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

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.

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).

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’).

Present address: Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany

Present address: Berlin Institute of Health, Berlin, Germany

These authors contributed equally: Caroline Fecher, Laura Trovò.

Institute of Neuronal Cell Biology, Technical University of Munich, Munich, Germany

Caroline Fecher, Laura Trovò, Nicolas Snaidero & Thomas Misgeld

German Center for Neurodegenerative Diseases, Munich, Germany

Caroline Fecher, Laura Trovò, Stephan A. Müller, Nicolas Snaidero, Wolfgang Wurst, Stefan F. Lichtenthaler & Thomas Misgeld

Graduate School of Systemic Neurosciences, Ludwig-Maximilians University, Munich, Germany

Neuroproteomics, School of Medicine, Klinikum rechts der Isar and Institute for Advanced Study, Technical University of Munich, Munich, Germany

Gene Center Munich, Ludwig-Maximilians University, Munich, Germany

Institute for Diabetes and Obesity, Helmholtz Zentrum München, Munich, Germany

Experimental Neuroimmunology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

Institute of Developmental Genetics, Helmholtz Zentrum München, Munich, Germany

Institute of Developmental Genetics, Technical University of Munich, Munich, Germany

Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland

Institute of Neuroscience, Technical University of Munich, Munich, Germany

Arthur Konnerth, Thomas Korn, Wolfgang Wurst, Stefan F. Lichtenthaler, Fabiana Perocchi & Thomas Misgeld

Division of Clinical Pathology, Geneva University Hospital, Geneva, Switzerland

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.

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.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

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