Those interested in ³¹P MRS, energy metabolism and Huntington’s disease.In this work we use localized ³¹P MRS, including progressive magnetization transfer (MT) experiments, to assess energy metabolism in a transgenic rat model of Huntington’s disease (BACHD [1]). Localized measurements of the ATP formation rate through creatine kinase and ATPsynthase are performed in the rat brain for the first time, and reveal an altered metabolic status in BACHD rats.

Animal preparation and in vivo ³¹P MRS: Seven 18 to 24 months old transgenic rats (BACHD) and six age-matched wild type (WT) littermates anesthetised with 1.5-2 % isoflurane were used to estimate forward reaction rates of both ATP synthase (k_f-ATPase) and creatine kinase (k_f-CK) using ³¹P magnetization transfer MRS [2]. All measurements were performed using a 11.7T Bruker BioSpec using a 1.5-cm double-tuned surface coil (Rapid Biomed GmbH®). Localisation of a large 11×8×12 mm (Fig. 1) voxel was achieved using an adiabatic ISIS module followed by a selective BISTRO progressive saturation of the γ-ATP peak. We decided to quantify peak (PCr and Pi) attenuation relative to an unsaturated spectrum rather than symmetric saturation, because: i) signal loss due to RF bleed-over was minimal (~5%) during selective symmetric saturation, as assessed on preliminary experiments (Fig. 2A); and ii) possible residual bias would be identical in both groups and thus wouldn’t affect intergroup comparison. Three different saturation times (tsat=0.55, 1.1 and 2.2 s, Fig. 2B) were used for γ-ATP saturation, as well as two unsaturated spectra, one acquired at the beginning and one at the end of each acquisition, which also ensured that signal remained stable throughout the experiment. Each spectrum was acquired in 17 min, making it possible to estimate k_f-CK at the individual level in less than 2 hours (including animal preparation) given the fact that symmetric-saturation spectra needed not to be acquired. Due to lower signal to noise ratio on the Pi peak, k_f-ATPase was estimated only at group level, with Pi being quantified on spectra averaged over all animals of each group.

Data Analysis: Data were analysed with LCModel using a simulated basis set, and metabolite concentrations were estimated in both groups on unsaturated spectra. PCr concentration was estimated relative to α-ATP concentration which was considered constant (and indeed yielded the same absolute signal on spectra in both groups, see Fig. 3). From the chemical shift difference between Pi and PCr, the pH was also derived. Individual concentrations of PCr, and group concentrations of Pi, were fitted as a non-linear function of saturation time according to modified Bloch equations accounting for chemical exchange. The standard deviation for k_f-ATPase was derived from Monte Carlo simulations over 500 repetitions.

The concentration of phosphocreatine (PCr/α-ATP) exhibits a moderate but significant 10% increase in the BACHD group compared to the WT group (1.84±0.15 vs. 1.67±0.11, p=0.026) (Fig. 3). Note that such an increase has also been reported during healthy aging [3]. No pH difference was found, contrasting with our previous results in 3NP-treated rats and Human patients [4], and suggesting a milder disease or a specific metabolic adaptation. There was no difference in k_f-CK (0.43±0.07 s-1 vs. 0.44±0.10 s-1 BACHD vs. WT) between the two groups (Fig. 4A). However, considering the increased PCr levels, the total flux of ATP generation through creatine kinase ([PCr]*k_f-CK) is increasing by ~10%. In parallel, a large decrease was measured for k_f-ATPase (0.23±0.02 s-1 vs. 0.48±0.10 s-1 BACHD vs. WT, p=0.05 based on permutation test on the distribution generated by Monte Carlo simulations) (Fig. 4B). Altogether these results suggest an adaptive mechanism in the transgenic rat model involving a larger PCr pool to compensate for the defect in oxidative phosphorylation reflected by k_f-ATPase.We demonstrate for the first time the use of localised ³¹P MT-MRS to quantify both k_f-CK and k_f-ATPase in the rat brain. This study also points towards a mitochondrial deficit in the BACHD rat model that can be quantified by ³¹P MT-MRS, with ATP synthase rate reduced by a factor 2. This decrease could be partly compensated by higher cerebral concentrations of phosphocreatine (the origin of which remains to be elucidated, but could be related to peripheral PCr formation) as a readily available energy source.