SHORT COMMUNICATION

The Proteasome Function Reporter GFPu Accumulates in YoungBrains of the APPswe/PS1dE9 Alzheimer’s Disease Mouse Model

Yanying Liu • Casey L. Hettinger • Dong Zhang •

Khosrow Rezvani • Xuejun Wang • Hongmin Wang

Received: 29 July 2013 / Accepted: 13 December 2013

� Springer Science+Business Media New York 2013

Abstract Alzheimer’s disease (AD), the most common

cause of dementia, is neuropathologically characterized by

accumulation of insoluble fibrous inclusions in the brain in

the form of intracellular neurofibrillary tangles and extra-

cellular senile plaques. Perturbation of the ubiquitin-pro-

teasome system (UPS) has long been considered an

attractive hypothesis to explain the pathogenesis of AD.

However, studies on UPS functionality with various

methods and AD models have achieved non-conclusive

results. To get further insight into UPS functionality in AD,

we have crossed a well-documented APPswe/PS1dE9 AD

mouse model with a UPS functionality reporter, GFPu,

mouse expressing green fluorescence protein (GFP) fused

to a constitutive degradation signal (CL-1) that facilitates

its rapid turnover in conditions of a normal UPS. Our

western blot results indicate that GFPu reporter protein was

accumulated in the cortex and hippocampus, but not stri-

atum in the APPswe/PS1dE9 AD mouse model at 4 weeks

of age, which is confirmed by fluorescence microscopy and

elevated levels of p53, an endogenous UPS substrate. In

accordance with this, the levels of ubiquitinated proteins

were elevated in the AD mouse model. These results sug-

gest that UPS is either impaired or functionally insufficient

in specific brain regions in the APPswe/PS1dE9 AD mouse

model at a very young age, long before senile plaque for-

mation and the onset of memory loss. These observations

may shed new light on the pathogenesis of AD.

Keywords Alzheimer disease � Ubiquitin-proteasome

system � Proteasome function reporter � GFPu � Protein

degradation � Ubiquitinated proteins

Abbreviations

AD Alzheimer’s disease

UPS Ubiquitin-proteasome system

GFPu Green fluorescence reporter for UPS

functionality

APP Amyloid precursor protein

PS Presenilin

NFT Neurofibrillary tangle

PolyUb Polyubiquitin

Tg Transgenic

Introduction

Alzheimer’s disease (AD), a prevalent neurodegenerative

disorder, is the most common type of dementia. Most AD

is sporadic and caused by a complex interaction of genetic

and environmental risk factors. In contrast, familial forms

of AD are caused by mutations in the genes encoding

amyloid precursor protein (APP), presenilin (PS), or other

proteins (Bird 2008). Neuropathologically, AD is featured

by the progressive accumulation of extracellular senile

plaques and intracellular neurofibrillary tangles (NFT)

(Perry et al. 1985). The NFT inclusions are positive in

ubiquitin immunoreactivity in human AD brains (Mori

et al. 1987; Layfield et al. 2005) and correlate well with the

degree of dementia (Braak and Braak 1991). It has been

speculated that impaired ubiquitin-proteasome system

(UPS)-dependent protein degradation may contribute to

AD pathogenesis (Upadhya and Hegde 2007; Chen et al.

2012). However, studies on UPS functionality with various

Y. Liu � C. L. Hettinger � D. Zhang � K. Rezvani � X. Wang �H. Wang (&)

Division of Basic Biomedical Sciences, Sanford School

of Medicine, University of South Dakota, Vermillion,

SD 57069, USA

e-mail: hongmin.wang@usd.edu

123

Cell Mol Neurobiol

DOI 10.1007/s10571-013-0022-9

methods and AD models have achieved conflicting results.

On one hand, some studies have shown that UPS func-

tionality is impaired in AD cells as well as animal models

(Keller et al. 2000; Ihara et al. 2012). On the other hand,

however, AD is linked to elevated proteasome activity

(Orre et al. 2013; Schubert et al. 2009).

Fluorogenic substrate-based biochemical assays have

been commonly used for examining proteasome activity in

Alzheimer’s disease cells or tissues (Keller et al. 2000;

Johnston et al. 1998). However, these assays do not fully

reflect UPS activities, as the substrates used do not pass

through all steps of physiologically relevant UPS-depen-

dent protein degradation pathways. An alternative method

for assessing UPS functionality has been the use of

recombinant probes containing green fluorescent protein

(GFP) fused to the CL-1 degron to generate the ‘‘GFPu’’

(also referred to as GFPdgn) (Bence et al. 2001; Kumara-

peli et al. 2005). The CL1 degron is a destabilizing

C-terminal 16 amino acid polypeptide (Bence et al. 2005).

Previous data have shown that GFPu is polyubiquitinated

and accumulates when the proteasome is suppressed,

indicating its reliability as a UPS activity reporter (Bence

et al. 2001). Indeed, GFPu has been utilized in assessing

UPS functionality in a number of studies, including studies

in neurodegenerative disorders such as Huntington’s dis-

ease (Bett et al. 2009) and other polyglutamine diseases

(Bennett et al. 2005; Khan et al. 2006). Here, we have

crossed the APPswe/PS1dE9 mouse overexpressing the

Swedish mutation of APP and PS1 deleted in exon9 (Jan-

kowsky et al. 2004), one of the most extensively used

transgenic (Tg) mouse models of Alzheimer’s disease

(AD), with GFPu Tg mice (Kumarapeli et al. 2005; Su

et al. 2011) to investigate potential impairment of the UPS

in AD. Our results indicate that GFPu accumulates in

specific brain areas in the AD mouse model at 4 weeks,

long before senile plaque formation and memory decline.

Materials and Methods

Animals

All studies were conducted with approval of the University

of South Dakota Animal Care and Use Committee and in

compliance with NIH guidelines for the use of experi-

mental animals. The APPswe/PS1dE9 AD mouse model on

the C57BL/6 J background was obtained from Jackson

Laboratory (Bar Harbor, Maine) via the Mutant Mouse

Regional Resource Center. The GFPu transgenic mice

(Kumarapeli et al. 2005) were on the FVB/N background

and crossed with the C57BL/6 J mice for five generations

before crossing with the AD mouse model. Mice were

maintained in a temperature and humidity controlled

environment with a 12 h light:12 h dark cycle and with

ad libitum access to food and water.

Western Blot Analysis

After sacrifice, different brain regions were isolated on an

ice pad and homogenized in a tissue lysis buffer as previ-

ously described (Lu and Wang 2012). Total protein quan-

tification and western blot analysis were performed

according to previously described methods (Lu and Wang

2012; Dong et al. 2012). Antibodies used in the studies

include anti-GFP (a mouse monoclonal antibody, Santa

Cruz Biotechnology, SC-9996, 1:1,000) and anti-actin (a

goat polyclonal antibody, Santa Cruz Biotechnology, SC-

1616, 1:1,000), anti-APP (a rabbit polyclonal antibody,

Cell Signaling, 2452S), anti-p53 (a rabbit polyclonal anti-

body, Santa Cruz Biotechnology, SC-6243, 1:1,000), anti-

proteasome 20S a7 subunit (a mouse monoclonal antibody,

Enzo Life Sciences, BML-PW8110-0025, 1:1,000), and

horseradish peroxidase (HRP)-linked anti-rabbit and anti-

goat antibodies (polyclonal antibodies, Santa Cruz Bio-

technology, SC-2004, SC-2020, 1:5,000). Western blot

bands were scanned and quantified by measuring pixel

density using a digitizing system (UN-Scan-it gel, version

6.1) as previously described (Dong et al. 2012).

Native GFP Imaging, Immunohistochemistry,

and Microscopy

Brain fixation and cryosection were based on previously

described methods (Lu and Wang 2012). Briefly, mice

were transcardially perfused with PBS followed by 4 %

paraformaldehyde (PFA). The brains were carefully iso-

lated and post-fixed in PFA and transferred to 30 % sucrose

(in PBS). Brains were embedded in OCT Compound

(Tissue-Tek) and coronally cryosectioned into a thickness

of 15 mm using a cryostat (Leica). After permeabilization

with 0.2 % Triton-X100 and blocking (in 5 % BSA in PBS,

1 h), brain sections were incubated with an anti-NeuN

antibody (Cell Signaling, 1:200) for 2 h, which was fol-

lowed by 45 min of incubation with Cy3-conjugated goat

anti-rabbit antibody (1:200; Jackson Immuno Research)

and 10 min of incubation with the nuclear dye Hoechst

33342 (Invitrogen, 1:1,000). Stained sections were viewed

on a confocal microscope and optimal settings were

obtained to limit background fluorescence and ensure

detected fluorescent signals were not saturated and

remained the same throughout the experiment.

Statistical Analysis

One-way analysis of variance was used for statistical

analysis of the experimental results and a t test was used for

Cell Mol Neurobiol

123

comparisons between two different groups. p \ 0.05 was

regarded as statistically significant.

Results and Discussion

To examine the possibility that mutant APP and PS1 cause

general impairment of the UPS in vivo, we crossed the

well-characterized APPswe/PS1dE9 mouse model of AD

(Jankowsky et al. 2004) with the cytomegalovirus pro-

moter-driven GFPu UPS reporter mice (Kumarapeli et al.

2005) to generate progeny of four genotypes: wild type,

AD, GFPu, and AD/GFPu double Tg mice. The AD/GFPu

double Tg mice did not show any evident behavioral or

morphological abnormalities compared to their GFPu lit-

termates at 4 weeks of age (data not shown). With the AD/

GFPu double Tg mice, we next examined whether co-

expression of the two mutant proteins, APPswe and

PS1dE9, in the mice impairs the UPS functionality at an

early age. We, therefore, analyzed GFPu fusion protein

levels in the 4-week-old AD/GFPu double Tg mice and

their GFPu littermates. Western blot analysis of tissue

lysates of different brain regions showed that GFPu protein

levels in the cerebral cortex (Fig. 1a, b) and hippocampus

(Fig. 1c, d) from the AD/GFPu mice were significantly

higher than those of the GFPu mice. However, GFPu level

in the striatum did not show a statistically significant dif-

ference between the AD/GFPu and GFPu mice (Fig. 1e, f).

Expression of the USP reporter protein, GFPu, did not

disrupt APP expression in the AD mouse model, as the AD/

GFPu double Tg mice showed striking overexpression of

APP protein in all brain regions examined (Fig. 1a–c). To

GFPu

Actin

a b

p53

α7

APP

GFPu AD/GFPu Cortex

*

0.5

0.6

0.7

0.8

0.9

1

1.1

APP GFPu p53

Pro

tein

Lev

els

(AU

)

GFPu AD/GFPu

* *

GFPu

Actin

f

c d

e

GFPu AD/GFPu

Hippocampus

p53

α7

APP

GFPu

Actin

p53

α7

APP

GFPu AD/GFPu Striatum

0.6

0.7

0.8

0.9

1

1.1

1.2

APP GFPu p53

Pro

tein

Lev

els

(AU

)

GFPu AD/GFPu

* *

*

0.4

0.6

0.8

1

1.2

APP GFPu p53

α7

α7

α7

Pro

tein

Lev

els

(AU

)

GFPu AD/GFPu

*

*

Fig. 1 Western blot analyses of

APP, GFPu, p53, and

proteasome 20S a7 subunit

protein levels in the cerebral

cortex (a), hippocampus (c), and

striatum (e) are shown. The

protein levels in each lane in a,

c, e are measured and

normalized against actin protein

levels, and are indicated in b, d,f (in arbitrary unit, AU),

respectively. Numerical data are

shown as mean ± SD; n = 4.

*p \ 0.05

Cell Mol Neurobiol

123

GF

Pu

AD

/GF

Pu

NeuNGFP MergeNucleusCortexa

b

0

20

40

60

80

GFPu AD/GFPu

GF

P F

luor

esce

nt

Inte

nsity

(A

U)

*

AD

/GF

Pu

Hippocampus

GF

Pu

c

d

0

20

40

60

80

100

GFPu AD/GFPu

GF

P F

luor

esce

nt

Inte

nsity

(A

U) *

NeuNGFP MergeNucleus

Fig. 2 Native GFPu fluorescence in APPswe/PS1dE9 AD mouse

brains at 4 weeks. Native GFPu fluorescence is notably increased in

the cortex (a, b) and hippocampus (c, d), but not in the striatum (e, f)of the AD mouse model. In the cerebral cortex and hippocampus, the

GFPu fluorescent intensity is higher in non-glial cells (neurons,

pointed by arrows) than glia (pointed by arrow heads) (g, h). Sections

were stained with the nuclear-specific fluorescent dye Hoechst44432

and a neuron-specific marker, NeuN, or a glia-specific marker, glial

fibrillary acidic protein (GFAP), antibody. Scale bars are 50 lm in a,

c, e, and 25 lm in g

Cell Mol Neurobiol

123

define whether the differences in GFPu protein levels in the

AD mouse model are caused by differential expression the

GFPu transgene, we monitored the expression of GFPu

mRNA using a previously described semi-quantitative RT-

PCR (Marone et al. 2001) and found that GFPu mRNA

expression was unchanged in AD/GFPu double transgenic

mice (data not shown). Interestingly, an endogenous pro-

teasome substrate, p53, also showed accumulation in the

brain regions examined (Fig. 1a–f). To further determine

whether accumulation of the proteasome substrates is due

to differential expression of the proteasome, we examined a

constitutive proteasome subunit of the 20S proteasome, a7.

As shown in Fig. 1a–f, the levels of 7a protein did not

show a significant change in the brain regions examined in

the two types of mice. These results suggest that UPS

functionality is impaired in specific brain regions in the

young AD mouse model.

To further determine the cellular and subcellular distri-

bution of GFPu in brain cells, we next performed fluores-

cence microscopy to brain sections of the 4-week GFPu

Striatum

GF

Pu

AD

/GF

Pu

e

0

20

40

60

80

GFPu AD/GFPu

GF

P F

luor

esce

nt

Inte

nsity

(A

U)f

NeuNGFP MergeNucleus

CortexGFP/GFAP/Nuclei

GF

Pu

AD

/GF

Pu

g

h

Hippocampus

* *

0

20

40

60

80

100

GFPu AD/GFPu GFPu AD/GFPu

Cortex Hippocampus

GF

P In

tens

ity (

AU

)

GliaNeurons

Fig. 2 continued

Cell Mol Neurobiol

123

aGFPu AD/GFPu

Cortex

GFPu AD/GFPuStriatum

GFPu AD/GFPuHippocampus

Ub UbUb

Actin Actin Actin

0.9

0.95

1

1.05

1.1

1.15

1.2

GFPu AD/GFPu

Ub-

Pro

tein

leve

ls (

AU

)

Cortex

0.9

0.95

1

1.05

1.1

GFPu AD/GFPu

Ub-

Pro

tein

Lev

els

(AU

)

Striatum

0.92

0.96

1

1.04

1.08

1.12

GFPu AD/GFPu

Ub-

Pro

tein

Lev

els

(AU

)Hippocampusb

c

d

e

f

* *

g WT AD

CortexWT AD

Striatum WT AD

Hippocampus

Ub UbUb

Actin Actin Actin

j

h

k

i

l

* *

0.9

0.95

1

1.05

1.1

WT AD

Ub-

Pro

tein

Lev

els

(AU

)

Hippocampus

0.9

0.95

1

1.05

1.1

WT AD

Ub-

Pro

tein

Lev

els

(AU

)

Striatum

0.95

1

1.05

1.1

WT AD

Ub-

Pro

tein

Lev

els

(AU

)

Cortex

Fig. 3 Western blot analyses of total ubiquitinated (Ub)-protein

levels in the cerebral cortex (a, g), hippocampus (c, h), and striatum

(e, i) in mice with the indicated genotypes at 4 (a, c, and e) or 2 weeks

(g, h, and i) are shown. Total Ub-protein levels in each lane in a, g, c,

h, and e, i were measured and normalized against actin protein levels,

and are indicated in b, j, d, k, and f, l, respectively. Numerical data

are shown as mean ± SD; n = 4. *p \ 0.05

Cell Mol Neurobiol

123

and AD/GFPu mice. Brain sections were prepared side-by-

side and images were captured with unchanged settings

after initial correction for background fluorescence in a

wild-type mouse brain. GFPu fluorescence intensity (green

in color in Fig. 2) in the cortex (Fig. 2a, b) and hippo-

campus (Fig. 2c, d) from the AD/GFPu mouse brains was

higher compared to the GFPu mouse brains, whereas

imaging of GFPu fluorescence in the striatum revealed very

comparable fluorescence intensity between GFPu and AD/

GFPu mice (Fig. 2e, f). These results support the obser-

vations obtained above, indicating distinct susceptibility of

impaired degradation of GFPu in different brain regions in

the young AD mouse model.

To define whether GFPu protein has a distinct cell type-

specific distribution between neurons and non-neuronal

cells, we stained the brain sections with NeuN (a neuron-

specific marker), or glial fibrillary acidic protein (GFAP, a

glia-specific marker), antibody. GFP fluorescence was

widespread in all regions examined, indicating that the

GFPu fusion protein is present throughout brain cells. In

each cell, GFPu showed distinct distribution, with low

intensity in nuclei, but high intensity in the non-nucleus

areas (Fig. 2a–c). In the cortex and hippocampus, GFP

fluorescence appeared to be less bright in glia (the GFAP-

positive cells) than in neurons (the GFAP-negative cells)

(Fig. 2g, h). Interestingly, compared to the neurons (NeuN-

positive cells), some of the non-neuronal cells had much

brighter GFPu fluorescence in the striatum (Fig. 2c). These

results suggest that UPS functionality is selectively

impaired in the neurons in the cortex and hippocampus in

the AD mouse model.

Selective accumulation of GFPu in the cortex and hip-

pocampus in the young AD mouse model suggests that

impaired or functionally insufficient UPS occurs in these

brain regions. To further examine whether ubiquitinated

(Ub) proteins accumulate in the brain regions, we per-

formed immunoblotting analysis of the total Ub-protein

levels. As shown in Fig. 3a–f, compared to the GFPu

control mice, Ub-protein levels were significantly elevated

in the cortex (Fig. 3a, b) and hippocampus (Fig. 3c, d) in

the AD/GFPu mice. However, the level of Ub-proteins in

the striatum from the AD/GFPu mice did not statistically

differ from the GFPu mice. To exclude the possibility that

the elevated Ub-protein levels in the AD/GFPu mice are

caused by expression of GFPu, we examined Ub-proteins

in the AD and wild-type mice at 4 weeks and obtained the

similar results; namely, the cortex and hippocampus of the

AD mice had higher levels of Ub-proteins than those of

wild type of mice (data not shown). To further define

whether the AD mouse model younger than 4 weeks also

shows increased Ub-proteins, we further examined Ub-

protein levels in the three brain regions of the AD mouse

model at 2 weeks of age. As shown in Fig. 3g–l, the AD

mouse model at 2 weeks showed significantly higher levels

of Ub-proteins in the cortex and hippocampus than the

wild-type littermates. Taken together, these data reveal that

the UPS is impaired or functionally insufficient in the

young AD mouse model in specific brain regions.

Previous data have shown that senile plaques are

detectable in the brains of AD mice at 4 months of age

(Garcia-Alloza et al. 2006). Our data reveal that impaired

UPS occurs long before the formation of beta-amyloid

plaques, suggesting that the formation of senile plaques

may be a progressive process and the neuropathological

alterations may take longer than previously thought.

Interestingly, we observed that the impaired UPS, which is

reflected by accumulations of GFPu and Ub proteins, is not

a generalized process, but occurs in selected brain regions.

Our results indicated that GFPu in the cortex showed the

most striking accumulation among the three brain regions

(Fig. 1b, d, e; and increase of 1.38 folds in the cortex

versus 1.09 in the hippocampus and 1.06 in the striatum in

the AD mouse model). This may be partially because the

double mutant transgenes, APPswe and PS1dE9, cause

increased production of reactive oxygen species (ROS)

selectively in the cerebrum. Previous studies have shown

that the cerebrum shows the most significant increase of

ROS with age (Baek et al. 1999) and the cerebral cortex is

the most vulnerable brain regions to ROS insult (Crivello

et al. 2007). It is also possible that the region-specific

accumulation of GFPu in the AD mouse model may reflect

distinct cellular susceptibility to the toxicity caused by the

expressions of the mutant genes APPswe and PS1deE9 in

the mice. This may have significant implications for the

pathogenesis of AD. It has long been well known that both

the cerebral cortex and hippocampus play a pivotal role in

memory. Accordingly, perturbation of the UPS in the two

brain regions may exacerbate oxidative stress (Hensley

et al. 1995) and lead to progressive synaptic dysfunction

and neurodegeneration, eventually resulting in AD.

Acknowledgments We would like to thank Dr. Robin Miskimins

for critical reading of the manuscript, Dr. Fran Day at the Imaging

Core of the University of South Dakota for help in fluorescence

microscopy, and Mr. Suleman said at the histopathology core for

assistance in preparation of brain sections. This work was supported

by Start-up Funds from the University of South Dakota (HW).

Conflict of interest The authors have declared no conflicts of

interest.

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