Zebrafish genetic model of neuromuscular degeneration associated with Atrogin-1 expression

Abstract

The degenerative loss of muscle associated with aging leading to muscular atrophy is called sarcopenia. Currently, practicing regular physical exercise is the only efficient way to delay sarcopenia onset. Identification of therapeutic targets to alleviate the symptoms of aging requires in vivo model organisms of accelerated muscle degeneration and atrophy. The zebrafish undergoes aging, with hallmarks including mitochondrial dysfunction, telomere shortening, and accumulation of senescent cells. However, zebrafish age slowly, and no specific zebrafish models of accelerated muscle atrophy associated with molecular events of aging are currently available. We have developed a new genetic tool to efficiently accelerate muscle-fiber degeneration and muscle-tissue atrophy in zebrafish larvae and adults. We used a gain-of-function strategy with a molecule that has been shown to be necessary and sufficient to induce muscle atrophy and a sarcopenia phenotype in mammals: Atrogin-1 (also named Fbxo32). We report the generation, validation, and characterization of a zebrafish genetic model of accelerated neuromuscular atrophy, the atrofish. We demonstrated that Atrogin-1 expression specifically in skeletal muscle tissue induces a muscle atrophic phenotype associated with locomotion dysfunction in both larvae and adult fish. We identified degradation of the myosin light chain as an event occurring prior to muscle-fiber degeneration. Biological processes associated with muscle aging such as proteolysis, inflammation, stress response, extracellular matrix (ECM) remodeling, and apoptosis are upregulated in the atrofish. Surprisingly, we observed a strong correlation between muscle-fiber degeneration and reduced numbers of neuromuscular junctions in the peripheral nervous system, as well as neuronal cell bodies in the spinal cord, suggesting that muscle atrophy could underly a neurodegenerative phenotype in the central nervous system. Finally, while atrofish larvae can recover locomotive functions, adult atrofish have impaired regenerative capacities, as is observed in mammals during muscle aging. In the future, the atrofish could serve as a platform for testing molecules aimed at treating or alleviating the symptoms of muscle aging, thereby opening new therapeutic avenues in the fight against sarcopenia.

Fig 1. Muscle-specific Atrogin-1 expression leads to muscle atrophy and affects locomotor function in zebrafish larvae.

(A) Schematic representation of the method used to express Atrogin-1 in muscle fibers using Tg(503unc:creERT2) and Tg(ubi:Lox-stop-Lox-atrogin1) transgenic lines (atrofish larvae). Efficient recombination of the loxP sites following 4-OHT treatment leads to Atrogin-1-overexpression-dependent muscle atrophy and degeneration, visualized by birefringence analysis using polarized light. Statistical significance is determined Mann-Whitney test. (B) Phalloidin incorporation in control and atrofish larvae after 72 hours of 4-OHT treatment reveals degenerative phenotypes in trunk skeletal-muscle tissue. Asterisk shows a site of muscle-fiber degeneration. (C) Quantification of muscle thickness in control (DMSO; n = 43) and atrofish (4-OHT; n = 51) larvae after 24 hours of 4-OHT treatment. Statistical significance is determined by t-test, two-tailed, unpaired. (D) Quantification of locomotor activity in control (DMSO; n = 12) and atrofish (4-OHT; n = 12) larvae for one hour after 24 hours of 4-OHT treatment. Statistical significance is determined by a Mann-Whitney test, two-tailed, unpaired. (E) Graphical representation of bulk RNAseq analysis and variation in gene-expression profiles in control (DMSO-treated) (n = 4; 50 larvae/sample) vs. atrofish (n = 4; 50 larvae/sample) after 24 hours of 4-OHT treatment. (F) Quantification of cortisol expression levels in control larvae (4-OHT-treated; n = 8 with 30 larvae/sample) and 4-OHT-treated atrofish larvae (n = 8 with 30 larvae/sample) larvae. Statistical significance is determined by t-test, two-tailed, unpaired. (G) Graphical representation of RNAseq analysis and variation in the cell populations based on gene-expression profiles in control vs. cortisol-treated. To map the effects of cortisol exposure to cell and tissue type, marker genes and cell types were identified within the clusters by matching the annotations from the full embryo atlas to the 5 dpf single-cell data (see method for details). Each differentially expressed gene is represented as a dot within the cluster. Error bars represent s.d. Scale bars: 100 μm.

Fig 2. Muscle-specific Atrogin-1 expression leads to myosin light-chain degradation and neuromuscular degeneration in atrofish larvae.

(A) Apotome sections of DMSO-treated atrofish larvae (left) or atrofish larvae treated with 4-OHT for 24 hours (right) at 6 dpf showing phalloidin incorporation (green) and F310 immunolabelling (purple) in trunk skeletal muscles. White arrow shows phalloidin incorporation in the absence of F310 expression. (B) Confocal sections of atrofish larvae treated with 4-OHT for 24 (top) or 48 (bottom) hours at 6 dpf showing GFP expression from Tg(murf1a:gfp) in muscle fibers (green) and F310 immunolabelling (purple) in trunk skeletal muscles. White arrow shows GFP expression in the absence of F310 expression. (C) Confocal sections of DMSO-treated atrofish larvae (left) or atrofish larvae treated with 4-OHT for 24 hours (right) at 6 dpf showing phalloidin incorporation (green) and znp-1 (purple) immunolabelling in trunk skeletal muscles. (D) Apotome sections of DMSO-treated atrofish larvae (left) or atrofish larvae treated with 4-OHT for 24 hours (right) at 6 dpf showing motoneuron degeneration using the mnx1:mCherry transgenic line. Asterisk indicates the site of degeneration in the spinal cord. Asterisks show sites of muscle-fiber degeneration. Scale bars: 100 μm.

Fig 3. Muscle-specific Atrogin-1 expression affects motoneurons in the central and peripheral nervous system.

(A) Confocal projections (top) and 3D reconstructions (bottom) of control or atrofish larvae treated with 4-OHT for 48 hours at 6 dpf. (A’, A”) Analysis of the motoneuron system reveals that motoneurons, axon branching, and neuromuscular connectivity and density (NMJ) are affected in atrofish (DMSO; n = 8 and 4-OHT; n = 7) but not in WT larvae treated with 4-OHT (DMSO; n = 20 and 4-OHT; n = 20). Each biological component was isolated and assigned a unique color label: white (spinal cord), red (axons), magenta (NMJs) and yellow (immune cells mpeg1.2 + macrophages in Fig 3B). Statistical significance was determined by t-test, two-tailed, unpaired with Welch’s correction. (B) Confocal sections of DMSO-treated atrofish larvae (top) or atrofish larvae treated with 4-OHT (bottom) for 48 hours at 6 dpf showing macrophages in close vicinity of the motoneuron axons and NMJ (mnx1:mCherry) in the degenerating muscle tissue (murf1a:GFP). Arrows show mpeg1 + macrophages expressing both GFP and mCherry (DMSO; n = 7 and 4-OHT; n = 7). Statistical significance is determined by t-test, two-tailed, unpaired with Welch’s correction. Error bars represent s.d. Scale bars: 100 μm.

Fig 4. Atrogin-1-dependent muscle dysfunction is reversible in atrofish larvae.

(A, B) Quantification of locomotion activity in zebrafish larvae. (A) WT larvae treated with 4-OHT (n = 8) and atrofish larvae treated with DMSO (n = 8) or 4-OHT (n = 8). Larvae were treated with DMSO or 4-OHT for 24 hours between 5 and 6 dpf before the locomotion test, and for 48 hours during the test. The figure shows only the 48-hour test results. (B) Atrofish larvae treated with DMSO or 4-OHT. Larvae were treated with DMSO (n = 12) or 4-OHT (n = 11) for 24 hours between 5 and 6 dpf before the locomotion test, and then placed in chemical-free medium for 48 hours during the locomotion test. The figure shows only the 48-hour test results. Statistical significance is determined by multiple t-test comparison with False Discovery Rate and individual variance for each time point, two-tailed, unpaired. (C, D) Quantification of heart volume after 3D reconstruction in control fish (n = 5) or atrofish (n = 5) (C) or heartbeat using the cmlc2:gfp transgene expressed in cardiomyocytes (D) at 6 dpf in control fish (WT fish) (n = 8) or atrofish (n = 8) treated with 4-OHT for 24 hours between 5 and 6 dpf. Statistical significance is determined by multiple t-test, two-tailed, unpaired. (E) Graphical representation of bulk RNAseq analysis and variation in gene-expression profiles in DMSO-treated control larvae (n = 4; 50 larvae/sample) vs. atrofish (n = 4; 50 larvae/sample) after 24 hours of 4-OHT treatment between 5 and 6 dpf, followed by 2 days of recovery until 8 dpf. (F) Quantification of cortisol expression levels in control larvae (4-OHT-treated; n = 3 with 30 larvae/sample n = 30) and 4-OHT-treated atrofish larvae (n = 3 with 30 larvae/sample) after recovery. Statistical significance is determined by t-test, two-tailed, unpaired. Error bars represent s.d. Scale bars: 50 μm.

Fig 5. Atrogin-1-dependent muscle dysfunctions are irreversible in chronically treated adult atrofish.

(A) Brightfield imaging of adult zebrafish trunk after tissue-clearing showing degeneration of skeletal-muscle tissue in one-month-old atrofish treated with tamoxifen (TAM) for 5 months (bottom) compared to TAM-treated control fish (control; top) at 6 months of age. Arrows indicate regions of degeneration. (B) Quantification of the volume of muscle tissue in TAM-treated control fish (n = 6) or atrofish treated with either DMSO (n = 3) or TAM (n = 10). Statistical significance is determined by t-test, two-tailed, unpaired. (C) Representative images of 3D reconstructed trunk skeletal-muscle tissue after chronic TAM treatment in control fish (top) or atrofish (bottom) at 6 months of age. (D) Quantification of the volume expected after 3D reconstruction of atrofish trunk skeletal-muscle tissue (n = 10). Statistical significance is determined by t-test, two-tailed, paired. (E) Representative images of 3D reconstructed trunk skeletal-muscle tissue after chronic TAM treatment in atrofish at 6 months of age. (F, G) Quantification of the number of focal injuries in TAM-treated control fish (n = 6) and TAM-treated atrofish (n = 6) at 6 months of age after 3D construction (F). Quantification of the average volume of injuries in TAM-treated control fish (n = 6) and TAM-treated atrofish (n = 6) at 6 months of age after 3D construction (G). Statistical significance is determined by t-test, two-tailed, unpaired with Welch’s correction. (H) Quantification of the distribution of the injuries identified in (G) in TAM-treated control fish (n = 6) and TAM-treated atrofish (n = 6) at 6 months of age after 3D construction. n in the graphic indicates the number of injuries per biological replicate. Injuries from 6 biological replicates for control or atrofish have been pulled together to determine statistical significance using t-test, two-tailed, unpaired. (I) Representative images of the numbers and sizes of injuries in trunk skeletal-muscle tissue after chronic TAM treatment in control (top) or atrofish (bottom) at 6 months of age. Each color represents a single injury, quantified in (H). (J) Quantification of swimming capacity of control fish and atrofish at 6 months of age treated with either DMSO or 4-OHT for 5 months (n = 19 for each condition). Statistical significance is determined by Mann-Whitney test, two-tailed, unpaired. (K) Quantification of swimming capacity of control fish (n = 9) and atrofish (n = 15) at 9 months of age, treated with TAM for 5 months, i.e., between 1 and 6 months of age, and then treated with either DMSO (ctrl, n = 5; atro, n = 4) or TAM (ctrl, n = 4; atro, n = 11) for an additional 3 months (i.e., until 9 months of age), to test for recovery of swimming function. Statistical significance is determined by a Two-way Anova. Error bars represent s.d. Scale bars: 1 mm.

Fig 6. Graphical representation of the biological processes underlying the neuromuscular degeneration phenotype associated with Atrogin-1 expression.

Created in BioRender. Menard, R. (2025) - https://BioRender.com/1kpf9fg.