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Silencing of p53 expression with Accell™ siRNA causes inhibition of the DNA damage response in IMR-32 neuroblastoma cells and protects primary cortical neurons from β-amyloid toxicityŽaklina Strezoska, Horizon Discovery, Lafayette, CO, USA Tamara Seredenina, Siena Biotech, Siena, Italy

IntroductionNeuroblastoma cell lines and primary neuronal cultures are commonly used as cellular model systems for studying cancer and neuronal development as well as being relevant cells for the study of neurodegenerative diseases. However, most neuroblastoma cell lines and primary neuronal cells suffer from low transfection efficiency due to the refractory nature of the cells to lipid-based transfection reagents. As such, application of small interfering RNA (siRNA) for inducing RNA interference (RNAi) has limited utility in these cell types, thus hindering the development of functional assays for screening and discovery of novel disease-relevant genes. Dharmacon™ Accell™ siRNA reagents enable efficient delivery in a wide range of cell lines and primary cells. Accell siRNA reagents carry a novel chemical modification pattern that facilitates the delivery of siRNA without a need for transfection reagents. To demonstrate the utility of Accell siRNA reagents in neuronal cells, the effects of silencing the expression of p53 was examined. p53 is a tumor suppressor protein that regulates the cellular response to different DNA damaging or cellular stress agents by controlling the expression of a wide variety of genes involved in cell growth, repair and survival or causing the cell to enter apoptosis if damage can not be repaired (Figure 1). Here we describe how application of Accell siRNA enabled the development of a high content assay for multiplex analysis of p53 and p21 expression in IMR-32 neuroblastoma cells, as well as a whole-culture cell viability assay in IMR-32 and primary rat cortical neurons. The ability to modulate gene expression in neuronal cell lines and primary neurons using Accell siRNA demonstrates potential new opportunities for functional genomic siRNA screens in the field of neuroscience.

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Figure 1. p53 is a a tumor suppressor protein that mediates cellular response to different stress agents. Cell exposure to damaging agents induces rapid increase of p53 protein levels in the nucleus, leading to the induction of its transcription targets controlling cellular responses such as cell cycle arrest, repair and cell survival (p21Waf1 Cip1, GAD45α, 14-3-3σ) and/or apoptosis (for example Bax, Apaf1, Casp-9).

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ResultsEfficient target mRNA knockdown in IMR-32 by Accell siRNAIn order to examine whether the Accell siRNA delivery protocol can be applied to IMR-32 neuroblastoma cells, several Accell siRNA controls were tested for silencing efficiency and maintenance of cell viability. This neuroblastoma cell line was chosen since it contains wild type p53 and could be further used for examination of the cellular effects of p53 knockdown on the DNA damage response. Accell GAPD and Non-targeting (NTC) control pools as well as Accell siRNA and Dharmacon™ SMARTpool™ reagents, and two individual siRNAs targeting p53 mRNA were delivered at 1 µM concentration in Accell siRNA Delivery Media. Target mRNA knockdown and cell viability were assessed at 72 hours post-transfection (Figure 2). Efficient target mRNA knockdown (> 75%) with no detrimental effect on cell viability was observed for all targeting siRNA.

p53 silencing by Accell siRNAs increases the survival of IMR-32 neuroblastoma cells after camptothecin treatmentThe function of p53 is to keep the cell from progressing through the cell cycle if there is DNA damage by either holding the cell at a checkpoint until repairs can be made and/or causing the cell to enter apoptosis if the damage cannot be repaired1,2. DNA damaging drugs, such as camptothecin, trigger rapid and extensive apoptosis in chemosensitive human neuroblastoma cell lines. Inactivation of p53, either by the human papillomavirus type 16 E6 protein or by a dominant-negative mutant p53 (R175H), protects cell lines with wild type p53 from drug-triggered apoptosis3. In order to demonstrate that knockdown of p53 by Accell siRNA would lead to similar protective effects, IMR-32 cells were incubated with Accell siRNAs against p53 or control siRNA pools (NTC, GAPD) for 72 hours and assayed for cell viability upon treatment with different camptothecin doses for the last 24 hours (Figure 3). The Accell SMARTpool and two individual Accell siRNAs targeting p53 caused a striking rescue from camptothecin-induced cell death, as seen in the phase contrast cell images (Figure 3A), and an observed 3-fold increase in cell survival (Figure 3B). p53 is normally maintained at low levels by continuous ubiquitination and subsequent degradation by the 26S proteasome. However, when the cell is stressed, p53 ubiquitination is suppressed and p53 accumulates in the nucleus, where it is activated and stabilized4.Once activated, p53 functions as a transcription factor to turn on or off various genes that affect cell cycle progression and repair (such as p21Waf1/Cip1, GAD45α, 14-3-3σ) and/or apoptosis (such as Bax, Apaf1, Casp-9; Figure 1)5.

Thermo Scientific™ HCS Reagent Kits for multiplex analysis of p53 and p21 expression was used to monitor the effects of down-regulating p53 by Accell siRNA. The reduction of the p53 signal and the induction on one of its downstream targets, p21 upon camptothecin treatment was quantified.

IMR-32 cells were found to be a challenging cell line for HCA due to their loose attachment to the tissue culture plates, so gentle fixing was necessary. This low adherence was further exacerbated by the response of IMR-32 cells to the Accell siRNA delivery conditions. Accell Delivery Media contains no serum, as high levels of serum are known to interfere with Accell siRNA efficacy. However, the addition of up to 3% serum to Accell Delivery Media is suggested for Accell siRNA application in cases where either the cell line or the assay is sensitive to prolonged serum-free conditions. Upon examination of IMR-32 cells under these modified Accell siRNA application conditions, it was found that the addition of 2% serum did not affect the target mRNA knockdown (Figure 4), but greatly improved cell adherence and fixation steps. The effects of p53 knockdown by Accell siRNA on the camptothecin-induced p53 and p21 protein levels were examined next. Treatment with 4 µM camptothecin for 18-22 hours was found to be the optimal concentration and time for induction of the p53 and p21 protein for the HCA assay in IMR-32 cells (data not shown).

IMR-32 cells were incubated with Accell control siRNA pools (NTC, GAPD), Accell SMARTpool, or two individual siRNAs against p53. At 52 hours post-delivery the cells were either treated with 4 µM camptothecin for 20 hours or left untreated. At 72 hours cells were fixed and stained with the Thermo Scientific™ Cellomics™ multiplexed p53 and p21 detection kit. Cells were analyzed with the Target Detection BioApplication software module. Mean fluorescent intensities of p53 or p21 staining in the nucleus were measured and quantified (Figure 5A). The results show that knockdown of p53 in IMR-32 cells inhibited the p53 and p21 induction following camptothecin treatment.

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Figure 2. Efficient target mRNA knockdown in IMR-32 by Accell siRNA. 1 µM Accell siRNA control pools (NTC or GAPD) and SMARTpool siRNA or individual siRNA duplexes against p53 (p53 pool, p53 siRNA 1, and p53 siRNA 2) were delivered in IMR-32 neuroblastoma cells in Accell Delivery Media. Knockdown of p53 and GAPD and cell viability were assessed at 72 hours post-transfection. Experiments were performed in biological triplicate for all samples.

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Figure 3. Knockdown of p53 increases the survival of IMR-32 after camptothecin treatment. 1 µM Accell siRNA control pools (NTC or GAPD) and SMARTpool reagent or individual siRNAs targeting p53 were delivered to IMR-32 neuroblastoma cells in Accell Delivery Media. At 48 hours post-transfection cells were treated with the indicated doses of camptothecin for 24 hours. Cell viability was assessed at 72 hours post-transfection by phase contrast cell images (A) or resazurin assay (B).

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In order to better visualize the effect of p53 knockdown on camptothecin-dependent induction of p53 and p21, 500 cells treated with either Accell NTC pool or Accell p53 SMARTpool siRNA reagent were selected and their nuclear intensity (Hoechst stain) was plotted versus the intensity for p21 stain or p53 stain (Figure 5B). As expected, knockdown of p53 by Accell siRNA resulted in a significant decrease of p53 and p21 staining intensities in the cell population, indicating that the cells do not enter cell cycle arrest upon the camptothecin treatment as they do when p53 is not silenced. Thus, we demonstrated that knockdown of p53 by Accell siRNA in IMR-32 neuroblastoma cell line results in inhibition of the p53 dependent DNA damage response upon camptothecin treatment.

Delivery optimization in primary Cortical NeuronsPrimary neuronal cultures are widely employed as a highly relevant cellular model system for the study of neurodegenerative diseases. Neurons are post-mitotic, differentiated cells that are cultured under specific conditions. Standard methods applied for modification of target expression by over-expression or by RNAi are often not efficient enough to obtain robust data employing common biochemical assays. The successful application of Accell siRNA for delivery into primary neurons would provide great opportunities for RNAi use in target discovery and validation in the field of neuroscience. For this reason, Accell siRNA delivery was tested and optimized in primary rat cortical neurons. Primary cortical neurons require specific culturing conditions and are extremely sensitive to medium changes. Therefore a series of experiments was performed in order to assess the effect of Accell delivery conditions on neuronal viability. Primary

cortical rat neurons6 harvested from 18-day embryos (E18) cultured 4 days in vitro (DIV) were incubated either in complete Neurobasal medium, Accell Delivery Media, Accell Delivery Media with B27 supplements or a 50:50 mix of Accell Delivery Media and complete Neurobasal medium. After 48 hours, neuronal viability was assessed by MTT assay (Figure 6). A decrease of cell viability was observed in Accell Delivery Media both with and without B27 supplements relative to Neurobasal medium alone. However, the survival of cells in the 50:50 mixture of Accell Delivery Media and complete Neurobasal medium was not dramatically affected and was further explored for supporting Accell siRNA delivery in primary cortical neurons.

To determine the optimal medium conditions for the assay, primary E18 rat cortical neurons at 4 DIV were incubated with 1 µM Accell NTC siRNA in either Accell (AM) medium alone, complete Neurobasal (NB) medium alone, or different ratios of Accell and Neurobasal Media. Following 48 hours incubation, neuronal viability was assessed by visual inspection (Figure 7A-D) and by MTT assay (Figure 7E). When the primary cortical neurons were incubated in the Accell Delivery Media a substantial decrease in cell viability was observed (Figure 7A, 7E). Cell morphology was not significantly affected by the different ratios of medium compared to Neurobasal medium alone (Figure 7B-D). A decrease in neuronal viability correlated with the quantity of Accell medium present in the delivery mix. Improved survival was obtained when no more than 50% of Accell medium was used in the delivery mix. In parallel, the effect of the different medium conditions on GAPD Accell siRNA ability to knockdown target mRNA was examined (Figure 7F). The expression of GAPD mRNA was significantly decreased in

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Figure 4. Efficient target mRNA knockdown in IMR-32 by Accell siRNA delivered in Accell Delivery Media supplemented with low quantity of serum. 1 µM Accell siRNAs control pools (NTC or GAPD) and SMARTpool siRNA reagent or individual siRNA duplexes against p53 were delivered in IMR-32 neuroblastoma cells in Accell Delivery Media or in Accell Delivery Media with 2% FBS. Knockdown of p53 and GAPD was assessed at 72 hours post-transfection.

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Figure 5. HCA shows reduction in p53 and p21 following camptothecin treatment when p53 is silenced. At 48 hours after siRNA delivery, cells were treated with 4 µM camptothecin for 20 hours or were left untreated. At 72 hours after siRNA delivery, cells were fixed and stained with the Cellomics Multiplexed p53 and p21 Detection Kit. A. Mean intensities of the nuclear p21 and p53 staining for different siRNA treatments. B. Shows the nuclear Hoechst versus p21 or p53 staining for each individual cell in the population (500 cells) for NTC and p53 siRNAs during camptothecin treatment.

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all conditions, with the least efficient knockdown (80%) in Neurobasal medium and the highest knockdown efficiency (> 90%) obtained in Accell Delivery Media. To strike a balance between optimal cell viability and target silencing, all subsequent Accell siRNA delivery experiments used a 50:50 ratio of Accell medium to Neurobasal medium.

In order to more closely examine the Accell siRNA delivery, neurons that were incubated for 48 hours with Accell Red GAPD siRNA (1 µM) in the optimal conditions (50:50 mix of AM:NB) were fixed and images were acquired using a confocal microscope. Almost all neurons were positive for Accell Red GAPD siRNA uptake (red, Figure 8A and B). Detailed analysis showed that siRNAs are localized in the cytoplasm of neuronal cell bodies and in neurites (Figure 8C and D).

Knockdown of p53 protects primary neurons from toxicity induced by β-amyloid peptideNeuronal apoptosis can be induced by β-amyloid peptides that play a major role in the pathogenesis of Alzheimer’s disease. p53 is a known mediator of β-amyloid peptide neurotoxicity7. Therefore we examine if silencing of p53 expression using Accell siRNA would provide a neuroprotective effect in primary cortical neurons from the β-amyloid peptide. Application of p53 Accell siRNA resulted in greater than 60% knockdown of p53 mRNA expression (Figure 9A). Different concentrations of β-amyloid peptide were added to the cells after 48 hours of incubation. The MTT assay was performed at both 48 hours (Figure 9B) and 72 hours (Figure 9C) in order to assess neuronal viability. Silencing of the pro-apoptotic gene p53 leads to a significant increase of neuronal survival as compared to NTC siRNA. The neuroprotective effect declined with increased concentration and exposure to β-amyloid. The strongest protective effect was observed at 5 µM β-amyloid at both time points. Reporter-based assays have been described for the validation of functional effects of modulated target expression in neurons8. However, reporter assays represent an indirect measure of neuronal viability. Moreover, increased toxicity associated with transfection makes it difficult to carry out an adequate evaluation of this phenotype. Here we demonstrate that Accell siRNA delivery technology enables the use of whole culture, homogeneous biochemical assays for functional target validation in primary cortical neurons.

Figure 6. Testing media compatibility for Accell siRNA delivery in primary neurons. Primary E18 rat cortical neurons at 4 DIV were treated for 48 hours with different medium conditions: complete Neurobasal medium (NB), Accell Delivery Media (AM), Accell Delivery Media with B27 supplements (AM + B27) or a 50:50 mix of Accell and Neurobasal medium (NB +AM). MTT assay was performed to assess neuronal viability.

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Figure 7. Testing cell viability and target mRNA knockdown in primary neurons upon Accell siRNA delivery in different medium conditions. Primary E18 rat cortical neurons at 4 DIV were treated for 48 hours with 1 µM Non-targeting Control (NTC) or GAPD Accell siRNA in different proportions of Accell Delivery Media and complete Neurobasal medium. Phase contrast images of cells treated with NTC siRNA in: A. Accell Delivery Media, B. a 50:50 mix of Accell and Neurobasal medium, C. a 25:75 mix of Accell and Neurobasal media, D. complete Neurobasal medium (NB) alone. E. Cell viability was also assessed by MTT assay, and F. siRNA silencing was examined by measuring GAPD mRNA knockdown.

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Figure 8. Analysis of siRNA cellular uptake. Primary E18 rat cortical neurons at 4 DIV were incubated for 48 hours with DY-547 labeled GAPD Accell siRNA (1 µM) in a 50:50 mix of Accell and Neurobasal media. Cells were fixed and images were acquired using a confocal microscope (Zeiss LSM 510). A, C. Detailed analysis shows that siRNAs (red) are localized in the cytoplasm in neuronal cell bodies and in neurites; blue staining (Hoechst) indicates nuclei. B, D. Phase contrast images of cells. Scale bar 10 µm.

Figure 9. Silencing of p53 by Accell siRNA causes significant increase of the survival of primary cortical neurons following β-amyloid peptide treatment. Primary E18 rat cortical neurons at 4 DIV were treated with 1 µM NTC Accell siRNA pool and Accell siRNA pool against p53 (in a delivery medium that represent a 50:50 mix of Accell and Neurobasal medium). A. p53 mRNA knockdown was assessed at 48 hours post transfection. At 48 hours post-transfection, cells were treated with different concentrations of β-amyloid peptide. MTT assay was performed after B. 48 hours and C. 72 hours in order to assess neuronal viability. ** p < 0.01; # p < 0.05, t-test.

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Cell viability assaysIMR-32 viability was assessed by resazurin assay. Resazurin was added to cells at a final concentration of 25 μg/mL 72 hours post-delivery. Cells were returned to the incubator for 1-3 hours. Plates were analyzed on a Wallac VICTOR 2 (Perkin Elmer Life Sciences) plate reader (Excitation 530 nM, Emission 590 nM and 1 second exposure). Viability of primary neurons was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay as described previously10.

High content screening assay and analysisIMR-32 cells at 72 hours post-transfection treated with or without 4 μM camptothecin for the last 20 hours were fixed with 4% paraformaldehyde and stained for p53 and p21 proteins. The plates were imaged and quantitatively analyzed on the ArrayScan VTI HCS Reader using the Target Detection Bio Application software module. Three replicate wells for each siRNA and treatment (-/+ campthotecin) were analyzed. Eight fields in each well were measured (> 100 cells/field). Parameters used in this analysis included Mean Object Average Intensity in Channel 1 and Mean Average Intensity in Channel 2 and Channel 3. The averages and standard deviations were calculated for each treatment. Numeric data generated by the ArrayScan VTI HCS Reader were evaluated with the vHCS Discovery Toolbox.

Confocal microscopyFor confocal analysis cells were fixed in 4% paraformaldehyde supplemented with Hoechst nuclear dye. Images were acquired using LSM510 confocal microscope (Zeiss).

References1. K.H. Vousden, X. Lu, Live or let die: the cell’s response to p53. Nat. Rev.

Cancer. 2, 594-604 (2002).2. E.S. Helton, X. Chen, p53 modulation of the DNA damage response. J. Cell.

Biochem. 100, 883-896 (2007).3. H. Cui, A. Schroering, p53 Mediates DNA Damaging Drug-induced

Apoptosis through a Caspase-9-dependent Pathway in SH-SY5Y Neuroblastoma Cells. Mol. Cancer Ther. 1, 679-686 (2002).

4. M.F. Lavin, N. Gueven, The complexity of p53 stabilization and activation. Cell Death Differ. 13, 941-950 (2006).

5. T. Riley, E. Sontag, Transcriptional control of human p53-regulated genes. Nat. Rev. Mol. Cell. Biol. 9, 402-412 (2008).

6. K. Goslin, G. Banker, Experimental observations on the development of polarity by hippocampal neurons in culture. J. Cell Biol. 108, 1507-1516 (1989).

7. M.P. Fogarty, E.J. Downer, A role for c-Jun N-terminal kinase 1 (JNK1), but not JNK2, in the beta-amyloid-mediated stabilization of protein p53 and induction of the apoptotic cascade in cultured cortical neurons. Biochem. J. 371, 789-798 (2003).

8. G. Pollio, R. Roncarati, A reporter assay for target validation in primary neuronal cultures. J. Neurosci. Methods. 172, 34-37 (2008).

9. W.B. Stine, Jr., K.N. Dahlgren, In Vitro Characterization of Conditions for Amyloid-beta Peptide Oligomerization and Fibrillogenesis. J. Biol. Chem. 278, 11612-11622 (2003).

10. T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65, 55-63 (1983).

Conclusions• The refractory nature of neuronal cells to lipid-based transfection

reagents can be overcome with the use of Accell siRNA reagents, enabling RNAi in these cell types.

• An optimization strategy is suggested for identification of conditions to maximize cell viability, and target gene silencing, while accounting for assay-specific requirements.

• Accell siRNA delivery technology permits functional target validation in neuroblastoma cell lines as well as primary cortical neurons.

Materials and methodsCell cultureIMR-32 cells were obtained from ATCC and cultured under recommended medium conditions. Cells were plated on collagen IV-coated 96-well plates for the purpose of improving the fixation of the cells for HCA analysis. Rat cortical neurons were obtained from E18 pups following Banker, Goslin and Brewer’s modified protocol6. The neurons were cultivated in Neurobasal medium with B27 supplements and used for siRNA delivery following 4 days in vitro (DIV).

Accell siRNA delivery in IMR-32 cells IMR-32 cells were plated at 20,000 cells per well in a 96-well plate and allowed to adhere overnight. Accell siRNAs used in IMR-32 cells include: Accell Non-targeting pool (Cat #D-001910-10), Accell GAPD Control Pool (Cat #D-001930-10) and Accell siRNA pool and individual siRNAs against human p53 (Accession #NM_000546; Cat #E-003329-00, A-003329-22 and A-003329-23). Accell siRNA was added to Accell siRNA Delivery Media (Cat #B-005000-100) for a final concentration of 1 μM. 100 μL of the Accell siRNA and media mixture was then added (per well) to the cells after the growth medium had been aspirated. For the purpose of improving the fixation of the cells for the HCA analysis, the Accell Delivery Media was supplemented with 2% FBS. Cells were incubated for 72 hours at 37 ºC and 5% CO2 at which point the plates were either analyzed for target mRNA knockdown, assessed for cell viability, or fixed for HCA analysis.

Accell siRNA delivery in primary rat neurons Neurons at 4 DIV were incubated for 48 hours with 1 µM Accell siRNA in medium containing different proportions of Accell Delivery Media in complete Neurobasal medium (100%, 50%, 25%, 0%) as indicated in the figure legends. Accell siRNAs used in primary rat neurons include: Accell GAPD Control siRNA (Cat #D-001930-03), Accell Non-targeting siRNA #1 (Cat #D-001910-01), Accell SMARTpool against rat p53 (Accession #NM_030989; Cat #E-080060-00-0020) and Accell Non-targeting pool (Cat #D-001910-10). The cells were analyzed for target mRNA knockdown at 48 hours although the protocol recommends a 72 hour time point. For the cellular uptake analysis DY547-labeled Accell GAPD Control siRNA and Accell Non-targeting siRNA were used. For the phenotypic assay the neurons at 4 DIV were treated with 1 μM Accell siRNA in the 50:50 mix of Accell Delivery Media and complete Neurobasal medium for 48 hours and then treated with different doses of β-amyloid peptide 1-42 (Californian peptides) for an additional 48-72 hours. After treatment, the cell viability was assessed by MTT. The β-amyloid peptide 1-42 was prepared according to the previously described protocol9.

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Cellomics is a trademark of Thermo Fisher Scientific All trademarks are the property of Horizon Discovery Company unless otherwise specified. ©2018 Horizon Discovery Group Company—All rights reserved. First published January 2015. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.