Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a severe decline of memory performance. A widely studied AD mouse model is the APPswe/PSEN1ΔE9 (APP/PS1) strain, as mice exhibit amyloid plaques as well as impaired memory capacities. To test whether restoring synaptic plasticity and decreasing β-amyloid load by Parkin could represent a potential therapeutic target for AD, we crossed APP/PS1 transgenic mice with transgenic mice overexpressing the ubiquitin ligase Parkin and analyzed offspring properties. Overexpression of Parkin in APP/PS1 transgenic mice restored activity-dependent synaptic plasticity and rescued behavioral abnormalities. Moreover, overexpression of Parkin was associated with down-regulation of APP protein expression, decreased β-amyloid load and reduced inflammation. Our data suggest that Parkin could be a promising target for AD therapy.

INTRODUCTION

Alzheimer's disease (AD) is the most prevalent form of neurodegenerative diseases and is characterized by a severe decline of memory performance and other cognitive abilities (1). The appearance of β-amyloid plaques and neurofibrillary tangles in the brain has been defined as pathological hallmark features of AD (2). It has been proposed that the decrease in amyloid clearance is due to impaired protein degradation by the ubiquitin–proteasome system (UPS). This hypothesis is supported by findings that the expression of several UPS components, including E1, E2, E3 ligase, and subunits of the proteasome is altered, and that proteasome activity is decreased during aging and AD (3,4). Thus, strategies aiming at restoring components of UPS might improve a clearance of β-amyloid plaques and help to recover memory performance in AD mouse models.

One component of the UPS is Parkin, a member of the Ring finger family of E3 ligases. Parkin was discovered in 1998 (5) and has been the subject of multiple studies related to Parkinson's disease (6,7), AD (8–11), lipid metabolism (12,13) and muscle degeneration (14). Moreover, it was found that Parkin levels are decreased in human AD brain (15) and that Parkin mediates ubiquitination and degradation of β-amyloid (15–17). Parkin deficiency causes cerebral and systemic amyloidosis (8), as well as aggregation of hyperphosphorylated Tau (11).

To test whether Parkin could represent a potential therapeutic target for Parkinson's disease or AD, we generated a Parkin transgenic mouse, in which overexpression of Parkin is restricted to neurons by using a neuron-specific enolase promoter. In our previous studies, we observed that Parkin overexpression reduced MPTP-induced dopaminergic neurodegeneration (18). To study the effect of Parkin overexpression on amyloid plaques and memory performance in AD mice, we crossed APPswe/PSEN1ΔE9 (APP/PS1) mice with mice overexpressing Parkin (Parkin OE) and analyzed their offspring (APP/PS1/Parkin OE). The APP/PS1 mouse strain is a double transgenic mouse strain expressing a chimeric human/mouse 695-amino acid APP isoform with the Swedish mutation and the exon-9-deleted variant (ΔE9) of PS1 and is characterized by increased levels of amyloid plaques and declined learning performance (19,20).

Here, we present data for APP, Parkin and additional components of the UPS, β-amyloid load, neuroinflammation, neurodegeneration, activity-dependent potentiation of synaptic transmission and behavioral performances in wild-type (WT), Parkin OE, APP/PS1 and APP/PS1/Parkin OE mice. Our results demonstrate that Parkin overexpression decreases β-amyloid load and gliosis in the cortex and the hippocampus of APP/PS1 mice. Moreover, Parkin overexpression protects against deficits in locomotion and neuropsychiatric behaviors in AD mice, and improves memory performance in Morris water maze.

RESULTS

Transcript expression levels of APP and UPS components in aging mice

Changes in UPS components, including E1, E2, E3, subunits of proteasome, and proteasome activity have been associated with brain aging and AD (3,4). To further specify this association in the cortex and the hippocampus, we analyzed mRNA levels of several E3 ligases, Parkin, SIAH-2, UCH-L1, Cbl-b, Cbl, CHIP, Itch, CDC23, Cul1 and Ccnf, E2 conjugation enzymes Ube2l6 and Ube2d1, and of the E1 activation enzyme Uba1. These genes were chosen for their distinct functions in the UPS. Real-time PCR results for distinct genes are summarized in Figure 1 for 3-, 6- and 9-month-old mice. Only 3 genes out of 13 were characterized by a steady decline in expression levels in the cortex and the hippocampus during aging. mRNA levels of UCH-L1, Cbl-b and Cbl were significantly reduced in 9-month-old mice, when compared with the expression level at 3 months of age (Fig. 1C–E). Several mRNAs for other genes were significantly up-regulated in the hippocampus but not in the cortex. The transcripts for SIAH-2, Itch, CDC23, Cul1 and Ube2l6 belong to this group and their expression levels were increased significantly in the hippocampus during aging (Fig. 1B–I and L). Interestingly, Parkin mRNA levels were up-regulated in the hippocampus with age, in contrast to their sharp decline in the cortex. A significant increase in Ccnf mRNA was found in the cortex as well as in the hippocampus. The two genes Uba1 and CHIP did not show significant changes in their transcript levels in the cortex or the hippocampus during aging. Our data show that the regulation of UPS-related gene transcripts exhibits not only a strong variability between the hippocampus and cortex, but also during aging.

Figure 1.

Age dependency of the transcript level of UPS-related genes in the cortex and hippocampus of C57BL/6J mice. Mice were 3-month- (white column), 6-month- (black column) and 9-month-old (gray column). (AJ) Bar diagrams summarize the relative expression levels of E3 ligases Parkin, SIAH-2, UCH-L1, Cbl-b, Cbl, CHIP, Itch, CDC23, Cul1 and Ccnf. (KM) Relative expression levels of E2 conjugation enzymes Ube2d1, Ube2l6 and E1 activation enzyme Uba1 are shown. mRNA expression levels were normalized to the amount of β-actin mRNA. Data are presented as means ± SEM (*P < 0.05 or **P < 0.01, n = 8–12).

Figure 1.

Age dependency of the transcript level of UPS-related genes in the cortex and hippocampus of C57BL/6J mice. Mice were 3-month- (white column), 6-month- (black column) and 9-month-old (gray column). (AJ) Bar diagrams summarize the relative expression levels of E3 ligases Parkin, SIAH-2, UCH-L1, Cbl-b, Cbl, CHIP, Itch, CDC23, Cul1 and Ccnf. (KM) Relative expression levels of E2 conjugation enzymes Ube2d1, Ube2l6 and E1 activation enzyme Uba1 are shown. mRNA expression levels were normalized to the amount of β-actin mRNA. Data are presented as means ± SEM (*P < 0.05 or **P < 0.01, n = 8–12).

To determine whether levels of UPS-related mRNAs as well as those for APP and BACE1 were further altered in AD mice, a real-time PCR investigation was conducted comparing the transcript level of distinct genes between WT and APP/PS1 mice at the age of 14–15 month (Table 1). We found that mRNAs for SIAH-2 and UCH-L1 were up-regulated in the hippocampus of APP/PS1 mice. Interestingly, the expression level of Parkin was significantly increased in the cortex but not in the hippocampus, indicating an inverse gene regulation from what was found in aging WT mice. Whereas many genes did not show significant changes in expression in the cortex or the hippocampus, a difference in relative expression level between the hippocampus and the cortex of APP/PS1 mice for Cbl-b, UCH-L1, Cul1, CDC23 and Uba1 was observed. In addition, the APP transcript level in APP/PS1 mice was found to be significantly different in comparison with WT mice in the cortex and the hippocampus. While BACE1 expression was not altered, its expression level was significantly different between the cortex and the hippocampus. The data suggest that only a moderate modulation of UPS-related gene expression takes place in 14–15-month-old APP/PS1 mice, although a significant up-regulation of Parkin was present in the cortex.

Table 1.

Transcript levels of APP, BACE1 and some selected UPS genes in the cortex and the hippocampus of APP/PS1 and WT mice

  Cortex
 
Hippocampus
 
WT APP/PS1 WT APP/PS1 
APP 1 ± 0.20 3.42 ± 0.49** 0.93 ± 0.02 2.96 ± 0.23** 
BACE1 1 ± 0.07 1.08 ± 0.08 1.59 ± 0.11 1.56 ± 0.17## 
Parkin 1 ± 0.16 1.82 ± 0.49* 0.95 ± 0.09 1.24 ± 0.11 
Cbl 1 ± 0.08 1.31 ± 0.25 1.41 ± 0.05 1.55 ± 0.10 
Cbl-b 1 ± 0.21 1.30 ± 0.18 2.02 ± 0.25 2.36 ± 0.25## 
UCH-L1 1 ± 0.13 1.28 ± 0.11 1.45 ± 0.08 1.82 ± 0.14*,## 
CHIP 1 ± 0.04 1.03 ± 0.04 1.32 ± 0.15 1.20 ± 0.09 
Itch 1 ± 0.08 1.19 ± 0.11 1.27 ± 0.04 1.44 ± 0.13 
SIAH-2 1 ± 0.09 0.99 ± 0.08 1.42 ± 0.10 1.13 ± 0.07* 
Ccnf 1 ± 0.10 0.85 ± 0.07 0.76 ± 0.03 0.74 ± 0.06 
Cul1 1 ± 0.07 1.03 ± 0.12 1.25 ± 0.09 1.53 ± 0.14## 
CDC23 1 ± 0.10 1.13 ± 0.09 1.40 ± 0.07 1.51 ± 0.11# 
Ube2l6 1 ± 0.14 1.14 ± 0.10 1.28 ± 0.26 1.34 ± 0.11 
Uba1 1 ± 0.03 1.07 ± 0.09 1.64 ± 0.14 1.71 ± 0.15## 
  Cortex
 
Hippocampus
 
WT APP/PS1 WT APP/PS1 
APP 1 ± 0.20 3.42 ± 0.49** 0.93 ± 0.02 2.96 ± 0.23** 
BACE1 1 ± 0.07 1.08 ± 0.08 1.59 ± 0.11 1.56 ± 0.17## 
Parkin 1 ± 0.16 1.82 ± 0.49* 0.95 ± 0.09 1.24 ± 0.11 
Cbl 1 ± 0.08 1.31 ± 0.25 1.41 ± 0.05 1.55 ± 0.10 
Cbl-b 1 ± 0.21 1.30 ± 0.18 2.02 ± 0.25 2.36 ± 0.25## 
UCH-L1 1 ± 0.13 1.28 ± 0.11 1.45 ± 0.08 1.82 ± 0.14*,## 
CHIP 1 ± 0.04 1.03 ± 0.04 1.32 ± 0.15 1.20 ± 0.09 
Itch 1 ± 0.08 1.19 ± 0.11 1.27 ± 0.04 1.44 ± 0.13 
SIAH-2 1 ± 0.09 0.99 ± 0.08 1.42 ± 0.10 1.13 ± 0.07* 
Ccnf 1 ± 0.10 0.85 ± 0.07 0.76 ± 0.03 0.74 ± 0.06 
Cul1 1 ± 0.07 1.03 ± 0.12 1.25 ± 0.09 1.53 ± 0.14## 
CDC23 1 ± 0.10 1.13 ± 0.09 1.40 ± 0.07 1.51 ± 0.11# 
Ube2l6 1 ± 0.14 1.14 ± 0.10 1.28 ± 0.26 1.34 ± 0.11 
Uba1 1 ± 0.03 1.07 ± 0.09 1.64 ± 0.14 1.71 ± 0.15## 

Mice were 14–15-month-old. Data are presented as means ± SEM

APP/PS1 versus WT: *P < 0.05, **P < 0.01.

Cortex versus hippocampus: #P < 0.05, ##P < 0.01, n = 4–8.

Parkin overexpression reduces post-translational levels of hippocampal APP and carboxy-terminal fragments in APP/PS1 mice

To determine whether Parkin OE could alter transcript levels of these genes in APP/PS1 mice additional real-time PCR studies were conducted. As shown in Figure 2A and B, expression levels of these genes were not altered with the exception of Parkin itself. Thus, Parkin is not modulating APP transcription. Since transcript levels of genes do not always correlate with the corresponding protein amount, we determined the protein levels for Parkin, BACE1, APP and carboxy-terminal fragments (CTFs). Parkin levels were significantly higher in the cortex and the hippocampus of Parkin OE and APP/PS1/Parkin OE mice in comparison with APP/PS1, indicating that increased Parkin levels were still present in APP/PS1/Parkin OE mice (Fig. 2C). No differences were found in BACE1 levels between the four strains of mice (Fig. 2D).

Figure 2.

Transcript and post-translational levels of Parkin, BACE1, APP and CTFs in the cortex and the hippocampus of 11–13-month-old mice. (A) Transcript levels of Parkin, BACE1, UCHL-1 and CHIP are presented for APP/PS1 and APP/PS1/Parkin OE mice. (B) mRNA levels of APP in the cortex and the hippocampus of four different mouse strains are shown. Relative protein expression levels for Parkin (C) and BACE1 (D) in the cortex and the hippocampus of different mouse strains are indicated. Expression levels of APP and CTFs in the cortex and the hippocampus are summarized in diagram (E) and (F), respectively. White, dark gray, light gray and black colored bars correspond to values from WT, Parkin OE, APP/PS1 and APP/PS1/Parkin OE mice, respectively. Positions of lanes of western blot inserts correspond with bar location. Data are normalized to corresponding β-actin levels and expressed as means ± SEM (brackets with *P < 0.05, **P < 0.01, n = 3–4).

Figure 2.

Transcript and post-translational levels of Parkin, BACE1, APP and CTFs in the cortex and the hippocampus of 11–13-month-old mice. (A) Transcript levels of Parkin, BACE1, UCHL-1 and CHIP are presented for APP/PS1 and APP/PS1/Parkin OE mice. (B) mRNA levels of APP in the cortex and the hippocampus of four different mouse strains are shown. Relative protein expression levels for Parkin (C) and BACE1 (D) in the cortex and the hippocampus of different mouse strains are indicated. Expression levels of APP and CTFs in the cortex and the hippocampus are summarized in diagram (E) and (F), respectively. White, dark gray, light gray and black colored bars correspond to values from WT, Parkin OE, APP/PS1 and APP/PS1/Parkin OE mice, respectively. Positions of lanes of western blot inserts correspond with bar location. Data are normalized to corresponding β-actin levels and expressed as means ± SEM (brackets with *P < 0.05, **P < 0.01, n = 3–4).

It has previously been shown that APP levels were increased in APP/PS1 mice, when compared with WT mice (21,22); however, levels of the carboxy-terminal fragments (CTFs) had not been characterized in the double mutant mice. To verify the expression level of APP and to learn more about CTFs regulation, we analyzed APP and CTFs levels in the cortex and the hippocampus of WT, APP/PS1 and Parkin OE mouse strains. In 12-month-old APP/PS1 mice, APP levels and related CTFs in the cortex (Fig. 2E) and the hippocampus (Fig. 2F) were significantly higher than those in WT or Parkin OE mice. In addition, APP levels in the cortex and the hippocampus of the triple transgenic mice were significantly decreased in comparison with those in the APP/PS1 group. Levels of the APP cleavage fragments, CTFs, were decreased significantly in the hippocampus (Fig. 2F), but not the cortex (Fig. 2E) in APP/PS1/Parkin OE mice, when compared with those in APP/PS1 mice. CTFs levels were also elevated in comparison with those in Parkin OE mice. In summary, these data indicate that Parkin overexpression has a strong effect on post-translational APP expression in the cortex and the hippocampus, reducing APP levels without altering APP transcription. Thus, we speculated that increased ubiquitination and altered proteasome activity might be responsible for the decrease in APP levels.

Overexpression of Parkin alters proteasome activity and p-Tau/Tau ratio

To better understand the mechanisms underlying the decrease in APP levels, we analyzed the 20S proteasome activity in WT, APP/PS1 and APP/PS1/Parkin OE mice. We found that in the hippocampus, but not in the cortex of APP/PS1 mice the proteasome activity was significantly decreased in comparison with WT mice (Fig. 3A). Overexpression of Parkin restored the proteasome activity. Thus, increased proteasome activity could be partly responsible for the decrease in APP levels in APP/PS1/Parkin OE mice. In addition, Parkin OE decreased phosphorylation levels of Tau in the hippocampus, as indicated by the significant decrease in the p-Tau/Tau ratio (Fig. 3B).

Figure 3.

Overexpression of Parkin alters proteasome activity and p-Tau/Tau ratio. (A) The hippocampal 20S proteasome activity was significantly reduced in APP/PS1 mice in comparison with WT mice and was found to be recovered to WT levels in APP/PS1/Parkin OE. (B) Western blotting of p-Tau and Tau revealed a significant difference of the p-Tau/Tau ratio in the hippocampus of APP/PS1 and APP/PS1/Parkin OE mice. Values from WT and Parkin OE mice served as a negative control. Data are expressed as means ± SEM and the significance of group differences is indicated with brackets and calculated P-values.

Figure 3.

Overexpression of Parkin alters proteasome activity and p-Tau/Tau ratio. (A) The hippocampal 20S proteasome activity was significantly reduced in APP/PS1 mice in comparison with WT mice and was found to be recovered to WT levels in APP/PS1/Parkin OE. (B) Western blotting of p-Tau and Tau revealed a significant difference of the p-Tau/Tau ratio in the hippocampus of APP/PS1 and APP/PS1/Parkin OE mice. Values from WT and Parkin OE mice served as a negative control. Data are expressed as means ± SEM and the significance of group differences is indicated with brackets and calculated P-values.

Overexpression of Parkin decreased β-amyloid load in the triple transgenic mice

Overexpression of Parkin in APP/PS1 mice caused a reduction in APP levels in the cortex as well as in the hippocampus. Since higher APP levels might result in increased generation of β-amyloid and amyloid plaques, we investigated the effect of overexpression of Parkin on β-amyloid levels. The amount of soluble and insoluble human β-amyloid was determined using a sandwich Elisa method. Values from WT and Parkin OE mice served as negative controls and depicted in Figure 4A. A clear expression of human β-amyloid was detected in the cortex and the hippocampus of APP/PS1 mice in comparison with that in WT and Parkin OE mice (Fig. 4A). Overexpression of Parkin decreased β-amyloid levels in the cortex significantly and moderately in the hippocampus when compared with APP/PS1 mice (Fig. 4A).

Figure 4.

β-Amyloid and amyloid plaques in the cortex and the hippocampus of transgenic and WT mice. (A) Parkin overexpression reduces β-amyloid levels in 6-month-old triple transgenic mice. Levels of total β-amyloid levels in the cortex and the hippocampus are expressed as pg/µg protein (n = 3–7 per group). (B) The appearance of β-amyloid plaques was evaluated using Congo-Red staining. Brightfield images show typical Congo-Red staining of the cortex and the hippocampus of 11–13-month-old mice as well amyloid plaques (black arrows). The number of dark spots was markedly reduced in sections of (D) APP/PS1/Parkin OE mice. (C) Plaques burden was determined by thioflavin-S staining. The area of amyloid plaques was determined and normalized to the total area. Fluorescence images indicate the distribution of amyloid plaques (white arrows) in the cortical and hippocampal area of 11–13-month-old mice. (D) Representative fluorescence image of thioflavin-S staining of sections from 6-month-old mice. Representative fluorescent images in (B, C and D) indicate sections from (a) WT, (b) Parkin OE, (c) APP/PS1 and (d) APP/PS1/Parkin OE mice (n = 4–13 per group). Scale bars: 500 µm. Data are expressed as means ± SEM (*/**P < 0.05).

Figure 4.

β-Amyloid and amyloid plaques in the cortex and the hippocampus of transgenic and WT mice. (A) Parkin overexpression reduces β-amyloid levels in 6-month-old triple transgenic mice. Levels of total β-amyloid levels in the cortex and the hippocampus are expressed as pg/µg protein (n = 3–7 per group). (B) The appearance of β-amyloid plaques was evaluated using Congo-Red staining. Brightfield images show typical Congo-Red staining of the cortex and the hippocampus of 11–13-month-old mice as well amyloid plaques (black arrows). The number of dark spots was markedly reduced in sections of (D) APP/PS1/Parkin OE mice. (C) Plaques burden was determined by thioflavin-S staining. The area of amyloid plaques was determined and normalized to the total area. Fluorescence images indicate the distribution of amyloid plaques (white arrows) in the cortical and hippocampal area of 11–13-month-old mice. (D) Representative fluorescence image of thioflavin-S staining of sections from 6-month-old mice. Representative fluorescent images in (B, C and D) indicate sections from (a) WT, (b) Parkin OE, (c) APP/PS1 and (d) APP/PS1/Parkin OE mice (n = 4–13 per group). Scale bars: 500 µm. Data are expressed as means ± SEM (*/**P < 0.05).

To verify the ELISA data, thioflavin-S staining was used to detect amyloid plaques in the cortex and the hippocampus. Amyloid plaques were not detectable in the cortex and the hippocampus of WT and Parkin OE mice at 11–13 months (Fig. 4C) and 6 months (Fig. 4D). In contrast, intense fluorescent labels were observed in the cortex and the hippocampus of APP/PS1 mice, indicating the presence of amyloid plaques (Fig. 4Cc). The relative fluorescent area was significantly reduced in the cortex and the hippocampus of APP/PS1/Parkin OE mice (Fig. 4Cd). The number of plaques in brain sections from 6-month-old APP/PS1 mice was lower than in sections from older mice, but a decrease by Parkin OE was still present (Fig. 4Dc and Dd). Finally, Congo-Red staining was used to confirm alterations in amyloid plaque load in the cortex and the hippocampus of the four different mouse strains (Fig. 4B). The results were similar to those found with thioflavin-S staining.

Effects of Parkin overexpression on inflammation in the cortex and the hippocampus

One of the most prevalent hypotheses for AD is the development of chronic inflammation. To evaluate inflammation processes in transgenic mouse strains, we analyzed the levels of microglia and astrocyte activation using Iba1 and GFAP as markers in sections from 11- to 13-month-old mice. As shown in Figure 5C, GFAP immunofluorescence was increased in the cortex and the hippocampus of APP/PS1 mice in comparison with that in WT and Parkin OE mice. Overexpression of Parkin in APP/PS1 mice markedly reduced GFAP levels. These observations were verified using the quantification of GFAP levels in western blot (Fig. 5A and B). GFAP levels were significantly reduced in the hippocampus of APP/PS1/Parkin OE mice in comparison with those in APP/PS1 mice (Fig. 5B). Similar analysis revealed a reduction in GFAP levels in the cortex (Fig. 5A). In contrast, levels of the microglia marker Iba1 in APP/PS1 mice were not altered by Parkin overexpression (Fig. 5C).

Figure 5.

Activation of astrocytes but not microglia is ameliorated in 11–13-month-old APP/PS1/Parkin OE mice. (A) Representative western blots (right) and the diagram show the GFAP expression level in the cortex for WT, APP/PS1/Parkin OE and APP/PS1 mice. (B) Blots and data are presented for the GFAP expression level in the hippocampus. Parkin overexpression reduced the GFAP level in comparison with APP/PS1 mice in the cortex and the hippocampus. Data are expressed as means ± SEM (*P < 0.05; #P < 0.01, n = 3). (C) Representative fluorescence images of GFAP (green) and merged with NeuN (red) for cortex (left) and hippocampus (right) of the indicated mouse strains are depicted. The immunosignal for GFAP was increased in APP/PS1 mice and reduced in APP/PS1/Parkin OE mice. Immunofluorescence images for the microglia marker Iba 1 (green) and merged with NeuN (red) are presented. (D) No difference in GFAP staining was seen in sections from the different strains of 6-month-old mice. (E) LPS injection into striatum-induced expression of Iba 1 and GFAP in 12-month-old WT, WT + LPS and Pakin OE + LPS. Scale bar: 100 µm.

Figure 5.

Activation of astrocytes but not microglia is ameliorated in 11–13-month-old APP/PS1/Parkin OE mice. (A) Representative western blots (right) and the diagram show the GFAP expression level in the cortex for WT, APP/PS1/Parkin OE and APP/PS1 mice. (B) Blots and data are presented for the GFAP expression level in the hippocampus. Parkin overexpression reduced the GFAP level in comparison with APP/PS1 mice in the cortex and the hippocampus. Data are expressed as means ± SEM (*P < 0.05; #P < 0.01, n = 3). (C) Representative fluorescence images of GFAP (green) and merged with NeuN (red) for cortex (left) and hippocampus (right) of the indicated mouse strains are depicted. The immunosignal for GFAP was increased in APP/PS1 mice and reduced in APP/PS1/Parkin OE mice. Immunofluorescence images for the microglia marker Iba 1 (green) and merged with NeuN (red) are presented. (D) No difference in GFAP staining was seen in sections from the different strains of 6-month-old mice. (E) LPS injection into striatum-induced expression of Iba 1 and GFAP in 12-month-old WT, WT + LPS and Pakin OE + LPS. Scale bar: 100 µm.

Investigation of astrocyte and microglia activation in 6-month-old mice did not reveal detectable alteration in expression levels of these markers between mouse strains (Fig. 5D).

In addition, we determined whether Parkin OE could reduce inflammation in general or whether this effect was due to a reduction in the amyloid load. Induction of inflammation by lipopolysaccharide (LPS) injection into the striatum increased Iba 1 and GFAP expression, and this effect was not altered by Parkin overexpression (Fig. 5E). Thus, reduction in the GFAP expression level in APP/PS1/Parkin OE mice is likely mediated by the reduction in amyloid load.

Neurodegeneration in APP/PS1 mice does not alter synaptophysin level but is slightly reduced by Parkin overexpression

Our previous data indicated significant increases in the APP level, β-amyloid and amyloid plaques as well as astrocyte activation, which were partially reduced by Parkin overexpression. All these processes had been linked to neurodegeneration. Thus, we analyzed neurodegeneration at the synaptic level by using the presynaptic vesicle-associated protein synaptophysin as a marker, and evaluated the amount of neuronal death using Fluoro-Jade B. As shown in Figure 6, expression levels of synaptophysin were not modified in all investigated mouse strains in the cortex (Fig. 6A) and the hippocampus (Fig. 6B).

Figure 6.

Synaptophysin expression and neurodegeneration in the cortex and the hippocampus of transgenic and WT mice. (A) The depicted western blot indicates the expression level of synaptophysin in the cortex of 11–13-month-old mouse strains with different genotypes. The relative expression level of synaptohpysin is summarized in the bar diagram. (B) Data are presented for synaptohpysin expression in the hippocampus. No changes in the expression level were detectable in the cortex or the hippocampus for the different mouse strains (n = 3 each group). β-Actin served as the internal control. (C) The level of neurodegeneration in the cortex and the hippocampus was evaluated using Fluoro-Jade B staining. White arrows indicate typical spots of degenerating neurons in the fluorescence images of the cortex and the hippocampus from (a) WT; (b) Parkin OE; (c) APP/PS1 and (d) APP/PS1/Parkin OE mice. Parkin overexpression reduced the amount of degenerating neurons only partially. Scale bars: 500 µm. All data are expressed as means ± SEM, *P < 0.05, **P < 0.01, n = 7–17.

Figure 6.

Synaptophysin expression and neurodegeneration in the cortex and the hippocampus of transgenic and WT mice. (A) The depicted western blot indicates the expression level of synaptophysin in the cortex of 11–13-month-old mouse strains with different genotypes. The relative expression level of synaptohpysin is summarized in the bar diagram. (B) Data are presented for synaptohpysin expression in the hippocampus. No changes in the expression level were detectable in the cortex or the hippocampus for the different mouse strains (n = 3 each group). β-Actin served as the internal control. (C) The level of neurodegeneration in the cortex and the hippocampus was evaluated using Fluoro-Jade B staining. White arrows indicate typical spots of degenerating neurons in the fluorescence images of the cortex and the hippocampus from (a) WT; (b) Parkin OE; (c) APP/PS1 and (d) APP/PS1/Parkin OE mice. Parkin overexpression reduced the amount of degenerating neurons only partially. Scale bars: 500 µm. All data are expressed as means ± SEM, *P < 0.05, **P < 0.01, n = 7–17.

In a further attempt to analyze the amount of neuronal death in the cortex and the hippocampus, we used Fluoro-Jade B staining in brain sections of 11–13-month-old mice. Whereas degenerating neurons were not detectable in sections from the cortex and the hippocampus of WT and Parkin OE mice, a large number of degenerating cells was observed in brain sections from APP/PS1 and APP/PS1/Parkin OE mice (Fig. 6C). The quantification of the fluorescence revealed a partial reduction in neurodegeneration in APP/PS1/Parkin OE mice in comparison with APP/PS1 (Fig. 6D).

Parkin rescues long-term potentiation impairment in APP/PS1 mice

Although our data indicated alterations in UPS-related genes, APP, amyloid, inflammation and neurodegeneration in different mouse strains, the functional consequences on synaptic transmission and animal behavior remained to be clarified. We first analyzed hippocampal synaptic transmission and activity-dependent synaptic plasticity in 6-month-old Parkin OE, APP/PS1 and APP/PS1/Parkin OE mice. We used a two-input stimulation paradigm to investigate potential changes in long-term potentiation (LTP; Fig. 7). LTP was induced in S1 and base line stability was evaluated by stimulation of a second synaptic input, S2. LTP was induced by three trains of 1 s 100 Hz tetanization with an inter-train interval of 10 min in acute hippocampal slices prepared from the various mouse strains. Values at the 5 and 180th min were 205 ± 6 and 172 ± 8% in WT mice (Fig. 7A). Overexpression of Parkin did not influence LTP expression compared with WT mice, and values at the 5 and 180th min were 200 ± 7 and 160 ± 7%, respectively (Fig. 7B). LTP in slices from APP/PS1 transgenic mice was significantly reduced (180th min: 121 ± 5%, Fig. 7C) in comparison with WT mice (P = 0.0001). We further determined whether overexpression of Parkin in APP/PS1 mice would restore LTP expression to the control level. High-frequency stimulation in hippocampal slices from APP/PS1/Parkin OE mice caused a field excitatory postsynaptic potential (fEPSP) potentiation that lasted over the 3h of recording at a level of ∼155% (180th min: 161 ± 5%, Fig. 7D). This fEPSP potentiation was significantly different from the one measured in slices from APP/PS1 mice (180th min: P = 0.002). Thus, Parkin overexpression in APP/PS1 mice restores LTP to a level not different from that found in WT or Parkin OE mice (Fig. 7E). In addition, we used a different LTP induction paradigm, which was based on theta-bust stimulation (2 times 5 bursts with 10 stimuli at 100 Hz and an interburst interval of 200 ms every 30 s). This paradigm induced a transient LTP in the hippocampus of APP/PS1 mice, which was prolonged by Parkin overexpression (Fig. 7F).

Figure 7.

Parkin overexpression restores hippocampal long-term potentiation in APP/PS1 mice. (A) The insert represents relative positions of electrodes within hippocampal slice. Activity-dependent synaptic plasticity was induced in input S1 by 1-s 100 Hz stimuli [3× high-frequency stimulation (HFS)]. Baseline stability was monitored at input S2. LTP in the acute slices of WT mice was stable for at least 180 min; (B) Parkin overexpression did not alter LTP; (C) fEPSP-slope potentiation in the slices from APP/PS1 transgenic mice declined significantly in comparison with the control and Parkin group; (D) Parkin overexpression prevented APP/PS1-related decay of fEPSP potentiation. fEPSPs of the non-tetanized input S2 remained stable in all groups. Inserts indicate fEPSP traces before tetanization and 180 min after. Scale bars: horizontal: 10 ms; vertical: 1 mV. (E) Bar diagram summarizes averaged group values at the 180th min for different mouse strains. Mice were 6–8-month-old, n = 5–10 per group. (F) Theta-burst stimulation (2 × 5 bursts with 10 stimuli at 100 Hz every 30 s) evoked a transient LTP in APP/PS1 mice (gray, n = 8), which was rescued by Parkin overexpression (black, n = 5). Brackets indicate a significant difference between groups.

Figure 7.

Parkin overexpression restores hippocampal long-term potentiation in APP/PS1 mice. (A) The insert represents relative positions of electrodes within hippocampal slice. Activity-dependent synaptic plasticity was induced in input S1 by 1-s 100 Hz stimuli [3× high-frequency stimulation (HFS)]. Baseline stability was monitored at input S2. LTP in the acute slices of WT mice was stable for at least 180 min; (B) Parkin overexpression did not alter LTP; (C) fEPSP-slope potentiation in the slices from APP/PS1 transgenic mice declined significantly in comparison with the control and Parkin group; (D) Parkin overexpression prevented APP/PS1-related decay of fEPSP potentiation. fEPSPs of the non-tetanized input S2 remained stable in all groups. Inserts indicate fEPSP traces before tetanization and 180 min after. Scale bars: horizontal: 10 ms; vertical: 1 mV. (E) Bar diagram summarizes averaged group values at the 180th min for different mouse strains. Mice were 6–8-month-old, n = 5–10 per group. (F) Theta-burst stimulation (2 × 5 bursts with 10 stimuli at 100 Hz every 30 s) evoked a transient LTP in APP/PS1 mice (gray, n = 8), which was rescued by Parkin overexpression (black, n = 5). Brackets indicate a significant difference between groups.

Parkin attenuates behavioral deficits of APP/PS1 mice in open-field test, elevated plus-maze test and Morris water maze

AD mouse models exhibit neuropsychiatric and learning abnormalities during aging (19,23,24). Various behavioral paradigms were used to test whether Parkin overexpression could improve behavioral impairments in APP/PS1 mice.

The open-field test is used to assess locomotion, exploratory and anxiety-like behavior. The open-field test was performed with 6-month-old mice. Total ambulatory distances within the first and second 15 min of observation were increased significantly in APP/PS1 mice in comparison with WT or Parkin OE mice (Fig. 8A and B). Moreover, the time APP/PS1 mice remained in the center was significantly decreased only during the second 15 min of observation in comparison with APP/PS1/Parkin OE mice (Fig. 8C). The longer travel path together with the same or shorter time APP/PS1 mice remained in the center indicates higher movement velocity and slightly elevated anxiety levels. Interestingly, Parkin overexpression in APP/PS1 mice restored total ambulatory distance and ambulatory distance in the center to values found in WT and Parkin OE mice.

Figure 8.

Parkin restores behavioral deficits of APP/PS1 mice in open-field test (AC), elevated plus-maze test (D) and Morris water maze (EG). The diagram (A) summarizes the total ambulatory distance (cm) for each of the four different mouse strains (6-month-old, n = 8–20 per group) in the open-field test for the first and second 15 min of observation. APP/PS1 mice showed a significant increase in travel distance, which was reduced by Parkin. (B) Similar results were obtained for their ambulatory distance in centre. (C) The time spent in the centre was only different for the second observation interval indicating a faster movement of the APP/PS1 mice. Data for the elevated plus-maze test are summarized in the diagram (D), presenting the ratio of open arm entries to total entries (9–10-month-old, n = 7–17 per group). Spatial learning and memory performance was tested using Morris water maze and data summarized in diagram (E) to (G) (n = 14–25). The bar diagram (E) represents the average swimming speed for different groups. (F) The average escape latency (four trails per day) for eight consecutive days and (G) crossing times over the former platform location in the probe test are presented. The escape latency was significant different (P < 0.05) at Day 8 between APP/PS1/Parkin OE versus APP/PS1, APP/PS1 versus Parkin OE and APP/PS1 versus WT. In diagram (F) calculated P-values are indicated for Day 8. Data are expressed as means ± SEM. Significance level for between certain groups are indicated with brackets (P < 0.05 or calculate P-values) and corresponding symbols.

Figure 8.

Parkin restores behavioral deficits of APP/PS1 mice in open-field test (AC), elevated plus-maze test (D) and Morris water maze (EG). The diagram (A) summarizes the total ambulatory distance (cm) for each of the four different mouse strains (6-month-old, n = 8–20 per group) in the open-field test for the first and second 15 min of observation. APP/PS1 mice showed a significant increase in travel distance, which was reduced by Parkin. (B) Similar results were obtained for their ambulatory distance in centre. (C) The time spent in the centre was only different for the second observation interval indicating a faster movement of the APP/PS1 mice. Data for the elevated plus-maze test are summarized in the diagram (D), presenting the ratio of open arm entries to total entries (9–10-month-old, n = 7–17 per group). Spatial learning and memory performance was tested using Morris water maze and data summarized in diagram (E) to (G) (n = 14–25). The bar diagram (E) represents the average swimming speed for different groups. (F) The average escape latency (four trails per day) for eight consecutive days and (G) crossing times over the former platform location in the probe test are presented. The escape latency was significant different (P < 0.05) at Day 8 between APP/PS1/Parkin OE versus APP/PS1, APP/PS1 versus Parkin OE and APP/PS1 versus WT. In diagram (F) calculated P-values are indicated for Day 8. Data are expressed as means ± SEM. Significance level for between certain groups are indicated with brackets (P < 0.05 or calculate P-values) and corresponding symbols.

In the elevated plus-maze test (EPM), the total time in the open arms was similar for the 6-month-old mice in the four experimental groups (data not shown). However, the ratio of open arm exploration time to total exploration time for the APP/PS1/Parkin OE mice increased significantly in comparison with the WT group (data not shown). The ratio of the number of entries in the open arms to the total number of entries was reduced in APP/PS1 mice at 9–10 months when compared with that in age-matched WT mice (Fig. 8D).

The Morris water maze has been widely utilized to assess spatial memory and learning related to hippocampal function (25,26). The average swimming speeds among the four genotypes of mice were similar (Fig. 8E). However, 9–10-month-old APP/PS1 mice required more time to find the platform from Day 1 to 8 when compared with WT mice. On the 8th day, both Parkin OE and APP/PS1/Parkin OE mice spent less time to reach the platform when compared with the APP/PS1 mice (Fig. 8F). In the probe trial, the platform-crossing time in APP/PS1 mice was significantly reduced when compared with the values from WT and Parkin OE mice (Fig. 8G).

DISCUSSION

AD mouse models are widely used to learn more about the underlying mechanism of AD and to find remedies to reverse the associated cognitive decline (20,27–30). In this study, we analyzed age-dependent regulation of UPS-related genes, which are thought to be associated with the onset of AD and the effects of Parkin overexpression in WT and APP/PS1 mice. Our data showed a strong alteration in E3 ligases and E2 conjugation enzymes during aging, which were only slightly altered by the expression of human mutant APP and PS1. Moreover, we show for the first time that Parkin overexpression in APP/PS1 mice is an effective way to reduce β-amyloid load and neurodegeneration and to restore activity-depended synaptic plasticity as well as behavioral deficits in APP/PS1 mice.

APP and UPS in aging mice

The UPS is involved in multiple signaling pathways regulating gene expression (31) and synaptic plasticity (32,33). UPS is also age-dependently altered, and modulation of UPS during aging might account for decline of cognitive abilities (3). In addition, amyloid plaques and Tau tangles in AD patients are heavily ubiquitinated (34,35).

During maturation and aging, UCH-L1 mRNA levels decreased in the cortex and the hippocampus in WT mice, but were increased in the hippocampus of old APP/PS1 mice. These data supplement findings describing that the E3 ligase UCH-L1 is down-regulated in AD brains (36) and that transduction of UCH-L1 protein restores synaptic function in APP/PS1 mice (37).

Other UPS members of the E2 conjugation enzymes and E3 ligases were also altered during aging. However, we could not find a general trend regarding these components, because their gene expression level was found to be either up- or down-regulated during aging and was often different in the cortex and the hippocampus. For example, Parkin was down-regulated in the cortex within 6 months but not in the hippocampus, where a significant up-regulation of gene expression was detected. However, in cortex of 14–15-month-old APP/PS1 mice, the transcript level of Parkin was significantly up-regulated in comparison with their WT counterparts. How APP and PS1 mutations mediate modulation of Parkin transcription remains unclear. Other genes of the UPS were only slightly altered in APP/PS1 mice, or were differently regulated in the cortex and the hippocampus of the APP/PS1 mice. In summary, UPS is age-dependently regulated; however, additional mutations of APP and PS1 did not alter UPS-related gene expression with the exception of Parkin, UCH-L1 and SIAH-2.

Post-translational levels of APP and CTFs in APP/PS1 mice

In APP/PS1 mice, the protein level of APP and CTFs was significantly higher than in WT or Parkin OE mice. Parkin overexpression in APP/PS1 mice efficiently reduced APP levels. The level of CTFs was also decreased in the hippocampus of APP/PS1/Parkin OE, but was still higher than that of Parkin OE mice. Interestingly, the transcript level of APP was not altered by overexpressed Parkin, indicating that reduction in post-translational levels of APP is mediated by different processes. Other studies indicated that inhibition of the proteasome increases APP processing and release of amyloid by interaction with gamma-secretase (38), in addition to the fact that APP is itself a proteasome substrate (39).

We also showed that Parkin overexpression in APP/PS1 mice increased the 20S chymotrypsin-like proteasome activity, which could likely reduce APP processing and decrease amyloid release. Our data are in line with findings that Parkin can directly alter proteasome activity in an ATP-dependent and independent manner (15,40).

Parkin reduces β-amyloid load in triple transgenic mice

A consequence of attenuated APP and CTFs might be a lower load of soluble and insoluble β-amyloid and amyloid plaques. We found using Elisa and amyloid plaque staining that Parkin overexpression causes a significant reduction in amyloid load especially in the cortex. Our data are in line with those of Moussa et al. (41), who showed that Parkin overexpression promotes ubiquitination and proteasomal degradation of β-amyloid in both the lentiviral models of intracellular β-amyloid and the 3xTg-AD mice (14–17).

Parkin alters neurodegeneration in APP/PS1 mice

Inflammation is another deleterious mechanism in AD pathogenesis. The increase in reactive astrocytes in the cortex and the hippocampus of APP/PS1 mice during aging has been reported (42). We also detected a severe gliosis in the hippocampus of 9-month-old APP/PS1 mice when compared with the levels in WT or Parkin OE mice. In line with the effects of Parkin to reduce amyloid load, Parkin also caused a normalization of the amount of reactive astrocytes, but not of the activation of microglia.

The neuronal death observed in APP/PS1 mice was attenuated by Parkin overexpression. One possible explanation for the promotion of cell survival might be related to protection of mitochondrial activity by Parkin (43,44). Mitochondrial dysfunction and oxidative stress are obvious in Parkin null mice and Drosophila (45,46). We have also reported that Parkin overexpression can ameliorate MPTP-induced damage to neuronal mitochondria in the substantia nigra (18). Moreover, in the triple transgenic AD mice, lentiviral-mediated Parkin overexpression prevented mitochondrial dysfunction and oxidative stress (16). Thus, we speculate that Parkin overexpression reduces oxidative stress and promotes mitochondrial function in APP/PS1/Parkin OE mice. This would cause a normalization of ATP production that could support protein degradation by ATP-dependent proteasome activity, and might reduce amyloid load. Our data showing that Parkin overexpression does not alter LPS-induced inflammation; further point into the direction that general inflammation-related process is not altered by Parkin, but that this effect is more likely indirect and due to reduced amyloid load (47).

Parkin rescues impairment of LTP in APP/PS1 mice

Synaptic plasticity is thought to represent one of the cellular mechanisms underlying learning and memory (48). Alteration of synaptic plasticity in the hippocampus or the frontal cortex is often correlated with deficits in memory performance (25,49). We compared LTP of fEPSPs in hippocampal slices from WT, Parkin OE, APP/PS1 and APP/PS1/Parkin OE. LTP was impaired in APP/PS1 mice and restored by Parkin overexpression. Parkin might have normalized LTP by prevention of amyloid load and astrocyte activation. Indeed, it was shown that amyloid application can prevent LTP induction (50–53) and that inflammation can reduce LTP (54). Our data indicate a weaker attenuation of LTP in APP/PS1 mice when compared with in vivo studies (55), which might also be correlated to the overall amyloid level and age of the mice. In addition, we observed a weaker LTP phenotype with the same strain and dependency on the LTP induction paradigm used. However, in both cases, Parkin overexpression was able to restore LTP.

Parkin and behavioral deficits of APP/PS1 mice

Besides memory decline, AD patients manifest behavioral and psychological symptoms of dementia (56,57). Similar observations were made in AD mice, in which besides the development of β-amyloid plaques, deficiencies in memory performance and alterations in neuropsychiatric behaviors are common. Here, we studied whether Parkin overexpression could restore behavioral performance in APP/PS1 mice. We found that 6-month-old APP/PS1 mice displayed hyperactivity and increased anxiety levels, as shown by longer ambulatory distances and shorter time spent in the center of the open-field area, which were restored by Parkin overexpression. The effect of Parkin on activity and anxiety of APP/PS1 mice was further confirmed in the elevated EPM. In addition, we found that Parkin overexpression improved significantly the memory performance of AD mice, as APP/PS1/ Parkin OE mice learned better than APP/PS1. However, an increased anxiety level might be to a certain degree responsible in alteration of learning abilities of mice (58).

We provided experimental observations that Parkin overexpression counteracts the increased levels of APP-CTFs, amyloid burden and reduced proteasome activity in APP/PS1 mice improving thereby synaptic plasticity and behavioral performance. In addition, our observations suggest that Parkin does not regulate gene expression of APP, but rather its translational level and also improves function of the proteasome in APP/PS1 mice. In addition, publications indicate, for example, that Parkin overexpression might act over a reduction of proteotoxicity and improvement of mitochondrial dynamics (59), interference with PINK1-phosphorylated mitofusin 2 to stabilize mitochondria (60), direct interactions with the Proteasome (40) and that Parkin-dependent ubiquitylome are altered by mitochondrial depolarization (61). However, the direct molecular interactions of Parkin and its potential targets require further clarification using a different set of methods.

In summary, Parkin overexpression restores LTP and reduces the anxiety level in AD mice. The beneficial effects of Parkin protein are associated with down-regulation of APP proteins, improvement of β-amyloid clearance and inhibition of inflammation.

MATERIALS AND METHODS

Ethics statement

The animal care and procedures were approved and performed under established standards of the Institutes of Brain Science and State Key Laboratory of Medical Neurobiology of Fudan University, Shanghai, China.

Animals

Parkin overexpression transgenic mice (Parkin OE) (18) were crossed with APP/PS1 mice [B6C3-Tg (APPswe, PSEN1dE9)85Dbo, The Jackson Laboratory, Bar Harbor, Maine, USA]. The offspring of either sex were genotyped by primers for APP, PS1 and Parkin as listed in Table 2. Triple transgenic mice are indicated as APP/PS1/Parkin OE. C57BL/6J mice were purchased from the Shanghai Branch of National Rodent Laboratory Animal Resources, China. Mice were housed in a 12 h light/dark cycle with food and water ad libitum.

Table 2.

Primers for APP, PS1 and Parkin transgenes

Parkin 
 Park2 5′-CATCACTTCAGGATCCTTGGAGAAG-3′ 
 Park7 5′-GTCCTTGTAGTCCACGTCAAACCAG-3′ 
APPswe 
 oIMR1597 5′-GACTGACCACTCGACCAGGTTCTG-3′ 
 oIMR1598 5′-CTTGTAAGTTGGATTCTCATATCCG-3′ 
PSEN1 A 
 oIMR1644 5′-AATAGAGAACGGCAGGAGCA-3′ 
 oIMR1645 5′-GCCATGAGGGCACTAATCAT-3′ 
PSEN1 B 
 oIMR0944 5′-CCTCTTTGTGACTATGTGGACTGATGTCGG-3′ 
 oIMR1588 5′-GTGGATAACCCCTCCCCCAGCCTAGACC-3′ 
Parkin 
 Park2 5′-CATCACTTCAGGATCCTTGGAGAAG-3′ 
 Park7 5′-GTCCTTGTAGTCCACGTCAAACCAG-3′ 
APPswe 
 oIMR1597 5′-GACTGACCACTCGACCAGGTTCTG-3′ 
 oIMR1598 5′-CTTGTAAGTTGGATTCTCATATCCG-3′ 
PSEN1 A 
 oIMR1644 5′-AATAGAGAACGGCAGGAGCA-3′ 
 oIMR1645 5′-GCCATGAGGGCACTAATCAT-3′ 
PSEN1 B 
 oIMR0944 5′-CCTCTTTGTGACTATGTGGACTGATGTCGG-3′ 
 oIMR1588 5′-GTGGATAACCCCTCCCCCAGCCTAGACC-3′ 

Tissue collection

For histochemistry, mice were anesthetized with chloral hydrate (400 mg/kg i.p.) and perfused transcardially with physiological saline followed by 4% paraformaldehyde in physiological saline. The brains were quickly removed and post-fixed overnight at 4°C in 4% paraformaldehyde. For RT–PCR and western blot experiments, mice were anesthetized with 10% chloral hydrate. Brains were carefully removed; the cortex and the hippocampus were isolated on ice for total protein preparation and total RNA extraction.

Real-time PCR

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, USA). Reverse transcription was carried out using random primer and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, USA). Real-time PCR was performed for the quantification of APP, Parkin, BACE1, Cbl, Cbl-b, UCH-L1, CHIP, Itch, SIAH-2, Ccnf, Cul1, CDC23, Ube2l6, Uba1 with a quantitative thermal cycler (Mastercycler ep realplex, Eppendorf, Germany). Relative expression values were calculated as the ratio of target cDNA to β-actin. The primers used in the real-time PCR are presented in Table 3.

Table 3.

Primers for real-time PCR

β-Actin forward 5′-ATGAGGTAGTCTGTCAGGT-3′ 
β-Actin reverse 5′-ATGGATGACGATATCGCT-3′ 
APP forward 5′-TGCTGGCAGAACCCCAGATCG-3′ 
APP reverse 5′-TTCTGGATGGTCACTGGCTGG-3′ 
BACE1 forward 5′-GCATCGCTACTACCAGAGGCA-3′ 
BACE1 reverse 5′-GGTCTGCTTCACCAGGGAGTC-3′ 
Parkin forward 5′-CCAAACCGGATGAGTGGTGAGTGC-3′ 
Parkin reverse 5′-ACACGGCAGGGAGTAGCCAAGTTG-3′ 
Cbl forward 5′-CCATTCTCATTGCCCTCACAAATGG-3′ 
Cbl reverse 5′-CAGTCACATGCTCGTGAACTCTGGG-3′ 
Cbl-b forward 5′-CATGGACAAAGTGGTAAGACTGTGC-3′ 
Cbl-b reverse 5′-CTGCCAGCATGTGACTGAAGATAAG-3′ 
UCH-L1 forward 5′-ATTGAGGAACTGAAGGGACAGGAAG-3′ 
UCH-L1 reverse 5′-TGGAAATTCACTTTGTCATCTACCC-3′ 
CHIP forward 5′-TGCTGAGCGAGAGAGGGAACTGGAG-3′ 
CHIP reverse 5′-CACACGCTGCAGGTGCTCCTCAATG-3′ 
Itch forward 5′-CTTCAGATCACTGTCATCTCAGC-3′ 
Itch reverse 5′-TCTCTGTTGGCTCTTTGTCACC-3′ 
Siah2 forward 5′-GTGTAACCAATGCCGCCAGAAG-3′ 
Siah2 reverse 5′-AGCCCCTGGCAGGTTAATGTCTG-3′ 
Ccnf forward 5′-TCAGCGACACCATGAGGTACATTC-3′ 
Ccnf reverse 5′-AGTGTCAGCAGGACCTCTTTATAG-3′ 
Cul1 forward 5′-AGCAGTGGGAAGATTACCGATTC-3′ 
Cul1 reverse 5′-TCAGCCCCAATTCCACATAAG-3′ 
CDC23 forward 5′-AGGATGTGGATGCTTACACCCTG-3′ 
CDC23 reverse 5′-ACCACCCCATACAGGTAAAGGC-3′ 
Ube2d1 forward 5′-CGTGGGAGATGACTTGTTCCAC-3′ 
Ube2d1 reverse 5′-TCTGGTACTAAGGGATCGTCTGG-3′ 
Ube2l6 forward 5′-AGTGGCGAAAGAGCTGGAGAGTC-3′ 
Ube2l6 reverse 5′-GAGGGCCTCCAAGACTTGATAAGG-3′ 
Uba1 forward 5′-CTCTCCTCCCAGTTTTACCTTCGG-3′ 
Uba1 reverse 5′-ATCACAGAAAAGTTGCCCAAACAG-3′ 
β-Actin forward 5′-ATGAGGTAGTCTGTCAGGT-3′ 
β-Actin reverse 5′-ATGGATGACGATATCGCT-3′ 
APP forward 5′-TGCTGGCAGAACCCCAGATCG-3′ 
APP reverse 5′-TTCTGGATGGTCACTGGCTGG-3′ 
BACE1 forward 5′-GCATCGCTACTACCAGAGGCA-3′ 
BACE1 reverse 5′-GGTCTGCTTCACCAGGGAGTC-3′ 
Parkin forward 5′-CCAAACCGGATGAGTGGTGAGTGC-3′ 
Parkin reverse 5′-ACACGGCAGGGAGTAGCCAAGTTG-3′ 
Cbl forward 5′-CCATTCTCATTGCCCTCACAAATGG-3′ 
Cbl reverse 5′-CAGTCACATGCTCGTGAACTCTGGG-3′ 
Cbl-b forward 5′-CATGGACAAAGTGGTAAGACTGTGC-3′ 
Cbl-b reverse 5′-CTGCCAGCATGTGACTGAAGATAAG-3′ 
UCH-L1 forward 5′-ATTGAGGAACTGAAGGGACAGGAAG-3′ 
UCH-L1 reverse 5′-TGGAAATTCACTTTGTCATCTACCC-3′ 
CHIP forward 5′-TGCTGAGCGAGAGAGGGAACTGGAG-3′ 
CHIP reverse 5′-CACACGCTGCAGGTGCTCCTCAATG-3′ 
Itch forward 5′-CTTCAGATCACTGTCATCTCAGC-3′ 
Itch reverse 5′-TCTCTGTTGGCTCTTTGTCACC-3′ 
Siah2 forward 5′-GTGTAACCAATGCCGCCAGAAG-3′ 
Siah2 reverse 5′-AGCCCCTGGCAGGTTAATGTCTG-3′ 
Ccnf forward 5′-TCAGCGACACCATGAGGTACATTC-3′ 
Ccnf reverse 5′-AGTGTCAGCAGGACCTCTTTATAG-3′ 
Cul1 forward 5′-AGCAGTGGGAAGATTACCGATTC-3′ 
Cul1 reverse 5′-TCAGCCCCAATTCCACATAAG-3′ 
CDC23 forward 5′-AGGATGTGGATGCTTACACCCTG-3′ 
CDC23 reverse 5′-ACCACCCCATACAGGTAAAGGC-3′ 
Ube2d1 forward 5′-CGTGGGAGATGACTTGTTCCAC-3′ 
Ube2d1 reverse 5′-TCTGGTACTAAGGGATCGTCTGG-3′ 
Ube2l6 forward 5′-AGTGGCGAAAGAGCTGGAGAGTC-3′ 
Ube2l6 reverse 5′-GAGGGCCTCCAAGACTTGATAAGG-3′ 
Uba1 forward 5′-CTCTCCTCCCAGTTTTACCTTCGG-3′ 
Uba1 reverse 5′-ATCACAGAAAAGTTGCCCAAACAG-3′ 

Protein extraction and western blot analysis

The method for protein extraction and western blot analysis has been previously described (62). Briefly, tissues were lysed in RIPA buffer (50 mm Tris–HCl, pH 7.5; 150 mm NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Calbiochem, San Diego, USA). Protein samples were separated on sodium dodecyl sulfate–polyacrylamide gels and blotted onto PVDF membrane (Immobilon-P; Millipore, Bedford, MA, USA). Membranes were blocked by 5% non-fat dried milk in TBS-T (10 mm Tris–HCl, 150 mm NaCl and 0.1% Tween, pH 8.0) at room temperature for 2 h and incubated with primary antibodies at room temperature for 2 h. Subsequently, the membranes were washed with TBS-T and incubated for 1 h with anti-rabbit or anti-mouse immunoglobulin G (1:10 000 dilutions in TBS-T) at room temperature. After three times washing with TBS-T, chemiluminescence was detected with a luminescence detection system (sc-2048; Santa Cruz, CA, USA). Protein levels were quantified by densitometry analysis using Quantity One 4.5.2 software (Bio-Rad, Hercules, USA).

Sandwich ELISA

To quantify levels of soluble and insoluble Aβ42, the cortex and the hippocampus from 6-month-old transgenic mice and their WT counterparts were homogenized in homogenization buffer (5 m guanidine HCl/50 mm Tris–HCl) and centrifuged. Protein concentrations of supernatants were determined with a BCA kit (Thermo Fisher Scientific, USA). Supernatant fractions were analyzed by the well-established human Aβ42 ELISA kit (KHB3441, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and a publication (63). Absorbance was determined for each well at 450 nm using a microplate reader (Bio-Rad, USA).

Thioflavine-S staining

Amyloid deposits in tissue sections were visualized with thioflavine-S staining and fluorescence microscopy (Olympus, Tokyo, Japan). Briefly, brain sections were rinsed with distilled water, and then placed in 1% thioflavine-S for 5 min and then in 70% alcohol for another 5 min. Sections were rinsed in distilled water twice and mounted in glycerin jelly.

Congo-Red staining

A different way to detect amyloid deposits in brain tissue was employed using Congo-Red staining (64). Brain sections were mounted on 2% gelatin-coated slides and air-dried, and incubated in saturated NaCl solution with 1% sodium hydroxide for 20 min at RT. Sections were further incubated in Congo-Red solution (2% Congo-Red in saturated NaCl) for 30 min at room temperature. Finally, brain sections were stained with Cresyl Violet. Amyloid deposits were detected under the microscope (Leica, Germany).

Fluoro-Jade B staining

The Fluoro-Jade B staining was performed to examine degenerating neurons in brain sections (65). Briefly, brain sections mounted on glass slides were successively immersed in solutions containing 1% sodium hydroxide in 80% alcohol (5 min), 70% alcohol (2 min) and 0.06% potassium permanganate (10 min). After rinsing with distilled water, slides were immersed in 0.004% Fluoro-Jade B solution for 20 min and washed. Fluoro-Jade B signals were detected at an excitation of 480 nm and an emission of 525 nm under an epifluorescence microscope (Olympus, Tokyo, Japan).

Fluorogenic 20S proteasome activity assay

20S chymotrypsin-like proteasome activity was measured according to the manufacturer recommendations (APT 280, Chemicon, Temecula, CA, USA) and as previously described (15,66). Briefly, freshly collected mouse cortex and hippocampus were homogenized by repeated cycles of a battery-operated pestle Motor Mixer (P7339–901, Argos, UK) in ice-cold assay buffer (25 mm HEPES, pH 7.5, 0.5 mm EDTA, 0.05% NP-40 and 0.01% SDS). Homogenates were centrifuged at 13 000g for 10 min at 4°C, supernatants were collected, and protein concentrations were determined with the BCA kit (MK164230, Pierce, USA). Assays were conducted using equal amounts of proteins from tissue lysates (80 µg). Levels of 7-amino-4-methylcoumarin (AMC) after cleavage from succinyl-LLVY-AMC were determined by the measurement of fluorescence intensity using a microplate reader (Infinite M200, Tecan, USA). The resulting fluorescence, reflecting activity of 20S proteasome, is presented as relative fluorescence units.

Intrastriatal lipopolysaccharide injection

Intrastriatal injection of LPS was performed according to a protocol published by Hunter et al. (67). Briefly, mice were anesthetized with avertin (T48402, Sigma, USA), and placed in a stereotaxic apparatus (SR-5M, Narishige, Japan). The stereotaxic coordinates from Bregma were anterior/posterior +1.18, medial/lateral +1.5 and dorsal/ventral −3.5 as well as anterior/posterior −0.34, medial/lateral +2.5 and dorsal/ventral −3.2. At each intrastriatal coordinate, either 1 µl of sterile saline or 1 µl of LPS (5 µg LPS/µl saline) was injected using a 1 µl microsyringe. The drug was delivered over 5 min and the needle was held in place for an additional 5 min to prevent reflux. Rectal temperature was monitored and maintained at 37.0°C. Four days after surgery, inflammation was assessed by immunohistochemistry using antibodies against the microglia marker Iba 1 and the astrocyte marker GFAP.

Immunohistochemistry and immunofluorescence

Immunohistochemistry of brain tissue was carried out according to a previously published method (62). Briefly, mouse brains were fixed in 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.2), stored in 30% sucrose solution for 24–48 h at 4°C, and sectioned into 30 µm sections with a cryomicrotome (Leica, Germany). Brain sections were permeabilized and blocked in 0.01 m PBS containing 10% goat serum and 0.2% Triton X-100 at 37°C for 45 min and then incubated at 37°C for 2 h and at 4°C overnight with rabbit polyclonal anti-GFAP (1:1000; #AB5804; Chemicon, USA) or rabbit polyclonal anti-Iba1 (1:500; #019-19741, Wako, Japan). Sections were incubated with biotinylated anti-rabbit secondary antibody (1:200; Vector Laboratories, USA) at 37°C for 45 min and then with avidin–biotin-peroxidase (1:200; Vector Laboratories, USA) at 37°C for 45 min. The peroxidase reaction was detected with 0.05% DAB (Sigma, USA) in 0.1 m Tris buffer and 0.03% H2O2. For immunofluorescence, sections were blocked and then incubated at 37°C for 2 h and at 4°C overnight with antibodies against NeuN (mouse monoclonal NeuN, 1:200; #MAB377, Chemicon, USA) and GFAP or NeuN and Iba1. Sections were then incubated with Alexa Fluor 488 conjugated donkey anti-mouse and with Alexa Fluor 647 conjugated donkey anti-rabbit secondary antibodies (1:500; Invitrogen, USA) at 37°C for 45 min. Then the sections were mounted with ProLong® Gold Antifade Reagent (Invitrogen, USA) and images were obtained with a Leica confocal microscope (TCS SP-2, Leica, Germany).

Field potential recording of EPSPs

Hippocampal slices were prepared as described previously (68–70). Briefly, after ether anesthesia, mouse brains were quickly removed and immersed in oxygenated ice-cold solution (composition in mm: 130 NaCl, 4.9 KCl, 1.5 CaCl2·2H2O, 0.3 MgSO4·7H2O, 11 MgCl2·6H2O, 0.23 KH2PO4, 0.8 Na2HPO4, 5 glucose, 25 HEPES, 22 NaHCO3, pH 7.32). Transverse hippocampal slices (350 µm) were prepared using a vibratome (Vibratome 3000, St Louis, MO, USA), transferred to a custom-made interface recording chamber and incubated for at least 2 h at 32°C under constant perfusion with oxygenated artificial cerebrospinal fluid (in mm: 110 NaCl, 5 KCl, 2.5 CaCl2·2H2O, 1.5 MgSO4·7H2O, 1.24 KH2PO4, 10 glucose, 27.4 NaHCO3, pH 7.3).

fEPSPs in the stratum radiatum (str. rad.) of field CA1 were evoked by stimulation of the Schaffer-collateral fibers with biphasic rectangular current pulses (100 µs/polarity) in a range of 15–30 µA through stainless steel electrodes (A-M Systems, Inc., Sequim, WA, USA) and recorded with stainless steel electrodes and a differential amplifier (EXT-20F; npi electronic GmbH, Tamm, Germany) using 3 kHz low-pass and 0.1 Hz high-pass filters. The recorded field potentials were digitized at a sample frequency of 10 kHz by a CED 1400plus AD/DA converter (Cambridge Electronics Design, Cambridge, UK). The stimulation strength was adjusted to generate 40% of the maximum fEPSP-slope value. A second stimulation electrode (S2) was placed opposite to the first stimulation electrode (S1) in str. rad. for recording of fEPSP baseline stability. LTP was induced by three 100 Hz trains of 1 s at an inter-train interval of 10 min (32).

Locomotor activity test (open-field test)

Basal locomotor activity was tested in 6-month-old transgenic and WT mice, as described previously (71). Tests were performed using an automatic-recording open-field working station (MED Associates, Georgia, VT, USA). The open-field box was 40 cm in width, 60 cm in height with white surrounding walls and blue bottom. The whole box was hidden in a light-free chest during the test. Before testing, mice were transferred to the behavioral room to adapt to the environment for at least 30 min. A mouse was then positioned in the center of the box in such a way that its long axis was parallel to a wall. After release, the behavior of the animal was observed and recorded using a video camera system for 30 min. The imaging software allowed determining the total walking distance, time of ambulatory movements and resting. Values for the central (72) and edged part of the box were calculated separately.

Elevated plus-maze test

The elevated plus-maze test was performed as described by Simonin et al. (73). The maze was a plastic cross-shaped box and installed at 74.5 cm above the floor (Med Associates, Inc., St Albans, Vermont, USA). Transgenic and WT mice were maintained in the room for 30 min. For the test, a mouse was placed in the center of the cross in such a way that the mouse faced the closed arms of the cross. The movements of the mouse were recorded for 5 min using a video system and then the exploratory time and entry frequency into the arms computed.

Morris water maze test

The Morris water maze test was performed as described previously (74,75). The water maze consisted of a circular pool (120 cm in diameter, 62.5 cm in height) with a white inner surface. The escape platform (10 × 10 cm) was fixed at equidistant from the center and the wall of the tank and submerged 1 cm below the surface. The tank was located in a test room surrounded with various visual cues on the wall. A video-tracking system (Actimetrics Co., Wilmette, IL, USA) recorded the swimming activities of the animal to the platform within 60 s. If an animal failed to find the platform within 60 s then it was gently guided to the platform and allowed to stay there for 30 s. Each mouse performed over 8 days four trials daily at randomly chosen starting points. Test sessions were performed 24 h after the last trial. Test sessions consisted of a single probe trial in which the platform was removed from the tank and each mouse was allowed to swim for 60 s in the maze.

Drugs, antibodies and primers

Thioflavine-S, Congo-Red and creysl violet were obtained from Sigma–Aldrich. Fluoro-Jade B was purchased from Chemicon. Eva green was obtained from Biotium. The antibodies were: rabbit anti-APP (A8717, Sigma–Aldrich, USA), rabbit anti-BACE1 (B690, Calbiochem or ab108394, Abcam, UK), mouse anti-β-actin (C4, Santa Cruz, USA), mouse anti-parkin (MAB5512, Chemicon, USA), mouse anti-t-Tau (Tau-5, Millipore, Billerica, MA, USA) and rabbit anti-p-Tau (pSer202, LifeSpan BioSciences, Seattle, USA). Primers were synthesized by Shanghai Shangon, China.

Statistical analysis

Data were analyzed using the SPSS software (version 17; SPSS, Chicago, USA). Values were expressed as means ± SEM. Statistical analysis was conducted using the t-test (when two groups were considered) or by one-way analysis of variance followed by multiple comparisons with the LSD post hoc test. LTP-data were normalized to baseline values and expressed as percentage as means ± SEM deviation. Comparisons of different time points and/or different groups were done by the Mann–Whitney U-test. P-values of <0.05 were considered to indicate a statistically significant difference between groups.

Conflict of Interest statement: None declared.

FUNDING

This work was supported by grants from the Ministry of Science and Technology of China (2012CB966300 to F.H.); National Natural Science Foundation of China (81171188 to F.H., 31271197, 31320103906 to T.B.) and Specialized Research Fund for the Doctoral Program of Higher Education (20110071110039 to F.H.). We thank Prof. Baudry (Western University of Health Sciences) for critical comments.

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Author notes

X.H., J.L. and G.Z. contributed equally to the work.