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. 2021 May 21;12:3027. doi: 10.1038/s41467-021-23188-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic showing specificity of antibodies used for western blot against protein domains of Fused in sarcoma (FUS). b Western blot of total FUS, full-length FUS and actin in synaptoneurosomes isolated from Fus^+/+ and Fus^ΔNLS/+ mice at 6 months of age. Source data are provided as a Source Data file. c Quantification of total FUS (p-value = 0.0091) and full-length FUS levels (p-value = 0.001) in synaptoneurosomes from Fus^+/+ and Fus^ΔNLS/+ at 6 months of age. Error bars represent mean ± SD. Each dot represents samples prepared from six different mice. n = 6, **p < 0.01, ***p < 0.001, two-tailed, unpaired t-test. d Confocal images of the hippocampal CA1 area from 6-month-old mice showing higher level of FUS in the dendritic tree and synaptic compartment in Fus^ΔNLS/+ mouse model. On the top, low magnification pictures show the dendritic area of pyramidal cells stained with FUS (green), microtubule-associated protein 2 (MAP2) (dendritic marker, magenta), Synapsin 1 (Syn, synaptic marker, Cyan), and DAPI (Blue). Red box indicates the area imaged in the high magnification images below. n = 5 independent Fus^+/+ and Fus^ΔNLS/+ mice. e Higher magnification equivalent to the area highlighted in red in d. f Representative images of staining using synaptic markers Synapsin 1, Vesicular GABA transporter (VGAT), postsynaptic GABAergic receptors containing α3 subunit (GABA_Aα3), and GluN1 (NMDAR submit) in Fus^+/+ and Fus^ΔNLS/+ at 1 and 6 months of age. Images were generated with Imaris and display volume view used for quantification with statistically coded surface area. Density and cluster area were analyzed. n = 6 independent Fus^+/+ and Fus^ΔNLS/+ mice at 1 month of age. At 6-month-old, n = 5 independent mice for Fus^+/+, n = 6 for Fus^ΔNLS/+. g Graph bar representation of the synaptic density of Synapsin 1, VGAT, GABA_Aα3, and GluN1 from Fus^+/+ and Fus^ΔNLS/+ at 1 and 6 months of age. Graph bar showing mean ± SD. *p < 0.05, two-tailed unpaired t-test. Graphs are extracted from the same analysis shown in Supplementary Fig. 3e, f. The statistical analysis can be found in Supplementary data 2. For 1-month-old group, n = 6 independent mice for Fus^+/+ and Fus^ΔNLS/+. For 6-month-old group, n = 5 independent mice for Fus^+/+, n = 6 for Fus^ΔNLS/+. For Synapsin 1 at 1-month-old, n = 14 fields of view for Fus^+/+ and 866013 synapses analyzed, 20 fields of view for Fus^ΔNLS/+ with 1,475,455 synapses analyzed. For Synapsin 1 at 6-month-old n = 19 fields of view for Fus^+/+ and 1590276 synapses analyzed, 24 fields of view for Fus^ΔNLS/+ with 2,138,361 synapses analyzed. For VGAT at 1-month-old, n = 19 fields of view for Fus^+/+ with 713,918 synapses analyzed, 23 fields of view for Fus^ΔNLS/+ with 782790 synapses analyzed. For VGAT at 6-month-old, n = 18 fields of view for Fus^+/+ with 553020 synapses analyzed, 24 fields of view for Fus^ΔNLS/+ with 1,273,325 synapses analyzed. For GABA_Aα3 at 1-month-old, n = 24 fields of view for Fus^+/+ with 1,062,095 synapses analyzed, 24 fields of view for Fus^ΔNLS/+ with 634,958 synapses analyzed. For GABA_Aα3 at 6-month-old, n = 20 fields of view for Fus^+/+ with 726,881 synapses analyzed, 22 fields of view for Fus^ΔNLS/+ with 804,732 synapses analyzed. For GluN1 at 1-month- old, n = 14 fields of view for Fus^+/+ with 704,322 synapses analyzed, 20 fields of view for Fus^ΔNLS/+ with 1,379,868 synapses analyzed. For GluN1 at 6-month-old, n = 19 fields of view for Fus^+/+ with 1,267,271 synapses analyzed, 24 fields of view for Fus^ΔNLS/+ with 1,866,397 synapses analyzed. h Colocalization analysis of GluN1 with Synapsin 1 to identify synaptic NMDAR (GluN1) and extrasynaptic NMDAR. Results were normalized by the control of each group. Graph bar showing mean ± SD. *p < 0.05., two-tailed unpaired t-test. ‘n’ values are as indicated in g. i Box and Whiskers representation (centered on the median with min to max value) of the average cluster area for each marker (Synapsin 1, VGAT, GABA_Aα3, and GluN1) from 1-month and 6-month-old Fus^+/+ and Fus^ΔNLS/+ mice. Box showing Min to Max, *p < 0.05 **p < 0.01, two-tailed, unpaired t-test. ‘n’ values are as indicated in g. Graphs are extracted from the same analysis shown in Supplementary Fig. 3f–i. The statistical analysis can be found in Supplementary data 3.