Based on size, the cells probably corresponded to nerve and glial cells. 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 Alzheimers disease (AD), these inclusions are made of hyperphosphorylated tau protein. Together with the extracellular -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, Picks disease, argyrophilic grain disease, Naltrexone HCl Parkinson-dementia complex of Guam, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).2,3 The identification of mutations in in FTDP-17 has established that dysfunction of tau protein is central to the neurodegenerative process.4C6 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 Rabbit polyclonal to INSL3 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 Naltrexone HCl 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 p19.7C13 Markers of the G1/S transition, such as cyclin E and Cdc25A, were also found to be abnormally expressed in degenerating nerve cells.13C15 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.8,14,16C27 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 cells30C33 and the phosphorylation of recombinant tau by CDKs for 15 minutes and the supernatants used for biochemical analysis. Protein concentrations were decided using the BCA kit (Pierce, Rockford, IL), Naltrexone HCl and 10 g of protein were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were blotted overnight at 4C onto a polyvinylidene difluoride membrane (Pierce). Membranes were blocked for 30 minutes at room heat in 0.1 mol/L phosphate buffer, pH 7.4, containing 5% milk and 0.1% Tween 20 (blocking buffer) and incubated overnight at 4C 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 Naltrexone HCl studies showed that none of the activators or co-activators of the cell Naltrexone HCl 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.