Transgenic mice expressing a Huntington's disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity

Abstract

Huntington's disease (HD) is a hereditary neurodegenerative disorder presenting with chorea, dementia, and extensive striatal neuronal death. The mechanism through which the widely expressed mutant HD gene mediates a slowly progressing striatal neurotoxicity is unknown. Glutamate receptor-mediated excitotoxicity has been hypothesized to contribute to the pathogenesis of HD. Here we show that transgenic HD mice expressing exon 1 of a human HD gene with an expanded number of CAG repeats (line R6/1) are strongly protected from acute striatal excitotoxic lesions. Intrastriatal infusions of the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid caused massive striatal neuronal death in wild-type mice, but no damage in transgenic HD littermates. The remarkable neuroprotection in transgenic HD mice occurred at a stage when they had not developed any neurological symptoms caused by the mutant HD gene. At this stage there was no change in the number of striatal neurons and astrocytes in untreated R6/1 mice, although the striatal volume was decreased by 17%. Moreover, transgenic HD mice had normal striatal levels of NMDA receptors, calbindin D28k (calcium buffer), superoxide dismutase activity (antioxidant enzyme), Bcl-2 (anti-apoptotic protein), heat shock protein 70 (stress-induced anti-apoptotic protein), and citrate synthase activity (mitochondrial enzyme). We propose that the presence of exon 1 of the mutant HD gene induces profound changes in striatal neurons that render these cells resistant to excessive NMDA receptor activation.

Figure 1

Micrographs of striatal sections prepared from brains 48 h after intrastriatal quinolinic acid injection, labeled with the two fluorescent cell death markers Fluoro-Jade (A, B, E, and F) or TUNEL (C, D, G, and H). Sections from wild-type mice (wt; A, C, E, and G) contain numerous stained cells. In sections from transgenic mice (tg; B, D, F, and H) only very few cells are labeled along the cannula track (arrowheads). [Bar = 720 μm (A and B), 250 μm (C and D), and 30 μm (E–H).]

Figure 2

Quantification of the number of striatal Fluoro-Jade- and TUNEL-positive cells 48 h after intrastriatal quinolinic acid injection. The number of positive cells was counted in the five coronal sections closest to the site of injection and is expressed as a mean (error bar = SD) percentage of the total number of cells in the same sections (n = 5 per group). ∗, P < 0.0001.

Figure 3

Micrographs of striatal sections prepared from brains 14 days after intrastriatal quinolinic acid injection, processed for DARPP-32 immunohistochemistry (A, B, E, and F) or cresyl violet staining (C, D, G, and H). Sections from wild-type mice (wt; A, C, E, and G) show massive loss of DARPP-32- and cresyl violet-stained cells. Sections from transgenic mice (tg; B, D, F, and H) reveal only a minor loss of DARPP-32- positive neurons (B and F) and an increased number of cresyl violet-stained cells (D and H) along the cannula track (arrowheads). [Bar = 1.0 mm (A–D) and 50 μm (E–H).]

Figure 4

Quantification of the number of striatal DARPP-32- and cresyl violet- labeled cells 14 days after intrastriatal quinolinic acid injection. The number of positive cells was counted in the five coronal sections closest to the site of injection and is expressed as a mean (error bar = SD) percentage of the number of positive cells on the contralateral unlesioned side (n = 9 transgenic mice; n = 7 wild-type mice). ∗, P < 0.0001.

Figure 5

Quantification of proteins possibly regulating susceptibility to excitotoxic damage. Striata of randomly chosen wild-type (wt) or transgenic (tg) mice were excised and homogenized. Proteins were separated by SDS/polyacrylamide gel electrophoresis and detected by immunoblot (Hsp-70, heat shock protein 70; NMDA-R, NMDA receptor subunit NMDA-NR1). The anti-calbindin D28k antibody recognized a nonspecific band at 26 kDa in addition to the 28-kDa calbindin band (arrow).