Aug 22, 2024

Public workspaceQuantification of pathological protein accumulation (aSyn and tau) in transplanted human iPSC-derived dopamine neurons

DOI
dx.doi.org/10.17504/protocols.io.n92ld86z9v5b/v1
  • 1University of Sydney
courtney.wright Wright
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DOI: dx.doi.org/10.17504/protocols.io.n92ld86z9v5b/v1
Protocol Citation: Giselle Sagredo, Hongyun Li, YuHong Fu, Glenda Halliday 2024. Quantification of pathological protein accumulation (aSyn and tau) in transplanted human iPSC-derived dopamine neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld86z9v5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2024
Last Modified: August 23, 2024
Protocol Integer ID: 106083
Keywords: ASAPCRN, iPSC, p62, alpha synuclein, tau, phosphorylated tau, phosphorylated alpha synuclein, xenotransplantation, microscopy immunofluorescent analysis
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
This protocol details the immunohistochemistry protocol and subsequent imaging analysis protocols for quantifying pathological protein accumulation in human derived iPSC dopaminergic neurons transplanted into athymic mice. Tissue sections were stained free floating for human dopamine cells (tyrosine hydroxylase antibody) and pathological markers (Table 1) as per protocol.
Materials
Materials

  • 0.1M PBS
  • 10mM sodium citrate (pH 6)
  • Triton X
  • normal donkey serum
  • bovine serum albumin
  • gelatin
  • Tween 20
  • Hoechst (# B2261; Sigma-Aldrich)
  • Anti-fade fluorescence mounting medium (#S3023, DAKO)
  • CoverGrip coverslip sealant (Cat.# 23005, Biotium


Antibody table
ABCD
Primary antibodies (type, Cat.#) Epitope (immunogen) Source Fluorophore
Tyrosine hydroxylase (TH) (mouse IgG2b mAb, Cat.# TA506549) Full length human recombinant protein of human TH ThermoFisher Scientific 647
a-Syn ((Syn204) mouse IgG2a mAb, Cat.# 838201) Syn 204 raised using recombinant a-syn and recognises aa.1-130 of a-syn Biolegend 568
Phospho-a-syn at S129 (rabbit mAb, Cat.# ab51253) Synthetic peptide aa.100-140 of human a-Syn Abcam 488
Tau ((SP70) rabbit mAb, Cat.# SAB5500182) Synthetic peptide N-terminus of human tau Sigma Aldrich 568
Phospho-tau (Ser202, Thr205) (AT8) (mouse IgG1 mAb, Cat.# MN1020)* Partially purified PHF-tau aa. 1-758 ThermoFisher Scientific 488
Secondary antibodies Epitope (immunogen) Source
Goat anti-mouse IgG2b- Alexa Fluor 647, A-21242 Mouse IgG 2b ThermoFisher Scientific
Goat anti-mouse IgG2a- Alexa Fluor 568, A-21134 Mouse IgG 2a ThermoFisher Scientific
Donkey anti-rabbit- Alexa Fluor 488, A-21206 Rabbit IgG ThermoFisher Scientific
Donkey anti-rabbit- Alex Fluor 568, A-21134 Rabbit IgG ThermoFisher Scientific
Goat anti-mouse IgG1- Alexa Fluor 488, A21206 Mouse IgG 1 ThermoFisher Scientific
Table 1: List of antibodies used in this study.
aa=amino acid; mAb=monoclonal antibody; pAb=polyclonal antibody
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet) for each chemical listed from the supplier.
Before start
For qualitative and quantitative analyses of iPSC-derived dopamine neurons, images of whole brain sections and higher magnification images of dopamine cells were captured using an Olympus VS200 slide scanner and a Nikon C2 confocal microscope.
  • Olympus VS200 slide scanner: For both qualitative and quantitative analyses of iPSC-derived dopamine neurons, images were captured at 20x magnification with a z-range and z-spacing of 12µm and 1.5µm, respectively, and at 60x at one z-plane on the slide scanner.

  • Nikon C2 confocal microscope: For z-stack qualitative analyses of iPSC-derived dopamine neurons, images were captured at multiple magnifications on the confocal microscope. A 10x overview image of the regions of interest (ROI) was captured, followed by multiple magnifications (60x-120x) of the ROI to capture cell morphology and any potential pathologies. Z-stacks at 120x (step size= 0.3µm) were also performed followed by Huygens deconvolution using Huygens Professional version 23.04 (Scientific Volume Imaging, The Netherlands, http://svi.nl) for image presentation. Parameters including laser, gain and offset were selected based on single channel labelling for each section.

For image analysis, FIJI, v. 2.9.0/1.53t (1), and QuPath, V 0.5.0 (2), software were used.
Tissue processing and immunofluorescence staining
Tissue processing and immunofluorescence staining
Day 1
Tissue Prep
  1. Pour sections into well insert in well-plate to separate the cryoprotectant storage solution from the sections.
  2. Move well-insert to well-plate containing approximately Amount6 mL of 1 x phosphate buffered saline (PBS) in each well.
  3. Wash sections for Duration00:30:00 in 1 x PBS on an orbital shaker changing the buffer every Duration00:05:00


35m
Antigen retrieval

  1. Place sections in glass vials containing Amount6 mL of Concentration10 millimolar (mM) sodium citrate buffer (pH 6.0).
  2. Place in the steamer at Temperature98 °C for ~Duration00:20:00 .
  3. Cool sections to room temperature and transfer back to corresponding wells.
  4. Wash sections in 1 x PBS for Duration00:05:00 and repeat.








25m
Blocking and primary antibody incubation
  1. Incubate sections in blocking buffer (containing 2% donkey serum, 1% bovine serum albumin solution, 0.2% Triton X-100, 0.1% gelatin and 0.1% Tween-20 in PBS (PBST)) for 2 hours on an orbital shaker at room temperature.
  2. Incubate sections in the cocktail of primary antibodies in blocking buffer as per Table 1 for ~72 hours at 4°C on the orbital shaker.
Day 2
Secondary antibodies
  1. Wash sections in 0.1% PBST forDuration00:30:00 , changing the buffer every 10 minutes.
  2. Incubate sections in corresponding secondary antibodies (Table 1) and bisbenzimide H 33342 trihydrochloride (Hoechst, Cat.# B2261; Sigma-Aldrich) in blocking buffer forDuration02:00:00 at TemperatureRoom temperature on the orbital shaker.
  3. Wash sections in 0.1% PBST for Duration00:30:00 , changing the buffer every Duration00:10:00 .
  4. Wash sections in 1 xPBS for 1Duration00:05:00 , changing the buffer everyDuration00:05:00 .





3h 20m
Mounting and sealing
  1. Place sections in a petri dish containing 1 x PBS and immerse slide at an angle to gently mount sections onto slides using a brush.
  2. Place the coverslip gently on the slide using anti-fade fluorescence mounting medium (Cat.# S3023, DAKO).
  3. Once dry, seal slides with CoverGrip coverslip sealant (Cat.# 23005, Biotium) and store slides away from light at Temperature4 °C .

Identification of protein pathologies
Identification of protein pathologies
Dopamine neurons were identified in scanned images and manually counted based on TH immunoreactivity together with a large dull nucleus (Fig. 1 and 2). Aggregate-like structures were identified, counted and analysed in 3D image reconstructions if phosphorylated a-synuclein (pS129) immunofluorescence appeared within or partially within TH+ neuronal somata. Similarly, aggregate-like structures labelled by phosphorylated tau (AT8) immunofluorescence were identified and further analysed in 3D images if they appeared within or partially within TH+ neuronal somata (Fig. 2). PS129 and AT8 tau immunofluorescence labelled morphologically diverse structures in neurons (Fig. 1 and 2).


Figure 1. Phospho-a-syn at S129 (pS129) aggregate-like structures apparent in transplanted human iPSC-derived TH+ neurons from controls (CT) and patients carrying SNCA, LRRK2 and PRKN mutations. Scanner images illustrating the various aggregate-like structures labelled by pS129 immunofluorescence (red arrows, Ai-Hii) apparent in transplanted human iPSC-derived TH+ neurons. Immunofluorescent images revealed Tyrosine Hydroxylase (TH), white; pS129, green; Hoechst (HO), blue. Images labelled with (i) show merged image, and (ii) show only pS129 and HO. Scale bar (10µm) in Hii applies to all panels. 

Figure 2. Phospho-tau (AT8) aggregate-like structures apparent in transplanted human iPSC-derived TH+ neurons from controls (CT) and patients carrying SNCA, LRRK2 and PRKN mutations.
Scanner images illustrating the various aggregate-like structures labelled by AT8 immunofluorescence (red arrows, Ai-Hii) apparent in transplanted human iPSC-derived TH+ neurons. Immunofluorescent images revealed Tyrosine Hydroxylase (TH), white; AT8, green; Hoechst (HO), blue. Images labelled with (i) show merged image, and (ii) show only AT8 and HO. Scale bar (10µm) in Hii applies to all panels. 

Imaging and analyses - QuPath analysis
Imaging and analyses - QuPath analysis
Contour
  1. Using the polygon annotation tool, contour the ROI based on pan a-syn and pS129 staining together with the outline observed from the camera images captured prior to staining. The ROI contour provides the area (µm2) of TH+ signals, which were used to detect total cell number (Hoechst), TH+ cell number, and intensity data as outlined below.

Cell detection and other counts

  1. Annotate a small region close to the ROI contour to test the accuracy of the cell detection parameters using the Hoechst channel, in different areas to account for different nuclei intensities and morphologies. Once the parameters (nucleus parameters including sigma, minimum and maximum area, intensity, and cell expansion) are used to result in a count within 10% variance of a manual count, these parameters may then be applied to the ROI annotation for cell detection.
  2. Using the acquired parameters, apply the cell detection tool to the ROI to identify the total cell number.
  3. Using the point tool, manually count the TH+ neurons and create an annotation.
  4. Using the point tool, manually count the TH+ neurons with pS129 immunofluorescence (see definition above) by toggling off individual channels together with the counted “TH+ cells point annotation”.
Intensity data and masking

  1. Extract intensity data from the ROI contour using the “add intensity features” function.
  2. Create thresholders using different intensity parameters guided by the detection measurements provided for the Cell: TH mean and standard deviation to generate masks within the ROI annotation.

a. Generate the first mask of the TH+ area fraction including all TH immunoreactivity within the ROI.
b. Generate the second mask utilized a much higher intensity to mask mainly the TH+ cells and major processes or projections.
c. Add intensity features to the masks created for the detection of mean intensities of other channels within the TH+ masks.
Statistical analyses
Statistical analyses
After identifying all TH+ neurons in the ROI, the TH+ cell proportion, density, projections and cell size were calculated. Non-parametric statistics were performed using Kruskal-Wallis tests and post-hoc Dunn’s multiple comparisons tests (Prism 10 (v.10.0.0)) to identify any differences in the above parameters between groups with p < 0.05 as the level of significance.
Protocol references
References
1.          Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 2012;9(7):676-82.
2.          Bankhead P, Loughrey MB, Fernández JA, Dombrowski Y, McArt DG, Dunne PD, et al. QuPath: Open source software for digital pathology image analysis. Scientific Reports. 2017;7(1):16878.