In the present study, we sought to immunohistochemically document the temporal and regional evolution of AD-related transgene expression, amyloid deposition, tau phosphorylation, astrogliosis, and microglial activation throughout the hippocampus, amygdala, primary motor cortex, and entorhinal cortex over a 26-month period in male 3xTg-AD mice (Antibodies employed in this study are shown in Table 1 and Nissl-stained brain regions of interest depicted in Figure 1). To sufficiently garner detailed insight into how AD-related pathologies arise in 3xTg-AD mice, animals were sacrificed at 2, 3, 6, 9, 12, 15, 18, and 26 months of age for subsequent immunohistochemical processing (N = 4 per time point). Amyloid pathology that develops in these mice derives from the proteolytic processing of the human APP transgene product that harbors the Swedish double mutation (K595N/M596L; 20) and M146V knock-in mutation in presenilin 1 2122 that, in combination, lead to the marked overproduction and progressive accumulation of the fibrillogenic peptide, Aβ_1–42 23. The hAPP^swe transgene, as well as the tau^P301L transgene, is under the transcriptional control of Thy1.2 gene promoter, which results in transgene expression specifically within neuronal populations beginning early in post-natal development and continuing into adulthood 24. Human APP^swe transgene expression, as assessed using the APP Y188 antibody (AbCam), which recognizes the NPXY amino acid motif of the hAPP protein localized amino terminal to the cleavage fragment of Aβ, was detectable in 3xTg-AD mouse brain beginning at the 2-month time point throughout the pyramidal neurons of the hippocampus (rostral, intermediate, and caudal; Figure 2), layer II and III neurons of the entorhinal cortex (Figure 3A–H), and primary motor cortex (Figure 4A–H). Staining intensities for hAPP^swe transgene product qualitatively appear to stabilize from 6 months and older in all of the regions of the hippocampus and entorhinal cortex examined (Figures 2 and 3A–H). Interestingly, in the amygdala, hAPP^swe expression is not detected by the Y188 antibody consistently until 6 months of age (Figure 5A–H).

Tau, which is expressed as 6 soluble isoforms from a genetic locus found on chromosome 17, is a microtubule-associated protein with numerous functions within the neuron 293031. One such cellular role is its ability to stabilize and promote the polymerization of microtubules 32333435. This function has led to the hypothesis that the inability of tau to adequately bind and promote polymerization of microtubules would result in diminished transport within a neuron. Since it has been shown that the abnormal morphologic entity in AD brains known as the neurofibrillary tangle is comprised primarily of tau 36, it has been proposed that abnormalities of tau, directly or indirectly, play a central role in the pathogenesis of AD by progressively leading to a loss of fast axonal transport. A number of abnormalities of tau have been identified or suggested in AD neurons. These abnormalities include formation of tau into abnormal straight filaments or paired helical filaments 373839, aggregations of paired helical filaments into the larger entities known as the neurofibrillary tangles (NFTs) 40, hyperphosphorylated tau 414243, truncated tau 44, and the inability of tau to bind microtubules due to phosphorylation of key epitopes within the binding domain 4546. The human tau^P301L mutation, which is included as one of the transgenes harbored in 3xTg-AD mice, is commonly used in mouse models for studying human tauopathies, such as progressive supranuclear palsy, corticobasal degeneration, and frontal temporal dementia (reviewed in 47). In these mouse models, intraneuronal inclusions of tau arise as a result of a number of progressive phosphorylation events on serine, threonine, and tyrosine residues 3148, and eventually evolve into NFTs.

Human tau^P301L transgene product can be detected using the HT7 antibody in a very limited number of 3xTg-AD pyramidal neurons in the CA1 of the hippocampus starting at 2 months of age (Figure 9A, I, Q). It was not until 6 months of age that a majority of pyramidal neurons harbored immunohistochemically detectable human tau. At both the 6 and 9-month time points (Figure 9C, K, S and 9D, L, T, respectively), the staining of axonal projections extending into the stratum radiatum began to intensify. Qualitatively, the staining for human tau^P301L transgene product in cell bodies and processes appeared to diminish starting at 12 months of age (Figure 9E, M, U), remaining relatively constant at subsequent ages. Such changes in apparent levels of human tau protein could correspond to age-related increases in the phosphorylation state of tau, leading to steric hindrance and/or structural alteration of the HT7 epitope. When we examined the entorhinal cortex, HT7-positive cells were not detectable until 12 months of age (Figure 10E), and no qualitative signs of human tau transgene product accumulation, as determined by intensification of immunopositive signal, were evident as the ages of the mice increased (Figure 10F, G, H). As in the case for hAPP^swe transgene expression, the human tau^P301L transgene product was detectable in the amydala beginning at 6 months of age and its levels did not appear to significantly fluctuate as the 3xTg-AD mice aged (Figure 11A–H). Primary motor cortex appeared to be the earliest of those examined to exhibit human tau^P301L transgene expression, where HT7-stained neurons were consistently detectable starting at 3 months of age (Figure 12A–H).

Oddo and colleagues recently showed evidence that numerous phospho-tau epitopes are immunohistochemically detectable in 3xTg-AD mice by 15 months of age 49. To assess the status of one important phospho-tau epitope not examined in that prior report and its evolution in the 3xTg-AD mouse brain as a function of age, we examined the phosphorylation of tau at residue Thr231 using the AT180 antibody. We could detect a limited number of immunopositive neurons in the pyramidal layer of the hippocampus as early as 6 months of age (Figure 13C, K), with a majority of AT180-positive cells residing in more caudal regions (Figure 13S). Robust AT180 positivity was apparent at 9 months of age in cells of the pyramidal layer, and fibers extending into the stratum radiatum of the hippocampus (Figure 13D, L, T). As with the HT7 detection there appears to be a waning of AT180 staining at 12 months. However, at more advanced ages (26 months), AT180-positive signals re-intensified, suggesting that these mice could exhibit a cycling phenomenon of tau phosphorylation profiles. AT180-positive cells were not detectable until 26 months in the entorhinal cortex of 3xTg-AD mice (Figure 10P), strongly suggesting that these mice are not experimentally suitable for studying the effects of pathogenic tau in neuronal networks comprising this brain region. The amygdala stains with AT180 beginning at 6 months and this immunopositivity remains high at later ages (Figure 11K–P), whereas the primary motor cortex less consistently stained for this phospho-tau epitope (Figure 12I–P), in that immunopositive signal could be detected only at 9, 12, and 26 months of age.

As tau transitions to a more hyperphosphorylated state, it undergoes a self-assembly process into intertwining 4-nm paired helical filament (PHF) structures, further diminishing the ability of tau to preserve microtubule network integrity 50. The monoclonal antibody PHF-1 (kindly provided by Dr. Peter Davies) recognizes PHF structural epitopes with robust affinity with trace reactivity towards unmodified normal human tau 51. We were able to detect limited tau PHFs beginning in 15 month-old mice within the caudal CA1 region and subiculum (Figure 14W), but it was not until mice reached 26 months of age that we were able to consistently detect PHF-1 positive structures throughout the hippocampus (Figure 14H, P, X). Limited numbers of PHF-1 immunopositive cells were detectable in the entorhinal cortex and amygdala only at 26 months of age (Figures 10X and 11X), while PHF-1 immunopositive structures were barely detectable in primary motor cortex only at 26 months of age (Figure 12X).

Inflammatory processes have long been posited as serving integral roles in initiating and/or propagating AD-associated pathology within the human brain, as the elaboration of inflammatory cytokine expression and other markers of inflammation is more pronounced in individuals with known AD pathology. We previously reported significant enhancement of pro-inflammatory cytokine and chemokine expression and concomitant increases in region-specific microglial cell numbers, prior to the onset of overt amyloid pathology in young 3xTg-AD mice 52. Herein, we assessed the status of two abundant non-neuronal cells traditionally activated in the setting of AD: microglia and astrocytes. The role of microglia and their accumulation at the sites of dense neuritic plaques has been described 535455. Immunohistochemical analysis of 3xTg-AD hippocampal brain tissue using an antibody specific for the microglia/macrophage surface marker, F4/80, revealed a qualitative enhancement of microglia staining from 2 (Figure 15A, I, Q) to 3 months of age (Figure 15B, J, R). The pattern of microglial distribution appeared rather uniform throughout the hippocampus from 3 to 15 months of age (Figure 15). Beginning at 18 months in the most caudal sections of hippocampus (Figure 15W) and continuing at 26 months (Figure 15X), there was a marked change in the distribution of microglia, with these cells appearing to assemble into dense aggregates, reminiscent of amyloid plaque-like structures. Microglial staining patterns in the entorhinal cortex were similar to those in hippocampus, except that aggregation of F4/80-positive cells was not overtly evident in 18 and 26 month-old 3xTg-AD mice (Figure 16A–H). F4/80-positive microglia in both the amygdala and primary motor cortex exhibited age-related distributions similar to those of the hippocampal formation (Figures 17A–H and 18A–H, respectively).

Astrocytes are involved in many different functions in the brain, including structural integrity of the blood brain barrier, support of neuronal synapses by ion regulation and removal of glutamate 56. Although it is believed that they are not directly responsive to primary insults, astrocytes react to inflammatory events in the brain, relying upon pro-inflammatory molecules elaborated from activated microglia 55. Once signaled to do so, astrocytes can perpetuate inflammatory events in the brain via expression of iNOS and the enzyme argininosuccinate synthetase 5758. Glial fibrillary acidic protein (GFAP) is often employed as a marker of astrocytic activation. GFAP-expressing astrocytes were readily visible in 3xTg-AD mice at 2 months of age throughout the hippocampus (Figure 19A, I, Q), with limited signs of activation in the entorhinal cortex, amygdala, and primary motor cortex at this age (Figures 16I, 17I, and 18I). There appeared to be a qualitative decline in staining intensity in rostral hippocampal regions beginning at 15 months of age (Figure 19F) and continuing through 26 months of age (Figure 19H). However, the overall pattern of activated astrocyte staining within the hippocampus remained relatively constant as a function of age. More robust GFAP-positive astrocyte staining in the entorhinal cortex was more apparent at the 18- and 26-month time points (Figure 16O, P), while GFAP staining was less detectable, but constant at ages greater than 18 months in amygdala and primary motor cortex (Figures 17O, P and 18O, P, respectively).

The 3xTg-AD mice (B1 line) were kindly provided by Frank LaFerla (University of California, Irvine; 17). All mice were housed and bred in accordance with University of Rochester requirements for animal welfare and care. Homozygous 3xTg-AD mice were monogamously mated to produce offspring, which were housed until sacrificed at the designated age. Mice were sacrificed via pentabarbitol overdose and subsequently transcardiac perfused with heperanized saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were removed and post-fixed overnight in 4% paraformaldehyde in 0.1 M PB, followed by equilibration in 20% sucrose in 0.1 M phosphate-buffered saline (PBS) and then 30% sucrose in 0.1 M PBS. Brains were coronally sectioned on a freezing stage sliding microtome (Microm, Walldorf, Germany) at 30 μm and stored in cryoprotectant at -20°C until immunohistochemical processing.

Brain sections were washed with 0.15 M PB for 2 h to remove the cryoprotectant, and mounted on Superfrost^® Plus slides (VWR International, West Chester, PA) and allowed to dry completely. The slides were subsequently hydrated in dH₂0 for 5 min. before being stained with 0.02% Cresyl violet Acetate in 0.25% Acetic acid for 30 min. Sections were rinsed in 3 changes of dH₂0, and placed in 50% ethanol for 1 min. followed by 70% for 1 min. to destain. Sections were allowed to dry and then cleared by dipping in xylene before being coverslipped.

The following antibodies were used at the designated working dilutions: anti-amyloid precursor protein A4, corresponding to the NPXY motif of hAPP, (Clone Y188; AbCam, Cambridge, MA, 1:750); anti-hAPP/amyloid-beta reactive to amino acid residue 1–16 of beta-amyloid (6E10; Covance, Berkeley, CA; 1:1000); anti-amyloid beta 1–42 clone 12F4 reactive to the C-terminus of beta-amyloid and specific for the isoform ending at amino acid 42 (Covance/Signet, Berkeley, CA, 1:1000); anti-amyloid beta 1–42 polyclonal antibody for intracellular amyloid-beta staining (Invitrogen, Carlsbad, CA, formerly Biosource, Hopkinton, MA 1:1000); anti-human tau HT7, reactive to residues 159 to 163 (Pierce, Rockford, IL; 1:200); anti-human phosphorylated tau AT180, specific for htau phosphorylated at the Thr231 residue (Pierce, Rockford, IL; 1:200); anti-human phosphorylated tau PHF-1 (gift from Dr. Peter Davies, Albert Einstein College of Medicine; 1:30); anti-glial fibrillary acidic protein GFAP (Dako Cytomation, Glostrup, Denmark; 1:1000); and an antibody specific for the microglial/monocytic cell surface marker F4/80 (AbD Serotec, Raleigh, NC; 1:500). Specificity of the anti-Aβ_1–42 antibodies (12F4 from Covance/Signet for extracellular Aβ staining and anti-Aβ_1–42 polyclonal from Biosource/Invitrogen for intracellular Aβ staining) was confirmed using peptide competition experiments. The results of these assessments are illustrated in Figures 7 and 8, respectively.

Brain sections were washed with 0.15 M PB for 2 h to remove the cryoprotectant, then incubated with 3% H₂O₂ in 0.15 M PB for 20 min. to quench endogenous peroxidase activity. For Aβ peptide-specific stains, the sections were treated with 70% formic acid for 15 min. for epitope retrieval. For the intracellular Aβ_1–42stain, we employed a microwave/Target buffer (Dako Cytomation, Glostrup, Denmark) epitope retrieval method as described previously 27. Briefly, the brain sections were washed and peroxidase activity quenched. The sections were mounted on to slides and allowed to dry. The Target buffer was heated to 98°C in a microwave (GE, Louisville, KY), the slides submerged into the buffer and placed in the microwave, twice for 3 min. at 450 W, and allowed to rest for 5 min. between microwave steps. Brain sections were similarly processed for immunohistochemistry as detailed below. The sections were washed and permeabilized in 0.15 M PB and 0.4% Triton X-100, followed by blocking in 0.15 M PB with 10% normal goat serum, and 0.4% Triton X-100. After blocking, the sections were incubated in 0.15 M PB with 1% normal goat serum, and 0.4% Triton X-100, with the designated primary antibody. The sections were washed with 0.15 M PB, followed by an incubation with the appropriate secondary biotin-conjugated secondary antibodies (Vector Labs, Burlingame, CA; 1:1000) in 0.15 M PB with 1% normal goat serum, and 0.4% Triton X-100. The sections were washed with 0.15 M PB with 1% normal goat serum and 0.4%Triton X-100, and incubated in the avidin-biotin complex (Vector Labs Vectastain ABC System as per manufacturer's protocol, Vector Labs, Burlingame, CA). Sections were washed in 0.15 M PB followed by rinses in dH₂O. The sections were developed with nickel-enhanced DAB (Vector Labs, Burlingame, CA). Sections were mounted on Superfrost^®Plus slides (VWR International, West Chester, PA) cover-slipped and viewed using an Olympus AX-70 microscope and motorized stage (Olympus, Center Valley, PA) and the MCID 6.0 Imaging software (Interfocus Imaging subsidiary of GE Healthcare, Cambridge, England).

Time points were compared to one another within a particular antibody staining group. Images were analyzed using the relative optical density (ROD) score in the MCID 6.0 Imaging software (Interfocus Imaging subsidiary of GE Healthcare, Cambridge, England). Sections of all the mice, corresponding to the areas of study, were scored according to the following schema: (-) indicates no staining present, (+/-) indicates limited number of cells/structures showing evidence of staining, (+) denotes consistent expression of the marker, (++) represents an elevated expression measured in ROD of Δ 0.075 to 0.250, and (+++) represents a further increase in staining as indicated by a change in ROD of Δ 0.251 greater than (+) staining intensities.