Ectopic cell cycle events (CCEs) in postmitotic neurons link the neurodegenerative process in human Alzheimer's disease (AD) with the brain phenotype of transgenic mouse models with known familial AD genes. Most reports on the mouse models use the appearance of brain amyloid pathology as a key outcome measure. In the current paper, we focus on the induction of neurodegeneration using CCEs as markers for impending neuronal loss. We compare 5 mouse models of familial AD for the appearance of CCEs in subcortical regions—deep cerebellar nuclei, amygdala, locus coeruleus, hippocampus, and dorsal raphe. We find that the models differ in their CCE involvement as well as in the appearance of phosphorylated tau and amyloid deposition, suggesting that each model represents a different disease phenotype. Comparison with the pattern of neuron death in human AD suggests that each may represent a distinctly different disease model when used in preclinical trials.
Alzheimer’s disease (AD) is a devastating late-onset neurodegenerative condition that affects many regions of the human brain. Although the most obvious disease symptoms involve the inability to form and store new memories, the neurological and psychiatric description of an individual with AD includes a wide range of symptoms such as depression, apathy, episodic behavioral outbursts, deteriorating executive functioning, and others. The biological substrates of these symptoms are only partially understood, but imaging and neuropathological studies have revealed important facets of their diverse and distributed nature. There is a clear loss of volume and pathologically visible degeneration in the brain’s memory centers, which include the entorhinal cortex, hippocampus, and basal forebrain nucleus. But there are also functional and structural abnormalities found in the locus coeruleus, dorsal raphe, cingulate gyrus, amygdala and prefrontal cortex as well as other cortical and subcortical regions [
Recent work from our laboratory and many others has explored the use of abnormal neuronal cell cycle processes as an additional pathological marker of disease [
Significantly, these unexpected attempts by neurons to reenter a cell cycle provide one of the few homologies observed between mouse models of AD and the pathogenesis of the human condition. A number of different AD models have been created, most of which rely on transgenes encoding the gene for
We have elected to focus on the pattern of neurodegeneration in APP transgenic mice in order to expand the characterization of this group of AD models, and we have used CCEs as outcome measures. Previously, where they have been studied in depth, the appearance of CCEs in many human disease models show an age-dependent increase in prevalence that often closely mimics the pattern of neuronal cell death in the human disease. For example, there is a significant correlation between the regional pattern of cell loss in human ataxia-telangiectasia and its mouse model [
Five transgenic mouse models of familial AD were used in the current studies. In each model, amyloid plaques induced by APP develop at different ages. Detailed information about the mouse strains is summarized in Table
Transgenic mouse lines used in this study.
Transgenic lines | Strain | Approach | Mutation | Promoter | A | Age of analysis (this study) |
---|---|---|---|---|---|---|
R1.40 | B6.129-Tg (APPSw) 40Btla/J | YAC Genomic | Swedish APP | Human APP | 14 months | 1 yr; 2 yrs |
Tg2576 | B6 SJL-Tg (APPsw) | cDNA (695) | Swedish APP | Hamster PrP | 9–12 months | 9–11 months |
Tg6799 (5xTg) | B6SJL-Tg (APPSwFlLon, PSEN1*M146L*L286V) 6799Vas/J | Pronuclear coinjection : APP and PS1 transgenes | Swedish, Florida and London APP & human PS1 | Murine Thy-1 | 3 months | 6 months |
APP8.9 | B6.129S2-Tg (APP) 8.9Btla/J | YAC Genomic | Wt huAPP | Human APP | N/A; same as mouse APP expression | 14 months |
APP/PS1 | B6.CgTg (APPswe, PSEN1dE9) 85Dbo/J | cDNA | Swedish/human PS1 | Murine PrP | 6 months | 6-7 months |
Animals were anesthetized with Avertin (0.02 cc/g body weight) and perfused through the heart with 0.1 M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M PBS solution. The brain was immediately removed from the skull and transferred to 4% paraformaldehyde at 4°C overnight. The brains were then cryoprotected by sinking in 30% sucrose in 0.1 M PBS at 4°C overnight. After cutting along the midline, the brains were embedded in OCT compound (Tissue-Tek). Cryostat sections were cut at 10 microns and air-dried on Superfrost/Plus glass slides overnight.
For hematoxylin staining, sections were washed in PBS with 0.25% Tween-20 and rinsed in PBS. They were exposed to hematoxylin for 45–60 sec then washed with double distilled water until clear. Sections were dehydrated through graded ethanol and two washes of xylene. VectaMount was used to mount slides.
A rabbit monoclonal antibody (Abcam, Cambridge, UK) to proliferating cell nuclear antigen (PCNA) was diluted 1 : 3000 in 10% goat serum/PBS blocking buffer before use. A rabbit polyclonal cyclin A antibody (Abcam, Cambridge, UK) was used at a working dilution of 1 : 500. The beta amyloid, 1–16 (6E10) mouse monoclonal antibody (Covance, Princeton, NJ), was used at a working dilution of 1 : 3000. The anti-PHF-tau antibody clone AT8 mouse monoclonal antibody (Thermo Scientific, Rockoff, IL) was used at a working dilution of 1 : 1000.
To perform fluorescent immunohistochemistry, sections were first rinsed twice in PBS, followed by pretreatment in Antigen Unmask Solution (Vector Laboratories, Burlingame, CA; working dilution 1 : 100) for 4-5 min at 95°C. After the slides had cooled in buffer for 10–20 min at room temperature, they were rinsed twice in double distilled H2O. For DAB staining, slides were subjected to an additional pretreatment step: 0.3% hydrogen peroxide in double distilled water for 30 min to remove endogenous peroxidase activity. Slides were then rinsed again in double distilled H2O followed by PBS. Subsequently, all sections were washed with PBS and incubated for 1 h at room temperature in 10% goat serum and 0.25% Tween-20 in PBS to block nonspecific binding. All primary antibodies, diluted in PBS containing 0.25% Tween-20 and 10% goat serum, were applied to sections and then incubated overnight at 4°C. After rinsing in three washes of PBS, they were incubated for 1 h with a secondary antibody, which was conjugated with fluorescent Alexa dyes (dilution, 1 : 500). The sections were rinsed with another three washes of PBS. Antifade with DAPI was applied before sealing the sections under a glass coverslip. For DAB staining, secondary antibody (1 : 500 dilution) was applied for 1 h at room temperature, washed three times in PBS, and incubated in Vectastain ABC Elite reagent (Vector Laboratories, Burlingame, CA) for thirty minutes. After three more PBS washes, slides were developed using diaminobenzidine (DAB), following the manufacturer’s protocol. Sections were dehydrated through double distilled water, graded ethanol, and washed twice in xylene. All sections were mounted in VectaMount under a glass coverslip. Control sections were subjected to the same staining procedure, except that primary antibody was omitted. Positive controls were obtained using cerebellar cortex of 15-day-old wild-type mice.
To analyze the immunocytochemical data in a more quantitative fashion, we developed a rating scale to rate the cell cycle, AT-8, and 6E10 markers. Scores were based only for their expression in the cell body of neurons. The values assigned to the rating scale were 0 (no staining or very little staining of cell cycle events), 1 (a few staining of cell cycle events 5–15%), 2 (low staining of cell cycle events, 15–30%), 3 (moderate staining of cell cycle events 30–50%), and 4 (>50% cell cycle events). Neuronal density was examined using hematoxylin staining.
We chose to study five different mouse models. The
We used both DAB and fluorescent immunostaining of proliferating cell nuclear antigen (PCNA) to study the regional variations of CCEs in dorsal raphe, hippocampus, cerebellum, pons, amygdala, and locus coeruleus. PCNA is known to play an essential role in positioning the DNA polymerase in replication and repair of DNA [
Comparison among the mouse lines studied with respect to the presence of cell cycle events (PCNA), tau-phosphorylation (AT8), and beta-amyloid plaque deposition (6E10) in the locus coeruleus. (a) Sagittal section of a wild-type mouse brain indicating the approximate location of the locus coeruleus. (b) PCNA-positive neurons are illustrated by their appearance in this representative section from the APP8.9 mouse model. (c) Minor PCNA immunostaining is seen in wild-type mice as illustrated in this representative micrograph. (d) Quantification of the extent of immunostaining for the cell cycle, phospho-tau, and beta-amyloid plaques in the five transgenic models plus wild type.
The results from the dorsal raphe are shown in Figure
Comparison among the mouse lines studied with respect to the presence of cell cycle events (PCNA), tau-phosphorylation (AT8), and beta-amyloid plaque deposition (6E10) in the dorsal raphe. (a) Sagittal section of a wild-type mouse brain indicating the approximate location of the raphe in our preparations. (b) PCNA-positive neurons are illustrated by their appearance in this representative section from the R1.40 mouse model. (c) Minor PCNA immunostaining is seen in wild-type mice as illustrated in this representative micrograph. The insets in both (b) and (c) are representative fields at higher magnification to illustrate the qualities of the cell cycle staining. (d) Quantification of the extent of immunostaining.
Our analysis of the amygdala was performed in sagittal sections, which makes the reliable identification of the specific subnuclei more difficult. To address this we divided the structure into three subregions as illustrated by the black boxes in Figure
Comparison among the mouse lines studied with respect to the presence of cell cycle events (PCNA), tau-phosphorylation (AT8), and beta-amyloid plaque deposition (6E10) in the amygdala. (a) Sagittal section of a wild-type mouse brain indicating the approximate location of the amygdala. The three black boxes indicate the regions identified as anterior (left), middle (center) and posterior (right). (b)–(g) Representative fields illustrating the involvement of cell cycle processes in the three regions. The insets are representative fields shown at higher magnification to illustrate the qualities of the cell cycle staining. Anterior amygdala for R1.40 (b) and wild-type (e) mice contains the lowest density of CCEs. Middle regions of the amygdala in R1.40 (c) and wild-type (f) mice show increased staining in most models. Posterior amygdala in R1.40 (d) but not wild type (g) animals also show cell cycle activity. (h) Quantification of the extent of immunostaining for the cell cycle, phospho-tau, and beta-amyloid plaques in the five transgenic models plus wild type.
All other regions of the CNS were substantially negative for cell cycle protein expression, as expected. The one exception to this was a small region of the ventral brainstem. In the pons and the more dorsal nucleus reticulari tegmentis pontis (NRTP), significant cell cycle protein expression was identified for all animals. Wild-type animals showed moderate levels of CCEs, with similar levels in the APP8.9 and Tg2576 APP/PS1 and Tg6799. The only exception to this pattern was found in the R1.40 model, which had low levels of CCEs in this region. The R1.40 showed high pontine levels of hyperphosphorylated tau, but low levels of 6E10-positive beta amyloid deposits. Tg6799 exhibited a very high level of 6E10 staining—at greater than 50%, the highest score of any of the models.
In addition to these results, there were observations of cell cycle staining that were more unexpected. For example, the deep nuclei of the cerebellum showed strong levels of CCEs in almost all the models (Figure
Comparison among the mouse lines studied with respect to the presence of cell cycle events (PCNA), tau-phosphorylation (AT8), and beta-amyloid plaque deposition (6E10) in the deep cerebellar nuclei. (a) Sagittal section of a wild-type mouse brain indicating the approximate location of the deep nuclei. (b) PCNA-positive neurons are illustrated by their appearance in this representative section from the Tg2576 mouse model. (c) Curiously, some PCNA immunostaining is also seen in wild-type mice as illustrated in this representative micrograph. The insets in both (b) and (c) are representative fields shown at higher magnification to illustrate the qualities of the cell cycle staining. (d) Quantification of the extent of immunostaining for the cell cycle, phospho-tau, and beta-amyloid plaques in the five transgenic models plus wild type.
The most unexpected finding, however, was in the hippocampal formation. CCEs have been reported in this region for two mouse models of AD, the PDAPP mouse and the R1.40 model [
Comparison among the mouse lines studied with respect to the presence of cell cycle events (PCNA), tau-phosphorylation (AT8), and beta-amyloid plaque deposition (6E10) in the hippocampus. (a) Sagittal section of a wild-type mouse brain indicating the approximate location of the areas illustrated in (b) and (c). (b) PCNA-positive neurons are illustrated by their appearance in this representative section from the R1.40 mouse model. Note the involvement of a subset of the dentate granule cells as well as a few CA4 pyramidal neurons (arrows). (c) Minor PCNA immunostaining is seen in wild-type mice. The insets in panels (b) and (c) represent higher magnification of the CA2 region of their respective mouse model. (d) Quantification of the extent of immunostaining for the cell cycle, phospho-tau, and beta-amyloid plaques in the five transgenic models plus wild type. Note that only the R1.40 model showed cell cycle protein expression at the ages we examined.
Transgenic mouse models have long been used to study the molecular mechanisms of disease. To be considered useful, such models must recapitulate the human disease in as many ways as possible, and by this criterion the mouse models of AD have been at least partially successful. No wild-type mouse has been reported to naturally develop amyloid plaques, neurofibrillary tangles, or an Alzheimer’s-like loss of neurons in any brain region. By contrast, mouse lines expressing
One of these concerns that we attempt to address here is the poor reproduction in the mouse of the neuronal cell death found in AD. In human AD, there is a massive degeneration of neurons and this is observed in a pattern with pronounced temporal and regional variability [
To the appearance of cell cycle events, we add the correlation with more traditional neuropathological indicators of Alzheimer’s disease. Our findings show that
In this study, we have expanded the range of biological responses in the 5 different AD mouse models to include cell cycle events (CCEs) as direct cell-autonomous indices of neuronal distress. Since CCEs have been observed in both the human AD brain and in the analogous regions of certain AD mouse models, characterizing CCE expression patterns provides a logical and independent outcome measure for the study of neuronal death process in human AD. It is significant, therefore, that the results reported here demonstrate clear differences among the mouse models examined in the pattern of CCE expression. We find a high level of CCEs in the brain stem of all 5 models, including the pons, locus coeruleus, dorsal raphe, and deep nuclei of the cerebellum. The consistent appearance of CCEs in these more caudal regions of the CNS despite differing transgene properties and
The pattern of CCEs in the amygdala suggests internal variations in AD pathology in this structure such as is seen in the cortex. In several AD models, the posterior and central amygdaloid nuclei showed stronger levels of CCE expression when compared to more anterior amygdaloid neurons, suggesting a common impact of APP transgene expression in this region. This variation is not apparent in the 6E10 or AT8 staining suggesting that specific neuroanatomical phenotypes may be uncovered when CCE markers are used. Our findings in the AD mouse models are consistent with the well-documented involvement of the structure in human AD pathology [
The R1.40 model showed the strongest staining for CCEs in most subcortical regions and was the only model in the current study to show significant CCE expression in the hippocampal region. The absence of CCE expression in the 4 other AD models we studied is noteworthy, albeit without explanation. Nearly all of the models we studied have been shown to have deficits in behavioral tasks that are known to involve the hippocampus. This discordance between function and pyramidal cell body neuropathology suggests several hypotheses, none of which are mutually exclusive. Perhaps the behavioral changes are due to synaptic loss or atrophy [
All of these alternatives are consistent with the proposal that Alzheimer’s begins as a synaptic disease. What is unknown at present is whether these synaptic problems precede the CCEs. This would appear to be the situation in hippocampus where slices, isolated in vitro, show impaired LTP [
Although no single mouse model provides a complete recapitulation of human AD, based on the regions we examined, the YAC R1.40 would appear to be the most reliable model, especially when using CCEs as an outcome measure. One possible explanation for R1.40’s strong fidelity as a model is the close reproduction of the pattern of transgenic
We have shown here that CCE markers are a reasonable way of studying AD mouse model fidelity to human AD. Since no transgenic mouse model is able to perfectly capture the complexities of the human AD pathology, using several phenotypic markers to study the effects of transgene insertions is well advised. Distinguishing the role of species differences and the effects of transgenes in AD pathogenesis through rigorous characterization of mouse models and AD will be important to uncovering the mechanisms of AD pathogenesis and lead to the more rapid identification of useful therapeutic targets.
L. Li and T. Cheung contributed equally to the work.