Distinct chronology of neuronal cell cycle re-entry and tau pathology in the 3xTg-AD mouse model and Alzheimer's disease patients

Abstract

Cell cycle re-entry in Alzheimer's disease (AD) has emerged as an important pathological mechanism in the progression of the disease. This appearance of cell cycle related proteins has been linked to tau pathology in AD, but the causal and temporal relationship between the two is not completely clear. In this study, we found that hyperphosphorylated retinoblastoma protein (ppRb), a key regulator for G1/S transition, is correlated with a late marker for hyperphosphorylation of tau but not with other early markers for tau alteration in the 3xTg-AD mouse model. However, in AD brains, ppRb can colocalize with both early and later markers for tau alterations, and can often be found singly in many degenerating neurons, indicating the distinct development of pathology between the 3xTg-AD mouse model and human AD patients. The conclusions of this study are two-fold. First, our findings clearly demonstrate the pathological link between the aberrant cell cycle re-entry and tau pathology. Second, the chronological pattern of cell cycle re-entry with tau pathology in the 3xTg-AD mouse is different compared to AD patients suggesting the distinct pathogenic mechanism between the animal AD model and human AD patients.

Keywords: Alzheimer's disease; animal model; cell cycle; retinoblastoma protein (Rb); tau; transgenic mouse.

Figure 1

With increasing age, neurons in the CA1 and subiculum express increasing levels of tau pathology, detected with MC1 (A,D,G). While no tau pathology have yet developed at 7 months of age (A–C), small numbers of tau pathology bearing neurons, detected with AT8, are found in a 14 month mouse (E), and the numbers of such neurons increase dramatically in the 18–20 month animals (H). A similar pattern to AT8 is seen using the cell cycle marker ppRb807 (C,F,I). In fact, even at the earliest age of tau pathology development, both AT8 and ppRb807 are present in all tau pathology bearing neurons (inset of boxed areas E and F show the same neurons from adjacent serial sections). Scale bar= 100 µm.

Figure 2

Double fluorescence immunostaining of the 3×Tg-AD mice find MC1 stains many neurons in a 19 month old mouse (A), with considerable, but not total overlap, with ppRb807 (B and merged image C). However, essentially all tau positive neurons contain both hyperphosphorylated tau stained with AT8 (D) and ppRb807 (E and merged image F). Scale bar= 100 µm.

Figure 3

In 3×Tg-AD mice over 18 months of age, many neurons and neurofibrillary tangles are prominent in the CA1 region of the hippocampus stained with MC1 (A). In adjacent sections of the same mouse, antibodies used to detect cell cycle proteins also stain some of the NFT including ppRb807 (B), BRCA-1 (C), and pMcm2 (D). Counterstain with hematoxylin. Scale bar= 100 µm.

Figure 4

Quantification of the large aging series of the 3×Tg-AD mice, found the percentage of neurons in the entire hippocampus expressing tau correlated significantly with age using MC1 (p<0.05). However, antibodies that detect only the neurons that contain NFT including AT8 and the cell cycle markers BRCA-1, pMcm2, and ppRb807 show variable numbers in the mice between 11 and 16 months of age, and then only increase dramatically after 18 months of age, a delayed increase relative to early tau accumulation detected by MC1.

Figure 5

Human AD serial adjacent hippocampus sections were stained with MC1 (A,C) and ppRb807 (B,D). In the CA1 (A,B), many of the same NFT contain both tau and cell cycle (arrows) and some pretangles or immature NFT with only diffuse weaker MC1 staining, still show strong ppRb807 (arrowheads). In the CA3, there are many ppRb807-positive NFT that have no increased MC1 staining (C,D). Adjacent serial sections stained for ppRb807 detected using DAB to ensure the same stained cells were visualized in both sections (E,H), were then stained using either AT8 or CP13 detected with Alexafluor 588-labelled secondary antibody. In most NFT, ppRb807 co-localized with both AT8 (F,G) and CP13 (I,J). Some neurons positive for ppRb807 did not contain hyperphosphorylated tau (asterisks, F,I).