{"paper_id":"775e9173-4274-4779-9ac5-0f6f25da963d","body_text":"System-Wide Proteomic Remodeling in Spinal Muscular Atrophy Reveals \nTissue-Specific Responses and Partial Rescue by SMN Restoration \nSofia Vrettou1,2, Stefan Müller2,3, Brunhilde Wirth1,2,4 \n1Institute of Human Genetics, University Hospital of Cologne, University of Cologne, \n50931 Cologne, Germany. \n2Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, \nGermany. \n3CECAD Proteomics Facility, 50931 Cologne, Germany \n4Center for Rare Diseases, University Hospital of Cologne, University of Cologne, 50931 \nCologne, Germany. \nCorresponding author: Sofia Vrettou, sofia.vrettou@uk-koeln.de  \n \nAbstract \nSpinal muscular atrophy (SMA), traditionally defined as a neuromuscular disorder \ncharacterized by degeneration of lower motor neurons, is increasingly recognized as a \nmulti-organ disease. SMA is caused by deficiency of the survival motor neuron (SMN) \nprotein below a critical threshold required for cellular homeostasis. While motor neurons \nare particularly vulnerable, the ubiquitous expression and fundamental functions of SMN \nresult in widespread perturbations across multiple tissues. \nHere, we generated a label-free quantitative proteomics atlas of spinal cord, heart, and \ngastrocnemius muscle from wild-type, heterozygous, and SMA mice at the symptomatic \nstage, including cohorts treated, at postnatal day 1 (P1), with a systemic suboptimal \ndose of SMN antisense oligonucleotides (SMN-ASOs), resulting in partial SMN \nrestoration. SMN deficiency induced pronounced, tissue-specific proteome remodeling, \nwith peripheral tissues exhibiting broader molecular alterations than spinal cord. Cross-\ntissue analyses revealed limited overlap, although heart and muscle showed partial \nconvergence in metabolic and mitochondrial-associated pathways. SMN-ASO treatment \npartially repositioned these proteomes toward control states; however, restoration was \nincomplete and strongly tissue-dependent, with persistent dysregulation of mitochondrial \nand metabolic pathways. \nThese findings demonstrate that SMN deficiency drives systemic yet heterogeneous \nproteome remodeling and that partial SMN restoration does not fully reverse established \nmolecular alterations. \nKeywords \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nSpinal muscular atrophy; SMN deficiency; Proteomics; Multi-organ disease; \nMitochondrial dysfunction; Antisense oligonucleotides (ASOs); Neuromuscular system \n \nGraphical Abstract \n \nIntroduction \nSpinal muscular atrophy (SMA) is caused by deficiency of the survival motor neuron \n(SMN) protein and is classically defined by progressive loss of lower alpha motor \nneurons [1-3]. Yet SMN is expressed in virtually all tissues [4], and increasing evidence \nshows that its deficiency perturbs cellular homeostasis far beyond the nervous system \n[5, 6]. Peripheral organs, including skeletal muscle and heart, exhibit early and sustained \nabnormalities, underscoring that SMA is not confined to the motor unit but represents a \nsystemic disorder with tissue-specific manifestations [7-11]. \nA central challenge in SMA research is understanding why organs respond so differently \nto a shared molecular deficit. Neuromuscular tissues are particularly vulnerable, but \neven among them, the magnitude and nature of molecular remodeling vary markedly [6, \n12, 13]. Spinal cord pathology dominates clinical presentation, while skeletal and cardiac \nmuscles show intrinsic metabolic and mitochondrial alterations that cannot be explained \nby denervation alone [8, 14-18]. These observations suggest that SMN deficiency \nengages organ-specific adaptive and maladaptive programs shaped by developmental \ntiming, metabolic demand, and compensatory capacity. Whether these divergent \nresponses are reflected in coordinated, tissue-specific proteomic programs remain \nunresolved. \n \nThe advent of SMN-restoring therapies, including antisense oligonucleotides (ASOs), \nhas fundamentally improved the clinical course of SMA, particularly when treatment is \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\ninitiated early [19-23]. However, therapeutic benefit is not uniform, and molecular \nnormalization across tissues is often incomplete [24]. This raises an important question: \nwhether early SMN restoration normalizes tissue proteomes or instead establishes \ndistinct, partially corrected states remains unclear. \n \nUnbiased proteomics offers a powerful means to address this question by capturing \ncoordinated protein-level changes across pathways and cellular compartments. While \nrecent targeted and single-organ studies have demonstrated organ-dependent molecular \nremodeling and partial correction following SMN-ASO treatment [24-26], a unified, cross-\ntissue proteomic analysis spanning both neuronal and muscular compartments within \nthe same experimental framework is lacking. \nHere, we present a label-free quantitative proteomics atlas of spinal cord, gastrocnemius \nmuscle, and heart from wild-type (WT), heterozygous (HET), and SMA mice at the \nsymptomatic stage (postnatal day 10; P10), including cohorts treated with SMN-ASOs at \npostnatal day 1 (P1), which led in partial SMN restoration. By jointly examining central \nand peripheral neuronal and muscular tissues within a unified experimental design, we \ndefine organ-specific proteomic signatures of SMN deficiency and evaluate how early \nSMN restoration reshapes these molecular states. This atlas establishes a cross-tissue \nproteomic framework for interpreting systemic SMN biology and for guiding future \nmechanistic and therapeutic studies in SMA. \n \nMaterials and Methods \nAnimal Model and Tissue Collection \nSevere Taiwanese SMA mice (Smn −/−; SMN2 tg/0), heterozygous carriers (Smn +/−; \nSMN2tg/0), and wild-type (WT) controls were generated and maintained as previously \ndescribed [24, 26-29].  \nFor SMN restoration, neonatal mice received a single subcutaneous injection of a splice-\nmodulating SMN-targeting antisense oligonucleotide (SMN-ASO) at P1, as previously \ndescribed based on validated protocols [24-26, 28, 30, 31]. This oligonucleotide acts on \nSMN2 pre-mRNA splicing to favor exon 7 inclusion and increase expression of full-\nlength SMN protein [32]. Because the SMN-ASO was administered systemically at a \nsuboptimal dose, the resulting rescue was partial and not expected to fully normalize \nSMN levels across tissues [24, 33]. Control WT animals were not injected. Treated and \nuntreated animals were sacrificed at P10, and tissues were rapidly dissected, snap-\nfrozen in dry ice, and stored at −80 °C until processing. \nMice were housed under controlled environmental conditions with a 12-hour light/dark \ncycle and had unrestricted access to food and water. All breeding, housing, and \nexperimental procedures were carried out in a specific pathogen-free environment. \nAnimal experiments were conducted in compliance with applicable institutional and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\ngovernmental guidelines and were approved by the Landesamt für Natur, Umwelt und \nVerbraucherschutz Nordrhein-Westfalen (LANUV) under application numbers: 81-\n02.04.2020.A196, 81-02.04.2019.A017, 81-02.04.2019.A138, §4.23.008, and §4.22.002. \n \nProtein Extraction and Digestion \nProtein extraction, reduction, alkylation, and enzymatic digestion were performed using a \nstandardized urea-based workflow as previously described [25, 26]. Briefly, tissues were \nhomogenized in 8 M urea buffer (50 mM TEAB, pH 8.0) supplemented with protease \ninhibitors. Protein lysates were reduced, alkylated, and sequentially digested with Lys-C \nand trypsin. Peptides were acidified and desalted using SDB-RPS StageTips prior to \nLC–MS/MS analysis. \n \nLC–MS/MS Data Acquisition and DIA-NN Processing \nPeptides were analyzed by nano LC–MS/MS on a Vanquish Neo system coupled to a \nThermo Orbitrap Exploris 480 mass spectrometer equipped with a FAIMS Pro interface, \nas previously described [26]. Data-independent acquisition (DIA) was performed across \nthe mass range of 400-1000 m/z. Instrument parameters and acquisition settings were \nkept consistent across all tissues to ensure comparability. \nRaw DIA data were processed using DIA-NN (v1.8.1) [34] with a predicted spectral \nlibrary generated from the Mus musculus UniProt canonical database, as described \npreviously [26]. Data were filtered at 1% false discovery rate (FDR) at both precursor \nand protein levels. Protein-level label-free quantification (LFQ) intensities were exported \nfor downstream analysis. \nComprehensive instrument parameters and DIA-NN processing settings are described in \ndetail in our accompanying Data in Brief publication on liver proteomics from the same \nanimal model, ensuring methodological transparency and reproducibility [26]. \n \nBioinformatics Analysis \n1. Statistical Analysis \nStatistical analysis was performed in Perseus (v1.6.15) [35] following established \nworkflows [25, 26]. Protein intensities were log2-transformed, filtered for valid values \nacross biological replicates, and missing values were imputed. Differential expression \nwas assessed using two-sample t-tests with permutation-based FDR correction (FDR = \n0.05; S0 = 0.1 unless otherwise specified). Principal component analysis was performed \non processed datasets. The number of biological replicates per group is indicated in the \ncorresponding figures and legends. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\n2. Proteomic Stratification and Cross-Organ Integration Analysis \nBecause proteomic responses to SMN-ASO treatment were heterogeneous within the \nSMA injected with SMN-ASOs (SMA+ASO) group, treated SMA samples were stratified \naccording to the degree of proteome repositioning observed by principal component \nanalysis (PCA) in each tissue. Samples showing a more pronounced shift away from \nuntreated SMA and toward the WT/HET proteomic space were considered to exhibit \ngreater proteomic normalization, whereas samples remaining closer to the untreated \nSMA cluster were considered to exhibit limited proteomic normalization. By contrast, \nHET+ASO samples showed comparatively homogeneous proteomic profiles across \nanimals within each tissue and were therefore analyzed as a single treatment group \nwithout additional stratification. \nUsing this stratified framework, we next assessed the extent to which SMN-ASO \ntreatment directionally normalized proteins significantly altered in SMA relative to WT. \nFirst, we sorted the proteins identified as significantly dysregulated in the WT versus \nSMA comparison and then evaluated their convergence (presence or absence; fold \nchange; significance thresholds) in the SMA versus SMA+ASO comparison. Proteins \nwere considered directionally rescued when their abundance shifted toward WT levels \nafter SMN-ASO treatment. Proteins that remained significantly altered without directional \nreversal were classified as persistent. Proteins significantly dysregulated in WT versus \nASO but not detected within the cohort of significantly altered in SMA versus SMA+ASO \nwere classified as no response in ASO. Proteins significantly altered in the SMA versus \nSMA+ASO comparison but not identified as significantly dysregulated in the WT versus \nSMA comparison were classified as ASO-responsive only. \nCross-organ integration was performed using UniProt identifiers to determine shared \nand tissue-specific protein alterations. Analyses focused on proportional rescue and \npathway convergence across tissues. \n3. Network and Functional Enrichment Analysis \nFunctional enrichment analysis was performed using STRING (v11.5; Mus musculus) \nand visualized in Cytoscape with the ClueGO plugin. Gene Ontology (Biological \nProcess, Molecular Function, Cellular Component), KEGG, and Reactome databases \nwere used for pathway analysis. Statistical significance was determined using two-sided \nhypergeometric testing with Benjamini-Hochberg correction (adjusted p < 0.05). A kappa \nscore threshold of 0.4 was applied for functional grouping. \n4. Data Visualization \nVolcano plots were generated using InstantClue [36] based on statistical outputs from \nPerseus. PCA and rescue classification visualizations were generated using SRplot [37]. \nVisualization tools were used exclusively for graphical representation and not for \nstatistical analysis. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nResults  \n1. Global proteome profiling reveals organ-specific remodeling in SMA \nTo define baseline proteome alterations associated with SMN deficiency across \nneuronal and muscular tissues, label-free quantitative proteomics was performed on \nspinal cord, heart, and gastrocnemius muscle isolated at P10 from WT, HET, and SMA \nmice. The goal was to quantitatively compare the extent and nature of proteome \nremodeling across neuromuscular tissues. After quality control and exclusion of samples \nwith insufficient signal or genotype-inconsistent clustering, final cohort sizes were: spinal \ncord (4 WT, 5 HET, 4 SMA), heart (4 WT, 5 HET, 5 SMA), and gastrocnemius (5 WT, 5 \nHET, 4 SMA). \nAcross tissues, 4,983 proteins were identified in spinal cord, 2,912 in heart, and 3,248 in \ngastrocnemius. After filtering for valid quantification, 4,787 proteins were retained in \nspinal cord, 2,761 in heart, and 3,164 in gastrocnemius. \nPCA revealed genotype-dependent separation across all three tissues (Figure 1A-C). In \nspinal cord and gastrocnemius, SMA samples segregated from WT, with HET samples \npositioned intermediately, consistent with SMN dosage-dependent proteome remodeling \n(Figure 1A, C). In heart, clear separation between genotypes was observed (Figure 1B). \nUnsupervised hierarchical clustering of global protein abundance (Figure 1D-F) \nsupported these findings, as samples grouped primarily according to genotype across \ntissues. \nDifferential abundance analysis between WT and SMA was performed using Perseus \n(FDR 0.05, S0 = 0.1). Volcano plots (Figure 1G-I) revealed marked differences in the \nextent of proteome remodeling across tissues. Spinal cord exhibited comparatively \nlimited proteome remodeling (Figure 1G), whereas heart and gastrocnemius displayed \nsubstantially broader sets of differentially abundant proteins (Figure 1H-I). Fold-change \ndistribution plots (Figure 1J-L) summarize the global magnitude and direction of \ngenotype-dependent shifts, demonstrating broader distribution changes in heart and \ngastrocnemius compared to spinal cord. Complete lists of significantly altered proteins \nfor all comparisons are provided in the Supplementary Tables S1-S3. \nVolcano plots for HET versus SMA are shown in Supplementary Figure 1. In spinal cord, \nno proteins passed the applied Perseus thresholds (Supplementary Figure S1A), \nconsistent with the PCA positioning of HET samples in this tissue (Figure 1A). In \ncontrast, heart and gastrocnemius exhibited significant alterations between HET and \nSMA (Supplementary Figure S1B-C). Comparison of WT versus HET revealed modest \ndifferences overall, with limited significant proteins detected (Supplementary Figure \nS1G-H). \nIntersection analysis across tissues demonstrated that proteome alterations were \npredominantly tissue-specific (Supplementary Figure S1G-H; Supplementary Tables S4-\nS5), with only limited overlap between spinal cord and peripheral tissues. By contrast, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nheart and gastrocnemius showed partial convergence in membrane-organization, \nmetabolic and mitochondrial-associated pathways (Supplementary Figure S2A). \nTo define the cellular architecture affected by SMN deficiency, 1D enrichment analysis \nwas performed for WT versus SMA in each tissue (Figure 2A-C). In spinal cord, enriched \ncategories included membrane-associated, nuclear, endoplasmic reticulum, and \nmitochondrial components (Figure 2A), supporting increasing recognition of \nmitochondrial involvement in SMA pathology [17, 18]. In heart, enrichment involved \nmitochondrial structures, ribosomal compartments, extracellular matrix–associated \nelements, and cytoskeletal assemblies (Figure 2B), consistent with the diverse molecular \nroles attributed to SMN in RNA metabolism [4, 38-40] and cytoskeletal regulation [41-\n45]. Likewise, in gastrocnemius, cytosolic, ribosomal, mitochondrial, and contractile-\nassociated components were enriched (Figure 2C). \nSTRING network analysis of significantly downregulated proteins in WT versus SMA \ncomparisons (Figure 2D-F) revealed tissue-specific organization. In spinal cord, \ndownregulated proteins formed interconnected clusters associated with mitochondrial \ncomponents, endoplasmic reticulum and Golgi compartments, axonogenesis, and glial \ncell differentiation (Figure 2D). In heart, downregulated proteins clustered in pathways \nrelated to DNA repair mechanisms, intracellular trafficking processes as well as clathrin-\nmediated endocytosis (Figure 2E), consistent with previous evidence linking endocytic \ndysregulation to SMA pathophysiology [33, 46-49]. In gastrocnemius, downregulated \nnetworks included phospholipid efflux pathways, DNA repair processes, mRNA \nprocessing, and neuromuscular junction development (Figure 2F), reflecting established \nneuromuscular junction vulnerability in SM A [50-53]. STRING network analysis of \nsignificantly upregulated proteins in WT versus SMA comparisons is shown in \nSupplementary Figure S3. \nA parallel 1D enrichment and STRING analysis was performed for HET versus SMA \ncomparisons (Figure 3; Supplementary Figure S4). In spinal cord, STRING analysis \nrevealed mitochondrial-associated clusters (Figure 3A, D) similar to those observed in \nWT versus SMA (Figure 2A, D). In heart, enrichment was dominated by extracellular and \nribosomal components (Figure 3B), and STRING networks showed prominent \nspliceosomal and snRNP-associated clusters together with endosome and vesicle \nlocalization pathways (Figure 3E). In gastrocnemius, enrichment involved mitochondrial \nand ribosomal compartments (Figure 3C), and STRING analysis identified a dominant \nphospholipid-associated cluster (Figure 3F) mirroring the WT versus SMA analysis \n(Figure 2C, F). Upregulated protein networks for HET versus SMA comparisons are \nshown in Supplementary Figure S4. \nTogether, these analyses demonstrate that SMN deficiency induces clear, genotype-\ndependent proteome remodeling at the symptomatic stage P10. These changes are \nlargely tissue-specific, with partial overlap between heart and gastrocnemius, and \ninvolve recurrent mitochondrial, trafficking, and RNA-related pathways. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\n2. Organ-specific pathway responses to SMN-ASO treatment \n2. 1. Global proteomic repositioning following partial SMN restoration \nTo assess whether SMN elevation alleviates tissue-specific proteome alterations, we \nanalyzed spinal cord, heart, and gastrocnemius muscle samples from SMN-ASO-treated \nanimals. Because proteomic responses within the SMA+ASO group were \nheterogeneous, subsequent analyses focused on the SMA+ASO samples showing the \nclearest proteomic shift away from untreated SMA and toward the WT/HET state in each \ntissue based on PCA and as it is discussed in Bioinformatics analysis section. This \napproach was used to define organ-specific molecular responses to partial SMN \nrestoration. \nPCA demonstrated organ-dependent responses to SMN-ASO treatment. In spinal cord \n(Figure 4A), heart (Figure 4B), and gastrocnemius (Figure 4C),\n SMA+ASO samples \nexhibited variable degrees of proteomic repositioning relative to untreated SMA, with a \nsubset of samples shifting toward the WT/HET proteomic space in each tissue. Even in \nthat subgroup, repositioning was incomplete across all tissues, indicating partial rather \nthan full proteome normalization following SMN elevation. \nHierarchical clustering and heatmap analysis further illustrated tissue-specific patterns of \nproteomic normalization and persistence (Figure 4D-F). \nTo systematically quantify treatment effects, proteins significantly altered in SMA relative \nto WT were classified according to their response to SMN-ASO treatment as rescued \n(shifted toward WT levels), persistent (remaining altered without directional reversal), or \nno change in ASO (when no significant treatment-associated change was detected). The \nrelative distribution of these categories per organ is summarized in Figure 4G. ASO-\nresponsive-only corresponds to significantly altered proteins following treatment but not \nsignificantly dysregulated in the WT versus SMA comparison. Complete protein lists for \neach classification in spinal cord, heart, and gastrocnemius are provided in \nSupplementary Tables S6-S9.  \nTo contextualize SMA-specific effects, PCA and heatmap analyses including WT, HET, \nSMA, HET+ASO, and SMA+ASO groups are shown in Supplementary Figure S5A-I. \nHET samples clustered closely with WT across tissues, and HET+ASO samples did not \nexhibit major global shifts relative to untreated HET controls (Supplementary Figure \nS5A-C). These findings indicate that SMN-ASO treatment exerts minimal proteomic \neffects in non-diseased contexts. Differential expression analysis between HET and \nHET+ASO groups was performed for each organ. Volcano plots are shown in \nSupplementary Figure S5G-I. Across spinal cord, heart, and gastrocnemius, a limited \nnumber of proteins were significantly altered following ASO administration, with \nsignificant changes detected only in spinal cord (Supplementary Figure S5G). The \nmagnitude of change was modest compared to the SMA versus SMA+ASO comparison. \nNotably, HET+ASO samples displayed comparatively homogeneous clustering behavior \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nacross tissues and were therefore considered as a single treatment group without further \nstratification. \n \n2. 2. Functional enrichment of rescued and persistent proteomic responses \nTo further define the molecular processes responsive or resistant to SMN elevation, \npathway enrichment analysis was performed separately for proteins rescued from SMA-\nassociated upregulation and downregulation (Figure 5A-B; Supplementary Figure S6A). \nBecause the ASO-responsive-only category reflects treatment-associated changes \noutside the set of baseline SMA-dysregulated proteins, downstream functional \nenrichment in the main analysis focused on rescued and persistent categories, while \nASO-responsive-only proteins are provided in Supplementary Tables S6-S9. \nRescue-associated pathways differed across tissues, reflecting organ-specific responses \nto SMN restoration (Figure 5A-B; Supplementary Figure S6A; Supplementary Tables S6-\nS9). In spinal cord (Figure 5B), rescued upregulated proteins were enriched for immune-\nrelated pathways. In heart (Figure 5B), rescued categories included vesicle-mediated \ntransport and cytoskeletal organization. In gastrocnemius (Figure 5B), rescued proteins \nwere enriched for synaptic and neuromuscular junction-related pathways as well as \nmetabolic processes. \nA substantial subset of SMA-associated proteins remained uncorrected following SMN-\nASO treatment (Figure 5A, C; Supplementary Figure S6B; Supplementary Tables S6-\nS9). Persistent downregulated proteins were enriched in mitochondrial respiratory chain \nand oxidative phosphorylation pathways in spinal cord and heart (Figure 5A, C; \nSupplementary Figure S6B). Persistent upregulated proteins were associated with \nstress-related and proteostasis pathways (Supplementary Figure S6B; Supplementary \nTables S6-S9).  \nThese findings indicate that SMN-ASO treatment selectively restores specific functional \npathways even within the subgroup of SMA+ASO showing the clearest proteomic shift \ntoward the WT/HET state, while key mitochondrial and stress-associated processes \nremain resistant to molecular correction. \n \nDiscussion \nPartial SMN restoration reshaped the proteomic landscape of SMA tissues in a tissue-\ndependent manner; however, this reorganization was selective rather than global. While \nsubsets of inflammatory, synaptic, and structural pathways showed directional \nnormalization, a substantial fraction of mitochondrial and metabolic alterations remained \nunresolved. These findings indicate that SMN-ASO treatment induces partial proteome \nreconfiguration rather than complete molecular restoration. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nThis incomplete reversibility was most evident in heart and spinal cord, where proteins \nassociated with oxidative phosphorylation and mitochondrial organization remained \npersistently dysregulated. Previous transcriptomic studies in SMA spinal cord have \nconsistently identified RNA-processing and splicing defects as primary consequences of \nSMN deficiency [54, 55], together with alterations in synaptic development and tissue \norganization [56, 57]. More recent multi-omics and single-cell analyses have expanded \nthis view to include impaired protein synthesis, metabolic dysfunction, and vascular-\nassociated signatures [58, 59]. Our data extend these observations to the protein level at \nthe symptomatic stage, revealing coordinated mitochondrial and ER/Golgi-associated \nmodules alongside axonogenesis- and glial differentiation-related networks. \nImportantly, metabolic and mitochondrial signatures identified in spinal cord were \nparalleled in heart and gastrocnemius, supporting a multi-organ dimension of SMA \npathology. Proteomic network analyses have previously implicated lipid metabolism and \nβ -oxidation pathways in SMA [60], and early transcriptomic studies have suggested \nbroader oxidoreductase and metabolic perturbations across tissues [61]. The present \natlas integrates these findings across central and peripheral compartments, highlighting \nconvergent metabolic vulnerability despite tissue-specific proteome remodeling. \nThe persistence of mitochondrial pathways following partial SMN restoration may \nindicate that secondary metabolic adaptations may not be fully reversible once \nestablished, particularly at symptomatic stages. This is consistent with biochemical \nobservations showing incomplete normalization of redox-regulatory systems despite \npartial reduction of oxidative damage [24], as well as with evidence from other tissues \nwhere mitochondrial regulatory programs remain uncoupled from SMN partial recovery \n[25]. However, this interpretation should also be considered in light of the treatment \nparadigm used here in which SMN-ASO was administered systemically at a suboptimal \ndose designed to achieve partial rather than complete restoration of SMN levels [24, 33]. \nThus, the incomplete proteomic rescue observed across tissues likely reflects both \nlimited molecular correction and tissue-specific differences in the capacity to recover \nfrom established SMN deficiency.  \nAn additional limitation of the rescue analysis is that downstream classification of \nrescued and persistent proteins was performed on the SMA+ASO samples showing the \nclearest proteomic shift toward the WT/HET state within each tissue. This stratified \napproach was chosen to assess treatment-associated molecular normalization in the \ncontext of heterogeneous responses within the SMA+ASO cohort, but it does not \ncapture the full spectrum of proteomic behavior across all treated SMA animals. \nAccordingly, the rescue patterns described here should be interpreted as reflecting a \nsubgroup with greater proteomic normalization rather than the entire SMA+ASO cohort. \nNevertheless, because the analysis was performed at the level of whole-organ unbiased \nproteomics, the coordinated repositioning of multiple proteins and pathways across \nindependent tissues supports the biological relevance of the observed rescue signatures \ndespite the limited number of SMA+ASO subgroup that shifted towards WT/HET \nproteome distribution.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nOverall, the heterogeneous degree of molecular rescue observed across tissues is \ncompatible with the clinical variability in response to SMN-ta rgeted therapies [19, 20, 62, \n63]. Differences in treatment timing, tissue-specific vulnerability, and the extent of pre-\nexisting remodeling are likely to influence the reversibility of molecular phenotypes [23]. \n \nConclusions \nThese data define a systemic yet heterogeneous proteomic response to SMN deficiency \nacross neuromuscular organs, with peripheral tissues exhibiting broader molecular \nalterations than spinal cord at the symptomatic stage. Although SMN-ASO treatment \npartially repositioned tissue proteomes, mitochondrial and metabolic pathways remained \nincompletely normalized. In the context of previous biochemical and transcriptomic \nstudies, these findings reinforce the view that mitochondrial and redox-associated \ndysfunction represent recurring components of SMA pathology and further support future \nstudies exploring combinatorial therapeutic strategies. \n \nList of Supplementary Materials \nSupplementary Figures S1-S6 \nSupplementary Table S1: Volcano matrices Spinal cord \nSupplementary Table S2: Volcano matrices Heart \nSupplementary Table S3: Volcano matrices Gastrocnemius \nSupplementary Table S4: WT vs SMA intersections for all organs \nSupplementary Table S5: HET vs SMA intersections for all organs \nSupplementary Table S6: ASO rescue summary and overlap per organ \nSupplementary Table S7: Rescue effect Spinal cord \nSupplementary Table S8: Rescue effect Heart \nSupplementary Table S9: Rescue effect Gastrocnemius \n \nData Availability \nRaw mass spectrometry data are available by the authors upon reasonable request. \nProcessed outputs are included in the Supplementary material accompanying this \npreprint. Detailed experimental protocols are available in our previous works [24-26]. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nAuthor contributions \nSV: involved in conceptualization, performed all experiments, performed the proteomics \nworkflow, performed statistical and bioinformatic analyses, wrote initial draft, generated \nthe figures. SM:  performed proteomics workflow, performed initial statistical analyses, \ninvolved in writing the methods, contributed to resources and funding. BW: involved in \nconceptualization, supervised the work, reviewed and edited the draft, contributed to \nfunding acquisition. \n \nAcknowledgements \nThe work has been funded by the European Union’s Horizon 2020 Marie Skłodowska-\nCurie Program (project 956185; SMABEYOND) and the Center for Molecular Medicine \nCologne (project C18) to BW and supported by the large instrument grant INST \n216/1163-1 FUGG by the German Research Foundation (DFG Großgeräteantrag), to \nSM. We thank IONIS Pharmaceuticals for providing the SMN-ASOs and Roman Rombo \nfor technical assistance in animal husbandry and treating the mice. The graphical \nabstract created using BioRender.com.  \n \nConflict of Interest \nThe authors declare no conflict of interest. \n \nReferences \n[1] Prior T , Leach M , Finanger E. Spinal Muscular Atrophy. 2000 Feb 24 [updated \n2024 Sep 19]. In: GeneReviews® [Internet]. Seattle (WA): University of Washington, \nSeattle. 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It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nFigure Legends \n \nFigure 1. Global proteome remodeling across neuromuscular tissues in SMA. (A-C) \nPrincipal component analysis (PCA) of label-free quantified proteomes from spinal cord (A), heart \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\n(B), and gastrocnemius muscle (C) at P10, showing genotype-dependent separation among WT, \nHET, and SMA samples. (D-F) Unsupervised hierarchical clustering of protein abundance across \nthe same tissues, demonstrating sample grouping primarily by genotype. (G-I) Volcano plots of \ndifferential protein abundance (WT vs SMA) generated in Perseus (FDR = 0.05, S0 = 0.1), \nhighlighting significantly downregulated (red) and upregulated (blue) proteins in spinal cord (G), \nheart (H), and gastrocnemius (I). (J-L) Distribution of log2 fold changes in protein abundance, \nillustrating the magnitude and direction of proteome-wide alterations, with broader shifts observed \nin heart and gastrocnemius relative to spinal cord. Complete statistical outputs are provided in \nSupplementary Tables S1–S5.  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nFigure 2. Functional enrichment and network organization of downregulated proteomes in \nWT versus SMA across tissues.  (A-C) One-dimensional enrichment analysis (Perseus) of \ncellular component categories (GOCC) for significantly downregulated proteins (WT vs SMA) in \nspinal cord (A), heart (B), and gastrocnemius muscle (C), highlighting subcellular compartments \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\naffected by SMN deficiency. (D-F) STRING network analysis of significantly downregulated \nproteins in spinal cord (D), heart (E), and gastrocnemius (F), visualized in Cytoscape with \nClueGO functional grouping. Networks reveal tissue-specific clustering of biological processes, \nincluding mitochondrial, endoplasmic reticulum and Golgi-associated pathways, axonogenesis \nand glial differentiation in spinal cord (D); DNA repair, intracellular trafficking, and clathrin-\nmediated endocytosis in heart (E); and phospholipid metabolism, mRNA processing, DNA repair, \nand neuromuscular junction-related pathways in gastrocnemius (F). \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\n \n \nFigure 3. Functional enrichment and network organization of downregulated proteomes in \nHET versus SMA across tissues.  (A-C) One-dimensional enrichment analysis (Perseus) of \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\ncellular component categories (GOCC) for significantly downregulated proteins (HET vs SMA) in \nspinal cord (A), heart (B), and gastrocnemius muscle (C), identifying subcellular compartments \nassociated with SMN dosage-dependent proteome alterations. (D-F) STRING network analysis of \nsignificantly downregulated proteins in spinal cord (D), heart (E), and gastrocnemius (F), \nvisualized in Cytoscape with ClueGO functional grouping. Networks reveal tissue-specific \nclustering of biological processes, including mitochondrial and membrane-associated \ncomponents in spinal cord (D); spliceosomal, ribonucleoprotein, and vesicle transport pathways in \nheart (E); and phospholipid metabolism, mitochondrial organization, and RNA-related processes \nin gastrocnemius (F). \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\n \nFigure 4. Organ-specific proteome repositioning and protein response classification \nfollowing SMN-ASO treatment.  (A-C) Principal component analysis (PCA) of spinal cord (A), \nheart (B), and gastrocnemius muscle (C) proteomes including WT, HET, SMA, and SMA+ASO \ngroups, showing organ-dependent repositioning of ASO-treated samples relative to untreated \nSMA. (D-F) Unsupervised hierarchical clustering of protein abundance across the same tissues, \nillustrating tissue-specific patterns of partial proteome normalization and persistence following \nSMN partial restoration. (G) Classification of SMA-associated proteins based on directional \nresponse to SMN-ASO treatment, shown as proportions of rescued (shifted toward WT), \npersistent (unchanged), and no change in ASO across tissues. Detailed target distribution per \ncategory and organ is provided in Supplementary Table S6. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\n \nFigure 5. Functional characterization of rescued and persistent proteomic responses \nfollowing SMN-ASO treatment.  (A) Directional response classification plots showing protein \nabundance changes across spinal cord, heart, and gastrocnemius. Proteins are categorized \nbased on combined WT versus SMA and SMA versus SMA+ASO comparisons into rescued (shift \ntoward WT), persistent (remaining altered without directional reversal), and no change in ASO. \n(B) STRING network analysis of proteins rescued from SMA-associated upregulation following \nSMN-ASO treatment in spinal cord, heart, and gastrocnemius, visualized in Cytoscape with \nClueGO functional grouping. Enriched pathways include immune-related processes in spinal \ncord, vesicle-mediated transport and cytoskeletal organization in heart, and synaptic, \nneuromuscular junction, and metabolic pathways in gastrocnemius. (C) STRING network analysis \nof proteins persistently downregulated in both SMA and SMA+ASO conditions, highlighting \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint \n\nresistant pathways across tissues. Networks reveal enrichment of mitochondrial and metabolic \nprocesses, including inner mitochondrial membrane organization in spinal cord, vesicle-\nassociated and redox-related pathways in heart, and cellular metabolic processes in \ngastrocnemius. Full list of rescued, persistent, no change in ASO and ASO-responsive-only \ntargets per organ is provided in Supplementary Tables S7-S9. \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.30.715402doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}