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. 2018 Jul;15(3):728-741.
doi: 10.1007/s13311-018-0628-1.

Pigment Epithelium-Derived Factor Plays a Role in Alzheimer's Disease by Negatively Regulating Aβ42

Mao Huang  1   2 , Weiwei Qi  1   2 , Shuhuan Fang  3 , Ping Jiang  2 , Cong Yang  3 , Yousheng Mo  3 , Chang Dong  2 , Yan Li  2 , Jun Zhong  1 , Weibin Cai  2 , Zhonghan Yang  2 , Ti Zhou  2 , Qi Wang  4 , Xia Yang  5   6   7 , Guoquan Gao  8   9   10   11
Affiliations

Pigment Epithelium-Derived Factor Plays a Role in Alzheimer's Disease by Negatively Regulating Aβ42

Mao Huang et al. Neurotherapeutics. 2018 Jul.

Abstract

Alzheimer's disease (AD) is the most common cause of dementia. Pigment epithelium-derived factor (PEDF), a unique neurotrophic protein, decreases with aging. Previous reports have conflicted regarding whether the PEDF concentration is altered in AD patients. In addition, the effect of PEDF on AD has not been documented. Here, we tested serum samples of 31 AD patients and 271 normal controls. We found that compared to PEDF levels in young and middle-aged control subjects, PEDF levels were reduced in old-aged controls and even more so in AD patients. Furthermore, we verified that PEDF expression was much lower and amyloid β-protein (Aβ)42 expression was much higher in senescence-accelerated mouse prone 8 (SAMP8) strain mice than in senescence-accelerated mouse resistant 1 (SAMR1) control strain mice. Accordingly, high levels of Aβ42 were also observed in PEDF knockout (KO) mice. PEDF notably reduced cognitive impairment in the Morris water maze (MWM) and significantly downregulated Aβ42 in SAMP8 mice. Mechanistically, PEDF downregulated presenilin-1 (PS1) expression by inhibiting the c-Jun N-terminal kinase (JNK) pathway. Taken together, our findings demonstrate for the first time that PEDF negatively regulates Aβ42 and that PEDF deficiency with aging might play a crucial role in the development of AD.

Keywords: Alzheimer’s disease; Aβ42; Pigment epithelium-derived factor; Presenilin-1.

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Figures

Fig. 1
Fig. 1
PEDF was reduced in AD patients and a mouse model of AD. (A) Serum PEDF content was quantified by ELISA in normal subjects and AD patient samples. The normal samples were divided into three groups by ages: the youth group (25–45 years; N = 121), the middle-aged group (46–60 years; N = 95), and the old-aged group (> 60 years; N = 55). The AD patients comprised one group (50–91 years; average = 70; N = 31). (B) Serum PEDF content was quantified by ELISA in C57BL/6 WT mouse samples. N = 6 in each group. (C) PEDF staining was performed in C57BL/6 WT mouse hippocampal tissue. Scale bar, 100 μm. (D) Serum PEDF content was quantified by ELISA in the SAMR1 and SAMP8 mice. N = 4 in each group. (E) PEDF staining was performed in the SAMR1 and SAMP8 mouse hippocampal tissue. Scale bar, 100 μm. Error bars represent the standard deviation (SD); one asterisk, p < 0.05; three asterisks, p < 0.001
Fig. 2
Fig. 2
The cognitive impairment in SAMP8 mice was attenuated by PEDF, as assessed through the Morris water maze. Effect of treatment with PEDF-induced alterations in acquisition trials in the Morris water maze test: (A) the track diagram and (B) escape latency. One asterisk, p < 0.05; three asterisks, p < 0.001, versus the SAMR1 normal control group at the same time point; one number sign, p < 0.05; three number signs, p < 0.001, versus the SAMP8 untreated group at the same time point. Effect of PEDF treatment with on probe trials in the Morris water maze test: (C) time to reach the target region (that previously contained the platform) and (D) number of times crossing the target region. Error bars represent the standard deviation (SD); one asterisk, p < 0.05, three asterisks, p < 0.001
Fig. 3
Fig. 3
Increased Aβ42 in SAMP8 mice was attenuated with PEDF treatment. (A) PEDF staining was performed on hippocampal tissue after the Morris water maze. Scale bar, 100 μm. (B) Assay of recombinant Rluc-PEDF crossing the blood–brain barrier (BBB) in vitro. Detection of luciferase in the lower chamber using an excitation wavelength of 488 nm. (C) Assay of recombinant Rluc-PEDF uptake by bEnd3 in vitro. Detection of luciferase in bEnd.3 cells using an excitation wavelength of 488 nm. Scale bar, 100 μm. (D) ELISA analysis of Aβ42 expression in hippocampal tissue. (E) Aβ42 staining was performed on mouse hippocampal tissue. The staining was quantified using Image-Pro Plus (IPP) (N = 3). Scale bar, 100 μm. (F) Protein levels of Aβ were determined by Western blot in hippocampal tissue. β-Actin served as a loading control. The bands were quantified using ImageJ software, and the data were transformed and normalized relative to β-actin (N = 3). Error bars represent the standard deviation (SD); one asterisk, p < 0.05; two asterisks, p < 0.01
Fig. 4
Fig. 4
The expression of Aβ42 was regulated by PEDF. (A) Aβ42 staining was performed in WT and PEDF KO hippocampal tissue. The staining was quantified using Image-Pro Plus (IPP) (N = 3). Scale bar, 100 μm. (B) ELISA analysis of Aβ42 expression in WT and PEDF KO hippocampal tissue. β-Actin served as a loading control. (C) Western blot analysis of Aβ expression in WT and PEDF KO hippocampal tissue. The bands were quantified using ImageJ software, and the data were transformed and normalized relative to β-actin (N = 3). (D) Aβ42 staining was performed in CHO and APP-PS1(M146L) cells. Scale bar, 100 μm. (E) ELISA analysis of Aβ42 expression in cell supernatants and lysates. (F) Protein levels of PEDF were determined by Western blot in cells. β-Actin served as a loading control. (G) Aβ42 staining was performed on APP-PS1(M146L) cells 72 h after transfection of adenovirus. Scale bar, 100 μm. (H) ELISA analysis of Aβ42 expression in cell supernatants and lysates 72 h after transfection of adenovirus. Error bars represent the standard deviation (SD); one asterisk, p < 0.05; two asterisks, p < 0.01, three asterisks, p < 0.001
Fig. 5
Fig. 5
PEDF regulated Aβ production through γ-secretase, not β-secretase. (A) γ-Secretase activity was analyzed in mouse hippocampal tissue. (B) β-Secretase activity was analyzed in mouse hippocampus tissue. (C) Protein levels were determined by Western blot in mouse hippocampal tissue. β-Actin served as the loading control. The bands were quantified using ImageJ software, and the data were transformed and normalized relative to β-actin (N = 3). (D) Immunofluorescence staining of PS1 on mouse hippocampal tissue. Scale bar, 100 μm. (E) γ-Secretase activity was analyzed in 8-month-old WT and PEDF KO mouse hippocampal tissue. (F) β-Secretase activity was analyzed in 8-month-old WT and PEDF KO mouse hippocampal tissue. (G) Protein levels were determined by Western blot in 8-month-old WT and PEDF KO hippocampal tissue. β-Actin served as the loading control. The bands were quantified using ImageJ software, and the data were transformed and normalized relative to β-actin (N = 3). Error bars represent the standard deviation (SD); one asterisk, p < 0.05; two asterisks, p < 0.01, three asterisks, p < 0.001
Fig. 6
Fig. 6
PEDF regulated the expression of PS1 through the JNK pathway. (A) APP-PS1(M146L) cells were infected with viruses expressing PEDF for 72 h followed by γ-secretase activity analysis. Ad-βgal served as a control. (B) Protein levels were determined by Western blot after APP-PS1(M146L) cells were infected with viruses expressing PEDF for 72 h. Ad-GFP served as control. (C) APP-PS1(M146L) cells were infected with viruses for 72 h followed by RT-PCR analysis of PS1. β-Actin served as an internal reference. (D) Western blot analysis of the key enzyme protein levels in the MAPK pathway. (E) APP-PS1(M146L) cells were treated with a JNK inhibitor or agonist for 2 h followed by Western blot analysis of protein levels. (F) Protein levels were determined by Western blot. APP-PS1(M146L) cells were infected with viruses expressing PEDF for 72 h followed by treatment with or without a JNK agonist for 2 h. Error bars represent the standard deviation (SD); one asterisk, p < 0.05; two asterisks, p < 0.01
Fig. 7
Fig. 7
Simplified model depicting the pathway of Aβ42 regulated by PEDF. (a) The phosphorylation of JNK is inhibited by high levels of PEDF in the physiological state. (b) Aβ42 is upregulated by JNK pathway activation lack of PEDF in the pathological state
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