Cholesterol‐loaded nanoparticles ameliorate synaptic and cognitive function in H untington's disease mice
2015; Springer Nature; Volume: 7; Issue: 12 Linguagem: Inglês
10.15252/emmm.201505413
ISSN1757-4684
AutoresMarta Valenza, Jane Chen, Eleonora Di Paolo, Barbara Ruozi, Daniela Belletti, Costanza Ferrari Bardile, Valerio Leoni, Claudio Caccia, Elisa Brilli, Stefano Di Donato, Marina Boido, Alessandro Vercelli, Maria Angela Vandelli, Flavio Forni, Carlos Cepeda, Michael S. Levine, Giovanni Tosi, Elena Cattaneo,
Tópico(s)RNA regulation and disease
ResumoResearch Article20 November 2015Open Access Source Data Cholesterol-loaded nanoparticles ameliorate synaptic and cognitive function in Huntington's disease mice Marta Valenza Marta Valenza Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Jane Y Chen Jane Y Chen Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Search for more papers by this author Eleonora Di Paolo Eleonora Di Paolo Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Barbara Ruozi Barbara Ruozi Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Daniela Belletti Daniela Belletti Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Costanza Ferrari Bardile Costanza Ferrari Bardile Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Valerio Leoni Valerio Leoni Neurological Institute C. Besta, Milan, Italy Laboratory of Clinical Chemistry, Ospedale di Circolo e Fondazione Macchi, Varese, Italy Search for more papers by this author Claudio Caccia Claudio Caccia Neurological Institute C. Besta, Milan, Italy Search for more papers by this author Elisa Brilli Elisa Brilli Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Stefano Di Donato Stefano Di Donato Neurological Institute C. Besta, Milan, ItalyDeceased on 12 November 2015 Search for more papers by this author Marina M Boido Marina M Boido Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin, Orbassano, Turin, Italy Search for more papers by this author Alessandro Vercelli Alessandro Vercelli Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin, Orbassano, Turin, Italy Search for more papers by this author Maria A Vandelli Maria A Vandelli Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Flavio Forni Flavio Forni Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Carlos Cepeda Carlos Cepeda Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Search for more papers by this author Michael S Levine Michael S Levine Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Search for more papers by this author Giovanni Tosi Giovanni Tosi Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Elena Cattaneo Corresponding Author Elena Cattaneo Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Marta Valenza Marta Valenza Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Jane Y Chen Jane Y Chen Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Search for more papers by this author Eleonora Di Paolo Eleonora Di Paolo Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Barbara Ruozi Barbara Ruozi Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Daniela Belletti Daniela Belletti Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Costanza Ferrari Bardile Costanza Ferrari Bardile Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Valerio Leoni Valerio Leoni Neurological Institute C. Besta, Milan, Italy Laboratory of Clinical Chemistry, Ospedale di Circolo e Fondazione Macchi, Varese, Italy Search for more papers by this author Claudio Caccia Claudio Caccia Neurological Institute C. Besta, Milan, Italy Search for more papers by this author Elisa Brilli Elisa Brilli Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Stefano Di Donato Stefano Di Donato Neurological Institute C. Besta, Milan, ItalyDeceased on 12 November 2015 Search for more papers by this author Marina M Boido Marina M Boido Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin, Orbassano, Turin, Italy Search for more papers by this author Alessandro Vercelli Alessandro Vercelli Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin, Orbassano, Turin, Italy Search for more papers by this author Maria A Vandelli Maria A Vandelli Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Flavio Forni Flavio Forni Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Carlos Cepeda Carlos Cepeda Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Search for more papers by this author Michael S Levine Michael S Levine Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Search for more papers by this author Giovanni Tosi Giovanni Tosi Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Elena Cattaneo Corresponding Author Elena Cattaneo Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Author Information Marta Valenza1,‡, Jane Y Chen2,‡, Eleonora Di Paolo1,‡, Barbara Ruozi3,‡, Daniela Belletti3, Costanza Ferrari Bardile1, Valerio Leoni4,5, Claudio Caccia4, Elisa Brilli1, Stefano Di Donato4, Marina M Boido6, Alessandro Vercelli6, Maria A Vandelli3, Flavio Forni3, Carlos Cepeda2, Michael S Levine2, Giovanni Tosi3 and Elena Cattaneo 1 1Department of BioSciences, Centre for Stem Cell Research, Università degli Studi di Milano, Milan, Italy 2Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA 3Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 4Neurological Institute C. Besta, Milan, Italy 5Laboratory of Clinical Chemistry, Ospedale di Circolo e Fondazione Macchi, Varese, Italy 6Neuroscience Institute Cavalieri Ottolenghi, Neuroscience Institute of Turin, Orbassano, Turin, Italy ‡These authors share first authorship ‡These authors share second authorship *Corresponding author. Tel: +39 02 50325842; E-mail: [email protected] EMBO Mol Med (2015)7:1547-1564https://doi.org/10.15252/emmm.201505413 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington's disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7-NPs-Chol), which per se is not blood–brain barrier (BBB) permeable, to obtain high-rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7-NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7-NPs-Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonstrate that cholesterol supplementation to the HD brain reverses functional alterations associated with HD and highlight the potential of this new drug-administration route to the diseased brain. Synopsis Cholesterol in brain is largely derived by local synthesis. One affected pathway in Huntington's disease (HD) implicates that reduced production and/or availability of brain cholesterol may be detrimental for neuronal function. Polymeric nanoparticles (NPs) loaded with cholesterol, modified with glycopeptides g7 (g7-NPs-Chol) to cross the BBB after systemic injection, have been used to deliver cholesterol to the brain of HD mice. These NPs, applied for the first time to a brain disorder, are made of PLGA, which is approved by FDA in various drug delivery systems in humans. Systemic administration of g7-NPs-Chol (i) rescued synaptic communication in striatal medium-sized spiny neurons, (ii) prevented cognitive decline, and (iii) restored the levels of proteins that compose the synaptic machinery in HD mice. g7-NPs or cholesterol itself did not induce inflammatory response in the periphery, where almost all g7-NPs are localized. Introduction Huntington's disease (HD) is a genetic neurological disorder caused by a CAG expansion in the gene encoding the huntingtin (HTT) protein (HDCRG, 1993). Clinically, HD is characterized by motor, cognitive, and psychiatric disturbances (Ross et al, 2014) and is associated with neuronal dysfunction, atrophy of the striatum and other brain regions, and progressive loss of striatal medium-sized spiny neurons (MSNs) and of cortical pyramidal neurons (Vonsattel & DiFiglia, 1998). Several molecular and cellular dysfunctions have been identified (Zuccato et al, 2010), and one affected pathway implicates brain cholesterol. The brain is the most cholesterol-rich organ in the body, with almost all of the cholesterol produced in situ, as circulating cholesterol is not able to cross the BBB (Dietschy & Turley, 2004). A large majority of cholesterol (> 70% of brain cholesterol mass) is present in myelin sheaths. Indeed, the rate of cholesterol synthesis is highest during post-natal stage to build myelin scaffolding. Cholesterol is also a structural component of glial and neuronal membranes and is concentrated in lipid rafts, specialized membrane microdomains that initiate, propagate, and maintain signal transduction events (Paratcha & Ibanez, 2002). Newly synthesized cholesterol is also required for vesicle assembly and fusion (Huttner & Zimmerberg, 2001; Lang et al, 2001), synapse formation, integrity, remodeling (Pfrieger, 2003), and neurotransmitter release (Thiele et al, 2000; Mauch et al, 2001). Accordingly, a breakdown of cholesterol synthesis causes brain malformations and impaired cognitive functions (Valenza & Cattaneo, 2006). HD is characterized by abnormal brain cholesterol homeostasis. Patients with HD show altered cholesterol homeostasis since pre- and early stages of disease as judged by the plasmatic measure of 24S-hydroxy-cholesterol (24OHC), the brain-specific catabolite of cholesterol able to cross the blood–brain barrier (BBB) (Leoni et al, 2008, 2013). Reduced cholesterol biosynthesis and levels are also found in the brain of several HD mouse models (Valenza et al, 2007a,b, 2010). On the contrary, others reported an increased accumulation of free cholesterol in brain tissues of HD mouse models (Trushina et al, 2006; del Toro et al, 2010) likely due to different sample preparation and less sensitive methods (colorimetric and enzymatic assays) to detect and quantify cholesterol compared to mass spectrometry (Marullo et al, 2012). Of note, more recently, some of the same groups have reported a decrease of lathosterol and cholesterol levels in the striatum of a HD mouse model by means of mass spectrometry (Trushina et al, 2014). Cholesterol dysregulation occurs in astrocytes (Valenza et al, 2015) and is linked to a specific action of mutant HTT on sterol regulatory-element-binding proteins (SREBPs) and its target genes, whose reduced transcription leads to less brain cholesterol produced and released and available to be uptaken by neurons (Valenza et al, 2005). Accordingly, an early decrease of cholesterol production in the HD brain might be detrimental for neuronal activities. Abnormalities in synaptic communication within the striatum and between the cortex and striatum occur long before, or in the absence of, cell death in HD animal models (Milnerwood & Raymond, 2010) and cognitive disturbances have been observed decades before predicted clinical diagnosis in HD gene carriers (Levine et al, 2004; Paulsen & Long, 2014). Similarly, brain cholesterol biosynthesis is significantly reduced before the onset of motor symptoms in all the HD animal models analyzed so far (Valenza et al, 2007a,b) and synaptosomes—a compartment dedicated to impulse transmission and neurotransmitter release—carry suboptimal levels of sterols in the early stages of HD in one mouse model (Valenza et al, 2010). However, a link between the reduced level of cholesterol and neuronal dysfunction in vivo in HD is still missing. Here, we explored the effects of cholesterol supplementation on synaptic communication and machinery, motor and cognitive behaviors, and neuropathology in the R6/2 mouse model, a well-established early onset transgenic mouse model of HD (Mangiarini et al, 1996). Since cholesterol does not cross the BBB, cholesterol was delivered using a new technology for drug administration in the brain (Vergoni et al, 2009; Tosi et al, 2010), that is, via biodegradable polymeric (polylactide-co-glycolide, PLGA) nanoparticles (NPs) modified with a glycopeptide (g-7) able to cross the BBB upon systemic injection in mice (Costantino et al, 2005; Tosi et al, 2007, 2011b). The development of new strategies to enhance brain delivery based on colloidal carriers is of great importance, since nanocarriers can protect drugs and deliver them across the BBB to target brain cells in a non-invasive way (Tosi et al, 2008). Notably, both FDA and EMA have approved PLGA in various drug delivery systems in humans (Mundargi et al, 2008), as confirmed by a number of market products (i.e., Lupron Depot®, Nutropin Depot ®). We report that, in contrast to unmodified NPs, g7-NPs efficiently crossed the BBB and within a few hours after systemic injection reached glial and neuronal cells in different brain regions. Importantly, repeated systemic delivery of g7-NPs-Chol rescued synaptic communication, protected from cognitive decline and partially improved global activity in HD mice. Results Chemical–physical and technological optimization of unloaded and cholesterol-loaded Nanoparticles The chemical formulation and features of unloaded NPs (u-NPs) herein employed have been largely described (Vergoni et al, 2009; Tosi et al, 2011a, 2014; Vilella et al, 2014). To optimize the production of NPs loaded with cholesterol (NPs-Chol), we first prepared u-NPs and NPs loaded with different amounts of cholesterol (1, 5, and 10 mg of Chol per 100 mg of polymer; herein defined as NPs-Chol1, NPs-Chol2 and NPs-Chol3, respectively) according to the nanoprecipitation procedure (Minost et al, 2012) (see 4). The composition of different NPs is described in Appendix Table S1, and details about their optimization and characterization are described in the Appendix. NPs were characterized by their chemical–physical properties, summarized in Appendix Table S2. The average diameter (Z-average) of u-NPs ranged from 170 to 192 nm. Z-average for NPs-Chol1 and NPs-Chol2 was lower than 210 nm, while size of NPs-Chol3 ranged between 200 nm and 300 nm. The polydispersity index (PDI value), a measure of the heterogeneity of NPs, was 0.08 ± 0.01 for u-NPs, suggesting a homogeneous and monomodal distribution population around the mean size. NPs-Chol1 and NPs-Chol2 showed a PDI value of 0.09 ± 0.01 and 0.11 ± 0.02, respectively, and a narrow dimension distribution, indicating that they are monomodal and monodisperse systems. On the contrary, NPs-Chol3 was characterized by a PDI value close to 0.3, accounting for a marked increase in sample heterogeneity. Zeta-potential (ζ-pot), a function of particle surface charges that influences cell interaction, was negative for all the NPs-Chol samples and similar to those of u-NPs. Moreover, ζ-pot of NPs-Chol3 displayed higher standard deviation (−12 ± 10 mV) with respect to those of NPs-Chol1 (−9 ± 4 mV) and NPs-Chol2 (−8 ± 4 mV), further highlighting the higher heterogeneity of this sample. To evaluate whether and how the incorporation of cholesterol influences the morphology, architecture and surface properties of NPs, atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses were performed on u-NPs and NPs-Chol (Fig 1A–C). In agreement with the chemical–physical properties (Appendix Table S2), the "height" AFM image (Fig 1A, left column), 3D reconstruction (Fig 1A, middle column), and TEM micrograph (Fig 1A, right column) of u-NPs highlighted well compact and defined spherical structures (Belletti et al, 2012). The AFM analysis for NPs-Chol1 confirmed the spherical shape, but shape and size were less homogeneous if compared with those of u-NPs (Fig 1B). Particles adopted an irregular frame, evident in the AFM 3D reconstruction, supporting the hypothesis that alteration of polymer organization and intimate interplay between cholesterol and PLGA occurred when cholesterol was added to the formulation. The greater complexity of these samples was confirmed by TEM microphotographs (right columns) emphasizing the less dense and compact structures of NPs-Chol1 with respect to u-NPs. NPs-Chol2 showed similar morphology and architecture of NPs-Chol1 (data not shown). Instead, the AFM images of NPs-Chol3 showed the presence of irregular structures and unformed material and a remarkable tendency to aggregate (Fig 1C). With respect to u-NPs and NPs-Chol1, NPs-Chol3 seemed to promote the formation of disorganized clusters characterized by heterogeneous dimensions (242 ± 52 nm) and by a roughness surface with evident fissuring. Similarly, TEM microphotographs showed the complexity of NPs-Chol3 that appeared with abundant adsorbed unformed material (likely unloaded cholesterol) and modified NPs' morphology. Figure 1. Characterization of NPs loaded with different concentrations of cholesterol A–C. AFM and TEM analysis of unloaded (u-NPs) and cholesterol-loaded NPs (NPs-Chol). AFM "height" images (left column), 3D reconstruction (middle column), and TEM micrograph (right column) of u-NPs (A), NPs-Chol1 (B), and NPs-Chol3 (C). D. Release profile in water of cholesterol (continuous line, —) and NBD-Chol (dotted line, - - -) from NPs-Chol1 and NPs-NBD-Chol1, respectively. The graph represents mean ± SEM. Data are from three independent experiments. E. In vitro release of NBD-Chol from NPs at different time intervals in NS cells. Data in the graph represent mean (μg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1. Data obtained from four independent experiments. N.T.: not treated cells. Download figure Download PowerPoint We also evaluated the content of cholesterol into NPs (loading capacity, LC%) and the encapsulation efficiency (EE%) (Appendix Table S2). About 0.7 ± 0.1 mg/100 mg of formulation, corresponding to an EE of 68%, were loaded in the NPs-Chol1, indicating that an important fraction of the initial cholesterol was stably incorporated into the NPs-Chol1. On the contrary, a decrease in EE value was observed as the amount of cholesterol used in the preparation increased. In NPs-Chol2 and NPs-Chol3, the EE remarkably decreased (about 20%) although the highest value of drug loading was observed in NPs-Chol3 (2.5 mg of Chol/100 mg of NPs). However, as previously pointed out, cholesterol in NPs-Chol3 was not completely embedded, but a remarkable fraction was absorbed onto the surface. Based on these analyses, NPs-Chol1 formulation was used in all experiments. Controlled release of cholesterol from NPs in physiological conditions and in vitro To explore the ability of the system to release cholesterol, we first carried out release studies in deionized water for 10 days (Fig 1D). The release profile of cholesterol from NPs-Chol1 (hereafter referred as NPs-Chol; solid line) showed an initial "burst release" (< 8%) during the first 3 days, followed by a second slow release phase. Chol release was detected close to values of 18% over 10 days owing to the poor water solubility of cholesterol (estimated to be 2 μg/ml). Moreover, during the second phase, the slow linear release kinetic of Chol from NPs-Chol between day 5 and day 10 could be ascribed to NPs degradation. In specific experiments, we also adopted a lead formulation prepared by replacing cholesterol with the fluorescent cholesterol derivative NBD-Chol to discriminate between endogenous and exogenous cholesterol released from NPs. We therefore characterized also the NBD-Chol-loaded NPs (NPs-NBD-Chol) in terms of their chemical–physical and technological properties (Appendix Table S2) and morphological features (Appendix Fig S1). The release of NBD-Chol from NPs in water showed a slow kinetic profile (Fig 1D, dotted line) similar to that observed for native cholesterol (Fig 1D, solid line). Similar findings were observed when the kinetic profile of drug release was evaluated in experiments conducted in cultured cells (Fig 1E). Spectrophotometric quantification of NBD-Chol in neural stem (NS) cells treated with 3 μg of NPs-NBD-Chol revealed that only 20% of the total NBD-Chol taken by the cells was released after 24 h (0.05 μg vs. 0.23 μg; Fig 1E, seventh and fourth columns, respectively). At 72 h, the amount of NBD-Chol released increased to about 35% of the total NBD-Chol taken up by cells (0.14 μg vs. 0.39 μg; Fig 1E, ninth and sixth columns, respectively), confirming the slow kinetic profile of cholesterol release from NPs. g7-NPs distribution in HD cells and brain The g7-NPs used in this study are designed to cross the BBB, and previous studies indicated that about 10% are estimated to penetrate the brain (Costantino et al, 2005; Tosi et al, 2007, 2011a,b, 2014). To verify that g7-NPs could penetrate HD cells, primary neurons from R6/2 mice and neurons and astrocytes from mouse NS cells carrying 140 CAG repeats (NS Q140/7) were exposed to g7-NPs labeled with rhodamine to allow their detection with fluorescence microscopy. Appendix Fig S2 shows that g7-NPs are taken up in vitro by different brain cells expressing mutant Htt. Importantly, 4 h after a single intraperitoneal (ip) injection into 8-week-old R6/2 mice and wild-type (WT) littermates, both control (unmodified) NPs (C-NPs) and g7-NPs were detected in the liver (Fig 2A) and in other peripheral tissues (Appendix Fig S3), but only g7-NPs were detected in the brain (Fig 2B). Quantification of g7-NPs yielded an approximate ratio of ~10:1 in the WT liver compared to striatum and cortex (Fig 2C). This quantification also revealed a reduced propensity of g7-NPs to reach the R6/2 brain compared to the WT brain, while g7-NPs were more prevalent in R6/2 liver compared to WT liver, suggesting that HD-related mechanisms may influence the BBB crossing of g7-NPs. g7-NPs were also found 24 h and 2 weeks after a single (Fig 2D) or multiple ip injections performed in the same week (Fig 2E). High-magnification confocal images indicated the presence of g7-NPs in different brain regions and in IBA1 immunoreactive microglial cells (Fig 2F) and in GFAP positive astrocytes (Fig 2G). Notably, g7-NPs were also detected in neuronal cells, as demonstrated by immunostaining against calbindin (Fig 2H; Appendix Fig S4) and DARPP-32 (Fig 2I). Figure 2. g7-NPs reach different brain cells and release cholesterol in R6/2 mice A, B. Representative confocal images of liver (A) and brain (B) slices from R6/2 mice ip injected with C-NPs (left) or with g7-NPs (right) and sacrificed after 4 h. C. Quantification of g7-NPs localized in the liver, striatum, and cortex of WT (n = 3) and R6/2 mice (n = 3). Data are expressed as the number of g7-NPs for 100 μm2 ± SEM. Statistics: *P < 0.05 determined by Student's t-test. D, E. g7-NPs in brain slices from R6/2 mice administered with a single ip injection and sacrificed after 24 h (D, left) or 2 weeks (D, right) and after multiple ip injections within 1 week (E). F–I. Representative confocal images of immunostaining against IBA1 (F), GFAP (G), calbindin (H), and DARPP-32 (I) on coronal sections of brains isolated from R6/2 mice ip injected with g7-NPs and sacrificed at the indicated time points. White arrowheads indicate intracellular g7-NPs. Data information: DAPI (A, B, D) or Hoechst 33258 (Ho) (F–I) was used to counterstain nuclei. Scale bars: 20 μm (A); 10 μm (B, D, E); 5 μm (F–I). Download figure Download PowerPoint Delivery and release of cholesterol in vivo in the R6/2 brain To track the delivery and intracellular release of cholesterol from g7-NPs, we employed rhodamine-labeled g7-NPs (Vergoni et al, 2009) loaded with the fluorescent cholesterol derivative NBD-Chol (g7-NPs-NBD-Chol). NBD-Chol closely resembles the structure of native cholesterol and is normally used to study cholesterol trafficking (Gimpl & Gehrig-Burger, 2007). Accordingly, NBD-Chol, injected into brain ventricles of mice, co-localizes with PMCA ATPase, a marker of plasma membrane, suggesting that exogenous cholesterol is incorporated on brain cells' membranes in vivo (Appendix Fig S5). We next monitored the distribution of g7-NPs as red spots and the distribution of released NBD-Chol as green signal. In vivo, at 12 and 24 h after a single ip injection of g7-NPs-NBD-Chol, g7-NPs and NBD-Chol co-localized in brain cells (Fig 3A and B). In particular, Fig 3B shows the distribution of g7-NPs (red signal) and NBD-Chol (green signal) in a brain section of a R6/2 mouse injected ip with g7-NPs-NBD-Chol and sacrificed 24 h later. Both g7-NPs and NBD-Chol signals co-localized as indicated by the scatterplot of red and green pixel intensities. However, g7-NPs and NBD-Chol were no longer co-localized after 14 days as demonstrated in Fig 3C. Similar results were found at 7 days after ip injection (data not shown). These findings indicate that NBD-Chol was partially released from NPs 1–2 weeks after injection, in parallel with a reduction in the signal from g7-NPs, probably due to their degradation. Quantification of g7-NPs in brain slices from injected mice confirmed a decreased number of NPs over time as determined after normalizing the red spots on the mean size of NPs (Fig 3D). In the liver, the kinetics of NBD-Chol release and g7-NPs degradation was faster (< 24 h) than in brain (Appendix Fig S6). Figure 3. Cholesterol delivery and release in vivo in the R6/2 brain A. Representative confocal image (crop) of brain slices from R6/2 mice ip injected with rhodamine-labeled g7-NPs-NBD-Chol and sacrificed after 12 h, showing co-localization of NBD-Chol (green) and rhodamine (NPs, red). Scale bar: 5 μm. B, C. Representative confocal image (low magnification) of brain slices from R6/2 mice ip injected with g7-NPs-NBD-Chol and sacrificed after 24 h (B) or 2 weeks (C) and relative co-localization of NBD-Chol and g7-NPs. Scale bar: 10 μm. D. g7-NPs quantification in brain slices at the same time points in (B, C). Data are expressed as number of g7-NPs (evaluated based their size) for 100 μm2 ± SEM. Statistics: **P < 0.01 (48 h vs. 7 days; 7 days vs. 14 days), ***P < 0.001 (24 h vs. 7 days; 7 days vs. 14 days) determined by one-way ANOVA followed by Newman–Keuls multiple comparison test. Data information: DAPI was used to counterstain nuclei. Download figure Download PowerPoint g7-NPs-Chol rescue synaptic activity in HD mice As synaptic transmission in striatal MSNs is altered in R6/2 mice during disease progression (Cepeda et al, 2003, 2004), we next explored whether cholesterol supplementation to the brain via systemic injection of g7-NPs-Chol restored synaptic parameters in HD mice. Pilot experiments with R6/2 animals that received only 1 or 2 injections of g7-NPs-Chol did not show any significant modifications in electrophysiological properties (data not shown). We therefore designed our trials in order to provi
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