DOI: 10.5068/d1bt3k
title: Dietary ketosis improves circadian dysfunction as well as motor symptoms in the BACHD mouse model of Huntington’s disease

description: 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.
description: 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. 
 <strong>Animals</strong> 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.
 <strong>Experimental groups and
 diet </strong> Male WT and BACHD mice (3 months
 old) were randomly assigned to either a Normal Diet (ND) or KD group. The
 mice had <em>ad</em> <em>libitum</em> 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. 
 <strong>Microbiome measurements</strong>
 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. 
 <strong>RNA extraction and Nanostring
 analysis</strong> 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<sup>TM</sup> TRIzol<sup>TM</sup> 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<sup>TM</sup> 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. 
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