p25/cyclin-dependent kinase 5 induces production and intraneuronal accumulation of amyloid beta in vivo

Abstract

Aberrant processing of the amyloid precursor protein (APP) and the subsequent accumulation of amyloid beta (Abeta) peptide has been widely established as a central event in Alzheimer's disease (AD) pathogenesis. The sequential cleavage steps required for the generation of Abeta are well outlined; however, there is a relative dearth of knowledge pertaining to signaling pathways and molecular mechanisms that can modulate this process. Here, we demonstrate a novel role for p25/cyclin-dependent kinase 5 (Cdk5) in regulating APP processing, Abeta peptide generation, and intraneuronal Abeta accumulation in inducible p25 transgenic and compound PD-APP transgenic mouse models that demonstrate deregulated Cdk5 activity and a neurodegenerative phenotype. Induction of p25 resulted in enhanced forebrain Abeta levels before any evidence of neuropathology in these mice. Intracellular Abeta accumulated in perinuclear regions and distended axons within the forebrains of these mice. Evidence for modulations in axonal transport or beta-site APP cleaving enzyme 1 protein levels and activity are presented as mechanisms that may account for the Abeta accumulation caused by p25/Cdk5 deregulation. Collectively, these findings delineate a novel pathological mechanism involving aberrant APP processing by p25/Cdk5 and have important implications in AD pathogenesis.

Figure 1.

Increased Aβ levels in p25 Tg mice. a, Aβ_x-42 and Aβ_x-40 levels were determined by ELISA using capture antibodies 21F12 and 2G3 that specifically recognize mouse Aβ_x-42 and Aβ_x-40, respectively. Data are mean ± SEM values for WT (n = 5) and CK–p25 Tg (n = 5) mice. APP^−/− indicates APP knock-out brain lysate used as negative control. b, Aβ levels are not increased in uninduced p25 Tg mice. Data are mean ± SEM values for WT (n = 3) and CK–p25 Tg (n = 3) mice. Aβ_x-42 levels were unchanged, whereas there was a small but significant decrease in Aβ_x-40 levels in uninduced p25 Tg mice compared with WT. c, Human Aβ levels in PD-APP/CK–p25 mice were determined by ELISA. Data are mean ± SEM values for PD-APP (n = 3) and CK–p25/PD-APP (n = 3) mice. d, Insoluble human Aβ levels were determined by ELISA from RIPA-insoluble pellet fraction from forebrain lysates of PD-APP and CK–p25/PD-APP mice induced for 8 weeks. Data are mean ± SEM values for PD-APP (n = 3) and CK–p25/PD-APP (n = 3) mice. **p < 0.005, by two-tailed, unpaired Student's t test.

Figure 2.

Intraneuronal accumulation of Aβ in p25 Tg mice in perinuclear pools. a, Increased 4G8 immunoreactivity was found in cell bodies of the cortex of CK–p25 mice compared with WT controls at 8 weeks of induction. b, Intraneuronal Aβ accumulation was detected by 6E10 and M1 antibodies. APP holoprotein levels, as detected by the R1736 APP N-terminal antibody, were similar between CK–p25/PD-APP and PD-APP mice. Cortex of mice induced for 8 weeks are shown. c, Immunoelectron microscopy with the 4G8 antibody demonstrate the aggregation of intraneuronal Aβ in fibril-like structures adjacent to the nucleus in CK–p25 mice (middle, arrows), which were more extensive and frequent in CK–p25/PD-APP mice (right, arrows). Also shown is a neuron without 4G8 immunoreactivity (left). Inset is a magnification of the area outlined by the smaller box. d, First panel, Perinuclear Aβ aggregates were also immunoreactive toward NF-H (smaller immunogold particles) and 4G8 (larger particles). In the CK–p25/PD-APP mice, aggregates stained positive for the Aβ-related antibodies 6E10 (second panel), M1 (third panel), and R1282 (fourth panel). Nuclear area (e) and percentage occupied by condensed chromatin (f) were calculated for neurons without (n = 16; blue squares) and with (n = 13; red triangles) perinuclear Aβ aggregations from immunoelectron micrographs from CK–p25/PD-APP mice. Results are displayed as a scatter plot, with error bars indicating SEM. Results were statistically significant (p = 0.0011 for e; p = 0.0002 for f) according to two-tailed, unpaired Student's t test. For c and d, immunoelectron microscopy was performed on 12 week induced CK–p25 and CK–p25/PD-APP mice, analyzing the subiculum region, which showed the greatest 4G8 staining (data not shown). Scale bar, 500 nm. g, Cortical neurons from 5–10 week CK–p25/PD-APP mice (n = 3) expressing p25–GFP with or without intraneuronal 6E10 staining were scored in a blinded manner for degenerative nuclear morphology, as indicated by condensed or invaginated nuclei. A minimum of 200 6E10-positive and 200 6E10-negative neurons were counted for each animal. Shown in the left are an example p25-positive, 6E10-positive neuron and a p25-positive, 6E10-negative neuron; the right indicates total quantification. DAPI, 4′,6′-Diamidino-2-phenylindole.

Figure 3.

CK–p25 and CK–p25/PD-APP Tg mice accumulate Aβ in swollen axons in the white matter tract of the dorsal hippocampal commissure. a, 4G8 immunoreactivity was observed in swollen axons in CK–p25 mice (second panel) and to a greater extent in PD-APP/CK–p25 mice (fourth panel) induced for 8 weeks but not in WT (first panel) and PD-APP (third panel) control mice. b, Swollen axons were clearly visible by Bielchowsky silver stain, showing various degrees of impregnation from orange to black (second panel) and by hematoxylin and eosin staining, appearing as well defined, eosinophilic structures (fourth panel) in CK–p25 mice. Swollen axons were not detected in age-matched WT (first and third panels) or PD-APP (data not shown) controls. c, Electron microscopy of the swollen axons, shown in cross section (left panel) and longitudinal sections (right panel), revealed an amassment of organelles and vesicles and deteriorating myelination. Electron micrographs are from CK–p25 mice induced for 9 weeks. d, 4G8 immunoelectron micrographs revealed immunoreactive clusters in electron-dense, vesicular structures within swollen axons in CK–p25/PD-APP mice induced for 12 weeks. Aggregations are indicated by arrows. Inset is a magnification of area outlined by the smaller box. Scale bars, 200 nm.

Figure 4.

Enhanced BACE1 and β-secretase processing of APP in p25 Tg mice. a, Increased BACE1 expression was shown by immunoblot analysis of RIPA-soluble forebrain extracts from 2 week induced CK–p25 mice. Data are mean ± SEM values for WT (n = 3) and CK-p25 Tg (n = 3) mice. BACE1 levels were not increased in uninduced p25 Tg mice (data not shown). b, Increased generation of APP C-terminal fragments C89 and C99 in CK–p25 and PD-APP/CK–p25 mice compared with their respective controls. Blots are representative from mice induced for 5–8 weeks. c, Densitometric analysis of C-terminal fragment bands plotted relative to WT (top) or PD-APP (bottom) C99 intensities. Data are mean ± SEM values; n = 3 for each genotype. *Statistically different C99 or C89 immunoreactivity by two-tailed, unpaired Student's t test from WT or PD-APP (top, p = 0.027 for C99 and p = 0.020 for C89; and bottom, p = 0.028 for C99 and p = 0.053 for C89). d, Increased BACE1 expression as shown by immunohistochemical analyses. Shown is a representative image from the cortex of a 2 week induced CK–p25 mouse. e, BACE1 and C-terminal APP fragments are concomitantly increased at 5 weeks of induction. DAPI, 4′,6′-Diamidino-2-phenylindole; CTF, C-terminal fragments.