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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2006 Mar;168(3):878–887. doi: 10.2353/ajpath.2006.050540

Cell-Cycle Markers in a Transgenic Mouse Model of Human Tauopathy

Increased Levels of Cyclin-Dependent Kinase Inhibitors p21Cip1 and p27Kip1

Patrice Delobel *, Isabelle Lavenir *, Bernardino Ghetti †, Max Holzer *, Michel Goedert *
PMCID: PMC1606514  PMID: 16507903

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.

The most common neurodegenerative diseases are characterized by the presence of abnormal filamentous protein inclusions in the brain.1 In Alzheimer’s disease (AD), these inclusions are made of hyperphosphorylated tau protein. Together with the extracellular β-amyloid deposits, they constitute the defining neuropathological characteristics of AD. Tau inclusions, in the absence of extracellular deposits, are characteristic of progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, argyrophilic grain disease, Parkinson-dementia complex of Guam, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).2,3 The identification of mutations in Tau in FTDP-17 has established that dysfunction of tau protein is central to the neurodegenerative process.4–6 In all these diseases, tau protein is hyperphosphorylated and in an abnormal filamentous form, with hyperphosphorylation at most sites appearing to precede assembly into filaments. It appears likely, therefore, that similar mechanisms lead to tau hyperphosphorylation in AD and other diseases with filamentous tau deposits. However, the mechanisms by which tau becomes hyperphosphorylated are not well understood.

Throughout the past decade, a body of work on AD has reported that cell-cycle markers are abnormally expressed in nerve cells with filamentous tau deposits. These markers include proteins involved in the G0/G1 transition, such as cyclin D and Cdk4/Cdk6; some of their substrates, such as the retinoblastoma protein; and the cyclin-dependent kinase inhibitors p15, p16, p18, and p19.7–13 Markers of the G1/S transition, such as cyclin E and Cdc25A, were also found to be abnormally expressed in degenerating nerve cells.13–15 Furthermore, regulators of the G2/M transition, such as cyclin B, Cdc2, Cdc25B, Polo kinase, Myt1/Wee1, and p27Kip1, and some mitotic epitopes, such as phosphorylated histone H3, phosphorylated RNA polymerase II, PCNA, Ki67, and MPM2, were found to co-localize with hyperphosphorylated tau protein.8,14,16–27 The expression of mitotic epitopes appeared to precede hyperphosphorylation and aggregation of tau protein, suggesting a possible cause-and-effect relationship.28,29 This was supported by studies showing AD-like phosphorylation of tau protein in mitotically active cells30–33 and the phosphorylation of recombinant tau by CDKs in vitro.34 Importantly, experimental studies have established that an inappropriate re-entry into the cell cycle results in nerve cell death. Thus, expression of SV40 T antigen in cerebellar Purkinje cells and rod photoreceptors of transgenic mice caused unscheduled cell cycle re-entry and cell death.35,36 Reactivation of the cell-cycle machinery is believed to play an important role in the apoptotic death of postmitotic neurons.

Taken together, these findings raised the possibility that an inappropriate re-entry into the cell cycle might be directly responsible for the hyperphosphorylation of tau and the neurodegeneration characteristic of AD. This may also apply to other diseases with tau deposits because an abnormal activation of cell-cycle markers has been described in progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, Parkinson-dementia complex of Guam, and FTDP-17.20

The above findings are correlational, having been obtained mostly using postmortem brain tissue from individuals with end-stage clinical disease of long duration. One way to throw fresh light on this issue may be to use experimental animal models to look at possible mechanistic links between the expression of cell-cycle markers, hyperphosphorylation of tau and neurodegeneration. In recent years, mice transgenic for human tau with missense mutations of FTDP-17 have been produced.37–44 Some of these lines replicate the essential molecular and cellular features of the human tauopathies, including hyperphosphorylation, abundant filament formation, and nerve cell death.37,41

Here we have used a line of mice transgenic for human P301S tau protein41 to study the expression of cell-cycle markers. We found no evidence for an abnormal expression or mislocalization of activators and co-activators of the cell cycle in brain and spinal cord of 5-month-old human P301S tau mice. However, two cyclin-dependent kinase (CDK) inhibitors, p21Cip1 and p27Kip1, were elevated in transgenic mouse brain and spinal cord. They accumulated in the cytoplasm of nerve cells, the majority of which contained tau inclusions. A similar staining pattern was observed in the frontal cortex from a case of FTDP-17 with the P301L tau mutation.

Materials and Methods

Animals and Antibodies

Five-month-old homozygous mice transgenic for human P301S tau protein and age-matched C57BL/6J control mice were used. We used the phosphorylation-dependent anti-tau antibodies AT8 (Innogenetics, Ghent, Belgium), AT100 (Innogenetics), and phospho-tau S422 (BioSource Europe, Nivelle, Belgium), as well as the phosphorylation-independent anti-tau antibody BR134. AT8 recognizes tau protein phosphorylated at S202 and T205, AT100 requires phosphorylation of both T212 and S214 in tau,45,46 and phospho-tau S422 recognizes tau protein phosphorylated at S422. BR134 recognizes all tau isoforms, irrespective of phosphorylation.47 In addition, a large panel of antibodies directed against cell-cycle proteins and related proteins was used (Table 1). They included antibodies to cyclins (A, B, D1, and E), CDKs 1 and 2, Cdc25A, Cdc25C, Cks1, Myt1/Wee1, Polo kinase, CDK inhibitors (p16Ink4a, p18Ink4c, p19ARF, p21Cip1, and p27Kip1), ORC1, Aurora2, MPM2, phospho-histone H3, mitotic cells, and the retinoblastoma (Rb) protein.

Table 1.

Antibodies Used

Antibodies Dilutions used for immunohistochemistry Dilutions used for immunoblots Source
Anti-Tau
 BR134 1/500 1/5000 M. Goedert
 AT8 1/500 1/1000 Innogenetics
 AT100 1/500 1/1000 Innogenetics
 Phospho Tau (Ser422) 1/500 1/1000 Biosource
Anti-CDKs
 Cdk2 1/100 1/200 H-298/Santa Cruz (Santa Cruz, CA)
 Phospho-Cdk2 (Thr160) 1/50 1/100 Santa Cruz
 Cdk1 (Cdc2) 1/250 1/1000 Upstate Biotechnology (Dundee, UK)
 Phospho-Cdk1 (Thr161) 1/200 1/1000 Cell Signaling (Danvers, MA)
Anti-Cyclins
 Cyclin A 1/100 1/200 H-432/Santa Cruz
1/100 1/200 ab7956/Abcam (Cambridge, UK)
 Cyclin B 1/100 1/200 H-433/Santa Cruz
1/250 1/2000 Cell Signaling
 Cyclin D1 1/70 1/200 H-295/Santa Cruz
 Cyclin E 1/70 1/200 M20/Santa Cruz
Anti-Cdk activators and inhibitors
 Cdc25 A 1/50 1/200 M-191/Santa Cruz
 Cdc25 C 1/50 1/200 TC-15/Upstate Biotechnology
 Phospho-Cdc25 C (Thr48) 1/100 1/1000 Cell Signaling
 CKs1 p9 1/50 n.d. FL-79/Santa Cruz
 Myt1/Wee1 1/200 1/1000 Cell Signaling
 Polo kinase (Plk) 1/200 1/1000 NT/Upstate Biotechnology
 p16 Ink4 1/50 n.d. M-156/Santa Cruz
 p18 Ink4 1/50 n.d. N-20/Santa Cruz
 p19 ARF 1/50 n.d. M-20/Santa Cruz
 p21 Cip1 1/50 1/200 F-5/Santa Cruz
1/50 1/200 Waf1 (Ab4)/Oncogene Research Products, (San Diego, CA)
 p27 Kip1 1/100 1/1000 Cell Signaling
1/50 1/200 DCS-72/Sigma-Aldrich (Poole, UK)
Other markers
 Anti-ATM 1/70 1/200 Sc-23922/Santa Cruz
 Anti-ORC1 1/50 n.d. 7F6/1/Neomarkers (Fremont, CA)
 Anti-Aurora 2 1/100 n.d. Cell Signaling
 Anti-MPM2 1/50 n.d. Upstate Biotechnology
 Anti-Phospho-histone H3 (Ser10) 1/50 n.d. Cell Signaling
 Anti-mitotic cells 1/100 n.d. ab8956/Abcam
 Anti-Rb 1/50 1/200 M153/Santa Cruz
 Anti-Phospho-Rb (Ser795) 1/100 1/1000 Cell Signaling
 Anti-β actin n.d. 1/5000 Sigma Clone AC-74
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n.d., not determined. 

Immunofluorescence

Mice were perfused transcardially with 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. Brains and spinal cords were removed, postfixed overnight at 4°C, and cryoprotected in 30% sucrose in 0.1 mol/L phosphate buffer for 24 hours. Sagittal brain sections and transverse spinal cord sections (30 μm) were cut on a Leica SM2400 microtome (Leica Microsystems, Bucks, UK) and stored at 4°C in 0.1 mol/L phosphate buffer containing 0.1% sodium azide. For single and double staining, sections were permeabilized and blocked in 0.1 mol/L phosphate buffer containing 3% bovine serum albumin and 0.1% Triton X-100 (blocking solution). This was followed by an overnight incubation at 4°C with the primary antibodies in blocking solution. After washing, the sections were incubated for 3 hours at room temperature in Alexa Fluor 488 (Molecular Probes, Leiden, The Netherlands) or Cy5 (Abcam, Cambridge, UK) secondary antibodies in blocking solution. After 4,6-diamindino-2-phenylindole staining, the sections were mounted using Vectashield (Vector Laboratories, Burlingame, CA). In control experiments, filamentous, sarkosyl-insoluble tau was extracted from human P301S tau-transgenic mouse brain and the tau filaments solubilized as described.48,49 The dialyzed material (50 μl) was incubated overnight at 4°C with antibodies directed against cell-cycle proteins or anti-tau antibody AT8 before addition to tissue sections. In additional control experiments, 1 to 10 μg of the p27Kip1 peptide antigen EQTPKKPGLRRQT (corresponding to amino acids 185 to 197 of mouse p27Kip1) was incubated overnight at 4°C with the p27 antibodies (1:100), followed by addition to tissue sections.

Paraffin-embedded sections (10 μm) of formalin-fixed brain tissue from the frontal cortex of a case of FTDP-17 with the P301L tau mutation50 were also used. Sections were deparaffinized and rehydrated. Antigen retrieval was performed by incubating the sections in 10 mmol/L sodium citrate buffer, pH 6, for 15 minutes in a microwave oven. Endogenous peroxidase activity was quenched using a 90-minute incubation in 20% methanol and 3% hydrogen peroxide. After permeabilization and blocking in Tris-buffered saline-0.2% Tween (TBST), the sections were incubated overnight at 4°C with the primary antibody in blocking solution. After washing, the sections were incubated for 2 hours at room temperature with the biotinylated secondary antibody in TBST containing 5% goat serum. After washing, the sections were incubated with streptavidin-Alexa 568 (Molecular Probes). The sections were then incubated overnight at 4°C with the second primary antibody in TBST containing 5% goat serum. The signal was revealed using the tyramide signal amplification kit (Molecular Probes). After 4,6-diamindino-2-phenylindole staining, sections were mounted using Vectashield.

Immunoblot Analysis

Brains and spinal cords of 5-month-old human P301S tau-transgenic mice and age-matched controls were homogenized in 3 vol of cold extraction buffer (25 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 5 mmol/L sodium pyrophosphate, 10 mmol/L β-glycerophosphate, 30 mmol/L sodium fluoride, 2 mmol/L sodium vanadate, 1 mmol/L phenylmethyl sulfonyl fluoride, and 10 μg/ml leupeptin, aprotinin, and pepstatin). The homogenates were spun at 80,000 × g for 15 minutes and the supernatants used for biochemical analysis. Protein concentrations were determined using the BCA kit (Pierce, Rockford, IL), and 10 μg of protein were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were blotted overnight at 4°C onto a polyvinylidene difluoride membrane (Pierce). Membranes were blocked for 30 minutes at room temperature in 0.1 mol/L phosphate buffer, pH 7.4, containing 5% milk and 0.1% Tween 20 (blocking buffer) and incubated overnight at 4°C with the primary antibodies in blocking buffer. The membranes were then washed in 0.1 mol/L phosphate buffer, pH 7.4, containing 0.1% Tween 20 and incubated for 45 minutes at room temperature in peroxidase-conjugated secondary antibody (Pierce) in blocking buffer. After washing, the blots were developed using enhanced chemiluminescence (Amersham Biosciences, Arlington Heights, IL).

Results

Analysis of Cell-Cycle Protein Expression

The expression and/or activity of proteins involved in cell-cycle regulation was analyzed in 5-month-old P301S tau-transgenic mice and age-matched controls (Table 2). Immunoblot and immunofluorescence studies showed that none of the activators or co-activators of the cell cycle tested were overexpressed or activated in brain and spinal cord of transgenic mice. Indeed, with the exception of antibodies directed against cyclin D1 and phospho-Rb, which stained the nuclei of a few cells in the spinal cord of transgenic mice, none of the proteins analyzed exhibited a different pattern of expression or activity in control and transgenic mice. This was true of cyclins, CDKs and their co-activators, as well as of substrates of CDKs and a number of their inhibitors and co-inhibitors. Similarly, antibodies specific for other markers of mitotic phosphorylation, such as ATM, MPM2, phospho-histone H3, and a nonspecific mitosis epitope, gave a similar staining pattern in control and transgenic mice. In parallel, we used several of the above antibodies [directed against CDK1, Cdc25A, cyclin B, cyclin D, and the retinoblastoma (Rb) protein] on sections of the temporal cortex from two cases of AD. As expected, we observed expression of these cell-cycle markers in many nerve cells with tau inclusions (not shown).

Table 2-6763.

Cell Cycle Markers in Human P301S Tau-Transgenic (Tg) Mice and Age-Matched Controls

Protein Effect on cell cycle G0/G1 progression Cellular location (Immunofluorescence)
Expression (Immunoblotting)
Controls Tg Controls Tg
Cyclin D1  Cdk4 Activator + + + +
G1/S progression
Cyclin E  Early S progression + + + +
Cyclin A  S/G2 transition + + + +
Cdk2  Activator + + + +
Phospho-Cdk2 (Thr160)  Activator (Active form) − − + +
Cdc25 A  Cdk2 and Cdc2 activator + + + +
G2/M progression
Cyclin B  Activator of Cdc2 + + + +
Cdk1  G2/M transition + + + +
Phospho-Cdk1 (Thr161)  Active form − − − −
Cdc25 C  Cdk1 Activator + + + +
Phospho-Cdc25 C (Thr48)  Cdk1 Activator + + + +
Myt1/Wee1  Cdc25 Inhibitor + + + +
Polo kinase  Cdc25 Activator + + + +
Cks1 p9  Cdk Activator − − n.d.
Cdk inhibitor
P16 Ink4a  G1/S (Cdk4/6) − − n.d.
P18 Ink4c  G1/S (Cdk4/6) − − n.d.
P19 ARF  G1/S (Cdk4/6) − − n.d.
p21 Cip1  Cdk inhibitor ++ ++ ++ +++
p27 Kip1  Cdk inhibitor + +++ + +++
Other markers
ATM  DNA damage + + + +
ORC1  S progression − − n.d.
Aurora2  Mitotic activator − − n.d.
MPM2  Mitotic epitope − − n.d.
Phospho-histone H3 (Ser10)  Mitotic indicator + + n.d.
Mitotic cell antibody  Mitotic indicator − − n.d.
Rb  Transcription factor + + + +
Phospho-Rb (Ser795)  Transcription factor + + + +
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−, no signal. 

+, medium signal. 

++/+++, strong signal. 

n.d., not determined. 

Expression and Cellular Localization of p21Cip1 and p27Kip1

Immunoblotting of brain and spinal cord extracts showed that p21Cip1 was expressed at approximately twofold higher levels and p27Kip1 at fourfold higher levels in 5-month-old human P301S tau-transgenic mice than in controls (Table 2, Figure 1). Immunoblotting with an anti-actin antibody was used to ensure equal loading. Sarkosyl-insoluble tau-rich fractions from the P301S tau-transgenic mice were not immunoreactive for p21Cip1 or p27Kip1 (data not shown). By immunohistochemistry, strongly p27Kip1-immunoreactive nerve cells were seen in transgenic mice, particularly in brainstem and spinal cord (Table 2, Figure 2). The same was also true of the cerebral cortex, with staining present in the cytoplasm. In age-matched controls, by contrast, antibodies directed against p27Kip1 only weakly labeled a few nerve cells and their processes. Identical results were obtained with both p27Kip1 antibodies. Moreover, p27Kip1 staining was completely abolished after preincubation of the primary antibody with the peptide antigen. It was not changed after preincubation of the p27Kip1 antibody with hyperphosphorylated tau protein extracted from the brain of human P301S tau-transgenic mice. Preincubation with the latter completely abolished staining with anti-tau antibody AT100.

Figure 1.

Figure 1

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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.

Figure 2

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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.

Similar numbers of nerve cells were immunoreactive for p21 Cip1 in brain and spinal cord from control and transgenic mice. However, the staining within nerve cells was different between the two groups. In control mice, p21Cip1-like immunoreactivity was present in the nucleus, with only weak cytoplasmic staining. By contrast, in transgenic mice, p21Cip1 staining was present in both nucleus and cytoplasm. Identical results were obtained with both p21Cip1 antibodies. Staining for p21Cip1 was not changed after preincubation of the p21Cip1 antibody with hyperphosphorylated tau protein extracted from the brain of human P301S tau-transgenic mice.

Co-Localization of p21Cip1 and p27Kip1 with Tau Protein

Double immunofluorescence was used to investigate the co-localization of p21Cip1 and p27Kip1 with tau protein in cerebral cortex, brainstem, and spinal cord of 5-month-old human P301S tau-transgenic mice. As shown in Figure 3, there was extensive co-localization of p27Kip1 staining with staining for anti-tau antibody AT100. In the spinal cord, ∼60% of cells immunoreactive for AT100 were also p27 Kip1-positive. A similar level of co-localization was observed when the different regions of the brainstem (autonomic, motor, tectal, and tegmental) were analyzed. Extensive co-localization of staining for p21Cip1 and phospho-tau S422 was also observed (Figure 4). Strong cytoplasmic staining for p21Cip1 was only observed in cells with an intracytoplasmic accumulation of tau protein. Similar findings were obtained in cerebral cortex and brainstem of transgenic mice. This contrasted with control mice in which p21Cip1 staining was concentrated in the nerve cell nucleus (Figure 4).

Figure 3.

Figure 3

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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.

Figure 4

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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.

Cellular Localization of p21Cip1 and p27Kip1 in a Case of FTDP-17 with the P301L Tau Mutation

The P301L tau mutation is known to cause the formation of intraneuronal and intraglial tau deposits.50 We found strongly p27Kip1-immunoreactive cells present in the frontal cortex from a human case of FTDP-17 with the P301L tau mutation. Based on size, the cells probably corresponded to nerve and glial cells. Double immunofluorescence was used to investigate the co-localization of p27Kip1 and tau protein. As shown in Figure 5, there was co-localization of p27Kip1 staining with staining for anti-tau antibody AT8. It is of note that staining for p27Kip1 was stronger in cells that were also AT8-positive than in singly labeled cells. In the frontal cortex of the P301L tau case, a number of nerve cells and glial cells was immunoreactive for p21Cip1. Cells immunoreactive for phospho-tau S422 were also p21Cip1-positive, with strong cytoplasmic staining of both markers (Figure 5).

Figure 5.

Figure 5

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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.

Discussion

We have analyzed the expression and cellular localization of proteins involved in cell-cycle progression in a mouse line transgenic for human P301S tau protein. None of the activators and co-activators of the cell cycle tested were overexpressed in 5-month-old transgenic mice when compared to controls. By contrast, the levels of CDK inhibitors p21Cip1 and p27Kip1 were increased in brain and spinal cord from transgenic mice. Both inhibitors accumulated in the cytoplasm of nerve cells, the majority of which contained inclusions made of hyperphosphorylated tau protein. Staining for p21Cip1 was also present in the nerve cell nucleus. To relate these findings to FTDP-17, we stained sections of frontal cortex from a human case with the P301L mutation in tau (brain tissue from human cases with the P301S mutation was not available to us) for p21Cip1 and p27Kip1. Consistent with the findings in the transgenic mice, strong staining for both proteins was observed in the human FTDP-17 case.

p21Cip1 and p27Kip1 are related proteins that inhibit cell-cycle progression by interacting with cyclin-CDK complexes in the nucleus.51–56 The presence of nuclear p21Cip1 in the human P301S tau-transgenic mice may thus be indicative of inhibition of cyclin-CDK activity. It is in line with our finding that activators and co-activators of the cell cycle were not overexpressed in these mice. Besides the nuclear localization of p21Cip1, both p21Cip1- and p27Kip1-like immunoreactivities were also observed in the cytoplasm of nerve cells with tau inclusions. Recent findings have shown that cytoplasmic Cip/Kip proteins have functions independent of cyclin-CDK inhibition.57,58 Cytoplasmic p21Cip1 has been reported to interact with stress-activated protein kinases and ASK1 kinase and to inhibit their catalytic activities, thus preventing apoptosis.59,60 p21Cip1 has also been shown to inhibit activation of caspase 3 and to resist Fas-mediated cell death.61 In the human P301S tau mice, nerve cell death occurs through a nonapoptotic mechanism,41 suggesting a possible link with the cytoplasmic expression of p21Cip1. Nonapoptotic nerve cell loss has also been described in cases of FTDP-1762 and in nerve cells of mice transgenic for human P301L tau.63,64 In addition to inhibiting apoptosis, cytoplasmic Cip/Kip proteins modulate actin dynamics by direct regulation of the RhoA pathway.65,66 It remains to be seen whether the actin cytoskeleton is abnormal in the human P301S tau-transgenic mice.

The mechanisms underlying increased expression and mislocalization of p21Cip1 and p27Kip1 in the human P301S tau-transgenic mice are unknown. A variety of anti-mitotic signals, such as cyclic AMP, rapamycin, transforming growth factor-β and glucocorticoids, have been shown to induce the expression of p21Cip1 and/or p27Kip1 in nonneuronal cells.67–69 Furthermore, in hippocampal granule cells of the rat, chronic stress paradigms have been shown to increase the expression of p27Kip1.70 It is conceivable that the aggregation of human P301S tau in affected nerve cells imposes a chronic stress that results in the overexpression and mislocalization of p21Cip1 and p27Kip1.

Little is known about the expression levels of p21Cip1 and p27Kip1 in human diseases with tau deposits. In AD, one study reported no change in the expression of p21Cip1 and p27Kip17 whereas a second study described a fourfold increase in p27Kip1 by Western blotting and co-localization with hyperphosphorylated tau in the cytoplasm of nerve cells.26 This stands in contrast to the extensive evidence showing re-expression of cell-cycle markers in nerve cells with tau inclusions in AD.7–29 Unlike FTDP-17 and the human P301S tau-transgenic mice, AD is characterized by two types of filamentous deposits made of Aβ and tau. It is therefore conceivable that the re-expression of cell-cycle markers in nerve cells in AD is related to the presence of Aβ deposits.

It has been reported that cell-cycle proteins are required for Aβ-induced nerve cell death in vitro.71 However, the re-expression of mitotic markers has also been described in neurodegenerative diseases that are characterized by the presence of filamentous tau inclusions and the lack of Aβ deposits.20 They include progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, Parkinsonism-dementia complex of Guam, and cases of FTDP-17 with the V337M mutation in Tau. It was concluded that human diseases with tau deposits are consistently associated with cell-cycle alterations and that the formation of an active mitotic kinase complex may be a necessary event upstream of tau hyperphosphorylation and filament formation. Furthermore, a recent study using a transgenic mouse model of tauopathy based on the expression of all six human brain tau isoforms in the absence of endogenous mouse tau has shown abnormal expression of a number of cell-cycle markers.72

The present findings show that hyperphosphorylation and filament formation of tau do not require the re-expression of cell-cycle markers in a mouse model of human tauopathy. The same residues are hyperphosphorylated in human diseases with tau deposits and in the human P301S tau-transgenic mice,41 suggesting that common mechanisms are involved. Phosphorylation at most of these sites precedes filament formation in the transgenic mice, consistent with a role upstream of filament formation. The protein kinases and/or protein phosphatases responsible for the hyperphosphorylation of soluble tau in the human P301S tau-transgenic mice are not known. The present findings show that CDKs and their associated cyclins are not involved, despite their abnormal re-expression in AD and other tauopathies. In the human diseases, the expression of cell-cycle markers in tangle-bearing nerve cells may therefore be unrelated to the events that trigger the hyperphosphorylation and filament formation of tau protein.

Acknowledgments

We thank T. Ignjatovic and M.G. Spillantini for assistance with immunofluorescence and K. Virdee for helpful comments.

Footnotes

Address reprint requests to M. Goedert, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. E-mail: mg@mrc-lmb.cam.ac.uk.

Supported by the Fédération pour la Recherche Médicale (fellowship to P.D.), The European Molecular Biology Organization (fellowship to P.D.), the European Commission (Marie Curie fellowship QLK6-1999-51519 to M.H.), the UK Medical Research Council, and United States Public Health Service grant P30AG10133.

Present address of M.H.: Paul Flechsig Institute of Brain Research, University of Leipzig, Leipzig, Germany.

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