Differential susceptibility to excitotoxic stress in YAC128 mouse models of Huntington disease between initiation and progression of disease

Abstract

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded CAG tract in the HD gene. Polyglutamine expansion of huntingtin (htt) results in early, progressive loss of medium spiny striatal neurons, as well as cortical neurons that project to the striatum. Excitotoxicity has been postulated to play a key role in the selective vulnerability of striatal neurons in HD. Early excitotoxic neuropathological changes observed in human HD brain include increased quinolinate (QUIN) concurrent with proliferative changes such as increased spine density and dendritic length. In later stages of the disease, degenerative-type changes are apparent, such as loss of dendritic arborization, a reduction in spine density and reduced levels of 3-hydroxykynurenine and QUIN. It is currently unknown whether sensitivity to excitotoxic stress varies between initiation and progression of disease. Here, we have assessed the excitotoxic phenotype in the YAC128 mouse model of HD by examining the response to excitotoxic stress at different stages of disease. Our results demonstrate that YAC128 mice display enhanced sensitivity to NMDA ex vivo and QUIN in vivo before obvious phenotypic changes. In contrast, 10-month-old symptomatic YAC128 mice are resistant to QUIN-induced neurotoxicity. These findings are paralleled by a significant increase in NMDAR-mediated membrane currents in presymptomatic YAC128 dissociated medium spiny neurons progressing to reduced NMDAR-mediated membrane currents with disease progression. These data highlight the dynamic nature of the mutant htt-mediated excitotoxic phenotype and suggests that therapeutic approaches to HD may need to be altered, depending on the stage and development of the disease.

Figure 1.

Increased sensitivity to excitotoxic stress in YAC128 mice before signs of illness. A, B, Primary neuronal cultures established from embryonic YAC128 (HD53) and WT striata were assessed for apoptotic cell death 24 h after NMDA. MSNs from YAC128 (HD53) striata demonstrate an increase in vulnerability to NMDAR-mediated excitotoxicity versus WT with increased cell death observed after NMDA (p < 0.01). C, Intrastriatal injection of QUIN in 1.5-month-old mice demonstrates an increase in apoptotic Fluoro-Jade positive cells in YAC128 (HD53) striatum compared with WT (p < 0.05). D, Quantification of lesion volume demonstrates a trend toward enhanced lesion volume in YAC128 (HD53) compared with WT striata (p < 0.07). Lesion volume and mean number of Fluoro-Jade positive cell is ±SEM. Mean percentage apoptotic cell death is given ±SD. *p < 0.05; **p < 0.01.

Figure 2.

Mutant huntingtin exacerbates neuronal dysfunction after stroke injury. A, Assessment of motor function in 1.5-month-old YAC128 (HD53) and WT mice 24 h after ischemia-induced injury reveals the presence of mhtt delays the recover of motor function in YAC128 (HD53) mice compared with WT (p < 0.05). No difference between genotypes is observed for baseline gross sensorimotor behavior 2 h after stroke injury. B, Percentage recovery from MCAO is significantly reduced in YAC128 (HD53) mice versus WT (p < 0.01). In contrast to WT mice, which demonstrate an improvement in motor function 24 h after ischemia (23%), YAC128 (HD53) continue to perform poorly (−4%). C, Representative images of infarct volume in YAC128 (HD53) versus WT coronal sections reveal the ischemic-induced damage by TTC staining. Quantification of lesion volume demonstrates a 39% increase in YAC128 (HD53) compared with control which approaches statistical significance (p < 0.09). Lesion volume is ±SEM. *p < 0.05; **p < 0.01.

Figure 3.

Aging influences vulnerability to excitotoxic stress in vivo. A, Effects of QUIN-induced striatal lesions on aging in FVB mice. An inverse correlation is observed between lesion volume and age of the animal. ANOVA analysis on 6- to 18-month-old mice reveals a significant difference in lesion volume with age (p = 0.02). B, Linear regression analysis of lesion volume and aging reveal a strong correlation between these outcomes (r² = 0.9501, p = 0.02). Lesion volume is ±SEM. *p < 0.05; **p < 0.01.

Figure 4.

Susceptibility to excitotoxic stress in YAC128 mouse models of HD is dependent on the stage of the HD phenotype. A, Levels of mhtt in YAC128 line HD55+/+ and HD53. A more severe phenotype is observed in the higher mhtt expressing line HD53. B, The presence of mhtt influences the response to QUIN-induced neurotoxicity with initial enhanced sensitivity to excitotoxic stress progressing to resistance later in the phenotype. HD55+/+ demonstrate an increase in lesion volume versus WT at 3 months of age (p < 0.01) and a trend toward resistance at 12 month. HD53, which demonstrates enhanced sensitivity at 1.5 months, are resistant to QUIN-induced excitotoxicity at 10 months of age (p < 0.05). A significant difference is observed in lesion volume between HD55+/+ and HD53 at 3 months of age (p < 0.05). C, Linear regression analysis reveals an increased rate of progression of the excitotoxic phenotype is observed in HD 53 mice as revealed by an increase in the slope of HD53 versus WT (p = 0.01). D, Onset of the resistance phenotype in HD53 occurs earlier than HD55+/+, at 6.9 and 8.6 months respectively. Lesion volume is ±SEM .*p < 0.05; **p < 0.01.

Figure 5.

Biphasic changes in NMDA receptor-mediated currents and current densities in acutely dissociated MSNs from YAC128 and WT mice. A, Examples of typical traces obtained from MSNs from WT and YAC128 (HD53) mice at 1.5 and 7 months. B, Bar graphs showing increased mean currents and current densities at 1.5 months and decreased mean currents and current densities at 7 months between YAC128 (HD53) and WT mice, respectively. *p < 0.05; **p < 0.01.

Figure 6.

Biphasic changes in NMDA receptor-mediated synaptically evoked currents in MSNs in slices from YAC128 and WT mice. A, B, Examples of typical synaptic responses at increasing stimulus intensities obtained from MSNs from WT and YAC128 (HD53) mice at 1.5 and 7 months, respectively. C, D, Graphs showing increased mean currents at 1.5 months and decreased mean currents at 7 months between YAC128 (HD53) and WT mice, respectively. *p < 0.05.