Exploring Neurodegenerative Diseases With Imaging Mass Cytometry

IMAGING WHAT’S INSIDE Graphical abstract Key takeaways Study design • Panel design • Imaging modes • Image analysis Results • Preview Mode • Cell Mode • Tissue Mode Conclusions Tips for success Methods Appendix Ordering information References Exploring Neurodegenerative Diseases With Imaging Mass Cytometry APPLICATION NOTE Key takeaways • Ready-to-use neurodegenerative panels compatible with human immuno-oncology IMC™ subpanels provide swift high-parameter panel design with preselected antibodies. • Multiple imaging modes for IMC enable simultaneous detection of protein targets, from whole tissue sections down to single-cell resolution. • Researchers now have access to the fastest and most comprehensive workflow to interrogate various neurodegenerative diseases by combining high-parameter panels with various IMC imaging modes. A Lewy body (1) and a Lewy neurite (2) in Parkinson’s brain READY-TO-GO IMC PANELS IMC IMAGING MODES Preview Mode Cell Mode Tissue Mode 2 mm Human Lymphoid IMC Panel Human Myeloid IMC Panel Human Cell Functional State IMC Panel Maxpar Neuro Phenotyping IMC Panel Kit Human Basic Tissue Architecture IMC Panel Maxpar IMC Cell Segmentation Kit and Cell-ID Intercalator Human Basic Immune IMC Panel Neurodegenerative IMC panels Alzheimer’s Disease Multiple Sclerosis Parkinson’s Disease Human Stromal Cell IMC Panel Choose one Interrogate various neurodegenerative diseases with the fastest, most comprehensive approach by utilizing: 2 | Application Note | Exploring Neurodegenerative Diseases With Imaging Mass Cytometry Figure 1. Main hallmark features of each neurodegenerative condition visualized in Tissue Mode: amyloid aggregates and tangles, Lewy bodies with Lewy neurites, and multiple sclerosis lesions. Diseased tissues stained with applicable neurodegenerative panels (Alzheimer’s Disease IMC Panel, Parkinson’s Disease IMC Panel, and Multiple Sclerosis IMC Panel) allow whole tissue visualization of the main protein contributors to disease pathology: amyloid precursor protein (APP) in amyloid plaques and Tau in tangles of Alzheimer’s disease, phosphorylated αSynuclein (p-αSyn) in Lewy bodies and Lewy neurites of PD, and large areas of lost myelin (lesion) of MS. Introduction Neurodegenerative diseases (NDs) are chronic progressive conditions of the central nervous system (CNS) leading to physical and cognitive disability in humans across the globe. NDs affect approximately 15% of the worldwide population and patient numbers have been steadily climbing over the past decades1 . Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS) represent the 3 most prominent contributors to the global ND population. AD, PD, and MS are currently incurable, with treatment focusing on palliative care and alleviating symptoms. Understanding the etiology of these diseases is a major challenge and requires deciphering the complex spatial biology processes that cause deterioration of neuronal tissue, such as formation of protein aggregates and tangles, neuroinflammation, and demyelination. To fully address this complexity, it is important to clarify the underlying molecular mechanisms driving neurodegeneration and identify new targets and therapies. Imaging Mass Cytometry™ (IMC) is a spatial biology technology that offers the ability to comprehensively visualize the pathological features of neurogenerative diseases. Unlike traditional cyclic fluorescent methods, IMC can uncover the spatial distribution of 40-plus distinct protein markers simultaneously without tissue degradation and autofluorescence artifacts usually observed in brain tissue. IMC offers various whole slide imaging (WSI) modes that range from visualization of an entire section to single-cell assessment, permitting in-depth exploration of tissue heterogeneity. WSI modes enable streamlined, comprehensive evaluation of human brain tissue using ready-to-use antibody panels specifically designed for neurological research. Three specialized panels are available to support neurodegenerative spatial biology studies (Figure 1): • The Alzheimer’s Disease IMC Panel, 3 Antibodies identifies amyloid aggregates and Tau tangles. • The Parkinson’s Disease IMC Panel, 3 Antibodies identifies Lewy bodies and Lewy neurites. • The Multiple Sclerosis IMC Panel, 3 Antibodies identifies the loss of major myelin components. When combined with other relevant IMC panels tailored for neurophenotyping, tissue architecture, and immune cell profiling, 3 different high-parameter 41-marker imaging panels for NDs can be easily assembled (Figure 2). The modular panel-building format facilitates rapid and effortless design of highparameter IMC panels tailored precisely to research objectives. Further, IMC WSI provides a streamlined approach that expedites the discovery of valuable biological insights into each of these important neurodegenerative conditions. Exploring Neurodegenerative Diseases With Imaging Mass Cytometry | Application Note | 3 In this application note, we demonstrate the spatial biology insights that IMC provides into NDs with a particular focus on AD tissue. We showcase the comprehensive utilization of a specialized AD panel and demonstrate its effectiveness in exploring different populations of neurons that contribute to aggregate formation. Importantly, the methodologies presented are equally applicable to other neurodegenerative tissues. Study design Panel design The 41-marker panel comprised of the following off-theshelf modular subpanels and kits was assembled to investigate AD brain tissue (Figure 2): • Human immuno-oncology IMC subpanels, 26 antibodies in total. Six human immuno-oncology IMC subpanels were used to interrogate structural, microglial, and neuroinflammation components involved in the causality, development, and progression of AD. Figure 2. Combination of 3 ND IMC panels with other compatible panels and kits for comprehensive interrogation of each disease. The modular panel design provides flexibility, allowing researchers to create customized IMC panels for investigating most neurodegenerative conditions, including AD, PD, and MS. When combined with other relevant IMC panels tailored for neurophenotyping, tissue architecture, and immune cell profiling, 3 different high-parameter 41-marker imaging panels for NDs can be easily assembled. This enables visualization of the main cell types in the CNS and provide insights into the contribution of the immune system in NDs. • Maxpar® Neuro Phenotyping IMC Panel Kit for phenotyping of major cell types in the CNS, such as neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells. The Maxpar Neuro Phenotyping IMC Panel Kit is essential for accurately identifying all major cell types and their interactions with other cells when analyzing brain tissue. • Alzheimer’s Disease IMC Panel to locate major protein markers associated with each corresponding pathology, such as APP, Tau, and phosphorylated Tau (pTau) • Maxpar IMC Cell Segmentation Kit (ICSK) to facilitate an end-to-end workflow for single-cell data analysis • Cell-ID™ Intercalator-Ir (191Ir/193Ir) for identification of nucleated cells • AD-relevant IMC catalog antibodies (αSyn, p-αSyn [S129]) to gain additional insights about αSyn participation in aggregate formation See Appendix, Table 1 for detailed panel configuration and clone information. 4 | Application Note | Exploring Neurodegenerative Diseases With Imaging Mass Cytometry The full-configuration panel was applied on sections of human AD brain. The tissue slides were prepared and stained according to the Standard BioTools™ Imaging Mass Cytometry Staining Protocol for FFPE Sections (400322) using optimized antibody concentrations for Tissue Mode, Preview Mode, and Cell Mode. Imaging modes Tissue slides were visualized with the Hyperion XTi™ Imaging System, using all 3 imaging modes (Figure 3): • Preview Mode enables quick visualization of all 41 markers within minutes across the whole tissue. This fast scan provides guidance for selecting regions of interest (ROIs) to be acquired on the same slide in Cell Mode. • Tissue Mode is a complete WSI mode to visualize 41 markers, revealing the heterogeneity of the whole tissue. • Cell Mode facilitates acquisition of ROIs selected in Preview Mode at subcellular resolution for detailed characterization of individual cells and cell populations. One serial section of AD tissue utilized Preview Mode and Cell Mode, while another sequential section of AD tissue exclusively employed Tissue Mode (Figure 3). Image analysis Qualitative data analysis, high-parameter image rendering, and single-channel image extractions were performed using MCD™ SmartViewer software. For AD tissue data obtained using Preview Mode and Cell Mode, quantitative single-cell analysis was performed using a 2-step data analysis pipeline: Cellpose was used for cell segmentation and PhenoGraph was used for clustering2. The data obtained using Tissue Mode was analyzed with a pixel-clustering analysis and neighborhood enrichment analysis approach using MCD SmartViewer software. See Methods for additional experimental details regarding samples, staining, ablation, and data analysis. Recommended Imaging Workflows Single-Cell Analysis PixelClustering Analysis Figure 3. WSI modes for IMC offer versatile workflows to accelerate quantitative spatial biology discoveries. Preview Mode rapidly scans the sample and generates useful data for guiding ROI placement used in Cell Mode single-cell analysis. Tissue Mode generates a high-quality scan of the entire tissue section in a matter of hours with higher spot-size ablations enabling pixel-clustering analysis of the whole tissue. Combining these new workflows with the Hyperion™ XTi Slide Loader streamlines IMC application and makes it a useful resource for high-throughput clinical and translational studies. Exploring Neurodegenerative Diseases With Imaging Mass Cytometry | Application Note | 5 Results Whole slide Preview Mode imaging enables the spatial localization of protein aggregates in AD tissue Multiple brain sections from a 73-year-old male donor diagnosed with AD were commercially obtained. The tissue sections were stained with a 41-marker assembly containing 3 markers from the Alzheimer’s Disease IMC Panel, then analyzed using the Hyperion XTi Imaging System. Tissue slides were first imaged using Preview Mode, with visualization of the whole post-central gyrus tissue measuring 3.9 mm x 5.2 mm completed in less than 5 min. One of the most notable benefits of visualizing brain tissue in Preview Mode is the ease of markerguided identification of specialized brain regions such as gray and white matter. For example, the utilization of MAP2 or NeuN markers visibly defines the gray matter area (Figure 4A). Localization of all 3 markers from the Alzheimer’s Disease IMC Panel (APP, total Tau protein, and phosphorylated [S202/T205] Tau protein) is also clearly visible in the tissue within the MAP2+ gray matter of AD brain (Figure 4A). The striking abundance of insoluble plaques containing APP and pTau (Figure 4A, left and middle images) suggests an advanced stage of the disease, possibly leading to irreversible cognitive decline3 . These large plaques scattered around the gray matter served as landmarks for selecting ROIs for visualization in Cell Mode using the same tissue section. By utilizing the same tissue slide for imaging using both Preview Mode and Cell Mode, we eliminated the need for additional stained slides, thereby conserving valuable patient samples. Figure 4. Qualitative and quantitative assessment of AD tissue using IMC Preview Mode and Cell Mode with subsequent single-cell and neighborhood enrichment analysis uncovers cell populations associated with aggregate-producing neurons. Preview Mode facilitates marker-guided ROI selection for subsequent visualization in Cell Mode. Cell Mode ROI was used to conduct single-cell analysis. (A) Three views of a single ROI. (B) Locations of the same aggregates with the most abundant presence of APP- and pTau-producing neurons are marked with arrowheads in a Cell Mode image and (C) cell population mask. Cell populations were identified using ICSK and Cell-ID Intercalator-Ir staining. Population names were assigned based on marker expression. (D) Subsequent quantitative neighborhood analysis demonstrates a heat map with an enrichment score (z-score) that indicates enrichment or depletion of the spatial proximity between all identified clusters. WM – white matter, GM – gray matter 6 | Application Note | Exploring Neurodegenerative Diseases With Imaging Mass Cytometry One region with high concentration of aggregates with both APP and pTau was selected to further investigate the tissue in Cell Mode (Figure 4B) using single-cell analysis (Figure 4C). Single-cell analysis of high-resolution Cell Mode images reveals diverse composition of cell populations within the aggregate vicinity High-resolution Cell Mode visualization of 1 ROI measuring 2.1 mm x 2.1 mm was acquired at 800 Hz and completed in 1 hr 43 min. Three functionally distinct populations of both neurons and astrocytes, 2 populations of microglia, and 1 population of oligodendrocytes were identified through singlecell analysis of the Cell Mode ROI. Most segmented nucleated elements were neurons, accounting for 56.9% of all cells, with roughly every 7 out of 10 being neurodegenerative neurons producing elevated levels of Tau and αSyn. Another population of neurons, the smallest at 1.5% of all cells, was observed to produce high levels of APP and pTau (Figure 4C). Interestingly, these neurons tended to gravitate toward the periphery of large aggregates (Figure 4B and 4C, arrowheads), suggesting their involvement in the process of actively producing toxic amyloid species that contribute to aggregate formation4 . Inhibiting this smallest population of “rogue” neurons producing high levels of APP and pTau to mitigate or slow the secretion of toxic amyloid species could potentially have a significant clinical impact. These aggregate-forming neurons were associated with populations of protoplasmic astrocytes and astrocytes expressing elevated levels of S100β as indicated by the neighborhood enrichment analysis heat map (Figure 4D, Box 1). S100β is an important inflammatory regulator of astrocytes by inducing their phenotype change to become reactive5, 6. Direct inhibition of S100β has been shown to have a neuroprotective effect in some populations of neurons and reduce neuronal loss in mouse AD models7 . Neighborhood analysis also suggests a close spatial proximity of aggregate-forming neurons with microglia that exhibit anti-inflammatory properties (Iba1+, CD163high), while activated phagocytic microglia (Iba1+, CD68high) appear to be located at a distance (Figure 4D, Boxes 2 and 3). Dissociation of phagocytic microglia from amyloid plaques may indicate microglial exhaustion due to prolonged exposure to aggregates, resulting in defective autophagy8. As microglia play a critical role in mediating the phagocytosis of aggregates, successful efforts were made to reverse microglial phagocytic deficits by treating microglia with anti-inflammatory agents9 . Overall, the data presented highlights the power of Preview Mode to rapidly locate desirable areas of the tissue for subcellular resolution imaging via Cell Mode on the same slide. Furthermore, combining this workflow with single-cell analysis effectively identifies diverse cellular organization and cellular interactions in the AD brain. While single-cell analysis provides valuable insights, nuclei-based segmentation of brain tissue is not sufficient to draw a complete picture of the disease. Additional complex analysis of neuronal, astrocytic, and microglial extensions that are not associated with DNA, especially of ramified phenotypes, is required to comprehensively understand the cellular population landscape of the diseased brain. Moreover, singlecell analysis is limited in capturing extracellular aggregates because they are devoid of membrane and nucleus markers. Therefore, pixel-clustering analysis offers a complementary perspective, allowing for the examination of non-nucleated extracellular aggregates at a tissue level. Exploring Neurodegenerative Diseases With Imaging Mass Cytometry | Application Note | 7 Pixel-clustering analysis of Tissue Mode images complements single-cell analysis by providing additional biological insights A slide with a sequential section was visualized in Tissue Mode (Figure 5A). Visualization of the whole tissue section of post-central gyrus measuring 3.9 mm x 5.2 mm was completed in 27 min. Subsequent pixel-clustering analysis using MCD SmartViewer software (Figure 5B) performed unsupervised clustering, automatically grouping pixels with similar characteristics by analyzing the intensity and distribution of 41 markers in each pixel. Pixel-clustering analysis unveiled numerous distinct morphology clusters, such as gray matter-associated and white matter-associated microglia, 3 populations of neurons, astrocytes, and oligodendrocytes, and vasculature, alongside the identification of 2 functional amyloid aggregate clusters. A cluster of neurodegenerative neurons expressing higher levels of Tau and αSyn encompassed a Figure 5. Visualization of Alzheimer’s disease tissue using IMC Tissue Mode with subsequent pixel-clustering analysis reveals distinct aggregate populations. (A) Tissue Mode imaging successfully captures the expression pattern of APP, pTau, and Tau in AD brain tissue. (B) Subsequent pixel-clustering analysis captures different types of cell clusters and aggregates based on the marker expression in each pixel. substantial portion of the whole tissue area (41.8% of total analyzed pixels). This is consistent with the observations from single-cell analysis implying a high degree of pathology, such as cellular dysfunction and disease progression. Aggregates of a relatively small size exhibited an exclusive enrichment of APP, whereas most of the larger aggregates demonstrated a spatial organization characterized by the central concentration of APP surrounded by a halo of Tau protein. The arrangement of APP and Tau hints at a potential aggregate stabilization and overall synergy among those proteins, alongside αSyn, all known to be prone to misfolding in the diseased brain10. Overall, these findings demonstrate the power of combining Tissue Mode imaging with pixel-clustering analysis to clearly unveil the heterogeneity of nonnucleated extracellular aggregates and nucleated cellular clusters in neurodegenerative conditions. 8 | Application Note | Exploring Neurodegenerative Diseases With Imaging Mass Cytometry Conclusions IMC captures key biological insights and provides a comprehensive multimodal approach to understanding the complex pathology of neurodegenerative conditions by utilizing ready-to-use antibody panels, synergistic WSI modes, and complementary data analysis pipelines. Preview Mode allows for rapid and simultaneous 40-plus-marker visualization across the entire tissue section, facilitating the identification of ROIs for use in Cell Mode analysis. Cell Mode and subsequent downstream single-cell analysis provide detailed insights at the highest resolution, uncovering distinct neuronal populations and their protein expression profiles, crucial for understanding disease mechanisms. Additionally, Tissue Mode enables comprehensive visualization of all markers, revealing the heterogeneity of aggregate distribution across the whole tissue. Furthermore, single-cell and pixel-based analyses complement each other effectively. Single-cell analysis elucidates the heterogeneity of nucleated elements associated with the aggregates, while pixel-based analysis offers a broader perspective by capturing different types of aggregates at a tissue level. The combination of these approaches allows for a comprehensive understanding of cellular dynamics. Researchers can now gain a more holistic understanding of neurodegeneration to discover new potential therapeutic targets and interventions. Besides the Alzheimer’s Disease IMC Panel, the Parkinson’s Disease IMC Panel and the Multiple Sclerosis IMC Panel effectively reveal Lewy bodies with Lewy neurites and lesions in PD and MS, respectively, facilitating a comprehensive high-parameter sample investigation with WSI modes and downstream analysis. Tips for success • For best results, use freshly cut FFPE tissue samples when possible. • Perform a 3-point titration and include positive control tissue for all antibodies when optimizing their working concentrations on tissue sections. Recommended dilution ranges for each antibody can be found in the technical data sheet. • Increase maximum threshold values in MCD SmartViewer software to better visualize small Lewy bodies and Lewy neurites in PD tissue. • After staining, samples should be stored at room temperature in slide holders inside a sealed bag in a non-humid environment. • Customers should reach out to their local Field Applications Scientist (FAS) for ordering and product support. To be connected to a FAS, contact technical support. Exploring Neurodegenerative Diseases With Imaging Mass Cytometry | Application Note | 9 Methods Staining Slide preparation and staining were conducted according to the Imaging Mass Cytometry Staining Protocol for FFPE Sections. Imaging Imaging was performed using the Hyperion XTi Imaging System with CyTOF® Software v9.0. Before ablation, instrument tuning was performed using a tuning slide. Ablation frequency was 800 Hz with optimized laser power for all imaging modes. Cell segmentation and analysis Qualitative data analysis, high-parameter image rendering, and single channel image extractions were performed using MCD SmartViewer software. For AD tissue data obtained using Preview Mode and Cell Mode, quantitative single-cell analysis was performed using a 2-step data analysis pipeline: Cellpose was used for cell segmentation and PhenoGraph was used for clustering. The data obtained using Tissue Mode was analyzed with a pixel-clustering analysis and neighborhood enrichment analysis approach using MCD SmartViewer software. Panel compatibility Neurodegenerative IMC panels are compatible with the following products: Product Name Part Number Human Stromal Cell IMC Panel, 4 Antibodies 201511 Human Lymphoid IMC Panel, 4 Antibodies 201512 Human Myeloid IMC Panel, 6 Antibodies 201513 Human Cell Functional State IMC Panel, 5 Antibodies 201514 Human Basic Tissue Architecture IMC Panel, 3 Antibodies 201517 Human Basic Immune IMC Panel, 4 Antibodies 201518 Maxpar Neuro Phenotyping IMC Panel Kit 201337 Maxpar IMC Cell Segmentation Kit 201500 Cell-ID Intercalator-Ir—125 µM 201192A 10 | Application Note | Exploring Neurodegenerative Diseases With Imaging Mass Cytometry Appendix Core panel Panel or Product Name Marker Clone Metal Concentration (µg/mL) Part Number Tissue Mode – Working Preview Mode and Cell Mode – Working Human Basic Tissue Architecture IMC™ Panel Collagen 1 Polyclonal 89Y 1 5 201517 Fibronectin EPR23110-46 171Yb 0.2 1 CD31 EPR3094 151Eu 1 5 Human Stromal Cell IMC Panel FAP E1V9V 161Dy 0.5 2.5 201511 Podoplanin D2-40 164Dy 0.5 2.5 αSMA 1A4 209Bi 0.125 0.5 CD44 IM7 153Eu 1 5 Human Basic Immune IMC Panel CD45 D9M8I 152Sm 0.25 2.5 201518 CD3ε D7A6E 170Er 1 5 CD20 H1 115In 1 5 CD68 KP1 159Tb 0.25 1 Human Lymphoid IMC Panel CD4 EPR6855 156Gd 1 5 201512 CD8a C8/1448 162Dy 0.5 2.5 CD45RO UCHL1 173Yb 0.2 1 CD57 NK/804 163Dy 0.5 2.5 Human Myeloid IMC Panel CD66b BLR111H 160Gd 0.25 1.25 201513 HLA-DR LN3 174Yb 0.2 1 CD163 EDHu-1 147Sm 1 5 CD14 EPR3653 175Lu 0.5 2.5 CD11b EPR1344 144Nd 0.25 1.25 CD11c D3V1E 154Sm 0.5 2.5 Human Cell Functional State IMC Panel Granzyme B EPR20129-217 176Yb 0.125 1 201514 PD-L1 73-10 166Er 5 10 PD-1 D4W2J 165Ho 5 10 FoxP3 PCH101 155Gd 5 10 Ki-67 B56 150Nd 0.5 2.5 Maxpar® Neuro Phenotyping IMC Panel Kit GFAP GA5 143Nd 0.125 0.25 201337 NeuN EPR12763 145Nd 2.5 5 S100β EP1576Y 146Nd 0.125 0.25 Olig2 EPR2673 168Er 2.5 5 Iba1 EPR16588 142Nd 0.63 1.25 CD34 EP373Y 167Er 2.5 5.0 MAP2 EPR19691 148Nd 0.25 0.42 Cell-ID™ Intercalator-Ir DNA1 191Ir 201192A DNA2 193Ir Maxpar IMC Cell Segmentation Kit ICSK1 195Pt 2.5 201500 ICSK2 196Pt 2.5 ICSK3 198Pt 5 Exploring Neurodegenerative Diseases With Imaging Mass Cytometry | Application Note | 11 Choice of 1 neurodegenerative panel IMC Panel Marker Clone Metal Concentration (µg/mL) Tissue Mode Cell Mode Part Number Parkinson’s Disease IMC™ Panel (9100006) αSyn E4U2F 141Pr 1.67 3.33 91H052141 p-αSyn [S129] EP1536Y 169Tm 1.25 2.5 91H053169 TH E2L6M 172Yb 1.25 2.5 91H054172 Alzheimer’s Disease IMC Panel (9100007) APP 4G8 141Pr 1.25 2.5 91H055141 Tau D1M9X 149Sm 0.25 0.63 91H056149 pTau [S202/T205] AT8 172Yb 0.31 0.63 91H057172 Multiple Sclerosis IMC Panel (9100008) NF SMI 312 141Pr 2.5 5.0 91H060141 MBP MBP101 149Sm 0.083 0.167 91H058149 MOG EP4281 158Gd 0.83 1.25 91H059158 Additional single antibodies used IMC Product Marker Clone Metal Concentration (µg/mL) Tissue Mode Cell Mode Part Number Single antibodies for Alzheimer’s Disease IMC™ Panel αSyn E4U2F 158Gd 0.63 1.25 91H052158 p-αSyn [S129] EP1536Y 169Tm 1.25 2.5 91H053169 Table 1. Antibodies used in this application note Standard BioTools Products Part Number Cell-ID™ Intercalator-Ir—500 µM 201192B Human Immune Cell Expansion IMC™ Panel, 7 Antibodies 201516 Human Immuno-Oncology IMC Panel, 31 Antibodies 201509 Maxpar® IMC Cell Segmentation Kit 201500 Maxpar PBS 201058 Maxpar Water 201069 Reagents From Other Suppliers Part Number Commercial Alcohols Ethyl Alcohol Anhydrous, USP P006EAAN Agilent® Dako Target Retrieval Solution, pH 9 (x10) S236784-2 Sigma-Aldrich® Bovine Serum Albumin solution, 10% in DPBS A1595 m-Xylene ReagentPlus®, 99% 185566-1L Thermo Fisher Scientific™ Triton™ X-100 Surfact-Amps™ Detergent Solution 85111 References 1. Van Schependom, J. and D’haeseleer, M. “Advances in neurodegenerative diseases.” Journal of Clinical Medicine 12 (2023): 1709. 2. Stringer, C. et al. “Cellpose: a generalist algorithm for cellular segmentation.” Nature Methods 18 (2021): 100–106. 3. Nelson, P.T. et al. “Neuropathology and cognitive impairment in Alzheimer disease: a complex but coherent relationship.” Journal of Neuropathology & Experimental Neurology 68 (2009): 1–14. 4. Ashraf, G.M. et al. “Protein misfolding and aggregation in Alzheimer’s disease and type 2 diabetes mellitus.” CNS & Neurological Disorders – Drug Targets 13 (2014): 1,280–1,293. 5. Rodríguez, J.J. et al. “Astrocyte S100β expression and selective differentiation to GFAP and GS in the entorhinal cortex during ageing in the 3xTg-Alzheimer’s disease mouse model.” Acta Histochemica 126 (2024): 152131. 6. Monterey, M.D. et al. “The many faces of astrocytes in Alzheimer’s disease.” Frontiers in Neurology 12 (2021): 619626. 7. Cirillo, C. et al. “S100B inhibitor pentamidine attenuates reactive gliosis and reduces neuronal loss in a mouse model of Alzheimer’s disease.” BioMed Research International (2015): 508342. 8. Pomilio, C. et al. “Microglial autophagy is impaired by prolonged exposure to β-amyloid peptides: evidence from experimental models and Alzheimer’s disease patients.” GeroScience 42 (2020): 613–632. 9. Mandrekar-Colucci, S. and Landreth, G.E. “Microglia and inflammation in Alzheimer’s disease.” CNS & Neurological Disorders – Drug Targets 9 (2010): 156–167. 10. Moussaud, S. et al. “Alpha-synuclein and tau: teammates in neurodegeneration?” Molecular Neurodegeneration 9 (2014): 43. 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