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Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature

Abstract

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex—Mof, Kansl2 or Kansl3—unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)–NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.

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Fig. 1: NSL complex deletion causes haemorrhaging in the developing brain.
Fig. 2: Neural depletion of the NSL complex disrupts neural metabolic networks and causes vascular inflammation.
Fig. 3: MOF regulates metabolic gene expression via H4K16ac.
Fig. 4: MOF deficiency leads to an altered neural metabolic environment.
Fig. 5: LCFAs activate NFκB in brain pericytes.
Fig. 6: Neural MOF depletion results in the breakdown of neural microvasculature.
Fig. 7: Free LCFAs trigger a TLR4-driven NFκB inflammatory response in pericytes.
Fig. 8: Pericytes sense extracellular metabolic changes caused by MOF loss.

Data availability

All sequencing data from this study have been uploaded to the NCBI GEO database. Raw data pertaining to RNA-seq experiments are deposited under the accession number GSE138981, scRNA-seq data under GSE133079, and H4K16ac and histone H3 ChIP-seq data under GSE138981. Previously published MOF, KANSL3 and MCRS1 ChIP-seq profiles used in this study are deposited under GSE51746. Identity of genes specifically enriched in different E14.5 brain cell populations were derived from: http://betsholtzlab.org/VascularSingleCells/database.html and https://mpi-ie.shinyapps.io/braininteractomeexplorer. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the Core Imaging facility (IMCF, University of Basel) and in particular W. Heusermann for technical assistance provided on the SPIM light-sheet microscope. We thank B. Joch, K. Seidel, H. Al-Hasani and J. Seyfferth for technical assistance, as well as T. Vogel, S. Weise (University of Freiburg), R. Adams, H. W. Jeong and E. Watson (MPI for Molecular Biomedicine, Münster) for their helpful discussions. We thank M. Shvedunova for help with writing and editing the manuscript. B.N.S. was funded by an Alexander von Humboldt fellowship. This work was supported by the CRC 992 and CRC 1381 awarded to A.A. and by the German Research Foundation (DFG) under Germany’s Excellence Strategy (CIBSS, EXC-2189, project ID 390939984).

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B.N.S. and A.A. conceived and initiated the project. B.N.S., S.G., T.H.T., M.S., B.H., H.H., T.S., N.A., O.M., O.K., W.R., A.C., L.J.B., D.v.E. and J.M.B. planned and carried out experiments. B.N.S., G.R., V.B., O.B., N.A. and J.M.B. carried out the bioinformatics analyses. J.T. and H.S. diagnosed, genetically tested and provided brain biopsies of patients with KdV. B.N.S., S.G., T.H.T., M.S., G.R., V.B., S.A., J.M.B. and A.A. analysed data and interpreted the results. B.N.S., T.B., T.B.H., D.G., D.V., M.P. and A.A. supervised the project. B.N.S. and A.A. wrote the manuscript. All authors corrected and approved the manuscript.

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Correspondence to Asifa Akhtar.

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Extended data

Extended Data Fig. 1 Nes-Cre depletes the NSL complex from the E14.5 brain.

a, Diagrams depicting the floxed Mof, Kansl2 and Kansl3 alleles. b, Survival of progeny from a Moffl/+ Nes-CreT/+ x Moffl/fl cross. Mof-nKO animals were found at Mendelian ratios before birth, but none survived until weaning. ***p=5x10–92 test). ^^denotes dead embryo. c, Quantification of Mof, Kansl2 and Kansl3 allele recombination in E14.5 brains. Knockout and floxed alleles were quantified using quantitative genomic PCR. n = 5 Mof-nKO, 5 Moffl/fl; 5 Kansl2-nKO, 3 Kansl2fl/fl; 4 Kansl3-nKO, 4 Kansl3fl/fl embryos. d, Mof, Kansl2 and Kansl3 mRNA levels in E14.5 nKO brains. n = 4 Mof-nKO, 4 Moffl/fl; 3 Kansl2-nKO, 3 Kansl2fl/fl; 5 Kansl3-nKO, 3 Kansl3fl/fl embryos. e, Protein expression of NSL complex members in E14.5 Mof-nKO brains. MOF protein was strongly reduced (quantification provided), but other NSL complex members were unchanged. n = 3 embryos per genotype. f, Expression of NSL complex members in Kansl2-nKO brains. KANSL2 protein was depleted and reduced levels of MCRS1, KANSL3 and MOF were observed. n = 3 embryos per genotype. g, Expression of NSL complex members in E14.5 Kansl3-nKO brains. KANSL3 protein was depleted, while MOF showed a mild reduction. n = 3 embryos per genotype. h, Levels of NSL complex members in WT versus Nes-CreT/+ E14.5 brains. Nes-Cre alone did not affect the levels of NSL complex members. n = 3 brains per genotype. i, Immunoprecipitation (IP) with antibodies raised against NSL complex members MOF, KANSL1, KANSL2, KANSL3 and MCRS1 as well as MSL complex member MSL3 were carried out, followed by Western blot analyses for MOF, KANSL1 and KANSL3. The experiment was repeated 3 times. Data in panels c, d and e are presented as mean ± s.e.m. and were analysed using a two-tailed Student’s t-test. L indicates ladder. Statistical source data and unprocessed blots are shown in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 Mof-nKO causes disruption of the ganglionic eminence.

a, Quantification of H4K16ac using immunofluorescence. Left panel: Representative H4K16ac staining in the E14.5 brain. Area indicated by the white box was quantified in Mof-nKO and Nes-CreT/+ brains. The white arrow signifies the occasional H4K16ac-positive cells in Mof-nKO. Right panel: enumeration of H4K16ac-positive cells. n = 4 animals per genotype, with 388 to 871 cells counted per sample. b, Activated caspase-3 staining showing extensive cell death in the ganglionic eminence region of the E14.5 Mof-nKO brain (marked by red arrow). 4 embryos per genotype were analysed. c, Staining of the E14.5 cortex with neuronal markers CTIP2 and SATB2 and cell death marker activated caspase-3. White squares represent the area from which the inset images are derived. Right panel: Quantification of activated caspase-3 and CTIP2 double positive cells. n = 4 animals per genotype, with 1310 CTIP2+ cells analysed from Mof-nKO, and 1597 from Nes-CreT/+ animals. d, Immunostaining of E16.5 Mof-nKO cortices for neuronal markers CTIP2 and SATB2. e, Transmission electron microscope (TEM) images of the ventricular zone lining the lateral ventricle at E14.5. f, Histological analysis of the choroid plexus. The choroid plexus is indicated with the red dotted line. 3 animals per genotype were analysed in d-f. g, Quantification of cell proliferation via phospho-histone H3 (pH3) staining in the ventricular zone. Positive cells were counted in the first three cell layers adjacent to the lateral ventricle. n = 3 animals per genotype; 1931 Mof-nKO, 937 Nes-CreT/+ cells analysed. Data in panels a, c and g are presented as mean ± s.e.m. and were analysed using a two-tailed Student’s t-test. Ctx – cortex, E – eye, GE – ganglionic eminence, LV – lateral ventricle. All scale bars are provided in μm. Statistical source data are shown in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Nes-Cre is specifically active in neural cells.

a, EMBRACE38 sorting strategy for the simultaneous isolation of cell populations enriched for neural cells (CD11b, CD45, CD41, PDGFRβ, PECAM1, CD102), pericytes (PDGFRβhigh, CD11b, CD45, CD41, PECAM1, CD102), endothelial cells (PECAM1+, CD102+, CD45, CD41) and microglia (CD11b+, CD45medium, PDGFRβ, PECAM1) from the E14.5 brain. b, Diagrammatic depiction of the mTomato-mGFP allele. Membrane-targeted (m) Tomato is expressed ubiquitously but is flanked by loxP sites. Following Cre-mediated recombination, mTomato is spliced out and mGFP expression activated. The histograms show mTomato and mGFP expression in E14.5 brain cells in the presence and absence of the Nes-Cre. As expected, Nes-Cre-mediated recombination results in lower levels of mTomato and activation of mGFP expression. 4 mT/mGT/+ Nes-CreT/+ E14.5 embryos were analysed. c, Exemplary FACS plots showing mTomato and mGFP expression in populations enriched for neural cells (PECAM1, CD11b), pericytes (PDGFRβhigh, CD13high), endothelial cells (EC, PECAM1+ CD11b) and microglia (CD11b+ CD45medium). 4 mT/mGT/+ Nes-CreT/+ E14.5 embryos were analysed. d, Quantification (median fluorescence intensity) of mTomato and mGFP expression in cell populations enriched for neural, pericytes, endothelial and microglial cells by flow cytometry. Increased mGFP expression and loss of mTomato expression was observed in neural cells. Only high mTomato expression was evident in the pericyte, endothelial and microglia enriched fractions. n = 4 mT/mGT/+ Nes-CreT/+ E14.5 embryos. Data are presented as mean ± s.e.m. Data were analysed using a two-tailed Student’s t-test via pair-wise comparisons of neural cells with each of the other cell populations. Asterisks signify statistical significance at ***p < 0.001. Precise p-values of all comparisons and statistical source data are shown in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 Neural deletion of the NSL complex causes metabolic changes.

a, Enrichment of marker genes in neural cells, pericytes, endothelial cells and microglia in cell populations isolated using the EMBRACE methodology. The enrichment score was calculated using standardized counts from the DESeq2 output with the formula [(C – average[C])/sd]. C represents the standardized read counts in the given cell population. Average[C] is the average counts of the gene across all four cell populations. sd is the standard deviation. The list of enriched genes for each population was extracted from http://betsholtzlab.org/VascularSingleCells/database.html71 and https://mpi-ie.shinyapps.io/braininteractomeexplorer38. Number of animals analysed: Neural cells, 3 per genotype; pericytes, 4 Nes-CreT/+, 3 Mof-nKO, 3 Kansl2-nKO, 4 Kansl3-nKO; endothelial cells (EC), 4 Nes-CreT/+, 4 Mof-nKO, 3 Kansl2-nKO, 3 Kansl3-nKO; microglia 3 per genotype. b, IGV profiles showing reads mapped via RNA-seq across the Mof locus. No reads were detected between the loxP sites in neural cells from Mof-nKO brains, where the Nes-Cre is active. In contrast, reads across the Mof locus in pericytes, endothelial cells and microglia isolated from Mof-nKO brains were unchanged compared to Nes-CreT/+ controls. Number of animals analysed: Neural cells, 3 per genotype; pericytes, 4 Nes-CreT/+, 3 Mof-nKO; endothelial cells (EC), 4 per genotype; microglia, 3 per genotype. c, KEGG pathways significantly enriched amongst genes upregulated in Mof-nKO neural cells. Data are presented on a log2 scale. n = 3 brains per genotype. Data were analysed using a Fisher exact test via the DAVID platform66. d, Correlation plots for gene expression changes in Mof-nKO, Kansl2-nKO and Kansl3-nKO neural cells. Gene expression changes in the presented comparisons were used to calculate the Pearson’s coefficient (r). n = 3 brains per genotype. e, Heatmaps showing the downregulation of OXPHOS and lysosome pathway genes in Mof-nKO, Kansl2-nKO and Kansl3-nKO neural cells. The gene lists were downloaded from the KEGG pathway database and converted to the corresponding mouse genes. n = 3 animals per genotype. Statistical source data are shown in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 Catalytic activity of MOF regulates levels of LCFAs.

a, Quantification of mitochondrial DNA via genomic qPCR. n = 4 brains per genotype. b, Mitochondrial respiration in cells acutely isolated from E14.5 brains and analysed using the Seahorse analyser. n = 3 Mof-nKO, 2 Moffl/+ Nes-CreT/+, 8 Cre-negative. A–antimycin A, F–FCCP, O–oligomycin, R–rotenone. c, Quantification of basal respiration rate from Seahorse profiles (panel b). d, Quantification of ATP-linked respiration from Seahorse profiles (panel b). e, Mof mRNA levels in Mof-KO and WT neurosphere cultures following 4-hydroxy-tamoxifen (4OHT) treatment. Exp.1 n = 6 Mof-KO, 8 WT; Exp.2 n = 5 Mof-KO, 8 WT neurosphere cultures. f, Western blot analysis of MOF, Flag-tagged MOF, Actin, H4K16ac and histone H3 protein levels after re-expression of WT and E350Q MOF in Mof-KO and control MEFs. L indicates ladder. Protein sizes are indicated in kDa. 4 independent MEF lines per genotype and per treatment were analysed. g-k, Quantification of g, Flag expression, h Total MOF, i endogenous MOF, j exogenous MOF and k H4K16ac levels in WT and Mof-KO cultures. Quantification was carried out from the blots shown in panel f. Total MOF levels are a combination of Flag-tagged MOF (exogenous, higher MW) and endogenous MOF. Flag-tagged and endogenous MOF levels were standardized to Actin. H4K16ac levels were standardized to histone H3. Asterisks signify statistical significance at *p < 0.05, **p < 0.01 and ***p < 0.001. Precise p-values are provided in Source Data Extended Data Fig. 5. n = 4 independent MEF lines per genotype and per treatment. l LCFA levels in WT and Mof-KO MEFS after the re-expression of WT and E350Q mutant MOF. 4 MEF lines per genotype and per treatment were analysed. Data are presented as mean ± s.e.m. and were analysed using a two-tailed Student’s t-test. Statistical source data and unprocessed blots are shown in Source Data Extended Data Fig. 5. Source data

Extended Data Fig. 6 Neural MOF depletion causes breakdown of neural microvasculature.

a, Heatmap showing enrichment of pericyte markers in cultured primary brain pericytes. The assay was repeated 3 times (represented by the 3 replicates). b, Examples of control Nes-CreT/+ capillaries. 63 capillaries of 2 Nes-CreT/+ brains were analysed. c, Electron microscopy images of select Mof-nKO capillaries. Images i and ii show collapsed capillaries with only remnants visible (marked by red asterisks). Capillaries from Mof-nKO brains were highly dilated (iv, v, vi, viii, xi and xii for example, marked with asterisks), often showed breakdown or thinning of the vessel-associated extracellular matrix (red arrows in images iii, iv, v, vii, ix and x), as well as detaching pericytes (black arrows in images vi, vii, ix and x). 79 capillaries of 2 Mof-nKO brains were analysed. Full quantifications are provided in Fig. 6c-e. d, Quantification of vessel branching (bifurcations). PECAM1 and PDGFRβ staining was undertaken. Four matching areas of the cortex were imaged, vasculature rendered using the Imaris software and the number of bifurcations determined. n = 3 animals per genotype; 2 sections per animal; 4 cortical areas per section. e, Quantification of mural cells at blood vessels. E14.5 brain sections were stained for PECAM1 and PDGFRβ and the ratio of PDGFRβ:PECAM1 staining intensity was used as a readout for the presence of mural cells. n = 2 animals per genotype. f, FACS identification and quantification of endothelial cells (PECAM1+, CD102+, CD45, CD41) and pericytes (PDGFRβhigh, CD11b, CD45, CD41, PECAM1, CD102) in Mof-nKO and Nes-CreT/+ brains. Endothelial cells: n = 4 Mof-nKO, 3 Nes-CreT/+; Pericytes: n = 10 Mof-nKO, 7 Nes-CreT/+ brains. Data in panels d and f are presented as mean ± s.e.m. and were analysed using a two-tailed Student’s t-test. Scale bars are provided in μm. E – endothelial cell, P – pericyte. Statistical source data are shown in Source Data Extended Data Fig. 6. Source data

Extended Data Fig. 7 Endothelial cells in Mof-nKO brains show normal zonation.

a, Experimental design. Endothelial cells (PECAM1+ CD102+) were isolated by FACS and scRNA-seq undertaken using mCEL-Seq268,72. Cells expressing more than 1,500 unique transcripts and the endothelial marker Flt1 were filtered. A total of 401 endothelial cells were subsequently analysed. b, Diffusion map showing zonation profile of endothelial cells isolated from Mof-nKO and WT littermates and analysed by scRNA-seq. Pseudotime is interpreted here as a spatial zonation coordinate along the arterial → capillary → venous axis. c, Self-organizing-map (SOM) depicting the expression of zonated genes in Mof-nKO and WT endothelial cells. The red (Mof-nKO) and black (WT) bars below the SOM represent the genotype of each endothelial cell along the arterial → capillary → venous zonation axis. In each of the four quadrants, no difference in the ratio of Mof-nKO:WT endothelial cells was observed (Fisher test). NES – normalized enrichment score. A NES of 1 refers to an equivalent ratio Mof-nKO and WT cells, a NES > 1 refers to fewer Mof-nKO cells, while a NES < 1 refers to increased Mof-nKO cells in each quadrant. d, Expression of the zonated marker genes Nr2f2, Mfsd2a, Tfrc, Sema3g and Vegfc across the arterial → capillary → venous axis. 3 animals per genotype were analysed in b-d. e, Summary of Tlr gene expression in microglia / macrophage (cluster 1), endothelial (cluster 6) and pericyte (cluster 19) populations derived from the study of Han and co-workers41. f, Presence of BODIPY FL-C16 in primary brain pericytes cultured for 18 h in increasing concentrations of BODIPY FL-C16. MFI – median fluorescence intensity. g, Experimental design for LCFA tracing experiment presented in Fig. 8b. Statistical source data are shown in Source Data Extended Data Fig. 7. Source data

Extended Data Fig. 8 Mof-nKO microglia display an NFκB pro-inflammatory signature.

a, Experimental outline. Microglia (CD11b+, CD45medium, PDGFRβ, PECAM1) were isolated from Mof-nKO and Nes-CreT/+ control brains and analysed via RNA-seq. With a false discovery rate (FDR) cut-off of 0.05, 165 genes were found to be significantly upregulated and 307 genes significantly downregulated. n = 3 animals per genotype. b, GO term (Biological Processes) analyses for differentially expressed genes (FDR < 0.05) in Mof-nKO microglia versus controls. Pathways related to inflammation such as “immune system processes” and “positive regulation of inflammatory response” were significantly enriched. Data were analysed using a Fisher exact test via the DAVID platform66. n = 3 animals per genotype. c, Expression of NFκB target genes in microglia derived from Mof-nKO brains compared to Nes-CreT/+ controls. Data are presented on a log2 scale. Expression of the majority of NFκB target genes was upregulated in microglia derived from Mof-nKO brains. n = 3 animals per genotype. Statistical source data are shown in Source Data Extended Data Fig. 8. Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: Genotyping primers. Supplementary Table 2: qRT-PCR primers. Supplementary Table 3: Primers used to quantify mitochondrial and nuclear DNA. Supplementary Table 4: Antibody list.

Supplementary Video 1

Neural vascular imaging using light-sheet microscopy. Exemplary wild-type E14.5 cleared brain stained with the endothelial marker PECAM1 and subsequently imaged with light-sheet microscopy. ‘Fly through’ visualization was made using the Arivis Vision4D software.

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Sheikh, B.N., Guhathakurta, S., Tsang, T.H. et al. Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature. Nat Cell Biol 22, 828–841 (2020). https://doi.org/10.1038/s41556-020-0526-8

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