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PML targets and resolves structured protein inclusions to mitigate neurodegeneration

Nature Cell Biology (2026)Cite this article

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Abstract

Intranuclear inclusions are defining features of many neurodegenerative diseases, yet their assembly mechanisms and pathological roles remain poorly understood. Here, we investigate polyglycine (polyG) inclusions in neuronal intranuclear inclusion disease (NIID) and show that they recruit intrinsically disordered proteins to form stratified, immobile condensates that disrupt nuclear protein quality control and DNA damage repair. Leveraging their ordered and stepwise assembly, we identify promyelocytic leukaemia protein (PML) as a key factor that actively recognizes and eliminates polyG inclusions through chaperone-mediated disaggregation and proteasome-dependent degradation. Engineered PML variants selectively clear both nuclear and cytoplasmic aggregates, including polyG, polyGA, polyQ, TDP-43 and SOD1. Systemic PML delivery alleviates cognitive and motor deficits in mouse models of NIID and TDP-43 proteinopathy. These findings uncover a conserved spatial organization of nuclear inclusions and establish PML as a therapeutic effector for neurodegenerative diseases linked to protein aggregation.

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Fig. 1: Organization and structure of nuclear polyG inclusions.
Fig. 2: Nuclear polyG inclusions sequester FUS and PML, disrupting cellular homeostasis.
Fig. 3: Ordered assembly gives rise to core-shell structured polyG inclusions.
Fig. 4: PML targets nuclear polyG inclusions and promotes their clearance by acting as molecular chaperone and disaggregase.
Fig. 5: PML protects against intranuclear inclusions and alleviates neurodegeneration in mouse models.
Fig. 6: PMLΔRBC truncation recognizes and dissolves polyG inclusions.
Fig. 7: Cytoplasmic PML variants protect against cytoplasmic protein aggregates.

Data availability

All data generated or analysed during this study are included in the article or Supplementary Information. Source data are provided with this paper. The details of participants with NIID used are listed in Supplementary Table 2. Detailed parameters for image acquisition and processing are provided in Supplementary Table 5. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Proteomics data that support the findings of this study have been deposited in the ProteomeXchange Consortium under accession code PXD072293.

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Acknowledgements

The authors sincerely thank the patients and their families for their collaboration. We also acknowledge Z. X. Wang and J. W. Deng from the Department of Neurology, Peking University First Hospital, Beijing, China, for kindly providing the human brain samples and the original uN2C-polyG104× plasmid. The polyQ plasmid (72Q-HTT) was generously gifted by B. X. Lu from the School of Life Sciences, Fudan University. This work was supported by the National Natural Science Foundation of China (NSFC: 82450108 to S.X.H., 32370739, 32200621 to Y.W., 82371255, 82071258 to L.C. and 22494700, 22477102 to X.Z.), National Key R&D Program of China (2023YFA1800202, 2024YFA1306000 to S.X.H.), Shanghai Rising-Star Program (23QA1400600 to Y.W.), Program for Shanghai Outstanding Academic Leaders (23XD1402500 to L.C.), Shanghai Science and Technology Innovation Action Plan (23DZ2291500 to L.C.), Program for Outstanding Medical Academic Leader of Shanghai (2022LJ011 to L.C.), Training program for research physicians of innovative translational ability (SHDC2022CRD037 to L.C.), Natural Science Foundation of Shanghai (24ZR1456900 to X.-H.L.), ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang (2025SDXHDX000 to X.Z.). Schematic figures (Figs. 3b, 6j and 7a,l,p and Extended Data Figs. 1i and 2i) were created in BioRender; Wang, Y. https://biorender.com/twm9tbl (2026).

Author information

Author notes

  1. These authors contributed equally: Yang Wang, Jia-Xin Zhu, Fei-Xia Zhan.

Authors and Affiliations

  1. State Key Laboratory of Genetics and Development of Complex Phenotypes, Department of Cell and Developmental Biology at School of Life Sciences, Institute of Metabolism and Integrative Biology, Zhongshan Hospital, Fudan University, Shanghai, China

    Yang Wang, Jia-Xin Zhu, Yuchen Xia, Jiaqi Liu, Peng Dai, Ying Hu, Yan-Hao Chen & Steven X. Hou

  2. Department of Neurology, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Fei-Xia Zhan, Xing-Hua Luan, Xi-Ya Shen, Yu-Wen Cao, Xiaojun Huang & Li Cao

  3. Shanghai Neurological Rare Disease Biobank and Precision Diagnostic Technical Service Platform, Shanghai, China

    Fei-Xia Zhan, Xing-Hua Luan, Xiaojun Huang & Li Cao

  4. Department of Chemistry and School of Life Sciences, Westlake University, Hangzhou, China

    Yinfeng Guo & Xin Zhang

  5. Department of Genetics and Rare Diseases, Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Li Cao

  6. Neurological Disorder Center, Haikou Orthopedic and Diabetes Hospital of Shanghai Sixth People’s Hospital, Haikou, China

    Li Cao

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  1. Yang Wang
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Contributions

Y.W., J.-X.Z., L.C. and S.X.H. conceived of and designed the study. Y.W., J.-X.Z., F.-X.Z., Y.G., Y.X., J.L., P.D., Y.H., Y.-H.C., X.-H.L., X.-Y.S., Y.-W.C. and X.H. performed experimental work. Y.W., J.-X.Z., F.-X.Z., Y.G., Y.X. and X.Z. performed analysis and/or interpreted the data. Y.W., J.-X.Z. and S.X.H. wrote the paper, with input from all authors.

Corresponding authors

Correspondence to Yang Wang, Li Cao or Steven X. Hou.

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The authors declare no competing interests.

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Nature Cell Biology thanks Jernej Ule and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Construction and characterization of nuclear polyG inclusions.

a, Representative fluorescence images of uN2C-polyG–eGFP (polyG–eGFP) or SV40-NLS-fused polyG–eGFP (npolyG-eGFP) in HEK293T cells, and quantification of the percentage of intranuclear inclusions in each group (n = 6 fields per group, 72 cells were analysed for polyG–eGFP and 139 cells for npolyG-eGFP). b, Representative fluorescence images of polyG–eGFP or npolyG-eGFP expressed in the midgut of Drosophila, and quantification of the percentage of intranuclear inclusions in each group (n = 4 fields from four Drosophila per group, 94 cells were analysed for polyG-mRuby3 and 93 cells for npolyG-mRuby3. c, Representative immunofluorescence images showing colocalization of P62 and SUMO2/3 with intranuclear polyG inclusions. d, Quantification of the size of polyG inclusions in HEK293T cells and NIID patient skin samples (n = 66 and 32 inclusions from 180, 246 cells, respectively, two NIID patient samples are used. e, Schematic of SV40-NLS-fused uN2C-polyG–eGFP constructs containing 9×, 60× and 104× GGN repeats. f, Representative fluorescence images of eGFP and SV40-NLS-fused uN2C-polyG–eGFP variants of different polyG lengths in HEK293T cells. g, Immunoblot of eGFP and SV40-NLS-fused uN2C-polyG–eGFP (9×, 60× and 104×) in lysate fractions from HEK293T cells. h, FRAP analysis of polyG–eGFP inclusions in HEK293T cells. ROI indicates the bleach region. i, Schematic showing the workflow for purification of intranuclear inclusions j, Comparison of proteins enriched in polyG inclusions identified through immunoprecipitation and biochemical purification. k, Comparison of the proportion of proteins containing intrinsically disordered regions (IDRs) in the human proteome versus those enriched in purified polyG inclusions. l, KEGG pathway analysis of proteins enriched in polyG inclusions. Enrichment was assessed using a one-sided Fisher’s exact test, with P values adjusted for multiple comparisons using the Benjamini–Hochberg method (FDR). The rich factor indicates the ratio of the number of enriched proteins in a given pathway to the total number of proteins annotated to that pathway. The y-axis lists the enriched KEGG pathways. m, n, Representative images of candidate proteins screened by exogenous expression of tagged constructs or by antibody staining in cells expressing polyG-mRuby3. Proteins were classified based on spatial distribution as either core-associated or shell-associated relative to polyG inclusions (m), or as proteins showing minimal or no colocalization with polyG (n). o, Representative FISH images showing poly(A) RNA and GGC RNA in cells expressing polyG-miRFP. p, Representative immunofluorescence images of SUMO2/3 and P62 in skin sections from an NIID patient. All data are presented as mean ± s.d. Statistical significance was determined using two-tailed unpaired Student’s t-test. Panel i created in BioRender; Wang, Y. https://biorender.com/twm9tbl (2026).

Source data

Extended Data Fig. 2 Dense polyG inclusions are independent of nucleolus.

a, Immunoblot of proteins identified from purified polyG–eGFP inclusions. b, Representative images of various nuclear membraneless organelle markers in cells with or without polyG-mRuby3 inclusions. Nuclear stress bodies were induced by 250 mM sodium arsenite (SA) treatment for 2 h. c, Representative immunofluorescence staining of NPM1 in cells expressing polyG, polyGA or polyGR (left). Line profiles show fluorescence intensities along dashed arrows (right). d, Quantification of the percentage of cells showing nucleolar (Nu.) or non-nucleolar (non-Nu.) localization of polyG, polyGA and polyGR (mean ± s.d.; n = 4 fields, 52, 61, 55 cells were analysed per group). e, Ratio of fluorescence intensity between nucleolar and non-nucleolar polyG–eGFP signals (n = 19 pairs analysed). f, Fractionation and immunoblot of eGFP in soluble and insoluble fractions of cells transfected with polyG–eGFP, polyGA–eGFP or polyGR-eGFP. g, FRAP analysis of polyG–eGFP, polyGA–eGFP and polyGR-eGFP with distinct localizations (mean ± s.e.m.; n = 10, 7, 10 and 7 for non-nucleolar polyG, nucleolar polyG, polyGA and polyGR, respectively). Each arrow indicates the bleach region. h, Live-cell imaging showing that polyG–eGFP inclusion formation is independent of the nucleolar marker mRuby3-NPM1. The time zero was defined as the moment before the formation of polyG–eGFP inclusions. The arrow indicates the site of polyG inclusion formation. i, Schematic illustrating the localization and aggregation dynamics of polyG depending on expression levels. j, Quantification of the percentage of cells exhibiting nucleolar or non-nucleolar localization of polyG–eGFP at different time points and expression levels (mean ± s.d.; n = 4 fields, for time-dependent analysis, 57, 52, 60 cells were analysed per condition; for dose-dependent analysis, 40, 44, 67 cells were analysed per condition). k, Representative immunofluorescence images of NPM1 with P62 or SUMO2/3 in skin samples from an NIID patient (left). Line profiles show fluorescence intensities along dashed arrows (right) show no colocalization between NPM1 and P62 or SUMO2/3. Panel i created in BioRender; Wang, Y. https://biorender.com/twm9tbl (2026).

Source data

Extended Data Fig. 3 FUS undergoes phase transition with polyG and does not respond to DNA damage.

a, b, Representative fluorescence images (a) and quantification (b) of cells co-transfected with polyG-mRuby3 and the indicated FUS truncations. n = 31, 27, 31, 34 and 28 cells were analysed for WT, ΔLCD, LCD, ΔGly-rich, ΔQSGY, respectively, pooled from three independent experiments. mEm, mEmerald. Line graphs of fluorescence intensity along each dashed arrow are shown on the bottom. c, Coomassie blue staining of His-polyG60×-eGFP and SUMO (SMT3)-mRuby3-FUS-His. d, Phase diagram of polyG60×-eGFP. e, Representative fluorescence images of phase-separated mRuby3-FUS in vitro, with or without different concentrations of polyG60×-eGFP. f, Quantification of the size and circularity of FUS droplets at different concentrations of polyG60×-eGFP (n > 2,000 droplets from 10 fields per group). g, Representative FRAP images (top) and fluorescence recovery curves (bottom) of 10 μM FUS droplets in vitro, with or without 10 μM polyG60×-eGFP (n = 11 droplets per group). h, Representative immunofluorescence images of γH2AX in cells expressing polyG-mRuby3 (top) and quantification of γH2AX intensity in cells with or without polyG inclusions (bottom) (n = 97 and 108 cells, respectively). i, Immunoblot of phosphorated ATM (pATM) and γH2AX in cells expressing eGFP or polyG–eGFP after etoposide (ETO) treatment (10 μM, 1 h) followed by a 2 h recovery period. j, Representative comet assay images (top) of cells expressing eGFP or polyG–eGFP after ETO treatment (10 μM, 1 h) and 2 h recovery, and quantification of tail DNA percentage (bottom; n = 40 cells per group). k, Representative images of microirradiation-treated cells expressing mEm-SFPQ with mRuby3 or polyG–mRuby3 (left) and time-course of normalized mEm-SFPQ fluorescence intensity at irradiated sites (right). White arrowheads indicate microirradiated regions (n = 6 and 5 events, respectively). l, Representative immunofluorescence images of γH2AX and 53BP1 in cells transfected with mRuby3 or polyG-mRuby3 under ETO treatment (10 μM, 1 h) followed by a 2 h recovery. Data in b are presented as mean ± s.d., data in f (top), g and k are presented as mean ± s.e.m., and data in f (bottom), h and j are presented as median ± quartiles. Statistical significance was determined using two-tailed unpaired Student’s t-test.

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Extended Data Fig. 4 Nuclear polyG and polyGA inclusions induce PML deficiency and are associated with TDP-43 pathology.

a, Representative immunofluorescence images of PML (left) and quantification of PML body number (right; n = 31 and 21 cells, respectively) in HEK293T cells transfected with polyG–eGFP. b, Representative immunofluorescence images (left) and quantification (right; n = 18 cells per group) of cells transfected with polyG–eGFP and the indicated HA-tagged PML truncations. c, Representative fluorescence images (left) and line profile (right) showing that mRuby3 fused to the SRS2 domain specifically localizes to polyG inclusions. Line graphs of fluorescence intensity along each dashed arrow are shown on the right. d, Representative immunofluorescence images of PML and mEm-TDP-43 in control and PML-knockout (KO) HEK293T cells. The percentage of cells with TDP-43 aggregates is indicated at the top of each image (n = 8 fields from two independent experiments). e, Representative FRAP images (left) and fluorescence recovery curves (right) of mEm-TDP-43 in control and PML-KO HEK293T cells. White arrowheads indicate bleached regions (n = 8 condensates per group). f, g, Immunoblot of TDP-43-HA (f) and pTDP-43 (g) in fractionated lysates from HEK293T cells expressing TDP-43-HA with eGFP or polyG–eGFP. h, Representative immunofluorescence images of endogenous TDP-43 in control and PML-KO HEK293T cells after heat shock (HS, 43 °C, 1 h) followed by 1 h recovery. i, Quantification of cells with TDP-43 aggregates after HS and recovery as in h (n = 4 fields, 131 and 161 cells were analysed for sgCtrl and sgPML, respectively). j, Representative immunofluorescence images and line profile analyses showing the interaction between mEm-TDP-43 and PML under HS (43 °C, 1 h) with or without polyG-miRFP. Line graphs of fluorescence intensity along each dashed arrow are shown on the right. k, Cells expressing TDP-43-HA with or without polyG were immunoprecipitated with anti-HA antibody following HS (43 °C, 1 h) or under normal conditions. l, m, Immunoblot of TDP-43-HA (l) and pTDP-43 (m) in fractionated lysates from HEK293T cells expressing TDP-43-HA with eGFP or polyG–eGFP under HS (43 °C, 1 h) or normal conditions. n, Immunoblot of PML in lysate fractions from HEK293T cells transfected with eGFP or polyGA–eGFP. o, p, Immunoblot of TDP-43-HA (o) and pTDP-43 (p) in fractionated lysates from HEK293T cells expressing TDP-43-HA with eGFP or polyGA–eGFP. Data in a, b, i are presented as mean ± s.d., data in d and e are presented as mean ± s.e.m. Statistical significance was determined using two-tailed unpaired Student’s t-test.

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Extended Data Fig. 5 Ordered architecture and assembly preference of nuclear inclusions.

a, Live-cell images (left) and cumulative fluorescence (right) of intranuclear polyGA inclusions. The time zero was defined as the moment before the appearance of the first polyG inclusion (#1). Each arrowhead indicates the site of polyGA inclusion formation. b, Chemical structure of S-SBD-SNAP showing the fluorophore core (green) and its correlation between lifetime and micropolarity. c, Schematic illustrating three distinct forms of inclusions classified by fluorescence lifetime (τ) distribution: homogeneous (H), layered (L) and reverse layered (RL). The outer (τ1) and inner (τ2) regions represent the average fluorescence lifetimes measured by FLIM. d, Representative FLIM images of polyGA inclusions in HEK293T cells expressing polyGA-SNAP at the indicated time points, labelled with 1 μM S-SBD-SNAP. e, Quantification of polyGA inclusion types at 48 h and 72 h (n = 86 and 39 inclusions, respectively). f, Dielectric constant of polyGA inclusions at the indicated time points (n = 66, 66, 8, 8, 12, 17, 17, 22 and 22 inclusions were analysed for L-out, and L-in, R-out, RL-in, H at 48 h, and L-out, L-in, RL-out, RL-in at 72 h, respectively, pooled from three independent experiments). g, Immunofluorescence and line profile analyses of PML (top) and FUS (bottom) in cells containing polyGA-mRuby3 inclusions. Line graphs of fluorescence intensity along each dashed arrow are shown on the right. h, Immunoblot of FUS in purified polyG–eGFP and polyGA–eGFP inclusions. i, 3D reconstruction showing active recognition of polyG–eGFP inclusions by mRuby3-PML (left), with quantification of touch and detach events per frame (right). White arrowheads indicate the PML body which interacts with polyG inclusions. j, Representative FRET images of polyG-mRuby3 with mEm-FUS or mNeonGreen (mNeo)-PML, and corresponding quantification. Red arrowheads indicate the bleach sites (n = 19 FRET events per group, pooled from three independent experiments). k, Coomassie staining of purified PMLΔCC-BFP-HRV3C-GST protein; the GST tag was cleaved before phase separation assays. l, Aged FUS–polyG droplets progressively recruit PMLΔCC-BFP to the shell region, whereas freshly formed droplets do not (n = 8 droplets per group). m, Knockout of PML (bottom), but not FUS (top), increases the insoluble fraction of polyG–eGFP. Data in f and j are presented as mean ± s.d., data in l are presented as mean ± s.e.m. Statistical significance was determined using two-tailed unpaired Student’s t-test.

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Extended Data Fig. 6 PML recognizes and dissolves nuclear inclusions, and its overexpression promotes their clearance.

a, Spatial relationship between PML localization and the polarity distribution of polyG inclusions. PML pseudocolor was adjusted for localization display with the polyG-FLIM image. CSLM, confocal scanning laser microscopy. b, Long-term live-cell imaging and cumulative intensity analysis of polyG–eGFP inclusions following recognition by mRuby3-PML, showing gradual dissolution of inclusions. Each arrowhead indicates a polyG inclusions. c, Schematic of pellet separation from HEK293T cells expressing polyG inclusions. PE., pellet fraction, SN., supernatant fraction. d, Fluorescence images of the pellet fraction from HEK293T cells expressing polyG–eGFP with or without PML–HA. e, Immunoprecipitation of eGFP and eGFP-tagged uN2C-polyG variants from HEK293T cells co-expressing PML–HA. Immunoblotting revealed that PML–HA specifically interacts with uN2C-polyG104×-eGFP. f, Immunoprecipitation of HA-tagged PML truncations from HEK293T cells co-expressing polyG–eGFP showing that the SRS2 domain of PML is required for its interaction with polyG–eGFP inclusions. g, Schematic indicating that PML-I lacks the complete SRS2 domain. h, Immunoblot of polyG–eGFP in lysate fractions from cells expressing PML-I or -IV variants showing PML-I exhibits reduced ability to eliminate polyG inclusions. i, Immunofluorescence (top) and quantification (bottom) of cells co-expressing polyG–eGFP and PML–HA (For intensity analysis, n = 15 coated and 20 diffused polyG aggregates were analysed; for proportion analysis, 8 fields from two independent experiments were measured). A.U., arbitrary unit. j, Fluorescence images (left) and quantification (right) of polyG–eGFP inclusions in cells expressing HA or PML–HA with or without MG132 treatment (10 μM, 6 h) (mean ± s.d.; n = 4 fields from two independent experiments per group). k, Schematic of WT PML and the SUMO E3 ligase-deficient mutant PML-M6 (top) and immunoblot of polyG–eGFP in lysate fractions from cells expressing PML-WT-HA or PML-M6-HA (bottom). l, Coomassie staining of purified PMLΔCC–GST protein. m, Microscale thermophoresis (MST) analysis showing that PMLΔCC binds polyG aggregates with a dissociation constant (Kd) of 23.8 μM (mean ± s.d.; n = 4 independent experiments). n, Thioflavin T (ThT) binding assay of preassembled polyG aggregates (25 μM, 37 °C, 24 h) treated with PMLΔCC–GST (mean ± s.d.; n = 3 independent experiments). o, Turbidity analysis of preassembled polyG aggregates (25 μM, 37 °C, 24 h) treated with 10 μM PMLΔCC–GST, monitored by optical density at 300 nm (blue) and 600 nm (red) (mean ± s.d.; n = 3 independent experiments). p, Representative fluorescence images of HEK293T cells expressing polyGA–eGFP together with HA or PML–HA at the indicated ratios. q, Quantification of the number and total area of polyGA inclusions shown in p (mean ± s.e.m.; n = 8 fields from two independent experiments per group). r, Fluorescence images of the pellet fraction from HEK293T cells expressing polyGA–eGFP with HA or PML–HA. s, Immunoblot of polyGA–eGFP in lysate fractions from HEK293T cells expressing HA or PML–HA. t, Representative immunofluorescence images of PSMB1 and DnaJB1 in cells co-expressing polyGA–eGFP and PML–HA. Line profiles show fluorescence intensities along the dashed arrows. u, Representative immunofluorescence images of HEK293T cells expressing polyGA–eGFP with or without PML–HA, followed by treatment with or without cytoskeleton-stripping buffer, which selectively removed polyGA–eGFP within PML–HA condensates. All statistical significance was determined using two-tailed unpaired Student’s t-test.

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Extended Data Fig. 7 PML recognizes nuclear polyG inclusions and facilitates their clearance in mice.

a, Silver staining of AAV preparations purified by iodixanol gradient ultracentrifugation. b, Representative immunofluorescence images of P62 and SUMO2/3 in brain sections from mice expressing polyG–eGFP, collected 15 days after AAV injection. c, Widespread intranuclear polyG–eGFP inclusions induced by AAV-polyG–eGFP in different brain regions 105 days post-injection. d, FUS colocalizes with intranuclear polyG inclusions in AAV-polyG–eGFP-infected mouse brain cells. Line profiles show fluorescence intensities along the dashed arrows. e, Validation of the colocalization between exogenously expressed PML–HA and polyG–eGFP in AAV-infected mice three months post-injection, as indicated by the white arrowheads. f, Immunoblot of polyG–eGFP in whole-brain lysates from AAV-injected mice (n = 3 per group). g, Representative immunofluorescence images of GFAP in brain regions of mice co-expressing AAV-polyG–eGFP with either AAV-HA or AAV-PML–HA. Mice co-expressing AAV-eGFP and AAV-HA served as controls. h, Quantification of GFAP-positive area shown in (g) (mean ± s.d.; n = 3 mice per group). Statistical significance was determined using two-tailed unpaired Student’s t-test. i, Representative immunofluorescence images of myelin basic protein (MBP) in the medulla of mice expressing AAV-polyG–eGFP. Solid and dashed arrows indicate MBP signals associated and unassociated with polyG–eGFP inclusions, respectively. j, Representative β-galactosidase staining images of brain sections from AAV-injected mice in the indicated groups. k, Validation of PML expression levels in cells transfected with increasing amounts of PML–HA plasmids. l, CCK-8 assay of cell proliferation in cells expressing different amounts of PML, showing that PML expression does not affect cell growth (mean ± s.d.; n = 4 independent experiments). Statistical significance was determined using two-tailed unpaired Student’s t-test; n.s., no significance. m, Schematic of the experimental design (top) assessing the rescue effect of PML overexpression in mice bearing polyGA inclusions, and validation (bottom) of polyGA inclusions in mouse brains 15 days after AAV-polyGA–eGFP injection.

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Extended Data Fig. 8 PMLΔRBC promote polyG inclusion clearance in vivo and in vitro.

a, Immunoprecipitation of HA-tagged PMLΔRBC from HEK293T cells co-expressing polyG–eGFP. b, Coomassie staining of purified WT and KR full-mutant PMLΔRBC-MBP-His proteins. c, d, Co-incubation with PMLΔRBC-MBP-His markedly decreased the formation of polyG aggregates (c) and reduced insoluble polyG (d) (polyG60×-eGFP, 10 μM; PMLΔRBC-MBP-His, 10 μM; 37 °C, 12 h). e, The SRS2 domain alone failed to remove polyG inclusions. f, g, Lysine-to-arginine (KR) mutations of PMLΔRBC suppressed polyG inclusion removal in a mutation number-dependent manner.

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Extended Data Fig. 9 Cytoplasmic PML clears cytoplasmic inclusions.

a, Schematic of WT PML and the NLS-deficient mutant (mPML) (top) and representative fluorescence images of mNeo-fused PML and mPML (bottom). b, Representative immunofluorescence images of HEK293T cells co-expressing mPML–HA with NES-polyGA–eGFP, Flag–TDP-43-CTF–eGFP–NES, Flag-mEm-FUS-P525L or Flag-SOD1-G93A-mNeo. mPML is detected by HA staining. Line profiles show fluorescence intensities of mPML–HA and the misfolded proteins along the dashed arrows. c–f, Immunoblot of NES-polyGA–eGFP (c), Flag–TDP-43-CTF–eGFP–NES (d), Flag-mEm-FUS-P525L (e) and Flag-SOD1-G93A-mNeo (f) in lysate fractions from HEK293T cells expressing HA or mPML–HA. g, Representative immunofluorescence images of PSMB1 and DnaJB1 in HEK293T cells expressing mNeo-mPML. Line profiles display fluorescence intensities along the dashed arrows. h, Schematic of full-length and truncated mPML variants tagged with HA. i, Immunoprecipitation of Flag–TDP-43-CTF–eGFP–NES or Flag-eGFP from HEK293T cells co-expressing HA-tagged mPML truncations. Immunoblotting shows that the SRS2 domain of mPML is required for its interaction with TDP-43-CTF. j, Representative immunofluorescence images of cells transfected with Flag–TDP-43-CTF–eGFP–NES and the indicated HA-tagged mPML truncations. Line profiles show fluorescence intensities along dashed arrows. k, Representative immunofluorescence images of Flag–TDP-43-CTF–eGFP–NES, mPML–HA and MAP2 in primary neuron cultures. Line profiles show fluorescence intensities of each protein along dashed arrows. l, Schematic of the experimental design assessing the rescue effect of mPML overexpression in Flag–TDP-43-CTF–eGFP–NES mice. m, Representative fluorescence images of TDP-43-CTF aggregates in brain sections from mice injected with AAV-Flag–TDP-43-CTF–eGFP–NES and either control or mPML–HA AAVs.

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Extended Data Fig. 10 mPMLΔRBC dissolves cytoplasmic TDP-43 and polyQ aggregates.

a, Representative fluorescence images (left) and quantification (right) of TDP-43-CTF-mRuby3-NES aggregates in HEK293T cells expressing HA or mPMLΔRBC-HA. Data are presented as mean ± s.d. n = 6 fields of view per group collected across two independent experiments. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. b, Coomassie staining of purified SUMO-mEm-TDP-43-His protein. c, Coomassie staining of purified mPMLΔRBC-MBP-His protein. d, Schematic of the sedimentation analysis of matured SUMO-mEm-TDP-43 fibres (20 μM, 37 °C, 24 h) treated with mPMLΔRBC-MBP-His (10 μM, 37 °C) for the indicated durations. e, mPMLΔRBC-MBP-His progressively dissolved pre-formed SUMO-mEm-TDP-43 fibres shown in d. f, Immunoblot analysis of Flag–TDP-43-CTF–eGFP–NES in fractions from HEK293T cells co-expressing mPMLΔRBC-HA with or without TAK-981 treatment (SUMOylation inhibitor, 500 μM, 6 h). g, Representative immunofluorescence images of htt72Q–KR-His and mPMLΔRBC in HEK293T cells. Line profiles show fluorescence intensities of each protein along dashed arrows.

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Supplementary information

Reporting Summary

Peer Review File

Supplementary Tables

Supplementary Table 1 MS list and KEGG pathway analysis of polyG interactomes. Supplementary Table 2 Information on the patients with NIID. Supplementary Table 3 Sequence used in this study. Supplementary Table 4 Information of antibodies and dyes. Supplementary Table 5 Light microscopy reporting table.

Supplementary Video 1

3D rendering of polyG inclusions with PML and FUS under SIM.

Supplementary Video 2

Microirradiation assay of FUS in control cells.

Supplementary Video 3

Microirradiation assay of FUS in cells with polyG inclusions.

Supplementary Video 4

Microirradiation assay of SFPQ in control cells.

Supplementary Video 5

Microirradiation assay of SFPQ in cells with polyG inclusions.

Supplementary Video 6

Live-cell imaging showing the organization of polyG inclusions.

Supplementary Video 7

Live-cell imaging showing the organization of polyGA inclusions.

Supplementary Video 8

Dual-colour live-cell imaging of FUS and polyG during the organization of polyG inclusions.

Supplementary Video 9

Dual-colour live-cell imaging of PML and polyG during the organization of polyG inclusions.

Supplementary Video 10

Dual-colour live-cell imaging of PML and polyG post-organization of polyG inclusions.

Source data

Source Data Fig. 1

Statistical source data for Figs. 1–7 and Extended Data Figs. 1–7 and 10.

Source Data Fig. 2

Unprocessed blots for Figs. 2, 4–7 and Extended Data Figs. 1–10.

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Wang, Y., Zhu, JX., Zhan, FX. et al. PML targets and resolves structured protein inclusions to mitigate neurodegeneration. Nat Cell Biol (2026). https://doi.org/10.1038/s41556-026-01894-z

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