Elimination of huntingtin in the adult mouse leads to progressive behavioral deficits, bilateral thalamic calcification, and altered brain iron homeostasis
Fig 1
Generation and evaluation of a tamoxifen inducible system for huntingtin ablation in the adult mouse.
(A) Schematic representation of wild-type allele (Hdh), Hdh loxP flanked allele (Hdhflox), and Hdh allele after tamoxifen-induced cre-mediated recombination (HdhΔflox). Exon 1 is represented by a black rectangle, and black ovals represent the loxP sites. Restriction sites shown on the schematic are: HindIII (H), XmnI (X) and KpnI (K). The location of oligonucleotides used for genotyping is indicated under arrows. (B) Western blot of total protein extracts from brains of 6 month-old Hdhflox/- (flox/-), and untreated CreER; Hdhflox/- (cKO noTM) mice at 6, 12, 15, 18, and 22 months of age was probed with mouse monoclonal anti-htt 2166 antibody (Chemicon), stripped and re-probed with anti-β-tubulin antibody (tub, lower panel) to ensure equal loading of the samples. Note that Htt expression in untreated cKO mice does not decrease significantly over time, and also does not differ from that of Hdhflox/- (flox/-) controls. (C) Western blot of total brain protein extracts from cKO noTM mouse (lane 1), cKO mouse TM-treated for 3 consecutive days (lane 2), and cKO mice TM-treated for 5 consecutive days (lanes 3 and 4) probed with mouse monoclonal anti-htt 2166 antibody (Chemicon) (Htt, top panel), stripped and re-probed with anti-β-tubulin antibody (tub, lower panel) to ensure equal loading of the samples. Note that the Htt band is significantly reduced after 3 days of TM (lane 2) and barely detectable after 5 days of TM administration (lanes 3 and 4). (D) Assessment of Hdh mRNA expression level by RT-PCR. Total RNA from cortex and striatum from Hdhflox/+ (flox/+); Hdhflox/- (flox/-); non-treated and TM-treated cKO mice was extracted and reverse transcribed. Semi-quantitative PCR amplification was performed using primers specific for Hdh coding regions spanning exons 11 and 12 (top panel) and 18S rRNA for internal control (bottom panel). (E) Total genomic DNA from cortex (ctx), striatum (str), and cerebellum (cb) from TM treated cKO mouse was submitted to PCR using primers that amplify the unrecombined (unrec) flox allele and recombined (rec) Δflox Hdh allele. Genomic DNA from Hdhflox/+ brain (flox: lane 1) and fully recombined Δflox (lane 5) were also submitted to PCR amplification with the same primers in parallel, as controls. Note that the unrecombined PCR product is barely visible in samples after TM treatment. (F) Quantification of htt protein levels using MSD electrochemiluminescence. Htt protein levels were independently evaluated and determined by the biopharmaceutical company BioFocus (A Galapagos Company). Cortices (left graph) and striata (right graph) from 3 month-old and 6 month-old C57BL/6 WT (3mo WT, 6mo WT accordingly), Hdhflox/+ (flox/+), Hdhflox/- (flox/-), untreated cKO (cKO noTM) and cKO TM-treated at 3 months of age and harvested at 4 months (4mo cKO TM@3mo) or cKO TM-treated at 6 months of age and harvested at 7 months (7mo cKO TM @6mo) were analyzed. For each time-point, genotype and treatment, 3 female and 3 male mice were analyzed. Individual data are depicted in these scatter plots and htt is expressed in fmol/mg of total protein. Note that the group 4mo cKO TM@3mo has only 5 samples, since one of the samples was wrongly genotyped and was therefore not included in the plots. Tamoxifen-induced cre-mediated recombination results in severe reduction of htt levels (***P<0.001, Student’s t-test).