PURPOSE

Huntington's disease (HD) is a progressive, neurodegenerative disorder caused by an abnormal expansion of a CAG repeat in the gene encoding the huntingtin protein (Htt). This mutation leads to the expression of a poly-glutamine stretch that changes the functions of mutant Htt (mHtt), inducing pathological processes eventually causing neurodegeneration. Previous MRS and MRI studies have shown that metabolic alterations i.e., reduction of N-acetylaspartate (NAA) and striatal atrophy can detected in HD patients and mouse models of HD^1,2. Newly available knock-in models (e.g., Q175) recapitulate the heterozygosity of human HD, the clinically observed pathophysiology and behavioral abnormalities including motor and cognitive deficits³. While older N-terminal and full-length transgenic HD models have been extensively characterized using ¹H-MRS and volumetry studies^2,4,5, data in knock-in models have only been assessed either in aged mice or in mixed-gender groups with volumetry limited to a few brain regions^6,7. Here we assessed striatal metabolites using ¹H-MRS and volumes of 30+ brain regions longitudinally in both male and female Q175 mice.

METHODS

¹H-MRS and MRI in Q175 male and female heterozygous (HET-m, HET-f), homozygous (HOM-m, HOM-f) and wild-type (WT-m, WT-f) littermates with longitudinal study design across 4 time points (2, 4, 7 and 10 months of age). Group size n=10 per genotype/gender. MRS/MRI: Bruker BioSpec 9.4T with 72 mm bird-cage resonator for excitation and surface coil for reception. Mice were sedated with isoflurane (2%). ¹H MR spectra were acquired using point-resolved spectroscopy PRESS from single voxel (1.8x1.8x2.0mm³) in the left striatum. Analysis was performed in an automated pipeline using custom scripts for the “LCModel” software. Volumetry: 31 axial T₂-weighted structural images (0.078x0.078x0.6mm³) were acquired using a RARE sequence. Volumes of anatomically predefined brain regions were determined by spatially registering an in-house digital brain atlas onto each individual brain at each visit (using “SPM5”).

RESULTS AND DISCUSSION

Our results confirm previous findings of a neurodegenerative phenotype in HOM Q175 mice⁷ reflected by an age-dependent decrease in striatal volume as well as a reduction in NAA when compared to age-matched controls (Fig.1+2). This phenotype was present in both male and female HOM mice and detectable from an age of 4 months. Table 1 provides an overview of significant differences between mutant (HET, HOM) and WT mice for both genders in striatal metabolite concentrations and volumes of brain regions. Remarkably, cortical volume loss together with reduction in taurine appeared to precede striatal atrophy and reduction in NAA and was present in HOM-m and HOM-f already at 2 months. Also, atrophy appeared to be restricted to certain brain regions. HET mice showed only a mild phenotype with no significant changes in striatal volume (unlike in previous assessment of Q175 mice⁷), and the reduction in NAA only starting at 7 months. The reduction in Glu and increase in Gln in HOM mice has been described earlier in HD models^7,8,9, albeit for mixed-gender groups. It appears that the increased Gln levels are stronger/earlier in female mice.
We also observed a remarkable feature not reported for HD mouse models so far, namely sub-groups of individuals with age-independent, abnormally high Gln values (usually twice the level of the standard concentrations), whose empirical incidence increased with increasing gene dose (1/20, 3/20 and 8/20 for WT, HET and HOM mice; p=0.0042, Cochran-Armitage trend test; no gender effect, Fig.3). A low incidence of about 1/25 in WT mice is supported by previous (unpublished) data from other in-house studies. The affected individuals also showed higher striatal volumes and deviant concentrations of other metabolites (e.g., higher NAA, lower Ins), although not as pronouncedly. Elevated Gln group levels have been reported previously in HD mouse models^7,8,9 and HD patients¹⁰, and have been attributed to a decrease in neuronal-glial Glu-Gln cycle and reduced glutaminase activity⁹. On the other hand, high Gln levels in the brain have also been found in patients with chronic hepatic encephalopathy, where excess ammonia diffuses into the brain and is subsequently metabolized to Gln^11,12. We hypothesize that excessive Gln present in some (“high-Gln”) individuals is a consequence of disordered hepatic metabolism, with the incidence related to mHtt dosage, whereas the age-dependent Gln increase in “low-Gln” individuals reflects locally disturbed Glu-Gln metabolism. Indeed, there is evidence for a widespread peripheral metabolic perturbation in HD¹³, albeit with unclear relevance to the core CNS symptomatology.

CONCLUSION

Apart from an age-dependent alteration in markers of neurodegeneration in homozygous Q175 mice, we identified sub-groups with abnormally high striatal Gln levels, whose incidence showed a mHtt dosage effect. This novel feature may represent a cerebral marker of peripheral metabolic perturbation described for mouse models of HD and HD patients.

Acknowledgements

We gratefully acknowledge Sebastien Debilly, Stephanie Schoeppenthau and Ciril Waelti for skilled animal handling and Thomas Bielser for data processing.

References

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