Muscle Exosome-Derived miR-126 Regulates Local Synthesis of TDP-43 and NMJ Integrity in ALS

Amyotrophic lateral sclerosis (ALS) is characterized by neuromuscular junction (NMJ) disruption and neurodegeneration. Recent findings highlight a pivotal role for TDP-43 in forming axonal pathological condensates and facilitating NMJ disruption through inhibition of local protein synthesis. However, the mechanisms which drive local TDP-43 accumulation remain unknown. Here we identify TDP-43 axonal accumulation in peripheral nerves of SOD1 patients and mice stemming from its aberrant local synthesis. This is a non-cell-autonomous process driven by muscle-derived miR-126a-5p exosomes. Inhibiting muscle secretion of miR-126a-5p prompts pre-synaptic TDP-43 synthesis and accumulation that disrupts axonal translation and causes NMJ degeneration. Introducing miR-126 to SOD1G93A mice, primary co-cultures, and human iPSC-derived co-cultures with ALS mutations exhibits neuroprotective effects and delays motor decline. These findings identify a transcellular communication axis between muscles and motor neurons that regulates axonal local synthesis and NMJ maintenance offering insights into ALS onset and progression. Small RNA Next Generation Sequencing For small RNA next generation sequencing (RNA-seq), libraries were prepared from 10-15 ng of total RNA using the QIAseq miRNA Library Kit and QIAseq miRNA NGS 48 Index IL (Qiagen), by an experimenter who was blinded to the identity of samples. Samples were randomly allocated to library preparation and sequencing in batches. Precise linear quantification of miRNA is achieved by using unique molecular identifiers (UMIs), of random 12-nucleotide after 3’ and 5’ adapter ligation, within the reverse transcription primers. cDNA libraries were amplified by PCR for 19 cycles, with a 3’ primer that includes a 6-nucleotide unique index, followed by on-bead size selection and cleaning. Library concentration was determined with Qubit fluorometer (dsDNA high sensitivity assay kit; ThermoFisher Scientific, Waltham, MA) and library size with Tapestation D1000 (Agilent). Libraries with different indices were multiplexed and sequenced on NextSeq 500/550 v2 flow cell or Novaseq SP100 (Illumina), with 75bp single read and 6bp index read. Fastq files were de-multiplexed using the user-friendly transcriptome analysis pipeline (UTAP). Mouse miRNAs, as defined by miRBase, were mapped using the GeneGlobe pipeline (https://geneglobe.qiagen.com/us/analyze). We defined "true positive" miRNAs and reduced the likelihood of considering “false positive” miRNAs by including only miRNAs with an average UMI counts > 100 across all samples and with at least a single UMI across all samples, similar to our previous works. Data were further normalized, and average read counts compared between groups using the DESeq2 package under the assumption that miRNA counts followed negative binomial distribution. Significance was determined using the Wald test. Axonal RNA sequencing RNA was extracted from primary MNs axons grown in radial microfluidic chambers and purified as described above. RNA yields were quantified using Qubit fluorometer (RNA broad range assay kit; ThermoFisher Scientific). RNA integrity was assessed by TapeStation. cDNA libraries were prepared from 10ng of axonal RNA by the genomic unit in Weizmann institute using MARS-seq pipeline. MARS-seq libraries with different UMIs were multiplexed and sequenced on Novaseq SP100 flow cell (Illumina), with 75bp single read and 15bp UMI read. After demultiplexing, fastq files were mapped to mouse genome using the GenCode annotations (vM25, 2020) in the UTAP piepeline. We defined "true positive" genes and reduced the likelihood of considering “false positive” genes by including only genes with an average UMI counts > 15 across all samples. Data was further normalized, and average read counts compared between groups using the DESeq2 package under the assumption that gene counts followed negative binomial distribution. Significance was determined using the Wald test.