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Delivery of large therapeutic payloads to the central nervous system is challenging owing to the presence of the blood brain barrier (BBB) that inhibits uptake of virtually any macromolecule. Targeted nanostructures could overcome this issue, allowing delivery of otherwise impermeant payloads without suffering serum protein adsorption during circulation in the bloodstream. Here we present our results on the realization of a rationally designed targeted nanostructure tailored to in vivo use. Our strategy relies on the development of a stealth shell that inhibits serum protein adsorption without using immunogenic PEG derivatives. A zwitterionic peptide (EKEKEKE) with known stealth properties was conjugated with lipid tails and used to prepare liposome-like self-assembled structures that outperform PEG in terms of serum protein adhesion, being stable up to 8 hours in pure serum.1 These structures were decorated with a targeting sequence that exploits transferrin receptor pathway. Transferrin (Tfn) is a promising target for delivery to the central nervous system (CNS) due to the overexpression of its receptor on the BBB. We designed and validated experimentally a new peptide aptamer (Tf2) able to recognize with good affinity transferrin without altering binding properties of the latter to its receptor.2 We shall show that the presence of Tf2 on the surface of a nanoparticle induces extensive modulation of the protein corona composition, with significant overrepresentation of Tfn. We will also show that Tf2- decorated nanoparticles are efficiently assimilated by the cells in a transferrin-dependent fashion.2 Finally, we shall present the applicability of our structure to enzyme replacement therapy for the treatment of Batten disease. Our nanostructures can be loaded with recombinant protein palmitoyl thiotransferase 1 fully retaining its activity. We shall discuss the effect of this administration on enzymatic activity recovery and on the phenotypic rescue in primary healthy and patient-derived fibroblasts.

Atypical chronic myeloid leukemia (aCML) is a clonal disorder belonging to the myelodysplastic/myeloproliferative syndromes. About 13% of aCML cases carry somatic mutations in ETNK1 gene, encoding for H243Y, N244S, and G245V substitutions. We previously showed that ETNK1 mutations cause a decreased catalytic activity of the enzyme. Despite this evidence however, their oncogenic role remained largely unexplained. Since ETNK1 activity is essential for the synthesis of phosphatidylethanolamine (PE) and given that PE is one of the most abundant phospholipids in the inner membrane of mitochondria, we focused our attention on mitochondrial activity. In order to characterize the oncogenic effect of ETNK1 variants we generated CRISPR/Cas9 clones carrying heterozygous N244S mutation and homozygous ETNK1 deletion (KO cells) on the 293 Flp-In™ cell-line. Both N244S and KO cells showed a significant increase in mitochondrial activity (1.78 and 2.13 fold increase, respectively; p= 0.0096 and p=0.0050) compared to WT, as assessed by MitoTracker™ Red. In line with this finding, electron microscopy revealed a significant modification in mitochondria morphology for N244S and KO cells, changing from an elongated, tubular shape to a round, swollen one. ATP (1.67 and 1.68 fold; p

Reversible photoswitching has been proposed as a way to identify molecules that are present in small numbers over a large, non-switching, background. This approach, called optical-lock-in-detection (OLID) requires the deterministic control of the fluorescence of a photochromic emitter through optical modulation between a bright (on) and a dark state (off). OLID yields a high-contrast map where the switching molecules are pinpointed, but the fractional intensities of the emitters are not returned. The present work presents a modified OLID approach (quantitative OLID or qOLID) that yields quantitative information of the switching (fSW) and non-switching (fNS) components. After the validation of the method with a sample dataset and image sequence, we apply qOLID to measurements in cells that transiently express the photochromic protein EYQ1. We show that qOLID is efficient in separating the modulated from the non-modulated signal, the latter deriving from background/autofluorescence or fluorophores emitting in the same spectral region. Finally, we apply qOLID to Förster (Fluorescence) Resonance Energy Transfer (FRET) imaging. We here demonstrate that qOLID is able to highlight the distribution of FRET intensity in a sample by using a photochromic donor and a non-photochromic acceptor. Reversible photoswitching has been proposed as a way to identify molecules that are present in small numbers over a large, non-switching, background. This approach, called optical-lock-in-detection (OLID) requires the deterministic control of the fluorescence of a photochromic emitter through optical modulation between a bright (on) and a dark state (off). OLID yields a high-contrast map where the switching molecules are pinpointed, but the fractional intensities of the emitters are not returned. The present work presents a modified OLID approach (quantitative OLID or qOLID) that yields quantitative information of the switching (fSW) and non-switching (fNS) components. After the validation of the method with a sample dataset and image sequence, we apply qOLID to measurements in cells that transiently express the photochromic protein EYQ1. We show that qOLID is efficient in separating the modulated from the non-modulated signal, the latter deriving from background/autofluorescence or fluorophores emitting in the same spectral region. Finally, we apply qOLID to Förster (Fluorescence) Resonance Energy Transfer (FRET) imaging. We here demonstrate that qOLID is able to highlight the distribution of FRET intensity in a sample by using a photochromic donor and a non-photochromic acceptor.

Quantum dots (QDs) and polymeric nanoparticles (NPs) are considered good binomials for the development of multifunctional nanomedicines for multimodal imaging. Fluorescent imaging of QDs can monitor the behavior of QD-labeled NPs in both cells and animals with high temporal and spatial resolutions. The comprehension of polymer interaction with the metallic QD surface must be considered to achieve a complete chemicophysical characterization of these systems and to describe the QD optical properties to be used for their unequivocal identification in the tissue. In this study, by comparing two different synthetic procedures to obtain polymeric nanoparticles labeled with QDs, we investigated whether their optical properties may change according to the formulation methods, as a consequence of the different polymeric environments. Atomic force microscopy, transmission electron microscopy, confocal and fluorescence lifetime imaging microscopy characterization demonstrated that NPs modified with QDs after the formulation process (post-NPs-QDs) conserved the photophysical features of the. QD probe. In contrast, by using a polymer modified with QDs to formulate NPs (pre-NPs-QDs), a significant quenching of QD fluorescence and a blueshift in its emission spectra were observed. Our results suggest that the packaging of QDs into the polymeric matrix causes a modification of the QD optical properties: these effects must be characterized in depth and carefully considered when developing nanosystems for imaging and biological applications.

Globoid cell leukodystrophy (GLD) is a rare, rapidly progressing childhood leukodystrophy triggered by deficit of the lysosomal enzyme galactosylceramidase (GALC) and characterized by the accumulation of galactosylsphingosine (psychosine; PSY) in the nervous system. PSY is a cytotoxic sphingolipid, which leads to widespread degeneration of oligodendrocytes and Schwann cells, causing demyelination. Here we report on autophagy in the human oligodendrocyte cell line MO3.13 treated with PSY and exploitation of Li as an autophagy modulator to rescue cell viability. We demonstrate that PSY causes upregulation of the autophagic flux at the level of autophagosome and autolysosome formation and LC3-II expression. We show that pretreatment with Li, a drug clinically used to treat bipolar disorders, can further stimulate autophagy, improving cell tolerance to PSY. This Li protective effect is found not to be linked to reduction of PSY-induced oxidative stress and might not stem from a reduction of PSY accumulation. These data provide novel information on the intracellular pathways activated during PSY-induced toxicity and suggest the autophagy pathway as a promising novel therapeutic target for ameliorating the GLD phenotype. © 2016 Wiley Periodicals, Inc.

Despite the recent identification of recurrent SETBP1 mutations in atypical chronic myeloid leukemia (aCML), a complete description of the somatic lesions responsible for the onset of this disorder is still lacking. To find additional somatic abnormalities in aCML, we performed whole-exome sequencing on 15 aCML cases. In 2 cases (13.3%), we identified somatic missense mutations in the ETNK1 gene. Targeted resequencing on 515 hematological clonal disorders revealed the presence of ETNK1 variants in 6 (8.8%) of 68 aCML and 2 (2.6%) of 77 chronic myelomonocytic leukemia samples. These mutations clustered in a small region of the kinase domain, encoding for H243Y and N244S (1/8 H243Y; 7/8 N244S). They were all heterozygous and present in the dominant clone. The intracellular phosphoethanolamine/phosphocholine ratio was, on average, 5.2-fold lower in ETNK1-mutated samples ( P < .05). Similar results were obtained using myeloid TF1 cells transduced with ETNK1 wild type, ETNK1-N244S, and ETNK1-H243Y, where the intracellular phosphoethanolamine/phosphocholine ratio was significantly lower in ETNK1-N244S (0.76 ± 0.07) and ETNK1-H243Y (0.37 ± 0.02) than in ETNK1-WT (1.37 ± 0.32; P = .01 and P = .0008, respectively), suggesting that ETNK1 mutations may inhibit the catalytic activity of the enzyme. In summary, our study shows for the first time the evidence of recurrent somatic ETNK1 mutations in the context of myeloproliferative/myelodysplastic disorders. © 2015 by The American Society of Hematology.

Globoid cell leukodystrophy (GLD, or Krabbe disease) is an autosomal recessive, neurodegenerative rare disease caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC). GALC degrades galactosylceramide, a major component of myelin, and other terminal b-galactose-containing sphingolipids, including the cytotoxic D-galactosylsphingosine (psychosine, PSY). GALC loss of function causes increased PSY levels in the neural tissues, leading to widespread degeneration of oligodendrocytes and Schwann cells and subsequent demyelination. Still little is known about the molecular mechanisms by which PSY imparts toxicity and no cure is currently available for GLD [Graziano et al., Gene 2015; Voccoli et al., Cell Death Dis 2014]. Unfortunately, the systemic GALC administration is not effective because of the presence of the blood brain barrier (BBB). The development of an enzyme replacement therapy (ERT) that takes advantage from the recent nanomedicine advancements to cross BBB would be one of the most promising options for curing GLD. In order to help in developing ERTs in the mouse GLD model, the Twitcher mouse (TWI), a study of GALC activity, PSY levels and viability in fibroblasts and different central and peripheral nervous system (CNS and PNS) cell types and tissues is here presented. We have set up cultures of primary wild-type (WT) and TWI fibroblasts and cells from CNS (i.e. astrocytes, oligodendrocytes and neurons) and characterized their GALC expression and activity by western-blotting and fluorimetric methods. These measurements were carried out both in cells lysates and in culture supernatants, allowing us to assess the level of enzyme exocytosis in different cell types. An analogous analysis is also reported for CNS and PNS tissues, such as: brain, optic and sciatic nerves. PSY quantification in WT and TWI homogenized brains, optic and sciatic nerves, and in different CNS cell types was performed by an LC-ESI-tandem-MS method. PSY accumulation in tissues was evaluated for TWI mice of different age. Moreover, data on cell viability of WT and TWI cells treated with different PSY concentrations are presented. Possible effect of autophagy modulators on cell viability will be finally discussed. A disturbance of autophagic pathways was indeed reported for many lysosomal storage disorders [Settembre et al., Autophagy 2008], and it has been linked to a deficiency of trafficking/processing of recombinant therapeutic enzymes, suggesting that autophagy dysfunction may have an impact in determining the efficacy of ERT.

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.