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Neurovascular dysfunction in GRN-associated frontotemporal dementia identified by single-nucleus RNA sequencing of human cerebral cortex

Nature Neuroscience volume 25, pages 1034–1048 (2022)Cite this article

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Abstract

Frontotemporal dementia (FTD) is the second most prevalent form of early-onset dementia, affecting predominantly frontal and temporal cerebral lobes. Heterozygous mutations in the progranulin gene (GRN) cause autosomal-dominant FTD (FTD-GRN), associated with TDP-43 inclusions, neuronal loss, axonal degeneration and gliosis, but FTD-GRN pathogenesis is largely unresolved. Here we report single-nucleus RNA sequencing of microglia, astrocytes and the neurovasculature from frontal, temporal and occipital cortical tissue from control and FTD-GRN brains. We show that fibroblast and mesenchymal cell numbers were enriched in FTD-GRN, and we identified disease-associated subtypes of astrocytes and endothelial cells. Expression of gene modules associated with blood–brain barrier (BBB) dysfunction was significantly enriched in FTD-GRN endothelial cells. The vasculature supportive function and capillary coverage by pericytes was reduced in FTD-GRN tissue, with increased and hypertrophic vascularization and an enrichment of perivascular T cells. Our results indicate a perturbed BBB and suggest that the neurovascular unit is severely affected in FTD-GRN.

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Fig. 1: Workflow of the experiment and sample validation.
Fig. 2: snRNA-seq of NEUNnegOLIG2neg nuclei.
Fig. 3: Altered cell–cell interaction network in the FC of FTD-GRN donors.
Fig. 4: Microglia are only mildly affected in FTD-GRN brains.
Fig. 5: Astrocytes are severely affected in the frontal and temporal cortices of FTD-GRN donors.
Fig. 6: Endothelial cells are affected and express BBB dysfunction gene modules in FTD-GRN.
Fig. 7: Loss of functional pericytes in FTD-GRN frontal and temporal cortices.

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Data availability

The data generated in this study are available through the Gene Expression Omnibus at https://www.ncbi.nlm.nih.gov/geo with accession number GSE163122. Spatial transcriptomics data were derived from 10x Genomics (https://support.10xgenomics.com/spatial-gene-expression/datasets/1.1.0/V1_Human_Brain_Section_1). snRNA-seq data from Grn−/− mouse thalami from Zhang et al.32 were downloaded from the Sequence Read Archive under accession number PRJNA507872.

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Acknowledgements

We would like to thank G. Mesander, J. Teunis and T. Bijma from the flow cytometry unit of the University Medical Center Groningen for sorting of the nuclei; L. Hesse and L. Apperloo for use of the 10x Genomics Chromium system; M. Meijer and H. van Weering for microscopy; J. Mulder for the WDR49 antibody; W. Baron and J. Dallinga-de Jonge for materials; the Research Sequencing Facility of the University Medical Center Groningen for sequencing; and the Center for Information Technology of the University of Groningen for their support and for providing access to the Peregrine high-performance computing cluster. We would like to thank the Netherlands Brain Bank and S. Leclère-Turbant of the Neuro-CEB brain bank for providing brain tissues. We would like to thank D. J. Irwin (University of Pennsylvania) for sharing the digital analysis protocol for pathology quantification using the open-source QuPath software. Several authors of this publication are members of the European Reference Network for Rare Neurological Diseases (project ID 739510). E.G. was funded by the Graduate School of Medical Sciences of the University of Groningen. I.L.B. received funding from the ‘Investissements d’avenir ANR-11INBS-0011’ and the ‘Programme Hospitalier de Recherche Clinique FTLD-exome’ (promotion AP-HP). This study was supported by ZonMw (project no. 733050513), the Bluefield Project to Cure Frontotemporal Dementia and Alzheimer Nederland (WE.03-2019-09). This project was co-financed by the Ministry of Economic Affairs and Climate Policy by means of the PPP allowance made available by the Top Sector Life Sciences & Health to stimulate public–private partnerships. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: John C. van Swieten, Bart J. L. Eggen.

  2. A list of members and their affiliations appears in the Supplementary Information.

Authors and Affiliations

  1. Department of Biomedical Sciences of Cells & Systems, Section of Molecular Neurobiology, University of Groningen and University Medical Center Groningen, Groningen, Netherlands

    Emma Gerrits, Nieske Brouwer, Erik W. G. M. Boddeke & Bart J. L. Eggen

  2. Department of Neurology & Alzheimer Center, Erasmus University Medical Center, Rotterdam, Netherlands

    Lucia A. A. Giannini, Shamiram Melhem, Harro Seelaar & John C. van Swieten

  3. Department of Neuropathology, Pitie Salpetriere Hospital, AP-HP Sorbonne Université, Paris, France

    Danielle Seilhean

  4. Paris Brain Institute – Institut du Cerveau (ICM), Sorbonne Université, Inserm U1127, CNRS UMR 7225, AP-HP – Hôpital Pitié-Salpêtrière, Paris, France

    Danielle Seilhean & Isabelle Le Ber

  5. Reference Center for Rare or Early Dementias, IM2A, Département de Neurologie, AP-HP - Hôpital Pitié-Salpêtrière, Paris, France

    Isabelle Le Ber

  6. MS Center Amsterdam, Department of Molecular Cell Biology and Immunology, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, Netherlands

    Alwin Kamermans, Gijs Kooij & Helga E. de Vries

  7. Center for Healthy Ageing, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark

    Erik W. G. M. Boddeke

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The Brainbank Neuro-CEB Neuropathology Network

Contributions

J.V.S. and B.J.L.E. conceived the study. E.G. and N.B. performed the RNA sequencing experiments. E.G. analysed and visualized the RNA sequencing data and wrote the manuscript. E.G., L.A.A.G., N.B., S.M., A.K., H.E.D.V., G.K. and H.S. performed and/or analysed immunohistochemistry. N.B. performed RT–qPCR experiments. L.A.A.G., S.M., I.L.B., D.S., H.S. and J.V.S. provided tissue collection and clinical information. H.S. and J.V.S. supervised L.A.A.G. B.J.L.E. and E.W.G.M. supervised E.G. J.V.S. and B.J.L.E. provided resources. All authors contributed to project design, organization of the experiments, interpretation of results and writing of the manuscript.

Corresponding author

Correspondence to Bart J. L. Eggen.

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Extended data

Extended Data Fig. 1 FANS sorting strategy and tissue validation.

(a) Fluorescent activated nuclear sorting (FANS). The DAPIpos nuclei population is segregated into three groups and these populations were collected separately: NEUNposOLIG2neg (neurons), NEUNnegOLIG2pos (oligodendrocyte lineage), NEUNnegOLIG2neg (other CNS cells). (b) Representative images of TDP43 IHC across the cortex. Scale bar is 250 μm. (c) Quantification of neuronal loss in Nissl staining per sample. Lines depict mean per group. Circles depict individual samples. Statistical analysis between CTR and FTD-GRN samples for each brain region was performed with unpaired two-samples Wilcoxon test. *:p < 0.05; **: p < 0.01; ***:p < 0.001. CTR = Control; FTD = Frontotemporal Dementia; FC = Frontal Cortex; TC = Temporal Cortex; OC = Occipital Cortex.

Extended Data Fig. 2 Bulk RNAseq neurons, OPCs/oligodendrocytes.

(a) Volcano plots depicting logFC (x-axis) versus -log(FDR) (x-axis) values per gene for OC versus FC in CTR and FTD-GRN separately. DEGs are defined as p < 0.05 and abs(logFC) > 1. The number of DEGs is indicated in the plot. (b) Fourway plot depicting log fold changes of FC versus OC in CTR (x-axis) and in FTD-GRN (y-axis). Green circles are DEGs between FC and OC in both CTR and FTD-GRN (region-associated DEGs). Purple circles are DEGs exclusively enriched in FTD-GRN-FC (enriched disease-associated). Yellow circles are DEGs exclusively depleted in FTD-GRN-FC (depleted disease-associated). (c) Principal component analysis of OLIG2pos bulk RNAseq. Colors depict brain regions, filled-circles are from CTR and open-circles are from FTD-GRN. (d) Barplot depicting imputed fractions of cell types in OLIG2pos nuclei samples. (e) Same PCA plot as in c), color scale depicts imputed OPC fraction in deconvolution analysis. (f) Fourway plot depicting log fold changes of FC versus OC in CTR (x-axis) and in FTD-GRN (y-axis). Green circles are DEGs between FC and OC in both CTR and FTD-GRN (region-associated DEGs). Purple circles are DEGs exclusively enriched in FTD-GRN-FC (enriched disease-associated). Yellow circles are DEGs exclusively depleted in FTD-GRN-FC (depleted disease-associated). (g) Barplot depicting imputed fractions of cell types in NEUNpos samples. OPC = Oligodendrocyte Progenitor Cell; FC = Frontal Cortex; TC = Temporal Cortex; OC = Occipital Cortex; CTR = Control; FTD = Frontotemporal Dementia. GM = Grey Matter; WM = White Matter; L1 = Layer 1; CAMs = CNS-associated Macrophages; SMCs = Smooth Muscle Cells. PC = Principal Component. DEG = Differentially Expressed Gene.

Extended Data Fig. 3 Vascular cell type identification and validation.

(a) UMAPs and violin plots depicting module scores of cell type specific mouse gene sets from Vanlandewijck et al. (b) Dot plots per group depicting RT-qPCR results from COL1A1 (fibroblasts) and PHLDB2 (mesenchymal) on total brain tissue,. Lines indicates mean per group. Circles depict individual tissue samples. CTR and FTD-GRN samples for each brain region was analyzed with an unpaired two-samples Wilcoxon test (c) Dot plot depicting top 10 most significantly enriched GO terms on marker genes of each cell type. (d) Heatmap depicting top 15 most enriched genes per cell type. Colors indicate scaled gene expression levels. FC = Frontal Cortex; TC = Temporal Cortex; OC = Occipital Cortex, SMC = Smooth Muscle Cell; GO = Gene Ontology; CTR = Control; FTD = Frontotemporal Dementia *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Extended Data Fig. 4 Hierarchy plots depicting selected differential signaling pathways between CTR and FTD-GRN frontal cortex.

(a-d) Hierarchy plot depicting TENASCIN (a), NRXN (b), COMPLEMENT (c) and PTN (d) signaling network pathways across all cell types for each group. Circle sizes are proportional to the number of cells in each group. Edge width is proportional to communication probability.

Extended Data Fig. 5 No clear increase in microglia numbers and slightly altered morphological changes in situ in FTD-GRN brains and statistical analysis of subcluster distributions.

(a) Representative images of IBA1 staining in the grey matter of CTR and FTD-GRN samples in FC, TC and OC. Scale bar is 100 μm. (b) Quantification of IBA1 staining. Top: Number of microglia per mm2; Bottom: Fraction IBA1-positive pixels. Lines depict mean per group. Circles depict individual samples. Statistical analysis between CTR and FTD-GRN samples for each brain region was performed with an unpaired two-samples Wilcoxon test. (c) Microglia subcluster distribution per group. (d) Violin plots per sample depicting module scores of a Grn-/- microglia enriched geneset from Zhang et al. (2020). Line depicts mean of all CTR samples. (e) GM-Astrocytes subcluster distribution per group. (f) Endothelial cells subcluster distribution per group. (g) Pericytes subcluster distribution per group. Statistical analysis was performed with a Chi-squared test on the group averages. Abbreviations: CTR = Control; FTD = Frontotemporal Dementia; FC = Frontal Cortex; TC = Temporal Cortex; OC = Occipital Cortex. Chi squared test *: p ≤0.05; **: p ≤0.01; ***: p ≤0.001.

Extended Data Fig. 6 Gene ontology analysis GM-Astrocytes.

(a) Dot plot of top 10 most significantly enriched GO terms of marker genes of each GM-Astrocyte sub cluster. (b) Representative GFAP images of the FC of a CTR donor and FC and TC of FTD-GRN donor. Scale bar is 50 μm. Dot plot of number of GFAPpos cells/0.5 mm2/group. Lines depicts mean/group, circles individual samples. CTR and FTD-GRN samples for each brain region were analyzed with an unpaired two-samples Wilcoxon test. (c) Dot plot of % GFAP-positive area per group. Lines depicts mean/group, circles individual samples. CTR and FTD-GRN samples for each brain region were analyzed with an unpaired two-samples Wilcoxon test. (d) Representative grey matter images for astrocyte marker GFAP. Dashed line shows grey-white matter junction. Scale bar is 250 μm. (e) Correlogram depicting Pearson correlations between different IHC measurements. (f) Dot plot depicting ratio of perivascular AQP4 area over vessel area per vessel per group. Lines depict mean per group. Circles depict individual samples. Statistical analysis between CTR and FTD-GRN samples for each brain region was performed with an unpaired two-samples Wilcoxon test. (g) Representative images of multiplexed IHC for proteins DAPI (blue), laminin (red), UAE-1 (ULEX; green) and AQP4 (white). Scale bar is 10 μm. CTR = Control; FTD = Frontotemporal Dementia; FC = Frontal Cortex; TC = Temporal Cortex; OC = Occipital Cortex; GM = Grey Matter. *: p≤0.05; **: p≤0.01; ***: p≤0.001.

Extended Data Fig. 7 WDR49 immunohistochemistry.

Representative immunohistochemical images of WDR49 in grey matter tissue of 4 CTR and 13 FTD donors. Circles depict clusters of WDR49pos cells. Each image is a different donor. Scale bar is 500 μm.

Extended Data Fig. 8 Immunofluorescent staining for WDR49 and GFAP.

Representative images of fluorescent double labelling of WDR49 and GFAP in FTD-GRN frontal cortex. Scale bar is 50 μm in (a) and 10 μm in (b).

Extended Data Fig. 9 Trajectory analysis of endothelial cell zonation, and neovascularization and inflammation in FTD-GRN.

(a) UMAPs depicting module scores for endothelial cell zonation genesets derived from Vanlandewijck et al. (2018) and trajectory analysis on zonation genes. (b) Dot plot depicting number of CLDN5pos and LNpos fragments per group per region. Lines indicate mean per group. Circles depict individual samples. Statistical analysis between CTR and FTD-GRN samples for each brain region was performed with unpaired two-samples Wilcoxon test. (c) Images of structures suggestive for neovascularization and inflammation in CLDN5 staining of FTD-GRN frontal cortex. Scale bar is 100 μm. (d) CLDN5/FN staining of raspberry and neovascularization structure in FTD-GRN frontal cortex. Nuclei are depicted in grey, CLDN5 in cyan and FN in magenta. Scale bar is 50 μm. (e) Dot plots depicting RT-qPCR result for CD31 on total brain tissue. Lines indicate mean per group. Circles depict individual samples. Statistical analysis between CTR and FTD-GRN samples for each brain region was performed with unpaired two-samples Wilcoxon test. Abbreviations: CTR = Control; FTD = Frontotemporal Dementia; FC = Frontal Cortex; TC = Temporal Cortex; OC = Occipital Cortex; CLDN5 = Claudin 5; LN = Laminin. *: p ≤0.05; **: p ≤0.01; ***: p ≤0.001.

Extended Data Fig. 10 Single-nucleus RNA sequencing of nuclei from Grn−/− mouse thalami.

(a) UMAP depicting 175,884 nuclei from 38 thalami from both WT and Grn−/− mice across 5 ages. Colors depict cell type clusters resulting from unsupervised (sub)clustering analysis. (b) Correlogram depicting correlations of gene expression profiles between cell types. Cell types are ordered by hierarchical clustering depicted on the left side. (c) Dotplot depicting representative marker genes for each cell type. Color scale depicts average gene expression level, size of the dots depicts fraction of nuclei expressing the gene. (d) Barplots depicting cell type distribution in each group per age. Data points represent individual samples. Bars depict mean, error bars depict standard error. Abbreviations: CAMs = CNS-associated Macrophages; SMCs = Smooth Muscle Cells; OPCs = Oligodendrocyte Progenitor Cells.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and membership list for the Brainbank Neuro-CEB Neuropathology Network

Reporting Summary

Supplementary Table

Table 1. Donor and sample information. Table 2. Disease-associated DEGs between occipital and frontal neuronal and oligodendrocyte/OPC nuclei. Table 3. Differential gene expression between all cell types and subtypes. Table 4. Imputed cell–cell communication. Table 5. Differential gene expression between subclusters within each cell type. Table 6. Antibodies used for IHC/IF. Table 7. RT–qPCR primer information

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Gerrits, E., Giannini, L.A.A., Brouwer, N. et al. Neurovascular dysfunction in GRN-associated frontotemporal dementia identified by single-nucleus RNA sequencing of human cerebral cortex. Nat Neurosci 25, 1034–1048 (2022). https://doi.org/10.1038/s41593-022-01124-3

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