Dataset Information

Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS.

ABSTRACT: Spinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA sequencing (RNA-seq), and bioinformatic quality control. We show that the axonal transcriptome is distinct from that of somas and contains fewer genes. We identified 3,500-5,000 transcripts in mouse and human stem cell-derived spinal motor axons, most of which are required for oxidative energy production and ribogenesis. Axons contained transcription factor mRNAs, e.g., Ybx1, with implications for local functions. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the SOD1^G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal function, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq provides an improved method for RNA-seq of axons, increasing our understanding of peripheral axon biology and identifying therapeutic targets in motor neuron disease.

SUBMITTER: Nijssen J

PROVIDER: S-EPMC6294264 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS.

Nijssen Jik J Aguila Julio J Hoogstraaten Rein R Kee Nigel N Hedlund Eva E

Stem cell reports 20181201 6

Spinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA sequencing (RNA-seq), and bioinformatic quality control. We show that the axonal transcriptome is distinct from that of somas and contains fewer genes. We identified 3,500-5,000 transcripts in mouse and human stem cell-derived spinal motor axo ...[more]

PMID: 30540963

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Project description:BackgroundThe characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated.MethodTwo sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference (p.H517D) in the fused in sarcoma (FUS) were constructed. By combining MN reporter lentivirus, MN specific phenotype was analyzed. Moreover, RNA profiling of isolated axons were conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo. The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology.FindingsWe identified aberrant increasing of axon branchings in FUS-mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. We identified increased level of Fos-B mRNA, the binding target of FUS, in FUS-mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo. The commonality of those phenotypes was further confirmed with other ALS causative mutation than FUS.InterpretationAnalyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS-mutant axon branching. FUND: Japan Agency for Medical Research and development; Japanese Ministry of Education, Culture, Sports, Science and Technology Clinical Research, Innovation and Education Center, Tohoku University Hospital; Japan Intractable Diseases (Nanbyo) Research Foundation; the Kanae Foundation for the Promotion of Medical Science; and "Inochi-no-Iro" ALS research grant.

Dataset Information

Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS.

Publications

Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS.

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