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Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes

Nature Protocols volume 17, pages 222–251 (2022)Cite this article

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Abstract

During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype– and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate that subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit ‘wiring’. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from the rodent central nervous system (CNS) in vivo. By using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is isolated by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting and established FACS of neurons and are suitable for downstream analyses of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes ~3 h, plus sorting time, and ~1–2 h for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, depending on the turnaround time of the core facility involved.

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Fig. 1: Paired isolation of subpopulation-specific GCs and somata enables mapping of subcellular proteomes and transcriptomes.
Fig. 2: A combination of fluorescent labeling of a neuronal subtype of interest; parallel preparation and isolation of labeled, axonal GCs and parent somata; and proteomic/transcriptomic analyses enables deep investigation of subtype-specific subcellular molecular machinery.
Fig. 3: In utero electroporation enables selective labeling of somata and GCs of projection neurons, in this case callosal projection neurons, in their endogenous anatomical context.
Fig. 4: Subcellular fractionation enables isolation of membrane-sealed GC particles, which protect their molecular cargo and pass stringent quality-control metrics.
Fig. 5: FSPS of GCF and FACS of dissociated somata enable isolation of subtype-specific GCs and parent somata.
Fig. 6: High-depth transcriptomic and proteomic analyses of paired GC and soma samples enables identification of compartment-specific transcriptomes/proteomes as well as subcellular localization analysis.
Fig. 7: Molecular candidates of interest identified in subcellular transcriptomic or proteomic analyses are validated and further investigated using in vitro and in vivo approaches.

Data availability

Most data included were first described in our earlier paper13, and the figure legends describe specifically where each piece of data included was published previously. Examples of datasets generated and analyzed by using the presented approaches are also available in the Harvard Dataverse repository (https://doi.org/10.7910/DVN/ISOEB6 (ref. 13).

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Acknowledgements

We dedicate this paper to the memory of John J. Hatch, who believed deeply in sharing resources, expertise, data and his excitement and exceptional abilities to enable others to deeply, rigorously and creatively investigate the subcellular biology of polarized cells, developing neurons and circuits in particular. We thank our laboratory colleague Dustin Tillman for a representative image of the hydrolysis protection assay. We thank Joyce LaVecchio and Nema Kheradmand of the HSCRB-HSCI Flow Cytometry Core; Emma White, Christian Daly and Claire Hartmann of the Harvard Bauer Sequencing Core; William Lane, John Neveu and Bogdan Budnik of the Harvard FAS CSB Mass Spectrometry and Proteomics Resource Lab; Anna-Katerina Hadjantonakis (Memorial Sloan Kettering) and Shankar Srinivas (Oxford) for reagents. Analytic tools and infrastructure in the Harvard Chan Bioinformatics Core were partially supported by funds from the Harvard NeuroDiscovery Center and the Harvard Stem Cell Institute. This work was supported by the following grants to J.D.M.: NIH Pioneer Award DP1NS106665; Paul G. Allen Frontiers Group—Allen Distinguished Investigator award and Brain Research Foundation Scientific Innovations Award; Max and Anne Wien Professor of Life Sciences fund; Emily and Robert Pearlstein Fund; and additional infrastructure support from NIH grants NS045523, NS104055, NS075672 and NS049553. J.J.H. was partially supported by NIH individual Training Grant F31 NS103262. A.K.E. was partially supported by the Jean-Jacques & Felicia Lopez-Loreta Foundation, the Travis Roy Foundation (to J.D.M.) and a Swiss National Science Foundation Fellowship. A.P. was partially supported by a European Molecular Biology Long-Term Fellowship and a Human Frontiers Science Program Long-Term Fellowship. A.J.M. was partially supported by NIH Training Grant T32 AG000222.

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Author notes

  1. Alexandros Poulopoulos

    Present address: Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA

  2. Alexander J. Murphy

    Present address: Takeda Pharmaceutical Company Limited, Cambridge, MA, USA

  3. These authors contributed equally: Anne K. Engmann, John J. Hatch.

  4. Deceased: John J. Hatch.

Authors and Affiliations

  1. Department of Stem Cell and Regenerative Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA

    Anne K. Engmann, John J. Hatch, Prakruti Nanda, Priya Veeraraghavan, Abdulkadir Ozkan, Alexandros Poulopoulos, Alexander J. Murphy & Jeffrey D. Macklis

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Contributions

A.K.E., J.J.H., A.P., P.V. and J.D.M. wrote the manuscript. A.P., A.J.M., J.J.H., A.O. and A.K.E. performed experiments and analyzed data. A.P., J.J.H., P.N. and A.K.E. designed figures.

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Correspondence to Jeffrey D. Macklis.

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Poulopoulos, A. et al. Nature 565, 356–360 (2019) https://doi.org/10.1038/s41586-018-0847-y

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Engmann, A.K., Hatch, J.J., Nanda, P. et al. Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes. Nat Protoc 17, 222–251 (2022). https://doi.org/10.1038/s41596-021-00638-7

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