Abstract
Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder stemming from deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene, leading to decreased levels of SMN protein, and subsequent motor neuron death and muscle atrophy. While traditionally viewed as a disorder predominantly affecting motor neurons, recent research suggests the involvement of various peripheral organs in SMA pathology. Notably, the liver has emerged as a significant focus due to the observed fatty liver phenotype and dysfunction in both SMA mouse models and SMA patients. Despite these findings, it remains unclear whether intrinsic depletion of SMN protein in the liver contributes to pathology in the peripheral or central nervous systems. To address this knowledge gap, we developed a mouse model with a liver-specific depletion of SMN by utilizing an Alb-Cre transgene together with one Smn2B allele and one Smn exon 7 allele flanked by loxP sites. We evaluated phenotypic changes in these mice at postnatal day 19 (P19), a time when the severe model of SMA, the Smn2B/- mice, typically exhibit many symptoms of the disease. Our findings indicate that liver-specific SMN depletion does not induce motor neuron death, neuromuscular pathology or muscle atrophy, characteristics typically observed in the Smn2B/- mouse at P19. However, mild liver steatosis was observed at this time point, although no changes in liver function were detected. Notably, pancreatic alterations resembled that of Smn2B/-mice, with a decrease in insulin producing alpha-cells and an increase in glucagon producing beta-cells, accompanied with a reduction in blood glucose levels. While the mosaic pattern of the Cre-mediated excision precludes definitive conclusions regarding the contribution of liver-specific SMN depletion to overall tissue pathology, our findings highlight an intricate connection between liver function and pancreatic abnormalities in SMA, adding a nuanced layer to our understanding of the disease’s complexities.
eLife assessment
This work presents a valuable mouse model for a liver-specific depletion of the Survival Motor Neuron (SMN) protein, where the liver retains 30% of functional full-length SMN protein. The authors provide a profile of phenotypic changes in liver-specific SMN depleted mice: while evidence supporting their claims are generally solid, the phenotype is mild and mechanistic understanding remains to be determined.
Significance of findings
valuable: Findings that have theoretical or practical implications for a subfield
- landmark
- fundamental
- important
- valuable
- useful
Strength of evidence
solid: Methods, data and analyses broadly support the claims with only minor weaknesses
- exceptional
- compelling
- convincing
- solid
- incomplete
- inadequate
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1. Introduction
Insufficient levels of Survival Motor Neuron (SMN) protein, primarily arising from mutations or deletions in the SMN1 gene, are the root cause of spinal muscular atrophy (SMA), a hereditary neuromuscular disorder widely acknowledged as one of the leading genetic causes of infant mortality1–3. Characterized by the loss of motor neurons, SMA presents as a progressive weakening of muscle strength, primarily impacting proximal muscles. If left untreated, this deterioration culminates in respiratory failure and premature mortality4.
The diverse phenotypes observed in SMA patients can be attributed to the genetic variations within the SMN gene1. Humans harbor two copies of the SMN gene, SMN1 and SMN2, which share a nearly identical sequence except for a critical C to T substitution at position 6 of exon 7 in SMN25. This single nucleotide change disrupts splicing of the gene, resulting in the predominant production of an unstable protein lacking exon 7, known as SMNΔ75. Despite this, approximately 10% of functional full-length (FL) SMN protein is still produced by the SMN2 gene2. Consequently, the SMN2 gene serves as a modifier of disease phenotype, with a higher copy number correlating with milder disease severity6. In contrast, mice possess a single copy of the Smn gene2. Notably, while homozygous deletion of both SMN genes has not been observed in humans, the homozygous deletion of the Smn gene in mice induces morphological alterations and degenerative changes in embryos post-morula stage, ultimately resulting in death at preimplantation stage7. This underscores the pivotal role of the SMN gene in early embryonic development8.
Recent research has unveiled the extensive impact of SMN depletion beyond motor neurons, implicating additional tissues such as the liver and pancreas in both SMA patients and mouse models of SMA9–12. Despite major therapeutic advancements focusing on restoring SMN levels, which have notably enhanced both lifespan and quality of life in SMA patients, complete phenotypic rescue remains elusive4,13,14. Additionally, existing therapies primarily target postnatal treatment, potentially overlooking crucial developmental changes stemming from SMN depletion. This underscores the likelihood of a broader role for the SMN gene in multiple non-neuronal cell types, necessitating further exploration.
Attention has been increasingly drawn to the liver as a critical area of investigation in SMA research. Both clinical studies and preclinical mouse models have unveiled disruptions in fatty acid metabolism, leading to an increased susceptibility among SMA patients to dyslipidemia and liver steatosis12,15–18. Given its multifaceted role in regulating numerous biological functions, including the storage and regulation of lipids, carbohydrates, amino acids, and iron, as well as the synthesis of essential growth factors and the clearance of toxic metabolites, any disruptions in liver function may have deleterious effects on other tissues19.
To explore the role of SMN in the liver, prior studies have utilized a transgenic mouse model featuring the Cre recombinase transgene driven by the α-fetoprotein promoter (Alfp-Cre). This approach led to near-total depletion of SMN protein specifically in the liver, resulting in severe impairment of liver development and late embryonic lethality8. In our study, to better represent the level of SMN depletion in the liver observed in SMA and elucidate the intricate interplay between SMN deficiency in the liver and its systemic impact in SMA-like pathology, we utilized a mouse model employing Alb-Cre, where the albumin promoter drives expression of Cre recombinase in a liver-specific manner. This model also harbors one Smn2B allele and one Smn exon 7 allele flanked by loxP sites. The Smn2B allele contains a 3-base pair substitution in the mouse Smn gene, resulting in aberrant splicing of exon 720,21. Consequently, this genetic approach results in the production of approximately 30% full-length functional SMN protein production in the liver.
Our findings reveal that our novel liver specific SMN depleted (herein referred to as AlbCre/+;Smn2B/F7) mouse model induces mild liver steatosis and disrupts pancreatic function, characterized by decreased insulin-positive and increased glucagon-positive cells, alongside reduced blood glucose levels. Remarkably, this mouse model does not exhibit an overt SMA phenotype, demonstrating normal lifespan and motor function. Nevertheless, our data suggests an intricate relationship between hepatic function and pancreatic abnormalities in SMA, thereby enhancing our comprehension of the disease’s pathophysiology.
2. Results
2.1 Generation of liver-specific SMN-depleted mice
To elucidate the impact of liver specific SMN depletion on both CNS and peripheral tissue pathology, we utilized mice expressing Cre recombinase under the control of the mouse albumin promoter, referred to as AlbCre. This strain is commonly employed for liver-specific gene knockout using the Cre/loxP system22. We generated the Smn2B/F7 genotype by crossing SmnF7/F7 with Smn2B/2B mice (Fig. 1A). The Smn2B/F7 genotype carries one allele harboring a three nucleotide switch in the exon splicing enhancer within exon 7 of the mouse Smn gene (known as Smn2B allele), mimicking the SMN2 gene in humans21, while the other allele features exon 7 flanked by loxP sites23. Additionally, crosses between AlbCre and SmnF7/F7yielded AlbCre/+;SmnF7/+ offspring.
The liver-specific mouse line was subsequentially established through further genetic crosses between AlbCre/+;SmnF7/+and Smn2B/F7, resulting in various genotypes, including +/+;Smn2B/F7(Fig. 1B), and AlbCre/+;Smn2B/F7 (Fig. 1C). As +/+;Smn2B/F7 mice lack Cre recombinase, they were used as heterozygous controls. In contrast, recombination occurs in AlbCre/+;Smn2B/F7animals resulting in the specific deletion of one allele of exon 7 in the liver, while the Smn2B allele remains unaffected.
Smn2B/- mice express approximately 15% residual full-length SMN24 (FLSMN) compared to Smn2B/+heterozygous mice. Therefore, both genotypes were included in this study as additional controls. Tissues were collected at postnatal day 19 (P19), corresponding to the symptomatic phase of the Smn2B/- mouse model. At this stage, Smn2B/- mice show increased liver steatosis and metabolic defects, ultimately culminating in multiple system defects and mortality, typically occurring around P2525.
We first assessed SMN protein levels in the liver and across CNS and peripheral tissues. Our findings confirmed a reduction in SMN protein levels across various tissues in Smn2B/- mice (Fig. 2A-E). However, in AlbCre/+;Smn2B/F7, there was a ∼70% decrease in FLSMN protein expression in the liver (Fig. 2A, A’) compared to littermate control counterparts (+/+;Smn2B/F7). Conversely, SMN protein levels in the brain (Fig. 2B, B’), muscle (Fig. 2C, C’), SC (Fig. 2D, D’) and pancreas (Fig. 2E, E’) remained unaltered, indicating that deletion of Smn exon 7 was restricted to the liver in AlbCre/+;Smn2B/F7mice.
2.2 AlbCre/+;Smn2B/F7 mice show mild yet important liver steatosis
We previously showed that Smn2B/- mice exhibit defects in fatty acid metabolism and lipid accumulation in the liver12,16–18. Additionally, SMA patients face an elevated risk of dyslipidemia and steatosis12. Hence, we explored whether the intrinsic decrease in SMN levels restricted to the liver would lead to increased lipid accumulation. Histology from AlbCre/+;Smn2B/F7 mice (Fig. 3A) qualitatively indicates some level of hepatic microvesicular steatosis and lipid accumulation. However, there was a noticeable variation in cell morphology and Oil Red-O retention among hepatocytes, suggesting incomplete recombination.
Consistent with the histological findings, AlbCre/+;Smn2B/F7mice showed variable triglycerides levels (n1=45.95, n2=10.10; n3=75.72; n4=21.91 μg/mg, mean±SEM 38.44±14.48) (Fig. 3B), and yet higher than those observed in +/+;Smn2B/F7 controls (n1=2.60, n2=0.15; n3=0.66; n4=1.44 μg/mg, mean±SEM 1.213±0.5332), although this difference did not reach statistical significance (p=0.0824). As expected, Smn2B/- controls exhibited increased lipid accumulation and triglyceride levels. Taken together, the data suggest an intrinsic role of SMN in regulating fat liver metabolism.
2.3 Liver-specific SMN depletion does not perturb liver homeostatic function
We next sought to identify whether AlbCre/+;Smn2B/F7mice display disturbed liver homeostatic function. Specifically, we evaluated parameters associated with iron metabolism (Fig. 4A,B), endoplasmic reticulum (ER) stress (Fig. 4C), and insulin-like growth factor (IGF-1) (Fig. 4D) levels, all of which have previously been identified as altered in Smn2B/- mice18 and were further confirmed in our study. Our analysis revealed no significant differences in the protein levels of heme oxygenase, transferrin, P62, and IGF-1 between the AlbCre/+;Smn2B/F7and +/+;Smn2B/F7 control groups. These findings suggest that liver-specific SMN depletion may not play a significant role in these pathways or that the mosaic nature of Cre-mediated excision resulted in a partial effect that was difficult to pick up at this time point.
2.4 Liver-specific SMN depleted mice display pancreatic defects
We have demonstrated pancreatic and glucose abnormalities in the Smn2B/- mouse model of SMA and type I SMA patients11,26. Therefore, we conducted immunohistochemistry to investigate the cellular profile of the pancreas, aiming to ascertain whether the targeted depletion of SMN in the liver contributes to pancreatic dysfunction. Surprisingly, double-labeling of insulin-producing β cells and glucagon-producing α cells revealed a significant decrease in the proportion of β cells and a notable increase in α cells in the pancreas of AlbCre/+;Smn2B/F7 mice compared to +/+;Smn2B/F7 and Smn2B/+ heterozygous controls (Fig. 5A-C). This loss of β cells and rise in α cells could suggest a functional impairment in glucose metabolism. Consequently, we evaluated non-fasting glucose levels in these mice (Fig. 5D), revealing reduced blood glucose levels in AlbCre/+;Smn2B/F7mice relative to +/+;Smn2B/F7 and Smn2B/+. However, blood glucose levels remained higher than those observed in Smn2B/- mice. Overall, these findings indicate imbalances in α and β cell fate within pancreatic islets in AlbCre/+;Smn2B/F7 mice, resembling those observed in Smn2B/- mice. Furthermore, the maintenance of SMN protein expression at levels comparable to those of heterozygous controls in the pancreas (Fig. 2E,E’) suggests that selective SMN depletion in the liver may be implicated in the dysfunction of the pancreas-liver axis.
2.5 Motor neurons, neuromuscular junction (NMJ) or motor fibers are unaffected in liver specific SMN depleted mice
The hallmark features of SMA include the degeneration of lower spinal motor neurons, neuromuscular junction (NMJ) pathology, and muscle atrophy10,25,27–33. While the exact mechanisms linking these changes in the motor unit to the clinical manifestations of SMA remain unclear, diminished levels of SMN protein are recognized as a pivotal factor1,2,25,33–35. Thus, we investigated the impact of liver-specific SMN depletion on these SMA-like features. Our findings show no significant differences in motor neuron cell body counts (Fig. 6A,B), neurofilament accumulation at NMJs (Fig. 7A,B), NMJ endplate occupancy (Fig. 7A,C), muscle fiber size (Fig. 8A,B), or muscle fiber frequency distribution (Fig. 8C) among AlbCre/+;Smn2B/F7 and +/+;Smn2B/F7 and Smn2B/+ heterozygous controls. However, as anticipated, Smn2B/- mice exhibited motor neuron loss, NMJ defects, and muscle atrophy.
2.6 Liver-specific SMN depleted mice have normal survival rate and motor function
We also conducted a comprehensive assessment of survival, weight, and motor function in AlbCre/+;Smn2B/F7 and +/+;Smn2B/F7 mice over a 60-day period (Fig. 9A). While the Smn2B/− mice typically have a mean survival of 25 days25, both AlbCre/+;Smn2B/F7 and +/+;Smn2B/F7 mice in our study survived up to 60 days (Fig. 9B), after which they were euthanized for tissue collection. Moreover, throughout the observation period, mice from both groups exhibited similar weight gain trends (Fig. 9C).
To further evaluate motor function at different stages, we implemented several age-specific behavioral tests commonly used to assess motor function36 (Fig. 9D-F). We previously showed Smn2B/-mice have reduced motor function scores, as seen through increased time to right, prolonged balancing time and diminished muscle strength26. Here, we found no alterations in AlbCre/+;Smn2B/F7 compared to +/+;Smn2B/F7 mice in time to right, as assessed by the righting reflex (Fig. 9D), nor in distal muscle strength measured through the inverted mesh grip test (Fig. 9E), or motor balance and coordination evaluated by the pen test (Fig. 9F). Overall, these findings collectively demonstrate that liver-specific SMN depletion does not impact survival, weight gain, or motor function.
3. Discussion
The liver is pivotal in regulating numerous biological functions in the body. These encompass, among others, the storage and balance of lipids, carbohydrates, and iron, as well as the synthesis of vital growth factors and the clearance of non-essential or harmful metabolites19. Given its multifaceted role, any alterations in liver function can potentially have cascading effects on other organs within the body. Hence, aside from elucidating the impact of liver specific SMN depletion on hepatic function, it is imperative to explore its effects on other tissues, as alterations in these tissues have been documented in various mouse models of SMA11,18,34,37.
Previous work on conditional knockout of Smn in the livers of mice resulted in severe impairment of liver development characterized by iron overload and subsequent liver atrophy, ultimately leading to late embryonic lethality8. In our study, we generated a novel mouse model where instead of a complete knockout of Smn in the liver, we created a scenario where SMN protein was depleted to 30% of full-length SMN production specifically within the liver, while SMN protein levels in other tissues remain comparable to those of heterozygous controls. Notably, this mouse line did not exhibit any signs of developmental delays, liver atrophy, or reduced lifespan. Nonetheless, within 19 days after birth, AlbCre/+;Smn2B/F7 mice showed a mild yet rapid onset of fatty liver disease and trends towards an increase in lipid levels when maintained on a normal chow diet, underscoring the importance of SMN in lipid metabolism.
Mild liver steatosis may be accompanied by disruptions in crucial liver function markers, notably those implicated in iron metabolism, autophagy, and growth factor regulation, such as IGF-118. Accordingly, we initially investigated the expression of HO-1, an enzyme responsible for heme degradation into iron, carbon monoxide, and biliverdin; and transferrin, a liver-synthesized glycoprotein involved in iron transport across tissues (reviewed in38). Prior studies have documented alterations in these proteins in Smn2B/- mouse livers18. While our current analysis did not reveal significant differences in AlbCre/+;Smn2B/F7mice, the observed upward trend in transferrin and HO levels suggests ongoing changes in iron metabolism, which may not be fully manifested at P19. In addition, incomplete Cre-mediated excision of SmnF7 allele may be hindering a comprehensive understanding of SMN’s role in liver function.
Furthermore, no alterations were observed for P62 levels, a multifunctional scaffold protein implicated in autophagy, formation of hepatic inclusion bodies, and hepatocyte cell death (reviewed in39) despite its increased presence in mouse models of SMA18. Similarly, there were no changes detected in IGF-1 protein levels, whose deficiency has been linked to liver pathology and non-alcoholic fatty liver disease (NAFLD)40,41, and its decrease is evident in Smn2B/- mice18.
Altogether, our findings underscore the necessity of conducting further investigations at later time points to unveil potential modifications in other pathways and their repercussions on liver physiology. Additionally, the variability in the data may be due to differences in Cre recombination efficiency from mouse to mouse due to inconsistent Cre recombination among littermates, or the absence of Cre expression in certain cells leading to recombination failure42.
Previously, our research unveiled metabolic abnormalities in SMA-like mice, including glucose intolerance, insulin hypersensitivity, and hyperglucagonemia, accompanied by changes in pancreatic islet composition characterized by an increase in glucagon-producing α-cells at the expense of insulin-producing β-cells11. Histopathological examination of pancreatic tissue from infants and children with SMA type I corroborated these findings, revealing a predominant presence of glucagon-producing α-cells within pancreatic islets11. Furthermore, one-year-old Smn+/-mice producing approximately 50% of SMN protein, when exposed to a high-fat diet, exhibited dysregulation in the proportion of glucagon-producing α-cells within pancreatic islets and heightened hepatic insulin and glucagon sensitivity9. Our study uncovers a novel intrinsic role of liver-specific SMN in pancreatic function, distinct from IGF-1 signaling, pancreatic SMN levels (which remained stable), or canonical SMA pathology.
In addition, while the hallmark pathological features of SMA include motor neuron loss, NMJ pathology, muscle atrophy, and consequent motor function alterations2,30,33–35, these were not evident in the AlbCre/+;Smn2B/F7 mouse model at P19. However, given that glucose metabolism defects are observed in various neurodegenerative and neuromuscular disorders, subsequent time points are crucial to discern whether additional alterations arise as the mice age. Although hepatic and pancreatic abnormalities likely occur independently of disease onset, given that SMN depletion in motor neurons and muscle alone23,43,44 is sufficient to induce a SMA-like phenotype, the potential influence of these anomalies on SMA progression warrants careful consideration.
Overall, our findings suggest that alterations in SMN production solely within the liver may suffice to induce pathological hepatic and pancreatic function, shedding light on a novel role of SMN in liver physiology.
4. Material and Methods
4.1 Mouse models
The Smn2B mouse, which harbors the Smn2Bmutation resulting from the substitution of three nucleotides within the exon splicing enhancer of exon 7, was developed and maintained on a C57BL/6 background in our laboratory21. The FVB.129(B6)-Smn1tm1Jme (SmnF7/F7) and B6.Cg-Speer6-ps1Tg(Alb-Cre)21Mgn/J (AlbCre) mice were acquired from The Jackson Laboratory. Liver-specific SMN-depleted mice were obtained through successive crosses between AlbCre/+;SmnF7/+and Smn2B/F7 mice (see Fig. 1). Line validation was conducted via genotyping of DNA extracted from mouse ear biopsies using PCR (see Table 1 for list of primers used). Both male and female mice were included in the studies. All mice were bred and housed in the University of Ottawa Animal Facility under protocol OHRI-3343, adhering to guidelines established by the Canadian Council on Animal Care. Additionally, we employed Smn2B/- mice (a severe SMA mouse model) and asymptomatic heterozygous Smn2B/+ mice as controls in our experiments.
4.2 Tissue harvesting and processing
Following euthanasia, liver, and tibialis anterior (TA) muscles underwent fixation in a 1:10 dilution of buffered formalin for 48 h at 4°C, followed by transfer to 70% ethanol at the same temperature until processing. The pancreas was fixed in 4% paraformaldehyde (PFA) for 48 h at 4°C, then similarly moved to 70% ethanol for processing. For the lumbar spinal cord (SC) and liver (for Oil Red-O staining), fixation was performed overnight at 4°C in 4% PFA, followed by immersion in 30% sucrose in PBS. Subsequently, the liver and SC were flash-frozen in optimal cutting temperature (OCT) embedding medium. Cryosections of the lumbar spinal cord were cut at a thickness of 16 μm, while liver sections were cut at 10 μm and stored at −80°C until immunohistochemical analysis.
The abdominal musculature was dissected and fixed in 4% PFA for 15 min at room temperature, followed by 3 washes in 1X PBS, after which the transversus abdominis (TVA) muscle was dissected from the abdominal musculature. TA, liver, and pancreas samples were handled at the University of Ottawa Department of Pathology and Laboratory Medicine. Paraffin blocks containing tissues were sectioned at 4 μm thickness using a microtome. Sections of TA and liver were stained with hematoxylin and eosin (H&E) using an XL CV5030 autostainer from Leica. Liver cryosections were stained with Oil Red-O and counterstained with hematoxylin using established protocols. Images of H&E and Oil Red-O-stained samples were scanned with a MIRAX MIDI digital slide scanner manufactured by Zeiss. Image acquisition was performed using Panoramic Viewer 1.15.4 software.
4.3 Immunoblotting
Total protein was extracted by homogenizing flash-frozen samples of spinal cord, brain, liver, pancreas and hindlimb muscles using RIPA lysis buffer (Cell Signaling), supplemented with phenylmethylsulfonyl fluoride (PMSF, Cell Signaling). The concentrations of the extracted proteins were determined using the Pierce BCA Protein Assay kit (ThermoFisher).
For liver, brain, and spinal cord tissues, 20 μg of protein per sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), while 30 μg were used for pancreas and muscle samples. Following electrophoresis, the proteins were transferred onto a PVDF membrane (Immobilon-P, Millipore) and blocked for 30 min at room temperature using intercept blocking buffer (LI-COR). Subsequently, the membranes were incubated overnight at 4°C with primary antibodies.
After incubation, the membranes were rinsed with tris-buffered saline containing 0.1% Tween-20 (TBS-T) and then incubated for 1 h at room temperature with IRDye 680R or 800CW (LI-COR) secondary antibody. Fluorescent signals were detected using the Odyssey Infrared Imaging System (LI-COR). Total raw values were normalized to α-tubulin (housekeeping protein) obtained from the same blot.
4.4 Immunohistochemistry
4.4.1 Pancreas
Pancreatic sections underwent deparaffinization using three washes of Histo-Clear (National Diagnostics, Atlanta, GA) for 5 min each, followed by two washes of a 50/50 mixture of absolute ethanol and Histo-Clear for 3 min each. Subsequently, the slides were gradually rehydrated in two washes of 100% (v/v) ethanol (3 min each), followed by one wash of 95% (v/v), 70% (v/v), 50% (v/v), and 0% (v/v) ethanol for 3 min each. The slides were then incubated in 0.5% Triton-X-100 (Millipore Sigma, Burlington, MA) in PBS for 5 min, followed by three washes with PBS. Afterward, the slides were blocked in a solution containing 20% goat serum and 0.3% Triton-X-100 in TBS for 2 h.
Primary antibodies targeting insulin and glucagon (Table 2) were applied to the slides in a solution containing 2% goat serum and 0.3% Triton-X-100 in TBS overnight at 4°C. The slides were then washed three times with PBS and subsequently incubated with secondary antibodies in a solution containing 2% goat serum and 0.3% Triton-X-100 for 1 h. Following this, the solution was removed, and DAPI (1:1,000) in PBS was added for 5 min at room temperature. The slides were washed three times with PBS and mounted in Fluoromount-G™ Mounting Medium (Invitrogen).
Images were taken using an Axio Imager M2 microscope (Zeiss) and captured with a 40X objective. The number of glucagon and insulin positive cells per islet was counted for each mouse.
4.4.2 Spinal cord (SC)
Lumbar spinal cord sections were prepared for choline acetyltransferase (ChAT) staining of motor neurons. The slides were air-dried at room temperature for 30 min and then rinsed in TBS-T for 5 min. Samples were permeabilized in 0.3% Triton X-100 in PBS for 30 min, followed by blocking in 1x Power Block (BioGenex, Fremont, CA) for 20 min at room temperature. Subsequently, the samples were incubated with a goat anti-ChAT (details in Table 2) antibody at a dilution of 1:100 in 1% BSA and 0.3% Triton X-100 in PBS for 3 nights at 4°C.
After the initial antibody incubation, slides were washed twice for 10 min with TBS-T at room temperature. Following the washes, samples were then incubated with Alexa Fluor 555 donkey anti-goat IgG at a dilution of 1:200 in 10% donkey serum and 0.3% Triton X-100 in PBS for 2 h at room temperature in a humid chamber. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) at a dilution of 1:1000 in PBS for 5 min.
Finally, the slides were rinsed 3X for 10 min with TBS-T and mounted with Fluoromount-G™ Mounting Medium (Invitrogen). SC were examined under fluorescence using an Axio Imager M2 microscope (Zeiss) and imaged at 20X magnification for quantification, and at 63x for representative pictures. The number of ChAT-positive motor neurons with a diameter of ≥20 μm per ventral horn was recorded for 7-10 different sections per animal, each separated by at least 100 μm to prevent re-counting of the same motor neuron. An average number of motor neuron cell bodies per section was determined.
4.4.3 Neuromuscular Junction (NMJ)
To evaluate neuromuscular innervation, we performed immunohistochemistry using established protocols26. After euthanasia, the TVA muscles were promptly dissected and fixed in 4% paraformaldehyde (Electron Microscopy Science) in PBS for 15 min. Post-synaptic acetylcholine receptors (AChRs) were labeled for 10 min with alpha-bungarotoxin (aBTX) conjugated to Alexa Fluor 488. Subsequently, the muscles were blocked in 4% bovine serum albumin (BSA) and 1% Triton-X-100 in PBS for 30 min.
Following blocking, the muscles underwent overnight incubation with primary antibodies for neurofilament and synaptic vesicle protein 2 (refer to Table 2). Visualization of the labeled structures was achieved using DyLight-conjugated secondary antibodies. The muscles were then whole-mounted in Dako Fluorescent mounting media and imaged with a 20x objective on a Zeiss Axio Imager M1 microscope.
A minimum of three fields of view (FOV) were quantified per muscle. For each FOV, the percentage of fully occupied endplates was noted. Fully occupied endplates were defined as motor endplates completely covered by the presynaptic terminal labeled with synaptic vesicle protein 2 (SV2) and neurofilament (NF).
4.5 Muscle fiber analysis
Muscle fiber analysis was performed using ImageJ software (version 2.9.0/1.53t). A total of one hundred fibers were evaluated per animal, covering various regions of the muscle section to ensure good representation. The area of each fiber was measured in square micrometers (μm²) to compute both the average and distribution of fiber sizes for each animal.
4.6 Glucose levels
Non-fasting blood glucose concentrations were assessed using a OneTouch Ultra2 Blood Glucose Test Strips (LifeScan Europe GmbH, Zug, Switzerland) immediately following blood collection. Approximately 2 μL of blood was applied to the test strip for glucose concentration measurement.
4.7 ELISA for Insulin-like Growth Factor I (IGF-1)
The concentration of IGF-1 in the liver was quantified using the Mouse/Rat IGF-I/IGF1 Quantikine ELISA Kit (MG100, R&D Systems, Inc., Minneapolis, MN, USA). Liver protein lysates were diluted at a ratio of 1:6 in calibrator diluent and the assay was performed following the manufacturer’s instructions.
4.8 Triglycerides quantification
Liver tissues from P19 mice were promptly dissected and flash frozen. Subsequently, triglyceride analysis was conducted at the Vanderbilt Mouse Metabolic Phenotyping Center, employing established protocols previously utilized by our team18.
4.9 Motor function, weight, and survival assessments
Motor function, weight, and survival were assessed according to established protocols36. Spinal reflexes were evaluated using the righting reflex test, while motor balance and coordination were evaluated with the pen test. Additionally, muscle strength was measured using the inverted mesh grip test (see Fig. 9A). 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. A maximum duration of 30 sec for the pen test and 60 sec for the inverted mesh grip represented the established threshold. Three consecutive measurements were obtained, and their average was recorded for subsequent analysis. Weight was measured every two days starting at day 7 until day 30, and then weekly until day 60. Animal welfare was monitored throughout the 60-day period during weight measurements.
4.10 Statistical analysis
Survival data were visualized using Kaplan-Meier survival curves, and intergroup differences were evaluated using the Mantel-Cox test. Brown-Forsythe and Welch ANOVA were employed to compare three or more sets of unpaired measurements, given the mosaic-like outcome observed in the AlbCre/+;Smn2B/F7 model, where assumptions of equal variances were not tenable. Two-way ANOVA, followed by Šídák’s method for multiple comparisons, was utilized to assess the influence of two factors on a response. Statistical analyses were conducted using GraphPad Prism V.10.2.1 (GraphPad Prism Software, San Diego, CA), with significance set at P < 0.05. Data were presented as mean ± standard error of the mean. Sample sizes (n) are provided in figure legends, indicating the number of biological replicates analyzed, with each data point corresponding to an individual mouse from at least two different litters. Detailed statistical information is outlined in the respective figure legends, where statistical significance is indicated by *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Data sharing
All authors had access to the study data and reviewed and approved the final manuscript. All data associated with this study are available in the main text or supplementary materials. Raw data can be provided upon request.
Acknowledgements
This work was supported by Muscular Dystrophy Association (USA) [grant number 963652 to R.K.]; the Canadian Institutes of Health Research [grant number PJT-186300 to R.K.]; and the University of Ottawa Brain and Mind Institute TRIMS Award to MMAA.
Declaration of interests
The authors disclose no conflicts of interest. The funders mentioned above were not involved in the study design, data collection and analysis, decision to publish, or manuscript preparation.
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