EMBO Molecular Medicine, Год журнала: 2015, Номер 7(12), С. 1547 - 1564

Опубликована: Ноя. 20, 2015

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

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