[en] In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.
Disciplines :
Neurology
Author, co-author :
Highet, Blake ; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Vikas Anekal, Praju; Biomedical Imaging Research Unit, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Ryan, Brigid; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Murray, Helen; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Coppieters't Wallant, Natacha ; Université de Liège - ULiège > GIGA > GIGA Neurosciences - Nervous system disorders and therapy
Victor Dieriks, Birger ; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Singh-Bains, Malvindar K; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Mehrabi, Nasim F; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Department of Pharmacology, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Faull, Richard L M; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Dragunow, Michael; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Department of Pharmacology, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Curtis, Maurice A ; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand ; Centre for Brain Research, Faculty of Medical and Health Science, University of Auckland, Auckland, New Zealand
Language :
English
Title :
fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.
Brain Research New Zealand Neurological Foundation of New Zealand Health Research Council of New Zealand
Funding text :
We are very grateful to Brain Research New Zealand (grant number: 3715777) and the Health Research Council of New Zealand who financially supported this research. Blake Highet is supported by a Brain Research New Zealand Doctoral scholarship. Brigid Ryan is supported by the Auckland Medical Research Foundation. Helen Murray is a Health Education Trust Postdoctoral Research Fellow. Natacha Coppieters is funded by the Neurological Foundation. Birger Victor Dieriks is funded by the Michael J Fox Foundation and Neuro Research Charitable Trust. Malvindar K. Singh‐Bains is funded by the Leo Nilon Huntington's Disease Fellowship. We are grateful to the Neurological Foundation for their support of the Brain Bank. POLR2A PPIB UBCWe are very grateful to Brain Research New Zealand (grant number: 3715777) and the Health Research Council of New Zealand who financially supported this research. Blake Highet is supported by a Brain Research New Zealand Doctoral scholarship. Brigid Ryan is supported by the Auckland Medical Research Foundation. Helen Murray is a Health Education Trust Postdoctoral Research Fellow. Natacha Coppieters is funded by the Neurological Foundation. Birger Victor Dieriks is funded by the Michael J Fox Foundation and Neuro Research Charitable Trust. Malvindar K. Singh-Bains is funded by the Leo Nilon Huntington's Disease Fellowship. We are grateful to the Neurological Foundation for their support of the Brain Bank. The authors thank the Neurological Foundation Brain Bank manager, Marika Eszes as well as Kristina Hubbard and Dr Remai Parker for their technical support in working with human brain tissue.
Allen, M., Carrasquillo, M. M., Funk, C., Heavner, B. D., Zou, F., Younkin, C. S., Burgess, J. D., Chai, H. S., Crook, J., Eddy, J. A., Li, H., Logsdon, B., Peters, M. A., Dang, K. K., Wang, X., Serie, D., Wang, C., Nguyen, T., Lincoln, S., … Ertekin-Taner, N. (2016). Human Whole Genome Genotype and Transcriptome Data for Alzheimer’s and Other Neurodegenerative Diseases. Scientific Data 3.
Alzheimer, A., Förstl, H., & Levy, R. (1991). History of Psychiatry On Certain Peculiar Diseases of Old Age Translated and with an Introduction By. https://journals.sagepub.com/doi/pdf/10.1177/0957154X9100200505 (February 6, 2019).
Antonell, A., Lladó, A., Sánchez-Valle, R., Sanfeliu, C., Casserras, T., Rami, L., Muñoz-García, C, Dangla-Valls, A., Balasa, M., Boya, P., Kalko, S. G., & Molinuevo, J. L. (2016). Altered blood gene expression of tumor-related genes (PRKCB, BECN1, and CDKN2A) in Alzheimer’s Disease. Molecular Neurobiology, 53(9), 5902–5911. https://doi.org/10.1007/s12035-015-9483-9
Barnes, A. M., Carter, E. M., Cabral, W. A., Weis, M., Chang, W., Makareeva, E., Leikin, S., Rotimi, C. N., Eyre, D. R., Raggio, C. L., & Marini, J. C. (2010). Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding. New England Journal of Medicine 362(6), 521–28. https://doi.org/10.1056/NEJMoa0907705
Barton, A. J. L., Pearson, R. C. A., Najlerahim, A., & Harrison, P. J. (1993). Pre-and Postmortem Influences on Brain RNA. Journal of Neurochemistry 61(1), 1–11.http://doi.wiley.com/10.1111/j.1471-4159.1993.tb03532.x (April 15, 2020).
Bloom, G. S. (2014). Amyloid-β and Tau. JAMA Neurology 71(4): 505. http://www.ncbi.nlm.nih.gov/pubmed/24493463 (February 6, 2019).
Coulson, D. T., Brockbank, S., Quinn, J. G., Murphy, S., Ravid, R., Irvine, G. B., & Johnston, J. A. (2008). Identification of valid reference genes for the normalization of RT QPCR gene expression data in human brain tissue. BMC Molecular Biology, 9(1), 46. https://doi.org/10.1186/1471-2199-9-46
Gadhave, K., Bolshette, N., Ahire, A., Pardeshi, R., Thakur, K., Trandafir, C., Istrate, A., Ahmed, S., Lahkar, M., Muresanu, D. F., & Balea, M. (2016). The ubiquitin proteasomal System: A potential target for the management of Alzheimer’s disease. Journal of Cellular and Molecular Medicine, 20(7), 1392–1407. https://doi.org/10.1111/jcmm.12817
Gebhardt, F. M., Scott, H. A., & Dodd, P. R. (2010). Housekeepers for accurate transcript expression analysis in Alzheimer’s disease autopsy brain tissue. Alzheimer’s and Dementia, 6(6), 465–474.
Grabinski, T. M., Kneynsberg, A., Manfredsson, F. P., & Kanaan, N. M. (2015). A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures. PloS one 10(3): e0120120. http://www.ncbi.nlm.nih.gov/pubmed/25794171 (January 30, 2019).
Gray, D. A. (2003). Ubiquitin, Proteasomes, and the Aging Brain. Science of Aging Knowledge Environment 2003(34): 6re–6. http://sageke.sciencemag.org/cgi/doi/10.1126/sageke.2003.34.re6 (April 7, 2020).
Hershko, A., & Ciechanover, A. (1998). The ubiquitin system. Annual Review of Biochemistry 67(1), 425–79.http://www.annualreviews.org/doi/10.1146/annurev.biochem.67.1.425 (April 7, 2020).
Liang, W. S., Dunckley, T., Beach, T. G., Grover, A., Mastroeni, D., Ramsey, K., Caselli, R. J., Kukull, W. A., McKeel, D., Morris, J. C., Hulette, C. M., Schmechel, D., Reiman, E. M., Rogers, J., & Stephan, D. A. (2008). Altered neuronal gene expression in brain regions differentially affected by Alzheimer’s disease: A reference data set. Physiological Genomics, 33(2), 240–256. https://doi.org/10.1152/physiolgenomics.00242.2007
Loring, J. F., Wen, X., Lee, J. M., Seilhamer, J., & Somogyi, R. (2001). A gene expression profile of Alzheimer's disease. DNA and Cell Biology, 20(11), 683–695.www.liebertpub.com (November 4, 2020).
Löw, P. (2011). The role of ubiquitin-proteasome system in ageing. General and Comparative Endocrinology, 172(1), 39–43. https://doi.org/10.1016/j.ygcen.2011.02.005
Lowe, J., Blanchard, A., Morrell, K., Lennox, G., Reynolds, L., Billett, M., Landon, M., & Mayer, R. J. (1988). Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man, including those of Parkinson’s Disease, Pick’s disease, and Alzheimer’s disease, as well as rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and mallory bodies in alcoholic liver disease. The Journal of Pathology 155(1), 9–15. https://doi.org/10.1002/path.1711550105
Manavalan, A., Mishra, M., Feng, L., Sze, S. K., Akatsu, H., & Heese, K. (2013). Brain site-specific proteome changes in aging-related dementia. Experimental and Molecular Medicine, 45(9), e39. https://doi.org/10.1038/emm.2013.76
Mathys, H., Davila-Velderrain, J., Peng, Z., Gao, F., Mohammadi, S., Young, J. Z., Menon, M., He, L., Abdurrob, F., Jiang, X., Martorell, A. J., Ransohoff, R. M., Hafler, B. P., Bennett, D. A., Kellis, M., & Tsai, L. H. (2019). Single-cell transcriptomic analysis of Alzheimer’s disease. Nature, 570(7761), 332–337.2020, doi: 10.1038/s41586-019-1195-2 (November 4).
Murdoch, A., Jenkinson, E. J., Johnson, G. D., & Owen, J. J. (1990). Alkaline phosphatase-fast red, a new fluorescent label. Application in double labelling for cell cycle analysis. Journal of Immunological Methods 132(1): 45–49.http://www.ncbi.nlm.nih.gov/pubmed/2118160 (April 8, 2020).
Narayan, P. J., Kim, S.-L., Lill, C., Feng, S., Faull, R. L. M., Curtis, M. A., & Dragunow, M. (2015). Assessing fibrinogen extravasation into Alzheimer’s disease brain using high-content screening of brain tissue microarrays. Journal of Neuroscience Methods, 247, 41–49. https://doi.org/10.1016/j.jneumeth.2015.03.017
Oddo, S. (2008). The ubiquitin-proteasome system in Alzheimer’s disease. Journal of Cellular and Molecular Medicine, 12(2), 363–373. https://doi.org/10.1111/j.1582-4934.2008.00276.x
Penna, I., Vella, S., Gigoni, A., Russo, C., Cancedda, R., & Pagano, A. (2011). Selection of candidate housekeeping genes for normalization in human postmortem brain samples. International Journal of Molecular Sciences, 12(9), 5461–5470. https://doi.org/10.3390/ijms12095461
Perrett, C. W., Marchbanks, R. M., & Whatley, S. A. (1988). Characterisation of messenger RNA extracted post-mortem from the brains of Schizophrenic, depressed and control subjects. Journal of Neurology Neurosurgery and Psychiatry, 51(3), 325–331. https://doi.org/10.1136/jnnp.51.3.325
Qing, H., Zhou, W. A., Christensen, M., Sun, X., Tong, Y., & Song, W. (2004). Degradation of BACE by the ubiquitin-proteasome pathway. The FASEB Journal, 18(13), 1571–1573. https://doi.org/10.1096/fj.04-1994fje
Rubinsztein, D. C. (2006). The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 443(7113), 780–86.http://www.ncbi.nlm.nih.gov/pubmed/17051204 (April 7, 2020).
Rustenhoven, J., Smith, A. M., Smyth, L. C., Jansson, D., Scotter, E. L., Swanson, M. E. V., Aalderink, M., Coppieters, N., Narayan, P., Handley, R., Overall, C., Park, T. I. H., Schweder, P., Heppner, P., Curtis, M. A., Faull, R. L. M., & Dragunow, M. (2018). PU.1 Regulates Alzheimer’s disease-associated genes in primary human microglia. Molecular Neurodegeneration 13(1): 44. https://molecularneurodegeneration.biomedcentral.com/articles/10.1186/s13024-018-0277-1 (April 2, 2020).
Rydbirk, R., Folke, J., Winge, K., Aznar, S., Pakkenberg, B., & Brudek, T. (2016). Assessment of brain reference genes for RT-QPCR studies in neurodegenerative diseases. Scientific Reports, 6(1), 1–11. https://doi.org/10.1038/srep37116
Sherwood, K. R., Head, M. W., Walker, R., Smith, C., Ironside, J. W., & Fazakerley, J. K. (2011). RNA integrity in post mortem human variant Creutzfeldt-jakob disease (VCJD) and control brain tissue. Neuropathology and Applied Neurobiology 37(6), 633–42. https://doi.org/10.1111/j.1365-2990.2011.01162.x
Speel, E. J. M., Schutte, B., Wiegant, J., Ramaekers, F. C., & Hopman, A. H. (1992). A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction. Journal of Histochemistry and Cytochemistry 40(9), 1299–1308. https://doi.org/10.1177/40.9.1506667
Stelzmann, R. A., Norman Schnitzlein, H., & Reed Murtagh, F. (1995). An english translation of Alzheimer’s 1907 Paper, ‘Uber Eine Eigenartige Erkankung Der Hirnrinde’. Clinical Anatomy 8(6), 429–31.http://doi.wiley.com/10.1002/ca.980080612 (April 15, 2020).
Toro, C. A., Zhang, L., Cao, J., & Cai, D. (2019). Sex differences in Alzheimer’s disease: Understanding the molecular impact. Brain Research, 1719, 194–207. https://doi.org/10.1016/j.brainres.2019.05.031
Tramutola, A., Domenico, F. D., Barone, E., Perluigi, M., & Butterfield, D. A. (2016). It is all about (U)biquitin: Role of altered ubiquitin-proteasome system and UCHL1 in Alzheimer disease. Oxidative Medicine and Cellular Longevity, 2016, 1–12.
Waldvogel, H. J., Curtis, M. A., Baer, K., Rees, M. I., & Faull, R. L. M. (2007). Immunohistochemical staining of post-mortem adult human brain sections. Nature Protocols, 1(6), 2719–2732. https://doi.org/10.1038/nprot.2006.354
Wang, F., Flanagan, J., Su, N., Wang, L.-C., Bui, S., Nielson, A., Wu, X., Vo, H.-T., Ma, X.-J., & Luo, Y. (2012). RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. Journal of Molecular Diagnostics, 14(1), 22–29. https://doi.org/10.1016/j.jmoldx.2011.08.002
Wang, Q. I., Ishikawa, T., Michiue, T., Zhu, B.-L., Guan, D.-W., & Maeda, H. (2012). Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: Comprehensive evaluation using GeNorm, NormFinder, and BestKeeper. International Journal of Legal Medicine, 126(6), 943–952. https://doi.org/10.1007/s00414-012-0774-7
Wintzerith, M., Acker, J., Vicaire, S., Vigneron, M., & Kedinger, C. (1992). Complete sequence of the human RNA polymerase II largest subunit. Nucleic Acids Research 20(4), 910. https://doi.org/10.1093/nar/20.4.910
Xu, P.-T., Li, Y.-J., Qin, X.-J., Kroner, C., Green-Odlum, A., Xu, H., Wang, T.-Y., Schmechel, D. E., Hulette, C. M., Ervin, J., Hauser, M., Haines, J., Pericak-Vance, M. A., & Gilbert, J. R. (2007). A SAGE study of apolipoprotein E3/3, E3/4 and E4/4 allele-specific gene expression in hippocampus in Alzheimer disease. Molecular and Cellular Neuroscience, 36(3), 313–331. https://doi.org/10.1016/j.mcn.2007.06.009
Yoo, Y., Byun, K., Kang, T., Bayarsaikhan, D., Kim, J. Y., Oh, S., Kim, Y. H., Kim, S., Chung, W., Kim, S. U., Lee, B., & Park, Y. M. (2015). Amyloid-beta-activated human microglial cells through ER-resident proteins. Journal of Proteome Research 14(1), 214–23. http://www.ingenuity. (October 22, 2020).
