Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions

Abstract

We have serendipitously established a mouse that expresses an N-terminal human huntingtin (htt) fragment with an expanded polyglutamine repeat (approximately 120) under the control of the endogenous human promoter (shortstop). Frequent and widespread htt inclusions occur early in shortstop mice. Despite these inclusions, shortstop mice display no clinical evidence of neuronal dysfunction and no neuronal degeneration as determined by brain weight, striatal volume, and striatal neuronal count. These results indicate that htt inclusions are not pathogenic in vivo. In contrast, the full-length yeast artificial chromosome (YAC) 128 model with the identical polyglutamine length and same level of transgenic protein expression as the shortstop demonstrates significant neuronal dysfunction and loss. In contrast to the YAC128 mouse, which demonstrates enhanced susceptibility to excitotoxic death, the shortstop mouse is protected from excitotoxicity, providing in vivo evidence suggesting that neurodegeneration in Huntington disease is mediated by excitotoxic mechanisms.

Fig. 1.

Characterization of shortstop fragment and comparison with full-length YAC128. (A) Western blot showing protein expression of shortstop fragment (arrowhead) probed with the htt-specific antibody BKP1 and the polyQ-specific antibody 1C2. Full-length, mouse-endogenous htt is shown (asterisk). (B) PCRs on genomic mouse DNA from shortstop (SS), YAC128 (53), and WT mice using primers within intron 2 of human htt before (9343F/9617R) and after (9343F/9680R) the shortstop breakpoint reveal the presence of part of intron 2 in shortstop. PCRs with primers from human intron 2 (9343F) and mouse chromosome 4 (m476R) reveal a band in shortstop, indicating integration into mouse chromosome 4. (C) Fragment analysis of PCR products amplified by using human-specific primers that border the CAG tract (including 69 bp of flanking sequence) demonstrate identical CAG size in YAC128 and shortstop mice. (D) Western blots with 1C2 show similar protein expression in shortstop and YAC128 mice across brain regions. (E) Western blots with 1C2 show similar levels of transgenic protein in shortstop and YAC128 cortex (n = 3). (F) Accompanying densitometry analysis of a shorter exposure of blot in E (in the linear range of detection) reveals that shortstop mice express 1.46 times the protein of YAC128 mice.

Fig. 2.

Htt inclusions in shortstop and YAC128 mice at 18 months and AF at 12 months. EM48 inclusions labeled with FITC (green) are present in NeuN-stained neurons labeled with Texas red in the striatum (A) and cortex (C) of YAC128 mice, but not in the hippocampus (E). Inclusions are present in most cells in the striatum (B), cortex (D), and hippocampus (F) in the shortstop (SS) mouse. At 12 months of age, YAC128 cortex (G) and hippocampus (K) demonstrate less labeling of AF compared with shortstop cortex (H) and hippocampus (L). Few AF are present in the striatum of the YAC128 (I) or shortstop (J) mice. Inclusions are indicated with white arrowheads; AF are indicated with black arrowheads. (Scale bar in D, which applies to A-D:10 μm; scale bar in F, which apples to E and F: 20 μm; scale bar in H, which applies to G-L: 20 μm.)

Fig. 3.

Shortstop mice do not manifest the neuronal dysfunction or degeneration of the YAC128 model. (A) Shortstop do not exhibit a deficit on an accelerating rotarod, whereas YAC128 mice demonstrate a deficit compared with WT littermates (P < 0.001). (B-D) The shortstop mice at 12 months do not exhibit significant brain weight decrease (B), striatal volume decrease (C), or striatal neuronal count decrease (D) when compared with WT mice, whereas YAC128 mice demonstrate significant decreases. (E and F) At 18 months, there is no significant decrease in brain weight (E) or striatal volume (F) of shortstop mice compared with WT mice; YAC128 mice exhibit significant deficits. Mean ± SD is shown in B-F, and mean ± SEM is shown in A. Data were analyzed by a one-way ANOVA, and P values between groups were calculated by using Tukey's posttest.

Fig. 4.

Shortstop mice demonstrate resistance to excitotoxicity compared with the YAC128 model. (A and B) Intrastriatal injection of the NMDA agonist QA in 6-month-old mice revealed larger fluorojade-stained (apoptotic) lesions in coronal sections of YAC128 mice compared with shortstop mice (B). Lesion volume was quantified and found to be significantly less in shortstop mice compared with YAC128 mice by Student's t test (A). (C) Primary striatal neuronal cultures established from YAC128 and SS postnatal day-0 pups were treated with NMDA. Apoptotic neurons were stained and quantified by using TUNEL and propidium iodide (PI). (D) YAC128 mice revealed a significantly increased percentage of apoptotic neurons compared with shortstop mice (C) by one-way ANOVA with Tukey's posttest. MK-801 reduces the level of apoptotic death in the YAC128 neurons to baseline. Mean ± SD is shown.