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Mice deficient in NRROS show abnormal microglial development and neurological disorders

Nature Immunology volume 18, pages 633–641 (2017)Cite this article

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Abstract

Microglia and other tissue-resident macrophages within the central nervous system (CNS) have essential roles in neural development, inflammation and homeostasis. However, the molecular pathways underlying their development and function remain poorly understood. Here we report that mice deficient in NRROS, a myeloid-expressed transmembrane protein in the endoplasmic reticulum, develop spontaneous neurological disorders. NRROS-deficient (Nrros−/−) mice show defects in motor functions and die before 6 months of age. Nrros−/− mice display astrogliosis and lack normal CD11bhiCD45lo microglia, but they show no detectable demyelination or neuronal loss. Instead, perivascular macrophage-like myeloid cells populate the Nrros−/− CNS. Cx3cr1-driven deletion of Nrros shows its crucial role in microglial establishment during early embryonic stages. NRROS is required for normal expression of Sall1 and other microglial genes that are important for microglial development and function. Our study reveals a NRROS-mediated pathway that controls CNS-resident macrophage development and affects neurological function.

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Figure 1: Nrros−/− mice develop spontaneous neurological disorders and early mortality.
Figure 2: Abnormal CNS astrocytes and myeloid cells in Nrros−/− mice.
Figure 3: Microglia in Nrros−/− mice display phenotypes similar to PVMs.
Figure 4: CNS-resident macrophages but not peripheral macrophages contribute to PLC development and neurological disorders.
Figure 5: NOX2 does not contribute significantly to the neurological phenotype in Nrros−/− mice.
Figure 6: Defective early microglial development in Nrros−/− mice.
Figure 7: Intrinsic role of NRROS in Cx3cr1+ lineage during early microglial differentiation.
Figure 8: PLCs in Nrros−/− mice display characteristics distinct from those of WT microglia.

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Acknowledgements

We thank K. Srinivasan and D. Hansen for assistance with flow cytometry–purified brain cells and fluidigm analysis; I. Peng for assistance with bone marrow transfer; B. Lauffer and P. Steiner for advice and help with microglia isolation and culture; C. Le Pichon, S. Dominguez and M. Weber for advice with behavioral studies; Z. Modrusan, C. Ha, J. Stinson and J. Guillory for assistance with RNA-seq; L. Diehl and P. Caplazi for advice and help with histopathological studies; and C. Allen, M. Thayer, M. Long, M. Lamoureux, T. Scholls and the Genentech Lab Animals facility for help and support with mouse colonies.

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Author notes

  1. Rajkumar Noubade, Paolo Manzanillo, Kimberly Scearce-Levie & Wenjun Ouyang

    Present address: Present addresses: Department of Inflammation and Oncology, Amgen Inc., South San Francisco, California, USA (R.N., P.M. and W.O.); Department of Preclinical Translational Biology, Denali Therapeutics, South San Francisco, California, USA (K.S.-L.).,

  2. Kit Wong, Rajkumar Noubade and Paolo Manzanillo: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology, Genentech, South San Francisco, California, USA

    Kit Wong, Rajkumar Noubade, Paolo Manzanillo, Naruhisa Ota, Rajita Pappu & Wenjun Ouyang

  2. Department of Pathology, Genentech, South San Francisco, California, USA

    Oded Foreman

  3. Department of Bioinformatics and Computational Biology, Genentech, South San Francisco, California, USA

    Jason A Hackney & Brad A Friedman

  4. Department of Neuroscience, Genentech, South San Francisco, California, USA

    Kimberly Scearce-Levie

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Contributions

K.W. and W.O. designed the study and wrote the manuscript. K.W., R.N., P.M., N.O. and O.F. performed experiments. R.N., P.M. and R.P. edited the manuscript. J.A.H. and B.A.F. analyzed RNA-seq data. K.W., P.M. and K.S.-L. designed experiments.

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Integrated supplementary information

Supplementary Figure 1 Nrros-/- mice show distended bladder and normal relative mass of major organs.

Representative H&E stained images of (a - b) bladder (bar = 500 μm), (c - d) ureter, (e - f) renal papillary epithelium (bar = 100 μm) and (g-h) renal glomerulus (bar = 50 μm) from Nrros+/+ and Nrros-/- animals. Graphs depicting the mass of gastrocnemius (i), tibialis (j), brain (k) and kidney (l) as percent body weight in Nrros+/+ and Nrros-/- animals. Arrow, outer smooth muscle wall; *, transitional epithelium. N ≥ 8. Error bar: ± SEM. Animals were 13 - 15 weeks old.

Supplementary Figure 2 Absence of immune infiltration and neurodegenerative signs in Nrros−/− mice.

Representative images of spinal cord cross sections from Nrros+/+ and Nrros-/- animals stained with (a) H&E (bar = 50 μm), (d) Nissl (bar = 500 μm) or (e) Luxol fast blue (bar = 1 mm). (b) Survival curve (n = 10) and (c) wire hang from WT and Nrros-/-Rag2-/- animals (12 - 14 weeks old; n = 5). Quantification of (f) number of axons per area, (g) total number of axons and (h) percent axons with indicated lumen area from p-Phenylenediamine staining on cross sections of sciatic nerve (n = 8). 15-week old Nrros+/+ and Nrros-/-animals are shown. Error bar: ± SEM. ***P < 0.001. Log rank test (b), One-way ANOVA (c), unpaired Student’s t-test (f - h).

Supplementary Figure 3 Signs of astrogliosis and altered expression of microglia signature genes in Nrros−/− brains.

RNA-seq comparing expression of whole brains from Nrros+/+ and Nrros-/- mice. Expression levels of (a - d) astrogliosis and (e - i) microglial markers. Itam (CD11b); Emr1 (F4/80). N = 5. (j) Full Western blot for protein expression of NRROS in the indicated cell types from WT brain as shown in Figure 3e. Actin as loading control. (k – p) Representative histograms of markers indicated in CD11b+ cells from Nrros+/+ and Nrros-/- brains. N = 3. Error bar: ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Unpaired Students’ t-test.

Supplementary Figure 4 Loss of Nrros from resident CNS macrophages but not peripheral myeloid cells causes neurological disorders.

(a - b) RT-qPCR analysis for Nrros expression in CD11b+ cells isolated from the brains of mice of the indicated genotype. Performance of (c - g) Nrrosfl/flVav-cre or (h - l) Nrrosfl/flLyz2-cre mice in rotarod test at (c, h) pretraining (9-week old), (d, i) fixed and (e, j) accelerating condition. S, session; T, trial. (f, k) Total beam breaks and (g, l) percent center beam breaks at open field tests. Animals were 10-11-week old unless specified otherwise. N = 16. (m-o) Nrros+/+ (WT) and Nrros-/- (KO) mice were sub lethally irradiated and reconstituted with donor marrow from either WT or KO mice (n = 15, 18, 14, 10). (m) Survival curve of WT and KO recipients grafted with indicated bone marrow stem cells. Quantification of percent engraftment in the blood (n) and brains (o) of these animals (n = 3). Error bar: ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (m) Log-rank test; all other analyses, unpaired Student’s t-test. ND, not determined.

Supplementary Figure 5 Loss of NOX2 fails to rescue neurological phenotype in Nrros−/− mice.

(a) mRNA expression of Cybb (NOX2) in Nrros+/+ and Nrros-/- brains from RNA-seq. (b - f) Performance of Nrros+/+, Nrros-/-, Cybb-/- and Nrros-/-Cybb-/- in rotarod test at (b) pretraining (9 weeks), (c) fixed and (d) accelerating conditions. S, session; T, trial. (e) Total beam breaks and (f) percent center beam breaks in open field test. Animals were 11-week old unless specified otherwise (n = 16). Error bar: ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA, (c, e, f); Unpaired Student’s t-test (a - c).

Supplementary Figure 6 Efficiency of Nrros deletion in Nrrosfl/flCx3cr1CreER mice.

Relative expression of Nrros in (a) E14.5 YFP+ FACS-sorted cells and (b) E21 CD11b+ cells from brains of E10.5 tamoxifen-treated animals. (c) Relative expression of Nrros in CD11b+ cells from the brain at week 9 after tamoxifen treatment at week 3. N = 3. Error bar: ± SEM. *P < 0.05. Unpaired Student’s t-test.

Supplementary Figure 7 Altered macrophage population in Nrros−/− brains.

(a) Gating strategy for FACS-sorting of microglia, PVM and PLCs. Expression level of (b) Nrros, (c - k) microglial, (l - o) PVM surface markers from RNA-seq comparing Nrros+/+ (WT) microglia, PVM and Nrros-/- PLCs (n = 3). Error bar: ± SEM. **P < 0.01; ***P < 0.001. Unpaired Student’s t-test. 12-week old animals were used.

Supplementary Figure 8 Nrros−/− PLCs do not resemble activated microglia.

(a - c) Gene expression of indicated cytokines from RNA-seq of Nrros+/+ (WT) microglia, PVM and Nrros-/- (KO) PLCs (n = 3). (d - f) Basal cytokine levels secreted by cultured myeloid cells isolated from WT and KO brains. (g) Heat map showing top 30 differentially expressed genes between WT PVM and KO PLCs. N = 3. Error bar: ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Unpaired Student’s t-test. 12-week old animals were used.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 2152 kb)

Supplementary Table 1

Differentially expressed genes in WT and Nrros−/− brains. (XLSX 8 kb)

Supplementary Table 2

Top 30 differentially expressed genes in WT PVMs and Nrros−/− PLCs. (XLSX 5 kb)

Supplementary Table 3

Differentially expressed gene in Nrros−/− PLCs and Sall1−/− microglia. (XLSX 11 kb)

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Wong, K., Noubade, R., Manzanillo, P. et al. Mice deficient in NRROS show abnormal microglial development and neurological disorders. Nat Immunol 18, 633–641 (2017). https://doi.org/10.1038/ni.3743

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