Proteomics approach to understanding tau co-aggregation and interacting partners in Alzheimer’s disease and related tauopathies Restricted; Files Only
Shapley, Sarah (Summer 2025)
Abstract
In Alzheimer’s disease (AD) and other tauopathies, tau dissociates from microtubules and forms toxic aggregates that contribute to neurodegeneration. Although some of the pathological interactions of tau have been identified from postmortem brain tissue, these studies are limited by their inability to capture transient interactions. To investigate the interactome of aggregate-prone fragments of tau, we applied an in vitro proximity labeling technique using split TurboID biotin ligase (sTurbo) fused with the tau microtubule repeat domain (TauRD), a core region implicated in tau aggregation. We characterized sTurbo TauRD co-expression, robust enzyme activity and nuclear and cytoplasmic localization in a human cell line. Following enrichment of biotinylated proteins and mass spectrometry, we identified over 700 TauRD interactors. Gene ontology analysis of enriched TauRD interactors highlighted processes often dysregulated in tauopathies, including spliceosome complexes, RNA-binding proteins (RBPs), and nuclear speckles. The disease relevance of these interactors was supported by integrating recombinant TauRD interactome data with human AD tau interactome datasets and protein co-expression networks from individuals with AD and related tauopathies. This revealed an overlap with the TauRD interactome and several modules enriched with RBPs and increased in AD and Progressive Supranuclear Palsy (PSP). The sTurbo TauRD interactome also highly mapped to insoluble proteins in detergent-insoluble tauopathies. These findings emphasize the importance of nuclear pathways in tau pathology, such as RNA splicing and nuclear-cytoplasmic transport, which are traditionally enriched in insoluble fractions in disease. It has thus far been difficult to parse tau co-aggregating partners, however, sTurbo TauRD system mapped to multiple disease-relevant insoluble proteins associated with insoluble tau levels in tauopathies, establishing the sTurbo TauRD system as a valuable tool for exploring the tau interactome.
Table of Contents
Table of Contents
1.0 Introduction: Tau and its co-aggregating partners in Alzheimer’s disease &
related tauopathies
1.1 Physiological and pathological tau
1.1.1 Isoform expression, structure, and localization
1.1.2 Tau homeostatic function:
1.2 Tauopathies: an overview
1.2.1 Primary & secondary tauopathies:
1.2.2 3R, 4R, and mixed tauopathies:
1.2.3 An introduction to pathological tau:
1.2.4 Tau post-translational modifications (PTMs):
1.2.5 The tau aggregation continuum:
1.3 Methods to investigate tauopathies
1.3.1 Proteomics of postmortem brain tissue
1.3.2 Cellular models
1.3.3 In vitro biochemical assay
1.3.4 Animal models
1.4 Investigating insoluble co-aggregating partners of tau
1.4.1 RNA-binding proteins role in neurodegeneration
1.4.2 Fibril formation LLPS/RBPs
1.5 Current therapeutics targeting tauopathies
1.5.1 FDA-approved therapies:
1.5.2 Tau therapeutic targets:
1.5.3 Developing therapeutics
1.6 Research aims & contributions
1.7 Figures
Figure 1.1 Tau spread, cell-type involvement, core tau fibril structure, and symptoms
across several distinct tauopathies.
Figure 1.2: Tau aggregation continuum
Figure 1.3: HEK-FRET TauRD seeding workflow
Figure 1.4: Proposed model of in vitro tau seeding
Figure 1.5: Capturing insoluble co-aggregating tau interactors
2.0 Marmosets as model systems for the study of Alzheimer's disease and related dementias: Substantiation of physiological tau 3R and 4R isoform expression
and phosphorylation
2.1 Introduction
2.2 Results
2.2.1 MAPT mRNA and protein expression in the marmoset brain
2.2.2 Mass spectrometry of 3R and 4R Tau
2.2.3 Visualization of 3R and 4R Tau expression in marmoset brain
2.2.4 Tau isoform expression in the synaptic region
2.2.5 Tau phosphorylation, oligomerization in marmoset brain
2.3 Materials and methods
2.4 Discussion
2.5 Figures
Figure 2.1: 3R and 4R tau expression in marmoset brain.
Figure 2.2. Quantification 3R Tau expression in marmoset brain.
Figure 2.3. Schematic mass spectrometry overview using a complementary dual
enzymatic digestion approach with Trypsin and LysargiNase (LysArg).
Figure 2.4: 3R and 4R tau expression and ratio quantification by LysArg digestion in marmoset brain by targeted mass spectrometry.
Figure 2.5. Light and heavy representative peptide peaks for quantitative mass spectrometry across human, marmoset, and mouse brain lysates.
Figure 2.6. 3R and 4R Tau expression by Trypsin in Marmosets by targeted mass spectrometry.
Figure 2.7. 3R Tau and 4R tau expression in hippocampus and entorhinal cortex of marmoset brain.
Figure 2.8. Negative control background staining of IF.
Figure 2.9. Immunohistochemistry of 12-year aged marmoset brain.
Figure 2.10. 4R tau and 3R tau are expressed in the synaptic region of the marmoset prefrontal cortex.
Figure 2.11. Phosphorylated tau in Sarkosyl soluble and insoluble fractions from
marmoset brain.
Table 2.1. Average demographics of post-mortem human brain tissue.
Table 2.2. Demographics of case traits for human tissues.
3.0 sTurbo TauRD capturing insoluble-enriched RNA-binding proteins related to tauopathies
3.1 Introduction
3.2 Result
3.2.1 Establishment of a Split-TurboID TauRD Proximity Labeling System in Cell
Culture
3.2.2 Development and Validation of a Bicistronic sTurbo TauRD Construct for
Uniform Expression and Functional Proximity Labeling in HEK293 Cells
3.2.3 Proximity Labeling of Tau MTBR Interactors and Mass Spectrometry Analysis
in Cells Reveals Established and Novel Tau-associated Pathways
3.2.4 Network Analysis of Human AD and Progressive Supranuclear Palsy Brain
Tissue Identifies Protein Modules Associated with Tau Pathology
3.2.5 Integrating the TauRD Interactome with AD and PSP Network Modules to
Identify Disease-Associated Modules Linked to Tau PPIs
3.3 Materials and methods
3.4 Discussion
3.5 Figures
Figure 3.1: sTurbo TauRD expression and biotin ligase activity in a human cell line.
Figure 3.2: Development and characterization of a bicistronic sTurbo TauRD construct
and functional proximity labeling.
Figure 3.3: 2A efficiently cleaves fragments and equally biotinylates sTurbo TauRD fragments.
Figure 3.4: Differential abundance analysis of TauRD proteomics demonstrates
nuclear protein enrichment.
Figure 3.5: Quality control western blots and total protein silver stains.
Figure 3.6. Intersection of tau-interactome datasets reveals core protein functions
shared between human AD Tau and sTurbo TauRD.
Figure 3.7: Network analysis of human AD and progressive supranuclear palsy (PSP)
brain tissue.
Figure 3.8: Integration of the TauRD interactome with a human tauopathy proteomic network identifies RNA-binding protein modules associated with AD and PSP that are enriched in insoluble proteins.
Figure 3.9: Insoluble disease mapping module classification across RBP localization,
key functions, and domains.
Figure 3.10: Network visualization of significant human RNA binding protein modules
that overlap with the sTurbo TauRD interactome.
Figure 3.11: Insoluble and RBP-enriched module hub proteins and BioGrid interaction Networks.
Figure 3.12: Tau-AP enriched proteins correlate with insoluble tau levels in disease
4.0 Discussion and Future Directions
4.1 Summary and contributions of Marmoset tau characterization
4.2 Summary and contributions of sTurbo TauRD interactome and integration
with human tauopathies
4.3 Additional experimental considerations
4.3.1 Development of additional in vivo & in vitro models of tauopathy
4.3.2 Mechanistic validation of candidate proteins:
4.3.3 Integrating proteomics from additional brain regions
4.4 Combined study contributions and future directions
4.5 Figures
Figure 4.1 In-depth TMT proteomic measurement of tryptic 3R and 4R tau peptides.
Figure 4.2: Summary of study contributions & future directions
5.0 References
Figure list Page
Figure 1.1 Tau spread, cell-type involvement, core tau fibril structure, and symptoms
across several distinct tauopathies. ............................................................................................ 24
Figure 1.2: Tau aggregation continuum ...................................................................................... 25
Figure 1.3: HEK-FRET TauRD seeding workflow ....................................................................... 26
Figure 1.4: Proposed model of in vitro tau seeding .................................................................... 27
Figure 1.5: Capturing insoluble co-aggregating tau interactors .................................................. 27
Figure 2.1: 3R and 4R tau expression in marmoset brain. ......................................................... 56
Figure 2.2. Quantification 3R Tau expression in marmoset brain. .............................................. 57
Figure 2.3. Schematic mass spectrometry overview using a complementary dual
enzymatic digestion approach with Trypsin and LysargiNase (LysArg). .................................... 58
Figure 2.4: 3R and 4R tau expression and ratio quantification by LysArg digestion in
marmoset brain by targeted mass spectrometry. ....................................................................... 60
Figure 2.5. Light and heavy representative peptide peaks for quantitative mass
spectrometry across human, marmoset, and mouse brain lysates. ........................................... 62
Figure 2.6. 3R and 4R Tau expression by Trypsin in Marmosets by targeted mass
spectrometry. .............................................................................................................................. 64
Figure 2.7. 3R Tau and 4R tau expression in hippocampus and entorhinal cortex of
marmoset brain. .......................................................................................................................... 66
Figure 2.8. Negative control background staining of IF. ............................................................. 68
Figure 2.9. Immunohistochemistry of 12-year aged marmoset brain. ........................................ 70
Figure 2.10. 4R tau and 3R tau are expressed in the synaptic region of the marmoset
prefrontal cortex. ......................................................................................................................... 72
Figure 2.11. Phosphorylated tau in Sarkosyl soluble and insoluble fractions from
marmoset brain. .......................................................................................................................... 74
Table 2.1. Average demographics of post-mortem human brain tissue. .................................... 75
Table 2.2. Demographics of case traits for human tissues. ........................................................ 76
Figure 3.1: sTurbo TauRD expression and biotin ligase activity in a human cell line. .............. 108
Figure 3.2: Development and characterization of a bicistronic sTurbo TauRD construct
and functional proximity labeling. .............................................................................................. 110
Figure 3.3: 2A efficiently cleaves fragments and equally biotinylates sTurbo TauRD
fragments. ................................................................................................................................. 112
Figure 3.4: Differential abundance analysis of TauRD proteomics demonstrates nuclear
protein enrichment. ................................................................................................................... 114
Figure 3.5: Quality control western blots and total protein silver stains. ................................... 117
Figure 3.6. Intersection of tau-interactome datasets reveals core protein functions shared
between human AD Tau and sTurbo TauRD. .......................................................................... 118
Figure 3.7: Network analysis of human AD and progressive supranuclear palsy (PSP) brain
tissue. ....................................................................................................................................... 120
Figure 3.8: Integration of the TauRD interactome with a human tauopathy proteomic
network identifies RNA-binding protein modules associated with AD and PSP that are
enriched in insoluble proteins. .................................................................................................. 122
Figure 3.9: Insoluble disease mapping module classification across RBP localization, key
functions, and domains. ............................................................................................................ 123
Figure 3.10: Network visualization of significant human RNA binding protein modules that
overlap with the sTurbo TauRD interactome. ........................................................................... 125
Figure 3.11: Insoluble and RBP-enriched module hub proteins and BioGrid interaction
Networks. .................................................................................................................................. 127
Figure 3.12: Tau-AP enriched proteins correlate with insoluble tau levels in disease .............. 129
12
Figure 4.1 In-depth TMT proteomic measurement of tryptic 3R and 4R tau peptides. ............. 142
Figure 4.2: Summary of study contributions & future directions ............................................... 143
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