Cyclin-dependent kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal lesion formation, and motor defects in Niemann-Pick Type C mice

Abstract

Dysregulation of cyclin-dependent kinases (cdks) and cytoskeletal protein hyperphosphorylation characterizes a subset of human neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, and Niemann-Pick Type C (NPC). It is thought that these cytoskeletal changes lead eventually to development of hallmark cytoskeletal lesions such as neurofibrillary tangles and axonal spheroids. Although many studies support an involvement of cdks in these neurodegenerative cascades, it is not known whether cdk activity is essential. The naturally occurring npc-1 mutant mouse mimics human NPC, in displaying activation of cdk5, mitotic cdc2, and cdk4, with concomitant cytoskeletal pathology and neurodegeneration. We availed of this model and specific pharmacological inhibitors of cdk activity, to determine whether cdks are necessary for NPC neuropathology. The inhibitors were infused intracerebroventricularly for a 2-week period, initiated at a pathologically incipient stage. While an inactive stereoisomer, iso-olomoucine, was ineffective, two potent inhibitors, roscovitine and olomoucine, attenuated significantly the hyperphosphorylation of neurofilament, tau, and mitotic proteins, reduced the number of spheroids, modulated Purkinje neuron death, and ameliorated motor defects in npc mice. These results suggest that cdk activity is required for neuropathology and subsequent motor impairment in NPC. Studies aimed at knocking down individual cdks in these mice will help identify the specific cdk(s) that are essential, and delineate their precise roles in the neurodegenerative process.

Figure 1

Roscovitine inhibits phosphorylation of cytoskeletal proteins in npc mice. Mice either treated with different doses of roscovitine or the vehicle only for 2 weeks were analyzed for the presence of various epitopes. The antibodies used are indicated on the right of the corresponding blots. A: Immunoblots of control or roscovitine-treated mice (duplicate animals from a total of 6 to 10 treated with 72 or 144 nmoles/day shown only). Cdk5 is the representative loading control for all antigens. B: Samples from the untreated (−/−), control mice (control), or 300 nmoles/day roscovitine-treated mice (300) are compared with samples from wild-type (+/+) to illustrate normalization of protein phosphorylation in npc mice after roscovitine treatment (NeuN, loading control). The bands corresponding to NF-H and NF-M are labeled as H and M, respectively.

Figure 2

Roscovitine treatment can rescue abnormal mitotic phosphoepitopes in npc mice. Mice treated either with the indicated doses of roscovitine (top) or the vehicle (control) for 2 weeks were analyzed for the presence of various cdc2 generated epitopes. Cdk5 has been used as loading control for both the blots. The antibodies used are indicated on the right of the corresponding blots.

Figure 3

Immunoblotting analyses of ERK (A), and GSK-3β (B), and their phospho-substates in control and inhibitor-treated mice. Mice treated with roscovitine for 2 weeks were analyzed for the presence or absence of either ERK or GSK3β-generated epitopes (cdk5, loading control). The epitopes being detected are indicated on the right of the corresponding blot. The figures on top indicate the inhibitor dose (nmoles/day). The bands corresponding to NF-H and NF-M are labeled as H and M, respectively.

Figure 4

Roscovitine reduces axon spheroid number in npc mice. A: Results of 2-week roscovitine infusion. a, Schematic showing 4 regions (regions 1 to 4) used for spheroid counting; b, axon spheroids (arrows) stained by SMI32 (brown, bar, 25 μm); c–h, SMI32 staining showing spheroid distribution in regions 1, 2, and 4 of control (c, e, and g) or roscovitine-treated (d, f, and h) mice (bar, 50 μm); i, summary of spheroid numbers from regions 1 to 4, normalized against the average spheroid number in the same sex controls: control (0 nmoles/day, blue), 72 nmoles/day (red), 144 nmoles/day (yellow, n = 10), 300 nmoles/day (green), and 600 nmoles/day (purple, n = 7). All of the photomicrographs were taken at ×20 magnification except for b which was taken at ×40. B: Results of 4-week roscovitine infusion. SMI32 staining shows spheroid distribution in control, a and c, and mice treated with 200 nmole/day roscovitine, b and d. Quantitative data for the 4-week roscovitine treatment is shown in e. Roscovitine caused a significant decrease in spheroids in all subregions.

Figure 5

Immunohistochemical analyses of mitotic phosphoepitopes in roscovitine-treated and control mice. A 2-week roscovitine treatment ameliorates MPM-2 phosphoepitopes (b), compared to the control animals treated with vehicle alone (a) (bar, 50 μm). Arrows indicate MPM-2-labeled spheroids. The photomicrographs were taken at ×40 magnification.

Figure 6

Roscovitine infusion effectively reduces Purkinje neuron loss in npc mice. Mice treated with either roscovitine or DMSO only (control) for 2 weeks (144 nmoles/day) (A) or 4 weeks (200 nmoles/day) (B) were analyzed for the presence or absence of Purkinje neurons Aa: H&E staining showing Purkinje neurons missing in control (arrow). Ab: H&E staining showing Purkinje neurons surviving in roscovitine-treated mice (arrows). Ac: Quantitative summary of Purkinje cell numbers in control and 2-week roscovitine-treated mice. The x-axis shows the dose of roscovitine treatment (nmoles/day). Note, mean values at 144, 300, and 600 nmoles/day roscovitine, are not statistically different from each other, but are statistically increased relative to control and 72 nmoles/day roscovitine. B: H&E staining showing missing Purkinje neurons (arrows) in 4-week control (a), and in roscovitine-treated mice (b). All of the photomicrographs were taken at ×40 magnification.

Figure 7

Roscovitine (ros) improves motor activity and inhibits weight loss in npc mice. The hanging time on a coat hanger and body weight were compared before (white bars) and after treatment (black bars) with either control or roscovitine-treated mice. A: Motor activity before and after 2-week infusion with roscovitine at the indicated concentrations (144 and 600 nmoles/day, n = 10 and 7 respectively, for the other doses tested, n = 6). B: Motor activity before and after 4-week roscovitine treatment at 200 nmoles/day, n = 8, P = 0.03. C: Body weight after 2- or 4-week roscovitine treatment at 200 nmoles/day, n = 8, P = 0.007.

Figure 8

Olomoucine can ameliorate the neuropathological phenotype in npc mice. Two-week olomoucine (olo), but not iso-olomoucine (iso), treatment improves motor activity (a). Olomoucine treatment (c) (bar, 50 μm), decreased SMI32-stained spheroid count compared to iso-olomoucine-treated mice (b). The results from regions (reg) 1 to 4 are summarized (d); spheroid numbers are expressed as percentage control; control mice, blue bars; iso-treated mice, red bars; olo-treated mice, yellow bars. Olomoucine treatment resulted in a decrease of SMI31 and PHF-1 immunoreactivity compared to iso-olomoucine treated mice (e). NeuN shows the loading control in this panel.