Bax deletion prevents neuronal loss but not
neurological symptoms in a transgenic model
of inherited prion disease
Roberto Chiesa†‡§, Pedro Piccardo¶?, Sara Dossena†, Lisa Nowoslawski††, Kevin A. Roth††, Bernardino Ghetti¶,
and David A. Harris‡§
†Dulbecco Telethon Institute and Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy;‡Department of Cell
Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110;¶Division of Neuropathology, Indiana University School of
Medicine, Indianapolis, IN 46202;?Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20852; and††Department of
Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0017
Edited by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved November 15, 2004 (received for review August 20, 2004)
Transgenic Tg(PG14) mice express a mutant prion protein contain-
with an inherited prion dementia. These mice develop a progres-
sive neurological disorder characterized by ataxia and cerebellar
atrophy, with massive apoptotic degeneration of granule neurons.
Bax, a proapoptotic gene of the Bcl-2 family, plays a key role in
regulating cell death in the nervous system. To analyze the role of
Bax in the Tg(PG14) phenotype, we crossed Tg(PG14) mice with
Bax?/?mice to obtain Tg(PG14)?Bax?/?offspring. Bax deletion
effectively rescued cerebellar granule neurons from apoptosis,
implying that these cells die via a Bax-dependent process. Surpris-
ingly, however, the age at which symptoms began and the dura-
Bax?/?mice. In addition, Bax deletion failed to prevent shrinkage
positive synaptic endings. Our analysis indicates that synaptic loss
makes a critical contribution to the Tg(PG14) phenotype. These
have important implications for the treatment of these disorders.
synapse ? apoptosis ? neurodegeneration ? cerebellum
hereditary, or idiopathic origin (1). The key event in the
pathogenesis of all forms of these diseases is the conversion of
a normal, cell surface glycoprotein (PrPc) into a conformation-
ally altered isoform (PrPSc) that has a high content of ?-sheet.
PrPScaccumulates in the brains of affected individuals in a
detergent-insoluble and protease-resistant form that is likely to
be the main component of infectious prion particles (2).
We have developed a transgenic (Tg) mouse model of a
familial prion disease by expressing the mouse PrP homologue
of a nine-octapeptide insertional mutation (PG14) described in
human patients (3). Tg(PG14) mice accumulate in their brains
a neurotoxic form of mutant PrP that possesses biochemical
properties reminiscent of the scrapie isoform of PrP, but is not
infectious (4). The mice develop a fatal neurological disorder
characterized clinically by ataxia and neuropathologically by PrP
deposition, astrogliosis, and massive apoptosis of cerebellar
granule neurons (3, 5). Granule cell apoptosis is a consistent and
dramatic feature of the Tg(PG14) phenotype, thus providing an
unprecedented setting for investigating the molecular triggers
for the apoptotic process and testing the therapeutic efficacy of
The Bcl-2 gene family includes both promoters and suppres-
sors of cell death (6). Within this family, the proapoptotic gene
Bax plays a major role in postmitotic neurons of the central
nervous system (7). BAX is a cytoplasmic protein that is
translocated to mitochondria in response to apoptotic signals,
where it promotes cell death by mediating release of cytochrome
of both humans and animals that can have an infectious,
c (8). The importance of BAX is demonstrated by the phenotype
of Bax?/?mice, which display reduced cell death during devel-
opment and after trophic factor deprivation or hypoxic-ischemic
injury (9–12). BAX has been implicated in regulating cell
autonomous and target-dependent cell death in several areas of
the brain, including the cerebellum (13–15).
In this study, we investigated the effect of inactivating the
Bax gene on the development of the neurological syndrome
and cerebellar pathology of Tg(PG14) mice. We found that
Bax inactivation markedly suppressed apoptotic death of gran-
ule neurons, but surprisingly, did not rescue synaptic degen-
eration or neurological symptoms. Our analysis indicates that
synaptic loss caused by accumulation of mutant PrP contrib-
utes significantly to neurological symptoms in Tg(PG14) mice.
These results suggest fundamental similarities in the patho-
genesis of prion diseases and other neurodegenerative disor-
ders, and they have important implications for the design of
Materials and Methods
Mice. Production of Tg(PG14) mice (3) and Bax?/?mice (16) has
been reported. Both types of mice were maintained on a
C57BL?6J ? CBA?J hybrid background. Tg(PG14) males of the
A3 line were crossed with Bax?/?females, and Tg(PG14?/?)?
(3, 16). The latter animals were intercrossed to generate mice of
the following genotypes: Tg(PG14)?Bax?/?, Tg(PG14)?Bax?/?,
Tg(PG14)?Bax?/?, and non-Tg littermates (Bax?/?, Bax?/?, and
Bax?/?). The zygosity of the PG14 transgene array was deter-
mined by Southern blot analysis. To monitor the development of
neurological symptoms, mice were scored according to a set of
objective criteria (3).
Biochemical Assays. Assays of detergent-insolubility and proteinase
K resistance of PrP in brain were carried out as described (3).
which selectively recognizes PrP encoded by the transgene (17),
anti-BAX polyclonal antibody (Chemicon), or anti-actin mAb C4
(Chemicon). DNA laddering was assessed as described (5).
Histology. Animals were perfusion-fixed with 4% paraformalde-
hyde in 0.1 M phosphate buffer (pH 7.2), after which brains were
embedded in paraffin and sectioned (3). Immunohistochemical
staining and fluorescent tyramide signal amplification were
This paper was submitted directly (Track II) to the PNAS office.
Tg, transgenic; GAP43, growth-associated protein-43.
§To whom correspondence may be addressed. E-mail: email@example.com or dharris@
© 2004 by The National Academy of Sciences of the USA
January 4, 2005 ?
vol. 102 ?
carried out as described (18) by using primary antibodies di-
rected against the following antigens: the 17-kDa fragment of
cleaved caspase-3 (Cell Signaling Technology, Beverly, MA),
glial fibrillary acidic protein (DAKO), synaptophysin (Chemi-
con), growth-associated protein-43 (GAP43; Chemicon), micro-
tubule-associated protein-2 (Sigma), and calbindin (Sigma). Cell
nuclei were labeled by incubating sections in 0.2 ?g?ml of
visualized with a Zeiss Axioskop microscope equipped with
epifluorescence. Immunohistochemical staining of PrP using
antibody 3F4 (3) and TUNEL (18) were performed as described.
Histological and Immunohistochemical Quantitation. Midsagittal
cerebellar area was measured on hematoxylin?eosin-stained
sections by using digital images captured with a Zeiss Axiocam
attached to a Zeiss Axioskop microscope and SCION IMAGE
software (Scion, Frederick, MD). Molecular and granule cell
layer widths were determined by averaging four independent
measurements taken at points midway between the base and
crest of at least two separate folia.
To quantitate TUNEL and cleaved caspase-3 reactivity, the
total number of labeled cells in complete midsagittal sections of
cerebellum was counted, and the data were expressed as number
of positive cells per cerebellar section without correcting for
differences in cerebellar area between specimens.
The density of synaptophysin immunoreactivity was quanti-
tated by converting digital images of each stained section into
binary images in which each pixel was considered positive if it
exceeded a threshold value established by staining in the absence
of primary antibody. The fraction of pixels possessing positive
reactivity was determined in six independent fields by using
SCION IMAGE software and was then averaged to obtain a single
measurement for each animal.
Statistical analysis was performed by using SAS V.6 software
(SAS Institute, Cary, NC).
Bax Deletion Does Not Rescue Neurological Illness in Tg(PG14) mice.
To determine whether Bax inactivation affected the neurological
phenotype of Tg(PG14) mice, we crossed these animals with Bax
knockout (Bax?/?) mice to produce Tg(PG14)?Bax?/?and
Tg(PG14)?Bax?/?offspring. As shown in Table 1, no significant
differences in the onset of symptoms or duration of illness were
found among Tg(PG14) mice carrying zero, one, or two Bax
alleles and that were hemizygous for the PG14 transgene array.
Additionally, two Tg(PG14)?Bax?/?mice that were homozygous
for the transgene array showed a disease onset comparable to
that previously described for homozygous Tg(PG14)?Bax?/?
mice (5) (data not shown). None of the non-Tg Bax?/?and
Bax?/?control mice developed neurological dysfunction, con-
sistent with previously published results (19). Tg(WT) mice that
express transgenically encoded WT PrP also remained healthy,
as reported (3).
Bax Deletion Does Not Modify the Levels or Biochemical Properties of
PG14 PrP. We used Western blotting to analyze the levels of PG14
PrP and BAX in the brains of Tg(PG14) mice. We did not
observe any differences in the amount of PG14 PrP among mice
on the Bax?/?, Bax?/?, and Bax?/?backgrounds (Fig. 5A Top,
which is published as supporting information on the PNAS web
site). As expected, no BAX protein was detected in Tg(PG14)?
Bax?/?mice, and reduced BAX levels were detected in
Tg(PG14)?Bax?/?mice (Fig. 5A Middle).
To determine whether Bax inactivation altered the biochem-
and protease resistance of PrP extracted from the brains of
Tg(PG14) mice on the Bax?/?, Bax?/?, and Bax?/?backgrounds.
As shown in Fig. 5 B and C, there was no effect of Bax status on
the proportion of insoluble PG14 PrP or on its degree of
proteinase K resistance.
Bax Deletion Rescues Loss of Granule Neurons, but Not Shrinkage of
the Molecular Layer in the Cerebellum. The most noticeable neu-
ropathological abnormality in Tg(PG14) mice is massive degen-
eration of cerebellar granule neurons, accompanied by shrink-
age of the molecular layer and severe atrophy of the cerebellum
(3, 5). In Tg(PG14) mice possessing at least one functional copy
of the Bax gene (Bax?/?or Bax?/?, designated Bax?/*), loss of
granule neurons was already apparent within the presymptom-
atic phase and progressed as the mice aged (Fig. 1 B and E;
compare with non-Tg control mice in Fig. 1 A and D). We did
not observe any difference in the time course of neuropatho-
logical changes between Tg(PG14) mice having one or two
copies of the Bax gene (data not shown). In contrast, Tg(PG14)?
Bax?/?mice showed a remarkable sparing of granule cells. The
number of granule neurons in mice at 91 and 320 days of age
(Fig. 1 C and F, respectively) appeared similar to that seen in
age-matched, non-Tg Bax?/?or Bax?/?mice, or in Tg(WT)?
Bax?/?mice (Fig. 1 A and D and data not shown). As expected,
Purkinje cells, where the transgene is not expressed (3), were
present in similar numbers in mice of all genotypes.
To quantitate these morphological changes (Table 2), we
measured midsagittal cerebellar area, granule cell layer width,
and molecular layer width in Tg(PG14) mice having either one
or two functional Bax genes (Bax?/*), Tg(PG14) mice lacking
functional Bax genes (Bax?/?), and Tg(WT) control mice. In
Tg(PG14)?Bax?/* mice, the early phase of cerebellar degener-
ation (?150 days of age) was characterized by modest decreases
in midsagittal cerebellar area, granule cell layer width, and
molecular layer width compared with control values. These
changes were much more pronounced in older mice (?150 days),
which exhibited a decrease of ?50% in the three morphological
measurements when compared with Tg(WT) mice.
Analysis of Tg(PG14)?Bax?/?mice confirmed selective
sparing of neuropathology in the granule cell layer (Table 2).
At ?150 days of age, granule cell layer width was increased by
?35% and molecular layer width by ?10% in Tg(PG14)?
Bax?/?mice compared with Tg(PG14)?Bax?/* mice. At this
stage, the width of the molecular layer in Tg(PG14)?Bax?/?
mice was similar to that in Tg(WT) mice. Midsagittal area was
actually slightly higher in Tg(PG14)?Bax?/?mice compared
with Tg(WT) mice, reflecting an increase in baseline brain
mass of Bax?/?animals (9). At older ages (?150 days),
Tg(PG14)?Bax?/?mice showed dramatic preservation of gran-
ule cell layer width, which remained only ?10% less than in
Tg(WT) mice. In contrast, there was continued shrinkage of
the molecular layer in Tg(PG14)?Bax?/?mice, which reached
a width comparable to that in Tg(PG14)?Bax?/* mice. Mid-
sagittal area also decreased, although not as much as in
Table 1. Clinical illness in Tg(PG14) mice with different
Age at onset
Age at death
235 ? 10 (61)
371 ? 21 (42)
154 ? 14 (35)
240 ? 28 (13)
353 ? 25 (6)
124 ? 28 (4)
255 ? 26 (5)
332 ? 24 (6)
113 ? 25 (3)
Entries show the mean number of days ? SEM. The number of animals in
each group is given in parentheses. The PG14 transgene array was hemizy-
†Data are from ref. 5.
Chiesa et al.PNAS ?
January 4, 2005 ?
vol. 102 ?
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Bax Deletion Inhibits Granule Cell Apoptosis. Several experimental
results indicated that granule cell apoptosis in Tg(PG14) mice
was blocked by Bax deletion. First, both early- and late-stage
Tg(PG14)?Bax?/?mice displayed greatly decreased numbers of
TUNEL-positive granule neurons compared with age-matched
Tg(PG14)?Bax?/* mice (Fig. 2 A–C and Table 2). Second, the
number of granule cells stained with an antibody to the activated
form of caspase-3 was also significantly reduced in Tg(PG14)?
analysis of chromosomal DNA extracted from the cerebella of
Tg(PG14)?Bax?/?mice at either the preclinical or clinical stage
of their illness showed an absence of DNA fragmentation (Fig.
3, lanes 6 and 10, respectively). In contrast, DNA from the
cerebella of Tg(PG14)?Bax?/?mice displayed a characteristic
200-bp ladder of fragments indicative of internucleosomal cleav-
age (Fig. 3, lanes 4 and 8). As expected, Tg(WT) and non-Tg
mice did not show DNA fragmentation (Fig. 3, lanes 2 and 12,
PrP Deposition Is Not Altered by Bax Deletion. We showed previ-
ously that mutant PrP accumulates in punctate, synaptic-like
deposits in the cerebellar cortex of Tg(PG14) mice as they age
(3, 5). We found that the amount and time course of PrP
deposition was similar in Tg(PG14)?Bax?/* and Tg(PG14)?
Bax?/?mice. In both kinds of mice, PrP immunolabeling in-
creased throughout the preclinical and clinical phases (Fig. 4 A
and B), but became less intense in terminally ill animals (Fig. 4
C and D). PrP deposits were present in both the granule cell and
molecular layers, but were most prominent in the latter.
and health status were stained with hematoxylin?eosin: non-Tg Bax?/?(70 days, healthy) (A); Tg(PG14)?Bax?/?(91 days, healthy) (B); Tg(PG14)?Bax?/?(91 days,
healthy) (C); non-Tg Bax?/?(323 days, healthy) (D); Tg(PG14)?Bax?/?(320 days, terminal) (E); and Tg(PG14)?Bax?/?(323 days, terminal) (F). gr, granule cell layer;
mo, molecular layer. (Scale bar: 200 ?m.)
Histological analysis of the cerebella of Tg(PG14) and control mice with different Bax genotypes. Sections from mice of the following genotypes, ages,
Table 2. Morphological measurements and labeling for apoptotic and synaptic markers in the cerebella of
TUNEL, no. of
Act. caspase-3, no.
of positive cells
% positive area
Early (?150 days)
Bax??* (n ? 8)
Bax???(n ? 6)
Late (?150 days)
Bax??* (n ? 6)
Bax???(n ? 3)
Tg(WT) (n ? 5)
5.1 ? 0.5
7.0 ? 0.7
90.2 ? 4.4†
123.5 ? 7.3‡
137.8 ? 6.2†
154.2 ? 8.0†
130.6 ? 32.9†
3.7 ? 0.6†‡
62.9 ? 9.8†
2.3 ? 0.9‡
50.0 ? 11.3
54.5 ? 9.9
3.0 ? 0.4†
4.2 ? 0.8
5.9 ? 0.6
55.6 ? 4.0†
113.3 ? 8.3‡
129.3 ? 5.3
95.6 ? 6.6†
92.9 ? 3.0†
180.0 ? 7.2
29.0 ? 14.6
5.0 ? 2.6
1.6 ? 0.5
8.7 ? 1.6†
0.7 ? 0.3‡
0.0 ? 0.0
32.3 ? 12.1
23.3 ? 8.2
61.2 ? 15.1
Entries show the mean value ? SEM. The number of animals in each group is given in parentheses. Bax??* refers to mice that were
either Bax???or Bax???. Values for Tg(WT) mice at different ages (62–451 days old) were similar and were therefore pooled. Act.,
†Significantly different from the corresponding value for Tg(WT) (P ? 0.05), two-tailed t test with Satterthwaite correction.
‡Significantly different from the corresponding value for Tg(PG14)?Bax??* (P ? 0.01), two-tailed t test with Satterthwaite correction.
www.pnas.org?cgi?doi?10.1073?pnas.0406173102Chiesa et al.
observation that the molecular layer of Tg(PG14) mice under-
goes marked shrinkage suggested that the density or structure of
synapses in this layer might be abnormal, even in Bax?/?animals
that display near-normal number of granule cells. To better
characterize synaptic pathology in the molecular layer, we
carried out immunostaining with several different markers,
including synaptophysin that stains presynaptic endings, micro-
tubule-associated protein-2 that stains dendrites, GAP43 that
stains axons, and calbindin that stains the cell bodies and
processes of Purkinje cells. We observed that immunoreactivity
in the molecular layer for all of these markers was relatively
preserved in Tg(PG14)?Bax?/* and Tg(PG14)?Bax?/?mice
during the early phase of illness (?150 days) (see Table 2 for
synaptophysin; other markers are not shown). As the animals
aged, however, there was a dramatic loss of synaptophysin and
GAP43 immunoreactivity in the molecular layer (see Fig. 2 G–I
and Table 2 for synaptophysin; GAP43, data not shown). Al-
though Purkinje neurons were spared, both calbindin and mi-
crotubule-associated protein-2 immunolabeling revealed de-
creased density and?or atrophy of dendritic arborizations in the
molecular layer (data not shown). No significant differences in
these parameters were observed between Tg(PG14)?Bax?/* and
Tg(PG14) mice model several key features of inherited prion
disorders in humans, including clinical symptoms, neuropathol-
ogy, and accumulation of abnormal PrP (3, 5). A dramatic and
consistent phenomenon observed in these mice is degeneration
In the present study, we have shown that inactivation of the Bax
gene in Tg(PG14) mice largely prevents the death of cerebellar
granule neurons, but does not affect the development of clinical
symptoms, nor synaptic loss and shrinkage in the molecular
layer. These results indicate that synaptic abnormalities induced
by accumulation of mutant PrP contribute significantly to the
neurological syndrome seen in Tg(PG14) mice.
Mechanism of Granule Cell Degeneration in Tg(PG14) Mice. Our
findings indicate that a Bax-dependent apoptotic pathway is
responsible for the granule cell death observed in Tg(PG14)
mice. We find that deletion of the Bax gene in these animals
greatly reduces degeneration of cerebellar granule neurons, as
demonstrated by a dramatic decrease in activated caspase-3 and
TUNEL labeling in the granule cell layer, as well as by the
absence of internucleosomal DNA fragmentation. Although we
have not carried out direct counts of neurons, the fact that the
thickness of the granule layer is significantly preserved indicates
Sections were stained by TUNEL (A–C) or by immunocytochemistry using antibodies directed against activated caspase-3 (Casp.-3) (D–F) or synaptophysin
(Synapto.) (G–I). Nuclei were counterstained blue with bisbenzimide. Mice were of the following genotypes, ages, and health status: Tg(WT)?Bax?/?mice (62
days, healthy) (A and D); Tg(PG14)?Bax?/?(91 days, healthy) (B and E); Tg(PG14)?Bax?/?(91 days, healthy) (C and F); non-Tg Bax?/?(323 days, healthy) (G);
Tg(PG14)?Bax?/?(320 days, terminal) (H); and Tg(PG14)?Bax?/?(323 days, terminal) (I). The arrows in B and E point to apoptotic cells. [Scale bars: 100 ?m in A
and D (applicable to B, C, E, and F) and 50 ?m in G (applicable to H and I).]
Bax inactivation blocks apoptosis of granule neurons but does not prevent synaptic loss in the molecular layer of the cerebellum of Tg(PG14) mice.
Chiesa et al. PNAS ?
January 4, 2005 ?
vol. 102 ?
no. 1 ?
that Bax deletion effectively prevents loss of granule neurons.
Thus, Bax-dependent apoptosis is a major cell death pathway
activated by the PG14 mutation. However, our data do not
exclude the possibility that a minor population of neurons in the
cerebellum or in other brain regions degenerate in Tg(PG14)?
Bax?/?mice, because of the activity of non-Bax-dependent
pathways. We have not observed obvious TUNEL reactivity or
activated caspase-3 staining outside of the cerebellum in
Tg(PG14) mice on either the Bax?/?or Bax?/* backgrounds
(data not shown).
Our results raise the possibility that Bax-dependent neuronal
apoptosis may play an important role in the pathology of prion
diseases. Although it is generally agreed that neuronal loss is a
cardinal feature of these disorders, the molecular and cellular
pathways that are activated remain unknown. There have been
several reports describing neuronal apoptosis in murine scrapie,
as well as in some cases of sporadic, inherited, and iatrogenic
Creutzfeldt–Jakob disease (20–23, ††). However, whether apo-
ptosis contributes significantly to neurodegeneration in prion
diseases and other neurodegenerative disorders has been con-
troversial (7, 25, 26).
Clinical Symptoms and Synaptic Abnormalities in Tg(PG14)?Bax?/?
Mice. Surprisingly, we find that Tg(PG14)?Bax?/?mice develop
a neurological syndrome that is clinically indistinguishable from
that of Tg(PG14)?Bax?/* mice, despite rescue of cerebellar
granule cell death in the former animals. In addition, Tg(PG14)
mice of both Bax genotypes display similar pathology in the
molecular layer of the cerebellum, including a reduction in the
thickness of this layer and marked loss of synaptophysin-positive
synaptic endings and GAP43-positive axons. These pathological
effects most likely are the result of damage to parallel fibers
projecting from granule cells, because these fibers represent the
majority of presynaptic inputs to the molecular layer. We also
observe a reduction in calbindin and microtubule-associated
protein-2 immunoreactivity in the molecular layer, indicating
that the dendritic arborizations of Purkinje neurons are reduced
and atrophic. Taken together, our results suggest that cerebellar
neural circuitry is severely disrupted in Tg(PG14) mice, even
when granule cell death is prevented, and that this effect
contributes to the ataxia and other clinical symptoms displayed
by these animals.
The synaptic loss observed in Tg(PG14)?Bax?/?mice in the
absence of neuronal cell death suggests that synapses may be a
primary pathogenic target of mutant PrP in these animals.
Consistent with this idea, we observe that mutant PrP is depos-
ited in a synaptic-like pattern in the molecular layer of the
cerebellum and in other brain regions of Tg(PG14) mice, and
PrP staining becomes less prominent as synaptic loss proceeds.
Electron microscopy will be necessary to determine the precise
localization of these PrP accumulations, for example, whether
they are presynaptic or postsynaptic, and whether they are
intracellular or extracellular.
How might mutant PrP damage synapses? One possibility is
that aggregation of PG14 PrP at synaptic sites damages the
synaptic membrane or interferes with synaptic function. Alter-
natively, misfolded or aggregated PrP may obstruct axonal or
dendritic transport, thereby inhibiting delivery of proteins to
synapses (27). A third possibility is that the PG14 mutation alters
a physiological function of PrP at synapses (28), leading to
have shown previously that mutant PrP in Tg(PG14) mice
accumulates in a noninfectious form, PG14spon, that is confor-
mationally altered, weakly protease-resistant, and aggregated
into small oligomers (4). We report here that the amount and
biochemical properties (protease resistance, detergent insolu-
bility) of PG14sponare not altered by deletion of the Bax gene.
Thus, PG14sponis likely to be cause of the synaptic loss seen in
††Migheli, A., Atzori, M., Srinivasan, A. N. & Ghetti, B. (2000) Neurobiol. Aging 21, S266
Tg(PG14) mice. Detergent extracts of cerebella were centrifuged at 16,000 ?
g, and DNA extracted from the pellet fraction (odd-numbered lanes) and the
supernatant fraction (even-numbered lanes) was subjected to Southern blot-
ting with restriction-digested mouse genomic DNA as a probe. Samples were
from a Tg(WT) mouse (lanes 1 and 2), Tg(PG14) mice of the indicated Bax
genotypes (lanes 3–10), and a non-Tg Bax?/?littermate (lanes 11 and 12). The
ages of the mice are indicated above the lanes. One-thirtieth of the DNA
extracted from the pellet fractions and the whole amount of DNA extracted
from the supernatant fractions was analyzed. Size markers are given in bp.
Bax inactivation prevents internucleosomal DNA cleavage in
Bax status. Sections from mice of the following genotypes, ages, and health
status were stained with anti-PrP antibody and lightly counterstained with
hematoxylin: Tg(PG14)?Bax?/?(134 days, healthy) (A); Tg(PG14)?Bax?/?(134
days, healthy) (B); Tg(PG14)?Bax?/?(320 days, terminal) (C); and Tg(PG14)?
Bax?/?(323 days, terminal) (D). gr, granule cell layer; mo, molecular layer.
(Scale bar: 50 ?m.)
PrP deposition in the cerebellum of Tg(PG14) mice is not affected by
www.pnas.org?cgi?doi?10.1073?pnas.0406173102Chiesa et al.
Tg(PG14)?Bax?/?mice,aswellasthegranulecellapoptosisseen Download full-text
in Tg(PG14)?Bax?/* mice.
Synaptic Dysfunction in Prion and Other Neurodegenerative Diseases.
A number of previously published observations are consistent with
the conclusion that synaptic dysfunction induced by abnormal PrP
is an important determinant of symptomatology and pathology in
prion diseases. For example, synaptic changes correlate with early
behavioral signs and precede neuronal degeneration in several
models of murine scrapie (29, 30). In addition, synaptic disorgani-
zation and loss have been described in sporadic and inherited
tide insertions (31). Accumulation of abnormal PrP at presynaptic
and postsynaptic sites has been found to precede neuronal dysfunc-
tion and death and be associated with loss of synaptic contacts in
the CNS of scrapie-infected mice and patients with Creutzfeldt–
Jakob disease (29, 32–34).
Our results suggest that synaptic pathology might be a com-
mon feature in the pathogenesis of prion diseases and other
function have been found to precede neuronal loss in Alzhei-
mer’s, Parkinson’s, and Huntington’s diseases (35, 36). In addi-
animal models of these disorders (37) and with cognitive decline
in Alzheimer’s disease patients (38). Finally, in vitro models
demonstrate that oligomeric forms of A? can directly impair
synaptic function (24).
Therapeutic Implications. The results reported here have impor-
tant implications for the therapy of prion diseases. Because
inhibition of neuronal apoptosis via Bax deletion fails to rescue
the neurological syndrome of Tg(PG14) mice, it seems unlikely
that antiapoptotic therapies alone will have a beneficial thera-
peutic effect unless associated with pharmacological interven-
tions aimed at preventing synaptic damage and neuronal dys-
function. Elucidating the structural properties of mutant PrP
responsible for its effect on synapses, and defining the cellular
mechanisms underlying this process, will be essential for design-
ing effective therapies for prion diseases.
We thank Richard Kascsak (Institute for Basic Research in Develop-
mental Disabilities, Staten Island, NY) for 3F4 antibody; Mae Gordon
and Brad Wilson for advice on statistical analysis; Cheryl Adles and
Michelle Kim for mouse colony maintenance and genotyping; Rose
Richardson and Constance Alyea for preparing histological specimens;
and Antonio Migheli for participation in the initial phase of this project.
This work was supported by Telethon-Italy Grant S00083 (to R.C.) and
National Institutes of Health Grants NS35107 (to K.A.R.), P30 AG10133
(to B.G.), and NS40975 (to D.A.H.). R.C. is an Assistant Telethon
Scientist (Dulbecco Telethon Institute, Fondazione Telethon). L.N. was
supported by the University of Alabama at Birmingham Medical Sci-
entist Training Program (Grant GM0831).
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