Immunohistochemistry

Abnormal phosphorylation and conformational changes on tau can be detected with monoclonal antibodies, including the following: (1) CP13, reactive with phosphorylation on serine 202 on tau in pretangles (Kimura et al., 1996) and later-stage NFTs (Augustinack et al., 2002); (2) MC1, recognizing a conformation (Jicha et al., 1997a,b, 1999) that is specific to abnormal tau (Weaver et al., 2000); and (3) PHF1 (paired helical filament), specific for phosphorylation at serines 396/404 (Ps396/404), reacting with later-stage NFTs (Otvos et al., 1994; Jicha et al., 1997b).

Immunohistochemistry was performed using these antibodies on htau mouse brain serial coronal vibratome sections (50 μm) collected from multiple mice, of both sexes, aged 1, 8, 12, 14, 16, and 18 months. Mouse brains were immunostained using standard streptavidin biotin peroxidase methods as described previously (Duff et al., 2000) and developed with 3,3′-diaminobenzidine (DAB). The following monoclonal antibodies were used to stain the free-floating brain sections: CP13 (1:25) (Ps202), MC1 (1:10) (detecting a conformational abnormality of tau), and PHF1 (1:25) (Ps396/404). Sections were also immunostained with antibodies to neuronal nuclei (NeuN) (Chemicon, Temecula, CA).

Electron microscopy

Tissue sections. Electron microscopy (EM) on tissue sections was performed as described previously (Andorfer et al., 2003) on mice aged 15, 18, 22, and 26 months stained with uranyl acetate (5% in 50% ethanol).

Sarkosyl-insoluble filaments. Isolation of Sarkosyl-insoluble tau filaments was performed as described previously (Andorfer et al., 2003). Ultracentrifugation and Sarkosyl extraction (30 min incubation in 1% Sarkosyl) was performed according to Greenberg and Davies (1990) to obtain insoluble fractions of tau from htau mice aged 15 months. Insoluble fractions (pellets) were washed one time with 1% Sarkosyl and resuspended in 100 μl of 1× TBS. A 6 μl aliquot of the suspension of the insoluble material was placed on each carbon-coated Formvar grid (Electron Microscopy Sciences, Fort Washington, PA) for 10 min, rinsed in distilled water for 10 min, placed in 1× TBS for 2 min, and then drained. Grids were blocked for 10 min with a 1× TBS solution containing 0.1% Tween and 0.1% gelatin from coldwater fish skin (Sigma, St. Louis, MO) and then drained. Primary antibodies (CP13 and PHF1) were diluted 1:100 in the blocking solution above, added to grids for 1 h, and then washed off in 1× TBS three times, 5 min each. Immunogold-conjugated (10 nm) goat anti-mouse IgG (heavy and light chain) (Ted Pella, Redding, CA) was diluted 1:50 in the blocking solution, placed on grids for 1 h, and then drained and washed three times for 5 min each in 1× TBS. Grids were then fixed in 2% glutaraldehyde (diluted in 1× TBS) for 5 min, washed three times for 5 min each in distilled water, stained for 15 min in uranyl acetate (5% in 50% ethanol), washed in distilled water, and allowed to dry.

Thioflavine-S staining

Briefly, free-floating vibratome sections were washed in distilled water for 1 min and then incubated for 10 min in 0.1% thioflavine-S (Sigma) made in distilled water. Sections were then dipped in 70% ethanol two times and incubated in 100 mm sodium citrate, pH 4.0, for 4 min. Sections were mounted (in the sodium citrate solution, pH 4.0) on glass slides, coverslipped in 80% glycerol at pH 4.0, and examined using a Bio-Rad (Hercules, CA) 2000 scanning laser confocal microscope. Incubation and mounting were performed at pH 4.0 to quench enhanced green fluorescent protein fluorescence (Nakanishi et al., 2001). Quenching was confirmed by examination of KO mice.

Comparison of cortical thickness

Vibratome sections (50 μm) collected from several ages of htau mice (1, 10, and 14 months) were stained with NeuN (Chemicon), an antibody that specifically labels neuronal nuclei. Cortical thickness and density of neuronal nuclei were qualitatively compared between closely matched sections and brain regions from htau mice of each of the ages.

Stereology

Tissue preparation. Brains were extracted from htau mice aged 8 and 17 months (five animals per age group) and immediately immersion fixed in 4% paraformaldehyde overnight at 4°C. Brains were cut in the coronal plane into 50-μm-thick sections using a vibratome. Sections were collected in order, mounted on slides, dried overnight at room temperature, stained with cresyl violet, dehydrated, and coverslipped.

Estimates of neuron loss. The optical fractionator (West et al., 1991) was used to obtain quantitative estimates of neuronal loss with age in the htau mice. After random selection of a starting point, six to eight sections, at equal intervals (800 μm), were collected through the length of the piriform cortex in each animal. We used an Axiophot IIc photomicroscope (Zeiss, Oberkochen, Germany) equipped with an ASI motorized stage, high-resolution Dage digital camera (Dage-MTI, Michigan City, IN), and StereoInvestigator software program (MicroBrightField, Wiliston, VT) running on a personal computer. The boundaries of the piriform cortex were delimited based on a mouse brain atlas (Hof, 2000). Neurons were identified using morphological criteria for nuclei and systematically and randomly sampled within counting frames of 20 × 20 μm, using a dissector height of 10 μm, across the entire extent of the piriform cortex. The average postprocessing thickness of sections was 18 μm. On average, 1000 neurons per animals were sampled. The coefficient of error was calculated according to Schmitz and Hof (2000). Volume estimates were calculated based on the outline of the region. Statistical analysis was performed with a Student's t test.

Immunoblotting

Immunoblotting was performed as described previously (Andorfer and Davies, 2000). Briefly, brains were extracted, weighed, and placed into a volume (in microliters) of homogenizing buffer (TBS, pH 7.4, containing 1 μm phenylmethylsulfonyl fluoride, 1 mm sodium vanadate, 2 mm EGTA, and 10 mm sodium fluoride) equal to five times brain mass (in milligrams). Samples were homogenized using a Polytron and stored at -80°C. Samples from a time course of htau brain homogenates (2, 5, 10, 13, 16, and 17 months of age) and an aged wild-type mouse control (20 months of age) were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% milk/1× TBS and probed with antibodies to caspase-3, -7, and -8 (Cell Signaling Technology, Beverly, MA) and caspase-6, -9, and -10 (Stressgen, Victoria, British Columbia, Canada), diluted at 1:1000 in 5% milk/1× TBS. Membranes were additionally probed with an antibody to SNAP-25 [synaptosome-associated protein of 25 kDa (SP-14)] (Honer et al., 1992) diluted 1:20 in 5% milk/1× TBS.

Paraffin sections

Antigen retrieval method. Sagittal brain hemispheres were incubated in 4% paraformaldehyde, embedded in paraffin, sectioned, and mounted on glass slides. Sections were deparaffinized and rehydrated, and endogenous peroxidase activity was quenched via serial incubation in xylene (two times for 10 min each), 100% Etoh (two times for 10 min each), 95% methanol/5% H₂O₂ (30 min), 95% Etoh (two times for 5 min each), and 80% Etoh (two times for 5 min each). Antigen retrieval was then performed by incubating sections in sodium citrate buffer, pH 6.0, for 20 min in a vegetable steamer (Black and Decker, Towson, MD). Sections were allowed to cool at room temperature for 30 min and then blocked in 5% milk/1× TBS for 1 h before antibody application.

Antibodies. Antibodies to the following cell-cycle regulatory molecules were used on the paraffin sections: proliferating cell nuclear antigen (PCNA) (1:100), Cdk5 (cyclin-dependent kinase 5) (1:1000), cyclin D₁ (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA), cyclin B₁ (1:1000), Cdk1/cdc2 (cell division cycle 2) (1:1000) (Upstate Biotechnology, Lake Placid, NY), ki67 (1:2000) (Vector Laboratories, Burlingame, CA), and cdc-2 (1:1000) (Cell Signaling Technologies). DAB was used as the chromogen, and, when double labeling was performed, reactivity was detected using the Vector Laboratories VIP substrate kit for peroxidase.

TUNEL assay. Detection of 3′-OH termini of DNA strand breaks was performed on paraffin sections of wild-type and htau mice aged 1, 8, 15, and 18 months using the ApopTag peroxidase kit (Intergen, Purchase, NY) following the recommendations of the manufacturer. Labeling was performed with DAB as the chromagen. Sections were counterstained with toluene blue.

Cell loss with advanced age in htau mice

Morphologic abnormalities indicative of cellular injury were evident in some neurons in the older htau mice that were also positive for cell-body accumulation of tau phosphorylated at serine 202 (CP13) and for the abnormal MC1 conformational change, particularly within the piriform cortex. Predominantly evident were enlarged vacuoles, irregularly shaped membranes, and distorted processes (Fig. 2A-C). Additionally, some neurons appeared ballooned (Fig. 2D), and still others had the character of a so-called “ghost tangle” or a late-stage tangle (Fig. 2E).

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Figure 2.

Gross changes in old htau brains, including abnormal neuronal morphology, a decrease in cortical thickness, and a dramatic enlargement of ventricle size. Irregularly shaped neurons were detected with CP13 (A-C) and MC1 (D, E). Note the presence of enlarged vacuoles, irregularly shaped membranes, and distorted processes (A-C). Additionally, some neurons appeared ballooned (D), and still others had the character of late-stage tangles (E). Comparison of cortical thickness from matched brain regions of 1-month-old (F), 10-month-old (G), and 14-month-old (H) htau mice indicated a decrease in thickness and an obvious reduction in the number of detectable nuclei with age. A comparison of ventricle size in level-matched coronal sections between htau mice aged 8 months (I) and 18 months (J) revealed a dramatic increase in the older mice. The red oval delineates the piriform cortex. Scale bars: A-E, 10 μm; F-H, 50 μm; I, J, 250 μm.

Gross morphological analysis of aging htau mouse brains compared with age-matched wild-type mice and younger htau mice revealed striking qualitative evidence of cell loss. Comparison of cortical thickness from level matched vibratome sections of 1-month-old (Fig. 2F), 10-month-old (Fig. 2G), and 14-month-old (Fig. 2H) htau mice stained with an antibody specifically reactive to neuronal nuclei indicated a decrease in thickness and a significant reduction in the number of detectable nuclei with age. Additionally, a comparison of ventricle size in level-matched coronal sections between htau mice aged 8 months (Fig. 2I) and 18 months (Fig. 2J) revealed a dramatic increase in the older htau mice, as well as a dramatic decrease in the thickness of the corpus callosum, both indicative of significant cell loss. This was not observed in aging wild-type control mice (data not shown). Considering these striking qualitative observations and the fact that neuronal injury and loss can be important features of diseases associated with NFT formation, quantitative stereologic evidence of cell loss in the htau mice was sought using the optical fractionator method.

Quantification of cell death in htau mice

We focused our examination of cell loss in the piriform cortex (Fig. 2J, surrounded by the red oval), a region in which older htau mice grossly display extensive accumulation of somatodendritic tau, morphologically abnormal cells, and a gross loss of cortical organization. The total number of neurons in the piriform cortex was estimated in 10 individual htau mice, aged 8 and 17 months. Three males and two females were examined per group, and the results were compiled in Figure 3A and presented graphically in Figure 3B. A significant reduction in the total number of neurons occurred between 8 and 17 months, from an average of 565,000 to 242,000, a decrease of 67.2% (p < 0.009). There was also a 23% reduction (p < 0.002) of the estimated volume of the piriform cortex between these age groups, consistent with the dramatic decrease in neuronal number and the gross brain observations described above.

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Figure 3.

Quantification of age-related neuronal loss in the piriform cortex of htau mice was performed using the optical fractionator method of stereology. A, Neuronal counts and region volume estimates in the piriform cortex provided for the individual htau mouse examined, aged 8 months (n = 5) or 17 months (n = 5). CE, Coefficient of error. B, Comparison of estimated total of neurons between the two ages of htau mice revealed a dramatic reduction in the total number of neurons between 8 and 18 months, from an average of 565,000 to 242,000, a decrease of 67.2% (p < 0.009). There was also a 23% reduction (p < 0.002) in the estimated volume of the piriform cortex between these age groups.

Electron microscopy of dying htau neurons

Ultrastructural morphological criteria are often used to discern the mode of cell death, particularly with respect to nuclear morphology (for review, see Leist and Jaattela, 2001). When examined by EM, neurons in the older htau mice display morphological evidence that is not easily classified into a single cell-death category. A number of dying neurons in the htau mice displayed features of apoptosis, including nuclear breakdown, chromatin condensation, membrane blebbing, and cytoplasmic shrinkage (Fig. 4A,B). Other neurons showed chromatin condensation into dark dispersed bodies, but there was no sign of cell shrinkage or cell fragmentation (Fig. 4C,D), suggesting that these cells were not dying by a classic apoptotic cell death. Further morphological diversity was seen in neurons that displayed no prominent chromatin condensation but extensive organelle swelling and cytoplasmic vacuolization more suggestive of necrotic cell lysis (Fig. 4E,G). Interestingly, the majority of the dying neurons with nuclear abnormalities in the old htau mice did not have significant accumulations of filamentous tau (Fig. 4A-E) (Andorfer et al., 2003), whereas many neurons with tau aggregates displayed intact nuclei (Fig. 4F). Local damage, swelling, and demyelination were evident in many of the axonal processes (Fig. 4H,I).

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Figure 4.

Nuclear abnormalities in neurons in the older htau mice detected by electron microscopy suggest multiple pathways of cell death. A, B, Features of apoptosis, including nuclear breakdown, chromatin condensation, membrane blebbing, and cytoplasmic shrinkage in a 22-month-old htau mouse cortex (A) and striatum (B). Chromatin condensation into dark dispersed bodies in the absence of cell shrinkage or cell fragmentation was also detected in 22-month-old htau hippocampus (C) and cortex (D). Other neurons in the cortex (E) and thalamus (G) of htau mice displayed no prominent chromatin condensation but extensive organelle swelling and cytoplasmic vacuolization suggestive of cell lysis. Local damage, swelling, vacuolization, and demyelination were visible in axonal processes (H, I), and many of these processes contained filamentous aggregates. Neurons were also detected in the 22-month-old htau brain that had little damage (F) and displayed dispersed chromatin and intact organelles but that had accumulated aggregates. Note that the neurons that displayed nuclear breakdown did not have significant accumulations of filamentous tau (A-F). Scale bars: A-G, 2 μm; H, I, 5 μm.

Cells in htau brains show evidence of DNA fragmentation but no activation of caspases

DNA fragmentation can be detected using the TUNEL method (Gavrieli et al., 1992; Sgonc and Wick, 1994) to label broken 3′-OH ends of DNA. When we used this method on 15-month-old wild-type mouse brain paraffin sections, we did not detect any TUNEL-positive cells (Fig. 5A), whereas age-matched htau mouse brain sections were TUNEL-positive in the hippocampus (Fig. 5B) and piriform cortex (Fig. 6C). Numbers of positive cells were low, with two to three positive cells per section. TUNEL has been classically used as evidence of caspase driven apoptosis; however, TUNEL has also been detected independently of caspase activation. We therefore looked for more direct biochemical evidence of caspase activation in Western blots of a time course of htau brain homogenates (2, 5, 10, 13, 16, 17, and 20 months of age) and an aged wild-type mouse control (20 months old) using antibodies to cleaved caspase-3, -6, -7, -8, -9, and -10. Caspases become active when cleaved (Hengartner, 2000), but, consistent with the small numbers of TUNEL-positive cells detected, we found no evidence of any caspase cleavage at the gross level in the htau mice, as shown in Figure 6A for caspase-3 (cleavage fragment would appear at 17/19 kDa). Similar results were obtained for other members of the caspase family, including caspase-6, -7, -8, -9, and -10 (data not shown).

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Figure 5.

DNA fragmentation was detected in the htau mice using TUNEL on paraffin sections. No TUNEL reactivity was detected in a 15-month-old control mouse brain (A), whereas TUNEL-positive cells were detected in an age-matched htau mouse hippocampus (B) and piriform cortex (C). Numbers of positive cells were low: two to three positive cells per section in mice aged 15-18 months. Scale bars, 10 μm.

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Figure 6.

Cell death in htau mice did not occur through a classic caspase-dependent apoptotic pathway. A Western blot of brain homogenates of a time course of htau mice (ht) (2, 5, 10, 13, 16, and 17 months of age) and an old aged wild-type (wt) mouse (20 months) probed with an antibody that detects both full length and cleaved (active) forms of caspase-3 revealed no cleavage of caspase-3 (A). Active caspase-3 would be detected as a band at 17/19 kDa. Similar negative results were obtained for other members of the caspase family, including caspase-6, -7, -8, -9, and -10 (data not shown). B, Blots were also probed with SP-14 and antibody that recognizes SNAP-25 to control for loading.

Expression of cell-cycle markers in htau mice

In light of the connection that has been drawn between abnormal expression of cell-cycle molecules and cell death in human tauopathies (for review, see Vincent et al., 2003), we hypothesized that a similar reexpression of some of these regulatory molecules might also occur in the htau mice. To test this, we performed immunohistochemistry on paraffin sections from htau mouse brains and wild-type control mice aged 12, 18, and 22 months with a series of antibodies to cell-cycle molecules that have been implicated in the human disease, including PCNA, cyclin D₁, ki67, cyclin B₁, and cdc2.

We found that the htau brains were positive for the cell-cycle regulating proteins cyclin D₁, ki67, and PCNA, but brains from age-matched wild-type mice were not reactive with antibodies to these proteins (Figs. ​(Figs.7,7, ​,8).8). The amount of ki67 is normally tightly regulated (Scholzen and Gerdes, 2000), and, although its exact function in the cell cycle is not known, it has been determined to be essential for cell proliferation (Schluter et al., 1993) and is aberrantly expressed in AD (Nagy et al., 1997). Select populations of neurons in htau cortex (Fig. 7B), thalamus (Fig. 7C), and hippocampus (data not shown) were positive for expression of ki67. The appearance of ki67 in these cells suggested a loss of regulation of ki67 expression, or degradation, and a possible reentry of these normally postmitotic cells into the cell cycle. Cyclin D₁ is another cell-cycle regulating molecule that is involved in controlling the progression of the cell cycle through G₁-phase to the boundary of the S-phase (for review, see Coqueret, 2002). Although cyclin D₁ is not normally expressed in postmitotic neurons, it has been detected in AD brains (Busser et al., 1998; Yang et al., 2003). Cyclin D₁-positive cells were detected in the htau mice, shown here in the cortex (Fig. 7E) and thalamus (Fig. 7F) of a 22-month-old mouse.

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Figure 7.

The S-phase associated cell-cycle molecules ki67 and cyclin D₁ are detectable in htau neurons. Brains from age-matched wild-type mice were not reactive with antibodies to ki67 (A), but sections from htau mice showed numerous positive neurons in the cortex (B) and thalamus (C). In wild-type mice, there was no reactivity with cyclin D antibodies (D), and select populations of neurons in htau mice were positive, shown here in the cortex (E) and thalamus (F) of a 22-month-old mouse. Scale bars, 10 μm. Very similar results have been obtained in 12- and 18-month-old htau mice.

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Figure 8.

Cell cycling and DNA synthesis in the htau mouse brain was detected with antibodies to PCNA and ki67 and by incorporation of the thiamine analog BrdU. No reactivity with the PCNA antibody was detected in wild-type mice (A) shown here at 14 months, but reactivity was detected in an age-matched htau mouse brain (B). Double labeling with PCNA (C, brown) and NeuN (C, purple) (a neuronal marker) showed that both neurons and glial cells expressed PCNA. Most cells that were positive for PCNA (D, brown staining) were not positive for CP13, reactive to phospho-serine 202 on tau (D, purple staining), with some rare exceptions. Incorporation of the thiamine analog BrdU (E, F, H, I), indicating DNA synthesis, was detected in some postmitotic brain regions of 12- and 18-month-old htau mice, including the somatosensory cortex (H, brown). Double labeling with antibodies to BrdU and ki67 (H, purple) revealed some cells that were positive for either marker and other cells that were positive for both (I).

Paraffin sections of htau brains were also reactive with an antibody to PCNA (Fig. 8B). PCNA is a cell-cycle regulatory molecule that appears to play a coordinating role in a number of interactions at the DNA replication fork but has also been shown to be involved in DNA repair (for review, see Maga and Hubscher, 2003). To determine which brain cell types were reactive to the PCNA antibody, we performed double labeling (Fig. 8C) with PCNA and NeuN, a neuronal marker. With this double labeling, we noted that both neurons and glial cells expressed PCNA, shown for a 22-month-old htau brain. Additionally, to determine whether there was any colocalization between PCNA and the abnormal tau phosphorylation in the htau brains, we performed double labeling with the PCNA antibody and CP13 (reactive with tau phosphorylated at serine 202). Additionally, we noted that most cells that were positive for PCNA were not positive for CP13 (Fig. 8D).

The cell-cycle regulators that we detected in the htau mice were for the most part associated with G₁-/S-phases of the cell cycle. Normally, the cell-cycle progresses in a highly stereotyped manner beginning with entry into G₁/S in which DNA replication occurs, progressing through mitosis, and ultimately ending in cell division. For mitosis to occur, cells must progress through the G₂/M transition that is initiated after the assembly of the maturation-promoting factor, composed of cyclin B and cdc2. We did not detect these mitosis-associated molecules cyclin B₁, and Cdk1 (cdc2) in the htau brains (data not shown), suggesting that the changes in cell-cycle regulation fall short of mitosis and cell division.