Loss of calbindin-D28K is associated with the full range of tangle pathology within basal forebrain cholinergic neurons in Alzheimer's disease

Abstract

Basal forebrain cholinergic neurons (BFCN) are selectively vulnerable in Alzheimer's disease (AD). We have shown that most of the BFCN in the human brain contain the calcium-binding protein calbindin-D28K (CB), a large proportion lose their CB in the course of normal aging, and the BFCN which degenerate in AD lack CB. Here, we investigated the relationship between CB in the BFCN and the process of tangle formation in AD using antibodies to tau epitopes that appear early, intermediate or late in the process of tangle formation. Very small percentages (0%-3.7%) of CB-positive BFCN contained pretangles and/or tangles, and very small percentages (0%-5%) of the total BFCN pretangles and/or tangles were in CB-immunoreactive neurons. The number of CB-positive BFCN which contained tau immunoreactivity was highest for the early epitope and lower for intermediate epitopes. A late appearing epitope was absent from CB-positive BFCN. Age-related loss of CB appears to coincide with tangle formation in the BFCN and is associated with the full range of tau pathology, including late appearing epitopes.

Keywords: Alzheimer's disease; Basal forebrain cholinergic neurons; Calbindin-D(28k); Selective neuronal vulnerability; Tangles; Tau epitope.

Figure 1. Calbindin staining and immunoreactivity for various Tau epitopes show little overlap in basal forebrain cholinergic neurons (BFCN)

Double immunohistochemistry demonstrates the presence of calbindin (brown, DAB) and Tau epitopes (granular blue, BDHC) (A) TOC-1, which appears early in Tau oligomers, (B) AT8 and (C) PHF-1, which are intermediate in the process of tangle formation, and (D) MN423, which appears late in this process, in BFCN. Immunoreactivity for all four Tau epitopes was observed within BFCN in all the AD cases investigated. Immunoreactivity for calbindin and Tau epitopes was virtually non-overlapping, with minor co-localization of calbindin with TOC-1, AT8 or PHF-1 (see Fig 3 and 4). CB was never co-localized with the late appearing Tau epitope MN423 in BFCN. Note that while neuropil threads stained for each tau epitope are present among the BFCN (elongated blue thread-like structures), no neuritic plaques are seen. Magnification 10X. Arrows point to single CB immunoreactive neurons and arrowheads to neurons singly stained for each Tau epitope.

Figure 2. Stereological quantitation of the number of tangles / pre-tangles in the basal forebrain cholinergic neurons containing various Tau epitopes

Unbiased stereological counting methods demonstrated that Thioflavin-S (Thio-S), which binds to β-pleated sheet conformation in mature tangles, is present in the largest number of tangles / pre-tangles in BFCN, followed sequentially by Tau epitopes PHF-1, MN423, TOC-1 and AT8. The number of Thio-S-positive BFCN tangles was significantly larger than the numbers of BFCN containing TOC-1 or AT8 immunoreactive tangles / pre-tangles (*p<0.05). Bars represent means and standard errors.

Figure 3. Abnormal Tau epitopes are rarely co-localized with calbindin immunoreactivity in basal forebrain cholinergic neurons (BFCN)

High power photomicrographs, showing examples of BFCN containing calbindin immunoreactivity only (brown, arrows), calbindin immunoreactivity and each Tau epitope (Tau epitopes in granular blue, arrowheads) or Tau epitope immunoreactivity only (granular blue, double arrowhead). BFCN were infrequently double-stained for calbindin and the early Tau epitope TOC-1 (A), or the intermediate Tau epitopes AT8 (B) and PHF-1 (C). Calbindin immunoreactivity was never co-localized with the late appearing Tau epitope MN423 in BFCN (D). Magnification 40X.

Figure 4. Presence of calbindin is associated primarily with early epitopes of tau in basal forebrain cholinergic neurons (BFCN)

While co-localization of calbindin with various Tau epitopes was infrequent when compared with total number of BFCN that contained calbindin or each Tau epitope, unbiased stereological counting methods demonstrated that the largest numbers of double stained calbindin immunoreactive BFCN contained the early appearing Tau epitope TOC-1, followed by the intermediate appearing epitopes AT8 and PHF-1. The late appearing Tau epitope MN423 was never co-localized with calbindin in BFCN. Thus calbindin-positive BFCN contain only tangles / pre-tangles that are in the initial stages of formation, and late stage tangles / pre-tangles are completely absent from these neurons. The number of BFCN double stained for calbindin and TOC-1 was significantly larger than the number containing calbindin and MN423 (* p< 0.05). Bars represent means with standard errors.