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. 2013 Dec 21;34(3):315–322. doi: 10.1007/s10571-013-0022-9

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

Yanying Liu 1, Casey L Hettinger 1, Dong Zhang 1, Khosrow Rezvani 1, Xuejun Wang 1, Hongmin Wang 1,✉
PMCID: PMC3954921  NIHMSID: NIHMS554826  PMID: 24363091

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 extracellular senile plaques. Perturbation of the ubiquitin-proteasome 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 striatum 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 suggest 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 formation 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

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 methods and AD models have achieved conflicting results. On one hand, some studies have shown that UPS functionality 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-dependent 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; Kumarapeli 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 disease (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 (Jankowsky 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 experimental 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 previously described (Lu and Wang 2012). Total protein quantification 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 antibody, Santa Cruz Biotechnology, SC-6243, 1:1,000), anti-proteasome 20S α7 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 Biotechnology, 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 isolated 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 followed 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 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 promoter-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 littermates 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 difference 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 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 proteasome 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, α7. As shown in Fig. 1a–f, the levels of 7α 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.

Fig. 1.

Fig. 1

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Western blot analyses of APP, GFPu, p53, and proteasome 20S α7 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

To further determine the cellular and subcellular distribution of GFPu in brain cells, we next performed fluorescence microscopy to brain sections of the 4-week GFPu 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 hippocampus (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 observations obtained above, indicating distinct susceptibility of impaired degradation of GFPu in different brain regions in the young AD mouse model.

Fig. 2.

Fig. 2

Fig. 2

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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 μm in a, c, e, and 25 μm in g

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 hippocampus 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 performed 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.

Fig. 3.

Fig. 3

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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

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.

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

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