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Cell-type-specific profiling of brain mitochondria reveals functional and molecular diversity

Abstract

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer’s disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum–mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.

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Fig. 1: MitoTag mice allow isolation of intact and functional mitochondria from cells in situ.
Fig. 2: Immunocapture from MitoTag mice enriches cell-type-specific mitochondria without mitochondrial cross-contamination from other cells.
Fig. 3: Proteomic profiling of cell-type-specific mitochondria in the adult mouse cerebellum.
Fig. 4: Cell-type-specific mitochondrial markers are conserved within the CNS and across species, and they enable mitochondrial studies under pathological conditions.
Fig. 5: Astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons.
Fig. 6: Granule cell mitochondria robustly buffer calcium via Mcu in contrast with Purkinje cell mitochondria.
Fig. 7: Rmdn3 mediates close ER–mitochondria juxtapositions in Purkinje cells.

Data availability

All proteomics data generated within this study are deposited to the ProteomeXchange Consortium via the PRIDE44 partner repository with the dataset identifiers PXD010772, PXD010774, PXD010781 and PXD013380. In addition, Supplementary Data provide information on the proteomics data analysis used for the conclusions in the main and supplementary figures. All additional data that support the findings of this study are available from the corresponding author upon reasonable request. The MitoTag mouse line is available from The Jackson Laboratory as JAX#032675 (Rosa26–CAG–LSL–GFP–OMM). The McuFL/FL and the Rbp4:Cre mouse strains are protected under a material transfer agreement with the MMRRC (C57BL/6N-Mcutm1a(EUCOMM)Hmgu/H; Tg(Rbp4-cre)KL100Gsat/Mmucd), and the Rmdn3−/− mouse strain is protected under a material transfer agreement with the KOMP Repository (KO mouse project 3U01HG004080).

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Acknowledgements

We thank M. Budak, N. Budak and S. Taskin for animal husbandry and A. Berghofer, A. Graupner, Y. Hufnagel, K. Wullimann and M. Schetterer for technical and administrative support. We thank U. Fünfschilling (Max Planck Institute Göttingen) for providing Gabra6:Cre mice, D. Crane (Griffith University) for the Pex14 antibody and N. Mizushima (Addgene plasmid #38249) for the pMXs-IP GFP–Omp25 plasmid. We are grateful to L. Jiang for help with bioinformatics analyses, Y.-H. Tai for neonatal virus injection and B. Zott for APPPS1 breeding. We thank L. Godinho, M. Schuldiner and O. Schuldiner for reading an earlier version of this article. This work was supported by the Deutsche Forschungsgemeinschaft through the Munich Center for Systems Neurology (SyNergy; grant no. EXC 1010 to A.K., T.K., S.F.L., T.M., F.P. and W.W.); the Center for Integrated Protein Science Munich (grant no. EXC 114 to A.K. and T.M.); the Collaborative Research Centers (CRC870 to T.M., CRC1054 to T.K. and CRC-TR128 to T.K.); research unit grant no. FOR2290 to S.F.L. and research grant no. Mi694/7-1/8-1 to T.M. F.P. and J.W. were supported by the Emmy-Noether program of the Deutsche Forschungsgemeinschaft (grant no. Pe2053/1-1 to F.P.). Further support came from the European Research Council under the European Union’s Seventh Framework Program (grant no. FP/2007-2013; European Research Council Grant Agreement nos. CoG 616791 to T.M. and CoG 647215 to T.K.) and the German Center for Neurodegenerative Diseases (DZNE Munich to S.F.L., T.M. and W.W.). L.T. was supported by an EMBO Long-Term Fellowship (no. EMBO ALTF 108-2013). Further support came from the Centers of Excellence in Neurodegeneration and the Helmholtz-Israel Program to S.F.L., the Swiss National Science Formation and HHS Foundation to D.M., the German Federal Ministry of Education and Research (BMBF) through ‘T-B interaction in NMO’ to T.K. and ‘Mitochondrial endophenotypes of Morbus Parkinson’ (grant no. 031A430E) to W.W. A.K. is supported by a Senior Hertie Professorship of Neuroscience. T.M. is supported by the Institute for Advanced Study, Technical University of Munich (Focus Group ‘Subcellular Dynamics in Neurons’).

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C.F., L.T. and T.M. devised the study. L.T., T.M., O.O., R.K. and W.W. designed and generated the MitoTag mice, which C.F. and L.T. characterized. C.F. designed the immunocapture protocol (with support from J.W. and F.P.) and performed most of the isolations. S.A.M. and S.F.L. performed sample preparation, mass spectrometry and primary data analysis. C.F. further analyzed the proteomics datasets. L.T. performed bioenergetics measurements and corresponding isolations (with support from J.W. and F.P.). C.F. performed calcium uptake assays (with support from F.P.). C.F., S.H. and T.K. performed and analyzed flow cytometry. C.F. performed western blot analysis and immunofluorescence stainings of candidates. N.S. and C.F. obtained and analyzed electron microscopy data. C.F. characterized the Rmdn3−/− and GC-specific McuFL/FL mouse models. J.H., R.M.K. and A.K. provided pilot experiments for single-cell characterization in the cerebellum. A.K. provided APPPS1 mice. I.W. and D.M. performed immunofluorescence staining on human tissue. C.F., L.T. and T.M. wrote the paper with input from all authors.

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Correspondence to Thomas Misgeld.

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Peer review information: Nature Neuroscience thanks Robert Friedlander, Daniel McClatchy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Figs. 1–18 and Supplementary Tables 1–4.

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Supplementary Data

Proteomics data from cell-type-specific mitochondria: Purkinje cells, granule cells and astrocytes.

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Fecher, C., Trovò, L., Müller, S.A. et al. Cell-type-specific profiling of brain mitochondria reveals functional and molecular diversity. Nat Neurosci 22, 1731–1742 (2019). https://doi.org/10.1038/s41593-019-0479-z

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