Research ArticleAlzheimer’s Disease

Activity of the poly(A) binding protein MSUT2 determines susceptibility to pathological tau in the mammalian brain

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Science Translational Medicine  18 Dec 2019:
Vol. 11, Issue 523, eaao6545
DOI: 10.1126/scitranslmed.aao6545
  • Fig. 1 Genetic ablation of Msut2 protects against tauopathy in mice.

    (A) Deletion of Msut2 decreases accumulation of neurofibrillary tangles (NFTs) in the CA1 region of the hippocampus of PS19 mice with tauopathy. Representative brain sections from 9-month-old PS19 mice with tauopathy are shown. NFTs were detected by Gallyas silver stain. Left: Number of Gallyas-positive NFTs in brain sections from Msut2 KO/PS19 transgenic (Tg) mice (n = 24) compared to PS19 Tg mice (n = 24) (P = 0.0129 by two-tailed t test). Scale bars, 100 μm. (B) Deletion of Msut2 decreases accumulation of pTau in the stratum lacunosum moleculare (SLM) of brains of PS19 mice with tauopathy. Representative brain sections from 9-month-old PS19 mice with tauopathy stained with AT180 monoclonal antibody to reveal pTau phosphorylated on Thr231 are shown. Left: Densitometry analysis of AT180-positive pathological tau deposits in Msut2 KO/PS19 Tg brain sections (n = 24) compared to PS19 Tg brain sections (n = 24) (P = 0.0156 by two-tailed t test). Scale bars, 100 μm. (C) Deletion of Msut2 decreases accumulation of pre-tangle tau species in the stratum oriens (SO) region of the hippocampus of PS19 mice with tauopathy. Pre-tangle pathological tau species were detected by the conformation-dependent anti-tau monoclonal antibody MC1. Representative brain sections from 9-month-old PS19 mice with tauopathy stained with MC1 antibody are shown. Left: Densitometry analysis of MC1-positive tau lesions in Msut2 KO/PS19 Tg mouse brain (n = 24) compared to PS19 Tg mouse brain (n = 24) (P = 0.0042 by two-tailed t test). Scale bars, 50 μm. (D) Deletion of Msut2 decreases loss of CA1 hippocampal neurons in PS19 mice with tauopathy. Representative brain sections from 9-month-old PS19 mice with tauopathy stained with cresyl violet to identify neuronal cell bodies are shown (black arrows indicate the pyramidal cell layer). Left: Densitometry analysis of pyramidal neuron density in Msut2 KO/PS19 Tg mouse brains (n = 21) compared to PS19 Tg mouse brains (n = 19) (P = 0.0062 by two-tailed t test). Photomicrographs in (A) to (D) were modified by equally adjusting brightness and contrast to optimize visualization of staining. (E) Deletion of Msut2 enhances performance of 8-month-old PS19 mice with tauopathy in the Barnes maze training test. PS19 Tg animals performed poorly on the Barnes maze test, taking longer to locate the escape hole compared to either wild-type (WT) or Msut2 KO/PS19 animals (***P < 0.001 by ANOVA). n.s., not significant.

  • Fig. 2 MSUT2 binds independently to both poly(A) RNA tails and PABPN1.

    (A) In vitro confirmation of the yeast two-hybrid interaction between the MSUT2 CCCH finger domain and PABPN1. 35S radiolabeled PABPN1 was tested against immobilized recombinant glutathione S-transferase (GST)–MSUT2 (ZF) fusion protein or recombinant GST alone using GST pulldown assays. RNase pretreatment of samples did not prevent MSUT2 and PABPN1 protein-protein interactions. (B) The proximity ligation assay detects MSUT2/PABPN1 interactions within intact HEK293 cell nuclei. Blue, DAPI nuclear stain; red, interactions detected by proximity ligation assay. Left: High-magnification stitched panel image of a large field of immunostained HEK293 cells. Scale bar, 100 μm. The white square in the left represents the magnified area shown in the right. (C) Surface plasmon resonance detection of MSUT2 and PABPN1 binding to poly(A) RNA. Dissociation constants were calculated for biotinylated poly(A)15 from the binding kinetics of each protein at five concentrations (1, 0.9, 0.8, 0.7, and 0.6 μM). MSUT2 KD = 60 ± 15 nM compared to PABPN1 KD = 237 ± 21 nM for poly(A)15. (D) MSUT2 and PABPN1 colocalization in SC35-positive nuclear speckles in HEK293 cells [Pearson coefficient of correlation (PCC) = 0.7976]. A representative single-channel image of nuclear MSUT2, PABPN1, and DAPI stain is depicted. Colocalization analysis of MSUT2 with SC35 (PCC = 0.7578) and PABPN1 with SC35 (PCC = 0.4347) is shown in fig. S4 (C and D). (E) MSUT2 and PABPN1 colocalization in nuclear speckles in neuronal nuclei from human frontal cortex brain tissue. Representative confocal image shows immunofluorescent staining for MSUT2 (green), PABPN1 (red), and DAPI (blue). Inset: Digital magnification of a single nucleus displayed as split channels for MSUT2 and PABPN1 and merged channels. MSUT2 and PABPN1 colocalized with a Pearson coefficient of correlation (PCC) = 0.88 in this image (see also fig. S4). Scale bar, 25 μm. Where necessary, image adjustments applied linear brightness and contrast changes.

  • Fig. 3 MSUT2 mediates effects on tauopathy through poly(A) tail length but not gene expression.

    (A) Western blot shows that synthetic siRNA treatment eliminated MSUT2 and PABPN1 protein in HEK293 cells. (B) Immunofluorescence staining of HEK293 cells overexpressing human tau. DAPI nuclear stain is blue, AT180 monoclonal antibody staining of pTau phosphorylated on Thr231 is green (86), and the positive staining control for total tau is red. Scale bar, 15 μm. (C) Immunofluorescence image of HEK293 cells overexpressing human tau shows MSUT2 knockdown by siRNA and a decrease in pTau accumulation. DAPI nuclear stain is blue, AT180 monoclonal antibody staining of pTau is green (86), and the positive staining control for total tau is red. Scale bar, 15 μm. (D) Immunofluorescence image of HEK293 cells overexpressing human tau shows PABPN1 knockdown by siRNA and an increase in pTau accumulation. DAPI nuclear stain is blue, AT180 monoclonal antibody staining of pTau is green (86), and the positive staining control for total tau is red. Scale bar, 15 μm. Image adjustments applied linear contrast and brightness changes. (E) Quantitation of immunostaining for pTau detected by AT180 monoclonal antibody, conformation-dependent tau detected by MC1 monoclonal antibody (87), and tau oligomeric complex-1 detected by TOC1 monoclonal antibody (38, 88). Error bars are SEM. Comparison of MSUT2 siRNA–treated to untreated HEK293 cells overexpressing human tau stained with AT180 antibody, P < 0.005; comparison of PABPN1 siRNA–treated to untreated HEK293 cells overexpressing human tau stained with AT180 antibody, P < 0.001. (F) Effect on swimming behavior of modulating poly(A) RNA tail length through ccr-4 mutation in tau transgenic C. elegans. Four-day-old tau transgenic (Tg) worms with or without the loss-of-function ccr-4 mutation were placed in liquid, and their swimming activity was recorded. Tau transgenic worms exhibited better locomotion relative to tau transgenic ccr-4 mutant animals (P < 0.0001 by two-tailed Student’s t test). (G) Total brain RNA from Msut2 KO and C57BL/6J mice was analyzed by RNA-seq. Multidimensional scaling and principal components analysis of RNA sequencing data are shown. (H) Transcriptomic changes in RNA sequencing data from total brain RNA of Msut2 KO and C57BL/6J mice (normalized mean read count by gene). Differentially regulated genes are depicted as red spots. See table S1 for listing of all differentially expressed genes. (I) Alternative splicing changes in RNA sequencing data from total brain RNA of Msut2 KO and C57BL/6J mice (normalized mean read count by exon). Two hundred one differentially spliced exons were detected (indicated by red spots). See table S2 for listing of all differentially spliced exons. (J) Volcano plot of alternative polyadenylation site selection changes in RNA sequencing data from total brain RNA of Msut2 KO and C57BL/6J mice. Analysis of polyadenylation site selection was conducted using Dynamic Analysis of Alternative Polyadenylation from RNA-seq (DaPars). Ten genes exhibited a change in percentage of distal poly(A) site usage (PDUI) of greater than 20% (see red dots). See table S3 for listing of all genes with significant alterations in polyadenylation site selection.

  • Fig. 4 MSUT2 potentiates neuroinflammation, tau pathology, and neurodegeneration in mouse brain.

    (A) Representative brain sections from Tau4RTg2652 mice injected with AAV-Msut2 or AAV-Gfp as control. Brain sections of 4-month-old Tau4RTg2652 mice with tauopathy were stained with the phospho-dependent anti-tau monoclonal antibody AT180 to detect pTau phosphorylated on Thr231 (arrows indicate the CA1 pyramidal cell layer). MSUT2 overexpression exacerbated pTau accumulation in the ipsilateral CA1 region of mouse brain compared to mice receiving an AAV-Gfp injection. Right: Densitometry analysis of AT180-positive pathological tau deposits in Tau4RTg2652 mice overexpressing Msut2 (n = 15) compared to GFP (n = 5) (P = 0.0044 by two-tailed t test). Scale bars, 100 μm. (B) Representative brain sections of the ipsilateral CA3 region of Tau4RTg2652 mice injected with AAV-Msut2 or AAV-Gfp as control. MSUT2 overexpression exacerbated neuronal loss in the CA3 region compared to GFP overexpression. Neuronal cell bodies were detected by cresyl violet staining (purple; arrows indicate CA3 pyramidal cell layer). Right: Densitometry analysis of pyramidal layer staining in Tau4RTg2652 mice overexpressing MSUT2 (n = 15) compared to GFP (n = 5) (P = 0.0001 by two-tailed t test). Scale bars, 100 μm. (C) Representative brain sections of the ipsilateral CA3 region of Tau4RTg2652 mice injected with AAV-Msut2 or AAV-Gfp. Microgliosis was detected by immunohistochemical staining of glia with the IBA1 monoclonal antibody in CA3 brain sections from 4-month-old Tau4RTg2652 mice. MSUT2 overexpression exacerbated microgliosis compared to GFP overexpression. Right: Densitometry analysis of IBA1 immunoreactivity in Tau4RTg2652 mice injected with AAV-Msut2 (n = 13) compared to AAV-Gfp (n = 5) (P = 0.024 by two-tailed t test). Scale bars, 100 μm. (D) Representative brain sections of the ipsilateral CA3 region of Tau4RTg2652 mice injected with AAV-Msut2 or AAV-Gfp. Astrocytosis was detected by immunostaining of brain sections from 4-month-old Tau4RTg2652 mice with anti-GFAP monoclonal antibody. MSUT2 overexpression exacerbated astrocyte reactivity compared to GFP overexpression. Right: Densitometry analysis of GFAP immunoreactivity in Tau4RTg2652 mice overexpressing MSUT2 (n = 14) compared to GFP (n = 5) (P = 0.022 by two-tailed t test). Scale bars, 100 μm. (E) Representative brain sections of the entorhinal cortex of PS19 transgenic (Tg) mice analyzed for astrocytosis by immunohistochemical staining with anti-GFAP monoclonal antibody. Right: Densitometry analysis of GFAP reactivity in the entorhinal cortex of Msut2 KO/PS19 Tg mice (n = 23) compared to PS19 Tg mice (n = 23) (P = 0.0104 by two-tailed t test). Scale bars, 100 μm. Photomicrographs in (A) to (E) were modified by adjusting brightness and contrast to optimize visualization of staining.

  • Fig. 5 MSUT2 activity potentiates neuroinflammation induced by tauopathy in human brain.

    (A) Representative brain section of postmortem brain frontal cortex from an AD case that was coimmunostained for pathological tau (red) and MSUT2 (green). White arrow indicates an MSUT2-positive neuron harboring tau tangles. Pathological tau was detected by immunofluorescence using the AT180 monoclonal antibody (86), and MSUT2 was detected using a polyclonal anti-MSUT2 antibody (32). Scale bar, 100 μm. (B) Representative brain sections of postmortem brain cortex from two AD cases and a neurologically normal control immunostained for MSUT2 (brown). Middle: AD cortex brain section with abundant MSUT2 protein. Right: AD cortex brain section with undetectable MSUT2 protein (see table S4 for characteristics of AD cases). Scale bar, 50 μm. (C) Representative brain sections of postmortem brain cortex from two AD cases and a neurologically normal control immunostained for PABPN1 (brown). PABPN1 protein was abundant in the normal control and one AD case (middle), whereas the other AD case had greatly reduced PABPN1. Scale bar, 50 μm. (D) Different amounts of MSUT2 in AD postmortem brain tissue depicted as a function of age at disease onset. AD cases with normal amounts of MSUT2 in postmortem brain (n = 14) had a later age of disease onset compared to those with depleted MSUT2 (n = 13) (P = 0.0306 by Student’s t test). (E) Different amounts of PABPN1 in AD postmortem brain tissue depicted as a function of age at disease onset. AD cases with normal amounts of PABPN1 in postmortem brain (n = 16) had a later age of disease onset compared to those with depleted PABPN1 (n = 11) (P = 0.0084 by Student’s t test). (F) Representative sections from postmortem brain cortex of an AD case with a normal amount of PABPN1/MSUT2 complexes (PMC+) compared to an AD case depleted of PABPN1/MSUT2 complexes (PMC depleted). Brain sections were stained with anti-GFAP monoclonal antibody to detect activated astrocytes. Right: Densitometry analysis of GFAP-positive astrocyte reactivity in PMC-normal AD cases (n = 9) compared to PMC-depleted AD cases (n = 6) (P = 0.0139 by two-tailed t test). Scale bars, 50 μm. (G) Representative brain sections from postmortem brain frontal cortex of an AD case with a normal amount of PABPN1/MSUT2 complexes (PMC+) compared to an AD case depleted of PABPN1/MSUT2 complexes (PMC-depleted). Brain sections were stained with anti-IBA1 monoclonal antibody to detect microgliosis. Right: Densitometry analysis of IBA1 reactivity in PMC-positive AD cases (n = 9) compared to PMC-depleted AD cases (n = 6) (P = 0.069 by two-tailed t test). Scale bars, 50 μm. (H) Representative brain sections from postmortem brain cortex of an AD case with a normal amount of PABPN1/MSUT2 complexes in the brain (PMC+) compared to an AD case depleted of PABPN1/MSUT2 complexes in the brain (PMC-depleted). Brain tissue from PMC-depleted AD cases exhibited more pathological tau as shown by immunostaining with the anti–phosphorylated tau antibody AT180. Right: Densitometry analysis of AT180-positive reactivity in PMC-positive AD cases (n = 14) compared to PMC-depleted AD cases (n = 13) (P = 0.0089 by two-tailed t test). Scale bars, 500 μm. (I) Representative brain sections from postmortem brain cortex of an AD case with a normal amount of PABPN1/MSUT2 complexes in the brain (PMC+) compared to an AD case depleted of PABPN1/MSUT2 complexes in the brain (PMC-depleted). Brain tissue from PMC-depleted AD cases exhibited decreased NeuN immunoreactivity (indicative of more neuronal loss) than did brain tissue from PMC-positive AD cases. Right: Densitometry analysis of NeuN reactivity in PMC-positive AD cases (n = 14) compared with PMC-depleted AD cases (n = 13) (P = 0.0039 by two-tailed t test). Scale bars, 500 μm. Photomicrographs in (B), (C), and (F) to (I) were modified by adjusting brightness and contrast to optimize visualization of staining.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/523/eaao6545/DC1

    Fig. S1. Characterization of Msut2 KO mice.

    Fig. S2. Msut2 KO decreases accumulation of pathological tau.

    Fig. S3. Msut2 KO mice have normal cognitive and locomotor abilities.

    Fig. S4. MSUT2, PABPN1, and poly(A) mRNA colocalize in nuclear speckles.

    Fig. S5. MSUT2 and PABPN1 have opposing effects on tau aggregation.

    Fig. S6. MSUT2 overexpression in mouse hippocampus using AAV-Msut2.

    Fig. S7. Effect of MSUT2 overexpression on astrocytes in the mouse hippocampus.

    Fig. S8. Msut2 deletion suppresses reactive microgliosis in PS19 mice.

    Fig. S9. Msut2 deletion does not protect against astrocytosis induced by kainic acid.

    Fig. S10. Schematic showing the potential molecular mechanism of MSUT2 activity.

    Table S1. Msut2 KO versus C57BL/6 RNA-seq gene expression changes.

    Table S2. Msut2 KO versus C57BL/6 RNA-seq exon inclusion changes.

    Table S3. Msut2 KO versus C57BL/6 RNA-seq poly(A) site selection.

    Table S4. Characteristics of human AD cases.

    Data file S1. Source data for Figs. 1D, 4, A to D, and 5, D to I.

  • The PDF file includes:

    • Fig. S1. Characterization of Msut2 KO mice.
    • Fig. S2. Msut2 KO decreases accumulation of pathological tau.
    • Fig. S3. Msut2 KO mice have normal cognitive and locomotor abilities.
    • Fig. S4. MSUT2, PABPN1, and poly(A) mRNA colocalize in nuclear speckles.
    • Fig. S5. MSUT2 and PABPN1 have opposing effects on tau aggregation.
    • Fig. S6. MSUT2 overexpression in mouse hippocampus using AAV-Msut2.
    • Fig. S7. Effect of MSUT2 overexpression on astrocytes in the mouse hippocampus.
    • Fig. S8. Msut2 deletion suppresses reactive microgliosis in PS19 mice.
    • Fig. S9. Msut2 deletion does not protect against astrocytosis induced by kainic acid.
    • Fig. S10. Schematic showing the potential molecular mechanism of MSUT2 activity.
    • Legends for tables S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Msut2 KO versus C57BL/6 RNA-seq gene expression changes.
    • Table S2 (Microsoft Excel format). Msut2 KO versus C57BL/6 RNA-seq exon inclusion changes.
    • Table S3 (Microsoft Excel format). Msut2 KO versus C57BL/6 RNA-seq poly(A) site selection.
    • Table S4 (Microsoft Excel format). Characteristics of human AD cases.
    • Data file S1 (Microsoft Excel format). Source data for Figs. 1D, 4, A to D, and 5, D to I.

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