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Aberrant Axon Branching via Fos-B Dysregulation in FUS-ALS Motor Neurons

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Aberrant Axon Branching via Fos-B Dysregulation in FUS-ALS Motor Neurons

Tetsuya Akiyama et al. EBioMedicine.

Abstract

Background: The 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.

Method: Two 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.

Findings: We 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.

Interpretation: Analyzing 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.

Keywords: Amyotrophic lateral sclerosis (ALS); Axon branching; Fos-B; Fused in sarcoma (FUS); Human-induced pluripotent stem cell (hiPSC)-derived motor neuron; Nerve organoid.

Conflict of interest statement

H.Ok. is a Scientific Advisor at SanBio Co.Ltd. and K Pharma Inc. Y.O. is a Scientific Advisor at Kohjin Bio Co., Ltd. Other authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
The FUS mutation significantly increased axon branching. (a) Details of hiPSCs derived from healthy donor (Control), a patient with familial ALS (FUS-ALS), and isogenic hiPSCs. FUSH517D/H517D and FUSRescued were established by modifying FUS gene using TALEN genome editing technology from Control and FUS-ALS each other. Therefore, we use two sets of isogenic pairs in the present study. See Supplementary Fig. 1 and Materials and methods (section 1.1. and 1.3.) for more detailed information. (b) The experimental schema for observing MN axon ends. HB9 reporter lentivirus-infected 2nd MPCs were plated onto the center of PO/M-gel coated culture dishes. The enlarged image of the white square in the upper figure presents an example of the axon end that is far from SDs (by at least 1 mm) for counting. Bars: 200 μm. (c) Representative ICC images at 40 DPP (refer to Supplementary Fig. 2). At 40 DPP, nuclear staining was detected only at the center of the well. Bar: 1 mm. (d) Representative images of the axon ends of each cell line at 10 DPP. Bar: 50 μm. (e–g) Quantification of axon branching [see Supplementary Fig. 3 and Materials and methods (section 1.12) for the counting and analysis methods].
Fig. 2
Fig. 2
MN axons were extracted with a microfluidic device. (a) The experimental scheme of MN culture using the microfluidic device. The 2nd MPCs with (for ICC) or without (for RNA-seq) HB9 reporter lentivirus infection were plated onto the device. The axons elongated in the microfluidics to the next well. After 20 DPP, the axons were divided from the SDs for RNA extraction. (b) Representative images of axon dividing. Axons were divided from the SDs by cutting the axon bundle at 450 μm away from the sphere to avoid contaminating the cell body and pushing out due to hydraulic pressure. (c) Representative ICC images of MNs on the microfluidic device. The lower four figures represent the enlarged images of white squares of the upper figures. Bars: 1 mm. (d,e) The RNA profiles of the SDs and axonal fractions were compared to check the fractional population. FPKM values were normalized with GAPDH FPKM values of each data set. The presented gene sets were extracted from the reference [19]. The 7SK in the present study indicated the mean FPKM of 7SK genes includes CCNT1, CCNT2, CDK9, DDX21, HEXIM1, and HEXIM2. Student's t-test was used for analysis. See also Supplementary Fig. 4, and Supplementary Table 1,2. (f) Inventory of RNA profile indicated in (d) and (e). Signs of inequality represent the statistically different genes with regard to SDs and axons. Gene profiles that matched with those previously reported are shown with orange highlights. Only ACTN4 exhibited a different profile between that previously reported and that reported in the present study. The other genes are indicated with gray highlights. The RNA profiles matched 84% in the gene sets. (g) Box plot of the SDs or axon-enriched genes. DEGs were extracted with fold changes (FC) |log 2| > 1, Q value <0.05 (Welch's T-test) by Subio platform (ver 1.21). (h,i) The global profiles of the SDs and axon-enriched genes in the present study were analyzed by GO term analysis using Subio platform. The top 5 terms are listed with a p-value (Fisher's exact test). See also Supplementary Table 3.
Fig. 3
Fig. 3
Global analysis of the FUS mutation in MNs revealed the accumulation of AP-1 related genes. (a) DEGs (FC difference of |log 2| > 1) of Control and FUSH517D/H517D MNs of the SDs and axon fractions were compared. (b,c) GO enrichment analysis with DEGs in (a). The top 5 terms are listed with a p-value (Fisher's exact test). (d) The detailed results of DEGs in (a). The genes are listed in Supplementary Table 4. (e) Visualization of the gene network using the set of 55 genes of upregulated DEGs in FUS-mutant MNs shown in (d). Using the GeneMANIA online tool (https://genemania.org/) [48], 17 of 55 genes were analyzed (represented by orange circles). The numbers of related genes (connected with lines described below) are denoted using superscript letters. Particularly, Fos-B-related genes are denoted using underlines. Purple lines represent co-expression connections. Red lines represent physical interactions. Yellow lines represent shared protein domains. Blue lines represent co-localization. Green lines represent genetic interactions. The accumulation of AP-1- (yellow circles) and IEG- (blue circles) related genes was observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Fos-B mRNA binds FUS and affects ECM in hiPSC-derived MNs. (a) RNA-seq results of Fos-B. Compared with Control, Fos-B was found to be upregulated in FUSH517D/H517D, particularly in the axon fraction (one-way ANOVA). (b) The top 3 gene sets that were observed to be increased in the axon fraction of FUSH517D/H517D. The gene sets were extracted with the findsimilar method of the cummeRbund package of the R (version 3.3.1) platform. (c) Confirming the expression level of Fos-B using qRT–PCR from two sets of isogenic MNs. The relative expression of Control SDs samples (with the ΔΔCt method) are presented; N = 3 independent experiments; one-way ANOVA. (d) Representative image of smFISH for detecting Fos-B mRNA in a FUS-mutant hiPSC-derived MNs. smFISH probes (red) for Fos-B mRNA were detected in the nucleus as well as at the neurites [white (left panel) and red (right panel) arrows]. White dotted lines represent the outline of neurites (see also Supplementary Fig. 6). (e) FUS purified using IP. (f) PCR results with RIP samples. Fos-B mRNA precipitated with the FUS protein. (g) The FUS protein was precipitated with the biotinylated Fos-B 3′UTR sequence. (h) The schema shows the site of the FUS binding sequence [49] on Fos-B. Compared with Fos-B mRNA that includes one binding sequence, the 3′UTR sequence contains four binding sites. (i) The schema of the microarray experiment for Fos-B overexpression. (j) Scatterplots of the transcripts of control MNs with EF-1α::Venus or EF-1α::Fos-B/Venus lentivirus infection. DEGs (FC difference of |log 2| > ± 0.58, Student's t-test <0.05) are represented by black dots (listed in Supplementary Table 5). Fos-B is highlighted with a red point and the low reads are illustrated in a column graph. N = 3 from three independent experiments were analyzed using Student's t-test. (k) The top 5 GO enriched terms and the p-values (Fisher's exact test with Subio platform) of the DEGs shown in (j). (l) Top 3 KEGG pathway analysis results and the p-values of DEGs shown in (j) with DAVID (https://david.ncifcrf.gov/) [66,67]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Intervention of aberrant Fos-B expression rescues the cellular phenotype of FUS-mutant MNs. (a–e) Abnormal increases in axon branching was rescued with si-Fos-B. a: Fos-B mRNA was suppressed by si-RNA. The relative expression of si-scrambled control (with ΔΔCt method) is presented. N = 3 independent experiments analyzed by Student's t-test. b: Representative images of the axon ends with or without si-Fos-B. c–e: Quantification of axon branching. To compare the normal condition, untreated data (same data from Fig. 1e–g) were presented and analyzed by one-way ANOVA. (f–i) The number of axon branches decreased with the AP-1 inhibitor, T5224, at 0.1 mM for 7 days. f; RNA expression levels did not differ with T5224 administration. The relative expression of DMSO administrated samples (with the ΔΔCt method) is presented. N = 3 independent experiments analyzed by Student's t-test. g–i; Quantification of axon branching.
Fig. 6
Fig. 6
hFos-B regulated axon branching in vivo. (a) Fos-B nucleotide and protein differences between human and zebrafish. Nucleotides correlated at 77%, whereas proteins correlated at 74%. The alignments of human and zebrafish Fos-B cDNA and amino acid (aa) sequences were compared using nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and eBioX, respectively. (b,c) The schema of injected hFos-B mRNA. (d,e) RNA expression levels were confirmed using qRT–PCR. The zf-Fos-B levels were not different among the three lines. (N = 20 embryos were analyzed in one experiment; qRT–PCR was triplicated and analyzEd.) (f) Representative images of the zebrafish axons at 24 hpf. Lower figures represent magnified images of the white squares of the upper figures. Abnormal spinal roots (red arrow) and axon branching (white arrowheads) were confirmed only in the hFos-B mRNA injected line. The pink asterisks represent the one set of spinal roots. Bars: 100 μm. (g) The % of branching axons at 20 axons/line at 24 hpf. Axon branching was significantly increased in the hFos-B mRNA-injected line. No branching was observed at 24 hpf in un-injected or mock control lines. N = 4, one-way ANOVA. (h) Quantification of axonal length at 24 hpf. The axonal length was adjusted by spinal lengths (the length among 20 spinal roots was defined as the spinal length). No differences in spinal length were observed among the three lines (N = 3, one-way ANOVA). (i) Quantification of the coiling rate/s. The coiling rate significantly decreased in the hFos-B-mRNA injected line. N = 28 (un-injected), 38 (mock), and 46 (hFos-B). The 40 s movies (Supplementary video 1, Supplementary video 2) were analyzed with duplication; one-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7
Fig. 7
Abnormal accumulation of Fos-B was confirmed in the ALS spinal cord. (a) Representative IHC images of autopsy samples from a patient with sporadic inclusion body myositis (sIBM; non-ALS) and one with FUSR521L/+ mutation. FUS was mislocalized in the FUSR521L/+ sample, whereas it was nuclear dominant in the non-ALS sample. Compared with non-ALS samples that did not stain with Fos-B, the cytoplasmic dominant staining of Fos-B was confirmed in FUSR521L/+ samples. Bars: 50 μm. (b) The quantification of cytoplasmic Fos-B-positive cells with three non-ALS [four sections each from one sIBM and two multiple system atrophy (MSA)], one FUSR521L/+, and three sporadic ALS (sALS) (four sections from one case and two sections from two cases) was analyzed using one-way ANOVA. See also Supplementary Fig. 9.

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