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L3MBTL1 regulates ALS/FTD-associated proteotoxicity and quality control

A Publisher Correction to this article was published on 04 June 2019

This article has been updated


Misfolded protein toxicity and failure of protein quality control underlie neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. Here, we identified Lethal(3)malignant brain tumor-like protein 1 (L3MBTL1) as a key regulator of protein quality control, the loss of which protected against the proteotoxicity of mutant Cu/Zn superoxide dismutase or C9orf72 dipeptide repeat proteins. L3MBTL1 acts by regulating p53-dependent quality control systems that degrade misfolded proteins. SET domain-containing protein 8, an L3MBTL1-associated p53-binding protein, also regulated clearance of misfolded proteins and was increased by proteotoxicity-associated stresses in mammalian cells. Both L3MBTL1 and SET domain-containing protein 8 were upregulated in the central nervous systems of mouse models of amyotrophic lateral sclerosis and human patients with amyotrophic lateral sclerosis/frontotemporal dementia. The role of L3MBTL1 in protein quality control is conserved from Caenorhabditis elegans to mammalian neurons. These results reveal a protein quality-control pathway that operates in both normal stress response and proteotoxicity-associated neurodegenerative diseases.

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Fig. 1: Identification of lin-61 as a robust suppressor that ameliorates the proteotoxicity in the C. elegans model of SOD1-associated ALS.
Fig. 2: Reduction of human L3MBTL1 accelerates proteasome-mediated protein degradation.
Fig. 3: SETD8 is upregulated in response to proteotoxicity-associated neurodegeneration and regulates protein quality control.
Fig. 4: Effects of protein quality control factors in Drosophila.
Fig. 5: L3MBT1 antagonist UNC669 protects against proteotoxicity in mammalian cells.
Fig. 6: L3MBTL1 antagonist UNC669 protects against proteotoxicity in neurons and mice.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Change history

  • 04 June 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


  1. Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    CAS  Article  Google Scholar 

  2. Prusiner, S. B. Cell biology. A unifying role for prions in neurodegenerative diseases. Science 336, 1511–1513 (2012).

    CAS  Article  Google Scholar 

  3. Van Langenhove, T., van der Zee, J. & Van Broeckhoven, C. The molecular basis of the frontotemporal lobar degeneration–amyotrophic lateral sclerosis spectrum. Ann. Med. 44, 817–828 (2012).

    Article  Google Scholar 

  4. Bosco, D. A. et al. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat. Neurosci. 13, 1396–1403 (2010).

    CAS  Article  Google Scholar 

  5. Wang, J., Xu, G. & Borchelt, D. High molecular weight complexes of mutant superoxide dismutase 1: age-dependent and tissue-specific accumulation. Neurobiol. Dis. 9, 139–148 (2002).

    CAS  Article  Google Scholar 

  6. Wang, J. et al. Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature. Hum. Mol. Genet. 12, 2753–2764 (2003).

    CAS  Article  Google Scholar 

  7. DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256 (2011).

    CAS  Article  Google Scholar 

  8. Renton, AlanE. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–68 (2011).

    CAS  Article  Google Scholar 

  9. Donnelly, C. J. et al. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415–428 (2013).

    CAS  Article  Google Scholar 

  10. Haeusler, A. R. et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507, 195–200 (2014).

    CAS  Article  Google Scholar 

  11. Ugolino, J. et al. Loss of C9orf72 enhances autophagic activity via deregulated mTOR and TFEB signaling. PLoS Genet. 12, e1006443 (2016).

    Article  Google Scholar 

  12. Ash, P. E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646 (2013).

    CAS  Article  Google Scholar 

  13. Mori, K. et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338 (2013).

    CAS  Article  Google Scholar 

  14. Zu, T. et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl Acad. Sci. USA 110, E4968–E4977 (2013).

    CAS  Article  Google Scholar 

  15. Periz, G. et al. Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription. PLoS Biol. 13, e1002114 (2015).

    Article  Google Scholar 

  16. Miyazaki, Y., Chen, L. C., Chu, B. W., Swigut, T. & Wandless, T. J. Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells. eLife 4, e07687 (2015).

    Article  Google Scholar 

  17. Wang, J. et al. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans. PLoS Genet. 5, e1000350 (2009).

    Article  Google Scholar 

  18. Harrison, M. M., Lu, X. & Horvitz, H. R. LIN-61, one of two Caenorhabditis elegans malignant-brain-tumor-repeat-containing proteins, acts with the DRM and NuRD-like protein complexes in vulval development but not in certain other biological processes. Genetics 176, 255–271 (2007).

    CAS  Article  Google Scholar 

  19. Hamer, G., Matilainen, O. & Holmberg, C. I. A photoconvertible reporter of the ubiquitin-proteasome system in vivo. Nat. Methods 7, 473–478 (2010).

    CAS  Article  Google Scholar 

  20. Bonasio, R., Lecona, E. & Reinberg, D. MBT domain proteins in development and disease. Semin. Cell Dev. Biol. 21, 221–230 (2010).

    CAS  Article  Google Scholar 

  21. Wang, J., Xu, G. & Borchelt, D. R. Mapping superoxide dismutase 1 domains of non-native interaction: roles of intra- and intermolecular disulfide bonding in aggregation. J. Neurochem. 96, 1277–1288 (2006).

    CAS  Article  Google Scholar 

  22. West, L. E. et al. The MBT repeats of L3MBTL1 link SET8-mediated p53 methylation at lysine 382 to target gene repression. J. Biol. Chem. 285, 37725–37732 (2010).

    CAS  Article  Google Scholar 

  23. Dantuma, N. P., Lindsten, K., Glas, R., Jellne, M. & Masucci, M. G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat. Biotechnol. 18, 538–543 (2000).

    CAS  Article  Google Scholar 

  24. Shi, X. et al. Modulation of p53 function by SET8-mediated methylation at lysine 382. Mol. Cell 27, 636–646 (2007).

    CAS  Article  Google Scholar 

  25. Gurney, M. E. et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772–1775 (1994).

    CAS  Article  Google Scholar 

  26. Wang, J. et al. Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS. Proc. Natl Acad. Sci. USA 106, 1392–1397 (2009).

    CAS  Article  Google Scholar 

  27. Watson, M. R., Lagow, R. D., Xu, K., Zhang, B. & Bonini, N. M. A Drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1. J. Biol. Chem. 283, 24972–24981 (2008).

    CAS  Article  Google Scholar 

  28. Ollmann, M. et al. Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell 101, 91–101 (2000).

    CAS  Article  Google Scholar 

  29. D’Brot, A., Kurtz, P., Regan, E., Jakubowski, B. & Abrams, J. M. A platform for interrogating cancer-associated p53 alleles. Oncogene 36, 286–291 (2017).

  30. Gupta, R. et al. The proline/arginine dipeptide from hexanucleotide repeat expanded C9ORF72 inhibits the proteasome. eNeuro (2017).

    Article  Google Scholar 

  31. Kramer, N. J. et al. CRISPR–Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nat. Genet. 50, 603–612 (2018).

    CAS  Article  Google Scholar 

  32. Mizielinska, S. et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345, 1192–1194 (2014).

    CAS  Article  Google Scholar 

  33. Xu, Z. et al. Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration. Proc. Natl Acad. Sci. USA 110, 7778–7783 (2013).

    CAS  Article  Google Scholar 

  34. Min, J. et al. L3MBTL1 recognition of mono- and dimethylated histones. Nat. Struct. Mol. Biol. 14, 1229–1230 (2007).

    CAS  Article  Google Scholar 

  35. Trojer, P. et al. L3MBTL1, a histone-methylation-dependent chromatin lock. Cell 129, 915–928 (2007).

    CAS  Article  Google Scholar 

  36. Koester-Eiserfunke, N. & Fischle, W. H3K9me2/3 binding of the MBT domain protein LIN-61 is essential for Caenorhabditis elegans vulva development. PLoS Genet. 7, e1002017 (2011).

    CAS  Article  Google Scholar 

  37. Herold, J. M. et al. Small-molecule ligands of methyl-lysine binding proteins. J. Med. Chem. 54, 2504–2511 (2011).

    CAS  Article  Google Scholar 

  38. Mojsilovic-Petrovic, J. et al. Protecting motor neurons from toxic insult by antagonism of adenosine A2a and Trk receptors. J. Neurosci. 26, 9250–9263 (2006).

    CAS  Article  Google Scholar 

  39. Kwon, I. et al. Poly-dipeptides encoded by the C9ORF72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345, 1139–1145 (2014).

    CAS  Article  Google Scholar 

  40. Subramaniam, S., Sixt, K. M., Barrow, R. & Snyder, S. H. Rhes, a striatal specific protein, mediates mutant-huntingtin cytotoxicity. Science 324, 1327–1330 (2009).

    CAS  Article  Google Scholar 

  41. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  42. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  43. Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strainw1118; iso-2; iso-3. Fly (Austin) 6, 80–92 (2012).

    CAS  Article  Google Scholar 

  44. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    CAS  Article  Google Scholar 

  45. Zhang, T., Mullane, P. C., Periz, G. & Wang, J. TDP-43 neurotoxicity and protein aggregation modulated by heat shock factor and insulin/IGF-1 signaling. Hum. Mol. Genet. 20, 1952–1965 (2011).

    CAS  Article  Google Scholar 

  46. Jensen, L. T. & Culotta, V. C. Activation of CuZn superoxide dismutases from Caenorhabditis elegans does not require the copper chaperone CCS. J. Biol. Chem. 280, 41373–41379 (2005).

    CAS  Article  Google Scholar 

  47. el-Deiry, W. S. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993).

    CAS  Article  Google Scholar 

  48. McCreedy, D. A. et al. A new method for generating high purity motoneurons from mouse embryonic stem cells. Biotechnol. Bioeng. 111, 2041–2055 (2014).

    CAS  Article  Google Scholar 

  49. Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992).

    CAS  PubMed  Google Scholar 

  50. Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    CAS  Article  Google Scholar 

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This work was supported by grants from the NIH (nos. NS074324, NS089616 and NS110098), the Packard Center for ALS Research at Johns Hopkins, the Muscular Dystrophy Association, the ALS Association and the Maryland Stem Cell Research Fund (to J.W.). R.G.K. and J. M.-P. were supported by grants from the NIH (nos. NS096746 and NS093439) and the Les Turner ALS Center at Northwestern. We thank H.-Y. Hwang for assisting in C. elegans genome deep sequencing and data analysis, C. Holmberg of the University of Helsinki for the Dendra2 C. elegans strain, V. Culotta of Johns Hopkins University for the C. elegans SOD1 antibody, A. Isaacs of University College London for the proline-arginine repeat plasmid template and members of the Wang laboratory for discussion.

Author information

Authors and Affiliations



J.L. performed the C. elegans suppressor screen, genome deep sequencing and data analysis, and the characterization of the C. elegans suppressor mutations. J.L. and G.P. performed most of the mammalian cell experiments. G.P. and Y.-N.L. performed the Drosophila experiments. Q.T., J.M.-P. and R.K. performed the mammalian neuronal experiments. Y.S., R.T., R.A. and W.L. performed the mouse experiments. Y.-N.L. and Y.L. performed the mouse and human tissue analyses. T.Z., Y.J. and K.J. performed additional experiments. G.P., J.L., Y.-N.L. and J.W. designed the studies and wrote the paper. All authors discussed the results and contributed to the preparation of the manuscript.

Corresponding author

Correspondence to Jiou Wang.

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The authors declare no competing interests.

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Journal peer review information Nature Neuroscience thanks Hande Ozdinler and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the lin-61 suppressor that ameliorates the locomotion defects in the C. elegans model of SOD1-associated ALS.

(a) Quantitative RT-PCR analysis of the mRNA expression levels of the SOD1G85R transgene in the presence of the loss-of-function mutation lin-61(n3809) as compared with the control background (WT) (n = 3; * P = 0.029). (b) A schematic graph that depicts the different folding states of proteins in relation to the solubility assay based on detergent extraction and centrifugation. The soluble fraction includes native proteins and the misfolded monomers and oligomers, while the insoluble fraction contains large protein aggregates. (c) Representative images showing the SOD1G85R-YFP protein aggregates in the WT or the lin-61(n3809) mutant background. The Iin-61(n3809) mutant worms show a marked decrease in protein aggregation. SOD1G85R-YFP is expressed in neurons. The head and nerve ring regions are shown. Scale bar: 5 μm. (d) Representative western blots of the SOD1G85R-YFP protein in the WT and lin-61(n3809) mutant worms (top). Quantification of SOD1G85R protein and mRNA levels (bottom). (n = 5 for proteins, *** P = 0.0005; n = 3 for mRNAs, P = 0.9862). (e) Western blots of C. elegans SOD-1 (C.e.SOD-1) in the WT (N2) and lin-61(n3809) C. elegans, showing no change in the level of the endogenous SOD-1 (top). Quantification of C. elegans SOD-1 (C.e.SOD-1) protein levels (bottom; n = 3; P = 0.3543). (f) Western blots of human wild-type SOD1 (SOD1WT) expressed in neurons of the N2 and lin-61(n3809) C. elegans (left). Quantification of human wild-type SOD1 (SOD1WT) protein level (right; n = 3; P = 0.8812). (g) Western blots of ploy-glutamine repeat (polyQ67-YFP) expressed in neurons of the N2 and lin-61(n3809) C. elegans (left). Quantification of polyQ67-YFP protein levels (right; n = 3; P = 0.5301). (h) Molecular phylogenetic analysis of L3MBTL1-related proteins. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (next to the branches). The analysis involved 5 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 619 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. Error bars represent ± SEM.

Supplementary Figure 2 UbG76V-Dendra2 fluorescence chase experiments to determine the rate of protein degradation in lin-61 mutant suppressor C. elegans.

Representative fluorescence images of live C. elegans before and after conversion of the green Dendra2 (Green) into photo-switched red Dendra2 (Red). Scale bar: 100 μm.

Supplementary Figure 3 Effects of L3MBTL1 on protein quality control.

(a) Representative western blots of SOD1 in total cell lysates after reduction of L3MBTL1 (left). Quantification of the WT and G85R mutant SOD1 western blots (right; n = 5, P(WT) = 0.1388, ** P(G85R) = 0.0038). (b) Representative western blots analyses of supernatants from HEK293 cells treated with non-targeting (CTRL), SETD8, or L3MBTL1 shRNAs. Unlike the misfolded SOD1 mutant G85R, the levels of wild-type SOD1 protein were not altered by the reduction of SETD8 or L3MBTL1 in the cells (top). Quantification of the SOD1 western blots (bottom; n = 3, SOD1WT: P = 0.46 (CTRL vs. SETD8), P = 0.80 (CTRL vs. L3MBTL1); SOD1G85R: * P = 0.024 (CTRL vs. SETD8)). (c) Co-immunoprecipitation analysis of Flag-tagged L3MBTL1 and endogenous p53 in HEK293 cells (left). A methyllysine-binding mutation, D355A, disrupts the interaction between L3MBTL1 and p53. Quantification of the co-IP western blots (right; n = 4; * P = 0.0114, *** P = 0.0009). (d) Western blots of cell lysates after treatments with non-targeting (CTRL) or L3MBTL1 shRNAs. Reduction of L3MBTL1 did not change the level of Flag-tagged polyQ82 proteins in both fractions from HEK293 cells (left). Quantification of the Flag-tagged polyQ82 western blots (right; n = 4, P(S) = 0.8355, P(P) = 0.9297). (e) Quantification of the SOD1WT protein clearance. The graph indicates the relative band intensity of SOD1WT at each chase time point in the L3MBTL1 shRNA-treated cells versus controls (bottom; n = 3, P = 0.3352). (f) Schematic representation of the proteasome degradation assay using destabilized fusion protein UbG76V-GFP as a readout. UbG76V-GFP is a reporter protein, formed by fusing a mutated uncleavable ubiquitin moiety with the N-terminus of GFP, which allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. (g) Schematic representation of the cell-based Proteasome-Glo activity assay. Cells are provided with a specific luminogenic proteasome peptide substrate, Suc-LLVY-aminoluciferin. When the substrate is cleaved by the proteasome, aminoluciferin is released and consumed by luciferase, resulting in a luminescent signal proportional to the proteasome activity in the cell. Error bars represent ± SEM.

Supplementary Figure 4 SETD8 interacts with p53 and regulates its protein level.

(a) Reduction of SETD8 by shRNAs decreases the level of a SETD8 enzymatic product, H4K20me1, while not changing the total level of histone H4 protein levels. (b) Co-immunoprecipitation analysis of V5-tagged SETD8 and endogenous p53 proteins in HEK293 cells (left). Quantification of the co-IP western blots (right; n = 3; ** P = 0.0087). (c) Quantification of the endogenous SOD1WT protein half-life in the SETD8 shRNA-treated HEK293 cells. The SETD8 shRNAs didn’t alter the half-life of the endogenous WT SOD1 protein (n = 3, P = 0.8315). (d) HEK293 cells were expressed with Flag-tagged human WT or K382R (methylation mutant) p53 with or without Flag-SETD8 co-expression or MG132 treatment (20 μM, 2hr), and the steady-state levels of p53 proteins were analyzed by western blotting (top). The presence of the mutation K382R, which disrupts methyllysine-binding, decreased the levels of p53 protein as well as the co-expressed SETD8 protein. The decrease of the p53-K382R or SETD8 protein was partially rescued with the MG132 treatment. Quantification of p53 and SETD8 protein levels (n = 3; P53: * P = 0.036 (p53WT vs. p53K382R), * P = 0.0245 (p53WT vs. p53WT with SETD8), **** P<0.0001(p53WT with SETD8 vs. p53K382R with SETD8), * P = 0.0309 (p53K382R with SETD8 vs. p53K382R with SETD8 and MG132); SETD8: ** P = 0.007 (p53WT with SETD8 vs. p53K382R with SETD8), * P = 0.0103 (p53K382R with SETD8 vs. p53K382R with SETD8 and MG132)). (e) Western blots of a representative cycloheximide chase experiment to determine the half-life of p53WT or p53K382R in HEK293 cells with SETD8-GFP co-expression (left). Quantification of p53 and SETD8 clearance with p53WT or p53K382R as measured by the western blots. The graph indicates the relative band intensity of p53 or SETD8 at each chase time point (right; n = 3; P53: P = 0.0494; SETD8: P≤0.048 for each time point). (f) Western blots of cell lysates after treatment with non-targeting (CTRL), SETD8, L3MBTL1, or double shRNAs. Reduction of SETD8 decreases the level of p53 protein, an effect that can be reversed by the reduction of L3MBTL1 (left). Quantification of the p53 and SOD1G85R western blots (right; n = 3, P53: *** P = 0.0006 (CTRL vs. ShSETD8), ** P = 0.0082 (ShSETD8 vs. ShSETD8+ShL3MBTL1), SOD1G85R: * P = 0.0298 (CTRL vs. ShSETD8), *** P = 0.0006 (CTRL vs. ShL3MBTL1), ** P = 0.0056 (CTRL vs. ShSETD8+ShL3MBTL1)). Error bars represent ± SEM.

Supplementary Figure 5 SETD8 is upregulated in response to stress and neurodegeneration.

(a) Treatment with the proteasome inhibitor MG132 (20 μM for 1 h) increases the level of SETD8 protein in MEF cells. (b) Quantification of SETD8 protein levels after treatment of MG132 (n = 5; * P = 0.0256). (c) Quantification of SETD8 mRNA levels after MG132 treatment on MEF cells (n = 3; *** P<0.0001). (d) Western blot analyses of spinal cord lysates from non-transgenic, SOD1WT-YFP, presymptomatic (Pre) and symptomatic (Sym) SOD1G85R-YFP, and SOD1G93A transgenic mice show an increase in the levels of H4K20me1 (an enzymatic product of SETD8-mediated methylation) and p53, occurring in a mutant SOD1- and symptom-dependent manner. Total H4 protein levels were not changed. (e) Additional ALS patients’ spinal cords were analyzed by western blotting for the expression levels of SETD8. The control and ALS samples were run on the same gel but separated by other samples. The full blot is shown in the supplementary information. (f) Western blot analyses of SETD8 in the brain region of occipital cortex from ALS patients and healthy controls (left). Unlike spinal cords of ALS patients, in which SETD8 is up-regulated, the occipital cortex from the same patients shows no significant change in the SETD8 protein levels (right; n = 3 controls and 5 patients, P = 0.19). (g) Western blot analyses of SETD8 in the brain region of inferior parietal cortex from Alzheimer’s disease (AD) patients and healthy controls show a reduction of the SETD8 protein in AD brains (n = 4 controls and 7 patients, * P = 0.03). The control and AD samples were run on the same gel but separated by other samples. The full blot is shown in the supplementary information. Error bars represent ± SEM.

Supplementary Figure 6 L3MBTL1 is upregulated in spinal cords of mutant SOD1 mice and a subset of patients with ALS.

(a) Western blot analyses of spinal cord lysates from age-matched non-transgenic (NTg) and symptomatic SOD1G93A transgenic mice show an increase in the levels of L3MBTL1 in the mutant SOD1 animals. (b) Quantification of L3MBTL1 protein levels by western blotting (n = 3, * P = 0.0354). (c) Immunofluorescence staining analysis shows increased levels of L3MBTL1 in neurons of symptomatic SOD1G93A transgenic mice compared to age- and gender-controlled non-transgenic mice. NeuN is a neuronal marker (left). Quantification of L3MBTL1 immunofluorescence signals in spinal cord neurons from three independent sets of mice (right; n = 3, * P = 0.036). (d) Representative western blot analyses of L3MBTL1 in the spinal cords from ALS patients and healthy controls (left). Quantification of L3MBTL1 protein levels as measured in the western blots (right; n = 6 controls and 10 patients, * P = 0.031). Error bars represent ± SEM.

Supplementary Figure 7 Control RNAi strains of Drosophila show normal eye phenotypes.

Representative images of Drosophila adult eyes in RNAi strains without the expression of poly(GR) or poly(PR) proteins, showing normal eye phenotypes. Scale Bar: 100 μm.

Supplementary Figure 8 The effects of L3MBTL1 inhibitor UNC669 on cells and mice.

(a) Western blot analyses of HEK293 cells treated with increasing concentrations of UNC669 showed that the level of endogenous SOD1WT protein was not affected by the treatment (n = 3; n.s. = non-significant; P(0 vs. 25) = 0.2035; P(0 vs. 50) = 0.0569; P(0 vs. 100) = 0.1514). (b) Mouse embryonic stem cell-derived motor neurons were transduced with GFP-expressing viral vectors and treated with vehicle or 100 µM UNC669 for six days. Cells were then loaded with calcein AM and imaged for cell viability using fluorescence microscopy (left). Quantification of the calcein AM by using a plate reader (right; n = 5; P = 0.36). There was no change in the survival of the GFP-expressing motor neurons. Scale Bar: 300 μm. (c) Rat primary spinal cord neuronal cultures were treated with 100 µM UNC669 or vehicle (EtOH, VEH). The cells were stained with a motor neuron-specific anti-NF-H antibody SMI-32 and imaged using fluorescence microscopy (left). Quantification of the counts of surviving rat motor neurons (right; n = 12, P = 0.95). Scale Bar: 300 μm. (d) Representative images of immunofluorescence staining using an antibody that recognizes the misfolded SOD1 protein, on spinal cord sections from SOD1G93A mice treated with UNC669 or vehicle controls. Arrows point to ventral horns of lumbar spinal cords. (e) Representative images of immunofluorescence co-staining using antibodies against both misfolded SOD1 and ChAT as a motor neuron marker on spinal cord from SOD1G93A mice treated with UNC669 or vehicle controls. Error bars represent ± SEM.

Supplementary Figure 9 Schematic summary of protein quality control programs modulated by L3MBTL1 and SETD8.

L3MBTL1 negatively regulates p53 activity, the proteasomal degradation, and the turnover of misfolded proteins. Accumulation of misfolded proteins leads to accumulation/activation of SETD8, which enhances protein quality control and clearance of misfolded proteins, an effect partially mediated by L3MBTL1. Together, these regulators constitute a protein quality control pathway that senses and controls the levels of misfolded proteins and associated toxicities.

Supplementary Figure 10

Scans of immunoblots presented in the study.

Supplementary Figure 11

Scans of immunoblots presented in the study.

Supplementary Figure 12

Scans of immunoblots presented in the study.

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Lu, J., Periz, G., Lu, YN. et al. L3MBTL1 regulates ALS/FTD-associated proteotoxicity and quality control. Nat Neurosci 22, 875–886 (2019).

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