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PMC10158546 | Figure 4.PrP is localized to TUBB3 expressing neurons in ReN 129M cells. | [
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"text": "ReN 129M"
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PMC10158546 | Immunofluorescence was used to image ReN WT, KO and 129M cells by staining for TUBB3 (yellow), GFAP (green) and PrP (Red). | [] |
PMC10158546 | Nuclei were counterstained with DAPI (Blue). | [] |
PMC10158546 | Representative images are shown at days 7 and 21 post-differentiation. | [] |
PMC10158546 | Images were acquired using the 63X oil immersion objective of a Zeiss LSM 700 instrument (scale bar = 10 µm). | [] |
PMC10158546 | PrP is localized to TUBB3 expressing neurons in ReN 129M cells. | [
{
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"text": "ReN 129M"
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] |
PMC10158546 | Immunofluorescence was used to image ReN WT, KO and 129M cells by staining for TUBB3 (yellow), GFAP (green) and PrP (Red). | [] |
PMC10158546 | Nuclei were counterstained with DAPI (Blue). | [] |
PMC10158546 | Representative images are shown at days 7 and 21 post-differentiation. | [] |
PMC10158546 | Images were acquired using the 63X oil immersion objective of a Zeiss LSM 700 instrument (scale bar = 10 µm). | [] |
PMC10158546 | The motivation for examining differentiation of ReN 129 M, WT and KO cells was to identify any unwanted effects from lentiviral transduction that could influence prion replication. | [
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"text": "ReN 129"
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PMC10158546 | As such, a comprehensive evaluation of the effects of PrP expression on the phenotype or function of ReN cells was beyond the scope of this study. | [
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PMC10158546 | However, we noticed regulation of PrP during ReN cell differentiation and observed modulated formation of spheroid-like structures by ReN cells that express different levels of PrP. We report these findings because they are consistent with the role of PrP as a regulator of neurogenesis and neuron differentiation . | [
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PMC10158546 | First, we noticed that protein abundance of PrP steadily increased throughout standard differentiation in both ReN 129 M and WT cells (Figure 3c). | [
{
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"text": "ReN 129 M"
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PMC10158546 | We also observed increased abundance of PRNP mRNA throughout differentiation in both ReN WT and empty cells, but not 129 M cells (Figure 3d). | [] |
PMC10158546 | These differences in PRNP RNA expression pattern between ReN 129 M and WT cells are likely explained by PRNP being under the control of the EF1 promoter in the lentiviral-transduced 129 M cells. | [] |
PMC10158546 | We also noticed that slower migrating species of PrP were lost upon differentiation in both ReN 129 M and WT cells, whereas faster migrating species became predominant (Figure 3b). | [
{
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"text": "ReN 129 M"
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PMC10158546 | The faster PrP migration pattern could imply that the di-glycosylated form of PrP is lost upon differentiation, or alternatively might correspond to truncation of PrP. The increased PRNP RNA abundance, increased PrP protein abundance, and altered PrP migration pattern throughout ReN cell differentiation imply regulation of PrP at the transcriptional, translational, and post-translational levels, respectively. | [
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"text": "ReN"
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PMC10158546 | We also visualized the size and number of 3D spheroid-like structures formed by the ReN 129 M, WT and KO cell lines that differed in their level of PrP expression. | [] |
PMC10158546 | Spheroids were visualized at day 21 post-differentiation using 5 × 5 tile Z-stacked images taken of ReN cells stained for TUBB3, GFAP and PrP (Figure 5a). | [
{
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PMC10158546 | We found both ReN 129 M and KO cells to form larger spheroids compared to WT (Figure 5b). | [] |
PMC10158546 | ReN empty cells formed fewer spheroids compared to ReN 129 M and WT cells (Figure 5c), and we noticed that while spheroids formed by the ReN KO cells took up a larger horizontal area compared to WT and 129 M, they did not occupy as much of a vertical distance within the Z-stacked images. | [] |
PMC10158546 | We concluded that the ReN KO cells were deficient in forming spheroids compared to ReN WT and 129 M cells. | [] |
PMC10158546 | When examining fluorescence signal intensity, we found ReN 129 M spheroids to express higher levels of GFAP, PrP and TUBB3 compared to WT and KO (Figure 5d). | [
{
"end": 64,
"label": "CellLine",
"start": 55,
"text": "ReN 129 M"
}
] |
PMC10158546 | Altogether our results suggest that ReN cells with higher levels of PrP expression may form larger spheroids that have higher expression of TUBB3. | [
{
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PMC10158546 | However, we noticed weaker localization of TUBB3 to neurites with more signal originating from cell bodies in the ReN KO and 129 M compared to WT cultures, making the spheroid-like structures more easily visible in the WT cell line. | [] |
PMC10158546 | This raises the possibility of differences between the ReN WT, KO and 129 M cell lines being influenced by selection during the CRISPR and lentivirus transduction steps and/or increased passage number. | [] |
PMC10158546 | Figure 5.Altered arrangement into spheroid-like structures by ReN cells that express different levels of PrP. (a) Representative Z-stack projections of 5x5 tile images taken with a 20x objective of ReN 129M, WT and KO cells after staining for TUBB3, GFAP and PrP at day 21 post-differentiation (scale bar = 200 µm). | [
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PMC10158546 | Area and fluorescence intensity within individual spheroids were analyzed with imagej. | [] |
PMC10158546 | Fluorescence of GFAP, PrP and TUBB3 was normalized to the fluorescence of DAPI. ( | [] |
PMC10158546 | Area per spheroid. ( | [] |
PMC10158546 | Number of spheroids per image. ( | [] |
PMC10158546 | Mean normalized fluorescence intensity per spheroid. * | [] |
PMC10158546 | p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001. | [] |
PMC10158546 | p-values were calculated using one-way ANOVAs. | [] |
PMC10158546 | Altered arrangement into spheroid-like structures by ReN cells that express different levels of PrP. (a) Representative Z-stack projections of 5x5 tile images taken with a 20x objective of ReN 129M, WT and KO cells after staining for TUBB3, GFAP and PrP at day 21 post-differentiation (scale bar = 200 µm). | [
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"text": "ReN"
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{
"end": 197,
"label": "CellLine",
"start": 193,
"text": "129M"
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] |
PMC10158546 | Area and fluorescence intensity within individual spheroids were analyzed with imagej. | [] |
PMC10158546 | Fluorescence of GFAP, PrP and TUBB3 was normalized to the fluorescence of DAPI. ( | [] |
PMC10158546 | Area per spheroid. ( | [] |
PMC10158546 | Number of spheroids per image. ( | [] |
PMC10158546 | Mean normalized fluorescence intensity per spheroid. * | [] |
PMC10158546 | p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001. | [] |
PMC10158546 | p-values were calculated using one-way ANOVAs. | [] |
PMC10158546 | ReN cultures that overexpress PrP within TUBB3 neurons might offer a promising paradigm for in vitro prion replication. | [] |
PMC10158546 | In initial attempts at optimizing this system, we did not observe convincing differences related to prion replication when comparing 2D-monolayer versus thin-3D differentiated cultures, or when inoculum was added at day 0 versus day 7 of differentiation (supplementary figure S4). | [] |
PMC10158546 | We ultimately decided to use the thin 3D method of differentiation because a larger number of cells can be used (more lysate for PrP detection assays) and because these cultures can be maintained longer (more time to accumulate replicated PrP). | [] |
PMC10158546 | These thin 3D cultures were inoculated on day 7 of differentiation to allow the cells time to recover from the initial shock of growth factor removal. | [] |
PMC10158546 | To assess prion replication in this system, we performed four different prion challenge experiments of ReN cells with sCJD MM1 and sCJD VV2 human prion isolates, as well as RML and 263K rodent adapted scrapie (Figure 6a). | [
{
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PMC10158546 | CJD inocula was sourced from clinical cases identified by the Canadian CJD Surveillance System and was selected because they represented typical cases of sCJD MM1 and sCJD VV2. | [] |
PMC10158546 | PrP amyloid seeding activity within inocula was characterized and quantified via RT-QuIC (supplementary figure S5). | [] |
PMC10158546 | Each culture made from approximately 2 × 10 ReN cells was exposed to 3.2 × 10, 5.1 × 10, 5.1 × 10 and 5.1 × 10 SD50 amyloid seeding equivalents of sCJD MM1, sCJD VV2, RML and 263K, respectively. | [
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"text": "MM1"
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"text": "VV2"
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PMC10158546 | Prion replication over time was assessed by measuring PrP amyloid seeding activity in lysate (detected via RT-QuIC) collected from the cells at bi-weekly time points out to 6 weeks post infection. | [] |
PMC10158546 | RT-QuIC reactions were seeded in quadruplicate with 5 × 10, 5 × 10 and 5 × 10 grams total protein of ReN cell lysate. | [] |
PMC10158546 | We detected positive seeding activity through increased ThT fluorescence over reaction time (40 h) across all of the conditions tested (Figure 6b). | [] |
PMC10158546 | ReN cells treated with MM1 and 263K inoculum generally eliciting stronger RT-QuIC signal compared to those treated with RML and VV2 inoculum – likely attributed to differences between the amounts of PrP present in the different inoculums used. | [
{
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PMC10158546 | Indeed, amyloid seeding activity within VV2 inoculum was approximately 2 logs lower compared to the other isolates (supplementary figure S5), explaining the inefficient seeding activity in ReN cells after challenge with sCJD VV2. | [
{
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"text": "ReN"
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PMC10158546 | Figure 6.Prion seeding activity is consistently detected within ReN cultures for 6 weeks following inoculation with four prion isolates. | [] |
PMC10158546 | To examine prion replication in differentiated ReN cultures, ReN-PrP, ReN-PrP and ReN-PrP cells were differentiated for one week, then inoculated with one of four prion isolates. | [
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PMC10158546 | Prion inocula included human clinical isolates of Scjd-MM1 and Scjd-VV2, the RML strain of mouse-adapted scrapie, and the 263K strain of hamster-adapted scrapie. | [
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"text": "RML"
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"text": "263K"
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PMC10158546 | ReN cultures were maintained for 6-week post-inoculation with lysate collections in triplicate every 2 weeks. ( | [] |
PMC10158546 | Schematic representation of the experimental workflow. | [] |
PMC10158546 | PrP amyloid seeding activity in ReN cell lysates was measured via RT-QuIC at each time point post-inoculation with the four prion isolates. ( | [
{
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"text": "ReN"
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PMC10158546 | ThT fluorescence signal is plotted against reaction time for RT-QuIC assays seeded with 5e-08, 5e-09 and 5e-10 g total protein of ReN cell lysate. | [] |
PMC10158546 | Prion seeding activity is consistently detected within ReN cultures for 6 weeks following inoculation with four prion isolates. | [] |
PMC10158546 | To examine prion replication in differentiated ReN cultures, ReN-PrP, ReN-PrP and ReN-PrP cells were differentiated for one week, then inoculated with one of four prion isolates. | [
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PMC10158546 | Prion inocula included human clinical isolates of Scjd-MM1 and Scjd-VV2, the RML strain of mouse-adapted scrapie, and the 263K strain of hamster-adapted scrapie. | [
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"start": 77,
"text": "RML"
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{
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"label": "CellLine",
"start": 122,
"text": "263K"
}
] |
PMC10158546 | ReN cultures were maintained for 6-week post-inoculation with lysate collections in triplicate every 2 weeks. ( | [] |
PMC10158546 | Schematic representation of the experimental workflow. | [] |
PMC10158546 | PrP amyloid seeding activity in ReN cell lysates was measured via RT-QuIC at each time point post-inoculation with the four prion isolates. ( | [
{
"end": 35,
"label": "CellLine",
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"text": "ReN"
}
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PMC10158546 | ThT fluorescence signal is plotted against reaction time for RT-QuIC assays seeded with 5e-08, 5e-09 and 5e-10 g total protein of ReN cell lysate. | [] |
PMC10158546 | To get a better sense of quantitative differences in amyloid seeding activity between cell lines and over time, we performed hierarchical clustering of average lag phase measurements for each sample (Figure 7a). | [] |
PMC10158546 | While we observed differences in lag phase measurements between inoculums used, we did not observe any clear clustering of samples that would suggest differences between cell lines or changes over time post-inoculation. | [] |
PMC10158546 | We also employed the Spearman-Karber transformation to obtain a robust quantitative measurement of amyloid seeding activity (SD50) within each sample. | [] |
PMC10158546 | While we again observed quantitative differences in amyloid seeding activity between inoculums used, we did not see convincing evidence of prion replication over time (Figure 7b) or any differences in SD50 measurements overall between cell lines used (Figure 7c). | [] |
PMC10158546 | Importantly, the amyloid seeding activity within PrP overexpressing cells (ReN 129 M, 129 V, mmu and ham) never rose above that seen in the PrP-deficient cells (ReN empty). | [
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"text": "ReN 129"
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"text": "129"
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PMC10158546 | From this, it is apparent that the amyloid seeding activity detected via RT-QuIC corresponds to residual inoculum present within the cultures for the entire duration of the 6-week time course. | [] |
PMC10158546 | The detection of PrP in ReN cell lysate implies successful exposure of cells to the inoculum, and so we concluded that ReN cultures do not promote prion replication under the conditions tested. | [] |
PMC10158546 | Figure 7.Prion seeding activity within ReN cultures does not surpass residual inoculum throughout 6-weeks post challenge with four prion isolates. | [] |
PMC10158546 | PrP-seeding activity was quantified via lag-phase measurements from each individual RT-QuIC reaction. | [] |
PMC10158546 | The mean lag-phase value for each dilution (5e-8, 5e-9 and 5e-10 g total protein of lysate) per sample was then visualized as a hierarchical-clustered heatmap (a). | [] |
PMC10158546 | RT-QuIC lag-phase measurements did not cluster based on timepoint post-inoculation, suggesting that prion-seeding activity did not change over time in any of the conditions tested. | [] |
PMC10158546 | We applied the spearman-karber transformation to the RT-QuIC data to obtain a single quantitative measurement of prion-seeding activity within each sample, expressed as log10(SD50) per gram of total protein. | [] |
PMC10158546 | To assess prion replication, RT-QuIC log10(SD50) was plotted against days post infection (b), and to compare overall seeding activity per condition, RT-QuIC log10(SD50) was plotted per each cell line and inoculum (c). | [] |
PMC10158546 | Prion seeding activity in ReN-PrP and ReN-PrP cells never surpassed ReN-PrP cells, which were included to account for signal from residual inoculum. | [] |
PMC10158546 | Prion seeding activity within ReN cultures does not surpass residual inoculum throughout 6-weeks post challenge with four prion isolates. | [] |
PMC10158546 | PrP-seeding activity was quantified via lag-phase measurements from each individual RT-QuIC reaction. | [] |
PMC10158546 | The mean lag-phase value for each dilution (5e-8, 5e-9 and 5e-10 g total protein of lysate) per sample was then visualized as a hierarchical-clustered heatmap (a). | [] |
PMC10158546 | RT-QuIC lag-phase measurements did not cluster based on timepoint post-inoculation, suggesting that prion-seeding activity did not change over time in any of the conditions tested. | [] |
PMC10158546 | We applied the spearman-karber transformation to the RT-QuIC data to obtain a single quantitative measurement of prion-seeding activity within each sample, expressed as log10(SD50) per gram of total protein. | [] |
PMC10158546 | To assess prion replication, RT-QuIC log10(SD50) was plotted against days post infection (b), and to compare overall seeding activity per condition, RT-QuIC log10(SD50) was plotted per each cell line and inoculum (c). | [] |
PMC10158546 | Prion seeding activity in ReN-PrP and ReN-PrP cells never surpassed ReN-PrP cells, which were included to account for signal from residual inoculum. | [] |
PMC10158546 | We report that the differentiated cultures of ReN cells were resistant to infection with multiple prion isolates despite a high level of PrP expression mediated through lentivirus transduction. | [
{
"end": 49,
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"text": "ReN"
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] |
PMC10158546 | Specifically, through repeated measurements of amyloid seeding activity in 6-week time course experiments, we failed to observe replication of MM1 and VV2 type sCJD, or RML and 263K rodent adapted scrapie. | [] |
PMC10158546 | Our findings are striking because others have reported that similar cultures derived from differentiated neural progenitor cells can be infected with prions [17–24]. | [] |
PMC10158546 | The lack of PrP conversion we observed in this paradigm is also counterintuitive, given the capacity of ReN cells to replicate misfolded proteins in models of Alzheimer’s disease [25–28]. | [] |
PMC10158546 | We were careful to verify the expression of PrP and confirm proper differentiation of the lentiviral-transduced ReN cell lines, finding PrP to be highly expressed by TUBB3 neurons following differentiation. | [
{
"end": 115,
"label": "CellLine",
"start": 112,
"text": "ReN"
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PMC10158546 | Although we did note an effect of PrP expression on the formation of 3D spheroid like structures in the differentiated ReN cultures, consistent with the role of PrP as a regulator of neurogenesis . | [
{
"end": 122,
"label": "CellLine",
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"text": "ReN"
}
] |
PMC10158546 | Nonetheless, had the ReN cultures successfully replicated prions, a system like this would be useful for characterizing a wide range of prion strains – providing a particularly valuable platform when applied to clinical isolates of human prion diseases. | [] |
PMC10158546 | We detected residual inoculum within the ReN cell cultures for as long as 6 weeks post-exposure (Figure 7), illustrating a major challenge of prion research – distinguishing de novo prion replication from PrP present within the inoculum. | [] |
PMC10158546 | Here, we designed our study to distinguish de novo prion replication by comparing PrP-overexpressing with PrP-deficient ReN cell lines, and so we can conclude with some certainty that the ReN cultures did not promote prion replication. | [
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