In vivo identification of astrocyte and neuron subproteomes by proximity-dependent biotinylation

Joselyn S. Soto, Yasaman Jami-Alahmadi, James A. Wohlschlegel, Baljit S. Khakh

Published: 2023-12-07 DOI: 10.1038/s41596-023-00923-7

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Extended Data Fig. 1 In vivo protein biotinylation in mouse.

a . Photo showing site of subcutaneous biotin injection in mouse for in vivo BioID2 protein biotinylation. b . A successful injection will produce a temporary pocket of biotin under the skin of the mouse (red arrows. The pocket should disappear within 10 minutes.

Extended Data Fig. 2 Microdissection setup and tissue homogenization.

a . Photos depicting the microdissection area for removal and isolation of the CNS region of interest. Because the sample must remain cold, the dissection is conducted with a pre-frozen TissueTek cold plate. Standard microdissection tools for striatum are shown. b . Photos of striata in a dounce homogenizer tube that was prefilled with 600 µL of lysis buffer 1 (Step 126) prior to homogenization with pestles. Photo on the right shows homogenate after homogenization with pestles (Step 127).

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References

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Allen, N. J. & Lyons, D. A. Glia as architects of central nervous system formation and function. Science 362, 181–185 (2018).
Khakh, B. S. & Sofroniew, M. V. Diversity of astrocyte functions and phenotypes in neural circuits. Nat. Neurosci. 18, 942–952 (2015).
Nagai, J. et al. Behaviorally consequential astrocytic regulation of neural circuits. Neuron 109, 576–596 (2021).
Khakh, B. S. & Deneen, B. The emerging nature of astrocyte diversity. Annu. Rev. Neurosci. 42, 187–207 (2019).
Ben Haim, L. & Rowitch, D. H. Functional diversity of astrocytes in neural circuit regulation. Nat. Rev. Neurosci. 18, 31–41 (2017).
Soto, J. S. et al. Astrocyte-neuron subproteomes and obsessive-compulsive disorder mechanisms. Nature 616, 764–773 (2023).
Uezu, A. et al. Identification of an elaborate complex mediating postsynaptic inhibition. Science 353, 1123–1129 (2016).
Heffernan, K. S., Rahman, K., Smith, Y. & Galvan, A. Characterization of the GfaABC1D promoter to selectively target astrocytes in the rhesus macaque brain. J. Neurosci. Methods 372, 109530 (2022).
Cahoy, J. D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).
Chai, H. et al. Neural circuit-specialized astrocytes: transcriptomic, proteomic, morphological, and functional evidence. Neuron 95, 531–549 (2017).
Burda, J. E. & Sofroniew, M. V. Reactive gliosis and the multicellular response to CNS damage and disease. Neuron 81, 229–248 (2014).
Linnerbauer, M., Wheeler, M. A. & Quintana, F. J. Astrocyte crosstalk in CNS inflammation. Neuron 108, 608–622 (2020).
Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).
Castle, M. J., Turunen, H. T., Vandenberghe, L. H. & Wolfe, J. H. Controlling AAV tropism in the nervous system with natural and engineered capsids. Methods Mol. Biol. 1382, 133–149 (2016).
Diaz-Castro, B., Gangwani, M. R., Yu, X., Coppola, G. & Khakh, B. S. Astrocyte molecular signatures in Huntington’s disease. Sci. Transl. Med. 11, eaaw8546 (2019).
Yu, X. et al. Context-specific striatal astrocyte molecular responses are phenotypically exploitable. Neuron 108, 1146–1162.e1110 (2020).
Yu, X. et al. Reducing astrocyte calcium signaling in vivo alters striatal microcircuits and causes repetitive behavior. Neuron 99, 1170–1187 e1179 (2018).
Cetin, A., Komai, S., Eliava, M., Seeburg, P. H. & Osten, P. Stereotaxic gene delivery in the rodent brain. Nat. Protoc. 1, 3166–3173 (2006).
Lee, Y., Messing, A., Su, M. & Brenner, M. GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia 56, 481–493 (2008).
Shigetomi, E. et al. Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses. J. Gen. Physiol. 141, 633–647 (2013).
Koerber, J. T. et al. Molecular evolution of adeno-associated virus for enhanced glial gene delivery. Mol. Ther. 17, 2088–2095 (2009).
Feist, P. & Hummon, A. B. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int. J. Mol. Sci. 16, 3537–3563 (2015).
Pajarillo, E., Rizor, A., Lee, J., Aschner, M. & Lee, E. The role of astrocytic glutamate transporters GLT-1 and GLAST in neurological disorders: potential targets for neurotherapeutics. Neuropharmacology 161, 107559 (2019).
Tong, X. et al. Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington’s disease model mice. Nat. Neurosci. 17, 694–703 (2014).
Zhang, X., Wan, J. Q. & Tong, X. P. Potassium channel dysfunction in neurons and astrocytes in Huntington’s disease. CNS Neurosci. Ther. 24, 311–318 (2018).
Cho, K. F. et al. Proximity labeling in mammalian cells with TurboID and split-TurboID. Nat. Protoc. 15, 3971–3999 (2020).
Rayaprolu, S. et al. Cell type-specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain. Nat. Commun. 13, 2927 (2022).
Lackner, D. H., Schmidt, M. W., Wu, S., Wolf, D. A. & Bähler, J. Regulation of transcriptome, translation, and proteome in response to environmental stress in fission yeast. Genome Biol. 13, R25 (2012).
Laurent, J. M. et al. Protein abundances are more conserved than mRNA abundances across diverse taxa. Proteomics 10, 4209–4212 (2010).
Liu, Y., Beyer, A. & Aebersold, R. On the dependency of cellular protein levels on mRNA abundance. Cell 165, 535–550 (2016).
Dowell, J. A., Johnson, J. A. & Li, L. Identification of astrocyte secreted proteins with a combination of shotgun proteomics and bioinformatics. J. Proteome Res. 8, 4135–4143 (2009).
Frese, C. K. et al. Quantitative map of proteome dynamics during neuronal differentiation. Cell Rep. 18, 1527–1542 (2017).
Basu, S., Campbell, H. M., Dittel, B. N. & Ray, A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J. Vis. Exp. http://doi.org/10.3791/1546 (2010).
Foo, L. C. Purification of astrocytes from transgenic rodents by fluorescence-activated cell sorting. Cold Spring Harb. Protoc. 2013, 551–560 (2013).
Pan, J. & Wan, J. Methodological comparison of FACS and MACS isolation of enriched microglia and astrocytes from mouse brain. J. Immunol. Methods 486, 112834 (2020).
Lange, S. C., Bak, L. K., Waagepetersen, H. S., Schousboe, A. & Norenberg, M. D. Primary cultures of astrocytes: their value in understanding astrocytes in health and disease. Neurochem. Res. 37, 2569–2588 (2012).
Foo, L. C. et al. Development of a method for the purification and culture of rodent astrocytes. Neuron 71, 799–811 (2011).
Sun, X. et al. Deep single-cell-type proteome profiling of mouse brain by nonsurgical AAV-mediated proximity labeling. Anal. Chem. 94, 5325–5334 (2022).
Shuster, S. A. et al. In situ cell-type-specific cell-surface proteomic profiling in mice. Neuron 110, 3882–3896.e3889 (2022).
Dumrongprechachan, V. et al. Cell-type and subcellular compartment-specific APEX2 proximity labeling reveals activity-dependent nuclear proteome dynamics in the striatum. Nat. Commun. 12, 4855 (2021).
Liu, G. et al. Mechanism of adrenergic Ca(V)1.2 stimulation revealed by proximity proteomics. Nature 577, 695–700 (2020).
Lobingier, B. T. et al. An approach to spatiotemporally resolve protein interaction networks in living cells. Cell 169, 350–360.e312 (2017).
Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).
May, D. G., Scott, K. L., Campos, A. R. & Roux, K. J. Comparative application of BioID and TurboID for protein-proximity biotinylation. Cells http://doi.org/10.3390/cells9051070 (2020).
Grant, M. K. O., Shapiro, S. L., Ashe, K. H., Liu, P. & Zahs, K. R. A cautionary tale: endogenous biotinylated proteins and exogenously-introduced protein a cause antibody-independent artefacts in western blot studies of brain-derived proteins. Biol. Proced. Online http://doi.org/10.1186/s12575-019-0095-z (2019).
Cho, K. F. et al. Split-TurboID enables contact-dependent proximity labeling in cells. Proc. Natl Acad. Sci. USA 117, 12143–12154 (2020).
Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).
Choi, H. et al. SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nat. Methods 8, 70–73 (2011).
Jami-Alahmadi, Y., Pandey, V., Mayank, A. K. & Wohlschlegel, J. A. A robust method for packing high resolution C18 RP-nano-HPLC columns. J. Vis. Exp. http://doi.org/10.3791/62380 (2021).
Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

In vivo identification of astrocyte and neuron subproteomes by proximity-dependent biotinylation

Extended

Extended Data Fig. 1 In vivo protein biotinylation in mouse.

Extended Data Fig. 2 Microdissection setup and tissue homogenization.

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