Fig 1.
Generation of N-terminal HTT knock-in mice.
(A) Structure of targeting vectors used for generating N160Q KI mice. Human HTT cDNA encoding the first 208 amino acids plus 160Q with a stop codon is inserted into the mouse Htt exon1. Neo selection marker is inserted into the intron after exon1 of the mouse HD gene. Two loxP sites also flank N-terminal HTT and neo cDNAs. (B) Genotyping of 160Q KI mouse tails showing the increased CAG repeat for 160Q. (C) RT-PCR analysis of N160Q KI mice (#30, #39, and #52). Primers specific to human HTT were used for PCR. WT and heterozygous full-length mutant HTT KI (F140Q KI) mice served as controls. Reverse transcripts (RT) were added or excluded in PCR reactions. (D) Quantitative RT-PCR shows the relative levels of N-terminal mutant HTT transcripts. The samples were obtained from the cortex, striatum, and cerebellum from heterozygous N160Q KI and F140Q KI mice. The relative levels of mutant HTT in N160Q KI mice were normalized by GAPDH levels and compared with full-length mutant HTT in heterozygous F140Q KI mice. (E) Expression of N-terminal mutant HTT in KI mouse brains via western blotting analysis. Arrows indicated N-terminal mutant HTT. The blot was probed with 1C2 antibody at 1:4000 dilution.
Fig 2.
Preferential accumulation of N-terminal mutant HTT in the striatum in KI mice.
(A) EM48 staining showing the more abundant distribution of mutant HTT in the striatum than the cortex in N160Q KI mice at 12 months of age. (B) EM48 staining also demonstrates the preferential distribution of mutant HTT in the striatum in full-length HTT KI (N140Q KI) and N160Q KI mice. (C) Age-dependent increases in HTT accumulation in N160Q mice at 6, 8, 12, and 16 months of age. Scale bar: (A and B): 100 μm; (C): 10 μm.
Fig 3.
Age-dependent increase in reactive astrocytes in N160Q-KI mouse brains.
(A) GFAP and HTT staining of N160Q-KI mice at 6 and 12 months of age. Note that increased GFAP occurs in the striatum and associates with the increased accumulation of mutant HTT in the striatum. HTT was stained by EM48 antibody. (B) More increased GFAP is seen in the striatum and cortex than cerebellum in 12-month-old N160Q-KI mice. (C) Quantification of the GFAP intensity in brain slices. The data are presented as mean±SE (n = 5–6 mice per group). ** P<0.01; *** P<0.001 compared with WT. Scale bars: 10 μm.
Fig 4.
Age-dependent neurological phenotypes of N-terminal HTT KI mice.
(A) The survival curve of N160Q-KI and wild type mice. (B) Body weight of N160Q KI mice. (C) Rotarod tests showing that motor deficits of N160Q KI mice increase with age. (D) Balance beam test showing a motor deficit of N160Q KI mice at 12 months of age. Age-matched WT mice (n = 19) and KI mice (n = 22) were examined. The data are presented as mean±SE. * P<0.05 compared with WT.
Fig 5.
Embryonic lethality of homozygous N160Q-KI mice.
(A) During mouse embryonic development, KI mouse embryos at embryonic day 8–9 were isolated for analysis of their morphology and genotypes, as well as numbers. (B) Summary of the numbers of normal and abnormal or re-absorbed embryos, their genotypes, and live pups from breeding N160Q KI mice.
Fig 6.
Expression of a truncated HTT in HEK293 cells.
(A) DNA structure of the truncated HTT (tHTT) that lacks N-terminal 237 amino acids of HTT. Full-length HTT (fHTT) and their amino acids were also presented. (B) Western blotting revealed the expression of truncated HTT (arrow) in HEK293 cells. The band above tHTT is full-length HTT in HEK293 cells. C is untransfected cells. (C) Immunocytochemical analysis showing the cytoplasmic distribution as full-length HTT (fHTT) and truncated HTT (tHTT), suggesting that loss of the N-terminal fragment of HTT does not affect the normal cytoplasmic distribution of HTT. (D) Transfected primary neurons from mouse brain cortex also showing the cytoplasmic distribution of tHTT. Anti-β-tubulin III was used to label neuronal cells. Scale bars: 10 μm.
Fig 7.
Truncated HTT lacking the N-terminal region is able to rescue neurite defects of PC12 cells.
(A) Western blotting showing that HTT shRNA depleted Htt expression in PC12 cells, which were also transfected with RFP-tHTT. (B) Immunofluorescent staining of transfected PC12 cells showing RFP-tHTT can reduce the neurite extension defect in cells (red cells with arrows) that also express shRNA-GFP (green) to suppress endogenous Htt, whereas cells with shRNA-GFP alone (green cells in left panel) show defective neurite extension. The cells were treated with NGF (50 ng/ml) for 48 h to induce neurite extension. (C) Transfection of nHTT was unable to prevent Htt loss-mediated neuritic defect. Arrows indicate PC12 cell that expressed nHTT and also shRNA-GFP. Arrowheads indicate PC12 cells with long neurites that only expressed nHTT but not shRNA-GFP. In (B) and (C), scale bars: 10 μm. (D) Quantitation of the number of PC12 cells with neurites longer than two cell bodies. The data are presented as mean±SE (n = 500 cells per group). *** P<0.001. (E). Expression of the truncated HTT (tHTT) or mutant HTT (dHTT) that lacks the dynein-binding region in HEK293 cells. HTT is tagged with Flag and linked to RFP via P2A, a self-cleaving peptide. Western blotting with anti-Flag antibody reveals the expression of tHTT and dHTT. (F). Quantitation of the number of PC12 cells with neurites longer than two cell bodies after inhibiting Htt expression by shRNA and transfection with tHTT or dHTT. The data are presented as mean±SE (n = 500 cells per group). *** P<0.001.
Fig 8.
Truncated HTT lacking the N-terminal region can rescue the selective degeneration of cultured neurons that have deleted endogenous Htt.
(A) Conditional Htt KO neuronal and glial cultures from floxed Htt mice were infected by adenoviral GFP-Cre to eliminate endogenous Htt expression in GFP-positive cells. Cultured striatal neurons and astrocytes were identified by antibodies to β-tubulin III and GFAP, respectively. Immunofluorescent staining shows that loss of Htt selectively causes neuronal, but not glial, degeneration. (B) Quantitative assessment of the surviving neurons and glial cells after the Htt gene is deleted by GFP-Cre expression. The control is cells infected with adenoviral-GFP without Cre. *** P<0.001. (C) Western blots with anti-HTT (2166) verified the depletion of Htt in primary cultures from the homozygous floxed Htt mouse brain cortex. (D) Transfection of nHTT, tHTT, or fHTT with 23Q into GFP-Cre-infected homozygous floxed Htt neurons demonstrating that tHTT and fHTT, but not nHTT (N-terminal HTT 1–208 amino acids), were able to rescue neurite degeneration caused by the loss of endogenous HTT. (E) Quantitative assessment of the surviving neurons after the endogenous Htt gene is deleted by GFP-Cre and transfection with different HTT forms. Adenoviral-GFP expression served as a control. *** P<0.001. Scale bars, (A and D): 10 μm.
Fig 9.
Truncated HTT lacking the N-terminal region can rescue the degeneration of cultured neurons by inducible depletion of the HTT gene.
(A) Schematic strategy to generate inducible HTT knockout neurons by tamoxifen. (B) HTT-inducible knockout neurons were transfected with tHTT or dHTT. Immunostaining showing that tHTT can increase neuronal processes, while dHTT is unable to protect against neuritic degeneration. The DIV 3 cortical neurons from floxed Htt mice with Cre were co-transfected with RFP-tHTT or RFP-dHTT and GFP. GFP immunostaining clearly reveals the processes of transfected cells. After 24 h transfection, the cells were treated with tamoxifen (1 g/ml) for 72 h to deplete endogenous mouse Htt. Immunofluorescent staining reveals the long processes of neurons expressing RFP-tHTT, but not RFP-dHTT, when endogenous mouse Htt is depleted. Scale bars: 10 m. (C) Western blotting confirming a dose-dependent depletion of Htt by tamoxifen in cultured cortical neuronal cells. (D) Quantitative measurement of total length of neurites per cell in each group after the endogenous Htt gene is deleted by tamoxifen. ** P<0.01.