Advertisement

Protocol for High-Content Screening for the Impact of Overexpressed MicroRNAs on Primary Motor Neurons

  • Tal Yardeni
  • Eran HornsteinEmail author
Protocol
Part of the Neuromethods book series (NM, volume 128)

Abstract

In this chapter we provide a protocol for the design and usage of automated, high-content microscopy screening that enables the investigation of microRNA (miRNA) impact on primary motor neuron. High-content screening (HCS) platforms facilitate superior precision in research and are scalable to study in parallel multiple genetic, molecular, or cellular conditions. miRNAs are critical for neuronal function and for brain integrity and are considered attractive candidate targets for therapy in many neuropathologies. Therefore, HCS platforms provide a novel paradigm for exploring the impact of miRNA expression, applicable for functional pathways discovery in an academic setting, or towards development of therapeutics in the pharma industry.

Keywords:

High-content screening Primary motor neuron Automated microscopy Morphometry MicroRNA miRNAs 

Notes

Acknowledgments

We thank Noga Kozer, Alexander Plotnikov, Haim M. Barr, Shir Katz, Hila Weiss, and Michal Eisenberg (Weizmann Institute of Science) for collaborations on developing the protocols. Raquel Fine and Cherill Banks for editing. This work was supported by grants from the ALS-Therapy Alliance, Israel Science Foundation, Legacy-heritage Fund, Bruno and Ilse Frick Foundation for Research on ALS, Yeda CEO fund, Minna-James-Heineman Stiftung through Minerva, ERC consolidator program, Nella and Leon Benoziyo Center for Neurological Diseases, Y. Leon Benoziyo Institute for Molecular Medicine, the Weizmann Institute of Science and A. Alfred Taubman via IsrALS, Kekst Family Institute for Medical Genetics, David and Fela Shapell Family Center for Genetic Disorders Research, Crown Human Genome Center, Yeda Sela Center, Helen and Martin Kimmel Institute for Stem Cell Research, the Maurice and Vivienne Wohl Biology Endowment, the Nathan, Shirley, Philip and Charlene Vener New Scientist Fund, Julius and Ray Charlestein Foundation, Celia Benattar Memorial Fund for Juvenile Diabetes, the Wolfson Family Charitable Trust, and Adelis Foundation. TY was supported by postdoctoral fellowship from Teva Pharmaceutical Industries Ltd as part of the Israeli National Network of Excellence in Neuroscience (NNE) and by the postdoctoral fellowship from the Sara Lee Schupf foundation. E.H. is Head of Nella and Leon Benoziyo Center for Neurological Diseases and the lab is further supported by Dr. Sydney Brenner and Friends.

References

  1. 1.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kim J et al (2007) A MicroRNA feedback circuit in midbrain dopamine neurons. Science 317(5842):1220–1224CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Campos-Melo D et al (2013) Altered microRNA expression profile in Amyotrophic Lateral Sclerosis: a role in the regulation of NFL mRNA levels. Mol Brain 6:26CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Haramati S et al (2010) miRNA malfunction causes spinal motor neuron disease. Proc Natl Acad Sci U S A 107(29):13111–13116CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Zock JM (2009) Applications of high content screening in life science research. Comb Chem High Throughput Screen 12(9):870–876CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gasparri F (2009) An overview of cell phenotypes in HCS: limitations and advantages. Expert Opin Drug Discov 4(6):643–657CrossRefPubMedGoogle Scholar
  7. 7.
    Kaltenbach LS et al (2010) Composite primary neuronal high-content screening assay for Huntington’s disease incorporating non-cell-autonomous interactions. J Biomol Screen 15(7):806–819CrossRefPubMedGoogle Scholar
  8. 8.
    Egawa N et al (2012) Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci Transl Med 4(145):145ra104CrossRefPubMedGoogle Scholar
  9. 9.
    Langlois SD et al (2010) Dissection and culture of commissural neurons from embryonic spinal cord. J Vis Exp. doi: 10.3791/1773 PubMedPubMedCentralGoogle Scholar
  10. 10.
    Milligan C, Gifondorwa D (2011) Isolation and culture of postnatal spinal motoneurons. Methods Mol Biol 793:77–85CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Rungta RL et al (2013) Lipid nanoparticle delivery of sirna to silence neuronal gene expression in the brain. Mol Ther Nucleic Acids 2:e136CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bhagat L et al (2008) Sodium arsenite induces heat shock protein 70 expression and protects against secretagogue-induced trypsinogen and NF-kappaB activation. J Cell Physiol 215(1):37–46CrossRefPubMedGoogle Scholar
  13. 13.
    Huang G et al (2013) Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis. Cell Death Dis 4:e841CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Nelson PT, Wang WX, Rajeev BW (2008) MicroRNAs (miRNAs) in neurodegenerative diseases. Brain Pathol 18(1):130–138CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Cogswell JP et al (2008) Identification of miRNA changes in Alzheimer’s disease brain and CSF yields putative biomarkers and insights into disease pathways. J Alzheimers Dis 14(1):27–41CrossRefPubMedGoogle Scholar
  16. 16.
    Sinha M, Mukhopadhyay S, Bhattacharyya NP (2012) Mechanism(s) of alteration of micro RNA expressions in Huntington’s disease and their possible contributions to the observed cellular and molecular dysfunctions in the disease. Neuromolecular Med 14(4):221–243CrossRefPubMedGoogle Scholar
  17. 17.
    Marcuzzo S et al (2015) Up-regulation of neural and cell cycle-related microRNAs in brain of amyotrophic lateral sclerosis mice at late disease stage. Mol Brain 8(1):5CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Molecular GeneticsWeizmann Institute of ScienceRehovotIsrael

Personalised recommendations