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Amyloid plaque deposition is recognized as the primary pathological hallmark of Alzheimer's disease(AD) that precedes other pathological events and cognitive symptoms. Plaque pathology represents itself with an immense polymorphic variety comprising plaques with different stages of amyloid fibrillization ranging from diffuse to fibrillar, mature plaques. The association of polymorphic Aß plaque pathology with AD pathogenesis, clinical symptoms and disease progression remains unclear. Advanced chemical imaging tools, such as functional amyloid microscopy combined with MALDI mass spectrometry imaging (MSI), are now enhanced by deep learning algorithms. This integration allows for precise delineation of polymorphic plaque structures and detailed identification of their associated Aß compositions. We here set out to make use of these tools to interrogate heterogenic plaque types and their associated biochemical architecture. Our findings reveal distinct Aß signatures that differentiate diffuse plaques from fibrilized ones, with the latter showing substantially higher levels of Aßx-40. Notably, within the fibrilized category, we identified a distinct subtype known as coarse-grain plaques. Both in sAD and fAD brain tissue, coarse grain plaques contained more Aßx-40 and less Aßx-42 compared with cored plaques. The coarse grain plaques in both sAD and fAD also showed higher levels of neuritic content including paired helical filaments (PHF-1)/phosphorylated phospho Tau-immunopositive neurites. Finally, the Aß peptide content in coarse grain plaques resembled that of vascular Aß deposits (CAA) though with relatively higher levels of Aß1-42 and pyroglutamated Aßx-40 and Aßx-42 species in coarse grain plaques. This is the first of its kind study on spatial in situ biochemical characterization of different plaque morphotypes demonstrating the potential of the correlative imaging techniques used that further increase the understanding of heterogeneous AD pathology. Linking the biochemical characteristics of amyloid plaque polymorphisms with various AD etiologies and toxicity mechanisms is crucial. Understanding the connection between plaque structure and disease pathogenesis can enhance our insights. This knowledge is particularly valuable for developing and advancing novel, amyloid-targeting therapeutics.
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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.
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Enfermedad de Alzheimer , Humanos , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Péptidos beta-Amiloides/metabolismo , Ratones Transgénicos , Encéfalo/metabolismo , Esfingolípidos/metabolismo , Placa Amiloide/metabolismo , Ceramidas/metabolismo , Modelos Animales de Enfermedad , Precursor de Proteína beta-Amiloide/metabolismoRESUMEN
Brain metastases represent an important clinical problem for patients with small-cell lung cancer (SCLC). However, the mechanisms underlying SCLC growth in the brain remain poorly understood. Here, using intracranial injections in mice and assembloids between SCLC aggregates and human cortical organoids in culture, we found that SCLC cells recruit reactive astrocytes to the tumour microenvironment. This crosstalk between SCLC cells and astrocytes drives the induction of gene expression programmes that are similar to those found during early brain development in neurons and astrocytes. Mechanistically, the brain development factor Reelin, secreted by SCLC cells, recruits astrocytes to brain metastases. These astrocytes in turn promote SCLC growth by secreting neuronal pro-survival factors such as SERPINE1. Thus, SCLC brain metastases grow by co-opting mechanisms involved in reciprocal neuron-astrocyte interactions during brain development. Targeting such developmental programmes activated in this cancer ecosystem may help prevent and treat brain metastases.
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Neoplasias Encefálicas , Neoplasias Pulmonares , Humanos , Animales , Ratones , Astrocitos/patología , Neoplasias Pulmonares/metabolismo , Ecosistema , Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Microambiente TumoralRESUMEN
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.
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In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for ß-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did ß-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.
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Acetilcoenzima A , Acetilcarnitina , Carnitina Aciltransferasas , Carnitina , Acetilcoenzima A/metabolismo , Acetilcarnitina/farmacología , Carnitina/metabolismo , Carnitina Aciltransferasas/metabolismo , Carnitina O-Acetiltransferasa/genética , Carnitina O-Acetiltransferasa/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Oxidación-Reducción , Humanos , Línea Celular TumoralRESUMEN
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.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Ratones , Animales , Humanos , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/patología , Placa Amiloide/diagnóstico por imagen , Placa Amiloide/patología , Enfermedades Neurodegenerativas/patología , Ratones Transgénicos , Encéfalo/patología , Imagen por Resonancia Magnética , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismoRESUMEN
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.
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Enfermedad de Alzheimer , Neuropatías Amiloides Familiares , Angiopatía Amiloide Cerebral , Demencia , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Angiopatía Amiloide Cerebral/genética , Demencia/patología , Dinamarca , Glicoproteínas de Membrana/metabolismo , Placa Amiloide , InglaterraRESUMEN
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.
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Metabolismo de los Lípidos , Neuroimagen/métodos , Placa Amiloide/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Microambiente Celular , Humanos , Lipidómica , Masculino , Ratones , Ratones Transgénicos , Microscopía ConfocalRESUMEN
Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) can infect the central nervous system (CNS) with dire consequences; in children and adults, HSV-1 may cause focal encephalitis, while HSV-2 causes meningitis. In neonates, both viruses can cause severe, disseminated CNS infections with high mortality rates. Here, we differentiated human induced pluripotent stem cells (iPSCs) towards cortical neurons for infection with clinical CNS strains of HSV-1 or HSV-2. Progenies from both viruses were produced at equal quantities in iPSCs, neuroprogenitors and cortical neurons. HSV-1 and HSV-2 decreased viability of neuroprogenitors by 36.0% and 57.6% (p < 0.0001), respectively, 48 h post-infection, while cortical neurons were resilient to infection by both viruses. However, in these functional neurons, both HSV-1 and HSV-2 decreased gene expression of two markers of synaptic activity, CAMK2B and ARC, and affected synaptic activity negatively in multielectrode array experiments. However, unaltered secretion levels of the neurodegeneration markers tau and NfL suggested intact axonal integrity. Viral replication of both viruses was found after six days, coinciding with 6-fold and 22-fold increase in gene expression of cellular RNA polymerase II by HSV-1 and HSV-2, respectively. Our results suggest a resilience of human cortical neurons relative to the replication of HSV-1 and HSV-2.
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Diferenciación Celular , Herpes Simple/virología , Herpesvirus Humano 1 , Herpesvirus Humano 2 , Neuronas/virología , Diferenciación Celular/genética , Supervivencia Celular , Sistema Nervioso Central , Regulación de la Expresión Génica , Herpes Simple/patología , Humanos , Células Madre Pluripotentes Inducidas , Neuronas/patología , Replicación Viral/fisiologíaRESUMEN
Point mutations in the amyloid precursor protein gene (APP) cause familial Alzheimer's disease (AD) by increasing generation or altering conformation of amyloid ß (Aß). Here, we describe the Uppsala APP mutation (Δ690-695), the first reported deletion causing autosomal dominant AD. Affected individuals have an age at symptom onset in their early forties and suffer from a rapidly progressing disease course. Symptoms and biomarkers are typical of AD, with the exception of normal cerebrospinal fluid (CSF) Aß42 and only slightly pathological amyloid-positron emission tomography signals. Mass spectrometry and Western blot analyses of patient CSF and media from experimental cell cultures indicate that the Uppsala APP mutation alters APP processing by increasing ß-secretase cleavage and affecting α-secretase cleavage. Furthermore, in vitro aggregation studies and analyses of patient brain tissue samples indicate that the longer form of mutated Aß, AßUpp1-42Δ19-24, accelerates the formation of fibrils with unique polymorphs and their deposition into amyloid plaques in the affected brain.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , HumanosRESUMEN
Since its discovery, amyloid-ß (Aß) has been the principal target of investigation of in Alzheimer's disease (AD). Over the years however, no clear correlation was found between the Aß plaque burden and location, and AD-associated neurodegeneration and cognitive decline. Instead, diagnostic potential of specific Aß peptides and/or their ratio, was established. For instance, a selective reduction in the concentration of the aggregation-prone 42 amino acid-long Aß peptide (Aß42) in cerebrospinal fluid (CSF) was put forward as reflective of Aß peptide aggregation in the brain. With time, Aß oligomers-the proposed toxic Aß intermediates-have emerged as potential drivers of synaptic dysfunction and neurodegeneration in the disease process. Oligomers are commonly agreed upon to come in different shapes and sizes, and are very poorly characterized when it comes to their composition and their "toxic" properties. The concept of structural polymorphism-a diversity in conformational organization of amyloid aggregates-that depends on the Aß peptide backbone, makes the characterization of Aß aggregates and their role in AD progression challenging. In this review, we revisit the history of Aß discovery and initial characterization and highlight the crucial role mass spectrometry (MS) has played in this process. We critically review the common knowledge gaps in the molecular identity of the Aß peptide, and how MS is aiding the characterization of higher order Aß assemblies. Finally, we go on to present recent advances in MS approaches for characterization of Aß as single peptides and oligomers, and convey our optimism, as to how MS holds a promise for paving the way for progress toward a more comprehensive understanding of Aß in AD research.
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Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/análisis , Química Encefálica , Espectrometría de Masas/métodos , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Humanos , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/líquido cefalorraquídeo , Placa AmiloideRESUMEN
ß-Amyloid (Aß) plaque formation is the major pathological hallmark of Alzheimer's disease (AD) and constitutes a potentially critical, early inducer driving AD pathogenesis as it precedes other pathological events and cognitive symptoms by decades. It is therefore critical to understand how Aß pathology is initiated and where and when distinct Aß species aggregate. Here, we used metabolic isotope labeling in APPNL-G-F knock-in mice together with mass spectrometry imaging to monitor the earliest seeds of Aß deposition through ongoing plaque development. This allowed visualizing Aß aggregation dynamics within single plaques across different brain regions. We show that formation of structurally distinct plaques is associated with differential Aß peptide deposition. Specifically, Aß1-42 is forming an initial core structure followed by radial outgrowth and late secretion and deposition of Aß1-38. These data describe a detailed picture of the earliest events of precipitating amyloid pathology at scales not previously possible.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Marcaje Isotópico , Cinética , Ratones , Ratones Transgénicos , Placa Amiloide/patologíaRESUMEN
MALDI mass spectrometry imaging (MSI) enables label-free, spatially resolved analysis of a wide range of analytes in tissue sections. Quantitative analysis of MSI datasets is typically performed on single pixels or manually assigned regions of interest (ROIs). However, many sparse, small objects such as Alzheimer's disease (AD) brain deposits of amyloid peptides called plaques are neither single pixels nor ROIs. Here, we propose a new approach to facilitate the comparative computational evaluation of amyloid plaque-like objects by MSI: a fast PLAQUE PICKER tool that enables a statistical evaluation of heterogeneous amyloid peptide composition. Comparing two AD mouse models, APP NL-G-F and APP PS1, we identified distinct heterogeneous plaque populations in the NL-G-F model but only one class of plaques in the PS1 model. We propose quantitative metrics for the comparison of technical and biological MSI replicates. Furthermore, we reconstructed a high-accuracy 3D-model of amyloid plaques in a fully automated fashion, employing rigid and elastic MSI image registration using structured and plaque-unrelated reference ion images. Statistical single-plaque analysis in reconstructed 3D-MSI objects revealed the Aß1-42Arc peptide to be located either in the core of larger plaques or in small plaques without colocalization of other Aß isoforms. In 3D, a substantially larger number of small plaques were observed than that indicated by the 2D-MSI data, suggesting that quantitative analysis of molecularly diverse sparsely-distributed features may benefit from 3D-reconstruction. Data are available via ProteomeXchange with identifier PXD020824.
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Enfermedad de Alzheimer/complicaciones , Elasticidad , Imagenología Tridimensional/métodos , Imagen Molecular , Placa Amiloide/complicaciones , Placa Amiloide/diagnóstico por imagen , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Animales , RatonesRESUMEN
One of the major hallmarks of Alzheimer's disease (AD) pathology is the formation of extracellular amyloid ß (Aß) plaques. While Aß has been suggested to be critical in inducing and, potentially, driving the disease, the molecular basis of AD pathogenesis is still under debate. Extracellular Aß plaque pathology manifests itself upon aggregation of distinct Aß peptides, resulting in morphologically different plaque morphotypes, including mainly diffuse and cored senile plaques. As plaque pathology precipitates long before any clinical symptoms occur, targeting the Aß aggregation processes provides a promising target for early interventions. However, the chain of events of when, where and what Aß species aggregate and form plaques remains unclear. The aim of this study was to investigate the potential of matrix-assisted laser desorption/ionization imaging mass spectrometry as a tool to study the evolving pathology in transgenic mouse models for AD. To that end, we used an emerging, chemical imaging modality - matrix-assisted laser desorption/ionization imaging mass spectrometry - that allows for delineating Aß aggregation with specificity at the single plaque level. We identified that plaque formation occurs first in cortical regions and that these younger plaques contain higher levels of 42 amino acid-long Aß (Aß1-42). Plaque maturation was found to be characterized by a relative increase in deposition of Aß1-40, which was associated with the appearance of a cored morphology for those plaques. Finally, other C-terminally truncated Aß species (Aß1-38 and Aß1-39) exhibited a similar aggregation pattern as Aß1-40, suggesting that these species have similar aggregation characteristics. These results suggest that initial plaque formation is seeded by Aß1-42; a process that is followed by plaque maturation upon deposition of Aß1-40 as well as deposition of other C-terminally modified Aß species.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides , Encéfalo/patología , Placa Amiloide/patología , Agregación Patológica de Proteínas/patología , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Imaging mass spectrometry (IMS) is a promising new chemical imaging modality that generates a large body of complex imaging data, which in turn can be approached using multivariate analysis approaches for image analysis and segmentation. Processing IMS raw data is critically important for proper data interpretation and has significant effects on the outcome of data analysis, in particular statistical modeling. Commonly, data processing methods are chosen based on rational motivations rather than comparative metrics, though no quantitative measures to assess and compare processing options have been suggested. We here present a data processing and analysis pipeline for IMS data interrogation, processing and ROI annotation, segmentation, and validation. This workflow includes (1) objective evaluation of processing methods for IMS datasets based on multivariate analysis using PCA. This was then followed by (2) ROI annotation and classification through region-based active contours (AC) segmentation based on the PCA component scores matrix. This provided class information for subsequent (3) OPLS-DA modeling to evaluate IMS data processing based on the quality metrics of their respective multivariate models and for robust quantification of ROI-specific signal localization. This workflow provides an unbiased strategy for sensitive annotation of anatomical regions of interest combined with quantitative comparison of processing procedures for multivariate analysis allowing robust ROI annotation and quantification of the associated molecular histology.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Agregado de Proteínas , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Amiloidosis , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila melanogaster , Humanos , Ratones , Fragmentos de Péptidos/genéticaRESUMEN
Amyloid-ß (Aß) pathology in Alzheimer's disease (AD) is characterized by the formation of polymorphic deposits comprising diffuse and cored plaques. Because diffuse plaques are predominantly observed in cognitively unaffected, amyloid-positive (CU-AP) individuals, pathogenic conversion into cored plaques appears to be critical to AD pathogenesis. Herein, we identified the distinct Aß species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated Aß polymorphism with amyloid conformation-sensitive dyes and a novel in situ MS paradigm for chemical characterization of hyperspectrally delineated plaque morphotypes. We found that maturation of diffuse into cored plaques correlated with increased Aß1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that Aß1-40 aggregates at the core structure of mature plaques, whereas Aß1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Aßx-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating Aß1-40 deposition. Experiments in tgAPPSwe mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of Aß1-42 and that Aß plaque maturation over time is associated with increases in Aß1-40. Finally, we found that Aß1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Aßx-42 pyroglutamation and Aß1-40 deposition are critical events in priming and maturation of pathogenic Aß from diffuse into cored plaques, underlying neurotoxic plaque development in AD.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Placa Amiloide/metabolismo , Ácido Pirrolidona Carboxílico/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Progresión de la Enfermedad , Humanos , Masculino , Ratones , Ratones Transgénicos , Modelos Animales , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
While the molecular mechanisms underlying Alzheimer's disease (AD) remain largely unknown, abnormal accumulation and deposition of beta amyloid (Aß) peptides into plaques has been proposed as a critical pathological process driving disease progression. Over the last years, neuronal lipid species have been implicated in biological mechanisms underlying amyloid plaque pathology. While these processes comprise genetic features along with lipid signaling as well as direct chemical interaction of lipid species with Aß mono- and oligomers, more efforts are needed to spatially delineate the exact lipid-Aß plaque interactions in the brain. Chemical imaging using mass spectrometry (MS) allows to probe the spatial distribution of lipids and peptides in complex biological tissues comprehensively and at high molecular specificity. As different imaging mass spectrometry (IMS) modalities provide comprehensive molecular and spatial information, we here describe a multimodal ToF-SIMS- and MALDI-based IMS strategy for probing lipid and Aß peptide changes in a transgenic mouse model of AD (tgAPPArcSwe). Both techniques identified a general AD-associated depletion of cortical sulfatides, while multimodal MALDI IMS revealed plaque specific lipid as well as Aß peptide isoforms. In addition, MALDI IMS analysis revealed chemical features associated with morphological heterogeneity of individual Aß deposits. Here, an altered GM1 to GM2/GM3 ganglioside metabolism was observed in the diffuse periphery of plaques but not in the core region. This was accompanied by an enrichment of Aß1-40arc peptide at the core of these deposits. Finally, a localization of arachidonic acid (AA) conjugated phosphatidylinositols (PI) and their corresponding degradation product, lyso-phosphatidylinositols (LPI) to the periphery of Aß plaques was observed, indicating site specific macrophage activation and ganglioside processing.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Glucolípidos/metabolismo , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Animales , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Masculino , Ratones Transgénicos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa de Ion SecundarioRESUMEN
Recent advances in the understanding of basic pathological mechanisms in various neurological diseases depend directly on the development of novel bioanalytical technologies that allow sensitive and specific chemical imaging at high resolution in cells and tissues. Mass spectrometry-based molecular imaging (IMS) has gained increasing popularity in biomedical research for mapping the spatial distribution of molecular species in situ. The technology allows for comprehensive, untargeted delineation of in situ distribution profiles of metabolites, lipids, peptides and proteins. A major advantage of IMS over conventional histochemical techniques is its superior molecular specificity. Imaging mass spectrometry has therefore great potential for probing molecular regulations in CNS-derived tissues and cells for understanding neurodegenerative disease mechanism. The goal of this review is to familiarize the reader with the experimental workflow, instrumental developments and methodological challenges as well as to give a concise overview of the major advances and recent developments and applications of IMS-based protein and peptide profiling with particular focus on neurodegenerative diseases. This article is part of the Special Issue "Proteomics".
Asunto(s)
Encéfalo/metabolismo , Espectrometría de Masas/métodos , Imagen Molecular/métodos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Proteómica/métodos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Humanos , Espectrometría de Masas/instrumentación , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteómica/instrumentaciónRESUMEN
BACKGROUND: Parathyroid hormone-related protein (PTHrP) is involved in intracellular calcium regulation, neural cell proliferation and synaptic transmission. To date, no studies have been performed to evaluate the potential of PTHrP concentrations in cerebrospinal fluid (CSF) as a biomarker of brain pathophysiology. In this study we evaluated the association between CSF concentrations of PTHrP with the core CSF biomarkers of Alzheimer's disease (AD). METHODS: PTHrP and calcium were analysed using validated mass spectrometry based methods in a set of CSF samples that tested positive (nâ¯=â¯45) and negative (nâ¯=â¯45) for the AD biomarkers, including total tau protein (T-tau), phosphorylated tau protein (P-tau) and amyloid-ß 42 (Aß42). The measured CSF concentrations of PTHrP and calcium (Ca) were evaluated for association with AD CSF biomarkers. RESULTS: PTHrP and Ca concentrations in CSF samples ranged between 25 and 137â¯pmol/L and 0.92-1.53â¯mmol/L, respectively. Higher concentrations of PTHrP were observed in association with increased concentrations of T-tau and P-tau in the AD and the control group; while a stronger correlation was observed in the control group (ρâ¯=â¯0.6, pâ¯<â¯0.0001; and ρâ¯=â¯0.72, pâ¯<â¯0.0001, for T-tau and P-tau, respectively). Negative correlation was observed between concentrations of PTHrP and Aß42 in the AD group (ρâ¯=â¯0.27, pâ¯=â¯0.015). A statistically significantly lower ratio Aß42/PTHrP was observed in the AD group (pâ¯<â¯0.0001). CONCLUSION: In the current study, we observed an association of PTHrP concentrations with concentrations of clinically used CSF biomarkers of AD. Concentrations of PTHrP were positively correlated with concentrations of T-tau and P-tau, suggesting an association with neuronal secretion and function, which is reduced upon progression to AD pathology. Our data suggest potential utility of the Aß42/PTHrP ratio in assessment of AD progression.