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. 2019 Dec 12;143(1):69–93. doi: 10.1093/brain/awz373

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© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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ZPR1 overexpression increases SMN levels in neuronal and non-neuronal tissues and leads to SMN-dependent amelioration of SMA phenotype. Proteins levels of SMN, ZPR1, Flag-ZPR1 and tubulin were examined by immunoblot analysis of mouse tissues. (A) spinal cord, (B) brain, (C) heart, (D) lung, (E) liver and (F) muscle from 7-day-old normal, SMA and Z-SMA mice using automated capillary Wes™ System (ProteinSimple) and quantification was performed using Compass software. Representative capillary-blot images of proteins are shown (full-length blots are provided in Supplementary Figs 2 and 3). Quantitative data are shown as a scatter plot with median, min and max; (median, min, max) range shows relative increase in SMN levels (%) in different tissues by ZPR1 overexpression in the: (G) spinal cord [SMA (25.96, 18.65, 26.98) and Z-SMA (77.32, 60.32, 85.63)], brain [SMA (19.96, 19.79, 21.24) and Z-SMA (57.93, 56.87, 73.67)], heart [SMA (15.76, 14.62, 22.36) and Z-SMA (49.61, 49.60, 60.32)], lung [SMA (15.61, 12.58, 26.19) and Z-SMA (55.69, 43.72, 65.36)], liver [SMA (14.82, 6.13, 30.98) and Z-SMA (20.20, 17.18, 28.65)] and muscle [SMA (14.78, 10.32, 20.36) and Z-SMA (20.36, 19.32, 25.63)] and statistical analysis (unpaired t-test) of protein levels (mean ± SEM, n = 3 mice/group) shows that ZPR1 overexpression resulted in statistically significant increase of SMN levels in the spinal cord of Z-SMA (74.42 ± 7.44, P = 0.0031) compared to SMA (23.86 ± 2.62), brain of Z-SMA (62.82 ± 5.43, P = 0.0015) compared to SMA (20.33 ± 0.45), heart of Z-SMA (53.18 ± 3.57, P = 0.0012) compared to SMA (17.58 ± 2.41), lung of Z-SMA (54.92 ± 6.25, P = 0.0080) compared to SMA (18.13 ± 4.12) but not in the liver of Z-SMA (22.01 ± 3.43, P = 0.5907) compared to SMA (17.31 ± 7.27) and muscle of Z-SMA (21.77 ± 1.95, P = 0.1317) compared to SMA (15.15 ± 2.90) relative to respective normal mice tissues protein levels (100%) normalized to tubulin. (H) Quantitative analysis of increase in relative levels (%) of total ZPR1 expression in the: spinal cord [SMA (50.36, 45.63, 66.36) and Z-SMA (153.47, 146.73, 187.32)], brain [SMA (59.34, 44.50, 70.12) and Z-SMA (171.80, 165.32, 178.36)], heart [SMA (61.36, 55.48, 62.36) and Z-SMA (163.06, 147.51, 164.91)], lung [SMA (61.89, 58.50, 63.35) and Z-SMA (170.76, 124.33, 175.32)], liver [SMA (59.99, 40.32, 69.97) and Z-SMA (80.36, 54.32, 90.32)] and muscle [SMA (50.32, 37.39, 65.95) and Z-SMA (75.66, 38.25, 92.04)]. Statistical analysis of increase in levels of total ZPR1 protein using antibody against ZPR1 shows increase in ZPR1 levels in the spinal cord of Z-SMA (162.50 ± 12.56, P = 0.0015) compared to SMA (54.12 ± 6.27), brain of Z-SMA (171.80 ± 3.76, P = 0.0002) compared to SMA (57.99 ± 7.42), heart of Z-SMA (158.50 ± 5.51, P = 0.0001) compared to SMA (59.73 ± 2.14), lung of Z-SMA (156.80 ± 16.29, P = 0.0043) compared to SMA (61.25 ± 1.43), liver of Z-SMA (75.00 ± 10.73, P = 0.2574) compared to SMA (56.76 ± 8.71) and muscle of Z-SMA (68.65 ± 15.92, P = 0.3861) compared to SMA (51.22 ± 8.25) relative to respective normal mouse tissue protein levels (100%) normalized to tubulin. (I) Quantitative data are presented as a scatter plot with median, min and max range shows relative increase in SMN levels in different tissues as fold change in Z-SMA mice compared to SMA mice; spinal cord (3.11 ± 0.31, P = 0.0031), brain (3.08 ± 0.26, P = 0.0015), heart (3.02 ± 0.20, P = 0.0012), lung (3.029 ± 0.34, P = 0.0080), liver (1.27 ± 0.19, P = 0.5911) and muscle (1.43 ± 0.13, P = 0.1333) relative to respective SMA mice tissues protein levels (100% or 1.0-fold) normalized to tubulin. (J) Quantitative data are shown as a scatter plot with median, min and max range shows relative expression (%) of recombinant Flag-ZPR1 in Z-SMA mice; spinal cord (100.027 ± 3.71), brain (84.36 ± 3.05), heart (64.86 ± 2.50), lung (61.17 ± 2.62), liver (24.98 ± 3.47) and muscle (20.78 ± 0.80) relative to Z-SMA spinal cord Flag-ZPR1 levels (100%) normalized to tubulin.