Muscle-derived miR-126 Regulates Axonal Local Synthesis of TDP-43 to Maintain Neuromuscular Junction Integrity in ALS Models — Review of a Nature Neuroscience Article

I. Academic Background and Research Motivation

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disease, primarily characterized by neuromuscular junction (NMJ) dysfunction, axonal degeneration, and motor neuron death. The majority of ALS cases are closely linked to pathological changes of TDP-43 (TAR DNA-binding Protein 43, a multifunctional DNA/RNA-binding protein), whose pathogenic mechanisms include its mislocalization from the nucleus to the cytoplasm and the formation of hyperphosphorylated aggregates, as well as interference with RNA splicing, transport, and local translation regulation. Notably, in ALS, TDP-43 frequently aberrantly aggregates in peripheral axons and NMJs, but the induction mechanisms underlying this aggregation remain incompletely understood.

Recent studies suggest that local protein synthesis is particularly important for the function and survival of long axons in neurons, and NMJ integrity also depends on spatially and temporally restricted protein synthesis processes at axon terminals. The traditional view recognizes ALS as an axonopathy in its early stages, and the aggregation of TDP-43 in peripheral nerves may impact NMJ protein renewal and energy metabolism, thereby accelerating motor neuron degeneration. At the same time, muscles do not merely passively receive neural input but also actively participate in NMJ maintenance and regulation through various molecular signals, especially miRNAs contained in exosomes.

The present study aims to elucidate the molecular mechanisms underlying the peripheral axonal and local NMJ aggregation of TDP-43 in ALS, with a particular focus on how muscle-secreted miRNA—miR-126a-5p—regulates local TDP-43 synthesis across cells via the exosomal pathway, and to explore the influence of this axon–muscle cross-cell communication on ALS onset and progression.

II. Paper Source and Author Information

This is an original research article published in the high-impact journal Nature Neuroscience (volume 28, November 2025, pp. 2201–2216), DOI: https://doi.org/10.1038/s41593-025-02062-6. The main authors include Ariel Ionescu, Lior Ankol, Anand Ganapathy Subramaniam, Topaz Altman, Iddo Magen, Yahel Cohen, Yehuda Danino, among others, affiliated with institutions such as Tel Aviv University and Sourasky Medical Center in Israel.

III. Detailed Research Workflow

1. Study Design and Subjects

This study integrated clinical ALS patient samples (including SOD1 gene mutants, sporadic ALS patients, and non-ALS controls), various ALS animal models (notably SOD1^G93A and SOD1^G37R mice), primary cell co-culture systems (including human induced pluripotent stem cell (iPSC)-derived neurons and muscle cells), and in vitro microfluidic chip systems. The experimental procedures encompassed histological analyses, molecular biology, exosome isolation and identification, single-molecule fluorescence in situ hybridization (smFISH), detection of local protein synthesis (puromycin-PLA and O-propargyl puromycin), gene knockdown, miRNA functional validation, lentiviral overexpression, in vivo AAV injection, behavioral assessments, and multi-omics data analysis.

2. Main Experimental Steps and Technical Route

(1) Detection of TDP-43 Aggregation at Peripheral Nerves and NMJs in ALS Patients and Mice

• Clinical Samples: Immunohistochemical staining was used to examine the distribution of phosphorylated TDP-43 (pTDP-43) aggregates in the sural nerve and intramuscular nerves of ALS patients (SOD1 mutants and sporadic ALS), compared to a non-ALS control group.
• Animal Models: In SOD1^G93A and SOD1^G37R mice, peripheral nerve and muscle tissue sections were analyzed using immunofluorescence to quantify pTDP-43 aggregation.
• NMJ Aggregation Analysis: 3D co-localization analysis of TDP-43 and NMJ markers (NFH, Synaptophysin, Bungarotoxin) revealed TDP-43 aggregation at the axon–NMJ interface in different muscle types (fast/slow fatigue muscles) and disease stages.

(2) Localization and Local Translation of TDP-43 mRNA in Axons and NMJs

• Microfluidic Primary Motor Neuron Culture System: Axonal and somatic RNAs were isolated, and qPCR was used to assess TDP-43 and control mRNA expression.
• Single-molecule FISH (smFISH): Visualized the localization of TDP-43 and β-actin mRNAs in neuronal axons and muscle NMJs.
• Puromycin-PLA: Detected locally synthesized TDP-43 protein in primary neurons and co-culture systems, validating the regulatory role of muscle on axonal local protein synthesis.

(3) Analysis of Muscle Extracellular Vesicles (EVs) and miRNA Delivery

• Immunodetection of EV Markers: Located classical EV markers CD63, CHMP2A, CD81 in muscle and NMJ regions.
• EV Functional Detection: Used real-time live imaging with CD63-phluorin and tracking with CD63-GFP to dynamically trace exosome transfer from muscle to axon across cells.
• Ultracentrifugation of Muscle Culture Medium to Isolate EVs, Nanoparticle Tracking Analysis (NTA), and TEM Identification of EV Characteristics.
• RNA-Seq and Proteomics Analysis: Studied the miRNAs and AGO2 protein encapsulated in muscle EVs, revealing that they carry RNA interference complexes (RISC) affecting the axonal transcriptome and protein synthesis.
• Enrichment Analysis of Muscle-specific miRNAs (myomiRs): Employed small RNA sequencing and FISH to isolate EV-enriched miRNAs (notably miR-126a-5p) and analyzed their expression distribution at the NMJ region.

(4) Mechanistic Validation of miR-126a-5p Regulation of TDP-43 mRNA

• Target Gene Prediction and Validation: Used tools such as TargetScan to predict miR-126a-5p targets among ALS-related genes, in which TDP-43 (TARDBP) contains binding sites in multiple isoforms.
• Dual Luciferase Reporter Assay: Constructed reporters containing wild-type and mutant TDP-43 3’UTRs to validate the direct binding of miR-126a-5p and its mediation of translational repression.
• Lentiviral Overexpression and Functional Detection in Primary Motor Neurons: Employed lentiviral vectors for miR-126 overexpression, quantitatively analyzed changes in TDP-43 transcript and protein expression, and verified the conservation of this regulatory effect in both human and mouse models.

(5) Expression Changes of miR-126a-5p in ALS Patients and Animal Models

• Quantification of miR-126-5p in Serum Exosomes of Human ALS Patients: Compared with healthy controls, confirmed significantly reduced exosomal miR-126-5p in ALS patients.
• Analysis of miR-126a-5p Expression in Muscle and Spinal Cord of ALS Model Animals: Observed that SOD1^G93A and SOD1^G37R mice show downregulation of miR-126a-5p in muscle (especially NMJ area), while spinal cord expression remains unchanged.

(6) Interventions on Exosome/miR-126a-5p Secretion to Validate NMJ Protective Function

• Conditional Rab27a Knockdown and Drug Inhibition: In microfluidic chip systems, used Tet-on shRNA against Rab27a or the GW4869 drug to conditionally knockdown/inhibit exosome secretion in primary muscle, assessing whether this leads to increased local axonal TDP-43 synthesis, declining NMJ protein synthesis, and structural and functional impairment.
• miR-126a-5p-specific Antisense Oligonucleotide (mir126i) Treatment: In co-culture systems, targeted inhibition of muscle miR-126a-5p prompted accumulation of abnormal axonal TDP-43 at NMJs and induced degeneration.
• Double Knockdown Experiment: Simultaneous application of miR-126i and TDP-43 siRNA revealed that inhibiting TDP-43 translation could reverse axonal degeneration induced by miR-126a-5p deficiency, confirming that miR-126a-5p mainly protects NMJs by regulating TDP-43.

(7) miR-126 Overexpression Improves ALS Model Phenotype

• In Vivo AAV-mediated miR-126 Overexpression: SOD1^G93A mice received PHP.EB AAV encoding miR-126-GFP into the central nervous system and one gastrocnemius muscle; behavioral assessments (e.g., Catwalk gait analysis, four-limb support) showed significant improvement during disease progression, while NMJ axonal TDP-43 aggregation decreased, though overall survival was not improved (as miR-126 levels were not fully restored).
• Human iPSC-ALS Model Co-culture System: In co-culture systems of human iPSC-derived SOD1^A5V and TDP-43^M337V mutant neuron–muscle pairs, lentiviral miR-126 expression likewise significantly reduced axonal pTDP-43 aggregation and protected against neurodegeneration.

IV. Detailed Research Results

TDP-43 Aggregation in Peripheral Axons and NMJs

The authors systematically demonstrated that TDP-43 aggregation occurs not only in the nuclei of spinal cord neurons in ALS patients and animal models but also as phosphorylated aggregates in peripheral nerves and NMJs, notably in SOD1 mutant cases (previously thought not to feature TDP-43 pathology). This aggregation is most pronounced at late stages of motor dysfunction and is strictly correlated with decreased local axonal protein synthesis capacity. There are differences in the susceptibility to aggregation among mouse strains and muscle types (e.g., more apparent in fast-fatigue muscles such as EDL).

Localization and Synthesis of TDP-43 mRNA in Axon and NMJ Regions

Through microfluidic separation and molecular detection, the authors confirmed that TDP-43 mRNA is not only localized in the soma but also abundantly and stably distributed in axonal terminals and NMJs. Puromycin-PLA detection verified that TDP-43 is actively locally translated at axon terminals, and that this local synthesis is significantly suppressed in primary muscle co-culture systems, indicating that muscle might regulate it via secreted factors such as miRNAs.

Muscle Exosome-mediated miRNA (Especially miR-126a-5p) Cross-cellular Regulation Mechanism

Muscle exosomes are highly enriched at NMJ regions, containing various miRNAs and RISC complex member AGO2, which can be taken up by motor axon terminals and act as hubs for intercellular material transfer and post-transcriptional regulation. A series of experiments revealed that miR-126a-5p is significantly enriched in EVs and localized at NMJs, rather than in the muscle cell body or other tissues, suggesting it is dedicated to intercellular regulation.

Direct Inhibitory Role of miR-126a-5p in Local TDP-43 Synthesis

Through genetic engineering and dual luciferase reporter assays, the authors clearly showed that miR-126a-5p directly binds to the 3’UTR site of the main axonal-specific transcript of TDP-43, suppressing translation and promoting mRNA degradation. This mechanism was confirmed in both mouse and human models, indicating its strong cross-species conservation.

Under ALS Conditions, miR-126a-5p Is Abnormally Downregulated, Promoting TDP-43 Aggregation and Neurodegeneration

Regardless of whether serum from ALS patients, muscle of ALS animal models, or the NMJ microenvironment was analyzed, miR-126a-5p was significantly downregulated, directly associated with increased local TDP-43 aggregation and decreased protein synthesis capacity in axons. Further interference experiments (knockdown of EV secretion, inhibition of miR-126a-5p) all led to NMJ dysfunction and structural degeneration, and only inhibition of TDP-43 synthesis could prevent these phenotypes.

miR-126a-5p Overexpression Mitigates ALS Model Phenotype

By AAV or lentiviral-mediated overexpression of miR-126a-5p, whether in mouse or human ALS models, axonal pTDP-43 aggregation was significantly reduced, NMJ protein synthesis capacity and structural function were restored, and motor function decline was delayed, although overall survival rates were not significantly improved.

V. Research Conclusions and Significance

This study innovatively reveals a new mechanism for cross-cellular regulation of protein synthesis at the NMJ in ALS—muscle-derived exosomal miR-126a-5p directly suppresses local axonal synthesis of TDP-43, maintaining protein synthesis at the neuronal terminals and the integrity of the NMJ. miR-126a-5p is highly enriched at the NMJ and is significantly downregulated in ALS, lifting its suppression on TDP-43, resulting in local abnormal accumulation, subsequent disruption of axonal protein synthesis, mitochondrial dysfunction, and NMJ degeneration. The elucidation of this mechanism not only provides a core molecular basis for understanding ALS pathogenesis and early pathology but also offers a theoretical framework and technical roadmap for future therapies targeting local axonal protein synthesis regulation (such as miRNA delivery or exosome engineering).

VI. Research Highlights and Innovations

• Mechanistic Innovation: For the first time, systematically clarifies that muscle-delivered exosomal miR-126a-5p regulates local axonal protein synthesis via a cross-cellular pathway, and this pathway undergoes systemic impairment in ALS.
• Methodological Advancement: Ingeniously combines microfluidic chips, single-molecule fluorescence in situ hybridization, in situ protein synthesis detection, exosome tracking, and behavioral evaluation, providing a new paradigm for studying microenvironmental signaling at the NMJ.
• Clinical Relevance: Confirms that serum exosomal miR-126a-5p is significantly downregulated in human ALS patients, offering promise as an early biomarker and guiding novel therapeutic exploration based on exosomal miRNA.
• Cross-species Validation: Highly consistent results in mouse and human iPSC models, greatly enhancing the translational potential of the findings.

VII. Research Outlook and Limitations

The authors candidly acknowledge the current limitations in ALS case and sample numbers, necessitating larger cohorts to validate the clinical significance of miR-126a-5p as a universal biomarker and intervention target. In addition, whether miR-126a-5p plays a special role in non-neuronal cells (e.g., Schwann cells) near the NMJ, the bottlenecks of targeted exosome delivery and long-term expression, are all issues worthy of further in-depth study.

VIII. Overall Evaluation and Significance

This research approaches early ALS pathology from a multidisciplinary perspective, bridging the “muscle–axon–NMJ” signaling cross-talk pathway, providing a solid foundation for understanding ALS molecular pathogenesis and precise intervention strategies. The decisive role of muscle exosome-enriched miRNAs in NMJ homeostasis paves new ways for early detection and personalized therapy of neurodegenerative diseases. Meanwhile, this study suggests that local protein synthesis regulation is not solely dependent on intrinsic neuronal mechanisms, but is greatly influenced by dynamic external tissues (such as muscle), broadening the systemic perspective on the pathogenesis of motor neuron diseases.