Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease

: Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors

A cutting-edge approach based on UHPLC-MS to simultaneously investigate oxysterols and cholesterol precursors in biological samples: Validation in Huntington's disease mouse model

Brain is most cholesterol-rich organ in the body. Since cholesterol does not cross the blood brain barrier, its metabolism is provided in situ by astrocytes and neurons, and it is crucial for maintaining sterol levels and neuronal integrity and function. Recent studies have shown that the levels of cholesterol precursors and metabolites are lower in the brains of animal models of Huntington's disease (HD) while reduced levels of its catabolite are detected in the plasma of patients. In this study, we introduce a novel analytical method designed to fulfill the complex analytical requirements associated with cholesterol metabolites detection in neurodegenerative disorders. The method allows for the simultaneous quantification of a specific set of oxysterols along with cholesterol precursors in biological samples.The proposed method uses an Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) system operating in multiple reaction monitoring (MRM). Since sterols can be found in biological matrices in either free form or esterified to various fatty acids, a three-step extraction procedure was devised, consisting of alkaline hydrolysis, liquid-liquid extraction and final concentration omitting the need for a solid-phase extraction (SPE) step.The validated method achieved a detection limit of 10 ng/mL in plasma and 1 ng/mg in brain tissue, reaching a comparable sensitivity to previously published LC-MS and GC–MS methods. All target analytes were separated on a reverse-phase column employing a segmented gradient and a temperature ramp. This strategy enabled the elution and separation of all selected metabolites within a 30-minutes timeframe. This innovative approach was employed to quantify cholesterol metabolites in both plasma and brain samples from wild-type (WT) and R6/2 mice, a mouse model of HD. The results obtained from the sample analysis highlighted a significant reduction in desmosterol levels in the R6/2 brain at 12 weeks.In conclusion, the proposed method paves the way for further development of high-sensitive and reproducible protocols to comprehensively investigate simultaneous alterations in both cholesterol biosynthesis and catabolism in HD samples

Insights into kinetics, release, and behavioral effects of brain-targeted hybrid nanoparticles for cholesterol delivery in Huntington's disease

Supplementing brain cholesterol is emerging as a potential treatment for Huntington's disease (HD), a genetic neurodegenerative disorder characterized, among other abnormalities, by inefficient brain cholesterol biosynthesis. However, delivering cholesterol to the brain is challenging due to the blood-brain barrier (BBB), which prevents it from reaching the striatum, especially, with therapeutically relevant doses. Here we describe the distribution, kinetics, release, and safety of novel hybrid polymeric nanoparticles made of PLGA and cholesterol which were modified with an heptapeptide (g7) for BBB transit (hybrid-g7-NPs-chol). We show that these NPs rapidly reach the brain and target neural cells. Moreover, deuterium-labeled cholesterol from hybrid-g7-NPs-chol is released in a controlled manner within the brain and accumulates over time, while being rapidly removed from peripheral tissues and plasma. We confirm that systemic and repeated injections of the new hybrid-g7-NPs-chol enhanced endogenous cholesterol biosynthesis, prevented cognitive decline, and ameliorated motor defects in HD animals, without any inflammatory reaction. In summary, this study provides insights about the benefits and safety of cholesterol delivery through advanced brain-permeable nanoparticles for HD treatment