Mutant huntingtin causes context-dependent neurodegeneration in mice with Huntington's disease

Abstract

Huntington's disease (HD) mouse models that express N-terminal huntingtin fragments show rapid disease progression and have been used for developing therapeutics. However, light microscopy reveals no significant neurodegeneration in these mice. It remains unclear how mutant huntingtin induces neurodegeneration. Using caspase staining, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, and electron microscopy, we observed that N171-82Q mice, which express the first 171 aa of mutant huntingtin, displayed more degenerated neurons than did other HD mouse models. The neurodegeneration was also evidenced by increased immunostaining for glial fibrillary acidic protein and ultrastructural features of apoptosis. R6/2 mice, which express exon 1 of mutant huntingtin, showed dark, nonapoptotic neurons and degenerated mitochondria associated with mutant huntingtin. In HD repeat knock-in mice (HdhCAG150), which express full-length mutant huntingtin, degenerated cytoplasmic organelles were found in both axons and neuronal cell bodies in association with mutant huntingtin that was not labeled by an antibody to huntingtin amino acids 342-456. Transfection of cultured cells with mutant huntingtin revealed that an N-terminal huntingtin fragment (amino acids 1-208 plus a 120 glutamine repeat) caused a greater increase in caspase activity than did exon 1 huntingtin and longer huntingtin fragments. These results suggest that context-dependent neurodegeneration in HD may be mediated by different N-terminal huntingtin fragments. In addition, this study has identified neurodegenerative markers for the evaluation of therapeutic treatments in HD mouse models.

Fig. 1.

GFAP staining of HD mouse striatum.A, Brain striatal sections from N171-82Q (4 months old), age-matched control [wild type (WT), 4 months old], R6/2 (3 months old), and HdhCAG150 knock-in (14 months old) mice were immunostained with an antibody against GFAP. Increased GFAP immunoreactivity of astroglial cell bodies and their fibrous processes is present in N171-82Q and HdhCAG150 mouse striatum. Scale bar, 84.6 μm. B, Density of GFAP staining in the striatum of different HD mouse models. The density was determined by counting grid points containing GFAP immunoreactive signal in micrographs at 630× magnification. Data are presented as the percentage (means ± SD) of a 5040 point grid in each micrograph and were obtained from three to four mice for each group. WT, Wild-type littermates (4 months old) of the same strain as N171-82Q mice. *p< 0.05 and **p < 0.01 compared with WT.

Fig. 2.

Immunostaining of HD mouse striatum.A, The striatum of N171-82Q (4 months old), HdhCAG150 knock-in (14 months old), R6/1 (8 months old), and R6/2 (3 months old) mice were stained with EM48, which specifically reacts with mutant huntingtin and its aggregates. Note that both nuclear inclusions and neuropil aggregates are present in all of the HD mouse brains examined, although they are more abundant in R6/2 and R6/1 mice.B, The striatum of HdhCAG150 was also labeled by the antibody EM121, which reacts with huntingtin amino acids 359–429. Only diffuse cytoplasmic staining was seen in HD mice, with a pattern similar to that of the wild-type (WT) mice. Scale bar, 10.8 μm.

Fig. 3.

Caspase-3-activated neurons in N171-82Q mouse brain. The cortex of a wild-type control mouse at 14 months of age (A) and the striatum (B) and cortex (C) of N171-82Q mice at 4.5 months of age were stained with an antibody against the activated form of caspase-3. Positively stained neurons (arrows) are present in the N171-82Q mouse brain regions. Inset, High-magnification micrograph of a neuron containing activated caspase-3. Scale bars, 32 μm.

Fig. 4.

TUNEL-positive neurons in N171-82Q mouse brain.A, TUNEL of the cortex of N171-82Q (5 months old), age-matched control (5 months old), R6/2 (12 weeks old), and Hdh150CAG (14 months old) mice. The brain sections were also counterstained with cresyl violet (blue). Arrows indicate TUNEL-positive neurons. The inset shows a high-magnification (630×) image in which a neuronal nucleus contains TUNEL-positive product. B, Quantitative measurement of the percentage of TUNEL-positive neurons in the cortex and striatum of HD mouse brains. The total number of neurons was assessed by cresyl violet staining in each micrograph (200×). N171-82Q mice at the age of 1.5, 3, and 5 months were examined. The ages of other HD mice are the same as those in A. KI, HdhCAG150 mice;WT, wild-type mice. *p < 0.05 and **p < 0.01 compared with wild-type control mice (n = 3–4).

Fig. 5.

Electron microscopy showing degenerated neurons in the cortex of N171-82Q mouse brain. A, B, An apoptotic neuron with condensed cytoplasm and abnormal nuclear shape showing margination and condensation of chromatin. Note that most cytoplasmic organelles remain intact. The rectangular area inA is shown in B at higher magnification, indicating that the mitochondria (arrowhead) appear normal, whereas the nuclear membrane has chromatin margination (arrow). C, The nucleus of an advanced stage of apoptosis showing chromatin condensation and fragmentation.D, A degenerating neuron with condensed nuclear chromatin (arrow) and an increased number of dark cytoplasmic bodies resembling lysosomes (arrowheads).E, EM48 immunogold labeling showing that a neuron contains mutant huntingtin and its inclusion (arrow) in the nucleus. Note that this neuron is undergoing early apoptosis, characterized by the disintegration of the nuclear membrane and mild chromatin margination (arrowheads). Scale bars:A, 1.54 μm; C, 1.77 μm;D, 0.92 μm: E, 1.45 μm.

Fig. 6.

Dark neurons and degenerated mitochondria in R6/2 mice. A, B, Dark degenerating neurons, which do not have typical chromatin margination and nuclear fragmentation, are present in an R6/2 mouse at 3 months of age (A) and in an R6/1 mouse at 8 months of age (B). A glial cell (gn) is also indicated. C, D, Degenerated mitochondria were observed in association with EM48 immunogold particles in the brain cortex at 8–10 weeks of age. Note that the cytoplasmic swelling, vacuolization, enlargement, and condensation of mitochondria (arrows) are associated with huntingtin immunogold particles. Degenerated mitochondria are also enclosed in a lysosome-like structure (D). Scale bars: A, 4 μm; B, 1.6 μm, C, D, 0.3 μm.

Fig. 7.

Degenerated neurons and axons in Hdh150CAG knock-in mice. A, B, Cytoplasmic organellar degeneration in HdhCAG150 mouse striatum. A, Several dark cytoplasmic organelles surround vacuoles (arrowheads). The nucleus that contains an intranuclear inclusion (arrow) appears intact, without nuclear fragmentation or condensation.B, A high-magnification micrograph shows clusters of electron-dense bodies and dark lysosomal structures that surround many cytoplasmic vacuoles. Some of these dense bodies show a mitochondrial origin with internal cristas (arrows). Others may represent secondary lysosomal bodies (arrowheads).C–E, Various types of degenerating axons are present in the striatum of HdhCAG150 mice. Axons are surrounded by disrupted myelin in which huntingtin aggregates (arrow) are trapped (C). Degenerated axons contain dark and swollen organelles (D, E), probably derived from mitochondria (arrowheads in D). Scale bars: A, 1 μm; B, 0.63 μm;C, 0.42 μm; D, 0.41 μm;E, 0.71 μm.

Fig. 8.

The effect of huntingtin context on the activation of caspases in transfected HEK293 cells. A, Western blot showing the expression of transfected huntingtin containing different protein sizes. The bracket indicates the stacking gel in which aggregated huntingtin is present.B, Immunofluorescence labeling of transfected HEK293 cells expressing 150Q-exon 1, 120Q-208, 120Q-508, and 120Q-945.C, Caspase-3 and caspase-9 activities of transfected cells that expressed different huntingtin fragments as indicated. The data were obtained from the 48 hr transfection of cells of three independent transfections and were presented as means ± SEM of control. The control is the activity of nontransfected cells.D, MTS assays of the viability of huntingtin-transfected cells. The control is the viability of nontransfected cells. The data (means ± SD) were obtained from three to four transfections for 72 hr. *p < 0.05 and **p < 0.01 compared with the activity of the vector-transfected cells.