Background
Alzheimer’s disease (AD) is an aging-related, progressive, and clinical incurable neurodegenerative disorder, ultimately leading to dementia. One typical pathological hallmark of AD is senile plaques, composed primarily of amyloid β-peptide (Aβ), which is produced by the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretase cleavage [
1‐
3]. It is recognized that increased production, oligomerization, and aggregation of Aβ are the crucial factors in the onset of AD [
4‐
6]. Although the precise mechanism is still unknown, it has been proved that Aβ mediates neuronal cytotoxicity which eventually leads to massive neuron loss and degeneration [
3,
7].
Recently, multiple studies showed that Aβ aggregation induces endoplasmic reticulum (ER) stress in neurons, which elicits the unfolded protein response (UPR) [
8,
9]. Many groups reported that UPR activation is increased in AD patients. ER stress marker immunoglobulin-binding protein (BiP)/GRP78 and UPR-transducer proteins, such as phosphorylated pancreatic ER kinase (p-PERK), eukaryotic initiation factor 2 (eIF2α), inositol-requiring enzyme 1 (IRE1), and activating transcription factor-6 (ATF6), were detected in the hippocampal neurons of AD [
10,
11]. If the provoking stress is prolonged, UPR can trigger the cell death program through the activation of C/EBP homologous protein (CHOP), a protein of the C/EBP family of transcriptional regulators, also known as growth arrest- and DNA damage-inducible gene 153 (GADD153) [
12]. As neurons are highly susceptible to the toxic Aβ, ER stress-mediated cell death might have an important role in the pathogenesis of AD.
Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to the fourth family of neurotrophic factors (NTFs) family, which is an important group of secreted proteins regulating the life and death of neurons during development [
13‐
15]. MANF and its homolog, the cerebral dopamine neurotrophic factor (CDNF), have been identified to protect and rescue midbrain dopaminergic neurons in the rat 6-OHDA model of Parkinson’s disease [
15‐
20]. More importantly, our previous studies show MANF to be a secreted protein, and ER stress upregulates its expression and secretion [
13,
14]. It was also found that MANF is widely expressed in mammalian tissues and differently regulated by various ER stress during disease processes [
13,
14,
21‐
26]. Our previous studies show that MANF protects against the neuron death induced by ischemia injury [
13,
27‐
29]. Intracerebral delivery of recombinant human MANF protein (rhMANF) or rAAV-mediated MANF gene also reduces cerebral infarction and neuronal cell apoptosis in stroke animals [
29,
30]. Additionally, cytoprotective effects of MANF are not restricted to neurons, also including cardiac myocytes [
31], pancreatic insulin-producing β cells [
24,
25,
32,
33], retinal cells [
26], and so on, suggesting that MANF may have important functions under different pathological conditions.
Owing to the ability of MANF to rescue neuronal loss in several nervous system diseases, we are interested at the profile of MANF induction and potential significance in Aβ pathology. In this study, we showed the increased expressions of MANF and ER stress markers BiP and CHOP in APP/PS1 transgenic mice as well as in the neuronal cells treated with Aβ1–42, assessed the protective effect of MANF against Aβ neurotoxicity, and the underlying mechanism was also investigated.
Methods
Materials and reagents
Aβ
1–42 peptide, 3-(4, 5dimemylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT), tunicamycin (TM), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma (St. Louis, MO, USA). Neurobasal medium, B27,
l-glutamine, Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, USA). Recombinant human MANF protein and monoclonal anti-MANF antibody have been described as we previously reported [
23,
28,
29].
Animals
PrP-hAPP/hPS1 double-transgenic mice were from Institute of experimental animals, Chinese academy of Medical Sciences (Beijing, China). The pregnant Sprague-Dawley (SD) rats (grade SPF) were obtained from Anhui Experimental Animal Center (Hefei, China). The animals were kept under standard conditions of temperature (25 ± 2 °C) and lighting (12 h light/dark cycle). The procedure for animal experiments was approved by the Animal Care and the Ethic Committee of Animal Usage of Anhui Medical University.
Preparation of oligomeric Aβ peptides
Aβ1–42 peptide powder was dissolved in phosphate-buffered saline (PBS) to a final concentration of 2 mM. Then, the peptides were snapping frozen in liquid nitrogen. The aliquoted peptide were incubated for 1 week at 37 °C before use, then were dissolved in culture medium to working concentration for treatment. The structure of Aβ peptides were examined under electron microscope. Aβ1–42 peptides contained rod and globular shape structure with about 100 nm in diameter.
Primary neuron cultures
Primary neurons from the cerebral cortex of E17 embryos of pregnant rats were cultured as described previously [
29]. Briefly, the embryos of rats were decapitated and the cerebral hemispheres were aseptically removed into Hank’s balanced salt solution (HBSS). After removal of the meninges, the cerebral cortices were cut into small pieces, digested with 0.25% Trypsin-EDTA for 20 min at 37 °C, mechanically dissociated by gentle pipetting with Pasteur pipette, and then centrifuged at 400 g for 5 min. The cell pellet was re-suspended in neurobasal medium supplemented with 2% B27 and 0.5 mM
l-glutamine and then cultured on 24-well plates pre-coated with poly-
l-lysine at 37 °C and 5% CO
2 in a humidified atmosphere. Primary cultured neurons at 10–14 days were treated with Aβ peptide or TM following by immunofluorescence staining.
Cell transfection
Human neuroblastoma SH-SY5Y cells and mouse neuroblastoma N2a cells were obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM supplemented with 10% FBS and maintained in 5% CO
2 at 37 °C. MANF-Flag plasmid [
13] or the MANF siRNA (5′-GGACCUC A AAGACAGAGAUTT-3′, Shanghai GenePharma Co., Ltd) was transfected into N2a cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Twenty-four hours after transfection, N2a cells were treated with Aβ
1–42 (10 μM) for 24 h or TM (2.5 μg/ml) for 12 h.
Cell viability assay
Cell viability was evaluated by MTT assay as described previously [
23,
34]. In brief, cells were plated into 96-well plates (1 × 10
4 cells per well) and incubated with Aβ
1–42 at 37 °C for indicated times. MTT (500 μl, 5 mg/ml) was added to each well and incubated at 37 °C for 4 h, then 100 μl DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader (Thermo, Varioskan Flash, Finland). Experiments were repeated three times with three replicates each time.
Flow cytometry with Annexin V/PI double staining
For assessment of apoptosis, cells were stained with Annexin V and propidium iodide (PI) using Annexin V-FITC/PI Apoptosis Detection kit (Bestbio, China) according to the manufacturer’s instructions and subjected to flow cytometry using FACSVerse flow cytometry (BD Biosciences, USA).
TUNEL assay
Cell apoptosis was measured by the TUNEL kit (Promega, Madison, Wisconsin, USA) according to the manufacturer’s instructions. TUNEL positive cells in five randomly selected fields in at least four independent slices were visualized and counted in a blinded manner under a fluorescent microscope (Olympus, Tokyo, Japan).
Quantitative real-time polymerase chain reaction
Total RNA was extracted from cells with Trizol reagent (Invitrogen, 15596018) following with reverse transcription to synthesize cDNA. The gene transcripts were quantified by quantitative PCR (qPCR) with SYBR green. The primers are listed as follows: Human BiP gene, forward 5′-ACCTGGGTTAGGGTGTGTG-3′ and reverse 5′-TTGCCTGAGT AAAGATGTGG-3′; human CHOP gene, forward 5′-GGAGCTGGAAGCCTGG TATGA-3′ and reverse 5′-TCCCTGGTCAGGCGCTCGATTT-3′; human MANF gene, forward 5′-TCACATTCTCACCAGCCACT-3′ and reverse 5′-CAGGTCGATCTGC TTGTCATAC-3′; human GAPDH gene, forward 5′-CCACTCCTCCACCTTTG-3′ and reverse 5′-CACCACCCTGTTGCTGT-3′. Expressions of gene transcripts were normalized to the levels of GAPDH mRNA. qPCR was carried out by using the ABI7500 instrument (Applied Biosystems, USA).
Immunohistochemistry
Acetone-fixed brain frozen sections were rehydrated in PBS, and endogenous peroxidase activity was quenched in 0.3% H2O2 on absolute methanol for 20 min. The sections were incubated with mouse anti-MANF antibody overnight at 4 °C. After washing in PBS, the sections were incubated with the appropriate biotinylated secondary antibodies for 1 h at 37 °C. This was followed by incubation with horseradish peroxidase conjugated streptavidin (HRP-SA) for 15 min at 37 °C. Immunohistochemistry was developed by application of 3,3-diaminobenzidine tetrahydrochloride (DAB) for about 1–3 min. Then the sections were counterstained with hematoxylin, dehydrated in graded ethanol, cleared in xylene, and then observed under light microscopy.
Immunofluorescent staining
Cells were fixed with paraformaldehyde, permeabilized/blocked in PBS containing 0.5% Triton X-100 and 5% BSA. The cells were incubated with following primary antibodies: rabbit anti-BiP antibody (1:500, proteintech, 11587-1-ap), rabbit anti-CHOP antibody (1:400, proteintech, 15204-1-AP), or mouse anti-MANF antibody overnight at 4 °C, followed by Alexa Fluor 488-conjugated or 568-conjugated IgG (1:500, Invitrogen, A11029, A11036) at 37 °C for 1 h; the nuclei of cells were stained with DAPI (5 mg/ml). Images were taken under fluorescent microscopy (Olympus, Tokyo, Japan) with constant parameters of acquisition. Immunofluorescent staining of brain slice was performed as described previously [
29]. The following primary antibodies were used: rabbit anti-NeuN antibody (1:100, Abcam, ab177487), rabbit anti-BiP antibody (1:500, proteintech, 11587-1-ap), rabbit anti-CHOP antibody (1:400, proteintech, 15204-1-AP), or mouse anti-MANF antibody.
Western blot
The cell lysate was prepared for SDS-PAGE as described previously [
23,
34]. The proteins were transferred to PVDF membranes and blocked in 5% nonfat milk at room temperature for 1 h, then incubated at 4 °C overnight with the following primary antibodies: rabbit anti-MANF antibody (1: 1000, Abcam, ab67271), rabbit anti-BiP antibody (1:1000, proteintech, 11587-1-ap), rabbit anti-CHOP antibody (1:1000, proteintech, 15204-1-AP), rabbit anti-phospho-eIF2α antibody (1:1000, CST, 3398 s), rabbit anti-phospho-IRE1 antibody (1:1000, Bioss, bs-4308R), rabbit anti-XBP1s antibody (1:1000, Biolegend, 619502), mouse anti-ATF4 antibody (1:1000, CST, 11815 s), rabbit anti-ATF6 antibody (1:1000, Proteintech, 24169-1-AP), rabbit anti-cleaved-caspase 3 antibody (1: 1000, CST, 9664S), mouse anti-α-tubulin (1: 1000, sigma, t6199), and rabbit-anti-GAPDH (1: 1000, Elabscience, E-AB-20059). After washing several times in TBST, membranes were incubated with horseradish peroxidase-conjugated secondary IgG for 1 h. Blots were developed using the enhanced chemiluminescence kit (Amersham Biosciences, NJ, USA). The densitometric analysis was performed using ImageJ software.
Statistical analysis
The data were expressed as mean ± SD from at least three independent experiments and analyzed using a one-way analysis of variance (ANOVA) with Tukey’s post-hoc tests. A value of p less than 0.05 was considered to be statistically significant.
Discussion
In this study, we demonstrated the roles of MANF in protecting neuronal cells from the toxicity induced by Aβ. We showed here that MANF expression is upregulated in cultured neuronal cells exposure to Aβ1–42 as well as in the brains of APP/PS1 mice. Over-expression of MANF or rhMANF treatment protected against Aβ1–42-induced cell death in N2a cells and SH-SY5Y cells, whereas MANF gene knockdown aggravated Aβ cytotoxicity. In addition, neuroprotective effect of MANF against Aβ1–42-induced cytotoxicity was associated with the inhibition of ER stress, which demonstrated by suppression of UPR signaling cascades and the decrease of CHOP expression and caspase-3 cleavage. These results suggested that MANF may serve as a plausible therapeutic target for the treatment of AD.
It was reported that Aβ induces ER stress leading to neuron apoptosis, and ultimately contributes to the pathogenesis of AD [
40‐
42]. In the present study, we found that Aβ
1–42 dose- and time-dependently decreased cell viability and increased cell apoptosis. Further investigation showed that the expressions of UPR-related proteins were upregulated both in primarily cultured rat neurons and in SH-SY5Y cells under Aβ exposure, including BiP, ATF6, phospho-IRE1, XBP1s, phospho-eIF2α, ATF4, and CHOP. These results indicate that Aβ
1–42 induces ER stress, which mediates neuronal cell apoptosis. Although molecular mechanisms of ER stress-mediated Aβ neurotoxicity still remain obscure, many studies have confirmed that Aβ triggers the UPR in neurons, which accompanies the activation of protective pathways such as BiP and PERK-eIF2a pathway, as well as the apoptotic pathways of the UPR such as CHOP and caspase-4 [
43].
Our previous studies had identified MANF as 1 of the 12 commonly UPR upregulated genes, and ER stress inducer TM induced MANF expression in the primarily cultured neurons [
13]. The mammalian MANF promoter contains ER stress-responsive elements ERSE and ERSEII, which are important for XBP1s and ATF6 binding leading to upregulation of MANF expression in response to ER stress [
14,
22,
44,
45]. In this study, Aβ
1–42 treatment significantly increased the expressions of XBP1s and ATF6, which at least partially contributes to the upregulation of MANF accompanied by subsequently increased expression of other UPR related proteins. Consistent with our results in cultured neuronal cells with Aβ, the expression of MANF was also dramatically upregulated in APP/PS1 mice compared with those of age- and sex-matched wild-type mice, especially in 6-month-old APP/PS1 mice, suggested that increased MANF expression is correlated with the severity of AD. But there is no difference of MANF level was observed between 4-month and 6-month-old wild-type mice, which suggested that aging is not likely to be responsible for the upregulation of MANF. Meanwhile, we found that MANF is mostly expressed in neurons in the brains of APP/PS1 mice, which is in accordance with the characteristics of MANF expression in rat ischemia model [
27,
29]. Though some NeuN-negative cells also presented MANF immunoreactivity, the function of MANF in these cells needs further study. In addition, we noticed that the immunoreactivity of MANF, BiP, and CHOP is weak and diffused in the cytoplasm of SY5Y cells without Aβ
1–42, whereas increased perinuclear MANF immune-positive signals were observed in most of the cell treated with Aβ
1–42 or TM as well as in the APP/PS1 mice brain. Meanwhile, we found the significant upregulated immunoreactivity of BiP in cytoplasm and nuclear translocation of CHOP in the cells treated with Aβ
1–42 or TM, which implies UPR activation. The cellular distribution of MANF under different pathology may be an interesting question. Our previous studies showed that MANF localization primarily in the juxta-nuclear region in the TM-treated cells, which proved to be localized in the ER and Golgi [
13]. Conversely, MANF induced by the stimulation of cerebral ischemia had no overlap with DAPI, suggesting that MANF localizes in cytosol and focal ischemia did not induce its relocalization [
29], whereas nuclear MANF immune-positive signals were observed in a few TM or LPS-treated primarily cultured fibroblast-like synoviocytes as well as in the synovial tissue of antigen-induced arthritis rabbit model [
23]. The reason for MANF exhibits different cellular distribution under different pathology worthy of further investigation.
MANF is a new member of neurotrophic factor families, which has been shown to be upregulated during ER stress and protected several cell populations from ER stress-induced cell death in vivo or in vitro [
13,
19,
31,
46], especially in PD and cerebral ischemia [
19,
47,
48]. In this study, we showed that over-expression of MANF attenuated Aβ
1–42-induced neuronal cell death, whereas MANF gene knockdown aggravated the Aβ
1–42 neurotoxicity. Whether MANF is directly involved in the regulation of UPR and thus play a key role in cell protection is still obscure. Previous studies reported that MANF may facilitate the formation of cysteine bridges and protein folding in the ER, thus reducing the ER stress caused by accumulation of unfolded or misfolded proteins [
49]. The most convenient and direct evidence is pancreas-specific Manf
−/− mice developed severe insulin-dependent diabetes due to progressive reduction of β cell mass and activation of UPR, including spliced Xbp1 and Chop mRNA, which implicates that MANF deficiency may lead to activation of the UPR following unresolved ER stress [
32]. Our previous studies also suggested that intraventricular injection of rhMANF reduced the elevated levels of BiP/Grp78, phosp-IRE1, and XBP1s induced by focal cerebral ischemia, but not affect CHOP expression [
28]. Glial cell line-derived neurotrophic factor (GDNF), another neurotrophic factor, was proved to prevent the translocation of CHOP into the nucleus in the brains of rabbits by intracisternal injections of Aβ [
50]. In this study, we observed that the levels of UPR-related molecules BiP, ATF6, phospho-IRE1, XBP1s, phospho-eIF2α, ATF4, CHOP, and apoptosis marker cleaved caspase-3 reduced in different degree in MANF over-expressed cells compared to vector-transfected cells, whereas MANF knockdown further increased these UPR relative proteins, suggesting that MANF alleviate Aβ-induced ER stress via suppressing UPR signaling activation, which lead to a decrease of CHOP and caspase-3 cleavage. In addition, our current data showed that all of the three UPR sensors were activated by Aβ in some extent, since the fact that CHOP is a transcriptional target of not only ATF4 but also XBP-1 and ATF6 provides an obvious possible link among all three branches [
51,
52], which pathway in Aβ pathology is the leading cause for CHOP induction and neuron death needs to further exploration.
On the other hand, the crystal structure analysis revealed that the C-terminal domain of MANF (C-MANF) shares the highest structural homology with the SAP domain of Ku70 protein, a well-known inhibitor of pro-apoptotic Bcl-2-associated X protein (Bax) [
53]. This was confirmed by the cellular studies that MANF protected neurons as efficiently as Ku70 did [
53,
54]. Based on this, we cannot exclude the possibility that the direct anti-apoptosis activity of MANF partially contributes to its protective effect against Aβ neurotoxicity.
Most importantly, MANF was reported to act as both a secreted neurotrophic factor and an intracellular protein [
55]. Previous studies have suggested a secretion-based protective role of MANF in cerebral ischemia and myocardial ischemia [
27‐
29]. Conversely, no protected effects against the Bax-dependent apoptosis were observed when rhMANF was added to the culture medium of cultured newborn mouse SCG neurons directly the neurons [
53]. Although rhMANF has been commonly used for the study regarding the function of secretory MANF protein, different concentrations of rhMANF was adopted in vivo and in vitro experiments depend on the different types of cells or the disease models [
23,
24,
26,
28‐
31,
38,
39,
48,
56‐
58]. Here, we tried several concentrations of rhMANF in preliminary experiments and finally we found that the concentrations of 0.5, 1, and 2 mg/ml rhMANF are sufficient to protect against the Aβ-induced neurotoxicity and alleviate ER stress. To date, signal transducing receptor for MANF has not been identified, although protein kinase C (PKC) signaling has been described to be activated downstream of MANF [
58]. Therefore, whether and how MANF acts in autocrine/paracrine manner will need more studies.