Mitotic epitopes are incorporated into age-dependent neurofibrillary tangles in Niemann-Pick disease type C

Abstract

The mechanism underlying neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) and other neurodegenerative disorders remains elusive. Niemann-Pick disease type C (NPC) is a kind of genetic neurovisceral disorder in which the intracellular sequestration of cholesterol and other lipids in neurons, NFT formation and neuronal degeneration in brain are the neuropathology hallmarks. The age of onset and progression of the disease vary dramatically. We have analyzed the hippocampus from 17 NPC cases, aged from 7 months to 55 years, to depict the temporal characteristics of NFT formation. Unexpectedly, classic NFT was observed in about 4-year-old NPC brain, suggesting that NFT is not aging dependent, and that juvenile brain neurons satisfy the requirements for NFT formation. NFT in the hippocampus of NPC was significantly increased in number with the advance of age. More importantly, multiple mitotic phase markers, which are not usually found in normal mature neurons, were abundant in the affected neurons and incorporated into NFT. The unusual activation of cdc2/cyclin B kinase and downstream mitotic indices are closely associated with the age-dependent NFT formation, signifying the contribution of abortive cell cycle to neurodegeneration. The cdc2 inhibitors may be therapeutically used for early intervention of neurodegeneration and NFT formation in NPC.

Figure 1

Progression of NFT formation with age in NPC. Paraffin‐embedded sections from hippocampus (for the 6‐year‐old case only the temporal cortex was available) were immunostained with the PHF‐1 and TG‐3 antibodies (brown), and each section was counter‐stained with Hematoxylin to visualize the nuclei (violet) of all cells in a given field. Light micrographs from NPC cases with different ages show the relative numbers and distribution of NFT in CA1 region of hippocampus, which look similar to those in a typical AD case (20×, scale bar = 80 microns).

Figure 2

NFT pathology in juvenile NPC. Sections from the hippocampus of the 7‐month‐old (A), the 4.5‐year‐old (B) and an 10‐year‐old NPC cases (C–F) were stained with PHF‐1 (C–D) or TG‐3 (A, B, E, F) and counter‐stained with hematoxylin (A, B, F, bar = 80 microns; C, D, bar = 40 microns; E, bar = 160 microns). PHF‐1 or TG‐3 did not stain the neurons in the 7‐month‐old NPC case (A). A mature NFT was developed in the CA1 region of the 4.5‐year‐old NPC case (B). Similarly, fully‐developed NFT were stained with PHF‐1 in the 10‐year‐old NPC case (C) and some neurons with diffuse PHF‐1 immunoreactivity were seen (arrowhead in C, and D). Increased numbers of NFT were common in the CA4 hippocampal region in the 10‐year‐old NPC case (E), and some neurons with only lipid storage were also noted (F).

Figure 3

NFT pathology in young adult NPC. Sections from the hippocampus of a 17‐year‐old NPC case were stained with PHF‐1 and TG‐3 and counter‐stained with hematoxylin. Numerous NFT and storage containing neurons were detected in the cortical layer of the parahipocampal cortex (Panels A, scale = 800 microns, and C, bar = 80 microns, both for TG‐3), and some NFT were present in the CA4 region (panel B, bar = 400 microns, PHF‐1). Deeper layers of the CA1/subiculum had scattered NFT and neuronal staining resembling pre‐tangle stages of pathology (panel D, PHF‐1, bar = 40 microns). Both antibodies reacted with NFT and storage material (arrows, panel C for TG‐3, and panel D for PHF‐1), although some storage‐containing neurons were also unstained (arrowheads, panels C and D).

Figure 4

NFT pathology in adult NPC. Hippocampal sections from a 32‐year‐old adult case of NPC were stained either with Bielschowsky silver reagent (panels A, C and D) or TG‐3 (panels B, E–I) (A, B, bar = 160 microns; C–G, bar = 40 microns; H, bar = 15 microns; I, bar = 80 microns). The number and distribution of NFT as visualized with Bielschowsky reagent appeared similar to those with TG‐3 (Panels A and B). Apart from the classic AD‐type NFT (panels C & D, arrows), reticular type NFT were also seen (panel D, arrowheads). A lot of distended neurons with storage were negatively stained by silver impregnation (arrowheads, panel C). Both NFT types were stained with TG‐3 (panels E & F, brown and green respectively for AD‐type; G, red for reticular type). Neurons with lipid storage were prominently stained with TG‐3 (panels E and H arrows), and although most had no visible NFT (panel H, inset), some rare neurons contained storage and small bundles of PHF (panel H, arrow). NFT were also frequent in the dentate granule layer of the hippocampus (panel I).

Figure 5

Mitotic epitopes demarcating NFT. Hippocampal sections from a 31‐year‐old case were immunostained with MPM‐2 (panels A, D), H14 (panel B), H5 (panels C, E), cdc2 (panel F) and cyclin B1 (G) and counter‐stained with hematoxylin (all panels, bar = 40 microns). Classic NFT were stained with every antibody (panels A and D for MPM‐2, and panel B for H14, and C and E, H‐5), but cdc2 and cyclin B1 staining of some neurons lacking obvious NFT was more prominent (panels F and G, respectively). The H5 staining shows a shift in distribution of the enzyme from the nucleus (panel C, arrowhead) to the cytoplasm (panel C, arrow, and inset). MPM‐2 (panel D, red) and H5 (panel E, green) colocalize in the same NFT.

Figure 6

Immunoblot analysis of NFT antigens. Lysates from frozen pieces of hippocampus taken from the 11 year and 31 year old NPC‐1 cases (NPC, juv and adt, respectively), a 23 year old control (C) and an AD case (AD) were subjected to immunoblotting with the indicated antibodies. The PHF‐1 antibody showed the predominant presence of PHF‐tau in the adult NPC case as in AD, but not in the juvenile case. Consistent with this increase in phosphorylation, the MC1 antibody displayed an upward shift in apparent molecular weight of tau in the adult case. The total tau‐immunoreactive protein as detected with TG‐5 antibody appeared no significant change NPC cases when compare to that in the control. A mitotic marker, MPM‐2 detected an expecting band of 110 kD in both NPC cases but not in control. Cdc2 PISTAIRE antibody stained a 34 band accumulated in NPC cases, in which the non‐specific binding band is same in all the samples. H‐14 immunolabeled bands in the adult case similar to those in AD.