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

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.

Comparative kinetic analysis of cholinesterase methods in rat and human erythrocytes and plasma.

A kinetic analysis of the substitution of 6,6'-dithiodinicotinic acid (DTNA) for 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) for the determination of rat and human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BuChE; EC 3.1.1.8) is presented. Increasing concentrations of DTNB, but not DTNA, significantly increased Km for the substrate acetylthiocholine but had little or no effect on Vmax for rat or human AChE. The coupling agent DTNA was more efficient than DTNB, as demonstrated by the higher Vmax/Km ratio for the former. DTNB, more so than DTNA, caused linear mixed-type inhibition of rat AChE. Poor precision was observed for the DTNB versus DTNA method. Reagent blanks were a significant component of rat, but not human, AChE activity. The use of DTNA in place of DTNB is recommended for quantitative mechanistic investigations of cholinesterases. The most practical aspect of the DTNA method is that it can be adapted to automated instruments which can monitor the change in absorbance at 340 nm, away from the hemoglobin peak.