Fig 2: Model and Expression of FUS Pathological Inclusion Components in Human(A) Model of the aberrant crosstalk between mutant FUS and ELAVL4 in ALS. (i) In normal conditions FUS (red) is mainly localized in the nucleus. ELAVL4 (green) is localized both in the nucleus and in the cytoplasm, where it has a diffuse localization punctuated by speckles. Human iPSC-derived FUSWT MNs would be representative of this situation. (ii) ALS mutations in the nuclear localization signal cause FUS partial de-localization in the cytoplasm. Mutant FUS binds ELAVL4 3'UTR. Moreover, as a consequence of FUS nuclear depletion, miR-375 levels decrease. These events gradually cause an increase of ELAVL4 protein levels. Human iPSC-derived FUSP525L MNs would be representative of this situation. (iii) Over time, ELAVL4 protein levels are increasingly augmented, forming bigger speckles in the cytoplasm. It is possible that protein-protein interaction with ELAVL4 results in further mutant FUS accumulation in the cytoplasm. This, in turn, would further increase ELAVL4 levels by the above-mentioned mechanisms. (iv) Increased levels of both ELAVL4 and mutant FUS in the cytoplasm and their interaction might lead to the formation of speckles containing both proteins, independently from cellular stress. This situation cannot be observed in human iPSC-derived MNs, which correspond to “young” MNs. However, mutant FUS/ELAVL4 speckles are formed in the cytoplasm of HeLa cells overexpressing both proteins. (v) In presence of cellular stress, e.g., oxidative stress, cytoplasmic mutant FUS and ELAVL4 proteins co-localize in stress granules. We observe this phenomenon in both transgenic HeLa cells and human iPSC-derived MNs. (vi) Alteration of granules/speckles biomechanical properties due to mutant FUS and/or increased ELAVL4 would lead over time to the formation of ALS pathological inclusions, in which mutant FUS, ELAVL4, and SG markers can be detected. As previously proposed (Bentmann et al., 2013), three modes of pathological inclusions formation are possible: SG-dependent, aggregation-dependent, aggregation- and SG-dependent.(B) Heatmap representing gene expression levels in different human tissues from the GTEx Portal (https://www.gtexportal.org/home/). Genes that are known components of FUS pathological inclusions (Blokhuis et al., 2013, Blokhuis et al., 2016) are reported. The ubiquitous ELAVL family member ELAVL1 (HuR) is also shown. Expression values are shown in TPM (transcripts per million), calculated from a gene model with isoforms collapsed to a single gene. See also Figure S9.

Fig 3: Increased HuD levels in human and mouse FUS-mutant MNs.a, b HuD protein levels analysis by western blot in FUSWT and FUSP525L hiPSC-derived spinal MNs (a) and Fus-?14 mouse model spinal cord (P81) (b). The molecular weight (kDa) is indicated on the left. The graphs show the average from three independent biological replicates, error bars indicate the standard deviation (a Student’s t-test, paired, two tails; b ordinary one-way ANOVA, multiple comparisons). TUBB3/Tubb3 signal was used for normalization. Protein levels are relative to FUSWT (a) or Fus+/+ (b) conditions. c HuD mRNA analysis by FISH in FUSWT and FUSP525L hiPSC-derived spinal MNs. The graphs show the average count of HuD mRNA puncta per cell from three independent differentiation experiments, error bars indicate the standard error of the mean (Student’s t-test; paired; two tails). d, e Combined PURO-PLA (HuD, magenta) and immunostaining (TUBB3, green) analysis in FUSWT and FUSP525L hiPSC-derived spinal MNs (d) and primary MNs from Fus-?14 mouse embryos (E12.5–13.5) (e). DAPI (blue) was used for nuclear staining. d FUSP525L hiPSC-derived spinal MNs treated with the eukaryotic protein synthesis inhibitor anisomycin (ANISO) were used as negative control of the PURO-PLA. The graphs show the average count of HuD PURO-PLA puncta per cell from three independent differentiation experiments (d) and three samples (e), error bars indicate the standard error of the mean (d Student’s t-test, unpaired, two tails; e ordinary one-way ANOVA, multiple comparisons). Scale bars (c–e): 10 µm.

Fig 4: FMRP is a post-transcriptional repressor of HuD expression.a Analysis of the protein levels of the indicated genes by western blot in FMRPWT and FMRPKO hiPSC-derived spinal MNs. The molecular weight (kDa) is indicated on the left. The graphs show the average from three independent differentiation experiments, error bars indicate the standard deviation (Student’s t-test; paired; two tails). TUBB3 signal was used for normalization. Protein levels are relative to FMRPWT conditions. b Analysis of the mRNA levels of the indicated genes by real-time qRT-PCR in FMRPWT and FMRPKO hiPSC-derived spinal MNs. For each experiment, values are shown as relative to the isogenic FMRPWT control, set to a value of 1. The graph shows the average from 3 to 5 independent differentiation experiments, error bars indicate the standard deviation (Student’s t-test; paired; two tails). c Luciferase assay in HeLa cells expressing RFP, RFP-FUSWT, or RFP-FUSP525L and transfected with the Renilla luciferase reporter construct containing the HuD 3'UTR (RLuc-HuD 3'UTR) alone (Mock) or in combination with plasmids overexpressing FMRP or eGFP as a control (Student’s t-test; paired; two tails). The drawing depicts the competition between mutant FUS and FMRP for HuD 3'UTR binding and its effects on the reporter construct.

Fig 5: Survival and differentiation of retinal organoid cells as analyzed by immunofluorescence microscopy after transplantation. (a, b) The SC121-positive cells expressing green fluorescent protein (GFP), which were used for construction of the retinal organoids, were clearly visible in the healthy eye (transplanted with a retinal organoid, but receiving no immunosuppressive treatment) and the Oz-eye (eye with ocular hypertension (OHT) transplanted with a retinal organoid and treated with dexamethasone), indicating that cells from the transplanted organoids were alive in the host retina. However, in (c) the RAP-eye (OHT eye transplanted with a retinal organoid and treated with rapamycin) and (d) the OHT-eye (OHT eye transplanted with a retinal organoid, but receiving no immunosuppressive treatment), no SC121-positive or GFP-positive cells were detected. HuD- and RBPMS-positive retinal ganglion cells (RGCs) were also observed in (e, i) the healthy eye and (f, j) the Oz-eye. (m) Tubulin- and (q) NEFL-positive axons were observed within and near the transplants in the healthy eye. (n) Tubulin- and (r) NEFL-positive axons were observed within and near the transplants in the Oz-eye. In (c, g, k, o, s) the RAP-eye and (d, h, l, p, t) the OHT-eye, no cells or axons expressing these markers were detected. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer.