Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that
Parkinson’s disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger
neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict
is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted
neuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and
The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model
neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or
The ubiquitin proteasome system (UPS) is the major regulated
protein degradation mechanism of the cell, involving covalent
tagging of unwanted proteins with polyubiquitin chains as a sig-
nal for their degradation by the multiprotein 26S proteasome
complex. This ATP-dependent complex is composed of a 20S
proteolytic core particle (CP), directly abutted by 19S regulatory
particles (RPs) (Voges et al., 1999; Pickart and Cohen, 2004;
? subunits stacked in a ?1–7–?1–7–?1–7–?1–7arrangement. The
N-terminal tails of the outer ? subunits restrict substrate entry
1997, 2000; Groll and Huber, 2003). Two multimeric arrange-
ments make up the 19S RP, the base and the lid, which play roles
essential for 26S proteasome function. A recent study into the
as well as the characterized six homologous AAA-ATPases
(Rpt1–6) are responsible for coassembly of the 19S RP with the
outer ?-rings of the 20S CP (Rosenzweig et al., 2008). Rpn1 and
Rosenzweig et al., 2008). Rpt2 (PSMC1/S4) has a unique role
during 26S proteasome formation and activation, opening the
Correspondence should be addressed to R. John Mayer at the above address. E-mail:
TheJournalofNeuroscience,August13,2008 • 28(33):8189–8198 • 8189
radation (Ko ¨hler et al., 2001; Smith et al., 2005, 2007). Polyubiq-
the 19S RP (e.g., non-ATPases Rpn1/S2, Rpn10/S5a, and Rpn13/
ARM1 and ATPase Rpt5/S6a), ensuring selective degradation by
al., 2008). The 19S RP also has components that deubiquitinate
proteins before their degradation (e.g., Rpn11/S13) (Koulich et
The UPS is necessary for intracellular homeostasis, not only
are involved in the cell cycle, transcription, and DNA repair, but
hanover, 2002; Welchman et al., 2005). UPS-regulated proteoly-
sis is emerging as part of neurodevelopment, synaptic function,
and plasticity and the survival of neurons (Yi and Ehlers, 2007).
Despite the recent interest in this field, a role for the ubiquitin
system in neuronal physiology was first highlighted in chronic
neurodegenerative disease. We have known for two decades that
neuropathological inclusions in the majority of neurodegenera-
ing impairment of the UPS (Lowe et al., 1988). Genetic evidence
also links ubiquitin system dysfunction to these diseases. The
parkin gene, which is mutated in autosomal recessive juvenile
Parkinson’s disease (PD), is a ubiquitin protein ligase (Giasson
and Lee, 2001). A functional role for proteasome impairment in
PD pathogenesis has been controversial because of conflicting
experimental data using inhibitors of the 20S proteolytic core
ubiquitin system are involved in Huntington’s disease neuropa-
thology (Bennett et al., 2007; Wang et al., 2008). Genetic disrup-
selective bulk intracellular protein degradation mechanism, in
mouse brain neurons leads to neurodegeneration with ubiquitin
the neuropathology of neurodegenerative disease (Hara et al.,
2006; Komatsu et al., 2006).
Notwithstanding the consistent links between the UPS, neu-
ing primary inhibition of ubiquitin-mediated protein degrada-
demonstrated. The aim of this work was to genetically disrupt
ing the 20S proteolytic core complex, which is involved in
ubiquitin-independent degradation, unaffected. Therefore, we
Cre/loxP method for an essential subunit of the 19S RP, PSMC1
(Rpt2/S4). Two Cre deletor mouse strains were used to spatially
restrict inactivation of Psmc1 to predominantly the forebrain or
substantia nigra, expressing Cre recombinase either under the
control of the calcium calmodulin-dependent protein kinase II?
(CaMKII?) promoter or from the tyrosine hydroxylase (TH) lo-
loss of PSMC1 leads to 26S proteasome depletion, which causes
neurodegeneration and the formation of intraneuronal Lewy-
the nigrostriatal pathway and forebrain. Our findings are signif-
Generation of floxed Psmc1 mice
onic stem (ES) cells (supplemental Fig. 1A, available at www.
jneurosci.org as supplemental material). The selection cassette was ex-
cised by transient expression of Cre recombinase in vitro. PCR amplifi-
cation identified correctly targeted ES cell clones. Male chimeric mice
morulae and bred with CD1 females to establish homozygous floxed
Psmc1 mice (Psmc1fl/fl).
Region-specific inactivation of Psmc1
Forebrain-specific ablation of Psmc1 was achieved by crossing Psmc1fl/fl
mice with CaMKII?-Cre mice (Lindeberg et al., 2002), generating
Psmc1fl/wt;CaMKII?-Wt and Psmc1fl/wt;CaMKII?-Cre mice. Female
Psmc1fl/wt;CaMKII?-Cre mice were mated with Psmc1fl/flmales to pro-
duce mice in which Psmc1 was selectively inactivated in calcium
calmodulin-dependent protein kinase II?-expressing cells (Psmc1fl/fl;
CaMKII?-Cre) and control mice (Psmc1fl/fl;CaMKII?-Wt or Psmc1fl/wt;
CaMKII?-Wt). For catecholaminergic neurone-specific inactivation of
Psmc1, Psmc1fl/flmice were crossed with THCremice (Lindeberg et al.,
2004) generating Psmc1fl/wt;THCreand Psmc1fl/wt;THwtanimals. Male
deficient in Psmc1 specifically in tyrosine hydroxylase-expressing cells
(Psmc1fl/fl;THCreor Psmc1fl/ko;THCre) and control mice (Psmc1fl/fl;
PCR amplification of ear biopsy DNA was used for genotyping.
Quantitative real-time RT-PCR
Total RNA was extracted using a QIAGEN RNeasy Micro kit. RNA was
500 ng of DNase 1 (Promega)-treated RNA provided the substrate for
cDNA synthesis using oligo-dT18and Invitrogen Superscript III Reverse
real-time RT-PCRs was run using Stratagene Brilliant SYBR Green
QPCR Master Mix and a Stratagene Mx3005P QPCR System. Primers
(100 nM) for Psmc1 and glyceraldehyde phosphate dehydrogenase
(GAPDH) were used. Analysis used Stratagene MxPro QPCR software,
generating a standard curve from plasmid dilutions. Experimental sam-
ples were analyzed in triplicate, and gene expression was normalized to
GAPDH and expressed as a fold change of the controls.
Sedimentation velocity analysis
This was performed as described previously (Tanahashi et al., 2000).
Microdissected brain regions were homogenized on ice in 50 mM Tris,
the separated proteins were transferred to nitrocellulose membrane. In-
cubation in the appropriate primary antibodies was for 1 h at room
temperature [ubiquitin, 1:1000 (in-house), or synaptophysin, 1:1000
(Calbiochem)], and visualization used horseradish peroxidise-
conjugated secondary antibodies (Sigma-Aldrich) and enhanced chemi-
luminescent substrate (Pierce). Protein analysis after adenoviral Cre re-
by recovering denatured protein from the cell lysate flowthrough during
QIAGEN RNeasy Micro purification of total RNA as described in the
handbook. Protein was subjected to SDS-PAGE as described above
[PSMC1, 1:1000 (in-house), or actin, 1:1000 (Sigma-Aldrich)].
For light microscopy, mice were perfusion-fixed with 4% paraformalde-
hyde in 0.1 M phosphate buffer, pH 7.4, and brains were embedded in
paraffin wax and sectioned (5 ?m) according to standard protocols.
General morphological examination used hematoxylin (Harris) and eo-
sin staining. Immunostaining was performed as directed in Vector Lab-
oratories M.O.M. immunodetection or Vectastain Elite rabbit IgG ABC
8190 • J.Neurosci.,August13,2008 • 28(33):8189–8198 Bedfordetal.•26SProteasomeDepletionCausesNeurodegeneration
kits. Antigen retrieval used microwave treatment in 0.01 M citrate buffer
containing 0.05% Tween 20, pH 6, or 10 mM EDTA followed by formic
acid treatment. Primary antibody incubation was for 1 h at room tem-
or 1:10,000 (Dr. Diane Hanger, Institute of Psychiatry, King’s College
London, London, UK) ?-synuclein, 1:1000 p62 (BIOMOL), 1:5
?-tubulin, 1:100 activated caspase-9 (Cell Signaling), 1:600 cytochrome
oxidase IV (cox IV) (Cell Signaling), 1:50 p53 (Calbiochem), and 1:1000
PSMC1 (BIOMOL). Analyses used an Olympus BX51 microscope and
camera. For electron microscopic analyses, 3.2% paraformaldehyde,
buffer was used. When cryosections were needed, brains were dissected
and frozen in precooled isopentane over liquid nitrogen. For laser cap-
ture microdissection, cryostat sections (10 ?m) were cut onto PEN
membrane slides (Carl Zeiss) and fixed in precooled acetone for 5 min.
The sections were immunostained using TH as described previously.
Immunopositive TH cells were microdissected and laser pressure cata-
pulted into adhesive caps using a PALM microbeam Microdissector
(Carl Zeiss). For thioflavin S staining, sections were stained in hematox-
ylin for 5 min, followed by washing in water and then incubating in 1%
thioflavin S for 10 min. Sections were washed in 50% ethanol and then
water for 5 min each before mounting in Gel Mount (Sigma-Aldrich).
Immunogold electron microscopy was performed by routine methods
with 10 nm gold (Agar Scientific) as described previously (La ´szlo ´ et al.,
Open field. Each mouse was monitored for 5 min in an open-field arena
(350 ? 300 ? 300 mm; three walls transparent, one wall and floor dark
gray). Activity in the open field was recorded and quantitated by a
computer-operated animal activity system.
Morris water maze. A hidden-platform test performed in an opaque
pool at ?25°C. Over 3 consecutive days, three blocks of six trials lasting
120 s each from two compass points located on the pool (18 trials total)
was recorded for each trial. The 120 s probe trial in which the platform
was removed was on day 4 from a start position opposite the original
Data analysis used two-tailed unpaired Student’s t test with unequal
variance. Statistical significance is indicated in the appropriate figure.
To study the direct effect of impaired 26S-mediated degradation
was genetically targeted, ATPase Psmc1 (Gene ID 19179) (Rubin
Psmc1 knock-out mice (Psmc1ko/ko) were embryonic lethal (data
not shown). Therefore, Psmc1 conditional knock-out mice
(Psmc1fl/fl) were generated (supplemental Fig. 1A, available at
www.jneurosci.org as supplemental material). To spatially re-
strict ablation of Psmc1, Psmc1fl/flmice were crossed with Cre
deletor mouse strains, expressing Cre recombinase under the
control of either the CaMKII? promoter (Psmc1fl/fl;CaMKII?-
Cre) or from the TH locus (Psmc1fl/fl;THCre) (Lindeberg et al.,
2002, 2004). The CaMKII? promoter directs expression to neu-
rons predominantly in forebrain regions (Burgin et al., 1990;
Mayford et al., 1996; Tsien et al., 1996). Expression is negligible
during prenatal and perinatal development, and then dramati-
mal CNS development before Cre-mediated recombination of
Psmc1 in postmitotic neurons. In Psmc1fl/fl;THCremice, TH is
(Bayer et al., 1995).
Cre-mediated recombination of Psmc1fl/flin the mouse brain
was confirmed by PCR analysis of Psmc1fl/fl;CaMKII?-Cre and
Psmc1fl/fl;THCremice (supplemental Fig. 1B,C, available at
www.jneurosci.org as supplemental material) (data not shown).
Because Cre deletor mice only target specific neurons, the mixed
cell population of the brain proved unsuitable for analysis of
Psmc1 mRNA expression. Therefore, we captured Psmc1-
targeted neurons using laser capture microdissection (e.g., TH-
of Psmc1fl/fl;THCremice). No cycle threshold value (Ct) was ob-
tained for Psmc1 expression by real-time RT-PCR at 3 weeks of
age. Immunostaining of cortex sections from 6-week-old
Psmc1fl/fl;CaMKII?-Cre and control mice showed deletion of
PSMC1 in cortical neurons of Psmc1fl/fl;CaMKII?-Cre mice. In
addition, sequential sections of 1 week substantia nigra were
stained with anti-TH and anti-PSMC1 antibodies, demonstrat-
ing the loss of PSMC1 in residual TH? cells of Psmc1fl/fl;THCre
mice (supplemental Fig. 1D, available at www.jneurosci.org as
population, we established an in vitro model of Psmc1 ablation
(mutant) MEFs were transduced with adenoviral Cre recombi-
nase green fluorescent protein (GFP) to inactivate the floxed
activated cell sorting, and real-time RT-PCR analysis demon-
strated loss of Psmc1 mRNA in mutant MEFs from 2 d after
confirmed that Cre-mediated recombination of floxed Psmc1 re-
sulted in loss of PSMC1 protein (supplemental Fig. 1E, available
at www.jneurosci.org as supplemental material).
subjected to glycerol density gradient centrifugation and frac-
tions were assayed for chymotrypsin-like activity. The character-
istic bimodal profile of 20S and 26S proteasome activity was evi-
dent in controls, with the 26S complex contributing the majority
of proteasomal activity (Fig. 1A). Western analyses confirmed
proteasome sedimentation positions (supplemental Fig. 2, avail-
able at www.jneurosci.org as supplemental material). At 2 weeks
of age, the proteasomal distribution was comparable in control
Psmc1fl/wt(control) and Psmc1fl/ko(mutant) MEFs were transduced on day 0 with adenoviral Cre recombinase
4 d. Representative proteasomal subunits are ?7 and chymotryptic ?5 of the 20S core complex, and SUG1/p45
Bedfordetal.•26SProteasomeDepletionCausesNeurodegenerationJ.Neurosci.,August13,2008 • 28(33):8189–8198 • 8191
and mutant cortex (Fig. 1A). In 3-week-old mice, less 26S com-
plexes were observed in the mutant compared with the control,
ing to 20S (Fig. 1A). The altered profile of proteasome activity in
the Psmc1fl/fl;CaMKII?-Cre cortex is clear at 4 and 6 weeks (Fig.
1A), at which 20S is the predominant complex. Because
CaMKII?-Cre activity is upregulated in mutant cortical neurons
during postnatal weeks 2 and 3, the data indicate that ensuing
CaMKII?-Cre (mutant) cortex with increasing age, shown by Western analysis of cortex homogenates with an anti-ubiquitin antibody. Anti-synaptophysin was usedas a loading control. C, D,
Immunohistological staining of cortex sections from 6-week-old control and Psmc1fl/fl;CaMKII?-Cre (mutant) mice using anti-ubiquitin (C) and anti-p53 (D) antibodies (40?). Higher-
8192 • J.Neurosci.,August13,2008 • 28(33):8189–8198 Bedfordetal.•26SProteasomeDepletionCausesNeurodegeneration
progressive depletion of PMSC1 disrupts 26S formation. Resid-
ual 26S proteasome activity is present because of nontargeted
neurons and gliosis (Fig. 2D). This is the first report of mamma-
lian 26S proteasome disruption in vivo (cf. Wo ´jcik and DeM-
artino, 2002; Koulich et al., 2008); the gradual change in protea-
somes between 2 and 4 weeks indicates a relatively long half-life
core formation, and therefore ubiquitin-independent proteaso-
mal degradation, was not affected in our model.
of high molecular weight polyubiquitinated proteins with in-
creasing age (Fig. 1B) and significantly increased ubiquitin im-
munostaining in affected neurons (Fig. 1C) in the Psmc1fl/fl;
of p53, which undergoes robust ubiquitin-dependent degrada-
tion, was also significantly elevated (Fig. 1D).
Increased 20S proteasome activity in the Psmc1fl/fl;CaMKII?-
Cre (mutant) cortex with age appears to be higher than would be
expected for a direct shift of the 20S activity that would normally
be associated with 26S complexes (Fig. 1A). This may be ex-
genesis of active 20S complexes in response to 26S proteasome
impairment (Wo ´jcik and DeMartino, 2002; Meiners et al., 2003;
Lundgren et al., 2005; Fuchs et al., 2008). In our MEF model, a
significant increase in representative proteasome subunit mR-
NAs was observed 3 d after transduction (Table 1). This is most
likely attributable to transcriptional activation (Meiners et al.,
2003). Together with the observations in vivo, these results sug-
gest that proteasome subunits are upregulated in an attempt to
maintain proteasomal homeostasis in the absence of PSMC1.
able and indistinguishable from their littermates at birth. Subtle
from 5 weeks of age (supplemental Fig. 3A, available at www.
jneurosci.org as supplemental material), and they die when aged
were significantly more anxious in open-field analysis and dis-
played obvious spatial learning deficits in the Morris water maze
task at 6 and 8 weeks, respectively, suggesting 26S proteasomal
function is essential for normal neurological function in mice
(supplemental Fig. 3B,C, available at www.jneurosci.org as sup-
plemental material). Psmc1fl/fl;THCremice became progressively
Gross histological analysis of brains from control and Psmc1fl/fl;
CaMKII?-Cre (mutant) mice revealed significant progressive at-
of age. A, Hematoxylin and eosin (H&E)-stained whole-brain sections, shown at higher magnification in B (40?). The arrows point to pyknotic and fragmented nuclei in the mutant cortex.
Immunohistochemistry with antibodies to cleaved caspase 9 (40?) (C) and GFAP (10?) (D) in cortex sections. Representative individual neurons are magnified and shown in the inset of
Bedfordetal.•26SProteasomeDepletionCausesNeurodegenerationJ.Neurosci.,August13,2008 • 28(33):8189–8198 • 8193
rophy of the forebrain, exemplified by re-
duced cortical thickness and expansion of
the ventricular cavities (Fig. 2A; supple-
mentalFig. 4, available
jneurosci.org as supplemental material).
At 2 weeks of age, there were no degener-
ative changes evident in the Psmc1fl/fl;
CaMKII?-Cre forebrain (supplemental
Fig. 4A, available at www.jneurosci.org as
supplemental material). However, we ob-
served numerous pyknotic nuclei with he-
matoxylin and eosin staining at 4 weeks of
age, which increased in 6-week-old mice,
preceding the extensive neuronal loss by 8
weeks (Fig. 2A,B; supplemental Fig. 4B,
available at www.jneurosci.org as supple-
mental material). Pyknotic nuclei sug-
gested an apoptotic mechanism of neuro-
degeneration. This was confirmed by
upregulation of the apoptosis signaling
molecule, cleaved caspase-9 (Fig. 2C). De-
creased Bcl-2 expression was observed in
the MEF model (Table 1). It remains pos-
sible that alternative death mechanisms
contribute to degeneration of these neu-
rons. Immunohistochemical staining with
GFAP demonstrated extensive astrocytic
gliosis accompanied the neuronal loss
in other CaMKII?-expressing regions of
the Psmc1fl/fl;CaMKII?-Cre brain, includ-
dala (data not shown). We conclude that
26S proteasomal depletion directly causes
the basal ganglia, as evidenced by TH immunostaining up to 3
adjacent sections revealed this was neurodegeneration and not
downregulation of TH expression (Fig. 2F). In neurochemical
support of the observations, significant catecholaminergic defi-
cits were evident in the Psmc1fl/fl;THCrebrain, including de-
creases in dopamine and norepinephrine in the striatum (Fig.
2G) and hypothalamus, and a decrease in norepinephrine in the
hippocampus and brainstem (data not shown). Figure 3 empha-
age. 26S proteasomal depletion in all catecholaminergic neurons
of the Psmc1fl/fl;THCremice causes autonomic dysfunction (data
not shown), explaining premature death.
using diagnostic antibodies for major human neurodegenerative
diseases. Diffuse accumulation of ubiquitinated proteins was ev-
at 2 weeks, but no other differences were observed at this age
(supplemental Fig. 4, available at www.jneurosci.org as supple-
mental material). By 4 weeks of age, coincident with progressive
thology. However, we also observed numerous eosinophilic in-
traneuronal paranuclear inclusions, containing ubiquitin,
?-synuclein, and p62, similar to Lewy bodies (LBs) seen in the
brains of patients with dementia with Lewy bodies (DLB) (Fig.
4A–D; supplemental Fig. 4, available at www.jneurosci.org as
supplemental material). Psmc1fl/fl;THCrenigral neurons also
PD (supplemental Fig. 5, available at www.jneurosci.org as sup-
plemental material). Although ?-synuclein-immunoreactive
neurites were not found in the mice, in contrast to human dis-
ease, some of these inclusions stained with ?-tubulin, PGP 9.5,
?B crystallin, and neurofilament protein, supporting their simi-
larity to Lewy bodies (supplemental Fig. 5, available at www.
jneurosci.org as supplemental material) (data not shown)
(Shults, 2006). No A? (?-amyloid)-containing amyloid or tau
protein deposits were evident on immunostaining (data not
shown). Therefore, Lewy-like inclusion formation and neuronal
loss are independent of amyloid formation in this model.
Electron microscopy of Lewy-like inclusions revealed spheri-
cal structures with a lamellar appearance (Fig. 4E; supplemental
Fig. 6, available at www.jneurosci.org as supplemental material).
Numerous mitochondria, fine filaments, and granular material
were evident in the central region, surrounded by a zone of clear
vesicular and membranous material, as well as double-
TH (A), ubiquitin (B), and GFAP (C) (10?). A, TH immunostaining identifies nigral neurons in control and Psmc1fl/fl;THCre
8194 • J.Neurosci.,August13,2008 • 28(33):8189–8198Bedfordetal.•26SProteasomeDepletionCausesNeurodegeneration
membraned autophagolysomes, suggesting the autophagic pro-
mental Fig. 6, available at www.jneurosci.org as supplemental
material). Immunogold labeling with anti-?-synuclein revealed
some filaments with 10 nm gold particles in the central region
(Fig. 4I). In support of this observation, a subset of inclusions
Fig. 6, available at www.jneurosci.org as supplemental material).
The pattern of ?-synuclein aggregate in our model, containing
sidered to be an early form of Lewy body (Gai et al., 2000). The
paucity of literature on mitochondria in human disease led us to
PD patients. This revealed the presence of mitochondria in pale
bodies, an ?-synuclein-containing inclusion thought to be an
early form of nigral Lewy body (Fig. 5A). In the example shown,
the larger mitochondria-containing PB is adjacent to a smaller
LB, which is devoid of mitochondria. Mitochondria-rich inclu-
sions were further investigated using cox IV immunostaining.
cox IV was seen in a proportion of human
cortical Lewy bodies as well as in nigral
pale bodies, but was absent from classical
dense core Lewy bodies (Fig. 5B–D).
resents filamentous ?-synuclein aggre-
gates containing mitochondria as “early-
inclusions do not progress to reveal Lewy
bodies with a dense core and lamellar pat-
tern as in human disease. The abundance
of mitochondria in mouse Lewy-like bod-
role in Lewy body formation and/or
neurons, enabling a definitive link be-
tween ubiquitinated protein degradation
by the 26S proteasome and neuronal pa-
thology to be established. Impaired 26S
proteasomal function causes neurodegen-
eration and intraneuronal inclusion body
formation, reflecting early forms of hu-
man Lewy bodies described as homoge-
neous Lewy bodies (Gai et al., 2000) or
pale bodies (Dale et al., 1992). The intran-
different from those seen in PD or DLB
does not form. The reasons for this are
rodegeneration proceeds before the time
that would be necessary to form a late-
stage concentric Lewy body. This is most
likely a consequence of the genetic ap-
proach taken in this model, which rapidly
ablates 26S proteasomal function. In con-
trast, progressive proteasomal inhibition
with aging most likely occurs in human
disease. Alternatively, there may be intrinsic differences in the
pathophysiology between the mouse model and human disease.
Based on mutational studies of the ATPase subunits in the
base of the 19S RP using yeast proteasomes as a model, we chose
bin et al., 1998; Ko ¨hler et al., 2001). Because the six base ATPases
are functionally nonredundant, deletion of PSMC1 would pre-
vent hexameric ring biogenesis in the base of the 19S RP and
coassembly with the 20S CP. PSMC1 also has a unique role in
proteolysis activity of the 20S core. Using glycerol gradient anal-
ysis, we demonstrate here in higher eukaryotes that ablation of
PSMC1 in mouse neurons disrupts 26S proteasome formation,
but does not affect the 20S CP. Therefore, in contrast to the use
of 20S proteasome inhibitors in models of PD, which do not
differentiate between ubiquitin-dependent and ubiquitin-
independent degradation or neurons and glia, the neuropatho-
logical changes evident in our mouse model are linked to neuro-
nal 26S proteasomal dysfunction. This model also reveals the
importance of the 26S proteasome versus the 20S proteolytic CP
Bedfordetal.•26SProteasomeDepletionCausesNeurodegenerationJ.Neurosci.,August13,2008 • 28(33):8189–8198 • 8195
in neuronal homeostasis. The 20S core is unable to maintain
a decrease in 20S proteolytic activity of the proteasome occurs
with aging in neurodegenerative disease. Importantly, this work
This model differs significantly from current models of neu-
rodegenerative disease, in which neuronal loss is minimal and
generally not seen in regions linked to disease (McGowan et al.,
2006; Melrose et al., 2006) (e.g., the substantia nigra in
?-synuclein-expressing transgenic mice). Neurodegeneration
may be an expected consequence of PSMC1 and thence 26S pro-
teasomal depletion in mouse neurons. The early Schizosaccharo-
dysfunction, with mutation of the homologous ATPase mts2 or
8196 • J.Neurosci.,August13,2008 • 28(33):8189–8198 Bedfordetal.•26SProteasomeDepletionCausesNeurodegeneration
rpt2, respectively, reflect the observations in our mouse model.
Lack of the functional ATPase caused accumulation of ubiquiti-
nated proteins and growth defects (Gordon et al., 1993; Seeger et
al., 1996; Rubin et al., 1998). More recently, as anticipated,
proteasome-dependent activities are being revealed as funda-
mental in the nervous system (Yi and Ehlers, 2007). Although a
buildup of polyubiquitinated proteins and degeneration may be
obvious effects of disrupted neuronal proteasomal homeostasis
in this model, previous work did not suggest that intracellular
paranuclear inclusion body formation would occur, resembling
early-stage Lewy bodies seen in human disease. This could be
and lower eukaryotes. The formation of inclusion bodies with
some of the currently accepted features of Lewy bodies (Shults,
2006) suggests 26S proteasome dysfunction is associated with a
cascade of specific neuropathological changes that occur in dis-
Much research into the synucleinopathies, particularly Par-
kinson’s disease, focuses on mechanisms involving ?-synuclein,
but more recently mitochondrial involvement in the biology of
Lewy body disorders has been recognized. We demonstrate both
?-synuclein and mitochondria are linked to neuropathological
changes after proteasomal dysfunction, which itself is consis-
tently associated with disease (Gandhi and Wood, 2005). Al-
in paranuclear aggresomes (Lewy-like bodies), another interpre-
tation is that ?-synuclein may be an essential protein involved in
the biogenesis of Lewy bodies and its accumulation is an active
event. This is supported by the disproportionate and early accu-
mulation of ?-synuclein in inclusion bodies after 26S proteaso-
mal dysfunction in neurons; ?-synuclein is not recognized as a
protein that has a high turnover mediated by the UPS, and has
been reported to undergo ubiquitin-independent degradation
(Tofaris et al., 2001).
Mitochondria are a striking component of the inclusion bod-
ies in mouse neurons and we show current evidence for mito-
chondria in some human cortical and nigral early-stage Lewy
bodies. Mitochondria have also been noted as a component of
Lewy-like aggresomes induced by proteasome inhibition (Abou-
Sleiman et al., 2006). The precise role of mitochondria in neuro-
degenerative disease remains uncertain. Evidence for mitochon-
drial dysfunction is provided by mutations in genes that are
2004; Poole et al., 2008)], and features of PD are caused by mito-
chondrialtoxins(Bove ´ etal.,2005).Theobservationsmadeinthe
us to propose a hypothesis that suggests that mitochondria are a
key component of an early phase in the biogenesis of the Lewy
Another mechanistic possibility for the findings described
here that will need additional investigation is that the roles of the
26S proteasome, 19S RP, and 20S catalytic core in gene expres-
the loss of PSMC1. Progranulin mutations result in neuronal
cytoplasmic inclusions containing TDP43, which is normally in-
volved in transcription and exon skipping and may cause neuro-
degeneration by errors in gene expression (Eriksen and Macken-
The known options for the isolation and/or degradation of
aggresomes (Johnston et al., 1998) and macroautophagy. The
neuroprotective function of macroautophagy has become an in-
teresting focus in neurodegenerative disease, exemplified in
Huntington’s disease (Menzies et al., 2006; Mizushima et al.,
2008), as well as in animal models (Hara et al., 2006; Komatsu et
way plays a role during dysfunction of the UPS is still unclear.
Proteasome function was found to be normal in autophagy-
deficient brain (Komatsu et al., 2006). Preliminary experiments
in 26S proteasome-ablated MEFs did not show significant up-
regulation of autophagic genes (Table 1), but in vivo EM analysis
revealed double-membraned autophagolysomes.
This is the first mouse model that permits investigation into
26S proteasomal dysfunction in neurons. We demonstrate that
impairment of neuronal 26S proteasomes in the neocortex or
substantia nigra causes florid neurodegeneration and Lewy-like
inclusion body formation, resembling pathophysiology evident
in the brains of patients with DLB and PD, respectively. The
models described here provide unparalleled novel platforms in
which to investigate and exploit therapeutic approaches to pre-
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