Significant upregulation of Alzheimer's β-amyloid levels in living system induced by extracellular elastin polypeptides.

Abstract Alzheimer's disease (AD) is a neurodegenerative disorder and the primary cause of age‐related dementia. The etiology of AD is complex and has not been completely elucidated. Herein, we report that treatment with elastin‐like polypeptides (ELPs), a component of the brain extracellular matrix (ECM), significantly increased the levels of AD‐related amyloid‐β peptides (Aβ) both in vitro and in vivo. Regarding the molecular mechanism(s), the upregulation of Aβ levels was related to increased proteolytic processing of the amyloid precursor protein. Furthermore, nesting tests demonstrated that the ELP‐treated animals showed significant neurobehavioral deficits with cognitive impairment. These results suggest that the elastin is associated with AD‐related pathological and behavioral changes. This finding presents a new aspect for Alzheimer's amyloidosis event and provides a great promise in developing ELP‐based model systems to better understand the pathogenesis of AD.


Materials
Major chemicals, kits and antibodies used in this study are listed in table S1 and table   S2. Water (typically 18.2 MΩ·cm at 25 °C) was from a Milli-Q ultrapure water system (Merck, Germany). Reagents for ELP expression, such as LB medium, salts, antibiotics as well as inducer compounds, were used as received (from Sigma-Aldrich) without any further purification. E. coli XL1-Blue competent cells for plasmid amplification were purchased from Stratagene (La Jolla, CA). Oligonucleotides for sequencing were from Sigma-Aldrich (St. Louis, MO).
Alpha-cyano-4-hydroxycinnamic acid was used as matrix during MALDI mass spectrometry and was purchased from Thermo Scientific (Waltham, MA). Animal experiments were in agreement with the guidelines of the Regional Ethics Committee for Animal and Clinical Experiments of Jilin University Institutional Animal Care and Use and the Second Hospital of Jilin University, respectively. Other reagents used in the work are analytical grade.

Protein expression and purification
The expression vectors for ELP30, ELP48 and ELP90 were from addgene.org (#67014, #68395, #68392). E. coli BLR (DE3) cells (Novagen) were transformed with expression vectors containing the ELP genes. For protein production, Terrific Broth medium (for 1 L, 12 g tryptone and 24 g yeast extract) enriched with phosphate buffer (for 1 L, 2.31 g potassium phosphate monobasic and 12.54 g potassium phosphate dibasic) and glycerol (4 mL per 1 L TB) and supplemented with 100 µg•mL -1 ampicillin, was inoculated with an overnight starter culture to an initial optical density at 600 nm (OD600) of 0. 1  Protein-containing fractions were dialyzed extensively against ultrapure water.
Purified proteins were frozen in liquid N2, lyophilized, and stored at -20 °C until further use.

Characterization of ELPs
The concentrations of purified polypeptides were able to be determined by measuring absorbance at 280 nm using a spectrophotometer due to the existence of a  Figure S2.

Protein characterization employing mass spectrometry
Mass spectrometric analysis was performed using a 4800 MALDI-TOF Analyzer in linear positive mode. The protein samples were mixed 1:1 v/v with Sinapinic Acid matrix (SIGMA) (100 mg•mL -1 in 70% MeCN and 0.1% TFA). Mass spectra were analyzed with the Data Explorer software (version 4.9).

Cell Culture and Cell Lysis
The

Studies at Animal Levels
To investigate whether ELP could induce AD-like pathology and symptoms in mice, system. A small hole was drilled a in the right hemisphere of the skull, then a micro-syringe (catalog 80383, Hamilton, Switzerland, needle size 30s) was slowly inserted the into brain at a coordinate of x:1.5mm, y:-1.7mm, z:1.8mm from bregma, and 1μL PBS or ELP90 was injected into the CA1 region of the hippocampus.
Following the injection, the needle was slowly pulled out and the wound was sutured.
Mice were placed in a warm environment until palinesthesia.

Nest Building Test
The Nest Building Test is one of the methods used to evaluate the cognitive state of mice. The tests began in the evening (17:00-18:00), all the mice was allocated to a single cage, together with new aseptic sawdust and two pieces of 5-centimeter square cotton in the center of the cage, and then the lights were turned off. After 16 hours three trained experimenters, who were given no information about the groups, graded all the mice independently . A score of 1 indicates that the cotton was not noticeably touched, a score of 2 indicates that the cotton was partially torn up, a score of 3 indicates that the nest was partly finished and lower than the mouse's head, a score of 4 indicates that the nest was perfect or nearly perfect. The average scores of individual mice were calculated. The p-value were analyzed by Student T-test.
Mann-Whitney U rank sum test were applied in non-normal distribution data.

Immunohistochemistry (IHC)
One brain hemisphere of each mouse was fixed with 4% paraformaldehyde for two days, then gradient dehydrated with alcohol from a concentration of 70% to 100%.
Samples were transparentized with dimethylbenzene twice for a total time of 5 hours, followed by wax immersion and paraffin embedding. Ten consecutive 5μm thick sagittal sections cut approximately 600-700 μm from the sagittal suture were used to examine Aβ plaques, phosphorylated tau and microglia. Succinctly, paraffin sections were submerged in citrate buffer (pH 6.0) and boiled for 3min in a pressure cooker to expose antigen, then blocked by hydrogen peroxide and 10% goat serum sequentially.
Diluted primary antibody was placed onto brain sections and incubated at 2-8 °C 150mM NaCl, 50mM Tris, 25mM EDTA, 1% Triton X-100, 0.5% deoxycholic and 0.5%(w/v) SDS, (pH 8.0)) with protease and phosphatase inhibitor MIX and centrifuged at 50000g for 20 min at 4 ℃ to collect the supernatant as the RIPA-soluble fraction (detergent soluble fraction). The pellet was suspended again with RIPA buffer, centrifuged and the supernatant was discarded. Then the pellets were further dissolved by 0.5mL 70% formic acid in 2-8℃ overnight as FA-soluble fraction (insoluble fraction). The three fraction samples were stored in Eppendorf tubes followed by cryopreserved in -80℃ before detection to avoid freeze/thaw.

Enzyme-linked immunosorbent assay (ELISA)
To monitor Aβ40 and Aβ42 in the plasma and brain homogenate of mice, ELISA kits for mouse Aβ40 (catalog CEA864Mu) and Aβ42 (catalog CEA946Mu) were employed. Plasma or brain homogenate samples of a single group were mixed in a ratio of 1:1 as a mixture sample for detection. Duplicate plasma samples were diluted and brain homogenate was 50-fold diluted before experiments. The concentration of Aβ40 and Aβ42 of plasma or brain samples were detected as described in the kit manual. Triplicate samples were used and the concentration was calculated according to the standard curve.

Western Blotting Analysis
Western blotting analysis has been described in previous work. 3 Briefly, equal amounts of fresh protein or brain homogenate were used for electrophoresis by the Novex NuPAGE SDS-PAGE Gel System. Then, the protein was transferred to membranes, and the membranes were blocked and incubated with antibody (G12A, sAPP, 6E10, and β-actin). β-actin or GAPDH antibody were used as an internal control. Finally, the membranes were developed using LI-COR Odyssey Fc imaging system (LI-COR, Lincoln, NE, USA). The analysis of protein used ImageJ (NIH, Bethesda, MD, USA) and Image Studio (LI-COR, Lincoln, NE, USA) software.
Briefly, 5×10 5 cell/well were added in 6 well plate, plates were incubated at 37•C in an incubator with 5% CO2 for 24h, then the culture media was aspirated and 2mL of analysis was used to monitor specificity of the PCR products. The negative control reactions contained nuclease-free water instead of template. The length of the products are designed to be between 100bp and 300bp. Relative expression levels of the selected target genes were calculated with the CFX Manager software method.
The cycle threshold (Ct) values determined for the target genes were normalized against the reference gene. All PCR fragments were analyzed on a DNA1000 Labchip (Agilent Technologies) to check amplification of a single product and sequenced to verify the obtained PCR product. Primers for each selected gene were designed using primer-BLAST software (www.ncbi.nlm.nih.gov/tools/primer-blast/) and listed in Table S3. Table S1. The chemicals used in this study. Table S2. Antibodies used in cell and animal experiments.    Figure S4. A scheme of the processing of mouse brain homogenates. Brain proteins were extracted progressively by RAB buffer (aqueous fraction), RIPA buffer (detergent soluble fraction) and FA buffer (insoluble fraction). Figure S5. Concentration of Aβ40 and Aβ42 in brain homogenate and plasma. Proteins in brain samples were extracted with RAB buffer, RIPA buffer and FA buffer sequentially as described in methods ("Preparation of brain homogenate"), which resulted in a hydrosoluble protein fraction, detergent soluble protein fraction and insoluble fraction, respectively. The concentration of Aβ40 and Aβ42 in these three fraction of brain extract were detected with an ELISA kit, and calculated by the standard curve as described in methods. (A) Concentration of Aβ40 and Aβ42 in RIPA fraction in 2 month brains. In i.v. groups, compared with the PBS subgroup, Aβ42 showed a significant increase in all the ELP-treated groups, conversely, the Aβ40 showed a significant dose dependent decline in ELP-treated groups. The fold-change of Aβ42 was higher than that of Aβ40, suggesting an increase cleavage level of APP by β-secretase. Compared with PBS treated mice in i.c.v. groups, the levels of both the Aβ40 and Aβ42 in the RIPA fraction increased with low fold-changes. The concentration of Aβ42 and Aβ42 in 50μg/mL subgroups and the concentration of Aβ42 in 200μg/mL groups showed statistically significant differences compared with PBS subgroup. Data are presented as mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Students' t test. (B) Concentration of Aβ40 and Aβ42 in the FA fraction in the 2 month groups. Both the concentrations of Aβ40 and Aβ42 in all groups were in the ranges of 5.5 to 7.4. No significant differences were seen among ELP-treated mice and PBS-treated mice in i.v. groups or i.c.v. groups. Data are presented as mean ± S.E.M. (C) Concentration of Aβ40 and Aβ42 of RAB fraction in 1 month mice. In the i.v. group, the concentration of Aβ40 showed little increase in three experimental groups, the 50μg/mL and the 800μg/mL showed statistically significant differences compared with the PBS group. The concentration of Aβ42 showed significant increase in all experimental groups compared with the PBS group, and the 200μg/mL subgroup had the highest concentration of Aβ42, which appeared to be a similar trend to that in 2 month (Fig.4D) The plasma Aβ40 in all ELP-treated subgroups was also higher than that in the PBS group, and the 50μg/mL and 200μg/mL subgroups appeared to show statistically significant differences. In the i.c.v group, both Aβ40 and Aβ42 levels in the ELP-treated subgroups showed significant increase compared to the PBS subgroup. Data are presented as mean ± S.E.M. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Students' t test.  Phospho-Tau (Ser396) protein was recognized by PHF13 antibody and the brown pixels in slides represented the protein immunosignal. 4,5 There was an increased tendency of the phospho-Tau protein accumulating at the soma and dendrite of neurons as the concentration of ELP90 rose, suggesting that the hyperphosphorylation of Tau protein also participated in the pathological changes of ELP-treated mice. Scale bar: 0.2 mm.