Animal Husbandry
Tg6799 transgenic mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were housed under a 12-h light–dark cycle with free access to food and water. Female Tg6799 mice (n = 8 in each group) were maintained until used for the experiment (4 months for early symptomatic stage and 8 months of age for late symptomatic stage). After breeding in an expansion colony, male Tg6799 transgenic mice were mated with female littermate mice. The genotype of each mouse was confirmed by PCR using the DNA from mouse tail tissue. The mice had free access to food and water. All animal studies were conducted in accordance with IACUC guidelines and were approved by the IACUC committee at Hanyang University (HY-IACUC-09-017).
RNA Sample preparation
RNA samples were isolated from the hippocampus (HC) using TRI-Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. The total RNA pellet was dissolved in nuclease-free water and RNA quality and quantity were assessed by Agilent Bioanalyzer 2100 analysis (Agilent, Santa Clara, CA). RNA with RNA Integrity Number (RIN) above 8 was used for microarray and sequencing. Gene expression was analyzed with GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA), which comprises over 45,000 probe sets representing approximately 28,700 well-characterized mouse genes. For each gene, eleven pairs of oligonucleotide probes were synthesized in situ on the arrays.
mRNA microarray hybridization and analysis
Biotinylated cRNA were prepared from 250 ng total RNA according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2001, Affymetrix). Following fragmentation, 15 μg of RNA were hybridized for 16 hr at 45°C on a GeneChip Mouse Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the Affymetrix Gene Chip Scanner 3000 7G. Eight microarray samples were normalized by the RMA method of R affy package [41]. DEG testing was accomplished using the 4 class limma test of 2 mutants and 2 aging factors. Finally, 2,197 probes were considered to have significantly differential expression by a cut-off p-value < 0.05.
miRNA sequencing and analysis
RNA quality was assessed by the absence of smear of 18S and 28S bands as analyzed by the Bio analyzer 2100 (Agilent Technologies, Santa Clara, CA). For the construction of micro RNA libraries, IonTotal RNA-Seq Kit v2 and Ion Xpress™ RNA-Seq Barcode kit (Life Technologies, Foster City, CA) were used. The Ion Total RNA-Seq Kit v2 (Life Technologies) was used for the preparation of micro RNA libraries according to the manufacturer’s instructions starting with 100 ng total RNA. The template was prepared from the libraries using the Ion OneTouch™ System (Life Technologies) and sequenced on an Ion PGM™ Sequencer (Life Technologies). Total read counts are shown in Additional file 5: Table S5. As a preprocessing step, the sequencing adapter was trimmed and read to be over the range of miRNA length (18–30 nucleotides) were excluded. We allowed bowtie2 with options of one mismatch and used mouse miRbase ver. 19 as a reference. [42, 43]. After alignment, a total of 527 miRNAs were identified. Raw level data from the miRNA read count were normalized using the quintile method and considered as miRNA expression. Differentially expressed miRNA (DEmiR) was also tested using limma-like microarray conditions. Finally 54 DEmiRs were detected within a cutoff p-value ≤ 0.1.
Gene expression network for miRNA target integration
For the genome wide study, we detected gene set modules from DEG and built a weight co-expression network. The network was constructed using WGCNA R packages with 2,197 DEGs from the limma test [44]. We detected 9 modules with block-wise module function. To evaluate significant modules related to the sample traits of AD mutation and aging, we performed scatter plots and correlations of gene significance and MM were measured. MM is a quantitative measure for the correlation of the module eigengene with the gene expression profile. MM and correlation allow quantification of gene member similarity for each module. We selected 8 significant modules using these correlations. The grey module was removed because of the small gene set count (27 probes, 23 identified genes). Finally for constructing a weighted network, a topological overlap measure (TOM), as a measure of the robustness of the network interconnectedness, was calculated based on the adjacency matrix from the 8 module gene set. From module TOMs with a weighted network threshold 0.25, a network dataset was exported to Cytoscape. Functional enrichment of each module was verified using the DAVID database (p-value < 0.1) [45].
An integrative network of miRNAs and genes was constructed after miRNA-target gene prediction. miRNA and target relationships were predicted by both expressional and sequential analysis. All miRNAs were considered for miRNA-target anti-correlated expression. However, we considered and calculated correlations in view of sample subsets in AD factors or aging factors. Additionally, the sequence-based target relation was predicted using both TargetScan and miRAN.org [46, 47]. miRNA priority was calculated using the PageRank algorithm to consider topological importance [48]. From the sequence and expression-based approaches, we removed insignificant miRNA and target gene relationships and ranked miRNA importance by PageRank score. The raw data were uploaded into the gene expression omnibus (GEO) database with an accession number of GSE52023.
RT-PCR
The SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) was used to synthesize first-strand cDNA from an equal amount (500 ng) of RNA. The following primer sequences were used for RT-PCR in this study: arrestin domain containing 3, 5′ -TTCTCAGTTTGCCCCTCGTC- 3′ (forward) and 5′ -TCCTCTGCAAACGTGTCTCC- 3′ (reverse); protein phosphatase 1, regulatory subunit 3, 5' -ACGATGGAAGTCCTTGGATG- 3' (forward) and 5' -TCCATGCGCCTTAATTCTTC- 3' (reverse); splicing factor proline/glutamine rich, 5' -ACGATGGAAGTVCTTGGATG- 3' (forward) and 5' - TCCATGCGCCCTTAATTCTTC- 3' (reverse); glyceraldehyde 3-phosphate dehydrogenase, 5′-AGAACATCATCCCTGCATCC- 3′ (forward) and 5′ -TCCACCACCCTGTTGCCTGTA- 3′ (reverse).
Protein sample preparation
Protein samples were prepared from the hippocampus in a homogenization buffer (50 mM Tris-Cl (pH8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors: 10 μg/ml aprotinin, 25 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml PMSF. Homogenates were centrifuged at 14,000 g for 30 min at 4°C and the supernatants were collected and stored at 80°C before use.
Western blot
Equal amounts (10 μg) of protein sample were used for western blot analysis [49]. Protein samples were loaded in 10% sodium dodecyl surfate (SDS)–polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane (BIO-RAD, Hercules, CA). The following antibodies were used for primary antibody reaction: polyclonal anti-ARRDC3 (1:2,000; Abcam, Cambridge, MA) and polyclonal anti-Sfpq (1:2,000; Abcam, Cambridge, MA) and for secondary antibody reaction: horseradish peroxidase (HRP)-linked anti-rabbit (1:2500; Vector, Burlingame, CA).
Co-immunoprecipitation (Co-IP)
Equal amounts (10 μg) of protein samples were immunoprecipitated using polyclonal anti-ARRDC3 (1:2,000; Abcam, Cambridge, MA) at 4°C for overnight, followed by binding anti-ARRDC3 to protein A agarose (Sigma, St. Louis, MO). Immunoprecipitated protein expression was confirmed by immunoblotting with used polyclonal anti-Sfpq (1:2,000; Abcam, Cambridge, MA).
Primary cell culture
Cortex and hippocampus were dissected from the brains of Tg6799 transgenic mice and their littermate controls at postnatal day 1 (p1). Neurons were cultured on glass cover slips or cell culture multi-well plates, both coated with poly-L-ornithine (Sigma, St. Louis, MO), fibronectin (Sigma, St. Louis, MO), and laminin (Sigma, St. Louis, MO). Culture media used was DMEM/F-12 with 10% fetal bovine serum (FBS, GenDEPOT, San Diego, CA), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St. Louis, MO), and 2 mM L-glutamin (Gibco, Carlsbad, CA) supplemented with 5% B-27 supplement (Gibco, Carlsbad, CA) and 10 ng/ml bFGF (Invitrogen, Carlsbad, CA) at densities of 3.63 × 104 cells/cm2.
Knock down or overexpression of ARRDC3
To analyze the role of ARRDC3, we transfected the primary cells with ARRDC3 cDNA on the 7th day of primary cell culture. The mammalian expression vector pCMV6-XL4 was used for the overexpression of full-length human ARRDC3 cDNA (NM 020801.1) (Origene Technologies, Inc., Rockville, MD). The plasmid was transiently transfected with standard calcium-phosphate preparation. After 24 hr, transfected cells were administered with 2 μM Aβ for 24 hr. Next, we transfected the primary cells on the 7th day of cell culture with ARRDC3 siRNA or scrambled ARRDC3 siRNA as negative control (data not shown). The sequence for ARRDC3 siRNA is 5′ -GACACCACUUGCUACCUCA- 3′ and the sequence for scrambled ARRDC3 siRNA is 5′ -ACACUCUAACCGGCUUACC- 3′. The siRNAs were transfected into the primary cells by Gene-mute siRNA Transfection Reagent (SignaGene laboratories, Gaithersburg, MD) for 48 hr, as described by the manufacturer.
ARRDC3 promoter-reporter construction
DNA fragments (−280 bp) upstream of the ARRDC3 gene were PCR-amplified using human SHSY-5Y cell gDNA as template with following primers [50]: (−280 bp) 5' -GGGGTACCGGGGTTGTTTTTCACAAAGCTG- 3' (forward) and 5' -ATCTCGGTCTTCACCTTTCCCAGCACC- 3' (reverse). The PCR fragments were purified, digested, and cloned into the luciferase vector pGL3-Basic (Promega, Medison, WI) with KpnI and XhoI restriction sites.
Transient transfection and luciferase assay
Transfection of ARRDC3 promoter was conducted into primary cells on the 7th day of culture using the standard calcium-phosphate preparation. After 24 hr, transfected cells were administered with 2 μM Aβ for 24 hr, lysed, then measured for luciferase activity using TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) by injecting 40 μl per tube of a buffer containing 100 mM Tris-Cl (pH 7.8), 15 mM MgSO4, 10 mM ATP, and 65 μM of luciferin. All transfections were carried out in triplicate.
Immunocytochemistry
After treatment, the primary cells on coated cover slips were post-fixed in 4% paraformaldehyde for 10 min. The cells were blocked for 30 min then incubated with polyclonal tau (Tau46) antibody (1:500; Cell Signaling, Danvers, MA) for 3 hr. After secondary antibody incubation, the cells were stained with blue alkaline phosphate substrate solution (Vector Laboratories Inc., Burilngame, CA) then mounted on gelatin-coated slides for microscopic analysis. Images were obtained using a Carl Zeiss microscope (Axio observer, Oberkochem, Germany). The number of tau-positive cells was counted from microscopic images and analysis was performed blindly by two different assessors.
Detection of miRNA expression
RNA samples were extracted from mouse hippocampal tissue using the miRCURY RNA Isolation Kit (Exiqon, Woburn, MA) according to the manufacturer’s protocol. cDNA was synthesized using the Universal cDNA synthesis kit II (Exiqon, Woburn, MA) and was used as a template for microRNA real-time PCR using ExiLENT SYBR Green master mix (Exiqon). The following primers were used for real-time PCR in this study: mmu-mir-139-5p, 5' -UCUACAGUGCACGUGUCUCCAG- 3'; mmu-mir-340-5p, 5' -UUAUAAAGCAAUGAGACUGAUU- 3'; mmu0mir-3470a, 5' -UCACUUUGUAGACCAGGCUGG- 3' and RNU5G control primer (Exiqon, Woburn, MA). Quantitative real-time PCR was performed for 10 min at 95°C followed by 10 sec at 95°C and 1 min at 60°C for 45 cycles. At the end of PCR cycles, melting curve analyses were performed for each PCR product.
Statistical analysis
All statistical analyses were accomplished using SPSS (version 17; SPSS Inc., IL). Data were objectively compared using independent-sample T-tests. Differences between groups were considered statistically significant when the p value was less than 0.05.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files. miRNA and mRNA microarray data have been deposited in GEO public repository with ID GSE52022 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52022), ID GSE52023 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52023), and ID GSE52024 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52024).