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Cited by 152Alzheimer's disease research and treatmentsDementia and Cognitive Impairment ResearchAmyotrophic Lateral Sclerosis Research

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RNA binding proteins co-localize with small tau inclusions in tauopathy

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Maziuk, Brandon F., Apicco, Daniel J., Cruz, Anna Lourdes, Jiang, Lulu, Ash, Peter E. A., da Rocha, Edroaldo Lummertz, Zhang, Cheng, Yu, Wai Haung, Leszyk, John, Abisambra, Jose F., Li, Hu, Wolozin, Benjamin·2018·Acta Neuropathologica Communications
DOI 10.1186/s40478-018-0574-5PMC PMC6069705
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Abstract

The development of insoluble, intracellular neurofibrillary tangles composed of the microtubule-associated protein tau is a defining feature of tauopathies, including Alzheimer's disease (AD). Accumulating evidence suggests that tau pathology co-localizes with RNA binding proteins (RBPs) that are known markers for stress granules (SGs). Here we used proteomics to determine how the network of tau binding proteins changes with disease in the rTg4510 mouse, and then followed up with immunohistochemistry to identify RNA binding proteins that co-localize with tau pathology. The tau interactome networks revealed striking disease-related changes in interactions between tau and a multiple RBPs, and biochemical fractionation studies demonstrated that many of these proteins including hnRNPA0, EWSR1, PABP and RPL7 form insoluble aggregates as tau pathology develops. Immunohistochemical analysis of mouse and human brain tissues suggest a model of evolving pathological interaction, in which RBPs co-localize with pathological phospho-tau but occur adjacent to larger pathological tau inclusions. We suggest a model in which tau initially interacts with RBPs in small complexes, but evolves into isolated aggregated inclusions as tau pathology matures.

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Fig. 1 TIA1 preferentially colocalizes with phosphorylated tau that is not in NFTs. Immunohistochemical analysis of the stress granule nucleating protein TIA1 (red) shows colocalization with phosphorylated tau (CP13 antibody against pSer202, green) in rTg4510 (a), PS19 (b) and human AD tissues (c) with DAPI in blue. d Immunohistochemistry of TIA1 on rTg4510 with and without immunosorbtion using TIA1 peptide, as well as TIA1 staining of TIA1 knockout mouse tissue, also show that the TIA1 antibody used for this analysis is specific for TIA1 and does not have off-target staining. e Further analysis of TIA1-tau colocalization in rTg4510 tissues indicates that TIA1 primarily has cytoplasmic colocalization with smaller tau aggregates (white arrow) over larger NFT-like tau aggregates (pink arrow). This is quantified in (f) using Imaris Bitplane software, where cytoplasmic TIA1 intensity negatively correlates with the size of tau tangles in neurons (R = − 0.1617 with a two tailed p value of 0.0404). g TIA1 also colocalizes with oligomeric tau stained using the TOC1 antibody specific for oligomeric tau
Fig. 1 TIA1 preferentially colocalizes with phosphorylated tau that is not in NFTs. Immunohistochemical analysis of the stress granule nucleating protein TIA1 (red) shows colocalization with phosphorylated tau (CP13 antibody against pSer202, green) in rTg4510 (a), PS19 (b) and human AD tissues (c) with DAPI in blue. d Immunohistochemistry of TIA1 on rTg4510 with and without immunosorbtion using TIA1 peptide, as well as TIA1 staining of TIA1 knockout mouse tissue, also show that the TIA1 antibody used for this analysis is specific for TIA1 and does not have off-target staining. e Further analysis of TIA1-tau colocalization in rTg4510 tissues indicates that TIA1 primarily has cytoplasmic colocalization with smaller tau aggregates (white arrow) over larger NFT-like tau aggregates (pink arrow). This is quantified in (f) using Imaris Bitplane software, where cytoplasmic TIA1 intensity negatively correlates with the size of tau tangles in neurons (R = − 0.1617 with a two tailed p value of 0.0404). g TIA1 also colocalizes with oligomeric tau stained using the TOC1 antibody specific for oligomeric tauCC BY · PubMed Central
Fig. 2 RNA binding proteins change in their association to tau in tauopathy. Using Tau13 antibody, tau was immunoprecipitated from the cortex of either 2.5 month old induced or uninduced rTg4510 brains.The co-immunoprecipitating proteins were subsequently identified by mass spectrometry. A variety of nucleotide binding and ribonucleoproteins show differential association to tau in rTg4510 tissue compared to WT tissue as indicated by significantly upregulated and downregulated annotation terms (a). Part (b) represents a network diagram of proteins enriched in the tau interactome. Red nodes denote proteins whose association with tau is ≥5-fold increased in Tg4510 vs uninduced control. Blue nodes denote proteins whose association with tau is ≥5-fold decreased in Tg4510 vs control. Node size is proportional to degree of replication (N = 4), non-replicating proteins excluded. Key proteins include EWSR1, HNRNPA0, PABP, PCBP2, DDX5, TAF15, FMR1, RPL and RPS family members, and eIF4a2
Fig. 2 RNA binding proteins change in their association to tau in tauopathy. Using Tau13 antibody, tau was immunoprecipitated from the cortex of either 2.5 month old induced or uninduced rTg4510 brains.The co-immunoprecipitating proteins were subsequently identified by mass spectrometry. A variety of nucleotide binding and ribonucleoproteins show differential association to tau in rTg4510 tissue compared to WT tissue as indicated by significantly upregulated and downregulated annotation terms (a). Part (b) represents a network diagram of proteins enriched in the tau interactome. Red nodes denote proteins whose association with tau is ≥5-fold increased in Tg4510 vs uninduced control. Blue nodes denote proteins whose association with tau is ≥5-fold decreased in Tg4510 vs control. Node size is proportional to degree of replication (N = 4), non-replicating proteins excluded. Key proteins include EWSR1, HNRNPA0, PABP, PCBP2, DDX5, TAF15, FMR1, RPL and RPS family members, and eIF4a2CC BY · PubMed Central
Fig. 3 RNA binding proteins become insoluble in the cortex of rTg4510 mice. (a, b) Immunoblots of the sarkosyl soluble (S3) and insoluble (P3) fractions isolated from rTg4510 cortical tissues indicate that many RBPs become insoluble as tau pathology develops. The fractions were also probed for TDP-43, which is not associated with tau aggregation. Quantification of these immunoblots (c, d) shows statistically significant RBP accumulation in the P3 fraction of induced rTg4510 mouse cortex using a two-tailed t-test (p = 0.00599 for TAOK1; p = 0.0007599 for EWSR1; p = 0.0122 for TAF15; p = 0.000252 for RPL7; p = 0.00195 for PABP; p = 0.0926 for DDX5; p = 0.0638 for HNRNPA0)
Fig. 3 RNA binding proteins become insoluble in the cortex of rTg4510 mice. (a, b) Immunoblots of the sarkosyl soluble (S3) and insoluble (P3) fractions isolated from rTg4510 cortical tissues indicate that many RBPs become insoluble as tau pathology develops. The fractions were also probed for TDP-43, which is not associated with tau aggregation. Quantification of these immunoblots (c, d) shows statistically significant RBP accumulation in the P3 fraction of induced rTg4510 mouse cortex using a two-tailed t-test (p = 0.00599 for TAOK1; p = 0.0007599 for EWSR1; p = 0.0122 for TAF15; p = 0.000252 for RPL7; p = 0.00195 for PABP; p = 0.0926 for DDX5; p = 0.0638 for HNRNPA0)CC BY · PubMed Central
Fig. 4 RNA binding proteins show significant colocalization with diffuse phospho-tau but not NFTs in the rTg4510 cortex. (a) Immunohistochemical analysis of rTg4510 tissue (n = 3) has also revealed a significant colocalization in the cortex between the RBPs DDX6, PABP, HNRNPA0, and eIF2a (red) with pathological phospho-tau stained using the CP13 antibody (green). However, the RBP and splicing factor U2AF2 does not show significant correlation. To the right of each merged image is a scatterplot of the pixel intensities for each pixel of the image in the red channel vs. the green (Pearson correlation coefficients r = 0.773 for DDX6, 0.791 for eIF2α, 0.325 for HNRNPA0, 0.798 for PABP, and − 0.14 for U2AF2). This colocalization is greatly reduced and/or completely lost as tau aggregates into large NFTs which are brightly fluorescent and fill the cell bodies of neurons (b) (r = 0.069 for DDX6, 0.372 for eIF2α, 0.481 for PABP, − 0.03 for HNRNPA0, and − 0.009 for U2AF2). c Staining of wild-type C57Bl/6 mice also indicates that HNRNPA0 is predominantly nuclear in healthy animals, while the rTg4510 staining shows significant cytoplasmic localization of HNRNPA0 (a, b). (d) Negative controls IHC using rabbit and mouse normal IgG indicates that there is no off target staining or fluorescence in our tissues. e Pearson coefficients of correlation between CP13 positive tau with RBPs DDX6, eIF2α, HNRNPA0, PABP, and U2AF2 are graphed for individual neurons using ImageJ. For all cases except U2AF2, neurons show heterogeneity in colocalization between phospho-tau and the RBPs stained, from no colocalization to fully overlapping reactivity patterns in individual neurons. The percent of neurons with r > 0.3 is graphed in (f) as the percentage of neurons showing moderate to strong correlations between green:red intensity (DDX6 = 36% of neurons; eIF2α = 54% of neurons; HNRNPA0 = 35% of neurons; PABP = 33% of neurons; U2AF2 = 0% of neurons)
Fig. 4 RNA binding proteins show significant colocalization with diffuse phospho-tau but not NFTs in the rTg4510 cortex. (a) Immunohistochemical analysis of rTg4510 tissue (n = 3) has also revealed a significant colocalization in the cortex between the RBPs DDX6, PABP, HNRNPA0, and eIF2a (red) with pathological phospho-tau stained using the CP13 antibody (green). However, the RBP and splicing factor U2AF2 does not show significant correlation. To the right of each merged image is a scatterplot of the pixel intensities for each pixel of the image in the red channel vs. the green (Pearson correlation coefficients r = 0.773 for DDX6, 0.791 for eIF2α, 0.325 for HNRNPA0, 0.798 for PABP, and − 0.14 for U2AF2). This colocalization is greatly reduced and/or completely lost as tau aggregates into large NFTs which are brightly fluorescent and fill the cell bodies of neurons (b) (r = 0.069 for DDX6, 0.372 for eIF2α, 0.481 for PABP, − 0.03 for HNRNPA0, and − 0.009 for U2AF2). c Staining of wild-type C57Bl/6 mice also indicates that HNRNPA0 is predominantly nuclear in healthy animals, while the rTg4510 staining shows significant cytoplasmic localization of HNRNPA0 (a, b). (d) Negative controls IHC using rabbit and mouse normal IgG indicates that there is no off target staining or fluorescence in our tissues. e Pearson coefficients of correlation between CP13 positive tau with RBPs DDX6, eIF2α, HNRNPA0, PABP, and U2AF2 are graphed for individual neurons using ImageJ. For all cases except U2AF2, neurons show heterogeneity in colocalization between phospho-tau and the RBPs stained, from no colocalization to fully overlapping reactivity patterns in individual neurons. The percent of neurons with r > 0.3 is graphed in (f) as the percentage of neurons showing moderate to strong correlations between green:red intensity (DDX6 = 36% of neurons; eIF2α = 54% of neurons; HNRNPA0 = 35% of neurons; PABP = 33% of neurons; U2AF2 = 0% of neurons)CC BY · PubMed Central
Fig. 5 RBPs show granularization and interfacing with NFTs in late stage human AD tissue. Immunohistochemical analysis of human AD frontal cortex tissue (n = 6) shows that RBPs (red) and NFTs stained with CP13 tau (green) do not colocalize (r = 0.001 for DDX6; 0.176 for eIF2α; 0.031 for HNRNPA0; and 0.222 for PABP). However, trace analyses across lines within the image (yellow bars) indicate that peak fluorescent intensities (reported as fluorescence intensity plots over the distance of the line for tau in green and the RBP in red) between phospho-tau and multiple RBPs are immediately adjacent to each other, indicating protein interfacing or interaction between the edges of NFTs and RBPs
Fig. 5 RBPs show granularization and interfacing with NFTs in late stage human AD tissue. Immunohistochemical analysis of human AD frontal cortex tissue (n = 6) shows that RBPs (red) and NFTs stained with CP13 tau (green) do not colocalize (r = 0.001 for DDX6; 0.176 for eIF2α; 0.031 for HNRNPA0; and 0.222 for PABP). However, trace analyses across lines within the image (yellow bars) indicate that peak fluorescent intensities (reported as fluorescence intensity plots over the distance of the line for tau in green and the RBP in red) between phospho-tau and multiple RBPs are immediately adjacent to each other, indicating protein interfacing or interaction between the edges of NFTs and RBPsCC BY · PubMed Central
Fig. 6 Model for the evolution of tau pathology a Stress elicits translocation of nuclear RBPs, such as TIA1 and HNRNPA0, to the cytoplasm where they distribute diffusely in the cytoplasm. b Core nucleating RBPs, such as TIA1, coalesce to form SGs, in a process that is stimulated by tau oligomers. Secondary nucleation brings in other RBPs, such as EWSR1, PABP, DDX6, EIFs, etc. c Persistent stress (such as occurs in disease) causes particularly insoluble RBPs (and tau) to consolidate to form persistent pathological stress granules. More soluble RBPs separate from the persistent pathological SG, and disperse without aggregating. d The tau oligomers evolve into fibrils forming pathological puncta, which contain classic markers of pathology such as hyper-phosphorylation and ubiquitination [43]. Other RBPs that remain associated with the persistent pathological SG, begin to form aggregated puncta around the tau puncta. e Tau fibrillizes forming neurofibrillary tangles, which are relatively inert, do not act in the translational stress response and contain few RBSs
Fig. 6 Model for the evolution of tau pathology a Stress elicits translocation of nuclear RBPs, such as TIA1 and HNRNPA0, to the cytoplasm where they distribute diffusely in the cytoplasm. b Core nucleating RBPs, such as TIA1, coalesce to form SGs, in a process that is stimulated by tau oligomers. Secondary nucleation brings in other RBPs, such as EWSR1, PABP, DDX6, EIFs, etc. c Persistent stress (such as occurs in disease) causes particularly insoluble RBPs (and tau) to consolidate to form persistent pathological stress granules. More soluble RBPs separate from the persistent pathological SG, and disperse without aggregating. d The tau oligomers evolve into fibrils forming pathological puncta, which contain classic markers of pathology such as hyper-phosphorylation and ubiquitination [43]. Other RBPs that remain associated with the persistent pathological SG, begin to form aggregated puncta around the tau puncta. e Tau fibrillizes forming neurofibrillary tangles, which are relatively inert, do not act in the translational stress response and contain few RBSsCC BY · PubMed Central

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Authors
Maziuk, B. F., Apicco, D. J., Cruz, A. L., Jiang, L., Ash, P. E. A., da Rocha, E. L., Zhang, C., Yu, W. H., Leszyk, J., Abisambra, J. F., Li, H., & Wolozin, B.
Affiliations
  • Boston University (US)
  • Mayo Clinic (US)
  • Columbia University Irving Medical Center (US)
  • University of Massachusetts Chan Medical School (US)
  • University of Kentucky (US)
Corresponding author
Wolozin, Benjamin
ORCID
Venue
Acta Neuropathologica Communications
Publisher
Springer Science and Business Media LLC
Published
2018-08-01
Volume
6
Issue
1
Pages
71-71
ISSN
2051-5960 (ISSN-L)
DOI
10.1186/s40478-018-0574-5
PMC
PMC6069705
OpenAlex
W2887193453
Cited by
151
References
45
Citations by year
2026: 18 · 2025: 17 · 2024: 14 · 2023: 20 · 2022: 18 · 2021: 17 · 2020: 25 · 2019: 17 · 2018: 5
FWCI
10.98
Type
journal article
Language
en
Open access
Licence
Locations
  • Acta Neuropathologica Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship@UMassChan (submittedVersion, cc-by)
  • UKnowledge (University of Kentucky) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institute on Aging (AG050471, AG056318, AG028383)
  • National Institute of Neurological Disorders and Stroke (NS089544, NS106751, NS091329)
  • National Institute of Environmental Health Sciences (ES020395)
  • BrightFocus Foundation
  • Alzheimer Association
  • Cure Alzheimer's Fund
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (MD009205)
  • U.S. Department of Defense (AZ140097)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
  • Health Sciences › Medicine › Clinical Neurology › Amyotrophic Lateral Sclerosis Research
Keywords
tauopathy, tau protein, RNA-binding proteins, stress granules, neurofibrillary tangles, EWSR1, poly(A)-binding protein, protein aggregation, phosphorylated tau, Alzheimer's disease, protein colocalization
MeSH terms
T-Cell Intracellular Antigen-1, Aged, Aged, 80 and over, Animals, Brain, Inclusion Bodies, Endodeoxyribonucleases, Humans, Male, Mice, Transgenic, Middle Aged, Phosphorylation, Mass Spectrometry, RNA-Binding Proteins, Neurofibrillary Tangles, tau Proteins, Cell Cycle Proteins, Tauopathies, Proteomics, Immunoprecipitation, Mice, Protein Interaction Maps, Protein Aggregation, Pathological

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