DOI: 10.5061/dryad.6m905qg89
title: Aggregate selective removal of pathological tau via clustering-activated degraders

description: Selective degradation of pathological protein aggregates while sparing
 monomeric forms is of major therapeutic interest. The E3 ligase TRIM21
 degrades antibody-bound proteins in an assembly state-specific manner,
 owing to the requirement of TRIM21 RING domain clustering for activation,
 yet effective targeting of intracellular assemblies remains challenging.
 Here, we fused the RING domain of TRIM21 to a target-specific nanobody to
 create intracellularly expressed constructs capable of selectively
 degrading assembled proteins. We evaluated this approach against histone
 2B-GFP and tau, a protein that undergoes pathological aggregation in
 Alzheimer’s and other neurodegenerative diseases. RING-nanobody degraders
 prevented or reversed tau aggregation in culture and in vivo, with minimal
 impact on monomeric tau. This approach may have therapeutic potential for
 the many disorders driven by intracellular protein aggregation.
description: Data presented in this publication has been collected via the
 following methods High content fluorescent
 microscopy Single molecule pull down (SiMPULL) super
 resolution microscopy Flow Cytometry
 Semi-quantitative immunoblotting
 Quantitative immunofluorescence
 ELISA Western blot band
 densitometry
description: # Aggregate-selective removal of pathological tau via clustering-activated
 degraders
 [https://doi.org/10.5061/dryad.6m905qg89](https://doi.org/10.5061/dryad.6m905qg89) Raw data from all graphs presented in the manuscript are collected here in excel format. The title of each file denotes the figure it relates to. Detailed experimental procedures used to collect data for each of the files presented can be found within the figure legends and methods section of the manuscript.  N/A in empty cells refers to not applicable. Used when data was not collected for a certain condition (i.e. mCherry fluorescence in non-transfected cells) or when the cells in that column are used to calculate an average from multiple cells in a previous column, therefore resulting in empty cells.  ## Extra Data Files Mass spectrometry raw files and data analysis files produced in Spectronaut have been uploaded to PRIDE ([https://doi.org/10.1093/nar/gkab1038](https://doi.org/10.1093/nar/gkab1038)) under the accession number PXD052957. ## **Description of Datasets and File Structure:** **Fig. 1F & S1D - Live imaging aggregated of Tau-Venus puncta and mCherry expression levels in TVA cells expressing DOX inducible constructs.** *Column Titles* TimeLapse Index(T): Time in hours since addition of Doxycycline to cells Well Name: The well of the 96 well plate that images and quantified data were collected from Tau Venus Puncta Count: The average number of Venus puncta across 3 images from one well mCherry Sum Intensity: The average sum mCherry signal intensity across 3 images from one well % Puncta compared to 0hr timepoint: Tau Venus Puncta Count normalised against 0 hour timepoint - used to plot the % of Tau-Venus aggregates remaining compared to before Doxycycline addition **Fig. 1K - Super Resulotion imaging analysis of TVA cell lysates following R-NbF8-2 expression.** *Column Titles* Image: The filename of the image being analysed Sample: The experimental condition the image belongs to Antibody: The antibody used to perform super resolution microscopy (Ht7 is a pan-tau antibody) Replicate: The replicate number of each condition the image belongs to average length (nm) with 50 nm filter: The average size of identified particles in nm, with particles shorter than 50nm being excluded from calculations. number: The number of particles detected in each image Mean number: The mean number of particles detected from technical replicates average length (nm) with 30 nm filter: The average size of identified particles in nm, with particles shorter than 30nm being excluded from calculations. number of clusters with 30 nm filter: The number of particles detected in each image, with particles shorter than 30nm being excluded Mean number of clusters with 30 nm filter: The mean number of particles detected from technical replicates, with particles shorter than 30nm being excluded **Fig. 2A & S3B - Fluorescent microscopy quantification of aggregated Tau-Venus puncta in TVA cells following transfection with various R-Nb constructs.** *Column Titles* Well Name: The well of the 96 well plate that images and quantified data were collected from Labels: The experimental condition each data point refers to - nanobodies used in R-Nb constructs are given, followed by which biological repeat they belong to mCherry Intensity: The sum mCherry signal Transfected Cells with aggregates (%): The percentage of mCherry positive cells (transfected) containing at least one tau-venus aggregate puncta All cells with aggregates (%): The percentage of all cells (transfected or not) containing at least one tau-venus aggregate puncta Average Red Cells Seeded: The average percentage of mCherry positive cells (transfected) containing at least one tau-venus aggregate puncta, from all technical repeats Average Green Cells Seeded: The average percentage of all cells (transfected or not) containing at least one tau-venus aggregate puncta, from all technical repeats Average mCh Fluorescence: The average sum mCherry signal across technical repeats **Fig. 2B & S3C - Flow cytometry quantification of soluble Tau-Venus levels in TVS cells following transfection with various R-Nb constructs.** *Column Titles* Condition: The experimental condition each data point refers to - nanobodies used in R-Nb constructs are given, followed by which biological repeat they belong to Mean mCherry Intensity: The average mCherry signal of mCh+ gated cells Mean Venus Intensity: The average Venus signal of mCh+ gated cells **Fig. 2E - Immunoblot quantification of soluble Tau-Venus levels in TVS cells expressing Doxycycline inducible R-NbF8-2.** *Column Titles* Sample: The experimental condition the image belongs to - the number following indicates which biological repeat each row represents GAPDH: The intensity of the GAPDH (GAPDH antibody) loading control band quantified in *Compass for SW* software from JESS immunoblot Tau: The intensity of the Tau (Tau12 antibody) band quantified in *Compass for SW* software from JESS immunoblot CypA: The intensity of the CypA (CypA antibody) band quantified in *Compass for SW* software from JESS immunoblot Tau Normalised to GAPDH, then to CypA: The intensity value of the Tau band was normalised to both the GAPDH and CypA loading controls Normalised to - DOX being 100%: The aforementioned values normalised against the loading controls was then divided by the average values for the -DOX conditions to generate data plotted as % tau remaining following DOX addition, relative to levels in -DOX cells \**Fig. 2F - Fluorescent microscopy quantification of aggregated Tau-Venus puncta in TVS cells (with or without DOX to induce R-NbF8-2 expression) following addition of varying concentrations of heparain assembled tau. ** *Column Titles* Well Name: The well of the 96 well plate that images and quantified data were collected from Labels: The experimental condition each data point refers to - Samples are either PLUS DOX or MINUS DOX. The first number following DOX indicates which biological replicate the data belongs to. The final number indicates what dose of heparin assembled tau has been added (in nM). LF is the negative control where no heparin assembled tau has been added DOX_3B2_Seeding.Green_Cells.Count: The count of green cells (TVS) identified DOX_3B2_Seeding.Green_Cells.MeanIntensity: The average Venus intensity of  TVS cells DOX_3B2_Seeding.red_cells.Count: The count of mCherry positive cells (reporter for R-NbF8-2 expression) DOX_3B2_Seeding.red_cells.MeanIntensity: The average mCherry intensity of cells DOX_3B2_Seeding.Puncta.Count: The count of Venus puncta DOX_3B2_Seeding.Seeded_Cells.Count: The count of green cells containing at least one Venus puncta  DOX_3B2_Seeding.Seeded_Red_Cells.Count: The count of mCherry positive cells containing at least one Venus puncta DOX_3B2_Seeding.pSeeded: The percentage of all TVS cells that contain at least one Venus puncta (DOX_3B2_Seeding.Seeded_Cells.Count/DOX_3B2_Seeding.Green_Cells.Count*100) DOX_3B2_Seeding.pRedSeeded: The percentage of mCherry positive TVS cells that contain at least one Venus puncta (DOX_3B2_Seeding.Seeded_Red_Cells.Count/DOX_3B2_Seeding.red_cells.Count*100) Average % Cells seeded per biological repeat: The average % of TVS cells containing at least one Venus puncta across biological repeats Average % Cells seeded - Background: The average % of TVS cells containing at least one Venus puncta across biological repeats, with background values from LF only conditions subtracted \**Fig. 2G & S4E & S4F - Fluorescent microscopy quantification of aggregated Tau-Venus puncta in TVA cells (10 hour after DOX treatment to induce R-NbF8-2 expression) following addition of varying UPS inhibitor drugs. ** *Column Titles* Condition: The experimental condition (inhibitor added) followed by biological repeat number % Aggregates compared to 0hr: The % of Tau-Venus aggregates in each condition at 10 hours post-DOX addition, compared to before the addition of DOX **Fig. 2H - Fluorescent microscopy quantification of aggregated Tau-Venus puncta in TVS cells following addition of various TVA cell lysates.** *Column Titles* Well Name: The well of the 96 well plate that images and quantified data were collected from Labels: The experimental condition each data point refers to - 0ug DOX indicates that lysates came from untreated cells, 1ug DOX indicates that lysates came from cells treated with DOX. F82, T21R or T21RF82 indicates that lysates came from cells with DOX inducible expression of the F8-2 nanobody, TRIM21 RING, or R-NbF8-2 respectively. 1to100 indicates the dilution of lysate that was applied to TVS cells. LF only and No LF are negative controls.  B2_Secondary_Seeding.Nuclei.Count: The count of DAPI nuclei  B2_Secondary_Seeding._GFP_Cells.Count: The count of Venus positive cells B2_Secondary_Seeding.Tau_Venus_Puncta.Count: The count of Venus puncta identified B2_Secondary_Seeding.Cells_with_Puncta.Count: The count of Venus positive cells containing at least one Venus puncta % Cells Seeded: The percentage of Venus positive cells containing at least one Venus puncta (B2_Secondary_Seeding.Cells_with_Puncta.Count/B2_Secondary_Seeding._GFP_Cells.Count*100) **Fig. 3A&B - GFP ELISA quantification of H2B-GFP protein levels in mouse hippocampal homogenates.** *Column Titles* Treatment: Indicates which AAV1/2 construct mice were treated with, and how long treatment was for Hemisphere/Mouse No.: Indicates whether the hippocampal hemisphere was injected or not (Left, L, was injected hemisphere) and which mouse the data is from Rep 1: GFP ELISA O.D value for repeat 1 Rep 2: GFP ELISA O.D value for repeat 2 Average: Average GFP ELISA O.D value from two technical repeats Average - 0: Average GFP ELISA O.D value from two technical repeats minus the blank value pg/ml: GFP-H2B protein levels calculated from standard curve of GFP standards. pg/ml*dilution: GFP-H2B protein levels calculated from standard curve of GFP standards - Adjusted for 1:100 dilution used in ELISA pg/ml*(mg tissue/ul lysis buffer)= pg GFP/mg tissue: Concentration of GFP-H2B adjusted against mg of hippocampal tissue used in ELISA GFP Levels in Injected vs Uninjected Hemisphere (%): Levels of H2B-GFP protein in injected hemispheres were divided by values in non-injected hemispheres to identify reduction in H2B-GFP following AAV1/2 injection. This was only performed for mice injected with AAV1/2 encoding the R-NbvhhGFP4 construct. **Fig. 3D - Fluorescent microscopy quantification of H2B-GFP and mCherry levels in mouse hippocampal slices.** *Column Titles* Condition: Indicates whether values are from injected or non-injected hippocampi, and which mouse they were taken from (3 biological repeats) T21RING-vhhGFP: The mean GFP intensity in hippocampal slices from mice injected with AAV1/2 encoding the R-NbvhhGFP4 construct T21RING: The mean GFP intensity in hippocampal slices from mice injected with AAV1/2 encoding the TRIM21 RING construct vhhGFP: The mean GFP intensity in hippocampal slices from mice injected with AAV1/2 encoding the vhhGFP4 construct **Fig. 4B - Immunofluorescent microscopy quantification of phospho-tau pathology in frontal cortices of aged Tg2541 mice injected with AAV1/2.** *Column Titles* Condition/Mouse Number: Gives details of which AAV1/2 delivered construct each mouse was injected with, and the number of the mouse that was injected (3 repeats) Injected hemisphere AT8 coverage (%): Percentage of the injected frontal cortex hemisphere that was positive when stained with the AT8 anti-pTau antibody. Uninjected hemisphere AT8 coverage (%): Percentage of the uninjected frontal cortex hemisphere that was positive when stained with the AT8 anti-pTau antibody. **Fig. 4E & S5C & S5D & S5F - Immunofluorescent microscopy quantification of phospho-tau in primary neurons.** *Column Titles* Well Name: The well of the 96 well plate that images and quantified data were collected from Labels: The experimental condition each data point refers to - 20K refers to 20,000vg of AAV used per cell. The name following this indicates whether the AAV used encoded the F8-2 nanobody, TRIM21 RING, or the R-NbF8-2 (T21RF82) construct.  GeneralAnalysis.Nuclei.Count: The count of DAPI nuclei GeneralAnalysis.MAP2.AreaFraction: The fraction of each image positive for MAP2 neuronal staining (expressed as a decimal value) GeneralAnalysis.S422_Puncta.Count: The count of aggregated phospho tau puncta (pS422 antibody staining)  GeneralAnalysis.S422_Bodies.Count: The count of large phosphotau (pS422 positive) aggregates which cover the neuronal soma GeneralAnalysis.Puncta_MAP2: The count of pS422 phospho tau puncta normalised against MAP2 neuronal coverage GeneralAnalysis.Bodies_MAP2: The count of pS422 phospho tau cell bodies normalised against MAP2 neuronal coverage Well Average pS422 Puncta over MAP2: The average value of pS422 phospho tau puncta normalised against MAP2 for all images from one well Well Averga Nuclei: The average DAPI nuclei count for all images from one well Well Average pS422 Cell bodies/MAP2: The average value of pS422 phospho tau cell bodies normalised against MAP2 for all images from one well Condition: A handy reminder of which condition each of the well average values represents \**Fig. 4F - Fluorescent microscopy quantification of aggregated Tau-Venus puncta in TVS cells following addition of neuronal lysates. ** *Column Titles* Well Name: The well of the 96 well plate that images and quantified data were collected from Labels: What the neurons were treated with prior to being lysed and applied to TVS cells. 0nM indicates unseeded neurons, all other conditions were seeded with 50nM heparin assembled tau. Dose of R-NbF8-2 AAV used to pre-treat neurons prior to seeding with tau is indicated. JB_3B2_Secondary_Seeding.Nuclei.Count: The count of DAPI nuclei JB_3B2_Secondary_Seeding.GFP_Cells.Count: The count of Venus positive cells JB_3B2_Secondary_Seeding.Puncta.Count: The count of Venus puncta JB_3B2_Secondary_Seeding.Cells_With_Puncta.Count: The count of Venus positive cells containing at least one Venus puncta % of TVS Cells Seeded: The percentage of Venus positive cells containing at least one Venus puncta (JB_3B2_Secondary_Seeding.Cells_With_Puncta.Count/JB_3B2_Secondary_Seeding.GFP_Cells.Count*100) **Fig. 4GF - Immunofluorescent microscopy quantification of phospho-tau in primary neurons.** *Column Titles* Condition: The replicate number No Seed: Relative levels of pS422 puncta (normalised against MAP2 coverage) in non-tau-seeded neurons (no AAV pre-treatment) compared to mean of "No AAV" condition No AAV: Relative levels of pS422 puncta (normalised against MAP2 coverage) in tau-seeded neurons (no AAV pre-treatment) compared to mean of "No AAV" condition R-NbF8-2: Relative levels of pS422 puncta (normalised against MAP2 coverage) in tau-seeded neurons, pretreated with AAV encoding R-NbF8-2, compared to mean of "No AAV" condition R(I18R+M72E)-NbF8-2: Relative levels of pS422 puncta (normalised against MAP2 coverage) in tau-seeded neurons, pretreated with AAV encoding catalytically dead R(I18R+M72E)-NbF8-2, compared to mean of "No AAV" condition **Fig. 5C&E - Immunofluorescent microscopy quantification of phospho-tau pathology in frontal cortices of aged Tg2541 mice injected with AAV9P31 R-NbF8-2** *Column Titles* Treatment: Indicates whether mice were treated with AAV9P31 R-NbF8-2 or were untreated controls Section: Indicates whether values were obtained from brain frontal cortex or spinal cord sections Mouse No.: Indicates which mouse (from each condition) values were obtained from Slice No.: Indicates which slice number values were obtained from  AT8 coverage (%): Percentage of the brain frontal cortex or spinal cord that was positive when stained with the AT8 anti-pTau antibody \**Fig. 5G - Fluorescent microscopy quantification of aggregated Tau-Venus puncta in TVS cells following addition of mouse brain lysates. ** *Column Titles* Well Name: The well of the 96 well plate that images and quantified data were collected from Condition: Indicates whether mice were treated with AAV9P31 R-NbF8-2 or were untreated controls Mouse No.: Indicates which mouse (from each condition) results were obtained from % of TVS Cells Seeded: The percentage of TVS cells treated with mouse brain lysates containing at least one Venus puncta **Fig. S3D - Fluorescent microscopy quantification of Tau-Venus levels in TVS cells following addition of AAV** *Column Titles* Time Post-Transduction (hours): Indicates at which time point, post addition of AAV, images were captured for analysis R-NbvhhGFP4_**1-5**: Each column represents an individual repeat from TVS cells treated with AAV encoding the R-NbvhhGFP4 construct. Values represent Venus fluorescence intensity normalised against the mean value of all repeats at 0hrs post AAV addition. T21R_**1-10**: Each column represents an individual repeat from TVS cells treated with AAV encoding the TRIM21 RING construct. Values represent Venus fluorescence intensity normalised against the mean value of all repeats at 0hrs post AAV addition. vhhGFP4_**1-5**: Each column represents an individual repeat from TVS cells treated with AAV encoding the vhhGFP4 construct. Values represent Venus fluorescence intensity normalised against the mean value of all repeats at 0hrs post AAV addition. **Fig. S3E - Fluorescent microscopy quantification of Tau-Venus levels in TVA cells following addition of AAV** *Column Titles* Time Post-Transduction (hours): Indicates at which time point, post addition of AAV, images were captured for analysis R-NbvhhGFP4_**1-5**: Each column represents an individual repeat from TVA cells treated with AAV encoding the R-NbvhhGFP4 construct. Values represent Venus fluorescence intensity normalised against the mean value of all repeats at 0hrs post AAV addition. T21R_**1-10**: Each column represents an individual repeat from TVA cells treated with AAV encoding the TRIM21 RING construct. Values represent Venus fluorescence intensity normalised against the mean value of all repeats at 0hrs post AAV addition. vhhGFP4_**1-5**: Each column represents an individual repeat from TVA cells treated with AAV encoding the vhhGFP4 construct. Values represent Venus fluorescence intensity normalised against the mean value of all repeats at 0hrs post AAV addition. **Fig. S3F - Fluorescent microscopy quantification of mCherry levels in TVS cells following addition of AAV** *Column Titles* Time Post-Transduction (hours): Indicates at which time point, post addition of AAV, images were captured for analysis R-NbvhhGFP4_**1-5**: Each column represents an individual repeat from TVS cells treated with AAV encoding the R-NbvhhGFP4 construct. Values represent mCherry fluorescence intensity normalised against the mean value of all repeats at 92hrs post AAV addition. T21R_**1-10**: Each column represents an individual repeat from TVS cells treated with AAV encoding the TRIM21 RING construct. Values represent mCherry fluorescence intensity normalised against the mean value of all repeats at 92hrs post AAV addition. vhhGFP4_**1-5**: Each column represents an individual repeat from TVS cells treated with AAV encoding the vhhGFP4 construct. Values represent mCherry fluorescence intensity normalised against the mean value of all repeats at 92hrs post AAV addition. **Fig. S3F - Fluorescent microscopy quantification of mCherry levels in TVA cells following addition of AAV** *Column Titles* Time Post-Transduction (hours): Indicates at which time point, post addition of AAV, images were captured for analysis R-NbvhhGFP4_**1-5**: Each column represents an individual repeat from TVA cells treated with AAV encoding the R-NbvhhGFP4 construct. Values represent mCherry fluorescence intensity normalised against the mean value of all repeats at 92hrs post AAV addition. T21R_**1-10**: Each column represents an individual repeat from TVA cells treated with AAV encoding the TRIM21 RING construct. Values represent mCherry fluorescence intensity normalised against the mean value of all repeats at 92hrs post AAV addition. vhhGFP4_**1-5**: Each column represents an individual repeat from TVA cells treated with AAV encoding the vhhGFP4 construct. Values represent mCherry fluorescence intensity normalised against the mean value of all repeats at 92hrs post AAV addition. \**Fig. S4C - Fluorescent microscopy quantification of adenovirus (GFP transgene) infection neutralisation via 9C12 antibody, with NMS-873 mediated VCP inhibition ** *Column Titles* Condition: Replciate number %ADV Neutralisation via 9C12 - DMSO (**three columns for three technical repeats**): The percentage reduction in adenovirus infection (green cells) when pre-incubating virus with DMSO and 9C12 antibody, compared to no antibody incubation.  %ADV Neutralisation via 9C12 - NMS 873 (**three columns for three technical repeats**): The percentage reduction in adenovirus infection (green cells) when pre-incubating virus with NMS-873 and 9C12 antibody, compared to no antibody incubation.  **Fig. S4G - Fluorescent microscopy quantification of aggregated Tau-Venus puncta in TVS cells following addition of TVA supernatant** *Column Titles* Condition: Gives repeat number %TVS Cells seeded with 50ul Media/well + DOX: The % of TVS cells containing at least one Venus puncta - Treated with supernatants from TVA cells treated with DOX for 72 hours %TVS Cells seeded with 50ul Media/well - DOX: The % of TVS cells containing at least one Venus puncta - Treated with supernatants from TVA cells treated without DOX for 72 hours **Fig. S5E - Fluorescent microscopy quantification of mCherry levels in primary neurons following addition of AAV** *Column Titles* Well Name: The well of the 96 well plate that images and quantified data were collected from Labels: The AAV delivered construct that neurons were treated with mCherry Mean Intensity: The average mCherry intensity of cells in each well **Fig. S5G - Fluorescent microscopy quantification of mCherry levels in primary neurons following addition of functional and catalytically dead R-NbF8-2 AAV** *Column Titles* Replicate number: Gives repeat number/identity  R-NbF8-2 Mean Cell mCherry fluorescence (a.u.): The average mCherry intensity of cells treated with R-NbF8-2 AAV in each replicate R(M72E+I18E)-NbF8-2 Mean Cell mCherry fluorescence (a.u.):  The average mCherry intensity of cells treated with catalytically dead R(M72E+I18R)-NbF8-2 AAV in each replicate **Fig. S6C,D,F&G - Quantification of western blot band intensities from brains and spinal cords of untreated mice, and mice treated with AAV9P31 R-NbF8-2** Results in the top half of the data set are from brain samples, with the bottom half from spinal cord samples.  *Column Titles* Condition: Gives treatment group of the mouse (R-NbF8.2 or untreated) followed by mouse number (1-3) and technical repeat number (a-b)  pTau: Intensity of AT100 antibody pTau signal Total Tau: Intensity of BR134 antibody total tau signal CypB: Intensity of CypB antibody loading control signal In the following columns technical repeats are averaged into one value for each mouse. pTau and total tau values are then normalised to the values for the CypB loading control. To create the data points plotted, these values are normalised respectively against the mean signal for pTau and total tau in the untreated mice, to give relative pTau and total tau levels compared to untreated control mice.