All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ The global burden of mental and neurological disorders has increased from 14.5% to almost one-fifth of the world's population during the past decades \[[@pone.0181211.ref001]\]. Alzheimer's disease (AD) is one of the most common neurodegenerative disorders in older age. It is characterized by the presence of extracellular amyloid-β deposits (Aβ), intracellular neurofibrillary tangles (NFTs), synaptic loss, and neuronal degeneration. Recent epidemiological studies have revealed that the risk of dementia due to AD increases by approximately two-fold with advancing age \[[@pone.0181211.ref002]\]. Thus, for ageing people, prevention of AD may have profound implications for human societies. Various factors including genetics, vascular changes, inflammation, and oxidative stress can all lead to AD. Many of these factors can cause the progressive formation of protein aggregates and impair their clearance, resulting in the degeneration of neuronal cells \[[@pone.0181211.ref003]\]. The most likely molecular causes of AD are the Aβ accumulation, hyperphosphorylated microtubule-associated tau protein (MAPT), and a deficiency of brain-derived neurotrophic factor (BDNF) that leads to neuronal dysfunction and finally to neuronal cell death \[[@pone.0181211.ref004]--[@pone.0181211.ref006]\]. It has been reported that the Aβ peptides in the brain are the most important causative factors of AD \[[@pone.0181211.ref007], [@pone.0181211.ref008]\]. Aβ peptides of 40 and 42 amino acids are generated by sequential cleavage of APP by β-secretase and γ-secretase \[[@pone.0181211.ref009]\]. The soluble monomers of the peptide Aβ are rapidly cleared from the central nervous system (CNS) by enzymes such as insulin-degrading enzyme (IDE), neprilysin, and plasmin \[[@pone.0181211.ref010]\]. However, these enzymes cannot degrade the oligomers of the Aβ peptide efficiently \[[@pone.0181211.ref010]\], so the formation of insoluble oligomers can have deleterious effects on the physiology of the CNS \[[@pone.0181211.ref011]\]. In AD, the formation of insoluble oligomers can cause neuroinflammation and eventually lead to neuronal death \[[@pone.0181211.ref012]--[@pone.0181211.ref015]\]. The APP processing enzymes are all associated with Aβ-mediated neurotoxicity \[[@pone.0181211.ref016]\]. It has been demonstrated that tau is a microtubule-associated protein (MAP) that binds to and stabilizes microtubules. Three protein forms of tau are found in the adult human brain, which are of different length: tubulin-bound (tau~3~), hyperphosphorylated (tau~3P~), and aggregated forms (tau~4~). Tau is a single molecule in tau~3P~, whereas tau~3~ is a multimeric protein that makes up the tau scaffold and tau~4~ is a non-functional aggregated form \[[@pone.0181211.ref017], [@pone.0181211.ref018]\]. There is some evidence that Aβ can bind to tau and promote its self-aggregation, which leads to the formation of paired helical filaments (PHFs) that finally result in neuronal degeneration \[[@pone.0181211.ref019]\]. Previous studies have revealed that NFTs and neurofibrillary tangles (NFTs) are formed in the brain of AD patients and that these formations are linked with cognitive dysfunction and Alzheimer's disease. However, the mechanism by which NFTs are formed and the formation sites within the brain remain elusive. Most of the previous studies have focused on the analysis of protein changes at certain time points, but little is known about the process of Aβ aggregation in the brain over time. We previously reported that *in vivo* Aβ-GFP fibrils in APP/PS1 mice were first observed in the cortex, hippocampus, and hypothalamus (a non-neuronal structure), and gradually invaded the brain parenchyma \[[@pone.0181211.ref020]\]. Thus, the formation of soluble Aβ fibrils is an early event that initiates Aβ aggregation. To study the process of Aβ aggregation in more detail, we carried out a time-course investigation of Aβ and tau expressions using immunohistochemistry and double immunofluorescence staining. Here, we demonstrated for the first time the spatial relationships between Aβ and tau. In addition, we report on the temporal relationships among soluble Aβ~1-40~ aggregates, NFTs, Aβ accumulation, and tau accumulation in the brains of 9-month-old APP/PS1 mice. Materials and methods {#sec002} ===================== Animals {#sec003} ------- APP/PS1 double-mutant mice (APPswe/PS1dE9; B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J) were obtained from The Jackson Laboratory (USA). APP/PS1 double-mutant mice were bred on a C57BL/6J background in the Department of Pathology at Xuanwu Hospital in Beijing (China) and in the Model Animal Research Center of Nanjing University (China). Experiments were performed using 1-, 5-, and 9-month-old APP/PS1 double-mutant mice (at 30 weeks of age), and wild-type C57BL/6J mice were used as controls (6 months of age). Mice were housed in pathogen-free, air-filtered, climate-controlled rooms with 12-hour light/dark cycles at 25°C and 50% humidity. Mice were provided food and water ad libitum. All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures were performed according to the Chinese Guidelines for Animal Welfare and approved by the Animal Care and Use Committee of the Model Animal Research Center of Nanjing University (the approval number: SYXK(su)2011--0056). Immunohistochemical staining {#sec004} ---------------------------- Four C57BL/6J and APP/PS1 double-mutant mice (at 4, 8, and 12 months of age) were anesthetized with 0.3% sodium pentobarbital and perfused intracardially with ice-cold 0.9% NaCl solution. Following perfusion, the left hemisphere was removed, fixed in 4% paraformaldehyde for 24 h at 4°C, then dehydrated in 20% sucrose and embedded in Tissue-Tek OCT (Sakura Finetek, CA, USA) for sectioning. Brain tissue sections (8 μm) were treated for 5 min in acetone at -20°C, followed by incubation in blocking buffer (5% donkey serum in 0.01 M phosphate-buffered saline, pH 7.2) for 1 h at room temperature. The sections were then incubated overnight at 4°C with primary antibodies against Aβ~1-40~ (1:150; Thermo Fisher Scientific, Waltham, MA, USA), Iba-1 (1:500; Abcam, Cambridge, MA, USA), APP/Aβ (1:2000; Millipore, Billerica, MA, USA), activated caspase 3 (1:50; Abcam), glial fibrillary acidic protein (GFAP) (1:2000; Abcam), C1q (1:1000; Abcam), Aβ oligomers (A11, 1:50; ABIN354600, Abgent, San Diego, CA, USA), and human tau (tau46, 1:100; Thermo Fisher Scientific). These antibodies were diluted in blocking buffer. Sections were incubated with secondary antibodies for 1 h at room temperature. The following secondary antibodies were used for double immunofluorescence: Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500; Invitrogen, Carlsbad, CA, USA), Cy3-conjugated donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:1000; Invitrogen), Cy3-conjugated donkey anti-rabbit IgG (1:1000; Jackson ImmunoResearch), Alexa Fluor 647-conjugated donkey anti-rabbit IgG (1:500; Invitrogen), and Alexa Fluor 647-conjugated donkey anti-rat IgG (1:200; Invitrogen). All samples were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Stained sections were analyzed with a laser scanning confocal microscope (LSM780; Zeiss, Oberkochen, Germany) equipped with Zen software (Zeiss). Measurement of Aβ plaque density {#sec005} -------------------------------- Brain sections (8 μm) from 8- and 12-month-old APP/PS1 double-mutant mice (n = 3 per group) were stained with the antibody against Aβ~1-40~. Staining intensity was quantified with NIH ImageJ software (NIH). Optical densities in the cortex and hippocampus (CA1, CA3, and dentate gyrus \[DG\]) were analyzed with ImageJ software (NIH). To quantify the amount of Aβ deposition, we selected two adjacent sections from each mouse brain. The total number of images (1:1) was calculated for each group. MTT cell viability assay {#sec006} ------------------------ SH-SY5Y cells were seeded into 96-well plates at a density of 4 × 10^5^ cells/well. The following day,