Expression of cell cycle proteins in frontal cortical layer V/VI in B6-R1.40 at 12 months. A–C, There was no evidence of cyclin D (A, green) observed in NeuN-positive cells (B, red) in 10-month-old B6-R1.40 animals (C, merge). At this age, neurons residing in frontal cortical layer II/III exhibit only two spots of hybridization (C, inset). D–F, Cyclin D (D, green) immunoreactivity was encountered in neurons (E, red) in R1.40 mice aged to 12 months. Overlaying the images reveals colocalization of both cyclin D and NeuN (F ). FISH indicates that neuronal re-expression of cyclin D is accompanied by DNA synthesis as evidenced by four spots of hybridization (inset). G–I, No evidence of cyclin D (G, green) was observed in NeuN+ cells (H, red) in age-matched non-transgenic controls. Cell nuclei were counterstained with DAPI (blue) throughout. For each age and genotype, a total of five animals were analyzed. Scale bar, 10µm.

Neuronal expression of cell cycle protein is delayed in D2-R1.40 animals. A–C, No evidence of cyclin A (green) immunoreactivity was apparent in NeuN-positive cells (red) in transgenic mice aged to 10 months. D–F, Cyclin A (D, green) was expressed in neurons (E, red) in 12-month-old D2-R1.40 animals. Overlaying the images reveals colocalization of both cyclin D and NeuN (F). G–I, There was no cyclin A found in comparable neuronal populations in age-matched non-transgenic control. Nuclei were counterstained with DAPI (blue throughout). The arrows indicate examples of cell cycle-positive neurons. Scale bar, 10µm.

Aβ oligomers induce neuronal BrdU incorporation in cortical neurons in vitro. A–I, Cultured cortical neurons were treated with Ham’s F12 vehicle (Veh) (A–C), 1000 nm of Aβ_{1– 42} monomer-rich preparations (D–F ), or 1000 nm Aβ_{1– 42} oligomer-rich preparations (G–I ) in the presence of BrdU for 24 h. Cells were fixed and coimmunostained with antibodies against Map2 (A, D, G) and BrdU (B, E, H), demonstrating the induction of BrdU incorporation in Map2-positive cells in the oligomer-rich preparations but not the monomer-rich or vehicle control (C, F, I, merged images). Scale bars, 10 µm. G–I, Insets, Higher magnification of the BrdU-positive, Map2-positive cell in the oligomer-rich treatment group. J, K, Quantification of the percentage of BrdU-positive/Map2-positive cells in Aβ monomer-rich and oligomer-rich preparations. Cortical neurons were exposed to either vehicle or increasing concentrations (50–1000 nm) of either monomer-rich (J ) or oligomer-rich (K ) preparations for 24 h. Treatment with oligomer-rich preparations at concentrations > 100 nm resulted in a statistically significant increase in the percentage of BrdU-positive cells ( p = 0.0001). L, Immunoneutralization of Aβ oligomers with the addition of the Aβ oligomer-specific antibody NU2 (100 nm) resulted in a five-fold reduction in BrdU incorporation (n = 3; p = 0.0325) in the neuronal cultures exposed to 100 nm of Aβ oligomer-rich preparations, to levels similar to the vehicle control (the same 100 nm control was used for experiments in K and L). At the same concentration, addition of nonspecific mouse IgG did not have a statistically significant effect on BrdU incorporation (L).

Appearance of cell cycle proteins and DNA synthesis in frontal cortical layers II/III in B6-R1.40 mice at 6 months. A–C, No evidence of cyclin A (A, green) immunoreactivity was apparent in NeuN-positive cells (B, red) in transgenic mice aged to 4 months (C, merge). At this age, neurons residing in frontal cortical layer II/III exhibit only 2 spots of hybridization (C, inset). D, E, Immunohistochemical profiles encountered at 6 months in B6-R1.40 animals reveal that cyclin A (D, green) is expressed in neurons (E, red). F, Overlaying the images reveals colocalization of both cyclin A and NeuN. FISH indicates that neuronal re-expression of cyclin A is accompanied by DNA synthesis as evidenced by three or four spots of hybridization (inset). G–I, There was no cyclin A (G, green) found in comparable neuronal populations (H, red) in age-matched non-transgenic controls. Nuclei were counterstained with DAPI (blue) throughout. The arrows indicate examples of cell cycle-positive neurons. Scale bar, 10µm.

Quantification of neuronal cell cycle activity within cortical layers. Percentages of neurons exhibiting immunoreactivity for cell cycle re-entry were tabulated in both male and female B6-R1.40 transgenic and non-transgenic mice aged 6 –12 months. A–C, Cell cycle re-entry was tabulated by scoring cyclin A (A) and cyclin D (B) immunoreactive neurons and polyploid neurons (C) in the frontal cortex.

B6-R1.40;Bace1^−/− animals do not display neuronal cell cycle re-entry at 6 months in frontal cortical layer II/III. A, Whole brain protein extracts were prepared from B6-R1.40 and B6-R1.40;Bace1^−/− animals. Western blotting was performed using a C-terminal antibody. B, D, Cyclin A (green; B) is expressed in neurons (red; D) in 6-month-old B6-R1.40 animals. Overlaying the images reveals colocalization of both cyclin A and NeuN (F). E, Neurons (red; E) in 6-month-old B6-R1.40;Bace1^−/− animals do not exhibit re-expression of cyclin A (green; C). Nuclei were counterstained with DAPI (blue throughout). The arrows indicate examples of cell cycle-positive neurons. Scale bar, 10µm.