Blood phosphorylated (p)-tau 181 and p-tau 217 have been proposed as accurate biomarkers of Alzheimer’s disease (AD) pathology. However, blood p-tau 181 is also elevated in amyotrophic lateral sclerosis (ALS) without a clearly identified source. We measured serum p-tau 181 and p-tau 217 in a multicentre cohort of ALS (n = 152), AD (n = 111) cases and disease controls (n = 99) recruited from four different centres. Further, we investigated the existence of both p-tau species using immunohistochemistry (IHC) and mass spectrometry (MS) in muscle biopsies of ALS cases (IHC: n = 13, MS: n = 5) and disease controls (IHC: n = 14, MS: n = 5) from one cohort. Serum p-tau 181 and p-tau 217 were higher in AD and ALS patients compared to disease controls. IHC and MS analyses revealed the presence of p-tau 181 and 217 in muscle biopsies from both ALS cases and disease controls, with ALS samples showing increased p-tau reactivity in atrophic muscle fibres. Blood p-tau species could potentially be used to diagnose both ALS and AD.

The quantification of amyloid-beta (Aβ) peptides in blood plasma as potential biomarkers of Alzheimer’s disease (AD) is hampered by very low Aβ concentrations and the presence of matrix components that may interfere with the measurements. We developed a two-step immunoassay for the simultaneous measurement of the relative levels of Aβ38, Aβ40 and Aβ42 in human EDTA plasma. The assay was employed for the study of 23 patients with dementia of the Alzheimer’s type (AD-D) and 17 patients with dementia due to other reasons (OD). We examined relationships with the clinical diagnosis, cerebral Aβ load as quantified by amyloid-positron emission tomography, apolipoprotein E genotype, Aβ levels and Tau protein in cerebrospinal fluid. Preconcentration of plasma Aβ peptides by immunoprecipitation substantially facilitated their immunological measurements. The Aβ42/Aβ40 and Aβ42/Aβ38 ratios were statistically significantly lower in the AD-D patients than in the OD group. The areas under the receiver operating characteristic curves reached 0.87 for the Aβ42/Aβ40 ratio and 0.80 for the Aβ42/Aβ38 ratio. The measurement of plasma Aβ peptides with an immunological assay can be improved by preconcentration via immunoprecipitation with an antibody against the Aβ amino-terminus and elution of the captured peptides by heating in a mild detergent-containing buffer. Our findings support the Aβ42/Aβ40 ratio in blood plasma as a promising AD biomarker candidate which correlates significantly with the validated core biomarkers of AD. Further studies will be needed for technical advancement of the assay and validation of the biomarker findings.

The deposition of neurotoxic amyloid-β (Aβ) peptides in plaques in the brain parenchyma and in cerebral blood vessels is considered to be a key event in Alzheimer’s disease (AD) pathogenesis. Although the presence and impact of full-length Aβ peptides such as Aβ1–40 and Aβ1–42 have been analyzed extensively, the deposition of N-terminally truncated Aβ peptide species has received much less attention, largely because of the lack of specific antibodies. This paper describes the generation and characterization of novel antibodies selective for Aβ4–x peptides and provides immunohistochemical evidence of Aβ4–x in the human brain and its distribution in the APP/PS1KI and 5XFAD transgenic mouse models. The Aβ4–x staining pattern was restricted mainly to amyloid plaque cores and cerebral amyloid angiopathy in AD and Down syndrome cases and in both AD mouse models. In contrast, diffuse amyloid deposits were largely negative for Aβ4–x immunoreactivity. No overt intraneuronal staining was observed. The findings of this study are consistent with previous reports demonstrating a high aggregation propensity of Aβ4–x peptides and suggest an important role of these N-truncated Aβ species in the process of amyloidogenesis and plaque core formation.

Senile plaques consisting of amyloid-beta (Aβ) peptides are a major pathological hallmark of Alzheimer’s disease (AD). Aβ peptides are heterogeneous regarding the exact length of their amino- and carboxy-termini. Aβ1-40 and Aβ1-42 are often considered to represent canonical “full-length” Aβ species. Using immunohistochemistry, we analyzed the distribution of Aβ1-x, Aβx-42 and Aβ4-x species in amyloid deposits in the subiculum, hippocampus and cortex in 5XFAD mice during aging. Overall plaque load increased in all three brain regions, with the subiculum being the area with the strongest relative plaque coverage. In the subiculum, but not in the other brain regions, the Aβ1-x load peaked at an age of five months and decreased thereafter. In contrast, the density of plaques positive for N-terminally truncated Aβ4-x species increased continuously over time. We hypothesize that ongoing plaque remodeling takes place, leading to a conversion of deposited Aβ1-x peptides into Aβ4-x peptides in brain regions with a high Aβ plaque burden.

Background: A reduced amyloid-β (Aβ)42/40 peptide ratio in blood plasma represents a peripheral biomarker of the cerebral amyloid pathology observed in Alzheimer’s disease brains. The magnitude of the measurable effect in plasma is smaller than in cerebrospinal fluid, presumably due to dilution by Aβ peptides originating from peripheral sources. We hypothesized that the observable effect in plasma can be accentuated to some extent by specifically measuring Aβ1–42 and Aβ1–40 instead of AβX–42 and AβX–40. Methods: We assessed the plasma AβX–42/X–40 and Aβ1–42/1–40 ratios in an idealized clinical sample by semi-automated Aβ immunoprecipitation followed by closely related sandwich immunoassays. The amyloid-positive and amyloid-negative groups (dichotomized according to Aβ42/40 in cerebrospinal fluid) were compared regarding the median difference, mean difference, standardized effect size (Cohen’s d) and receiver operating characteristic curves. For statistical evaluation, we applied bootstrapping. Results: The median Aβ1–42/1–40 ratio was 20.86% lower in amyloid-positive subjects than in the amyloid-negative group, while the median AβX–42/X–40 ratio was only 15.56% lower. The relative mean difference between amyloid-positive and amyloid-negative subjects was −18.34% for plasma Aβ1–42/1–40 compared to −15.50% for AβX–42/X–40. Cohen’s d was 1.73 for Aβ1–42/1–40 and 1.48 for plasma AβX–42/X–40. Unadjusted p-values < 0.05 were obtained after .632 bootstrapping for all three parameters. Receiver operating characteristic analysis indicated very similar areas under the curves for plasma Aβ1–42/1–40 and AβX–42/X–40. Conclusions: Our findings support the hypothesis that the relatively small difference in the plasma Aβ42/40 ratio between subjects with and without evidence of brain amyloidosis can be accentuated by specifically measuring Aβ1–42/1–40 instead of AβX–42/X–40. A simplified theoretical model explaining this observation is presented.

Here we present a water-in-air droplet platform for micro-compartmentalization for single molecule guided synthesis and analysis consisting of a flow-system hosting dense arrays of aqueous microdroplets on a glass surface surrounded by air. The droplets are formed in a few seconds by passing a waterfront over the array of hydrophilic spots surrounded by a hydrophobic coating, thus forming a micro-droplet array (MDA). The droplet volumes are tunable from approximately 50 femtoliter to 20 picoliter by adjusting the size of the hydrophilic spots. MDAs consisting of femtoliter volume droplets were stable for more than 24 hours in air at 37 °C in a reversibly sealed flow-system, thus allowing us to perform assays that require long incubations in the droplets. Using differently fluorescing liquids, it was further shown that droplets can be reformed on the same MDA several times by passing a new liquid plug over the surface, and that fluorescence from one reaction can be washed away with little to no carry-over, hence allowing for multistep reactions to be carried out on the system. The MDA created by an air/water interface supported digital immunoassays as was demonstrated by measuring the Aβ₄₂ peptide in cerebrospinal fluid of Alzheimers patients and control patients. To demonstrate a two step droplet assay, first, histidine tagged peptides were expressed in the droplets and bound to the droplet-enclosed surface. Subsequently, the his-tagged peptides were detected using enzyme-conjugated antibodies in a second droplet generation step. As such, the chip demonstrates features necessary for library preparations for high throughput screening applications.