Q175 Mouse Model

[Marilina Crawn]

Copyright2013 Loh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study was supported by the CHDI Foundation ( ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To study the underlying mechanisms of sleep-wake disturbances associated with HD, we first need to identify suitable animal models that recapitulate as many symptom sets of HD as possible. While there are numerous mouse models of HD, no single model has yet been determined to be the ideal mirror of human HD [17]. Two examples of transgenic insertion HD mouse models are the exon 1-fragment model (R6/2 [18]) and the stable 90Q-repeat with full-length human HTT gene model (BACHD [19]). Circadian deficits begin in the R6/2 line at 10 weeks, starting with imprecise activity onset and progressing to a complete loss of rhythms in activity [10], [20]. The BACHD model exhibits a decline in the power of circadian rhythms of activity at 3 months [20], [21]. Of the current complement of HD mouse models, the knock-in of 140 CAG repeats into exon 1 of the mouse Htt gene (CAG140 KI [22], [23]) has considerable benefits as the genetically precise insertion of the mutation into the Htt locus rules out position and copy number effects that may affect the other transgenic models. The age-related decline in circadian rhythms in the CAG 140 KI line was not distinguishable from the age-related decline also observed in WT mice at 12 months of age, suggesting that the effects of the targeted CAG repeats are subtle in the heterozygote mutants [20]. A spontaneous expansion mutant that arose in this line of mice, with over 175 CAG repeats (Q175), has motor and cognitive deficits that have earlier onsets than the CAG140 line [24], and thus may also exhibit sleep and circadian rhythm disruption that can be detected before WT rhythms also decline.

We examined the spontaneous expansion Q175 model for sleep and circadian rhythm disruptions over the course of 12 months of age. We also examined the decline in motor function and the impact of the mutation on circadian gene expression in the SCN.

The experimental protocols used in this study were approved by the UCLA Animal Research Committee (ARC 2009-022), and all recommendations for animal use and welfare, as dictated by the UCLA Division of Laboratory Animal Medicine and the guidelines from the National Institutes of Health, were followed. A line of mice with a spontaneous expansion of the CAG repeats in the CAG140 KI line, the Q175 mutation, was obtained from the CHDI colony of Q175 mice at the Jackson Laboratories (Bar Harbor, Maine). The Q175 mice have previously been shown to have around 188 CAG repeats [24]. We obtained an age-matched cohort of male mice on the C57Bl6/J background that were wild-type (WT), heterozygote (Het) and homozygote (Hom) for the Q175 allele.

We employed both the accelerating rotarod test [29] and challenging beam test [30] to determine the progression of motor dysfunction in the mice. All tests were performed at the light:dark transition (ZT 11 to 12; one hour prior to lights-off) to minimize effects of sleep deprivation.

At 6, 9 and 12 months, the mice were trained on the rotarod (Ugo Basile, Varese, Italy) with 5 trials on the first day, as previously described [29]. The accelerating rotarod went from 5 to a maximum of 38 rpm and the maximum length of each trial was 600 sec. On the second day, mice were placed on the rotarod and the latency to fall from the rotarod was recorded from 5 trials, and averaged between trials.

We employed a modified version of the traditional beam walking test, as described in detail by Fleming and colleagues [30]. The premise of this task, named the challenging beam test, is to determine the ability of mice to cross a bridge of decreasing width, with re-entry into the home cage as the motivating factor [30]. The beam narrows in 4 intervals from 33 mm >24 mm >18 mm >6 mm, with each bridge spanning 253 mm in length. Mice at 9 and 12 months of age were trained on the beam for 5 consecutive trials at ZT 11 on two consecutive days. During each trial, each mouse was placed on the widest end of the beam and allowed to cross with minimal handling by the experimenter. On the third day, the mice were further challenged during testing by the application of a metal grid (1010 mm spacing) overlaid onto the bridge. A second experimenter was on hand to record the progress of the animal using a hand held camcorder and five consecutive trials were recorded. The videos were scored by two independent observers blind to the experimental conditions for the time to cross the beam and touch the home cage, the number of steps taken by one designated limb, and the number of mis-steps (errors) made by each mouse. We considered an error to have occured when more than half of the foot in question dipped below the grid. Time to cross, number of steps, and number of errors were averaged across the 5 trials per mouse to give the final reported values.

To determine cell counts and the expression of a core circadian gene, we perfused 13 month old WT, Q175 Het and Hom mice at either ZT 2 (2 hours after lights on; PER2 trough in SCN) or ZT 14 (2 hours after lights off; PER2 peak in SCN). Coronal sections (20 m) were cut, and the sections containing the SCN were analyzed. Alternate sections of SCN were chosen for either Nissl staining to determine cell counts or IHC with an antibody against PER2 to determine the peak/trough of the molecular oscillator.

Neuronal cell bodies through the middle of the rostro-caudal axis of the SCN were identified using Nissl stain (0.1% Cresyl Violet). Sections through the SCN were photographed at 10X magnification, and counts of cell bodies within a defined area were determined by averaging the numbers from two investigators blind to the conditions. The surface area of the SCN and the lateral ventricles were determined using Axiovision (Carl Zeiss).

For comparison of WT, Q175 Het and Hom locomotor activity and sleep parameters that passed normality and equal variance tests within each age group, we applied one way analysis of variance (ANOVA) for which we report the F statistic, and deemed differences as significant if P

A. Free-running period (tau) is no different between the three genotypes. B. Power, as measured by the X2 periodogram, declines dramatically in Q175 Hom mutants at 9 months. C. The cycle-to-cycle activity onset is less precise in Q175 Hom mutants from 9 months. D. The amount of activity (wheel revolutions per hour, rev/hr) plummets in Q175 Hom mutants at 9 months. * indicates significance to P

The reduced nocturnality of the Q175 Hom mice suggests deficits in the light response, which we tested by exposing the mice to discrete pulses of light to induce phase shifts in their free-running rhythms. The light-induced phase delay increased in magnitude in Q175 Hom mice, beginning at 9 months and increasing further at 12 months compared to WT and Het mice (Table 3). Housing mice under a skeleton photoperiod also revealed deficits in the temporal patterning of activity that began at 6 months in the Q175 Hom mice (Table 4).

We conclude from these wheel running assays that the Q175 Hom mutants show a rapid decline of rhythms in locomotor activity from 9 months of age, with poor precision and abnormal temporal patterning of activity. The Q175 Het mutants also exhibit an age-related decline in the power of activity rhythms, but to a lesser degree than the Hom mutants, suggesting an effect of gene dosage.

We wished to determine if the Q175 line recapitulated the HD-like symptoms observed in its preceding line, the CAG140 knock-in model. As such, we performed two motor function tests previously found robust enough to detect motor deficits in mouse models of neurodegeneration: the accelerating rotarod test (e.g. [28]) and a modified version of the beam walking test: the challenging beam test [29].

A. The accelerating rotarod test revealed that the age-related motor deficits in the Q175 model take up to 12 months to appear. B. 9 month old Q175 Hom mutants take longer to traverse the challenging beam test than age-matched WT and Q175 Het mice. C. The number of errors made while crossing each segment of the beam are shown, with the widest part of the beam on the left (33 mm) and narrowing towards the right (6 mm). The beam lengths are not to scale. The number of errors made while crossing the challenging beam are higher in Q175 Hom mutants. * indicates P

We can thus confirm that the Q175 Hom mice showed a decline in motor performance at 9 months as revealed by the challenging beam test, and that both Q175 Het and Hom mice showed deficits compared to age-matched WT controls on the accelerating rotarod test at 12 months.

We used video recording to measure sleep as defined by time spent immobile, in combination with automated mouse tracking analysis software. Average waveforms of hourly immobility-defined sleep clearly show a change in the distribution of sleep in the Q175 Hom mice at both 9 (Fig. 7A top) and 12 months (Fig. 7A bottom). As nocturnal creatures, mice typically spend majority of the daylight hours inactive, and the reduction in nocturnality that we previously observed of Q175 Hom mutants using wheel running activity is replicated in these video measurements of freely behaving mice.

The impact of the Q175 mutation appears to have a selective effect on daytime sleep in the Hom mutants, with a specific reduction of the amount of time spent asleep in the day, as well as increased fragmentation of already reduced sleep.

The pathophysiology of HD is neurodegeneration followed by neuronal cell death in vulnerable cell populations like the striatum. The CAG140 KI line exhibited loss of striatal neurons as the disease progressed [32], [33]. Striatal volume was found to be reduced in the Q175 Het and Hom mutant mice from 4 months [34]. We examined the SCN to ascertain the impact of the Q175 mutation on the neuronal cell counts in WT, Het and Hom mice.

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