Cell-cycle markers in a transgenic mouse model of human tauopathy: increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1

Abstract

Recent evidence has suggested that an abnormal reactivation of the cell cycle may precede and cause the hyperphosphorylation and filament formation of tau protein in Alzheimer's disease and other tauopathies. Here we have analyzed the expression and/or activation of proteins involved in cell-cycle progression in the brain and spinal cord of mice transgenic for mutant human P301S tau protein. This mouse line recapitulates the essential molecular and cellular features of the human tauopathies, including hyperphosphorylation and filament formation of tau protein. None of the activators and co-activators of the cell cycle tested were overexpressed or activated in 5-month-old transgenic mice when compared to controls. By contrast, the levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 were increased in brain and spinal cord of transgenic mice. Both inhibitors accumulated in the cytoplasm of nerve cells, the majority of which contained inclusions made of hyperphosphorylated tau protein. A similar staining pattern for p21Cip1 and p27Kip1 was also present in the frontal cortex from a case of FTDP-17 with the P301L tau mutation. Thus, reactivation of the cell cycle was not involved in tau hyperphos-phorylation and filament formation, consistent with expression of p21Cip1 and p27Kip1 in tangle-bearing nerve cells.

Figure 1

Levels of p21Cip1 and p27Kip1 in spinal cord and brain of human P301S tau-transgenic mice and age-matched controls. A: Immunoblotting for p21Cip1 and p27Kip1 of spinal cord (Sc) from six control (Co) and six human P301S tau-transgenic (Tg) mice. Immunoblotting with antibody AT8 was used to document the presence of hyperphosphorylated tau, and anti-β-actin antibody was used to ensure equal loading. B: Quantitative analysis of the immunoblots shown in A. The results were normalized relative to β-actin and are expressed as percentage of controls (taken as 100%) and represent the means ± SEM (n = 6). C: Immunoblotting for p21Cip1 and p27Kip1 of brain (B) from six control (Co) and six human P301S tau-transgenic (Tg) mice. D: Quantitative analysis of the immunoblots shown in C.

Figure 2

p27Kip1 and p21Cip1 immunofluorescence staining in spinal cord, brainstem, and cerebral cortex of human P301S tau-transgenic (Tg) mice and age-matched controls (C). Nerve cell bodies and processes were strongly immunoreactive for p27Kip1 in spinal cord, brainstem, and cerebral cortex of 5-month-old human P301S tau-transgenic mice. In age-matched control mice, only a small number of weakly p27Kip1-immunoreactive cells was seen. The number of p21Cip1-positive nerve cells was similar in control and transgenic mice. Whereas the staining was predominantly nuclear in control mice, p21Cip1-like immunoreactivity was both nuclear and cytoplasmic in human P301S tau-transgenic mice. Scale bar, 60 μm.

Figure 3

Double-labeling immunofluorescence staining for p27Kip1 and tau protein in spinal cord, brainstem, and cerebral cortex of human P301S tau-transgenic mice. Nerve cells in 5-month-old human P301S tau-transgenic mice were strongly immunoreactive for p27Kip1 (green) and tau protein (antibody AT100, red). Merged pictures demonstrated co-localization of p27Kip1 and hyperphosphorylated tau (yellow). Some nerve cells were singly stained for either p27Kip1 or hyperphosphorylated tau. Scale bar, 60 μm.

Figure 4

Double-labeling immunofluorescence staining for p21Cip1 and tau protein in spinal cord (Sc), brainstem (BS), and cerebral cortex (Cx) of human P301S tau-transgenic (Tg) mice and single-labeling for p21Cip1 in age-matched controls. Nerve cells in 5-month-old human P301S tau-transgenic mice were strongly immunoreactive for p21Cip1 (green) and tau protein (S422-P, red). Merged pictures demonstrated co-localization of p21Cip1 and tau (yellow). In control mice, p21Cip1 staining was concentrated in the nerve cell nucleus, with weaker cytoplasmic staining. In transgenic mice, strong p21Cip1 staining was present in both cytoplasm and nucleus. Scale bar, 60 μm.

Figure 5

Double-labeling immunofluorescence staining for p27Kip1 or p21Cip1 and tau protein in frontal cortex from a case of FTDP-17 with the P301L mutation in tau. Nerve cells and glial cells were strongly immunoreactive for p27Kip1 (green) or p21Cip1 (green) and tau protein (antibodies AT8 and S422-P, red). Merged pictures demonstrated co-localization of p27Kip1 or p21Cip1 and hyperphosphorylated tau (yellow). Some cells were only singly stained. Scale bars, 60 μm.