Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid β protein on brain tissues of Alzheimer's disease and cerebral amyloid angiopathy samples using MALDI-IMS.
The 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 40 amino acids, are generated from amyloid precursor proteins (APP) by β- and γ-secretases. Aβ is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of Aβ peptides were identified, the detailed production and distribution of individual Aβ peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 µm. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various Aβ species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 µm) imaging of an AD brain and severe CAA sample clearly shows that Aβ1-36 to Aβ1-41 were deposited into leptomeningeal vessels, while Aβ1-42 and Aβ1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.
In order to make the diagnosis and to understand the pathogenesis of neurodegenerative disorders, precise molecular identification of pathological deposits is essential1. In the course of AD, Aβ is produced to make SPs in the cerebral parenchyma and deposited in vessels long before disease onset2,3,4,5,6. While Aβ1-42 is the predominant peptide in the SP of AD brains, other Aβ variants, such as N-terminal or C-terminal truncated or modified Aβs, are also identified in affected AD brains7,8,9,10. A full picture of the broad range of Aβ species in human brains, especially with AD and cerebral amyloid angiopathy (CAA)11, will help scientists understand Aβ production, metabolism, and deposition.
As the classical approach of neuropathology, immunohistochemistry (IHC) has been the most conclusive method to determine the location of Aβs in the brain tissues12,13,14,15. In general, IHC cannot distinguish molecules when several epitopes coexist simultaneously. By contrast, an emerging mass spectrometry-based proteomic analysis is a valuable approach especially for analyzing a variety of Aβ species in brain tissues, which cannot be differentiated with antibodies16,17. The conventional mass spectrometry-based analysis of brain lysates and immuno-precipitated samples fails to detect minor Aβ peptides and loses distribution information of Aβ in the brain tissue.
In earlier works, visualizing Aβ deposits in mouse brains has been successful using a transgenic animal model of AD, such as APP23. However, this process still needs technical advancements to compare IMS and IHC with respect to resolution and sensitivity18,19,20. AD neuropathology should be studied on human brains, and we used MALDI-IMS technology on human autopsy brain tissues to obtain comprehensive protein mapping21. For this purpose, we developed a protocol for an advanced type of mass spectrometry that has advantages in its rapidity, sensitivity, and reproducibility.
Here, tissue samples were collected at the Tokyo Metropolitan Geriatric Hospital, which provides community-based medical services to the elderly population in Japan. The brain autopsy samples used in the study were registered to the Brain Bank for Aging Research (BBAR) with the informed consent of the deceased’s relatives. The BBAR is approved by the ethics committee of the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology. For all brains registered at the brain bank, we obtained written informed consents for their use for medical research from the patients or their families. The patients were placed in a cold (4 °C) room within 2 h after death to reduce postmortem changes in the brain. All methods described here have been approved by Doshisha University and the Brain Bank for Aging Research, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology.
1. Preparation of Tissue Sections for IMS
2. MALDI-IMS
3. Data Processing
Sporadic AD patients with CAA (n = 5; mean age = 83.2 y) and aged subjects with senile plaque free (SP O) and CAA (n = 5; mean age = 77.2 y) were analyzed (Table 1). The CAA phenotypes of patient #3 were most prominent in this study. Distributions of Aβ1-40 and Aβ1-42 deposits in the brain tissue from patient #3 were visualized with MALDI-IMS (Figure 1). There were no significant signals in nonpathological control brains (patients #9 and #10), as shown in Figure 1. Here, MALDI-IMS clearly visualized that Aβ1-42 was preferentially deposited as SPs in the cerebral parenchyma. By contrast, shorter Aβs, such as Aβ1-36 to 1-41, were preferentially deposited on the leptomeningeal vascular areas (Figure 1).
Distributions of Aβ40 and Aβ42 were further validated with IHC using adjacent frozen sections of the tissues. The anti-Aβ40 antibody labeled CAA (arrows in Figure 2B), which is in clear contrast to the distribution of Aβ42 in the cerebral parenchyma as SPs. For Aβ1-41, we are the first to detect this fragment in human brains, and we have generated specific antibodies that can differentiate Aβ41 from Aβ40 andAβ4221.
The spatial resolution of the MALDI-IMS is generally the most important factor to be improved upon. In Figure 1 and Figure 2 show MALDI-IMS with a 100 µm pitch resolution and obtain an overall distribution profile for a relatively wide area21. It is difficult to define amyloid deposition exactly at the subarachnoid space, including vascular structure. To portray fine tissue structures of subarachnoid vessels and the surface of the cortex, high-resolution MALDI imaging (40 µm: Figure 3; 20 µm: Figure 4 and Figure 5) was performed. As a result, MALDI-IMS clearly demonstrated that shorter Aβs, such as Aβ1-36 to 1-41, are distributed in the walls of arteries, which is comparable with IHC.
MALDI-IMS demonstrated the detailed distributions of both Aβx-40 and Aβx-42 (x = 2, 4, 5, 6, 7, 8, 9, and 11pE) in AD accompanied with moderate CAA brain. For example, while Aβx-40 species showed a similar distribution profile to Aβ1-40, Aβx-42 species showed a different distribution pattern with Aβ1-4221. By the current protocol, single ion images of the individual Aβ peptides observed with MALDI-IMS are assigned to Aβ species. By contrast, acquired image data can be investigated using unsupervised multivariate statistics in order to obtain image segmentation of anatomical regions of interest for the further analysis of undefined proteins. Figure 5 shows a segmentation map obtained with a bisecting k-means analysis applied to the same section from patient #321. This clustering method successfully identified plaque-like structures in the parenchyma (blue in Figure 5C) and vascular structures in the subarachnoid space (green in Figure 5C). It is interesting to find a small circular area in the parenchyma, which is detected just around a small arteriole in the parenchyma, as well as in the subarachnoid space (purple in Figure 5C). This is verified by single ion images of these individual Aβ peptides in Figure 5D-5F.
Figure 1: MALDI-IMS of a frozen AD/CAA brain section. Various C-terminal truncated Aβ peptides in AD accompanying severe CAA (patient #3) are visualized in the left panel and controls (patient #9 on the right and patient #10 on the left in all images) in the right panel. Aβ1-36 to Aβ1-41 are preferentially deposited in leptomeningeal blood vessels, while Aβ1-42 and Aβ1-43 are deposited in the cerebral parenchyma as senile plaques in case #3, while there was no signal in the control patients' brains (cases #9 and #10). Resolution = 100 μm. Scale bar = 5 mm. This figure has been modified with permission from Kakuda et al.21. Please click here to view a larger version of this figure.
Figure 2: MALDI-IMS of frozen AD/CAA brain sections and adjacent sections of the occipital cortex from AD brains. (A – C) The frozen AD/CAA brain sections and (D and E) adjacent sections of the occipital cortex from AD brains were immune-stained and focused on arteriole and cerebral parenchyma, using antibodies against Aβ40 (D: BA27) or Aβ42 (E: anti-Aβ42 polyclonal). Both analyses demonstrated that Aβ40 is preferentially deposited in leptomeningeal blood vessels (arrows in panel D) and arterioles in the subarachnoid space and the cerebral parenchyma forming CAA. In contrast, Aβ42 is mainly deposited in SPs. For IMS, Resolution = 100 μm. Scale bar = 5 mm (A, B, and C) = 5 mm, 500 (D and E). This figure has been modified with permission from Kakuda et al.21. Please click here to view a larger version of this figure.
Figure 3: MALD-IMS of frozen AD/CAA brain sections (patient #3) at a resolution of 40 µm. Various C-terminal and N-terminal truncated and modified Aβ peptides in AD accompanying severe CAA (patient #3). Aβ1-36 to Aβ1-41 are preferentially deposited in leptomeningeal blood vessels, while Aβ1-42 and Aβ1-43 are deposited in the cerebral parenchyma as senile plaques. Scale bars = 1 mm (A-B), 2 mm (upper left). Resolution = 40 μm. Please click here to view a larger version of this figure.
Figure 4: MALDI-IMS of frozen AD/CAA brain sections (patient #3) at a resolution of 20 µm. This panel shows various C-terminal and N-terminal truncated and modified Aβ peptides in AD accompanying severe CAA (patient #3). Aβ1-36 to Aβ1-41 are preferentially deposited in leptomeningeal blood vessels, while Aβ1-42 and Aβ1-43 are deposited in the cerebral parenchyma as senile plaques. Scale bars = 200 μm (a, b, c), 1 mm (upper left). Resolution = 20 μm. Please click here to view a larger version of this figure.
Figure 5: Segmentation map, obtained by MALDI-IMS of a frozen AD brain section, reveals putative senile plaque, large subarachnoid vessel structures, and small parenchymal arterioles. (A) Segmentation map obtained from a multivariate image analysis of MALDI-IMS data. (B) Bisecting k-means based clustering analysis identified plaque-like and vessel-like structures in the occipital cortex. The clusters and substructures and their relations are shown as nodes (e.g.,1-0-0). (C) Distinct Aβ peptide localization patterns resembling plaques (blue), subarachnoid vessels (green), and arteriole (red) structures. Note that a few red clusters are also distributed in the subarachnoid space. This is verified by single ion images of these individual Aβ peptides at a 20 µm resolution: (D) Aβ 1-40, (E) Aβ 1-41, and (F) Aβ 1-42. Please click here to view a larger version of this figure.
Case | Gender | Age at death | Braak SP | CAA |
1 | M | 83 | C | 0.5 |
2 | M | 88 | C | 1 |
3 | M | 84 | C | 2 |
4 | M | 78 | C | 1 |
5 | M | 83 | C | 1 |
6 | M | 84 | O | 0 |
7 | M | 78 | O | 0 |
8 | M | 70 | O | 0 |
9 | M | 73 | O | 0 |
10 | M | 81 | O | 0 |
Table 1: Clinical and pathological data of AD with CAA cases and aged SP O subjects. Human cortical specimens for IMS and IHC were obtained from the Brain Bank at Tokyo Metropolitan Institute of Gerontology. Each brain specimen was taken from the occipital cortex of five AD patients and five age-matched controls. The extent of amyloid deposition as shown by an Aβ monoclonal antibody was defined by Braak SP (amyloid) stages. At stage O, there are almost no senile plaques throughout the isocortex. At stage C, virtually all the isocortical areas are affected. AD brains are invariable at stage C. This table has been modified with permission from Kakuda et al.21.
Here we demonstrated a detailed protocol and results of the visualization of Aβ and its isoforms from several autopsied brains with AD and CAA with MALDI-IMS. The deposition profile of Aβs was drastically changed from Aβ1-42 to its N- and C-terminal variations. Aβ1-41 was first identified and visualized in human brains with the current protocol and was further validated with IHC21. Considering that the morphology of deposited Aβ by IMS with a high-resolution analysis (20 μm) must be in good agreement with IHC, IMS and IHC equally contribute to distinguishing Aβ deposits by their location and protein contents, as well as by their morphology. As the entire experiment described here was conducted in the occipital cortex, studying the localization of different beta-amyloid species across all brain regions will be generalized with future experiments using the current protocol.
Critical steps within the protocol are tissue preparation steps to obtain an effective ionization of aggregated proteins in human brain tissues. A matrix layer is required to absorb the laser energy and induce desorption and ionization of analytes. In this process, an entire tissue section is homogeneously coated with small crystals. Homogenous cocrystallization of the analyte with the matrix is crucial for high-sensitivity and artifact-free imaging. Each of the three spray methods has its own advantages. Manual spray coating is one of the most frequently used methods. An airbrush is convenient; however, it requires skillful operation. As precise and reproducible experimental technique is essential, using an ultrasonic sprayer and/or an automatic sprayer as described in the current protocols is recommended. With respect to an ultrasonic sprayer, it will not be influenced by the humidity and temperature of the room because it is sprayed into a chamber. Meanwhile, with an automatic sprayer, relatively preserved spatial resolution with good reproducibility is obtained. Generally, the spatial resolution obtained with the three methods increase in the order of 1) airbrush, 2) automatic device, and 3) ultrasonic sprayer.
Most importantly, this protocol was originally generated to detect and visualize Aβs in human autopsied brain samples. The visualization of Aβs with MALDI-IMS for APP23 mice, which is generated as an animal model of AD based on the Swedish-type mutation of human APP, has been reported earlier by others with an existing method18,19. However, the former protocol applied to APP23 was not sufficient to visualize Aβ in its lateral resolution and sensitivity. Earlier works discussed that high Aβ concentrations outside the tissue boundary are clearly artifacts in APP23 imaging with their protocol18,19. That means the so-called 'blur' between real SPs and IMS images due to the matrix extraction step was inevitable with MALDI-type imaging. However, in the current protocol, the blur disappeared and each spectrum represented every single SP in the brain parenchyma.
As shown here, we can trace Aβ1-41 for the first time in AD and CAA brains with MALDI-IMS, as well as with IHC, with a specific antibody by our own generation21. According to the Aβ processing model, Aβ1-38 derives from Aβ1-45 via Aβ1-42, while Aβ1-41 derives from Aβ1-45 by γ-secretase stepwise cleavage27,28,29,30,31. This means that the current protocol supports this model. As for the limitations of this technique, we must consider the heterogeneity of the samples from human autopsy brain. The most critical step, in a sense, is to assess qualified autopsy brain tissue with ethical proof. With the current protocol on those qualified autopsy brain tissues, MALDI-IMS can individually track the whole distribution of the complex molecules having multiple modifications, as well as unknown factor(s) regulating the pathogenesis of AD, that are yet to be defined. Furthermore, in understanding the overall pathogenesis of various neuropathology in aged human brains, it must be feasible to adopt MALDI-IMS as a standard approach, in combination with clinical, genetic, and pathological observations in neurological diseases.
Another critical step of MALDI-IMS is the data mining process from the obtained data set, which is always time-consuming. Manual data mining of each peak distribution requires the users to click through every image and look for distributions that may correlate to the morphology of the analyzed sample. Automatic spatial segmentation can be used as the first step of data mining, providing an overview of the data set and allowing the quick detection of prominent features. In this approach, similarities between spectra of a given region are statistically determined, and similar spectra are grouped into one cluster. All pixels are color-coded according to their cluster assignment (Figure 5). In the present AD/CAA study, the area of interest is the space of Aβ depositions in parenchymal plaque and the subarachnoid and parenchymal vascular structures. The two distinctive peaks which were further validated with IHC were the m/z values from Aβ1-40 and Aβ1-4221. Thus, it is easy to find colocalized m/z with Aβ1-40 and Aβ1-42 which was already annotated to N- and C-terminal truncated Aβ, as well as unknown peptides, for further analysis.
Weller and colleagues have reported that Aβ accumulates in vessel walls, more around arteries than around veins21. Moreover, it has been proposed that the interstitial fluid (ISF) includes Aβs excreted from the cerebral parenchyma to the lymph node via a perivascular drainage pathway22,23,24,25,26. The current protocol to generate a segmentation map based on a MALDI-IMS data set at a 20 µm resolution supports the possible existence of perivascular drainage pathways of the brain (Figure 5), which contribute significantly to CAA in AD21,32. Furthermore, we can discover marker proteins colocalized with plaque and subarachnoid vasculature by calculating the correlation of each m/z values. In understanding the overall pathogenesis of various neuropathology in aged human brains, it must be feasible to adopt MALDI-IMS as a powerful approach in combination with established IHC data of clinical, genetic, and pathological observations in neurological diseases.
The authors have nothing to disclose.
This work was supported in part by the Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control 26117004; to M.I. and T.M.). This research was partially supported by the Strategic Research Program for Brain Sciences from the Japan Agency for Medical Research and Development (AMED). All experiments were conducted in compliance with the ARRIVE guidelines.
Cryostat | Leica Microsystems, Wetzlar, Germany | CM1950 | |
Indium tin oxide (ITO)-coated microscope glass slide | Bruker Daltonics | #237001 | |
Blade (disposable) | Leica Microsystems, Wetzlar, Germany | #117394 | |
O.C.T. Compound | Leica Microsystems, Wetzlar, Germany | FSC 22 Blue | |
Scanner | EPSON | GT-980 | |
Air-Brush | GSI Creos | PS274 | |
Compressor | MRHOBBY | Mr.Linear compressor L5 | |
Ultrasonic sprayer | Bruker Daltonics | ImagePrep | |
Automatic sprayer | HTX Technologies | TM Sprayer | |
Confocal-laser-scanning-microscope | Carl Zeiss Inc. | LSM 700 | |
Ultra high speed MALDI instrument | Bruker Daltonics | rapifleX MALDI Tissuetyper | |
MALDI control software | Bruker Daltonics | FlexControl 3.8 | |
Data analysis software | Bruker Daltonics | FlexImaging 5.0 | |
Molecular histology software | SCiLS, Bremen, Germany | SciLS Lab 2016a | |
Statistical software | SCiLS, Bremen, Germany | SciLS Lab 2016a | |
Sinapinic acid (SA) | Nacalai tesque | 30494-91 | |
Alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA) | Wako | 037-19261 | |
Calibration standard | Bruker Daltonics | ||
Biotinylated anti-mouse IgG antibodies | Vector Laboratories, Inc., Burlingame, CA | ||
Biotinylated anti-rabbit IgG antibodies | Vector Laboratories, Inc., Burlingame, CA | ||
Avidin and biotinylated HRP complex. | Vector Laboratories, Inc., Burlingame, CA | Vectastain Elite ABC kit | |
3,3-diaminobenzidine (DAB) | Vector Laboratories, Inc., Burlingame, CA | Vectastain Elite ABC kit |