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Jenna Kausner PBIO 427 5/13/11

RNA interference improves motor and neuropathologival abnormaliies in a Huntinton’s disease mouse model. Jenna Kausner PBIO 427 5/13/11. Huntinton’s disease. Dominant neurodegenerative disease

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Jenna Kausner PBIO 427 5/13/11

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  1. RNA interference improves motor and neuropathologivalabnormaliies in a Huntinton’s disease mouse model Jenna Kausner PBIO 427 5/13/11

  2. Huntinton’s disease • Dominant neurodegenerative disease • Polyglutamine repeat expansions (CAG, codon, Q) in exon 1 of huntingtin gene (htt). Usually >35 CAG repeats. • Toxic gain of function mutation causing gradually damage to certain areas in brain

  3. Symptoms • Behavioral and cognitive disturbances • Involuntary movements (chorea) • Neuronal inclusions • Striatal and cortical neurodegeneration

  4. Huntingtin Protein (htt) • Expressed in all mammalian cells, highest in brain and testes • Function not entirely clear in humans • Interacts with proteins involved in transcription, cell signaling, and intracellular transporting

  5. Huntington Protein (htt) • Glutamine is polar and causes interactions with other proteins when overproduced in htt • Then, htt forms H-bonds with each other, resulting in a protein aggregate instead of normal folding protein. • Aggregates over time result in neuronal inclusions.

  6. Hypothesis • By directly inhibiting the expression of mutant htt, HD associated symptoms may be reduced or prevented. • This study tested if RNAi induced by short hairpin RNAs (shRNAs) could improve HD-associated abnormalities by reducing expression of mutant htt in a transgenic HD mouse model.

  7. Plasmids • HD-N171-82Q gene expressed from the pCMV-HD-N171-82G plasmid. • HD-N171-82Q is a truncated htt fragment • shRNAs and U6 promoter were amplified with PCR to target human htt (shHD2.1), eGFP (shGFP) or E. coli β-galactosidase (shLacZ).

  8. RNA sequence of shHD2.1

  9. Adenoassociated Virus Constuction • PCR products were cloned, sequenced, and inserted into adenoassociated virus (AAV) plasmid pAAV.CMV.hrGRP with AAV serotype 2 inverted terminal repeats, CMV-humanized Renilla GFP (hrGFP)-simian virus 40 poly(A) reporter cassette.

  10. Htt expression in Vitro • HEK293 cells were transfected with pCMV-HD-N171-82G and plasmid expressing shHD2.1, shGFP, or shLacZ. • RNA was isolated 48 hours after transfection and Northern blot analysis performed with human htt probes or human GAPDH probes as a control.

  11. Northern blot analysis of RNA from transfected HEK293 cells

  12. Htt expression in Vitro • HEK293 transfected cells were lysed to recover total protein and Western blot analysis was performed with actin as a control.

  13. Expression of shRNA in mouse brain • Mice were injected with AAV plasmids containing U6-driven shHD2.1 or shLacZ at four-weeks old and analyzed at four-months. • After injection into mouse striatum, shHD2.1 expression was analyzed by isolating total RNA from grGFP-positive striata using Northern blot analysis.

  14. Northern blot and mouse brain

  15. AAV.shHD2.1 reduces HD-N171-82Q expression in Vivo • To test the effect of RNAi on neuronal inclusions associated with HD, tissues were harvested from mice at about 5.5 months old and RNA was isolated. • In striata from mice injected with AAV.shHD2.1, htt-reactive inclusions were absent and mutant htt expression was reduced.

  16. Photomicrographs of htt-reactive inclusions

  17. Western blot analysis of striatal extracts

  18. HD-N171-82Q mRNA reduced in AAV.shHD2.1-injected mice

  19. Immunofluorescence • Coronal sections were isolated from mice and stained with mEM48 antibody followed by goat anti-mouse secondary antibody • Images were captured using fluorescent microscopy

  20. Immunofluorescence of cerebellum direct injection

  21. Behavioral analysis • Stride length measurements were taken by injected mice walking across a paper-lined chamber and into an enclosed box and measuring footprint tracings. • There was a noticable weight difference between HD-N171-82Q and wild type mice that was not normalized by RNAi directly to the striatum.

  22. Improved stride length in AAV.shHD2.1 infected mice

  23. Rotarod performance test • Mice were injected at 4 weeks of age and tested at 10 and 18 weeks old. • Amount of time it took mice to fall was measured.

  24. Rotarod performance test results

  25. Conclusion • Motor and neuropathological abnormalities in a HD mouse model are significantly improved using AAV delivered shRNA to reduce striatal expression of pathogenic htt allele. • Suggests feasibility of treating HD with direct reduction of mutant htt gene expression using RNAi.

  26. References • Harper, Q. S. et al. (2005) RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. PNAS, 102: 5820-5825. • http://www.animalstudies.bayer.com/en/introduction-animals.aspx • http://www.animalstudies.bayer.com/en/introduction-animals.aspx

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