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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.

Background Cholesterol (Chol), an essential molecule for brain function, is produced locally in the brain as the blood-brain barrier (BBB) prevents its uptake from blood. Huntington disease (HD) is associated with reduced synthesis within the brain, especially in the striatum (Shankaran 2017). Previous studies showed that the delivery of a low dose of Chol (20µg) to the brain of R6/2 mice via systemic injection of brain-permeable polymeric nanoparticles (NPs-Chol_1.0) improved synaptic and cognitive but not motor defects (Valenza 2015). Recently, by infusing three doses of Chol (20-250-500µg over 4 weeks) into the striatum of R6/2 mice through osmotic minipumps, we identified the optimal dose (500µg) to ameliorate both motor and cognitive defects, rescue morphological and functional abnormalities of striatal neurons, and reduce mutant huntingtin aggregates (Birolini 2020). Aims and Methods To develop a cholesterol-based strategy for HD, a new generation of brain-permeable NPs (NPs-Chol_2.0), with enhanced drug loading capacity, has been developed. Distribution, kinetics, drug release, efficacy and safety have been explored in two HD mouse models. Results NPs-Chol_2.0 rapidly target neural cells. Chol is released in a controlled manner and accumulates over time in the brain, while being rapidly removed from peripheral tissues and plasma. Systemic and repeated injections of NPs-Chol_2.0 prevent cognitive decline and ameliorate motor defects in R6/2 mice, without any inflammatory reaction (Birolini 2021). Different therapeutic regimens are ongoing in Q175 mice to explore the long-term effects of Chol on behavioral, molecular, and functional parameters. Preliminary data show that NPs-Chol_2.0 prevents cognitive decline and muscular strength loss, and rescues paw clasping behavior and late-onset motor defects in these HD mice. Conclusions This study provides insights on the therapeutic potential of Chol delivery to the HD brain through advanced brain-permeable NPs.

Background ADAM10 is a major protease of the human brain and regulates the strength and activity of the glutamatergic synapse by cleaving a large repertoire of synaptic substrates. In Huntington’s Disease (HD), active ADAM10 accumulates at the postsynaptic density and causes proteolysis of the cell adhesion protein Ncadherin, loss of excitatory synaptic contacts and cognitive decline. Aims and Methods To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed a system-level study of ADAM10 interactors in the brain of HD mice. We then integrated our ADAM10 dataset with the HTT interactome curated in HDinHD database (www.hdinhd.org), to look for common protein partners. Results We found that ADAM10 and HTT share presynaptic proteins as putative interactors, including Bassoon, Piccolo, ERC2 and liprin-alpha3. We therefore speculated that ADAM10 may be involved in the organization of synaptic vesicle (SV) pools, and that this function is affected in HD. Here we show that, in the cortex, active ADAM10 forms a complex with Piccolo, a key player in the recycling and maintenance of SVs. Though the level of active ADAM10 increases in the HD cortical presynaptic fraction, we observed reduced ADAM10/Piccolo immunoprecipitation. We also found that SVs density is reduced in HD cortical neurons and depleted SVs stores are restricted to presynaptic regions actively engaged in SVs release and recycling. Notably, restoring ADAM10 activity to control level in the brain of HD mice restored the ADAM10/Piccolo complex and replenished SVs in HD cortical neurons. Conclusions We conclude that increased level of active ADAM10 in HD affects the cortical presynaptic terminal through the reduction of SVs pools involved in recycling and release. The results indicate that presynaptic ADAM10 is a relevant component of HD synaptic dysfunction and reinforces the role of the cortex and the presynaptic compartment in HD.

Background Cholesterol is a multifaceted molecule essential for brain function (Martin 2014). In the adult brain, cholesterol is produced locally by astrocytes and transferred to neurons through apoE-containing lipoproteins (Jurevics & Morell 1995; Mauch 2001). Disruption of brain cholesterol pathways has been linked to several neurological disorders, including Huntington’s disease (HD), a genetic, neurodegenerative disorder caused by a CAG expansion in the gene encoding the Huntingtin protein (Valenza & Cattaneo 2011). Brain cholesterol biosynthesis and content are reduced in several HD models (Valenza 2005; 2007; 2010; Shankaran 2017). The underlying molecular mechanism relies on reduced nuclear translocation of SREBP2, the transcription factor that controls the transcription of several genes involved in cholesterol biosynthesis (Valenza 2015; Di Pardo 2020). We have recently shown that cholesterol supplementation to the brain, with different delivery systems, ameliorates synaptic and behavioral defects in the R6/2 mouse model (Valenza 2015; Birolini 2020; Birolini 2021). Aims and Methods Here, we used recombinant adeno-associated virus 2/5 to deliver exogenous SREBP2 specifically in astrocytes in order to enhance the endogenous cholesterol biosynthesis in the striatum of R6/2 mice. Results We found that exogenous SREBP2 stimulates the transcription of key cholesterol biosynthesis genes resulting in fully restoration of synaptic transmission, reversal of Drd2 transcript levels, clearance of mutant Huntingtin (muHTT) aggregates and rescue of behavioral deficits. Conclusions These results demonstrate that stimulating cholesterol biosynthesis in striatal astrocytes has a positive effect on behavioral decline and disease-related phenotypes in HD mice. Furthermore, we have demonstrated that glial SREBP2 participates in HD pathogenesis in vivo, highlighting the translational potential of cholesterol-based strategies for this 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 bloodbrain 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.

Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington’s Disease (HD). One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP-controlled genes in the cholesterol biosynthesis pathway.Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the N-terminal fragment of human SREBP2 (hSREBP2).Robust hSREBP2 expression in striatal glial cells in HD mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared muHTT aggregates and attenuated behavioral deficits. We conclude that glial SREBP2 participates in HD brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of HD.

Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington’s disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element-binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP2-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2 (hSREBP2). Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington’s disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed dopamine receptor D2 (Drd2) transcript levels decline, cleared mutant huntingtin aggregates and attenuated behavioural deficits. We conclude that glial SREBP2 participates in Huntington’s disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.

Synaptic dysfunction and cognitive decline in Huntington’s disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification–mass spectrometry (IP–MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.

A variety of pathophysiological mechanisms are implicated in Huntington’s disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.

The current pharmacological treatment of Huntington's disease (HD) is palliative, and therapies to restore functions in patients are needed. One of the pathways affected in HD involves brain cholesterol (Chol) synthesis, which is essential for optimal synaptic transmission. Recently, it was reported that in a HD mouse model, the delivery of exogenous Chol to the brain with brain-permeable nanoparticles protected animals from cognitive decline and rescued synaptic communication, indicating Chol as a therapeutic candidate. We examined whether nose-to-brain delivery, already used in human therapy, could be an alternative, noninvasive strategy to deliver Chol to the adult brain and, in the future, replenish Chol in the HD brain. We gave wild-type (WT) mice a single intranasal (IN) dose of liposomes loaded with deuterium-labeled cholesterol (Chol-D6, to distinguish and quantify the exogenous cholesterol from the native one) (200 μg Chol-D6/dose). After different intervals, Chol-D6 levels, determined by LC-MS in plasma, striatum, cortex, and cerebellum, reached a steady-state concentration of 0.400 ng/mg between 24 and 72 h. A subsequent acute study confirmed the kinetic profiles of Chol-D6 in all tissues, indicating correspondence between the dose (two doses of 200 μg Chol-D6/dose) and the calculated brain area concentration (0.660 ng/mg). Finally, in WT mice given repeated IN doses, the average Chol-D6 level after 24 h was about 1.5 ng/mg in all brain areas. Our data indicate the effectiveness of IN Chol-loaded liposomes to deliver Chol in different brain regions, opening the way to future investigations in HD mice.

There are numerous studies showing a reduction in cholesterol biosynthesis in the brain of different HD animal models of HD since pre-symptomatic stages, with the striatum being more affected compared to other brain tissues (Valenza et al., 2005; 2007; 2010; Shankaran et al., 2017). Abnormalities in brain cholesterol homeostasis are also present in HD patients: the specific brain cholesterol metabolite 24-hydroxy-cholesterol (24OHC) is progressively reduced in the plasma of HD patients and in pre-HD manifesting individuals who are close to the onset of the disease (Leoni et al., 2008; 2013). On the other hand, we reported that supplementation of a low dose (˜20µg) of cholesterol to the brain, through polymeric nanoparticles (NPs) modified with a peptide to cross the BBB, ameliorates synaptic and cognitive defects in R6/2 mice (Valenza et al., 2015), providing the proof of concept that providing exogenous cholesterol to the brain is beneficial in HD. In a more recent study we used three escalating doses of cholesterol (20, 250 and 500µg) infused directly into the striatum of R6/2 mice through osmotic minipumps, to identify the optimal dose of cholesterol from a therapeutic point of view. After a 4-week-period of intrastriatal infusion, all three doses of cholesterol were able to protect R6/2 mice from cognitive decline. With respect to motor abilities, only the highest dose of cholesterol was found to significantly ameliorate motor abnormalities. This dose also rescued functional and ultrastructural synaptic defects, enhanced endogenous cholesterol biosynthesis and significantly reduced mutant Huntingtin aggregates in the striatum of HD mice. Based on these findings, a second generation of g7-NPs (Belletti et al., 2018), with a different formulation and an increased cholesterol loading capacity compared to the previous ones (Valenza et al., 2015), have been recently developed and will soon be tested in order to move forward towards the clinic.