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Photoproximity labeling of endogenous receptors in the live mouse brain in minutes

Nature Chemical Biology (2024)Cite this article

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Abstract

Understanding how protein–protein interaction networks in the brain give rise to cognitive functions necessitates their characterization in live animals. However, tools available for this purpose require potentially disruptive genetic modifications and lack the temporal resolution necessary to track rapid changes in vivo. Here we leverage affinity-based targeting and photocatalyzed singlet oxygen generation to identify neurotransmitter receptor-proximal proteins in the live mouse brain using only small-molecule reagents and minutes of photoirradiation. Our photooxidation-driven proximity labeling for proteome identification (named PhoxID) method not only recapitulated the known interactomes of three endogenous neurotransmitter receptors (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), inhibitory γ-aminobutyric acid type A receptor and ionotropic glutamate receptor delta-2) but also uncovered age-dependent shifts, identifying NECTIN3 and IGSF3 as developmentally regulated AMPAR-proximal proteins in the cerebellum. Overall, this work establishes a flexible and generalizable platform to study receptor microenvironments in genetically intact specimens with an unprecedented temporal resolution.

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Fig. 1: PhoxID enables light-triggered PL in the live mouse brain.
Fig. 2: PhoxID can profile the AMPAR-proximal proteome in the live mouse brain in minutes.
Fig. 3: Extension of PhoxID to the GABAAR-proximal proteome.
Fig. 4: PhoxID can distinguish different neurotransmitter receptor neighborhoods.
Fig. 5: PhoxID can reveal shifts in the AMPAR-proximal proteome in postnatal development.
Fig. 6: NECTIN3 and IGSF3 are developmentally regulated AMPAR-proximal proteins.

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Data availability

The MS raw data and analysis files have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the jPOST partner repository (http://jpostdb.org) with data set identifier JPST002162 (ref. 65). For the search of ligands targeting neurotransmitter receptors, the IUHPAR/BPS Guide to PHARMACOLOGY (https://www.guidetopharmacology.org/) and Uniprot (https://www.uniprot.org/) were used. All data supporting the findings of this study are available within the paper and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

The authors thank K. Uchida, M. Ishikawa, Y. Yabuki and E. Kusaka for their support in chemical synthesis and compound characterization; K. Nishimura and K. Matsuba for mass analysis; K. Nishizawa and T. Gonda for mouse experiments; H. Utsunomiya for early-stage evaluation of nanobody-photosensitizer conjugates. We also thank Radu Aricescu and Michisuke Yuzaki for their contributions to the preparation of nanobodies. Super-resolution imaging was performed at the Analysis Center, Institute for Integrated Cell-Material Sciences, Kyoto University Institute for Advanced Study. This work was supported by Japan Science and Technology Agency ERATO grant JPMJER1802, the Japan Society for the Promotion of Science Grant-in-Aid for Specially Promoted Research 23H05405 and the Japan Science and Technology Agency CREST grant JPMJCR1854 to I.H.; Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research on Innovative Areas ‘Integrated Bio-metal Science’ 19H05764 and the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) 21H02058 to T.T.; the Japan Society for the Promotion of Science Grant-in-Aid for JSPS Fellows 21J23228 to M.T.; and the AMED/MEXT Science and Technology Platform Program for Advanced Biological Medicine grant JP22am0401006 to H.N.

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Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan

    Mikiko Takato, Seiji Sakamoto, Hiroshi Nonaka, Fátima Yuri Tanimura Valor, Tomonori Tamura & Itaru Hamachi

  2. JST-ERATO, Hamachi Innovative Molecular Technology for Neuroscience, Kyoto, Japan

    Seiji Sakamoto, Hiroshi Nonaka, Tomonori Tamura & Itaru Hamachi

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  1. Mikiko Takato
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Contributions

M.T., T.T. and I.H. conceived the project and designed the experiments. M.T., T.T., S.S., H.N. and I.H. designed the compounds. T.T. synthesized the compounds. H.N. advised the injection of reagents into the mouse brain. M.T., S.S. and F.Y.T.V. performed the anchoring of the photosensitizers in mice and evaluated the reaction. M.T. and T.T. designed the in vivo PhoxID workflows with input from S.S., H.N. and I.H. M.T. performed the PhoxID experiments and the LC–MS/MS data acquisition and analyses. M.T. and H.N. performed the validation experiments for the developmental proteins. M.T., T.T. and I.H. supervised the work and wrote the manuscript with input from all authors.

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Correspondence to Tomonori Tamura or Itaru Hamachi.

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Extended data

Extended Data Fig. 1 Estimation of the 1O2 quantum yield (ΦΔ) of MBF.

ΦΔ values for MBF were estimated by a relative spectrophotometric method using chemical traps for 1O2 and DBF as the reference photosensitizer. (a, c) Absorption spectra showing the photoirradiation time-dependent decrease in absorbance of (a) 1,3-diphenylisobenzofuran (DPBF) in 95% ethanol/0.01 M KOH and (c) 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) in phosphate buffer (pH 7) in the presence of DBF or MBF. Insets in (c) show magnified views of the ABDA peaks. (b, d) MBF- and DBF-dependent decrease in (b) DPBF and (d) ABDA absorbances plotted against irradiation time. Data are shown from three independent replicates and the linear line of best fit is shown for mean values at each time point. Error bars represent ± s.d. (e) ΦΔ values for MBF were calculated from the slopes of the plots in (b) and (d) and photosensitizer absorbances as described in the Methods. †ΦΔ values for DBF are given in the literature32.

Extended Data Fig. 2 Covalent modification of AMPAR with MBF and subsequent light-triggered proximity labeling in the live mouse brain.

(a) Western blot of brain tissue isolated from mice injected to the lateral ventricles with 1 and probed with anti-fluorescein, which recognizes MBF. Red triangle indicates the band corresponding to AMPAR subunits covalently modified with MBF. (b) Confocal laser scanning microscopy images of brain slices prepared from mice injected to the lateral ventricles with 1 and stained with anti-fluorescein or anti-GRIA2. Scale bars, 2 mm. (c) Streptavidin blot of cerebellum proteins isolated and enriched from mice subjected to PhoxID. Asterisks indicate endogenously biotinylated proteins. Triangle indicates the expected molecular weight of AMPAR subunits. Anti-fluorescein blot shows anchoring of MBF to AMPARs in samples prior to enrichment. All experiments shown are representative of at least two independent measurements.

Source data

Extended Data Fig. 3 PhoxID is not biased towards highly abundant proteins.

The PhoxID fold change of the hippocampal AMPAR-proximal hits is plotted against their abundance in the brain as reported in PAXdb 4.2 to show that PhoxID fold changes do not correlate with protein expression levels.

Extended Data Fig. 4 Light intensity and irradiation time dependence of PhoxID results.

Scatterplots showing the degree of AMPAR-PhoxID labeling in the hippocampus under different irradiation conditions.

Extended Data Fig. 5 Data in support of Extended Data Fig. 4.

(a) Fraction of hit proteins detected in each photoirradiation condition that are annotated to synaptic and cell-peripheral GOCC terms. Proteins that were annotated to both synaptic terms and the cell periphery were classified here as synaptic proteins. (b) Heatmap showing the fold change of proteins under various photoirradiation conditions. Data only shown for proteins that met the hit protein criteria in at least one of the conditions. The hierarchical clustering was performed by Ward’s method. Mitochondrial/nuclear contaminant proteins were omitted.

Extended Data Fig. 6 PhoxID can be performed in acute brain slices.

(a) Chemical structure of Hyd-PEG4-Bt bearing biotin as the enrichment handle. (b) Experimental workflow for PhoxID in acute brain slices. (c) Western blot analysis of MBF anchoring onto AMPARs in the cerebral cortex. The data is representative of at least two independent measurements. (d) Streptavidin blot of the proteins labeled by PhoxID in acute cortical and hippocampal slices. Western blotting was performed after NeutrAvidin enrichment. The data is representative of at least two independent measurements. (e) Fold change plot of the proteins identified by LC-MS/MS in a single biological replicate.

Source data

Extended Data Fig. 7 PhoxID with a photosensitizer-nanobody conjugate.

(a) Schematic of the conjugation reaction between DBF and a nanobody for ionotropic glutamate receptor delta-2 (GRID2) modified with a tandem GS linker and Cys residue at the C-terminus. The target specificity of GRID2 Nb-DBF has been confirmed in our previous work30. (b) Fold change plot of the proteins identified by PhoxID in the cerebellum with GRID2 Nb-DBF and Hyd-PEG4-dBt. Upper-right panel, schematic diagram of the brain injection and photoirradiation sites. Black dots represent the injection sites of GRID2 Nb-DBF and the green dot represents the site of Hyd-PEG4-dBt injection and photoirradiation. (c) Cartoon depiction of the localization and topology of representative enriched proteins. All components of the GRID2-CBLN1-NRXN1 transsynaptic complex46 were successfully labeled.

Extended Data Fig. 8 Western blot analysis of AMPAR-proximal PhoxID in the postnatal cerebellum.

(a) Streptavidin blots of proteins labeled by PhoxID in the P8, P13, and 5-week-old cerebellum. Western blotting was performed after NeutrAvidin enrichment. The data is representative of at least two independent measurements. (b) Western blot analysis of MBF anchoring in the mouse cerebellum isolated from P8, P13, and 5-week-old mice injected with 1. n1, n2, and n3 denote biological replicates. (c) Mean intensity of the bands in (b). Data are normalized to the mean band intensity in 5-week-old mice. Error bars represent ± s.d. (n = 3 biological replicates).

Source data

Extended Data Fig. 9 Network diagrams of known and predicted interactions between the AMPAR-proximal PhoxID hit proteins at each postnatal age studied.

Interaction information was obtained from STRING 11.5 (text mining and co-expression excluded) and several manual annotations.

Extended Data Fig. 10 Cartoon depictions of AMPARs and NECTIN3 in the developing cerebellum.

(a) Changes in the regional distribution of AMPARs and NECTIN3 in the cerebellum as revealed by immunostaining. PC, Purkinje cell. PF, parallel fiber. CF, climbing fiber. EGL, external granular layer. ML, molecular layer. PCL, Purkinje cell layer. IGL, internal granular layer. GL, granular layer. (b) Hypothesis regarding the spatial dynamics of AMPARs and NECTIN3 in the cerebellum. We postulate that NECTIN3 populates both synaptic junctions and puncta adherens in the neonatal period but are depleted and/or sequestered to puncta adherens by adulthood.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10, source data and note.

Reporting Summary

Supplementary Data 1

Supplementary Data 1.

Supplementary Data 2

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Supplementary Data 3

Supplementary Data 3.

Supplementary Data 4

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Supplementary Data 6.

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Supplementary Data 8.

Source data

Source Data Fig. 3

Unprocessed western blots of Fig. 3b.

Source Data Fig. 5

Unprocessed western blots of Fig. 5e.

Source Data Extended Data Fig. 2

Unprocessed western blots of Extended Data Fig. 2a,c.

Source Data Extended Data Fig. 6

Unprocessed western blots of Extended Data Fig. 6c.

Source Data Extended Data Fig. 8

Unprocessed western blots of Extended Data Fig. 8b.

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Takato, M., Sakamoto, S., Nonaka, H. et al. Photoproximity labeling of endogenous receptors in the live mouse brain in minutes. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01692-4

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