coronal sections at the level of the motor cortex shown in (D) from aged (17-month-old) p73+/+ and p73+/
brains.
(F) Mean cortical thickness as determined by measuring the distance from the corpus callosum to the pia in photomicrographs similar to those shown in (E) (n = 5
mice of each genotype).
(G) Mean density of Nissl-stained neurons in the motor cortex of aged p73+/+ versus p73+/ brains (n = 5 per group).
(H) Absolute number of primary motor cortex neurons in p73+/+ and p73+/ brains as determined using the optical fractionator method. (*p < 0.05, n = 3 per group).
(I) Absolute number of glial cells in the primary motor cortex of p73+/+ and p73+/ brains as determined using the optical fractionator method (n = 3 per group).
(J) Representative photomicrograph of a Nissl-stained section through the p73+/+ motor cortex showing condensed cells (arrow).
(K) Mean density of small, condensed cells in the motor cortex of young and aged p73+/+ and p73+/ brains (*p < 0.05, n = 5 per group).

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mice were normal but acquired this behavior with time, so that
almost 75% were positive by 16–18 months of age (Figure 1B).
We then performed a battery of neurological and sensory-motor assays on young and aged p73+/ versus p73+/+ mice, including the sticky dot test, the elevated body swing test, the righting
reflex text, the negative geotaxis test, the paw grip endurance
test, a cylinder rearing test, a grid walking test, the open-field
test, and gait analysis. Aged, but not young, p73+/ mice performed poorly on two, the paw grip endurance and open-fieldtests. In the former, aged p73+/ mice maintained their grip fora significantly shorter time than any other group, including
aged p73+/+ mice (Figure 1C). In the open-field test, aged
p73+/ mice spent significantly more time in the center of theopen field than any other group (Figures 1D and 1E), although
the distance they traveled was similar (Figure 1F), suggestinga reduction in overall anxiety levels.
Since spatial learning and memory are also perturbed in mousemodels of neurodegeneration, we asked whether aged p73+/
mice were impaired in this regard using the hidden platform version of the Morris water maze (Kobayashi and Chen, 2005). In thistest, an invisible escape platform is submerged just below waterlevel, and white curtains are marked with distinct cues, thereby
allowing mice to triangulate the hidden platform location. Mice
were trained to find the platform and assessed on their ability
to spatially learn and remember the platform location. Agedp73+/ mice were significantly impaired in their ability to discriminate the platform location (as measured by percent time in theplatform zone) and in the length of time (latency) it took to findthe platform (Figures 1G and 1H), although they swam at a speed
similar to aged p73+/+ mice (Figure 1I). In contrast, young p73+/
and p73+/+ mice behaved similarly on this test (data not shown).
Thus, aged p73+/ mice displayed a number of behaviors diagnostic of age-related neurodegeneration.
p73+/ Mice Show Anatomical Evidence of Age-Related
Neurodegeneration
To ask about neuroanatomical perturbations that might cause
these behavioral abnormalities, we performed 3D magnetic resonance imaging (MRI) and computer analysis. Digital images
from within the skulls of perfused young and aged p73+/+ andp73+/ murine mice were acquired with a multichannel 7.0 Tesla
MRI scanner, allowing for resolution and volume analysis of finenuclei and white matter tracts. Computer analysis created an
unbiased model-independent atlas of all MR scans, which were
deformed into exact alignment with each other in an unbiased
fashion; quantitative volume differences of individual mouseneuroanatomical features were determined based upon this collective brain atlas. This analysis revealed that, relative to aged
p73+/+ mice, aged p73+/ mice displayed a 5%–16% reduction
in volume of selected brain regions, including the motor cortex,
corpus callosum, hippocampal dentate gyrus, and posterior cerebellum (Figures 2A and 2B and Movie S2). In contrast, young

p73+/ versus p73+/+ brains were statistically similar in all theseregions (Figure 2C). The volume perturbations in motor cortex
and cerebellum could explain the aberrant clasping and grip
test behaviors, and the dentate gyrus atrophy could explain
the poor water maze performance.
To ask about the cellular basis of this atrophy, we analyzed cytoarchitecture of Nissl-stained sections through one affected region, the motor cortex. Measurement of coronal sections at
equivalent rostrocaudal levels revealed that width of the motor
cortex from corpus callosum to the pia (see boxed region in
Figure 2D) was thinner in aged p73+/ versus p73+/+ mice (Figures 2E and 2F). Neuronal density was also decreased in aged
p73+/ mice (Figure 2G). To confirm this, we performed unbiased
stereology using the optical fractionator method (Gundersen
et al., 1988; West et al., 1996; Luk and Sadikot, 2003; Luk
et al., 2001). The mean coefficient of error (Gundersen andJensen, 1987) of Nissl-stained neuronal counts was 0.9 ±0.005 (n = 4). In aged p73+/ primary motor cortex, there was a
reduction (29%) in total neuron number (743,531 ± 83,667 versus
1,054,281 ± 30,512 in aged controls, mean ± SEM, n = 3;
p < 0.05) (Figure 2H) and in total volumetric density (28%;
129,144 ± 7,967 versus 179,535 ± 12,601 in aged controls;
number/mm3, mean ± SEM, n = 3; p < 0.05). In contrast, total glial
cell number was unchanged (Figure 2I).
We also observed an increase in density of small, condensed
cells in the aged p73+/ motor cortex (Figures 2J and 2K). Someof these expressed the neuronal marker NeuN (Figure 2L), but
not the astrocytic marker GFAP or the microglial marker Iba1
(data not shown). To ask whether these were dying neurons,
we performed silver staining for degenerating cells. This analysis
revealed, in aged p73+/ but not p73+/+ brains, condensed, silver-stained, degenerating cells, many with dystrophic neurites(Figure 2M), identifying them as neurons. There were also
many silver-stained neuritic processes (Figure 2M). In contrast,
young p73+/ and p73+/+ brains did not contain degenerating
cells (data not shown), and both genotypes were similar with regard to cortical thickness (p > 0.05) and density of neurons andcondensed cells (Figures 2G and 2K). Thus, neuronal degeneration likely accounts for the atrophy of aged p73+/ brains.
Neurodegenerative disorders like Parkinson’s disease and AD
also manifest in the PNS (Hawkes, 2006; Goldstein, 2003). In thisregard, we observed that 67% of aged p73+/ , but not p73+/+,mice displayed prolapse of the penis and/or rectum (12/21 penis
only, 4/21 rectum only, 2/21 both; Figure S1A). To ask whether
this was due to peripheral neurodegeneration, we characterizedparasympathetic neurons of the major pelvic ganglion, which
innervates the penis. Immunostaining for the parasympathetic
marker nNOS and the panneuronal marker PGP9.5 revealed
that pelvic ganglia from aged prolapsed p73+/ mice contained
many fewer neurons (Figures S1B and S1C), and those neurons
that were present were shrunken. Thus, p73+/ mice displayed
phenotypes arising from degeneration of both the CNS and PNS.

(L) Immunostaining for NeuN (red) in the motor cortex showing that some of the condensed cells are shrunken, potentially degenerating, neurons (arrow). Section
was counterstained with Hoechst to show nuclei (blue).
(M) Photomicrographs of silver-stained p73+/ cortices, showing argyrophilic, degenerating condensed neurons with processes (left panel), larger degenerating
cells (middle panel), and degenerating neuritic processes (right panel).
In all panels, arrowheads denote cell bodies, and arrows degenerating neuritic processes. In all panels, WT = p73+/+, and HET = p73+/ .

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Figure 3. Aged p73+/ Brains Display Hallmarks of Neurodegeneration(A) Mean density of microglia in the motor cortex of aged p73+/+ versus p73+/ mice (n = 5 per group).
(B) Representative photomicrographs of sections through the aged p73+/+ (Aged WT) and p73+/ (Aged HET) motor cortex immunostained for the reactive
astrocyte marker GFAP (green).
(C) Relative mean lipofuscin particle size in the motor cortex of aged p73+/+ versus p73+/ mice (n = 5 per group).
(D) Photomicrograph of neurons within the motor cortex immunostained for NeuN (green) and PCNA (red). Arrows indicate double-labeled cells.
(E) Representative flow cytometry plots of cortical cells from aged p73+/+ versus p73+/ brains stained with neuron-specific NeuN (y axis) and with propidium
iodide (PI, x axis) to assess neuronal DNA content. The antibody control, where primary antibody was omitted, but propidium iodide was included, is shownfor comparison. The red boxes indicate the relevant NeuN-positive population, and numbers to the right of these boxes indicate the percentage of total cells
that were NeuN positive and that had a DNA content indicative of G2 or M phase.
(F) Flow cytometry plots similar to those shown in (E) were used to determine the normalized relative percentage of neurons in the G2/M phase of the cell cycle(n = 6 per group). The proportion of neurons in the aged p73+/+ cortex that were in G2/M phase was considered to be 100%.
(G) Photomicrograph of neurons in the motor cortex stained for SA-b-galactosidase (blue) and then immunostained for NeuN (brown).
(H) Representative photomicrographs of staining for the senescence marker SA-b-galactosidase (blue) in aged p73+/+ and p73+/ motor cortex, showing the more
lightly staining cells that were quantified.

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Aged p73+/ Brains Display Microglial Activationand Lipofuscin Accumulation
Since neurodegenerative disorders such as AD display activation of microglia and astrocytes, we asked whether similarchanges might occur in aged p73+/ brains. Immunostaining
for Iba-1 revealed increased microglial density throughout
aged p73+/ versus p73+/+ brains, and quantification within
a 325 mm wide strip of motor cortex spanning from the corpus
callosum to the pia (see Figure 2D) demonstrated an increaseof 3-fold (Figures 3A and S2A). Similarly, immunostaining for
GFAP showed increased reactive astrocytes in the aged p73+/
motor cortex (Figure 3B).
Another hallmark of neurodegeneration is intralysosomal accumulation of the oxidative stress biomarker lipofuscin, a causeof premature dementia in Batten’s disease (Seehafer andPearce, 2006; Haltia et al., 2006). Lipofuscin autofluorescence
was increased throughout aged p73+/ versus p73+/+ brains(Figure S2B), and quantification demonstrated an 2.5-fold increase in lipofuscin particle size in the aged p73+/ motor cortex(Figure 3C). Thus, normal levels of p73 are necessary to prevent
brains from degenerating as they age.

Normal Levels of p73 Are Essential to Prevent Neuronal
Cell Cycle Reentry and Cellular Senescence duringAging
One pathological event observed in AD is aberrant re-expressionof cell cycle proteins and re-entry of neurons into the cell cycle(Herrup and Yang, 2007). Since p73 can directly regulate thecell cycle (Talos et al., 2007), we asked whether this also occurredin aged p73+/ mice. Immunostaining of forebrain sections revealed a subpopulation of NeuN-positive neurons that aberrantly
expressed the cell cycle marker PCNA in the motor cortex and adjacent lateral cortical region of aged p73+/ but not p73+/+ mice
(Figure 3D). To confirm and quantify this result, we isolated cerebral cortices, immunostained the triturated cortical tissue for
NeuN, stained with propidium iodide to monitor cell cycle status,and performed flow cytometry, gating for single cells. Approximately 0.8% of wild-type neurons had a DNA content indicativeof the G2 or M phase of the cell cycle (Figure 3E), a number thatwas increased as much as 2-fold with p73+/ neurons (Figures
3E and 3F). In contrast, the percentage of NeuN-negative nonneuronal cells in G2/M phase was similar in both genotypes (p >0.05). Thus, p73 regulates neuronal cell cycle reentry as mice age.
Cellular senescence also occurs as cells and animals age, andthe p53 family, including p73, is implicated in regulation of senescence (Keyes et al., 2005; Talos et al., 2007; Papazoglu and Mills,2007). We therefore double labeled forebrain sections for NeuNand for a marker of senescing cells, senescence-associatedb-galactosidase (SA-b-Gal) (Serrano et al., 1997). Two populationsof SA-b-Gal-positive cells were seen in the aged cortex; NeuNpositive neurons (Figure 3G) and NeuN-negative, vasculature-associated cells that were more darkly stained for SA-b-Gal. Quantification revealed a significant increase in the more lightly stained
cells in aged p73+/ versus p73+/+ motor cortex (Figures 3H and

3I). Thus, p73 haploinsufficiency causes increased neuronal expression of a senescence marker as animals age.
Haploinsufficiency for p73 Causes Formationof Aberrant Phosphorylated Tau Filamentsin the Aged Brain
In humans, high levels of filamentous hyperphosphorylated tau
protein are thought to be one of the underlying causes of neurodegeneration (Ballatore et al., 2007). We therefore immunostained sections from brains of young and aged p73+/ andp73+/+ mice with three antibodies that recognize phosphotau-containing paired helical filaments (P-PHF-tau), AT-8 (phospho-Ser202 and phospho-Thr205; Goedert et al., 1995), AT-180(phospho-Thr231), and AT-100 (phospho-Ser212 and phosphoThr214). No P-PHF-tau immunoreactivity was observed in brainsof young animals or of aged p73+/+ mice (Figure 4A). Unexpectedly, all of the aged p73+/ brains contained abundant P-PHFtau immunoreactivity, as visualized with the three antibodies(Figures 4A–4D). Phosphatase treatment abolished this immunostaining (Figure S3A), confirming the labeling specificity. This
P-PHF-tau immunoreactivity was localized throughout the brain,including the motor cortex (Figures 4A–4D), the hippocampus,
the subiculum, (Figure 4E), and the piriform cortex (data notshown). P-PHF-tau immunoreactivity was localized within cells
and their neuritic processes (Figures 4A–4E), and many positive
cells coexpressed bIII-tubulin (data not shown), confirming theiridentity as neurons. P-PHF-tau immunoreactivity was alsopresent within neuropil threads and dystrophic neurites. Quantification within a defined region of the lateral cortex where
P-PHF-tau structures were consistently present (Figure 4F)
demonstrated the specificity of this immunoreactivity for aged,
p73+/ brains (Figures 4G and 4H). In contrast, b-amyloidpositive plaques, as detected by thioflavin-T staining, were notpresent in brains of either genotype (data not shown).
Two different approaches confirmed that this staining reflected P-PHF-tau filaments. First, immuno-electron microscopy
with AT-8 on aged p73+/ cortices revealed immunoreactive
filaments, either scattered or in bundles, within neuronal cellbodies (Figure 4I) and axons (Figure 4J). Second, we fractionatedp73+/ versus p73+/+ brains to enrich for P-PHF-tau filaments(Ikeda et al., 2005); western blots confirmed the presence ofAT-8-immunopositive P-PHF-tau filaments in these fractions(Figure S3B). Thus, p73 haploinsufficiency causes formation of
P-PHF-tau tangle-like structures similar to those seen in neurodegenerative disorders.
Haploinsufficiency for p73 Causes Neurodegenerationand Early Formation of Phospho-Tau-Containing
Filaments in an Amyloid-Based Alzheimer’s DiseaseModel
The hallmarks of human AD are b-amyloid-positive plaques,
P-PHF-tau-positive tangles, and neurodegeneration. However,most amyloid precursor protein (APP)-based mouse models
show plaques, but not neurodegeneration, and if they do(I) Mean density of SA-b-galactosidase-positive cells in the motor cortex (n = 5 per group).
All values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. In all panels, WT = p73+/+, and HET = p73+/ . Scale bar, 10 mm.

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Figure 4. p73 Haploinsufficiency Leads to Accumulation of P-PHF-Tau Filaments in the Aged Brain(A) Representative photomicrographs of coronal sections of
the aged p73+/+, young and aged p73+/ lateral cortex immunostained with P-PHF-tau AT-8 antibody.
(B–D) Aged p73+/ brain sections labeled with three different
antibodies for P-PHF-tau (red) and Hoechst nuclear dye(blue), AT-8 (B), AT-180 (C), and AT-100 (D). Scale bar,
60 mm (A), 10 mm (B and C), 2.5 mm (D).
(E) Photomicrographs of hippocampal neurons displaying somatodendritic localization of P-PHF-tau (AT-180, red) within
the subiculum (left panel) dentate gyrus (center panel), andCA1 pyramidal layer (right panel).
(F–H) Quantification of P-PHF-tau immunoreactivity in thecortex (region shown in [F]) in young and aged p73+/+ versus
p73+/ brains, as assayed using the AT-8 (G) and AT-180 (H)antibodies (n = 4 per group). Only aged p73+/ brains werepositive for P-PHF-tau.
(I and J) Immuno-electron microscopy was performed to detect P-PHF-tau ultrastructurally in the cerebral cortex ofaged p73+/ mouse brains using the P-PHF-tau AT-8 antibody
visualized with 10 nm gold conjugated secondary antibodies.
Immunoreactivity was detected within neuronal cell bodies (I)and myelinated axons (J). In panel (I), the arrow indicates goldlabeling of cell body filament aggregates, and the inset showsa higher-magnification view of these filaments. The arrowhead
indicates another immunolabeled structure. In panel (J), thearrow indicates a myelinated axon, with the immunopositive
filaments shown at higher magnification in the inset. The arrowhead indicates immunolabeled aggregates in a neuronalcell body. Scale bar, 0.2 mm. ER, endoplasmic reticulum;
mito, mitochondrion.
All values are mean ± SEM. *p < 0.05. In all panels, WT =p73+/+, and HET = p73+/ .

develop tangles, this only occurs long after plaque formation.
Since p73 haploinsufficiency causes both neurodegeneration
and P-PHF-tau positive filaments, we asked whether it might
be a susceptibility factor for AD. To do this, we crossed p73+/
mice with the transgenic CRND8 (TgCRND8+/ø) mouse modelof AD (Chishti et al., 2001). These mice encode a double-mutant
form of APP 695 (KM670/671NL+V717F) under the control of the
PrP gene promoter and develop plaques, but not tangles, by 3months and do not display neuronal loss. We analyzed thesecrosses at 1.5–2 months, a time point early during plaque formation, first quantifying plaque density by thioflavin T staining andimmunocytochemistry with two antibodies, the 4G8 antibody
specific for human APP (Figure 5C) and the 6E10 human APP

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C-terminal-specific antibody (data not shown).
This analysis demonstrated a small number of plaques in 1.5- to 2-month-old TgCRND8+/ø brains, regardless of p73 genotype (Figures 5A–5D).
We then immunostained brains with AT-8 and
AT-180 to reveal P-PHF-tau. As reported (Bellucci
et al., 2007), there was no immunoreactivity detected by either antibody in young TgCRND8+/ø
brains (Figures 5E and 5F). In contrast, all of the
p73+/ ; TgCRND8+/ø brains displayed abundant
P-PHF-tau filaments/deposits at 1.5–2 months ofage (Figures 5E and 5F). These P-PHF-tau structures were consistently present in the lateral cortex and, toa lesser degree, in other forebrain regions and in the hippocampus and subiculum (Figure S4). Quantification in the lateral cortex demonstrated the specificity of these P-PHF-tau filaments
for the p73+/ ; TgCRND8+/ø brains (Figures 5G and 5H). Thus,
p73 haploinsufficiency and overexpression of mutant APP together cause P-PHF-tau accumulation early during the ADtype neuropathology.
We then asked whether p73 heterozygosity led to neuronalloss in these mice. Stereological analysis, using the optical fractionator method, demonstrated that total primary motor cortex
neuron number was reduced by 22% in p73+/ ; TgCRND8+/ø
mice versus their TgCRND8+/ø littermates (721,617 ± 50,757

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Figure 5. p73 Haploinsufficiency Causes Neuronal
Degeneration and Early Formation of Phospho-TauPositive Filaments in TgCRND8 Mice, an AD Mouse
Model(A–D) Quantification of amyloid plaques in the cortex of
2-month-old p73+/+;TgCRND8+/B (WT;AD) versus p73+/ ;TgCRND8+/B (HET;AD) mice, as detected and quantified using(A and B) Thioflavin T or (C and D) immunocytochemistry forhuman APP-specific antibody 4G8 (red), where the insets
show higher-magnification micrographs of amyloid plaques.
In (C), sections were counterstained with Hoechst to show
nuclei (blue). Scale bar, 500 mm.
(E and F) Representative photomicrographs of coronal sections of the lateral cortex of 1.5- to 2-month-old TgCRND8
mice immunostained with two antibodies for P-PHF-tau(red), AT-8 (E) and AT-180 (F). Some panels were counterstained with Hoechst (blue). Scale bar, 60 mm (left and center
panels in [E] and [F]), 10 mm (right panels in [E] and [F]).
(G–I) Quantification of P-PHF-tau immunoreactivity in the cortex (region shown in [G]) in 1.5- to 2-month-old TgCRND8+/B
brains that were wild-type or heterozygous for the p73 gene,as assayed with the AT-8 antibody. p73+/+ and p73+/ brainsof the same age that did not carry the APP transgene (NTg)were also characterized. Only p73+/ ;TgCRND8+/B brains
were positive for P-PHF-tau. n = 3 each for p73+/+, p73+/ ,p73++-;TgCRND8+/B, and 4 for p73+/ ;TgCRND8+/B. (I) Total
number of primary motor cortex neurons was quantified bystereological methods for p73+/ ;TgCRND8+/B versus
p73+/ ;TgCRND8+/B brains. n = 5 for each group.
(J and K) Silver staining was performed to detect argyrophilic,degenerating neurons in (J) 2-month-old and (K) 12-month-old
p73+/ ;TgCRND8+/B brains. In all panels, arrowheads denotesilver stain-positive cell bodies, while the arrows denote silver
stain-positive neuritic processes. In older brains, the amyloid
plaques were also silver stain-positive ([K], right panel).
All values are mean ± SEM. *p < 0.05, **p < 0.01. In all panels,
WT = p73+/+, and HET = p73+/ ; AD = TgCRND8+/B; NTg = animals that did not carry the TgCRND8 transgene.

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Neuronp73 in Neurodegeneration

Figure 6. p73+/ Loss of p73 Causes an Increase in JNK Activation and JNK-MediatedTau Phosphorylation(A–C) Quantitative RT-PCR analysis for p53,
Tap63 and PUMA, and p21, two targets of the
p53 family on cerebral cortices from (A and B)
16- to 17-month-old p73+/+ and p73+/ mice (C)12-month-old p73+/+ and p73+/ mice, n = 3 foreach genotype at each time point.
(D) Western blot analysis of equal amounts of protein from 8 day sister cultures of p73+/+, p73+/ ,and p73 / embryonic cortical neurons probed
for phosphorylated, activated JNK (P-JNK). The
blot was reprobed for total JNK, and ERK as loading controls. Similar results were obtained in three
independent experiments.
(E and F) p73+/+, p73+/ , and p73 / cortical neurons (n = 6 per group from three separate experiments) were exposed to glutamate (Glu) for 24 hr
and (E) percent survival was calculated (Hoechstpositive nuclei before and after glutamate), and (F)lactate dehydrogenase (LDH) released into themedium was measured.
(G) Western blot analysis was performed for phosphorylated tau (P-tau) with the AT-8 antibody in embryonic day 16 p73+/+ and p73 / cortices, and the blot was
reprobed with ERK as a loading control.
(H and I) Cultured cortical neurons were treated with JNK inhibitor (SP 6000125, 20 mM for 90 min). JNK activity was assessed by western blot analysis for phosphorylated c-jun (P-cJun) (H). The effects of JNK inhibition on tau phosphorylation were determined by probing similar western blots with the AT-8 antibody (I). In
both cases, blots were reprobed for PLC-g.
In all panels, WT = p73+/+, HET = p73+/ , KO = p73 / . Values indicate mean ± SEM. *p < 0.05, ***p < 0.001.

versus 927,555 ± 25,798, respectively; mean ± SEM, n = 4; p <0.05) (Figure 5I). Silver staining also revealed the presence of degenerating cells, many with dystrophic neurites identifying themas neurons, in the 2-month-old p73+/ ; TgCRND8+/ø brains (Figure 5J). By 12 months of age, this degeneration was even more
dramatic (Figure 5K), with many condensed, dystrophic neurons
present throughout the brain and numerous silver stain-positive
neurites (Figure 5K). In contrast, only the very occasional silver
stain-positive cell was observed in the 12-month-old
TgCRND8+/ø brain, although we did observe silver-stained plaques (Figure 5K; data not shown). Thus, p73 haploinsufficiency
causes neuronal loss and degeneration coincident with accumulation of P-PHF-tau filaments in an APP-based mouse model ofAD.
p73 Modulates JNK Activity to Regulate Neuronal
Survival and Tau Phosphorylation
Since DNp73 antagonizes proapoptotic p53 family members,
one potential explanation for these findings is upregulation of
the levels and/or activity of p53 or p63 (Jacobs et al., 2005). However, quantitative RT-PCR (QRT-PCR) of aged p73+/ versus
p73+/+ cortices demonstrated that levels of p53 and p63 mRNAs,
and their downstream targets p21 and PUMA were unaltered by
p73 haploinsufficiency (Figures 6A–6C). A second potential explanation involves JNK, since we previously showed that
DNp73 can bind to and inhibit JNK (Lee et al., 2004), and JNK
has been implicated in both neurodegeneration and aberrant
tau phosphorylation (reviewed in Johnson and Nakamura,2007). We therefore measured JNK activity in cultured p73+/+,
p73+/ , and p73 / cortical neurons. Western blot analysis revealed that levels of the phosphorylated, activated form of JNK

716 Neuron 59, 708–721, September 11, 2008 ª2008 Elsevier Inc.

increased as the number of wild-type p73 alleles decreased(Figure 6D). Coincident with this increase in JNK activity, weobserved that p73+/ and p73 / cortical neurons were more
vulnerable to death in response to mildly excitotoxic concentrations of the neurotransmitter glutamate (Figures 6E and S5).
Measurement of lactate dehydrogenase (LDH) release into themedium, a biochemical indicator of necrosis, demonstrated
that, unexpectedly, LDH release increased as p73 copy number
decreased (Figure 6F). Thus, decreased p73 copy number
causes increased JNK activity and sensitizes neurons to oxidative damage-induced necrosis.
Since JNK directly phosphorylates tau on residues known tobe hyperphosphorylated in P-PHF-tau (Reynolds et al., 1997,
2000; Yoshida et al., 2004), we asked whether JNK dysregulation
might also explain the aberrant P-PHF-tau filaments. Western
blot analysis revealed increased tau phosphorylation at Ser202/
Thr205, targets of JNK-mediated phosphorylation, in neonatal
p73 / versus p73+/+ cortices (Figure 6G). To ask whether thistau phosphorylation was JNK dependent, we cultured corticalneurons and treated them for 90 min with the JNK inhibitor
SP6000125. SP6000125 decreased phosphorylation of the
JNK target, c-jun, confirming the efficacy of the inhibitor(Figure 6H). The JNK inhibitor significantly decreased tau phosphorylation in both p73+/+ and p73 / neurons (Figure 6I).
These data indicate that loss of p73 causes increased JNK activity and tau phosphorylation in cultured neurons. To ask
whether JNK dysregulation might also explain the phenotypes
documented here, we performed double-label immunocytochemistry for phospho-JNK and P-PHF-tau in the 2-month-old
p73+/ ; TgCRND8+/ø cortex. Few phospho-JNK-positive neurons were seen in the TgCRND8 brains at this age, as previouslyNeuronp73 in Neurodegeneration

Figure 7. p73 Haploinsufficiency Causes Aberrant
JNK Activation in P-PHF-Tau-Positive Neurons in the
Aged and AD Brain(A) Representative photomicrographs of coronal sections of
the 2-month-old p73+/ ;TgCRND8+/B lateral cortex double labeled for P-JNK (red; left panel), and P-PHF-tau with the AT-8
antibody (green, center panel; right panel is the merge, including the Hoechst counterstain in blue). Arrows denote doublelabeled cells, the arrowhead a P-PHF-tau-positive cell, andthe asterisk a P-JNK-positive cell.
(B–D) Cortical sections immunostained as in (A) were quantified to determine the number of cells that were positive for(B) P-JNK alone, (C) P-tau alone, and (D) P-JNK and P-tau
double-labeled cells.
(E) Representative photomicrographs of coronal sections of
the 16- to 17-month-old p73+/ lateral cortex double labeledfor P-JNK (red; left panel), and P-PHF-tau with the AT-8 antibody (green, center panel; right panel is the merge, including
the Hoechst counterstain in blue). Arrows denote doublelabeled cells, and arrowhead P-PHF-tau-positive cells.
(F–H) Cortical sections immunostained as in (A) were quantified to determine the number of cells that were positive for(B) P-JNK alone, (C) P-tau alone, and (D) P-JNK and P-tau
double-labeled cells. *p < 0.05, **p < 0.001. n = 3 animals
from each genotype.

reported (Bellucci et al., 2007). However, the p73+/ ; TgCRND8+/ø
brains contained many phospho-JNK-positive cells (Figure 7A);
quantification revealed a 20-fold increase in their density within
the lateral cortex (Figure 7B). Most of these phospho-JNK-positive cells were also positive for P-PHF-tau Ser202/Thr205 phosphorylation (Figures 7A–7D). Similar results were obtained in theaged p73+/ brains; many more phospho-JNK-positive cells
were present in aged p73+/ versus p73+/+ cortices (Figures 7E
and 7F), and many of these colocalized with P-PHF-tau
Ser202/Thr205 (Figures 7E, 7G, and 7H). Thus, p73 haploinsufficiency causes increased neuronal JNK activity that likely participates in the genesis of P-PHF-tau pathology.
DISCUSSION
The findings presented here support three major conclusions.
First, our data demonstrate that haploinsufficiency for p73 leads
to behavioral and anatomical changes indicative of age-related
neurodegeneration. Some of these changes, such as increased
microglial activation and enhanced lipofuscin, are likely second-

ary to neuronal degeneration. However, the neuronal cell loss, increased neuronal senescence, increased reentry of neurons into the cell cycle, andaccumulation of P-PHF-tau filaments are all likely
to be direct effects of the loss of p73. Second, we
demonstrate that p73 haploinsufficiency causes
the amyloid-based TgCRND8 AD mouse model tocoincidentally develop the three primary pathologies associated with AD—plaques, P-PHF-tau deposits, and neuronal degeneration—at the same
early time point. This accumulation of P-PHF-tau
deposits is seen as early as 1.5 months, which ismuch sooner than in either the p73+/ mice or in
the TgCRND8 AD mice, indicating that these two phenotypes
synergize. These animals therefore represent a new mousemodel for AD. Third, our mechanistic studies indicate that haploinsufficiency for p73 likely causes both neuronal degeneration/loss and P-PHF-tau filament accumulation by directly regulating JNK. These findings, together with recent reports that
decreased p73 expression occurs in some cases of AD (Liet al., 2004) and that some individuals show loss of one copyof the genomic locus encompassing the p73 gene (Wong et al.,2007), suggest that haploinsufficiency for p73 may represent
a previously unrecognized susceptibility factor for AD.
One surprising result of our study was the occurrence in aged
p73+/ mice of multiple hallmarks of neurodegeneration, including phosphorylated tau tangle-like structures, lipofuscin deposits, activation of microglia, cellular senescence, and aberrant entry of neurons into the cell cycle. Aberrant tau
regulation is prominent in dementias such as AD (reviewed inJohnson and Stoothoff, 2004), and buildup of lipofuscin causes
Batten’s disease, an inherited disease with premature dementia(reviewed in Haltia et al., 2006). Microglia likely contribute to

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Neuronp73 in Neurodegenerationneurodegeneration, since immunosuppression decreases tangles and neuronal loss and increases lifespan in mice expressing mutant tau (Yoshiyama et al., 2007). Reentry of neurons into
the cell cycle has been noted in early-stage AD (reviewed in
Herrup and Yang, 2007), and postmitotic neurons forced to reenter the cell cycle die by p53-dependent apoptosis (reviewed
in Jacobs et al., 2006). Cellular senescence is also associatedwith aging in mammals. While neurons are postmitotic, andsenescence involves cell cycle arrest, these two states are notnecessarily equivalent, and it is possible that postmitotic neurons directly enter a senescent state as a consequence of accumulated DNA damage. Alternatively, damaged neurons may
aberrantly reenter the cell cycle, as observed here, and perhaps
it is this population that then enters a senescent state. In either
case, these senescent neurons are abnormal and may wellcontribute to the pathology described here. Thus, aged p73+/
brains show all of the hallmarks of a neurodegenerative
phenotype.
How does p73 haploinsufficiency cause neurodegeneration?
A characteristic of the aging brain is increased vulnerability to
toxic insults such as oxidative stress and the resultant DNA damage. In this regard, we show here that loss of one or two alleles ofp73 markedly enhances the vulnerability of CNS neurons to excitotoxicity-induced necrotic cell death, and we have previously
shown that overexpression of truncated DNp73 protects neurons from DNA damage-induced apoptosis (Pozniak et al.,
2002; Walsh et al., 2004). We propose that the neurodegeneration phenotypes reported here are the result of reduced DNp73
levels, which is the only detectable p73 isoform in the postnatal
mouse CNS (Pozniak et al., 2000). DNp73 binds to and suppresses the activity of two important regulators of neural death,p53 (Pozniak et al., 2000) and JNK (Lee et al., 2004), and data
presented here indicate that it is the latter pathway that is perturbed in the aged p73+/ brain. This aberrant JNK activation
would sensitize cells to oxidative damage, ultimately causing
cell senescence and loss (Yang et al., 1997). JNK is also a tau kinase (Reynolds et al., 1997, 2000; Yoshida et al., 2004), and, as
seen here, aberrant upregulation of tau phosphorylation likely
causes genesis of P-PHF-tau filaments, which may themselvescause neuronal degeneration.
A second surprising finding of our study is that the combination of p73 haploinsufficiency and overexpression of mutant
APP causes neurodegeneration and P-PHF-tau accumulationmuch earlier than is seen in either genotype alone, suggestingthat these two insults synergize. The resultant mouse therefore
mimics human AD, demonstrating coincident neuronal loss,
and plaque and P-PHF-tau filament formation, without the necessity of tau overexpression. Interestingly, genetic deletion of
Pin1, a prolyl isomerase that stabilizes p73, also causes neurodegeneration and accumulation of P-PHF-tau (Liou et al.,2003). Why do these two genetic alterations together cause an
early AD-like phenotype? We propose that p73 haploinsufficiency and b-amyloid accumulation converge to upregulate
JNK activity in neurons. This convergent JNK dysregulation
would presumably cause early dysregulation of tau phosphorylation and increased sensitivity to the toxic effects of b-amyloid
and other oxidative stresses (reviewed in Johnson and Nakamura, 2007). The net predicted result would be neurodegenera-

718 Neuron 59, 708–721, September 11, 2008 ª2008 Elsevier Inc.

tion at time points much earlier than seen with either genetic insult individually, as reported here.
These data, and the recent association between decreased
DNp73 and AD (Li et al., 2004), suggest that those individuals
with copy number variations at the p73 genomic locus (Wonget al., 2007) may be at enhanced risk for development of AD orother neurodegenerative disorders. We predict that this population would also be more vulnerable to age-related neurodegeneration or other degenerative disorders such as Parkinson’s
disease. It will be interesting to test this prediction and to determine whether haploinsufficiency for p73 will exacerbate thedegeneration phenotype of mouse models of other humandiseases.
EXPERIMENTAL PROCEDURES
Animals
This study was approved by The Hospital for Sick Children’s Animal Care
Committee, and use was in accordance with CCAC guidelines. p73+/ transgenic mice (Yang et al., 2000) were maintained on a C129SvJae background,as described (Pozniak et al., 2000, 2002). TgCRND8 hemizygous mice containing the Swedish (K670N/M671L) and Indiana (V717F) APP mutations weremaintained on a C129 background, as described (Chishti et al., 2001).
TgCRND8 hemizygous mice were crossed with p73 +/ transgenic mice togenerate F1 p73 +/ ; TgCRND8+/B and p73 +/+; TgCRND8+/B mice for analysis.
Primary Neuron Cultures
Embryonic cortical cultures were prepared as described (Lee et al., 2004). For
cell death experiments, 50 mM glutamate was added at 7 DIV, and nuclei werelabeled with Hoechst 33258 (1:2000). LDH release was assayed as described(Wetzel et al., 2003). JNK was inhibited with SP6000125 (AG Scientific, San
Diego, CA).
Behavioral Studies
All behavioral testing took place under dim lighting and constant backgroundnoise. Mice were handled 4 min a day for 7 days and habituated to the test environment prior to testing. Groups of 15 to 20 mice were tested sequentiallyand were handled, habituated, trained, and tested at the same time of day
for each experiment. Behavioral tests were run in order from least to moststressful, i.e., limb clasping, paw grip endurance, open-field, water maze. Details of behavioral tests are described in Supplemental Data.
Neuroanatomy
For histology, mice were sacrificed by sodium pentobarbital overdose andtranscardially perfused with PBS followed by 4% paraformaldehyde. Tissues
were cryoprotected and sectioned at 16 mm or 20 mm for brains and penises,
respectively. Nissl staining and immunocytochemistry were performed as described (Pozniak et al., 2002). Endogenous lipofuscin autofluorescence wasquenched using Autofluorescence Eliminator (Chemicon, Temecula, CA). Antibodies are listed in Supplemental Data. Immunoreactivity was visualized using CY-3 or FITC conjugated secondary antibodies (1:250, Jackson ImmunoResearch Labs, West Grove, PA) and nuclei labeled with Hoechst 33258(1:2000, Sigma). SA-b-gal staining was performed as described (Dimri et al.,1994). To detect argyrophilic, degenerating neurons, silver staining was performed using the FD NeuroSilver kit II (FD Neurotech, Ellicott City, MD). Neuroanatomical parameters were measured in the motor cortex at the same rostrocaudal level and were quantified in a strip of cortical tissue 325 mm wide
spanning from corpus callosum to pial surface as shown in Figure 2B. Celland fluorescence density were calculated using these area, cell count, andfluorescence measurements (Pozniak et al., 2002). All digital image acquisition
was performed using Northern Eclipse software (Empix Inc.) linked to a Sony
XC-75CE CCD video camera (Tokyo, Japan), and processed by ImageJ analysis software (National Institute of Health).

Neuronp73 in Neurodegeneration

Stereology
Total neuron number and volumetric density of primary motor cortex neurons
were obtained by applying unbiased stereology using the optical fractionator(Gundersen et al., 1988; West et al., 1996; Luk and Sadikot, 2003; Luk et al.,2001) with Stereo Investigator software (Micobrightfield, VT, USA). The rostraland caudal limits of the primary motor cortex were determined (equivalent toBregma 2.46 to 1.94 mm; Paxinos and Franklin, 2001), and every 55th serial
section of 16 mm within this volume was examined. Typically, six coronal sections at 880 mm intervals were analyzed throughout the reference volume.
Mean section thickness after Cresyl Violet processing, mounting, and coverslipping was 10 mm (tissue shrinkage effect), as measured with a z axis microcator. Sampling of the primary motor cortex was performed by randomly translating a grid with 300 3 300 mm squares onto the section of interest andapplying an optical dissector consisting of an 80 3 80 3 6 mm brick. Sections
were analyzed by using a 3100 lens (oil, numerical aperture of 1.3, with matching condenser). The most prominent nucleolus was taken as the unique identifier in all sections stained for Nissl substance.
Magnetic Resonance Imaging
For MRI, mice were perfusion fixed, decapitated, and skinned skulls were incubated in ProHance contrasting reagent (Henkelman et al., 2006; Spring
et al., 2007). Prior to imaging, samples were removed from the contrast agent
solution, blotted, and placed into 13 mm diameter plastic tubes filled witha proton-free susceptibility-matching fluid (Fluorinert FC-77, 3M Corp., St.
Paul, MN). Digital anatomical images of brains within skulls were acquiredwith a multichannel 7.0 Tesla MRI scanner (Varian Inc., Palo Alto, CA) witha 6 cm inner bore diameter insert gradient set. Three brains were imaged inparallel using custom-built, 14 mm diameter solenoid coils with a length of
18.3 cm and over wound ends. The parameters used in the scans were optimized for gray/white matter contrast: a T2-weighted, 3D fast spin-echo
sequence, with TR/TE = 325/32 ms, four averages, field-of-view 12 3 12 3
25 mm, and matrix size = 432 3 432 3 780 giving an image with 32 mm isotropic voxels35. Total imaging time was 11.3 hr. Geometric distortion due toposition of the three coils inside the magnet was corrected using an MR phantom. An unbiased model-independent atlas of the MR scans was createdas previously described (Spring et al., 2007) and detailed in Supplemental
Data.
Flow Cytometry of DNA Content
One cortical hemisphere per animal (n = 6 per group) was minced, dissociatedwith papain (2.5 U/ml, Worthington Biochemicals, Lakewood, NJ), dispase(1 U/ml, Sigma), and DNase I (250 U/ml, Sigma) for 30 min at 37 C, triturated
in DMEM containing 10% FBS, and fixed with 70% ethanol. Cells were
blocked using the Mouse-on-Mouse (MOM) kit (Vector labs, Burlington, ON,
Canada), and immunolabeling was performed with monoclonal mouse antiNeuN (1:100; Chemicon), biotinylated anti-mouse IgG secondary, and Strepavidin conjugated FITC (1:400; Becton Dickson, Mississauga, ON, Canada).
Samples were incubated with propidium iodide (50 mg/ml, Sigma) and RNase
A (2 mg/ml, Fermentas, Burlington, ON, Canada), and analyzed using a FACScan (Becton Dickinson, Franklin Lakes, NJ) and FlowJo software (Tree Star,
Inc., Ashland, Oregon), gating for single cells.
Electron Microscopy
Mice were sacrificed by sodium pentobarbital overdose and transcardiallyperfused with 4% PFA and 0.1% glutaraldehyde in 0.1 M phosphate buffer,
pH 7.4. The tissue was dehydrated, infiltrated with LR White, 80 nm sections
were placed on Formvar-coated nickel grids, incubated with P-PHF-tau (AT8; 1:10), which was detected using goat anti-mouse gold conjugate secondary
antibodies (10 nm gold; 1:20, Jackson ImmunoResearch Labs), fixed 10 min
with 2% glutaraldehyde, stained with uranyl acetate, and lead citrate. The sections were examined using an FEI Tecnai 20 transmission electron microscope(FEI Company, Hillsboro, OR), and images were captured using a Gatan Dualview digital camera (Gatan Inc, Pleasanton, CA).
Quantitative RT-PCR
Total RNA was extracted from each cortex using Trizol reagent (Invitrogen) and
the RNeasy kit (QIAGEN). cDNA was prepared with SuperScript III First-Strand

Synthesis SuperMix for qRT-PCR (Invitrogen). Real-time PCR was performed
according to the manufacturers’ specifications using Chromo4 Real-TimePCR Detection System (Bio-Rad) and a Platinum Quantitative PCR SuperMix-UDG (Invitrogen). Samples were analyzed in triplicate and were normalized to b-actin for each reaction. The primers are detailed in Supplemental
Data. All PCR products were single bands with predicted molecular weights.
Phosphatase Treatment
To determine specificity of P-PHT-tau antibodies, fixed brain sections were
dephosphorylated and then labeled with P-PHF-tau antibody AT-180 as previously described (Papasozomenos and Binder, 1987; Yang et al., 2003) anddescribed in Supplemental Data.
Biochemistry and Western Blot Analysis
Western blot analyses of cultured cortical neurons were performed as previously described (Lee et al., 2004). Antibodies are in Supplemental Data. To detect P-PHF-tau in aged mouse brain, a P-PHF-tau enriched fraction was generated from 18-month-old p73+/ and p73+/+ mice as previously described(Ikeda et al., 2005).
Statistics
Data are expressed as means ± SEM. Significant differences between means
were determined using t tests or one-way analysis of variance.
SUPPLEMENTAL DATAThe Supplemental Data include figures, movies, and Supplemental Experimental Procedures and can be found with this article online at http://www.
neuron.org/cgi/content/full/59/5/708/DC1/.
ACKNOWLEDGMENTS
This work was funded by grants from the CIHR and NeuroScience Canada. We
thank R. Temkin and the Advanced Bioimaging Centre of the HSC ResearchInstitute for immuno-EM, Frank McKeon (Harvard University) for the generous
gift of the p73 knockout mice, and David Westaway (University of Alberta) for
the generous gift of the TgCRND8 mice. M.K.W. has a fellowship from CIHR/
Heart & Stroke and J.A.B. and J.P.L. from CIHR. F.D.M. is an HHMI International Research Scholar, and R.M.H., F.D.M., and D.R.K. hold CRC chairs.

Accepted: July 8, 2008
Published: September 10, 2008
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