Vijayaragavan K, Cannon BJ, Tebaykin D, Bossé M, Baranski A, Oliveria JP, Bukhari SA, Mrdjen D, Corces MR, McCaffrey EF, Greenwald NF, Sigal Y, Marquez D, Khair Z, Bruce T, Goldston M, Bharadwaj A, Montine KS, Angelo RM, Montine TJ, Bendall SC. Single-cell spatial proteomic imaging for human neuropathology. Acta Neuropathol Commun. 2022 Nov 4;10(1):158. PubMed.

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  1. This study by Ajami and colleagues shows that a modification of CO-Detection by indEXing (CODEX) called CODEX-CNS identified several microglial phenotypes that varied depending on their surrounding microenvironment (gray and white matter) both in healthy and in AD brain tissue.

    This impressive technology was additionally able to detect different cells, such as astrocytes and macrophages, each with specific phenotypes around Aβ plaques.

    It was well known that microglia adopt different morphologies and express certain markers that are often expressed in distal segments of their processes but not within the cytoplasm adjacent to the nucleus. These distal segment expression patterns vary in disease and it is therefore especially important to take them into account.

    So, the technology presented in this paper not only improves our understanding of different microglial types but also their interactions with the environment, suggesting that there may be more detailed characteristics of their morphology that might be relevant for Alzheimer’s disease. This will certainly have an important impact in the field, which is increasingly recognizing the role played by microglial cells in neuroinflammation in neurodegenerative and other diseases.

    View all comments by Joana Pereira

  2. The array of tools available for spatial proteomics is continually expanding, yet autofluorescence remains a significant impediment to fluorescence-based techniques in the human brain. These two recently published offer innovative solutions to this issue.

    Ajami et al. introduces a method they have dubbed CODEX-CNS. Building on the well-established CODEX method, they've incorporated a simple chemical and light autofluorescence reduction step borrowed from Du et al. (2019). This approach is complemented by digital background subtraction. For robust antigens such as GFAP, Aβ, and MAP2, they managed to produce exceptionally clear images using an epifluorescence microscope. Leveraging a suite of open-source tools, including QuPath and their newly developed segmentation method, OTSM (pronounced "awesome"), they were able to identify unique clusters of plaque-associated CD163 positive+ IBA1+ cells.

    In contrast, Vijayaragavan et al. employed metal-conjugated antibodies and multiplex ion beam imaging. They analyzed 36 antibodies on their tissue samples, including pathology and cell type markers. Despite their cellular resolution not matching that of the CODEX-CNS method, they identified unique populations of neurons expressing the mitochondrial protein MFN2.

    These two publications add to the growing body of literature on spatial proteomics, including our method, QUIVER (Shahidehpour et al., 2023). It's an exciting time to study human neuropathology. As the field progresses, I anticipate a move beyond well-known markers, such as IBA1, GFAP, and MAP2. Instead, I foresee these technologies used to study proteins that can provide deeper insights into the functional state of cells and their interactions with neuropathology. This pursuit will necessitate integrating and expanding digital pathology tools, such as HALO, OTSM, histoCAT, and CytoMAP, among others, to quantify staining patterns.

    The field is advancing rapidly, and the potential to increase our understanding of devastating neurodegenerative diseases by examining neuropathological changes using these spatial proteomic tools is truly exciting.

    References:

    Du Z, Lin JR, Rashid R, Maliga Z, Wang S, Aster JC, Izar B, Sorger PK, Santagata S. Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging. Nat Protoc. 2019 Oct;14(10):2900-2930. Epub 2019 Sep 18 PubMed.

    Shahidehpour RK, Nelson AS, Sanders LG, Embry CR, Nelson PT, Bachstetter AD. The localization of molecularly distinct microglia populations to Alzheimer's disease pathologies using QUIVER. Acta Neuropathol Commun. 2023 Mar 18;11(1):45. PubMed.

    View all comments by Adam Bachstetter

  3. Both studies illustrate commercial, multiplexed immunohistochemistry techniques and their application toward postmortem AD brain tissue. While only a few cases are studied, the kind of high-dimensional data generated with those approaches is intriguing. Similar efforts have been illustrated for spatial transcriptomics (Wood et al., 2022), as well as for combined spatial transcriptomics and proteomics (Calafate et al., 2023) in other biomedical applications (He et al., 2022).

    The codex technique is interesting because it provides cost-efficient cyclic immunohistochemistry analysis. The imaging mass cytometry (IMC) approach is much faster and represents truly correlative imaging but requires quite delicate equipment. As for all antibody-based techniques, extensive validation is required and the systems are limited to targets that can be delineated with antibodies as well as by antibody availability. It will be very interesting to see the application of those systems toward more functional studies in, for example, AD model systems, and their expansion beyond of the shelf panels toward more tailored targets, such as different tau epitopes in concert with the corresponding neural cell architecture.

    References:

    Wood JI, Wong E, Joghee R, Balbaa A, Vitanova KS, Stringer KM, Vanshoiack A, Phelan SJ, Launchbury F, Desai S, Tripathi T, Hanrieder J, Cummings DM, Hardy J, Edwards FA. Plaque contact and unimpaired Trem2 is required for the microglial response to amyloid pathology. Cell Rep. 2022 Nov 22;41(8):111686. PubMed.

    Calafate S, Özturan G, Thrupp N, Vanderlinden J, Santa-Marinha L, Morais-Ribeiro R, Ruggiero A, Bozic I, Rusterholz T, Lorente-Echeverría B, Dias M, Chen WT, Fiers M, Lu A, Vlaeminck I, Creemers E, Craessaerts K, Vandenbempt J, van Boekholdt L, Poovathingal S, Davie K, Thal DR, Wierda K, Oliveira TG, Slutsky I, Adamantidis A, De Strooper B, de Wit J. Early alterations in the MCH system link aberrant neuronal activity and sleep disturbances in a mouse model of Alzheimer's disease. Nat Neurosci. 2023 Jun;26(6):1021-1031. Epub 2023 May 15 PubMed.

    Zeng H, Huang J, Zhou H, Meilandt WJ, Dejanovic B, Zhou Y, Bohlen CJ, Lee SH, Ren J, Liu A, Tang Z, Sheng H, Liu J, Sheng M, Wang X. Integrative in situ mapping of single-cell transcriptional states and tissue histopathology in a mouse model of Alzheimer's disease. Nat Neurosci. 2023 Mar;26(3):430-446. Epub 2023 Feb 2 PubMed.

    He S, Bhatt R, Brown C, Brown EA, Buhr DL, Chantranuvatana K, Danaher P, Dunaway D, Garrison RG, Geiss G, Gregory MT, Hoang ML, Khafizov R, Killingbeck EE, Kim D, Kim TK, Kim Y, Klock A, Korukonda M, Kutchma A, Lewis ZR, Liang Y, Nelson JS, Ong GT, Perillo EP, Phan JC, Phan-Everson T, Piazza E, Rane T, Reitz Z, Rhodes M, Rosenbloom A, Ross D, Sato H, Wardhani AW, Williams-Wietzikoski CA, Wu L, Beechem JM. High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging. Nat Biotechnol. 2022 Dec;40(12):1794-1806. Epub 2022 Oct 6 PubMed.

    View all comments by Jörg Hanrieder

  4. The study by Sanchez-Molina and collaborators offers a notable multiplexing approach that is, in principle, capable of visualizing up to 100 different proteins simultaneously in brain tissue, using a novel methodology called CODEX-CNS, a variation of the currently existing CODEX technology (Black et al., 2021). The authors designed a panel of 32 oligonucleotide-conjugated antibodies that were incubated simultaneously on fixed, paraffin-embedded postmortem human brain tissue from Alzheimer’s disease patients and age-matched, otherwise healthy individuals. The primary antibodies were thereafter detected using complementary oligonucleotide-conjugated fluorophores, incubated on the tissue in cycles of three fluorophores. After image acquisition in each cycle, the fluorophores were removed by isothermal wash. Tissue autofluorescence due to lipofuscin and fixative, among others, was dramatically reduced by incubating the tissue with H2O2 while simultaneously exposing it to broad-spectrum LED light prior to antibody staining, allowing for the acquisition of images with dramatically increased signal-to-noise ratios. The method is convenient not only for its multiplexing capabilities but also for the high image quality capable of rendering.

    Simultaneously using specific markers such as Iba-1, NeuN, Olig2, GFAP, and Collagen IV, the authors were able to identify and differentiate brain-cell populations and tissue features such as blood vessels. Abundant claudin-5 was detected in blood vessels as well as astrocytic end feet using GFAP. The combined use of these and additional markers could constitute a novel avenue of research in blood-barrier dysfunction during AD. The authors also describe the presence of CD163-positive, TMEM119-negative cells co-localizing with Aβ plaques, which could indicate monocyte/macrophage infiltration. Given the multiplexing capacity of CODEX-CNS, it should be possible to additionally stain these tissues with markers of phagocytic activity (CD68 staining was included in the study) and lysosomal function such as v-ATPase, Clc-7 and LAMPs, which combined would provide insight into the degradative capacity of resident microglia versus presumably infiltrating myeloid cells. This is important, since microglia do not seem particularly good at degrading Aβ in cell culture, whereas macrophages are (Boissonneault et al., 2009; Majumdar et al., 2011). 

    The authors showed images of ApoE and GFAP staining near Aβ plaques. In these areas, ApoE staining seems most intense around plaques with reduced astrogliosis, which is an interesting observation. Astrocytes have been suggested to phagocytose Aβ in mouse brain, and ApoE-deficient astrocytes fail to respond or internalize Aβ deposits when compared with wild-type (Gomez-Arboledas et al., 2018; Koistinaho et al., 2004). 

    Using a threshold-based segmentation approach, the authors dissected nearly 50,000 microglia cells, which could be sorted into a number of different subpopulations based on expression of activation markers. One of the subpopulations, with high CD68 expression, seemed to predominantly exist away from Aβ deposits, suggesting that proximity to Aβ reduced phagocytic capacity. This could well be the case. Microglia near Aβ deposits may trim diffuse structures by extracellular digestion in a process called digestive exophagy, rather than by phagocytosis (April 2023 conference news). Although controversial, microglia could also internalize some Aβ material by phagocytosis, rapidly reach their peak degradative capacity, then, no longer being able to internalize additional material, downregulate phagocytosis markers.

    Another subpopulation of interest expressed high levels of CD163, which spatially correlated with Aβ deposits. This specific subpopulation might be associated with cerebral amyloid angiopathy, and might also even represent infiltrating myeloid cells—some of the plaques associated with these cells presented the typical morphology of vascular Aβ.

    It would be fantastic to see this methodology eventually applied to the study of lysosomal markers. For instance, the study of v-ATPase and Clc-7 expression in microglia near to and far from plaques would provide insight into the effect of fibrillar, extracellularly deposited Aβ on microglial degradative capacity. This is important, because lysosomal acidification is reduced during AD (Majumdar et al., 2011; Lee et al., 2022). 

    References:

    Black S, Phillips D, Hickey JW, Kennedy-Darling J, Venkataraaman VG, Samusik N, Goltsev Y, Schürch CM, Nolan GP. CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nat Protoc. 2021 Aug;16(8):3802-3835. Epub 2021 Jul 2 PubMed.

    Boissonneault V, Filali M, Lessard M, Relton J, Wong G, Rivest S. Powerful beneficial effects of macrophage colony-stimulating factor on beta-amyloid deposition and cognitive impairment in Alzheimer's disease. Brain. 2009 Apr;132(Pt 4):1078-92. Epub 2009 Jan 17 PubMed.

    Gomez-Arboledas A, Davila JC, Sanchez-Mejias E, Navarro V, Nuñez-Diaz C, Sanchez-Varo R, Sanchez-Mico MV, Trujillo-Estrada L, Fernandez-Valenzuela JJ, Vizuete M, Comella JX, Galea E, Vitorica J, Gutierrez A. Phagocytic clearance of presynaptic dystrophies by reactive astrocytes in Alzheimer's disease. Glia. 2018 Mar;66(3):637-653. Epub 2017 Nov 27 PubMed.

    Koistinaho M, Lin S, Wu X, Esterman M, Koger D, Hanson J, Higgs R, Liu F, Malkani S, Bales KR, Paul SM. Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides. Nat Med. 2004 Jul;10(7):719-26. PubMed.

    Lee JH, Yang DS, Goulbourne CN, Im E, Stavrides P, Pensalfini A, Chan H, Bouchet-Marquis C, Bleiwas C, Berg MJ, Huo C, Peddy J, Pawlik M, Levy E, Rao M, Staufenbiel M, Nixon RA. Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. Nat Neurosci. 2022 Jun;25(6):688-701. Epub 2022 Jun 2 PubMed.

    Majumdar A, Capetillo-Zarate E, Cruz D, Gouras GK, Maxfield FR. Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes. Mol Biol Cell. 2011 May 15;22(10):1664-76. PubMed.

    View all comments by Santiago Sole-Domenech

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