Unraveling amyloid beta-pathology in Alzheimer's Disease
Abstract
Neuropathology of Alzheimer’s disease (AD) is characterized by the accumulation and aggregation of Amyloid β (Aβ) peptides into extracellular plaques of the brain. The Aβ peptides, composed of forty of amino acids, are generated from amyloid precursor proteins (APP) by β- and γ-secretases. Aβ deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls. This has been known as cerebral amyloid angiopathy (CAA). While a variety of Aβ peptides were identified, detailed production and distribution of individual Aβ peptides in pathological tissues of AD and CAA is not fully addressed. In recent years, Matrix- assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has entered the field of tissue-based research by providing unique advantages for analyzing tissue specimen in an unprecedented detail. A broad spectrum of analytes ranging from proteins, peptides, protein modification over small molecules, drugs and their metabolites as well as pharmaceutical components, endogenous cell metabolites, lipids, and other analytes are made accessible by this in situ technique in tissue. With MALDI-IMS, here we have succeeded in comprehensive visualization of Aβ species in human autopsied brains from AD and CAA. By MALDI-IMS, the depositions of Aβ1-42 was characteristic to senile plaque in brain parenchyma and depositions of shorter Aβ peptides such as Aβ1-40 were characteristic to leptomeningeal blood vessel walls which was consistent with immunohistochemical analysis. Furthermore, distinct depositions of N-terminal truncated Aβ40 and Aβ42 including pyroglutamylation (pE) at Glu-3 were also clarified. These observations were not covered with immunohitochemical analysis so far because specific antibodies are not available. With current analysis, it must be feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic and pathological observations for neurological diseases. MALDI-IMS with standardized protocols, further development of the analysis softwear, and the accumulating comprehensive database, would replace inventional immunocytochmistry in future.
Visualization of aldosterone on adrenal frozen sections
Abstract
Background: Primary aldosteronism (PA) results not only in blood high pressure as well as subsequent cardiovascular diseases. Aldosterone-producing adenomas (APAs) is one of the major lesion of PA that frequently occurred with aldosterone-producing cell clusters (APCCs) where aldosterone synthase (CYP11B2) expressing small lesions, in adrenal glands specimens of PA-patients. Moreover, it has been shown that hyperaldosteronism can be caused only by multiple APCCs. However, whether the CYP11B1/2 enzymes in these lesions are actively producing aldosterone or not, has not been demonstrated.
Methods: We employed on-tissue derivatization of steroid hormones slightly modified from as described in Cobice et al (Anal. Chem. 2013, 85, 11576). Prior to matrix coating, Girard-T reagent (1.0 mg/mL in 20% acetic acid) was manually spray-coated tissue sections with the solution using an artistic airbrush (Procon Boy FWA Platinum 0.2-mm caliber airbrush, Mr. Hobby, Tokyo, Japan), and then DHB as a matrix (50 mg/mL, dissolved in 80% ethanol containing saturated ammonium sulfate [AS]) was sprayed onto the tissues. We note that the addition of AS reduced ion suppression effect of analyte caused by excess presence of derivatization-reagent. MALDI-IMS was performed using a 7T FT-ICR-MS (Solarix Bruker Daltonik, Bremen, Germany) equipped with an Nd:YAG laser.
Results:
More than 8 molecular species of steroid hormones including cortisol, progesterone and aldosterone were successfully detected and visualized with mass accuracy of 5ppm, in the specimen of PA patients. Intriguingly, in adrenal glands with multiple APCCs, aldosterone was found to be strongly localized within APCCs, demonstrating that small APCC is functionally aldosterone producing lesion. Moreover, in large APAs (≥5 mm), aldosterone was detected only in limited parts of CYP11B2 positive tumor areas, where only progesterone co-localized area.
The results suggested that (1) APCCs may be a precursor of small (micro) APAs, and thus grown APAs still retain steroid producing features of APCCs, and (2) the heterogeneous steroid localization pattern in large APAs may be due to dysregulation of steroidogenic enzymes by insufficient supply of a substrate, i.e., progesterone.
Novel aspect:
Novel functionally aldosterone producing lesion was identified by on-tissue derivatized coupled imaging MS, in specimens of PA patient.
Defining and Supporting Integrated Strategies for Integrated Systems Biology through High Performance MALDI Imaging
Abstract
Introduction of advanced ionization techniques supporting complex biomolecules served to draw the disciplines of mass spectrometry and biology together creating a wealth of molecular based information related to biological processes. For years, the aptitude of mass spectrometry laboratories worldwide increased through the development of sample cleanup and isolation steps for introduction to the mass spectrometer.
One missing element of these efficient pathways was spatially localizing detected biochemical processes to organ structures or tissue regions. While the general spatial location could be anecdotally inferred, biochemical changes along or within these structures was missing, leaving a gap in understanding between the molecular data traditional histological presentation.
Imaging mass spectrometry (IMS) has emerged as a bridge tool for the biological sciences that leverages the high sensitivity and selectivity of mass spectrometry with the ability to describe molecular results in the context of traditional histopathology. Recent improvements in workflows and instrumentation have combined to help overcome several of the many limitations and challenges of this emerging technique. Within the scope of these improvements comes the need for selecting the most optimized platform for addressing the question or testing the hypothesis at hand which can be daunting even with a direct knowledge of the technique.
We will show several examples of how MALDI imaging was successfully used to chase the “omics” cascade in cellular systems to help complete the picture of systems biology assembled from “vial based” mass spectrometric methods. The greater perspective will be offered that describes evaluating project needs and properly defining and selecting platform requirement for the project examples offered. Getting a proper handle on the wide variable space available to the MALDI imaging researcher is vital to project success and will be discussed in detail from sample preparation to data evaluation strategies.
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