M6A reduction relieves FUS-associated ALS granules

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m⁶A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m⁶A downregulation. Notably, cells expressing mutant FUS were characterized by higher m⁶A levels suggesting a possible link between m⁶A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

Fig. 1. METTL3 downregulation restores the physiological RNA composition of stress granules in ALS cellular models.

a Schematic representation of the experimental flow used for comparing stress granule transcriptomes. b–d Scatter plot depicting RNA differential enrichment in SG in FUS^WT vs FUS^WT METTL3-KD (b) or FUS^P525L vs FUS^P525L METTL3-KD (c), or FUS^WT vs FUS^P525L METTL3-KD (d) conditions. Axes describe log₂FC of SG RNA enrichment in the indicated conditions. Red dots indicate ΔMETTL3-enriched RNAs. Blue dots indicate ΔMETTL3-depleted RNAs. Black dots indicate commonly enriched RNAs. Gray dots indicate invariant RNAs. n = 2 biologically independent replicates. e Pie charts displaying the percentage of meRIP-Seq enriched RNAs among invariant, commonly enriched, and relocated defined in the comparison between FUS^P525L vs FUS^P525L METTL3-KD. n = 2 biologically independent replicates. Source data are provided as a Source Data file.

Fig. 2. METTL3 downregulation reduces the number of stress granules in ALS cellular models.

a Representative images of the indicated stressed SK-N-BE cells. G3BP1 antibody staining is depicted in green, FUS-Flag in red. The nuclei are stained with DAPI (blue). The merge of the signal is shown. The scale bar is 10 µm. (n = 3 biologically independent replicates.) b Representative single focal planes captions of mFISH for CLSTN1 transcript (red), immunofluorescence for stress granule marker G3BP1 (green) and DAPI (blue) in the indicated SK-N-BE cells (top). In the right panel, the digital magnification of G3BP1 granules is highlighted by dashed squares. All scale bars correspond to 3 µm. Bar plot showing representing the percentage of CLSTN1 smFISH signals colocalizing with stress granules as mean of replicates with standard deviation. Dots represent independent replicates. From 356 to #498, cells were analyzed for each condition. n = 3 biologically independent replicates. The ratio of each sample versus its control was tested by a two-tailed Student’s t-test. P values are indicated. c, d Box plots illustrating the number of stress granules per cell in the indicated SK-N-BE cell lines (n = 3 biologically independent replicates.) or iPS-derived MN (n = 4 biologically independent replicates). The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average number of granules in each sample after 1 h stress. Each black dot represents the number of granules in a single field. Seven fields were acquired for each biological replicate. The ratio of each sample versus its control was tested by a two-tailed Student’s t-test. P values are indicated. e Box plots illustrating the number of stress granules per cell in SK-N-BE cells either overexpressing METTL3 (“oeMETTL3”) or downregulated for ALKBH5 (“ALKBH5 KD”) with respect to a control condition (“Ctrl”). The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average number of granules in each sample after 1 h stress. Each black dot represents the number of granules in a single field. Seven fields were acquired for each biological replicate. n = 3 biologically independent replicates. The ratio of each sample versus its control was tested by a two-tailed Student’s t-test. P values are indicated. Source data are provided as a Source Data file.

Fig. 3. METTL3 decrease restores SG recovery rate in ALS cellular models.

a Representative images of the indicated SK-N-BE cells after 1 h stress followed by 4 h recovery. G3BP1 antibody staining is depicted in green, FUS-Flag in red. The nuclei are stained with DAPI (blue). The merge of the signal is shown. The scale bar is 20 µm. (n = 4 biologically independent replicates) b Box plots showing the percentage of unrecovered indicated SK-N-BE cells after 1 h stress followed by 4 h recovery. The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average percentage in each sample. Each black dot represents the percentage in a single field. Seven fields were acquired for each biological replicate. n = 4 biologically independent replicates. The ratio of each sample versus its experimental control was tested by a two-tailed Student’s t-test. P values are indicated. c Box plots showing the percentage of unrecovered SK-N-BE cells either overexpressing METTL3 or downregulated for ALKBH5 after 1 h stress followed by 4 h recovery. The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average percentage in each sample. Each black dot represents the percentage in a single field. Seven fields were acquired for each biological replicate. n = 3 biologically independent replicates. The ratio of each sample versus its experimental control was tested by a two-tailed Student’s t-test. P values are indicated. Source data are provided as a Source Data file.

Fig. 4. METTL3 chemical inhibition relieves FUS-containing SG.

a Representative images of the indicated stressed patients-derived fibroblasts. G3BP1 antibody staining is depicted in green and FUS in red. The nuclei are stained with DAPI (blue). The merge of the signal is shown. The scale bar is 10 µm. (n = 3 biologically independent replicates.) b, c Box plots illustrating the number of stress granules per cell in stressed iPS-derived MN expressing FUS^P525L or patient-derived fibroblasts treated either with DMSO or STM-2457. The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average number of granules in each sample. Each black dot represents the number of granules in a single field. Seven fields were acquired for each biological replicate. n = 4 biologically independent replicates. The ratio of each sample versus its experimental control was tested by a two-tailed Student’s t-test. P values are indicated. d Box plots showing the percentage of unrecovered patients-derived fibroblasts treated either with DMSO or STM-2457. The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average percentage in each sample. Each black dot represents the percentage in a single field. Seven fields were acquired for each biological replicate. n = 3 biologically independent replicates. The ratio of each sample versus its experimental control was tested by a two-tailed Student’s t-test. P values are indicated. e, f Box plots illustrating the diffusion coefficients (D) of FUS^P525L in living SK-N-BE cells treated with either DMSO or STM-2457 in stress or recovery conditions. The box plots are defined by minima, 25% percentile, median, 75% percentile, and maximum (See Source Data file for values). The red dot indicates the average diffusion coefficient value. Each black dot is a single measurement. n = 2 biologically independent replicates. From 16 to 26, single measurements were carried out for each condition. The ratio of each sample versus its experimental control was tested by a two-tailed Student’s t-test. P values are indicated. Source data are provided as a Source Data file.