Plasma p-tau212 antemortem diagnostic performance and prediction of autopsy verification of Alzheimer's disease neuropathology.

Blood phosphorylated tau (p-tau) biomarkers, including p-tau217, show high associations with Alzheimer's disease (AD) neuropathologic change and clinical stage. Certain plasma p-tau217 assays recognize tau forms phosphorylated additionally at threonine-212, but the contribution of p-tau212 alone to AD is unknown. We developed a blood-based immunoassay that is specific to p-tau212 without cross-reactivity to p-tau217. Here, we examined the diagnostic utility of plasma p-tau212. In five cohorts (n = 388 participants), plasma p-tau212 showed high performances for AD diagnosis and for the detection of both amyloid and tau pathology, including at autopsy as well as in memory clinic populations. The diagnostic accuracy and fold changes of plasma p-tau212 were similar to those for p-tau217 but higher than p-tau181 and p-tau231. Immunofluorescent staining of brain tissue slices showed prominent p-tau212 reactivity in neurofibrillary tangles that co-localized with p-tau217 and p-tau202/205. These findings support plasma p-tau212 as a peripherally accessible biomarker of AD pathophysiology.

Spatial Neurolipidomics at the Single Amyloid-ß Plaque Level in Postmortem Human Alzheimer's Disease Brain.

Lipid dysregulations have been critically implicated in Alzheimer's disease (AD) pathology. Chemical analysis of amyloid-ß (Aß) plaque pathology in transgenic AD mouse models has demonstrated alterations in the microenvironment in the direct proximity of Aß plaque pathology. In mouse studies, differences in lipid patterns linked to structural polymorphism among Aß pathology, such as diffuse, immature, and mature fibrillary aggregates, have also been reported. To date, no comprehensive analysis of neuronal lipid microenvironment changes in human AD tissue has been performed. Here, for the first time, we leverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) through a high-speed and spatial resolution commercial time-of-light instrument, as well as a high-mass-resolution in-house-developed orbitrap system to characterize the lipid microenvironment in postmortem human brain tissue from AD patients carrying Presenilin 1 mutations (PSEN1) that lead to familial forms of AD (fAD). Interrogation of the spatially resolved MSI data on a single Aß plaque allowed us to verify nearly 40 sphingolipid and phospholipid species from diverse subclasses being enriched and depleted, in relation to the Aß deposits. This included monosialo-gangliosides (GM), ceramide monohexosides (HexCer), ceramide-1-phosphates (CerP), ceramide phosphoethanolamine conjugates (PE-Cer), sulfatides (ST), as well as phosphatidylinositols (PI), phosphatidylethanolamines (PE), and phosphatidic acid (PA) species (including Lyso-forms). Indeed, many of the sphingolipid species overlap with the species previously seen in transgenic AD mouse models. Interestingly, in comparison to the animal studies, we observed an increased level of localization of PE and PI species containing arachidonic acid (AA). These findings are highly relevant, demonstrating for the first time Aß plaque pathology-related alteration in the lipid microenvironment in humans. They provide a basis for the development of potential lipid biomarkers for AD characterization and insight into human-specific molecular pathway alterations.

Correlative Chemical Imaging and Spatial Chemometrics Delineate Alzheimer Plaque Heterogeneity at High Spatial Resolution.

We present a novel, correlative chemical imaging strategy based on multimodal matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), hyperspectral microscopy, and spatial chemometrics. Our workflow overcomes challenges associated with correlative MSI data acquisition and alignment by implementing 1 + 1-evolutionary image registration for precise geometric alignment of multimodal imaging data and their integration in a common, truly multimodal imaging data matrix with maintained MSI resolution (10 µm). This enabled multivariate statistical modeling of multimodal imaging data using a novel multiblock orthogonal component analysis approach to identify covariations of biochemical signatures between and within imaging modalities at MSI pixel resolution. We demonstrate the method's potential through its application toward delineating chemical traits of Alzheimer's disease (AD) pathology. Here, trimodal MALDI MSI of transgenic AD mouse brain delineates beta-amyloid (Aß) plaque-associated co-localization of lipids and Aß peptides. Finally, we establish an improved image fusion approach for correlative MSI and functional fluorescence microscopy. This allowed for high spatial resolution (300 nm) prediction of correlative, multimodal MSI signatures toward distinct amyloid structures within single plaque features critically implicated in Aß pathogenicity.

Tetramodal Chemical Imaging Delineates the Lipid-Amyloid Peptide Interplay at Single Plaques in Transgenic Alzheimer's Disease Models.

Beta-amyloid (Aß) plaque pathology is one of the most prominent histopathological feature of Alzheimer's disease (AD). The exact pathogenic mechanisms linking Aß to AD pathogenesis remain however not fully understood. Recent advances in amyloid-targeting pharmacotherapies highlight the critical relevance of Aß aggregation for understanding the molecular basis of AD pathogenesis. We developed a novel, integrated, tetramodal chemical imaging paradigm for acquisition of trimodal mass spectrometry imaging (MSI) and interlaced fluorescent microscopy from a single tissue section. We used this approach to comprehensively investigate lipid-Aß correlates at single plaques in two different mouse models of AD (tgAPPSwe and tgAPPArcSwe) with varying degrees of intrinsic properties affecting amyloid aggregation. Integration of the multimodal imaging data and multivariate data analysis identified characteristic patterns of plaque-associated lipid- and peptide localizations across both mouse models. Correlative fluorescence microscopy using structure-sensitive amyloid probes identified intra-plaque structure-specific lipid- and Aß patterns, including Aß 1-40 and Aß 1-42 along with gangliosides (GM), phosphoinositols (PI), conjugated ceramides (CerP and PE-Cer), and lysophospholipids (LPC, LPA, and LPI). Single plaque correlation analysis across all modalities further revealed how these distinct lipid species were associated with Aß peptide deposition across plaque heterogeneity, indicating different roles for those lipids in plaque growth and amyloid fibrillation, respectively. Here, conjugated ceramide species correlated with Aß core formation indicating their involvement in initial plaque seeding or amyloid maturation. In contrast, LPI and PI were solely correlated with general plaque growth. In addition, GM1 and LPC correlated with continuous Aß deposition and maturation. The results highlight the potential of this comprehensive multimodal imaging approach and implement distinct lipids in amyloidogenic proteinopathy.

Correlative Chemical Imaging Identifies Amyloid Peptide Signatures of Neuritic Plaques and Dystrophy in Human Sporadic Alzheimer's Disease.

Objective: Alzheimer's disease (AD) is the most common neurodegenerative disease. The predominantly sporadic form of AD is age-related, but the underlying pathogenic mechanisms remain not fully understood. Current efforts to combat the disease focus on the main pathological hallmarks, in particular beta-amyloid (Aß) plaque pathology. According to the amyloid cascade hypothesis, Aß is the critical early initiator of AD pathogenesis. Plaque pathology is very heterogeneous, where a subset of plaques, neuritic plaques (NPs), are considered most neurotoxic rendering their in-depth characterization essential to understand Aß pathogenicity. Methods: To delineate the chemical traits specific to NP types, we investigated senile Aß pathology in the postmortem, human sporadic AD brain using advanced correlative biochemical imaging based on immunofluorescence (IF) microscopy and mass spectrometry imaging (MSI). Results: Immunostaining-guided MSI identified distinct Aß signatures of NPs characterized by increased Aß1-42(ox) and Aß2-42. Moreover, correlation with a marker of dystrophy (reticulon 3 [RTN3]) identified key Aß species that both delineate NPs and display association with neuritic dystrophy. Conclusion: Together, these correlative imaging data shed light on the complex biochemical architecture of NPs and associated dystrophic neurites. These in turn are obvious targets for disease-modifying treatment strategies, as well as novel biomarkers of Aß pathogenicity.

Plasma p-tau212: antemortem diagnostic performance and prediction of autopsy verification of Alzheimer's disease neuropathology.

Blood phosphorylated tau (p-tau) biomarkers, including p-tau217, show high associations with Alzheimer's disease (AD) neuropathologic change and clinical stage. Certain plasma p-tau217 assays recognize tau forms phosphorylated additionally at threonine-212, but the contribution of p-tau212 alone to AD is unknown. We developed a blood-based immunoassay that is specific to p-tau212 without cross-reactivity to p-tau217. Thereafter, we examined the diagnostic utility of plasma p-tau212. In five cohorts (n=388 participants), plasma p-tau212 showed high performances for AD diagnosis and for the detection of both amyloid and tau pathology, including at autopsy as well as in memory clinic populations. The diagnostic accuracy and fold changes of plasma p-tau212 were similar to those for p-tau217 but higher than p-tau181 and p-tau231. Immunofluorescent staining of brain tissue slices showed prominent p-tau212 reactivity in neurofibrillary tangles that co-localized with p-tau217 and p-tau202/205. These findings support plasma p-tau212 as a novel peripherally accessible biomarker of AD pathophysiology.

Chemical traits of cerebral amyloid angiopathy in familial British-, Danish-, and non-Alzheimer's dementias.

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2-derived amyloidogenic peptides, ABri and ADan that resemble APP/ß-amyloid (Aß) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C-terminal amino acids, CAA in FDD is characterized by co-aggregation of ADan with Aß, while in contrast no Aß deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aß co-aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N-terminally truncated- and pyroglutamate-modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C-terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co-deposition with Aß x-42 and Aß x-40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aß. When compared with FDD, Aß in CAA+ showed more C-terminal and less N-terminally truncations. In FDD, ADan showed spatial co-localization with Aß3pE-40 and Aß3-40 but not with Aßx-42 species. This suggests an increased aggregation propensity of Aß in FDD that promotes co-aggregation of both Aß and ADan. Further, CAA maturity appears to be mainly governed by Aß content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP-derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non-AD-related vascular amyloid pathology. Cover Image for this issue: https://doi.org/10.1111/jnc.15424.

Structural amyloid plaque polymorphism is associated with distinct lipid accumulations revealed by trapped ion mobility mass spectrometry imaging.

Understanding of Alzheimer's disease (AD) pathophysiology requires molecular assessment of how key pathological factors, specifically amyloid ß (Aß) plaques, influence the surrounding microenvironment. Here, neuronal lipids have been implicated in Aß plaque pathology, though the lipid microenvironment in direct proximity to Aß plaques is still not fully resolved. A further challenge is the microenvironmental molecular heterogeneity, across structurally polymorphic Aß features, such as diffuse, immature, and mature, fibrillary aggregates, whose resolution requires the integration of advanced, multimodal chemical imaging tools. Herein, we used matrix-assisted laser desorption/ionization trapped ion mobility spectrometry time-of-flight based mass spectrometry imaging (MALDI TIMS TOF MSI) in combination with hyperspectral confocal microscopy to probe the lipidomic microenvironment associated with structural polymorphism of Aß plaques in transgenic Alzheimer's disease mice (tgAPPSWE ). Using on tissue and ex situ validation, TIMS MS/MS facilitated unambiguous identification of isobaric lipid species that showed plaque pathology-associated localizations. Integrated multivariate imaging data analysis revealed multiple, Aß plaque-enriched lipid patterns for gangliosides (GM), phosphoinositols (PI), phosphoethanolamines (PE), and phosphatidic acids (PA). Conversely, sulfatides (ST), cardiolipins (CL), and polyunsaturated fatty acid (PUFA)-conjugated phosphoserines (PS), and PE were depleted at plaques. Hyperspectral amyloid imaging further delineated the unique distribution of PA and PE species to mature plaque core regions, while PI, LPI, GM2 and GM3 lipids localized to immature Aß aggregates present within the periphery of Aß plaques. Finally, we followed AD pathology-associated lipid changes over time, identifying plaque- growth and maturation to be characterized by peripheral accumulation of PI (18:0/22:6). Together, these data demonstrate the potential of multimodal imaging approaches to overcome limitations associated with conventional advanced MS imaging applications. This allowed for the differentiation of both distinct lipid components in a complex micro-environment as well as their correlation to disease-relevant amyloid plaque polymorphs. Cover Image for this issue: https://doi.org/10.1111/jnc.15390.

Oral gabapentin suppresses pentylenetetrazole-induced seizure-like behavior and cephalic field potential in adult zebrafish.

We report the effect of orally administered gabapentin (GBP) on pentylenetetrazole (PTZ)-induced seizure-like activity in adult zebrafish. Zebrafish were pretreated with vehicle or GBP using a novel method of precise oral administration, followed by an intraperitoneal administration of PTZ. Behavioral assessment was carried out using locomotion-based video-tracking analysis and seizure score assignment using visual observation. Cephalic field potential recordings of the zebrafish brain were conducted using an electrical data acquisition system. Orally administered GBP significantly suppressed the seizure-like locomotor activity and strong slow-wave (~3Hz) activity in the cephalic field potential caused by PTZ. This work is the first report of the activity of an orally delivered anticonvulsant in adult zebrafish. Our study provides behavioral and physiological evidence in support of an adult zebrafish model for studying seizures including excitotoxic brain injury and a novel in vivo framework for the evaluation of pharmacological modulators of epilepsy.