Acta Neuropathologica https://doi.org/10.1007/s00401-021-02263-w ORIGINAL PAPER Distinct amyloid‑β and tau‑associated microglia profles in Alzheimer’s disease Emma Gerrits1 · Nieske Brouwer1 · Susanne M. Kooistra1 · Maya E. Woodbury2 · Yannick Vermeiren3,4,5,6 · Mirjam Lambourne7 · Jan Mulder7 · Markus Kummer8 · Thomas Möller2 · Knut Biber8 · Wilfred F. A. den Dunnen9 · Peter P. De Deyn3,4,10 · Bart J. L. Eggen1 · Erik W. G. M. Boddeke1,11 Received: 19 September 2020 / Revised: 6 January 2021 / Accepted: 6 January 2021 © The Author(s) 2021 Abstract Alzheimer’s disease (AD) is the most prevalent form of dementia and is characterized by abnormal extracellular aggre- gates of amyloid-β and intraneuronal hyperphosphorylated tau tangles and neuropil threads. Microglia, the tissue-resident macrophages of the central nervous system (CNS), are important for CNS homeostasis and implicated in AD pathology. In amyloid mouse models, a phagocytic/activated microglia phenotype has been identifed. How increasing levels of amyloid-β and tau pathology afect human microglia transcriptional profles is unknown. Here, we performed snRNAseq on 482,472 nuclei from non-demented control brains and AD brains containing only amyloid-β plaques or both amyloid-β plaques and tau pathology. Within the microglia population, distinct expression profles were identifed of which two were AD pathology- associated. The phagocytic/activated AD1-microglia population abundance strongly correlated with tissue amyloid-β load and localized to amyloid-β plaques. The AD2-microglia abundance strongly correlated with tissue phospho-tau load and these microglia were more abundant in samples with overt tau pathology. This full characterization of human disease-associated microglia phenotypes provides new insights in the pathophysiological role of microglia in AD and ofers new targets for microglia-state-specifc therapeutic strategies. Keywords Microglia · Alzheimer’s disease · Single-nucleus RNA sequencing · Amyloid-β · Tau Bart J. L. Eggen and Erik W. G. M. Boddeke have contributed equally to this work. * Erik W. G. M. Boddeke 6 Division of Human Nutrition and Health, Chair group [email protected] of Nutritional Biology, Wageningen University & Research, Wageningen, the Netherlands 1 Department of Biomedical Sciences of Cells and Systems, 7 Department of Neuroscience, Karolinska Institute, Section Molecular Neurobiology, University of Groningen Stockholm, Sweden and University Medical Center Groningen (UMCG), Antonius Deusinglaan 1, 9713AV Groningen, 8 Neuroscience Discovery, AbbVie Deutschland GmbH & the Netherlands Co. KG, Ludwigshafen, Germany 2 Foundational Neuroscience Center, AbbVie Inc, Cambridge, 9 Department of Pathology and Medical Biology, University MA, USA Medical Center Groningen (UMCG), University of Groningen, Groningen, the Netherlands 3 Department of Biomedical Sciences, Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, 10 Department of Neurology, Memory Clinic of Hospital University of Antwerp, Wilrijk, Antwerp, Belgium Network Antwerp (ZNA), Middelheim and Hoge Beuken, Antwerp, Belgium 4 Department of Neurology and Alzheimer Center, University of Groningen and University Medical Center Groningen 11 Center for Healthy Ageing, Department of Cellular (UMCG), Groningen, the Netherlands and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark 5 Faculty of Medicine & Health Sciences, Translational Neurosciences, University of Antwerp, Antwerp, Belgium Vol.:(0123456789)1 3 Acta Neuropathologica Introduction Materials and methods Alzheimer’s disease (AD) is the most prevalent cause of Human brain tissue and neuropathology dementia, afecting about 35 million people worldwide. AD is neuropathologically characterized by abnormal aggrega- Brain tissue for snRNAseq was obtained from the Neuro- tion of extracellular amyloid-β and hyperphosphorylation of Biobank of the Institute Born-Bunge (NBB-IBB), Wilrijk neuronal tau. These pathological abnormalities exert stress (Antwerp), Belgium (ID: BB190113) and donors gave on various cell types in the brain, including neurons, oligo- informed consent to donate their brain to the NBB-IBB. dendrocytes, astrocytes, microglia, and vascular cells [31]. Ethical approval was granted by the medical ethics com- Microglia, the tissue-resident macrophages of the central mittee of the Hospital Network Antwerp (ZNA, approval nervous system (CNS) [21], are important for CNS homeo- numbers 2805 and 2806). The study was compliant with stasis and are implicated in AD pathology and single-cell the World Medical Association Declaration of Helsinki on profling of human microglia has frst been reported by Mas- Ethical Principles for Medical Research Involving Human uda et al. (2019). In amyloid mouse models of AD, a phago- Subjects. cytic/activated microglia phenotype was identifed (known Neuropathological evaluation of the brain was per- as DAM/ARM/MGnD) [8, 18, 30]. It is unclear whether a formed on the formalin-fxated right hemisphere. A stand- similar microglia phenotype is present in the human brain. ard selection of 10–13 regionally dissected brain regions, Recently, three single-cell transcriptomics studies based including frontal, temporal and occipital lobes (at the level on human tissue indicated that neurons, oligodendrocytes, of Brodmann area 17, area striata) of the neocortex, amyg- astrocytes and microglia are afected by AD pathology [11, dala, hippocampus (at the level of the posterior part of the 22, 26]. However, in these studies, changes associated with amygdala and the lateral geniculate body), basal ganglia, disease progression and transcriptional afects linked to thalamus, brainstem, substantia nigra, pons at the level amyloid-β and phospho-tau pathology on cellular transcrip- of the locus coeruleus and cerebellum (including dentate tional profles were not reported. gyrus), was embedded in parafn and routinely stained The complex morphology of brain cells and the low avail- with hematoxylin and eosin, cresyl violet and Bodian’s ability of ‘fresh’ material (biopsies/necropsies) complicate method, allowing neuropathological confirmation or single-cell RNA sequencing (scRNAseq) of human brain rejection of the clinically-established diagnosis. Further- tissue. As an alternative, brain banks contain high numbers more, routine examination of immunoreactivity against of frozen brain tissue samples, from which transcriptomic amyloid-β (clone 4G8) and P-tau181-P (clone AT8) was data can be generated. From frozen tissue, single nucleus can performed, as well as detection of hyperphosphorylated be analyzed as a reliable proxy for the cellular transcriptome TAR DNA-binding protein-43 (TDP)-43 and ubiquitin. [10]. Multiple frozen samples can be processed simultane- When the presence of Lewy bodies was suspected based ously allowing for balanced experimental designs, mini- on the hematoxylin and eosin and ubiquitin immunoreac- mizing technical variation between experimental groups. In tivity, an anti-α-synuclein staining was included to rule addition, frozen tissue samples allow for neuropathological out Parkinson’s disease. examination prior to sample preparation, which does not The staining procedures of the NBB-IBB are standardized always confrm the clinical diagnosis [1]. and as follows: parafn-embedded tissue sections were de- In the current study, amyloid-β and tau-pathology-associ- parafnized in xylene followed by an ethanol series from 100 ated transcriptional changes in AD were investigated through to 70% and rinsed in tap water. Antigen retrieval was per- single-nucleus RNA sequencing (snRNAseq). snRNAseq formed for the amyloid-β staining by incubation in 80% for- has previously been used to successfully characterize human mic acid for 5 min. For the phospho-tau staining, no antigen brain tissue from donors with AD, multiple sclerosis and retrieval was performed. Peroxidase blocking was performed autism spectrum disorder [11, 22, 29, 34]. In these studies, with 1% H2O2 in methanol for 30 min. The sections were put unsorted nuclei were profled, resulting in datasets largely in TBS (pH 7.4) and blocked with normal goat serum (1:25) composed of neurons and oligodendrocytes and with rela- in 1% BSA/TBS. Then, the sections were incubated with pri- tively low numbers of microglia and other less abundant cell mary antibody in 1% BSA/TBS overnight (AT8 1:10,000 for types. To overcome this limitation, we improved the isola- tau, own production; 4G8 1:10,000 for amyloid-β, Senetek). tion of nuclei of less abundant cell types from the far more Slides were then washed in TBS and incubated for 30 min numerous neuronal and oligodendrocyte nuclei in the total with secondary antibody (goat-anti-mouse IgG, 1:500) in CNS pool, increasing the statistical power to detect disease- 1% BSA/TBS. After washing with TBS, the sections were induced transcriptomic changes and progressive cell-state incubated for 30 min with Avidin–Biotin Complex. The sec- shifts in microglia and astrocytes. tions were incubated in a DAB solution (0.05% in TBS with 1 3 Acta Neuropathologica six drops NaOH and 12.5 μL of H2O2). Sections were rinsed Nuclei isolation in water and counterstained with Hematoxylin for 1 min. The sections were dehydrated in graded ethanol (70–100%) Fresh frozen brain tissue of the left hemisphere was used followed by xylene. Lastly, coverslips were mounted with for snRNAseq. Nuclei isolation and sorting were per- HistoRAL. formed on multiple days with two donors (one CTR and AD patients