Expression of Huntington's disease protein results in apoptotic neurons in the brains of cloned transgenic pigs

Abstract

Neurodegeneration is a hallmark of many neurological diseases, including Alzheimer's, Parkinson's and the polyglutamine diseases, which are all caused by misfolded proteins that accumulate in neuronal cells of the brain. Although apoptosis is believed to contribute to neurodegeneration in these cases, genetic mouse models of these diseases often fail to replicate apoptosis and overt neurodegeneration in the brain. Using nuclear transfer, we generated transgenic Huntington's disease (HD) pigs that express N-terminal (208 amino acids) mutant huntingtin with an expanded polyglutamine tract (105Q). Postnatal death, dyskinesia and chorea-like movement were observed in some transgenic pigs that express mutant huntingtin. Importantly, the transgenic HD pigs, unlike mice expressing the same transgene, displayed typical apoptotic neurons with DNA fragmentation in their brains. Also, expression of mutant huntingtin resulted in more neurons with activated caspase-3 in transgenic pig brains than that in transgenic mouse brains. Our findings suggest that species differences determine neuropathology and underscore the importance of large mammalian animals for modeling neurological disorders.

Figure 1.

Generation of cloned HD transgenic pigs. (A) DNA construct for expression of transgenic N-terminal mutant htt (N208-105Q or N208-160Q). CAG: the cytomegalovirus enhancer and chicken beta-actin promoter. (B) PCR analysis of the CAG repeats (105 and 160) in transgenic htt in pig fibroblast cell clones. (C) Expression of transgenic htt (N208-105Q) and ECFP in cloned Tibetan miniature pig fibroblast cells. (D) PCR reveals the presence of transgenic htt in tail tissues of all cloned transgenic pigs. (E) A live Tibetan miniature pig (7-1-3) transgenic for N208-105Q htt at 10 days after birth. (F) Expression of transgenic ECFP in cultured cells isolated from transgenic HD pigs (7-9 and 7-1-3).

Figure 2.

Genomic Southern blotting of cloned cells. Genomic DNAs were isolated from wild type and transgenic fibroblast cells from transgenic HD pigs (6-15, 7-9, 7-1-1, 7-1-2 and 7-1-3). The DNAs were digested with EcoRI and probed with the ECFP probe. Asterisks indicate strong bands that represent the integration sites of the transgene. Weak bands may be those that were not completely digested, degraded products or cross-reactive products.

Figure 3.

Expression of mutant htt in transgenic HD pigs and mice. (A) Western blots of cultured fibroblast cells isolated from HD transgenic or wild-type (WT) pigs. The blots were probed with antibodies to htt (mEM48) and polyQ domain (1C2). Arrowhead indicates the uncleaved transgenic N208-105Q-F2A-ECFP protein. Arrow indicates the cleaved N208-105Q protein (105Q). (B) Western blotting of the brain cortical tissues from transgenic pigs (7-9, 7-1-2 and 7-1-3) and wild-type (WT) pig. Sample (Cell) from cultured fibroblast cells of a 7-1-2 pig was also included. Arrowhead indicates the uncleaved N208-105Q-F2A-ECFP. Arrow indicates the N208-105Q protein. The blots were probed with mEM48 for htt and 1C2 for the expanded polyQ tract. (C) Western blots of the brain cortical tissues from F1 transgenic HD mice (2 months old) of lines 11 and 23. Some mice (35, 55, 56) express the N208-105Q protein (arrow) and the uncleaved N208-105Q-F2A-ECFP protein (arrowhead), which are not seen in other littermates.

Figure 4.

Apoptotic neurons in the brains of transgenic HD pigs. (A) 1C2 immunocytochemistry of the brain regions (cortex and striatum) of wild-type (WT) mouse, N208-105Q transgenic mouse or the 7-9 HD pig. Arrows indicate apoptotic cells in the transgenic pig brain. (B) 1C2 immunocytochemistry of the brain cortex and striatum of HdhCAG140 knock-in mice. (C) Anti-htt (EM48) immunostaining of the brain striatal sections of the HD pig (7-9), 3-month-old N208-105Q transgenic mouse and 12-month-old Hdh140CAG knock-in mouse. Arrows indicate EM48-positive cells in the HD transgenic pig and mouse brain sections. Scale bars: (A) 10 µm; (B) 5 µm; (C) 20 µm.

Figure 5.

mEM48 immunostaining of the brain striatum of wild-type and HD pig brains. (A) mEM48 immunocytochemistry revealed the presence of mutant htt in the neurons of HD transgenic pig (7-1-2), but not in the wild-type pig. (B) mEM48 immunocytochemistry also revealed apoptotic neurons (arrows) in transgenic HD pigs (7-1-1, 7-1-2, 7-9 and 6-15). Scale bars: 10 µm.

Figure 6.

Electron microscopy of the striatum of wild type (WT) transgenic HD pig brain (6–15) at low (A) and high (B–D) magnifications. (A) Degenerated neuron (arrow) shows nuclear chromatin condensation and is larger than a glial cell (double arrows). (B–C) Apoptotic cells (arrows) in the striatum of the HD pig brain show typical chromatin condensation and fragmentation. (D) The nuclei of glial cell (double arrows) also shows slight chromatin condensation or fragmentation. Scale bars: (A) 5 µm; (B) 2 µm; (C) 1 µm; (D) 2 µm.

Figure 7.

Immunohistochemistry with an antibody to the activated form of caspase-3 of the mouse brain striatal tissues. (A) The brain striatum of wild-type (WT), HdhCAG140 knock-in (140Q KI), N208-105Q mice and N208-105Q transgenic pig (7–9) were immunostaininged with anti-caspase-3. Arrows indicate caspase-3-positive cells. (B) High power images showing caspase-3-positive neurons, which are distinct from negative cells and small glial nuclei. Scale bars: (A) 20 µm; (B) 10 µm.

Figure 8.

Increased numbers of activated caspase-3-positive neurons in the brains of transgenic HD pig brains. (A) Increased numbers of caspase-3-positive cells (arrows) in the brain striatal tissues of transgenic HD pigs (7-9, 7-1-2 and 6-15) but not in wild-type pig (control). (B) Active caspase-3-positive neurons (arrows) in transgenic HD pig (7-1-1 and 7-9) brains show the DNA fragmentation feature of apoptosis. (C) Quantification of the relative number of caspase-3-positive cells in the transgenic HD pig and mouse brains. The images of HD mice are presented in Figures 4 and 7. The data were collected by examining the wild-type and HD (7-1-2, 7-9 and 6-15) pig brains and are presented as mean ± SE. **P<0.01 compared with control. Scale bars: 5 µm.