Disturbances in sleep/wake cycles are common among patients with neurodegenerative diseases including Huntington’s disease (HD) and represent an appealing target for chrono-nutrition-based interventions. In the present work, we sought to determine whether a low-carbohydrate, high-fat diet would ameliorate the symptoms and delay disease progression in the BACHD mouse model of HD. Adult WT and BACHD male mice were fed a normal or a ketogenic diet (KD) for 3 months. The KD evoked a robust rhythm in serum levels of β-hydroxybutyrate and dramatic changes in the microbiome of male WT and BACHD mice. NanoString analysis revealed transcriptional changes driven by the KD in the striatum of both WT and BACHD mice. Disturbances in sleep/wake cycles have been reported in mouse models of HD and are common among HD patients. Having established that the KD had effects on both the WT and mutant mice, we examined its impact on sleep/wake cycles. KD increased daytime sleep and improved the timing of sleep onset, while other sleep parameters were not altered. In addition, KD improved activity rhythms, including rhythmic power, and reduced inappropriate daytime activity and onset variability. Importantly, KD improved motor performance on the rotarod and challenging beam tests. It is worth emphasizing that HD is a genetically caused disease with no known cure. Life-style changes that not only improve the quality of life but also delay disease progression for HD patients are greatly needed. Our study demonstrates the therapeutic potential of diet-based treatment strategies in a pre-clinical model of HD.

The work presented in this study followed all the guidelines and regulations of the UCLA Division of Animal Medicine that are consistent with the Animal Welfare Policy Statements and the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. 

Animals

The BACHD mouse model used in this study expresses the full length of the human mutant HTT gene encoding 97 glutamine repeats under the control of the endogenous regulatory machinery (Gray et al., 2008). BACHD dams, backcrossed on the C57BL/6J background for a minimum of 12 generations, were bred with C57BL/6J (WT) males from The Jackson Laboratory (Bar Harbor, Maine) in our facility at UCLA. Data were collected from male WT and heterozygous for the BACHD transgene littermates. Genotyping was performed at 15 days of age by tail snips, and after weaning at postnatal day 21, littermates were group housed, until otherwise noted. All animals were housed in soundproof chambers with controlled temperature, humidity and lighting conditions, 12 hr light, 12 hr dark cycle (12:12 LD, intensity 350 lux) for at least two weeks prior to any experimentation or change in diet. For all experiments, a light meter (BK precision, Yorba Linda, CA) was used to measure light-intensity (lux).  Each chamber holds 8 cages of mice, grouped together by feeding treatment. The animals received cotton nestlets and had water available at all times.

Experimental groups and diet 

Male WT and BACHD mice (3 months old) were randomly assigned to either a Normal Diet (ND) or KD group. The mice had ad libitum access to either a custom KD (Teklad diet TD.10911.PWD, Envigo, Madison, WI) or ND (Teklad diet 7013, NIH-31 Modified Open Formula diet, Envigo) for 3 months. The KD used (77.1% fat, 22.4% protein, 0.5% carbohydrate) has moderately high protein, no sugars and predominantly healthy fats (with a 2:1 ratio of n-3 to n-6 fatty acids from medium chain triglycerides with a little flax and canola oil). The level of proteins in the KD diet is consistent with recommendations for optimal health. The food was refreshed every 4 days, and new and unconsumed food were weighed. At the end of the treatment, the animals (6-7 months old) were used to assess motor functions and then euthanized for tissue collection. 

Microbiome measurements

Fecal samples were collected at ZT 16 from WT and BACHD mice (n=5 animals/genotype) that had been on ND or KD for at least 3 months. The fecal samples were then sent to TransnetYX (TransnetYXyx, Inc., Cordova, TN) for sequencing of the gut microbiome. The composition of the gut microbiome and the species' relative abundances were analyzed using the One Codex (One Codex, San Francisco, CA) platform. 

RNA extraction and Nanostring analysis

WT and BACHD (6-7 months old) mice (n=5-6 animals/genotype) on either ND or KD were euthanized with isoflurane at ZT 14. The left and right striati were rapidly dissected out, separately frozen and stored at -80°C. Samples were lysed using the Invitrogen^TM TRIzol^TM reagent (ThermoFisher; Carlsbad, CA). Total RNA was extracted using the RNeasy® Mini kit (Qiagen). Concentration and purity of the samples were assessed using a ThermoScientific^TM NanoDropTM One Microvolume UV-Vis Spectrophotometer (Canoga Park, CA). Gene analysis was performed in 150ng of total RNA (at a concentration of 20ng/ml) at the UCLA Center for Systems Biomedicine (genetic engineering platform) using the Nanostring nCounter® Neuropathology Panel designed to interrogate 770 transcripts specific for neurodegenerative processes/disease. Data quality and normalization of the sample signals were performed using the nSolver analyses software. The Rosalind® software (https://rosalind.bio/) was used to identify differentially expressed genes, as well as to obtain fold changes and p-values within two groups comparison (4 combinations) with genotype and diet as attributes as described in the ROSALIND® Nanostring Gene Expression Methods. The Reactome database was used to identify the top enriched biological pathways (Rosalind interactive analysis: https://app.rosalind.bio/interactive-analysis). The identified genes along with selected genes known to be circadian regulated were further analyzed by two-way ANOVA followed by the Holm-Sidak’s multiple comparisons test using the relative expression values obtained with the nSolver software. 

Excel