Progression of tau pathology in cholinergic Basal forebrain neurons in mild cognitive impairment and Alzheimer's disease

Abstract

Tau is a microtubule-associated protein that forms neurofibrillary tangles (NFTs) in the selective vulnerable long projection neurons of the cholinergic basal forebrain (CBF) in Alzheimer's disease (AD). Although CBF neurodegeneration correlates with cognitive decline during AD progression, little is known about the temporal changes of tau accumulation in this region. We investigated tau posttranslational modifications during NFT evolution within the CBF neurons of the nucleus basalis (NB) using tissue from subjects with no cognitive impairment, mild cognitive impairment, and AD. The pS422 antibody was used as an early tau pathology marker that labels tau phosphorylated at Ser422; the TauC3 antibody was used to detect later stage tau pathology. Stereologic evaluation of NB tissue immunostained for pS422 and TauC3 revealed an increase in neurons expressing these tau epitopes during disease progression. We also investigated the occurrence of pretangle tau events within cholinergic NB neurons by dual staining for the cholinergic cell marker, p75(NTR), which displays a phenotypic down-regulation within CBF perikarya in AD. As pS422+ neurons increased in number, p75(NTR)+ neurons decreased, and these changes correlated with both AD neuropathology and cognitive decline. Also, NFTs developed slower in the CBF compared with previously examined cortical regions. Taken together, these results suggest that changes in cognition are associated with pretangle events within NB cholinergic neurons before frank NFT deposition.

Figure 1

Difference between clinical diagnosis and neuropathological score. Histograms demonstrating the percentage of cases in each clinical group (NCI (light gray), MCI (black), and AD (dark gray), versus Braak score (A) and NIA Reagan diagnosis (B). These histograms indicate that there are several NCI and MCI cases with higher than expected Braak scores and NIA Reagan diagnoses.

Figure 2

p75^NTR/pS422 Immunolabeled neurons. Sections containing the NB subfield of the CBF dual immunostained for p75^NTR+ (brown) and pS422+ (blue) show a decrease in the number of p75^NTR+ neurons with an increase of pS422+ neurons as disease progression continues from NCI (A and B), to MCI (C and D) and AD (E and F). In NCI (B), most NB neurons were single-labeled for p75^NTR+ (asterisk), few neurons were double-labeled for p75^NTR+/pS422+ (black arrow), whereas only an occasional neuron was single-labeled for the early tau pathological marker, pS422+ (green arrow). In the MCI (D) and AD (F) cases, there was a decrease in the p75^NTR+ neurons (asterisk) and an increase in both p75^NTR+/pS422+ (black arrow) and pS422+ (green arrow) neurons. In some p75^NTR+/pS422+ neurons, it can be seen that the pS422 immunoreactivity begins in the center of the neuron (blue arrow). Images in A, C, and E were taken at magnification ×4. Scale bar = 250 μm. Images in B, D, and F were taken at magnification×20. Scale bar is 50 μm. The panels to the right were taken at magnification ×60. Scale bar is 25 μm.

Figure 3

Quantification of the p75^NTR and pS422 double-stain. Box plots showing an increase in the number of pS422+ (A) and p75^NTR+/pS422+ (B) neurons from NCI to MCI and AD. C: p75^NTR+ Neurons decreased from NCI to MCI and AD.

Figure 4

Neuropathological and clinical correlates in p75^NTR and pS422 double-stain. Bar charts plotting mean and SEM show an increase in number of pS422+ (A) and p75^NTR+/pS422+ (B) compared with a decrease in p75^NTR+ (C) neurons across Braak stage of NFT pathology. The increase in pS422+ (A) and p75^NTR+/pS422+ (B) neurons showed a strong positive correlation with Braak score, and the loss of p75^NTR+ neurons showed a negative correlation with Braak score (C). Scatterplots showed that a strong negative correlation was also observed between pS422+ (D) and p75^NTR+/pS422+ (E) neurons and GCS. r = Spearman rank correlation.

Figure 5

pS422 and TauC3 immunolabeled neurons. NB tissue immunohistochemically stained for pS422+ (brown) and TauC3+ (blue) within the CBF demonstrating an increase the number of pS422+, pS422+/TauC3+, and TauC3+ neurons from NCI (A and B), to MCI (C and D) and to AD (E and F). In most NCI cases (B), single-label pS422+ neurons were sparse (asterisk), and many pS422 immunoreactive neurons displayed very light staining (red arrow). In MCI (D), there was an increase in pS422+ (asterisk) neurons and the appearance of both pS422+/TauC3+ (black arrow) and TauC3+ neurons (green arrow). In AD (F), there was an increase in all three neuronal phenotypes for pS422+ (asterisk), pS422+/TauC3+ (black arrow) and TauC3+ (green arrow). Images in A, C, E taken at × 10 and scale bar is 250 μm. Images in B, D, F were taken at × 20 and scale bar is 50 μm. Images to the right were taken at × 60 and scale bar is 25 μm.

Figure 6

Quantification of pS422 and TauC3 double-stain. Box plots showing an increase in the number of pS422+ neurons (A) and pS422+/TauC3+ neurons (B) in MCI and AD. C: Appearance of TauC3+ neurons was variable across the cognitive diagnosis groups.

Figure 7

Quantification and correlations of pS422+ NT length. Photomicrograph showing an example of pS422+ NTs within the NB in an AD case (A) Scale bar = 25 μm. The box plots demonstrate an increase in pS422+ NT length in MCI and AD (B). C: Bar chart plotting mean and SEM shows that the increase in NT length significantly correlated with severity of Braak stage. D: Scatterplot showing a significant negative correlation between GCS and pS422+ thread length.

Figure 8

Dual immunofluoresence of p75^NTR and pS422. Immunofluoresence of p75^NTR+ (green) and pS422+ (red) neurons in the NB revealed a large number of p75^NTR+ neurons (A) and pS422+ NTs (B) most commonly seen in NCI cases (A–C). In MCI, p75^NTR+ neurons (D) were also labeled with pS422 (E and F). In AD, p75^NTR+ neurons were still seen (G), but there was an increase in the number of pS422+ neurons (H) that co-labeled with p75^NTR (I). All images taken at magnification ×63. Scale bar = 25 μm.

Figure 9

p75^NTR and TauC3 do not colocalize. p75^NTR+ (green) and TauC3+ (red) immunofluorescence within the NB revealed that most neurons were p75^NTR+ (A). Virtually no TauC3+ neurons (B and C) were found in NCI. In MCI, there were many p75^NTR+ neurons (D) and the appearance of, TauC3+ neurons (E) that did not colocalize with p75^NTR (F). In AD, p75^NTR+ neurons were still present (G) but there were many more TauC3+ neurons (H). In most cases, TauC3+ cells lacked p75^NTR reactivity (I). All images taken at magnification ×63. Scale bar = 25 μm.