Purpose

Huntington disease (HD) is an inherited polyglutamine disease characterized by involuntary abnormal movements, cognitive and psychiatric symptoms, associated with preferential atrophy of the striatum.¹ Evidence suggests that energy deficit plays a critical role in the disease pathophysiology.² Evaluating metabolic dysfunction can help identify functional biomarkers for therapeutic trials. However, existing methods are unable to fully capture the dynamic metabolism of the brain with their static measurements. This study thus aims to measure dynamic parameters of brain energy metabolism to decipher the mechanisms underlying brain energy deficit in HD and identify novel functional biomarkers to be used in clinical trials such as those targeting the Krebs cycle.

Methods

The study was divided into two phases. The first phase involved recruiting 10 healthy individuals for method validation. The second phase is still ongoing and seeks to recruit 20 presymptomatic individuals, 20 patients at the early stage of HD and 20 controls with similar age and body mass index. Following our observation of an abnormal energy profile in the occipital cortex in previous studies,^3,4 we performed ³¹P magnetization transfer (³¹P MT, pulse acquire, T_R = 15 s) in the occipital cortex to measure the rate of brain creatine kinase. A 25 x 25 x 25 mm³ voxel covering the calcarine was used for shimming. Frequency calibration was performed for each subject in order to determine the voltage needed to achieve the maximum signal. Saturation of the γATP resonance was performed using 8 cycles of B1-insensitive train to obliterate signal (BISTRO)⁵ pulse (duration 3.5 s). A symmetric saturation was performed at the opposite side of the phosphocreatine (PCr) resonance to correct for RF bleedover. Data were collected for 8 min each at rest, during visual stimulation with red-black checkerboard flashes, and recovery after visual stimulation. Spectra were analyzed using LCModel as explained.⁶ Diffusion weighted spectroscopy (DWS) was also used to evaluate the diffusion properties of metabolites that reflect distinct metabolic compartments, i.e. neuronal versus glial. DWS was performed in the occipital cortex (VOI = 25 x 25 x 25 mm³, b value = 0, 3550 s/mm²) and the corpus callosum (VOI = 8 x 15 x 32 mm³, parallel diffusion: b values = 0, 482, 772, 1737, 3088 s/mm²; perpendicular diffusion: b values = 0, 964, 1544, 3474, 6176 s/mm²). Spectra were analyzed with LCModel and the apparent diffusion coefficient of total N-acetylaspartate (tNAA), total creatine (tCr) and total choline (tCho) were calculated. Furthermore, resting state functional MRI (rsfMRI, T_R = 1000 ms, slice thickness = 3 mm isotropic, number of measurements = 600, multiband acceleration factor = 3, acquisition time = 10 min) was performed to capture the impact of functional connectivity on neurometabolism and vice versa. The data were analyzed using robust and reproducible algorithms⁷ developed by our collaborators at the Neuroscience Research Australia in Sydney, Australia to incorporate temporal correlations, remove spurious spatial correlations, deal with artifacts, and the ability to apply on single subjects. Multi-shell diffusion weighted imaging (b values = 2500 s/mm² (60 directions), 900 s/mm² (32 directions) and 300 s/mm² (8 directions)) was also included in the protocol to evaluate the effect of neurometabolism on the microstructural integrity in HD.

Results

The first phase of the study was successfully completed and we are currently on phase two and hence will present preliminary results. Preliminary analyses of ³¹P MT data showed that we fully saturated γATP resonance (Figure 1). We observed decreased CK rate at rest in patients compared to controls (p < 0.05) (Figure 2) but are yet to observe any change during visual stimulation and the recovery phase. The DWS data showed increase in the parallel diffusion of tCho (p < 0.05) in the corpus callosum and may signify increased gliosis (Figure 3A). There was also an increased perpendicular diffusion of tNAA, tCr and tCho (p < 0.05) in the corpus callosum (Figure 3B). These may point to possible axonal damage, energetic compensatory mechanism and increased gliosis respectively. No change was observed in the visual cortex. The rsfMRI protocol was well tolerated – e.g. no induction of peripheral nerve stimulation – and the data was of high quality. Preliminary analyses showed that we are able to extract pertinent information related to the networks (Figure 4). Since data acquisition is still ongoing, we are yet to perform statistical analyses of rsfMRI and diffusion data between the subject cohorts.

Conclusion

Our protocols were able to capture the dynamic alterations in neurometabolism in HD. They are thus suitable for finding functional biomarkers for use in therapeutic interventions targeting the Krebs cycle.

Acknowledgements

We are very grateful to the patients and volunteers who participated in this study.