Generation of liver specific SMN depleted mice.

A. Schematic representation of the breeding scheme to generate liver specific SMN depletion. In these mice, the Smn gene carries one Smn2B allele that harbors a three-nucleotide switch in exon 7, and one Smn allele whose exon 7 is floxed by loxP sites (SmnF7). The Alb-Cre transgene has also been crossed into this background to provide liver-specific expression of the Cre recombinase. B. Littermate controls do not harbor Cre recombinase but do carry one Smn2B allele and are thus used as heterozygous controls. C. Upon crossing AlbCre/+;SmnF7/+with Smn2B/F7 animals, Cre recombination will induce specific excision of exon 7 in the liver, leading to reduced SMN protein production only in the liver.

Liver-specific SMN depletion in AlbCre/+;Smn2B/F7 mice.

A–E. Immunoblots were performed to assess SMN protein levels in various tissues from Smn2B/+, Smn2B/-, +/+;Smn2B/F7, and AlbCre/+;Smn2B/F7 mice at P19. Membranes were probed for SMN and then reprobed for α-tubulin (loading control). A’-E’. Bar graphs show quantification of SMN protein levels in the liver (A’), brain (B’), muscle (C’), spinal cord and pancreas (E’), normalized to α-tubulin. n ≥ 3, mean ± SEM. Statistical significance indicated by *p < 0.05, **p < 0.01 or p values, following Brown-Forsythe and Welch ANOVA.

Impact of liver specific SMN depletion in hepatocyte morphology and lipid accumulation. A. Representative images of H&E (top row, scale bar 100 μm) and Oil Red-O (bottom row, scale bar 50 μm) stained liver sections from Smn2B/+, Smn2B/-, +/+;Smn2B/F7, and AlbCre/+;Smn2B/F7 mice at P19. B. Bar graph shows quantification of liver triglycerides. n ≥ 3, mean ± SEM. Statistical significance indicated by *p < 0.05, **p < 0.01, following Brown-Forsythe and Welch ANOVA.

Levels of key proteins involved in liver homeostatic function are unchanged in liver-specific SMN-depleted mice.

A-C. Immunoblots were performed to assess heme oxygenase (HO, A-A’), transferrin (B-B’) and P62 (C-C’) protein levels in the liver. α-tubulin was used as a loading control. A’-C’ Bar graphs show quantification of HO (A’), transferrin (B’), and P62 (C’) levels. D. Bar graph depicts quantification of liver IGF-1. n ≥ 3, mean ± SEM. Statistical significance indicated by *p < 0.05, ***p < 0.001, following Brown-Forsythe and Welch ANOVA.

Contribution of liver specific SMN depletion to pancreatic pathology.

A. Representative immunofluorescent images of pancreatic islets stained for glucagon (red) and insulin (green) from Smn2B/+, Smn2B/-, +/+;Smn2B/F7, and AlbCre/+;Smn2B/F7 mice at P19. Scale bar 50 μm. B-C. Bar graphs show quantification of insulin (B) and glucagon (C) positive cells relative to the total number of DAPI positive cells within the pancreatic islet. D. Bar graph depicts non-fasting blood glucose levels from P19 mice across different genotypes. n ≥ 3, mean ± SEM. Statistical significance indicated by *p < 0.05, **p < 0.01, ***p < 0.001, following Brown-Forsythe and Welch ANOVA.

Impact of liver specific SMN depletion on motor neuron cell body numbers.

A. Representative images of lumbar spinal cord anterior horns stained for ChAT (red) and DAPI (blue) from Smn2B/+, Smn2B/-, +/+;Smn2B/F7, and AlbCre/+;Smn2B/F7 mice at P19. B. Bar graph shows quantification of motor neuron cell body numbers. n ≥ 3, mean ± SEM. Statistical significance indicated by **p < 0.01, ***p < 0.001, following Brown-Forsythe and Welch ANOVA. Scale bar = 50 μm.

Impact of liver specific SMN depletion on neuromuscular junction pathology.

A. Representative images of transversus abdominis (TVA) muscle stained with bungarotoxin (red), neurofilament (NF) (green) and synaptic vesicle protein 2 (green) from Smn2B/+, Smn2B/-, +/+;Smn2B/F7, and AlbCre/+;Smn2B/F7 mice at P19. *Depicts occupied endplate, whereas **depicts normal neurofilament distribution; arrowheads show NF accumulation and arrows show unoccupied endplate/denervation. B-C. Bar graphs show quantification of neurofilament accumulation (B) and endplate occupancy (C). n = 3, mean ± SEM. Statistical significance indicated by **p < 0.01, ***p < 0.001, following Brown-Forsythe and Welch ANOVA. Scale bar = 50 μm.

Impact of liver specific SMN depletion on muscle fiber size and distribution.

A. Representative images of H&E-stained tibialis anterior muscle sections from Smn2B/+, Smn2B/-, +/+;Smn2B/F7, and AlbCre/+;Smn2B/F7 mice at P19. B. Bar graph shows quantification of muscle fiber cross-sectional area. C. Graph demonstrating frequency of muscle fiber size across different genotypes. n ≥ 3, mean ± SEM. Statistical significance indicated by **p < 0.01, following Brown-Forsythe and Welch ANOVA. Scale bar = 20 μm.

Impact of liver specific SMN depletion on motor function.

A. Schematic representation of experimental design. The righting reflex test was conducted from P7 to P13, the inverted mesh grip test from P13 to P25, and the pen test from P19 to P25. Assessments were performed every two days. Weight was measured every two days until day 30, and then weekly until day 60. Animal welfare was monitored throughout the 60-day period during weight measurements. B. Kaplan–Meier survival curve comparing AlbCre/+;Smn2B/F7 and +/+;Smn2B/F7 mice up to 60 days. C-F. Graphs show weight (C), righting reflex (D), inverted mesh grip (E) and pen test (F). n = 5 per genotype, mean ± SEM. B: Kaplan–Meier survival analysis; C-F: two-way ANOVA, followed by Šídák’s method.

List of primers used for genotyping

List of antibodies used.