Quantifying the accretion of hyperphosphorylated tau in the locus coeruleus and dorsal raphe nucleus: the pathological building blocks of early Alzheimer's disease

Abstract

Aims: Hyperphosphorylated tau neuronal cytoplasmic inclusions (ht-NCI) are the best protein correlate of clinical decline in Alzheimer's disease (AD). Qualitative evidence identifies ht-NCI accumulating in the isodendritic core before the entorhinal cortex. Here, we used unbiased stereology to quantify ht-NCI burden in the locus coeruleus (LC) and dorsal raphe nucleus (DRN), aiming to characterize the impact of AD pathology in these nuclei with a focus on early stages.

Methods: We utilized unbiased stereology in a sample of 48 well-characterized subjects enriched for controls and early AD stages. ht-NCI counts were estimated in 60-μm-thick sections immunostained for p-tau throughout LC and DRN. Data were integrated with unbiased estimates of LC and DRN neuronal population for a subset of cases.

Results: In Braak stage 0, 7.9% and 2.6% of neurons in LC and DRN, respectively, harbour ht-NCIs. Although the number of ht-NCI+ neurons significantly increased by about 1.9× between Braak stages 0 to I in LC (P = 0.02), we failed to detect any significant difference between Braak stage I and II. Also, the number of ht-NCI+ neurons remained stable in DRN between all stages 0 and II. Finally, the differential susceptibility to tau inclusions among nuclear subdivisions was more notable in LC than in DRN.

Conclusions: LC and DRN neurons exhibited ht-NCI during AD precortical stages. The ht-NCI increases along AD progression on both nuclei, but quantitative changes in LC precede DRN changes.

Keywords: Alzheimer disease; Human; brain stem; dorsal raphe nucleus; locus coeruleus; unbiased stereology.

Figure 1

A 60-μm-thick horizontal section through the pons showing a portion of the locus coeruleus immunostained for phospho-tau (CP-13) and counterstained with gallocyanin. The arrow points to a ht-NCI+ neuron. The arrowhead points to a neuron free of ht-NCI displaying neuromelanin.

Figure 2

(a) An overview of a 60-μm-thick horizontal section through the pons showing locus coeruleus immunostained for phospho-tau (CP-13) and gallocyanin. (b) An overview of a 60-μm-thick horizontal section through the midbrain showing dorsal raphe nucleus at the level of the supratrochlear nucleus immunostained for phospho-tau (CP-13) and gallocyanin. (c and d) Horizontal sections, 300-μm-thick, adjacent to the sections in (a) and (b), respectively, stained for gallocyanin (Nissl staining). Note that thick sections provide optimal cytoarchitectural clarity and are ideal to assist with tracing the nuclei’s borders as a reference for the analysis of the 60 μm sections.

Figure 3

Features and descriptive statistics (intervals represent one standard deviation) for the 48 cases used in this study with subsets broken down by LC subanalysis. Abbreviations: CDR, Clinical Dementia Rating; ht-NCI, hyperphosphorylated tau neuronal cytoplasmic inclusions; LC, locus coeruleus.

Figure 4

Features and descriptive statistics (intervals represent one standard deviation) for the 48 cases used in this study with subsets broken down by DRN subanalysis. Abbreviations: CDR, Clinical Dementia Rating; DRN, dorsal raphe nucleus; ht-NCI, hyperphosphorylated tau neuronal cytoplasmic inclusions.

Figure 5

Box plots depicting ht-NCI burden between subjects at early Braak stages. A significant difference was detected between Braak stages 0 and I in the number of ht-NCI+ neurons in the locus coeruleus (LC), with no significant difference between Braak stages I and II (a). No significant difference was detected between any Braak stages in the proportion of neurons with ht-NCI in LC (b). Likewise, no significant difference was detected in the number (c) or proportion (d) of ht-NCI+ neurons in the dorsal raphe nucleus (DRN).

Figure 6

Box plots depicting the burden of ht-NCI between subjects at early and late Braak stages. While there is no significant change in the number of neurons positive for ht-NCI in the locus coeruleus (LC) (a), a significant increase can be detected in the proportion of neurons with ht-NCI (b). The ht-NCI burden in the dorsal raphe nucleus (DRN) increases both as a number (c) and proportion (d) of neurons.

Figure 7

In Braak stages 0, I and II cases the middle third of the locus coeruleus (LC) has more ht-NCI+ neurons than the rostral and caudal thirds (a, c and e). When considering the proportion of the total neuronal population, the rostral third had significantly more ht-NCI than the caudal third, with no difference compared to the middle third (b). In Braak stages I and II cases we failed to observe any differences in the proportions of LC ht-NCI+ neurons in the dorsal raphe nucleus (DRN) subdivisions (d and f).