Deregulation of cdk5, hyperphosphorylation, and cytoskeletal pathology in the Niemann-Pick type C murine model

Abstract

NPC-1 gene mutations cause Niemann-Pick type C (NPC), a neurodegenerative storage disease resulting in premature death in humans. Spontaneous mutation of the NPC-1 gene in mice generates a similar phenotype, usually with death ensuing by 12 weeks of age. Both human and murine NPC are characterized neuropathologically by ballooned neurons distended with lipid storage, axonal spheroid formation, demyelination, and widespread neuronal loss. To elucidate the biochemical mechanism underlying this neuropathology, we have investigated the phosphorylation of neuronal cytoskeletal proteins in the brains of npc-1 mice. A spectrum of antibodies against phosphorylated epitopes in neurofilaments (NFs) and MAP2 and tau were used in immunohistochemical and immunoblotting analyses of 4- to 12-week-old mice. Multiple sites in NFs, MAP2, and tau were hyperphosphorylated as early as 4 weeks of age and correlated with a significant increase in activity of the cyclin-dependent kinase 5 (cdk5) and accumulation of its more potent activator, p25, a proteolytic fragment of p35. At 5 weeks of age, the development of axonal spheroids was noted in the pons. p25 and cdk5 coaccumulated with hyperphosphorylated cytoskeletal proteins in axon spheroids. These various abnormalities escalated with each additional week of age, spreading to other regions of the brainstem, basal ganglia, cerebellum, and eventually, the cortex. Our data suggest that focal deregulation of cdk5/p25 in axons leads to cytoskeletal abnormalities and eventual neurodegeneration in NPC. The npc-1 mouse is a valuable in vivo model for determining how and when cdk5 becomes deregulated and whether cdk5 inhibitors would be useful in blocking NPC neurodegeneration.

Fig. 1.

NF abnormalities in npc-1 −/− mice. Immersion-fixed, paraffin-embedded sagittal brain sections from 7-week-old +/+ (A, C, E, J) and −/− (B, D, F, G–I, K) mice were immunolabeled with antibodies SMI 31 (A–I) or SMI 32 (J, K). For some sections, antibody binding was visualized with DAB (brown) and hematoxylin counterstain for highlight ing the nuclei of all cells (blue-purple) or with Cy-3 (red) and DAPI counterstain for nuclei (blue). In the +/+ mouse, SMI 31 positively stained bundles of processes throughout the brain [shown for the basal ganglia (A), brainstem (C), and hippocampus (E)]. In sharp contrast, intense spheroid-like structures were observed in vast numbers in the basal ganglia (B) and brainstem (D) and sparsely in the hippocampus (F, arrows). The absence of nuclei within the spheroidal structures (G) and their size suggest that they are cross sections of swollen axons. Some rarer, larger spots containing a nucleus resembled perikarya (H, arrow in cortex). The gigantically enlarged neurons containing storage were not stained with SMI 31 (I, arrow), in contrast to axonal staining with a translucent core (I,arrowhead). SMI 32 stained a few large neurons and small fibers in the pons of the +/+ mouse (J) and axonal spheroids principally in the brainstem of −/− mice (K, arrows). Magnification:A–F, 4×; G–K, 40×.

Fig. 2.

Increased levels of NF-H and increased phosphorylation of NF-M in npc-1 −/− mice. Whole-brain lysates containing 10 μg of protein from 8-week-old +/+ and 4- and 8-week-old −/− mice were resolved on 8% gels, transferred to nitrocellulose, and blotted with the indicated antibodies. The pan NF antibody R39 identified the three major NF isoforms (marked NF-H, NF-M, and NF-L, respectively). The intensities of the bands visualized with SMI 31 and R39 were quantitated, and the results indicated a significant increase in the levels of all three isoforms and a marked increase in phosphorylation of the NF-M isoform. Equivalent loading of all samples is demonstrated with the NeuN antibody recognizing the 46–48 kDa neuronal-specific antigens.

Fig. 3.

Tau and MAP2 abnormalities in thenpc-1 −/− mouse brain. Sagittal brain sections from +/+ (A, D, F, H) and 8-week-old −/− mice (B, C, E, G, I, J) were immunostained with PHF-1 (A–C), CP-22 (D, E), CP-10 (F, G), and AP-18 (H–J). PHF-1 stained bundles of processes in the brainstem and white matter of +/+ mice (A, pons) but numerous axonal spheroids (B, arrows) and some perikarya (C) of neurons in similar regions of the −/− mouse brain. CP-22 displayed much weaker staining of processes in +/+ mice (D) and a similar pattern of axonal spheroids (arrows) in the brainstem of −/− mice (E, pons). CP-10 exhibited weak staining of neuronal cell bodies in +/+ mice (F, pons), but in −/− mice there was a dramatic increase in the staining of neurons both in the cytoplasm and in the nucleus (G, large arrow). Some neurons containing foamy material were conspicuously unstained (G, asterisk), but others were CP-10 positive (arrowhead). AP-18 was primarily negative in the +/+ mouse brain (H) but labeled the soma and dendrites of several neurons (arrows) in the thalamus (I). J, One such neuron with prominent staining of the soma and apical dendrite. Magnification:A, B, D–G, I, J, 40×; C, H, 100×.

Fig. 4.

Increased phosphorylation of microtubule-associated proteins in npc-1 −/− mice. Ten micrograms of whole-brain, heat-stable protein from +/+ and −/− mice and a typical AD case were blotted with the indicated antibodies (supernatant only for AP-18). Ten and 8% gels were used for detection of tau and MAP2, respectively. An increase in intensity and in the apparent molecular weight of tau was observed with PHF-1 and CP-13 in 4-week-old and even more so in 8-week-old −/− mice, consistent with hyperphosphorylation of tau. The pattern of tau bands at 8 weeks of age resembled that of AD-tau. CP-10-detected phosphorylated tau was increased threefold in −/− mice. CP-22 raised against and shown to recognize phosphorylated tau did not react with tau in the BALB/c strain of mouse. However, the antibody detected a molecule of ∼180 kDa that was more highly phosphorylated in −/− mice. The tau sequence antibodies TG-5 and ALZ-50 displayed equivalent amounts of tau in alllanes. AP-18 immunoreactivity showed increased phosphorylation of the 280 kDa MAP2a and MAP2b and 70 kDa MAP2c protein −/− mice, particularly at 4 weeks of age. AP-20 did not recognize MAP2c but showed invariable amounts of total MAP2 (2a+2b) protein in all lanes.

Fig. 5.

cdk5 and p25 abnormalities in thenpc-1 −/− mouse brain. A, cdk5/p35 immunoblots. Whole-brain lysates containing 40 μg of protein from +/+ and −/− mice were resolved on 12% gels and blotted with the indicated antibodies. cdk5 levels remained unchanged in −/− mice compared with controls. The p35 C-terminal antibody showed no difference in p35 levels but an enrichment of the 25 kDa p35 fragment (p25) in −/− mice (left panel, mouse). Similar preparations containing 100 μg of protein from a human control and an NPC hippocampus were immunoblotted with cdk5 and p35C antibodies. cdk5 and p35 levels were invariant, but p25 was elevated in NPC (right panel, human).B, cdk5 IP/kinase activity. Whole-brain lysates (left panel) from +/+ and −/− mice were immuno- precipitated with cdk5 (C-8) antibody. Immunoprecipitate (cdk5 IP) was immunoblotted with a different cdk5 antibody to show that the relative recoveries of enzyme in the samples were similar (Cdk5). Catalytic aliquots of cdk5 immunoprecipitate were incubated with kinase buffer containing histone H1 and [γ-³²P]-ATP for 20 min at 30°C, and the reaction mixtures were resolved on 12% gels. The incorporation of [γ-³²P]ATP into histone H1 (H1) was significantly higher with cdk5 IP from −/− mice compared with +/+ mice (left panel), on the basis of equivalent H1 loading demonstrated by staining of the gel with Coomassie blue (gel). The cdk5 activity in 5- to 10-week-old −/− mice was increased 1.5-fold over that seen in +/+ mice (p < 0.01; n = 12;left panel). Right panel, The increase in cdk5 activity in the forebrain (Forebr.) and brainstem (B.stem) but not in the cerebellum (Cblm.) of a 5-week-old −/− mouse compared with those of a 5-week-old +/+ mouse (all scales, ×1000). C, P-GSK-3β immunoblots. Whole-brain lysates containing 50 μg of protein from +/+ and −/− mice were resolved on 10% gels and blotted with P-GSK-3β antibody (P-GSK-3) or primary sequence-dependent antibody (GSK-3). The intensities of the resulting bands were quantitated densitometrically, and no significant differences (p = 0.11) were observed between the npc-1 and control mice.

Fig. 6.

p35 accumulates and colocalizes with phosphorylated NF in axonal spheroids of npc-1 −/− mice. Perfusion-fixed, paraffin-embedded sections from 7-week-old +/+ (A, C–E) and −/− (B, F–H) mice were immunohistochemically labeled with cdk5 (brown) and counterstained with hematoxylin (blue-purple) or immunofluorescently labeled with p35 (Alexa 488, green) and SMI 31 (Cy-3, red) and counterstained with DAPI (blue). cdk5 barely stained neurons in +/+ mice (A, pontine nucleus). In contrast, cdk5 immunoreactivity was increased in the soma of some deformed neurons (arrow) and in the axonal spheroids (arrowhead) in the brainstem of −/− mice (B, pontine nucleus). p35 faintly labeled the soma of neurons in the brainstem of +/+ mice (arrows inC and E, pons) and displayed little colocalization with SMI 31 immunoreactivity (D andmerged image in E). In the −/− mouse brain, intense p35 immunoreactivity was seen in the axonal spheroids (F, pons), which perfectly matched the accumulation of SMI 31 immunoreactivity (G and arrows inmerged image in H).Inset in H, Gigantically enlarged axons containing p35 (green) and SMI 31 (red) immunoreactivity in the cerebral cortex. Magnification: A, B, 100×; C–H, 40×.