Dominant neurodegenerative disease Polyglutamine repeat expansions (CAG, codon, Q) in exon 1 of huntingtin gene (htt). Usually >35 CAG repeats.

RNA interference improves motor and neuropathologival abnormaliies in a Huntinton’s

disease mouse model

Jenna KausnerPBIO 427

5/13/11

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

Symptoms

Behavioral and cognitive disturbances

Involuntary movements (chorea)

Neuronal inclusions

Striatal and cortical neurodegeneration

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

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.

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.

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).

RNA sequence of shHD2.1

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.

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.

Northern blot analysis of RNA from transfected HEK293 cells

Htt expression in Vitro HEK293 transfected cells were lysed to

recover total protein and Western blot analysis was performed with actin as a control.

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.

Northern blot and mouse brain

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.

Photomicrographs of htt-reactive inclusions

Western blot analysis of striatal extracts

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

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

Immunofluorescence of cerebellum direct injection

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.

Improved stride length in AAV.shHD2.1 infected mice

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.

Rotarod performance test results

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.

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