Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

shRNA-induced saturation of the microRNA pathway in the rat brain

Gene Therapy volume 21, pages 205–211 (2014)Cite this article

Subjects

Abstract

RNA interference (RNAi) is a powerful strategy for unraveling gene function and for drug target validation, but exogenous expression of short hairpin RNAs (shRNAs) has been associated with severe side effects. These may be caused by saturation of the microRNA pathway. This study shows degenerative changes in cell morphology and intrusion of blood vessels after transduction of the ventromedial hypothalamus (VMH) of rats with a shRNA expressing adeno-associated viral (AAV) vector. To investigate whether saturation of the microRNA pathway has a role in the observed side effects, expression of neuronal microRNA miR-124 was used as a marker. Neurons transduced with the AAV vector carrying the shRNA displayed a decrease in miR-124 expression. The decreased expression was unrelated to shRNA sequence or target and observed as early as 1 week after injection. In conclusion, this study shows that the tissue response after AAV-directed expression of a shRNA to the VMH is likely to be caused by shRNA-induced saturation of the microRNA pathway. We recommend controlling for miR-124 expression when using RNAi as a tool for studying (loss of) gene function in the brain as phenotypic effects caused by saturation of the RNAi pathway might mask true effects of specific downregulation of the shRNA target.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3

Similar content being viewed by others

References

  1. Bish LT, Sleeper MM, Reynolds C, Gazzara J, Withnall E, Singletary GE et al. Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines. Hum Gene Ther 2011; 22: 969–977.

    Article  CAS  Google Scholar 

  2. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006; 441: 537–541.

    Article  CAS  Google Scholar 

  3. Borel F, van Logtenstein R, Koornneef A, Maczuga P, Ritsema T, Petry H et al. In vivo knock-down of multidrug resistance transporters ABCC1 and ABCC2 by AAV-delivered shRNAs and by artificial miRNAs. J RNAi Gene Silencing 2011; 7: 434–442.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Ahn M, Witting SR, Ruiz R, Saxena R, Morral N . Constitutive expression of short hairpin RNA in vivo triggers buildup of mature hairpin molecules. Hum Gene Ther 2011; 22: 1483–1497.

    Article  CAS  Google Scholar 

  5. McBride JL, Boudreau RL, Harper SQ, Staber PD, Monteys AM, Martins I et al. Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi. Proc Natl Acad Sci 2008; 105: 5868–5873.

    Article  CAS  Google Scholar 

  6. Martin JN, Wolken N, Brown T, Dauer WT, Ehrlich ME, Gonzalez-Alegre P . Lethal toxicity caused by expression of shRNA in the mouse striatum: implications for therapeutic design. Gene Therapy 2011; 18: 666–673.

    Article  CAS  Google Scholar 

  7. Boudreau RL, Martins I, Davidson BL . Artificial microRNAs as siRNA shuttles: improved safety as compared to shRNAs in vitro and in vivo. Mol Ther J Am Soc Gene Ther 2009; 17: 169–175.

    Article  CAS  Google Scholar 

  8. Ulusoy A, Sahin G, Björklund T, Aebischer P, Kirik D . Dose optimization for long-term rAAV-mediated RNA interference in the nigrostriatal projection neurons. Mol Ther J Am Soc Gene Ther 2009; 17: 1574–1584.

    Article  CAS  Google Scholar 

  9. Khodr CE, Sapru MK, Pedapati J, Han Y, West NC, Kells AP et al. An alpha-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson’s disease, but displays toxicity in dopamine neurons. Brain Res 2011; 1395: 94–107.

    Article  CAS  Google Scholar 

  10. Ehlert EM, Eggers R, Niclou SP, Verhaagen J . Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system. Bmc Neurosci 2010; 11: 20.

    Article  Google Scholar 

  11. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BRG . Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 2003; 5: 834–839.

    Article  CAS  Google Scholar 

  12. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003; 21: 635–637.

    Article  CAS  Google Scholar 

  13. Tiesjema B, la Fleur SE, Luijendijk MCM, Adan RAH . Sustained NPY overexpression in the PVN results in obesity via temporarily increasing food intake. Obesity (Silver Spring) 2009; 17: 1448–1450.

    CAS  Google Scholar 

  14. De Backer MWA, la Fleur SE, Adan RAH . Both overexpression of agouti-related peptide or neuropeptide Y in the paraventricular nucleus or lateral hypothalamus induce obesity in a neuropeptide- and nucleus specific manner. Eur J Pharmacol 2011; 660: 148–155.

    Article  CAS  Google Scholar 

  15. Frayling TM, Timpson NJ, Weedon MN, Zeggini E, Freathy RM, Lindgren CM et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 2007; 316: 889–894.

    Article  CAS  Google Scholar 

  16. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz A-L, Iggo R . Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 2003; 34: 263–264.

    Article  CAS  Google Scholar 

  17. Yi R, Doehle BP, Qin Y, Macara IG, Cullen BR . Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs. RNA 2005; 11: 220–226.

    Article  CAS  Google Scholar 

  18. Tung Y-CL, Ayuso E, Shan X, Bosch F, O’Rahilly S, Coll AP et al. Hypothalamic-specific manipulation of Fto, the ortholog of the human obesity gene FTO, affects food intake in rats. PLoS One 2010; 5: e8771.

    Article  Google Scholar 

  19. Hayes MR, Skibicka KP, Leichner TM, Guarnieri DJ, DiLeone RJ, Bence KK et al. Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation. Cell Metab 2010; 11: 77–83.

    Article  CAS  Google Scholar 

  20. Choi DL, Davis JF, Magrisso IJ, Fitzgerald ME, Lipton JW, Benoit SC . Orexin signaling in the paraventricular thalamic nucleus modulates mesolimbic dopamine and hedonic feeding in the rat. Neuroscience 2012; 210: 243–248.

    Article  CAS  Google Scholar 

  21. Hommel JD, Trinko R, Sears RM, Georgescu D, Liu Z-W, Gao X-B et al. Leptin receptor signaling in midbrain dopamine neurons regulates feeding. Neuron 2006; 51: 801–810.

    Article  CAS  Google Scholar 

  22. Yi R, Qin Y, Macara IG, Cullen BR . Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 2003; 17: 3011–3016.

    Article  CAS  Google Scholar 

  23. Lund E, Güttinger S, Calado A, Dahlberg JE, Kutay U . Nuclear export of microRNA precursors. Science 2004; 303: 95–98.

    Article  CAS  Google Scholar 

  24. Diederichs S, Jung S, Rothenberg SM, Smolen GA, Mlody BG, Haber DA . Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites. Proc Natl Acad Sci USA. 2008; 105: 9284–9289.

    Article  CAS  Google Scholar 

  25. Grimm D, Wang L, Lee JS, Schürmann N, Gu S, Börner K et al. Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver. J Clin Invest 2010; 120: 3106–3119.

    Article  CAS  Google Scholar 

  26. de Backer MW, Brans MA, van Rozen AJ, van der Zwaal EM, Luijendijk MC, Garner KG et al. Suppressor of cytokine signaling 3 knockdown in the mediobasal hypothalamus: counterintuitive effects on energy balance. J Mol Endocrinol 2010; 45: 341–353.

    Article  CAS  Google Scholar 

  27. Broekman MLD, Comer LA, Hyman BT, Sena-Esteves M . Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain. Neuroscience 2006; 138: 501–510.

    Article  CAS  Google Scholar 

  28. Kan AA, van Erp S, AAHA Derijck, de Wit M, Hessel EVS, O’Duibhir E et al. Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response. Cell Mol Life Sci Cmls 2012; 69: 3127–3145.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by TIPharma, project T5-210-1, and by a VIDI grant from the Netherlands Organization for Health Research and Development and a grant from Neuroscience and Cognition Utrecht (to RJP). We acknowledge Eljo van Battum, Rudolf Magnus Institute of Neuroscience, for her technical assistance and Dr Joost Verhaagen, Netherlands Institute for Neuroscience, for critically reading the manuscript.

Author information

Authors and Affiliations

  1. Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands

    M A van Gestel, S van Erp, L E Sanders, M A D Brans, M C M Luijendijk, M Merkestein, R J Pasterkamp & R A H Adan

Authors
  1. M A van Gestel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S van Erp
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L E Sanders
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M A D Brans
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M C M Luijendijk
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M Merkestein
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R J Pasterkamp
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. R A H Adan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R A H Adan.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

van Gestel, M., van Erp, S., Sanders, L. et al. shRNA-induced saturation of the microRNA pathway in the rat brain. Gene Ther 21, 205–211 (2014). https://doi.org/10.1038/gt.2013.76

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2013.76

Keywords

This article is cited by

Search

Advanced search

Quick links