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Focusing on the caprylic acid (C8:0), this study aimed at investigating the discrepancy between the formerly described beneficial effects of dietary medium chain fatty acids on body weight loss and the C8:0 newly reported effect on food intake via ghrelin octanoylation. During 6 weeks, Sprague-Dawley male rats were fed with three dietary C8:0 levels (0, 8 and 21% of fatty acids) in three experimental conditions (moderate fat, caloric restriction and high fat). A specific dose-response enrichment of the stomach tissue C8:0 was observed as a function of dietary C8:0, supporting the hypothesis of an early preduodenal hydrolysis of medium chain triglycerides and a direct absorption at the gastric level. However, the octanoylated ghrelin concentration in the plasma was unchanged in spite of the increased C8:0 availability. A reproducible decrease in the plasma concentration of unacylated ghrelin was observed, which was consistent with a decrease in the stomach preproghrelin mRNA and stomach ghrelin expression. The concomitant decrease of the plasma unacylated ghrelin and the stability of its acylated form resulted in a significant increase in the acylated/total ghrelin ratio which had no effect on body weight gain or total dietary consumption. This enhanced ratio measured in rats consuming C8:0 was however suspected to increase (i) growth hormone (GH) secretion as an increase in the GH-dependent mRNA expression of the insulin like growth Factor 1 (IGF-1) was measured (ii) adipocyte diameters in subcutaneous adipose tissue without an increase in the fat pad mass. Altogether, these results show that daily feeding with diets containing C8:0 increased the C8:0 level in the stomach more than all the other tissues, affecting the acylated/total ghrelin plasma ratio by decreasing the concentration of circulating unacylated ghrelin. However, these modifications were not associated with increased body weight or food consumption.

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Huntington disease (HD) is a devastating neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Disrupted cortico-striatal transmission is an early event that contributes to neuronal spine and synapse dysfunction primarily in striatal medium spiny neurons, the most vulnerable cell type in the disease, but also in neurons of other brain regions including the cortex. Although striatal and cortical neurons eventually degenerate, these synaptic and circuit changes may underlie some of the earliest motor, cognitive, and psychiatric symptoms. Moreover, synaptic dysfunction and spine loss are hypothesized to be therapeutically reversible before neuronal death occurs, and restoration of normal synaptic function may delay neurodegeneration. One of the earliest synaptic alterations to occur in HD mouse models is enhanced striatal extrasynaptic NMDA receptor expression and activity. This activity is mediated primarily through GluN2B subunitcontaining receptors and is associated with increased activation of cell death pathways, inhibition of survival signaling, and greater susceptibility to excitotoxicity. Abbreviations: AHA, azidohomoalanine; AP5, amino-5-phosphonovaleric acid; a competitive NMDA receptor antagonist; BACHD, Transgenic mouse model of Huntington disease containing the full-length human mutant huntingtin gene with 97 mixed CAA-CAG repeats; BDNF, brain-derived neurotrophic factor; C6R, Transgenic mouse model expressing full-length human mutant huntingtin bearing a point mutation at the D586 caspase cleavage site; CaMKII, calcium/calmodulindependent protein kinase II; CB, cerebellum; CK2, casein kinase 2; COS-7, fibroblast-like cell line derived from monkey kidney tissue; CREB, cAMP response element-binding protein; CS, cortico-striatal; CTX, cortex; cortical; DAPK1, death-associated protein kinase 1; DARPP32, dopamine-and cAMP-regulated neuronal phosphoprotein; DIV, day-in vitro; DKI, DAPK1 inhibitor; also referred to as TC

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Background: Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington’s disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. Objective: To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. Methods: We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. Results: We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine differences (ΔQs 22–179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. Conclusion: This quantitative immunoblotting method can be used to reliably resolve full length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.

Introduction: Huntington disease is an autosomal dominant neurodegenerative disorder which is caused by a CAG repeat expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. Huntingtin is subjected to multiple post-translational modifications which regulate its cellular functions and degradation. We have previously identified a palmitoylation site at cysteine 214 (C214), catalyzed by the enzymes ZDHHC17 and ZDHHC13. Reduced palmitoylation level of mutant huntingtin is linked to toxicity and loss of function. Moreover, we have described N-terminal myristoylation by the N-myristoyltransferases of a short fragment of huntingtin (HTT553-586) at glycine 553 (G553) following proteolysis at aspartate 552 (D552).Results: Here, we show that huntingtin is palmitoylated at numerous cysteines: C105, C433, C3134 and C3144. In addition, we confirm that full-length huntingtin is cleaved at D552 and post-translationally myristoylated at G553. Importantly, blocking caspase cleavage at the critical and pathogenic aspartate 586 (D586) significantly increases posttranslational myristoylation of huntingtin. In turn, myristoylation of huntingtin promotes the co-interaction between C-terminal and N-terminal huntingtin fragments, which is also protective.Discussion: This suggests that the protective effect of inhibiting caspase-cleavage at D586 may be mediated through post-translational myristoylation of huntingtin at G553.