Mutant huntingtin in glial cells exacerbates neurological symptoms of Huntington disease mice

Abstract

Huntington disease (HD) is caused by an expansion of the polyglutamine (polyQ) repeat (>37Q) in huntingtin (htt), and age of onset is inversely correlated with the length of the polyQ repeat. Mutant htt with expanded polyQ is ubiquitously expressed in various types of cells, including glia, but causes selective neurodegeneration. Our recent study demonstrated that expression of the N-terminal mutant htt with a large polyQ repeat (160Q) in astrocytes is sufficient to induce neurological symptoms in mice (Bradford, J., Shin, J. Y., Roberts, M., Wang, C. E., Li, X.-J., and Li, S. H. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 22480-22485). Because glia-neuron interactions are critical for maintaining the normal function and survival of neurons in the brain and because mutant htt is more abundant in neurons than in glial cells, it is important to investigate whether glial htt can still contribute to HD pathology when mutant htt is abundantly expressed in neuronal cells. We generated transgenic mice that express mutant htt with 98Q in astrocytes. Unlike our recently generated htt-160Q transgenic mice, htt-98Q mice do not show obvious neurological phenotypes, suggesting that the length of the polyQ repeat determines the severity of glial dysfunction. However, htt-98Q mice show increased susceptibility to glutamate-induced seizure. Mice expressing mutant htt in astrocytes were mated with N171-82Q mice that express mutant htt primarily in neuronal cells. Double transgenic mice expressing mutant htt in both neuronal and glial cells display more severe neurological symptoms and earlier death than N171-82Q mice. These findings indicate a role of glial mutant htt in exacerbating HD neuropathology and underscore the importance of improving glial function in treating HD.

FIGURE 1.

Expression of mutant htt in glial cells in HD mouse brains. A, EM48 immunostaining of HD 150CAG knock-in (KI) mouse brains that express full-length mutant htt at the endogenous level. HD KI mice at the age of 4, 10, and 15 months, and WT mice at 12 months of age were examined. The upper panels (×10 magnification) show brain sections containing the cortex (Ctx), white matter (WM) of the corpus callosum, and striatum (Str). Scale bar: 50 μm. The lower panels (×40) show the striatum in which the regions containing glia and neurons are indicated. Scale bar: 20 μm. B, high magnification images (×63) showing that mutant htt forms smaller aggregates in glial cells (arrows) than neuronal htt aggregates in HD KI mouse brains. C, Western blots of cultured astrocytes from WT, R6/2, N171–82Q, and HD KI mouse brains showing the expression of mutant htt in astrocytes from R6/2, and HD KI, but not N171–82Q mice. 1C2 was used to detect expanded polyQ-containing proteins. Note that full-length (arrow) and multiple N-terminal htt fragments containing an expanded polyQ domain are evident in HD KI astrocytes.

FIGURE 2.

Expression levels of transgenic htt in htt-98Q transgenic mice. A, Western blot analysis of the expression of transgenic htt (N-terminal htt (208 amino acids) containing 98Q) in whole brain lysates using anti-htt antibody (EM48). Mouse brain extracts of htt-98Q mice were obtained from two independent transgenic mouse lines (98Q-217 and 98Q-218 lines). Transfected htt in HEK293 cells served as a control (arrow). B, Western blots with EM48 reveals the low level of transgenic htt-98Q compared with transgenic htt in R6/2 mouse brain. The arrow indicates htt-98Q. C, EM48 Western blotting of different brain regional tissues of htt-98Q transgenic mice. D, RT-PCR analysis of the transcript levels of transgenic htt in WT and HD (htt-98Q, N171–82Q, and R6/2) mouse brain cortex tissues. Primers that can amplify both mouse and transgenic htt were used for PCR. GAPDH was also amplified and served as an internal control. RT, reverse transcriptase. E, RT-PCR analysis of cultured astrocytes showing lower levels of transgenic htt-98Q (arrowhead) than endogenous mouse htt (arrow). Primers that amplify the CAG repeat were used in RT-PCR. F, real-time PCR of cultured astrocytes from wild type and transgenic mice (htt-23Q, htt-98Q, htt-160Q) that express mutant htt in astrocytes. The relative expression levels of transgenic htt were normalized to endogenous GAPDH levels and were obtained from three independent real-time PCR assays.

FIGURE 3.

Age-dependent accumulation of mutant htt in glial cells of htt-98Q transgenic mice. A, immunohistochemical staining with 1C2 shows the presence of mutant htt in the white matter (WM) of the corpus callosum in htt-98Q, but not in WT mice at the age of 9 months. B, wild-type and htt-98Q mice at 19 months of age were examined using 1C2 immunocytochemical staining. Note that mutant htt is expressed in glial cells in the WM of the corpus callosum and brain stem (B.S.) in htt-98Q mice. Scale bars: 10 μm.

FIGURE 4.

Age of htt-98Q mice influences accumulation of soluble or aggregated transgenic htt in astrocytes. A, double immunofluorescent staining of mouse brain sections containing the Str and WM from wild-type, htt-98Q, and N171–82Q mice. The staining was performed with mouse 1C2 antibody to htt (red) and rabbit antibody to GFAP (green). Note that mutant htt staining is enriched in GFAP-positive cells in the WM of htt-98Q mice or in neuronal cells of N171–82Q mice. Merged images also show nuclear staining (blue). B, age-dependent accumulation of mutant htt in astrocytes of htt-98Q transgenic mice. Note that mutant htt is diffuse or forms small aggregates in the cytoplasm of astrocytes in young (3 and 9 month) mouse brain and can form nuclear inclusions (arrow) in old (16 month) mouse brain. Scale bars: 10 μm.

FIGURE 5.

Increased glutamate-induced seizure in htt-98Q mice. A, Western blot analysis showing decreased levels of GLT-1 in various brain regions of htt-98Q mice as compared with WT mice. The ratios of GLT-1 to tubulin on the same blots are presented beneath the blots. B, quantitative analysis of the ratios of GLT-1 to tubulin from Western blots. Data were obtained from two independent Western blot experiments with 5–6 mouse brain samples for each group. *, p < 0.05 as compared with WT. C, glutamate intraperitoneal injection induced excitotoxicity in wild type and htt-98Q mice. Time (min) for the onset latency and duration of hyperexcitability (wild running, jumping, or circling) and the duration of chronic seizure (tonic-clonic) are presented. *, p < 0.05; **, p < 0.01 (n = 10–11 each group).

FIGURE 6.

Exacerbating effects of glial htt on neurological symptoms of HD mice. A, EM48 immunostaining of brain sections from WT, htt-98Q, N171–82Q, and double transgenic (htt-98Q/N171–82Q) mice. The brain sections contain the WM and Str. Arrows indicate EM48-positive glial cells. B, rotarod performance of WT, htt-98Q, N171–82Q, and double transgenic mice at the ages of 15–22 weeks. Three-day examination (four trials each day) was performed. One-way ANOVA Newman-Keuls Multiple Comparison Test indicates that double transgenic mice show a significant decrease (p < 0.01) in latency to fall than mice of other genotypes.

FIGURE 7.

Neurological phenotypes of double transgenic mice expressing mutant htt in neuronal and glial cells. A, comparison of latency to fall of mice of different genotypes, including double transgenic mice expressing htt-98Q or htt-160Q. Mice at the age of 11–12 or 18–19 weeks (n = 8–12 each group) were compared. *, p < 0.05; **, p < 0.01. B and C, body weight (B, n = 8–10) and survival plot (C, n = 7–11) of mice of different genotypes showing that double transgenic mice lose more body weight (p < 0.05 after 14 weeks) and die earlier than N171–82Q mice.