Labeling and exocytosis of secretory compartments in RBL mastocytes by polystyrene and mesoporous silica nanoparticles

Maneerat Ekkapongpisit1,*, Antonino Giovia1,*, Giuseppina Nicotra1, Matteo Ozzano1, Giuseppe Caputo2,3, Ciro Isidoro1 1Laboratory of Molecular Pathology and Nanobioimaging, Department of Health Sciences, Università del Piemonte Orientale "A. Avogadro", Novara, Italy; 2Department of Chemistry, University of Turin, Turin, 3Cyanine Technology SpA, Torino, Italy *These authors contributed equally to this workBackground: For a safe ‘in vivo’ biomedical utilization of nanoparticles, it is essential to assess not only biocompatibility, but also the potential to trigger unwanted side effects at both cellular and tissue levels. Mastocytes (cells having secretory granules containing cytokines, vasoactive amine, and proteases) play a pivotal role in the immune and inflammatory responses against exogenous toxins. Mastocytes are also recruited in the tumor stroma and are involved in tumor vascularization and growth.Aim and methods: In this work, mastocyte-like rat basophilic leukemia (RBL) cells were used to investigate whether carboxyl-modified 30 nm polystyrene (PS) nanoparticles (NPs) and naked mesoporous silica (MPS) 10 nm NPs are able to label the secretory inflammatory granules, and possibly induce exocytosis of these granules. Uptake, cellular retention and localization of fluorescent NPs were analyzed by cytofluorometry and microscope imaging.Results: Our findings were that: (1) secretory granules of mastocytes are accessible by NPs via endocytosis; (2) PS and MPS silica NPs label two distinct subpopulations of inflammatory granules in RBL mastocytes; and (3) PS NPs induce calcium-dependent exocytosis of inflammatory granules.Conclusion: These findings highlight the value of NPs for live imaging of inflammatory processes, and also have important implications for the clinical use of PS-based NPs, due to their potential to trigger the unwanted activation of mastocytes.Keywords: secretory lysosomes, inflammation, nanoparticles, vesicular traffi

Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates

Knockdown of cathepsin D in zebrafish fertilized eggs determines congenital myopathy

CD (cathepsin D) is a ubiquitous lysosomal hydrolase involved in a variety of pathophysiological functions, including protein turnover, activation of pro-hormones, cell death and embryo development. CD-mediated proteolysis plays a pivotal role in tissue and organ homoeostasis. Altered expression and compartmentalization of CD have been observed in diseased muscle fibres. Whether CD is actively involved in muscle development, homoeostasis and dystrophy remains to be demonstrated. Zebrafish (Danio rerio) is emerging as a valuable ‘in vivo’ vertebrate model for muscular degeneration and congenital myopathies. In this work, we report on the perturbance of the somitic musculature development in zebrafish larvae caused by MPO (morpholino)-mediated silencing of CD in oocytes at the time of fertilization. Restoring CD expression, using an MPO-non-matching mutated mRNA, partially rescued the normal phenotype, confirming the indispensable role of CD in the correct development and integrity of the somitic musculature. This is the first report showing a congenital myopathy caused by CD deficiency in a vertebrate experimental animal model

Role of cathepsin D in eye development.

<p>Hematoxylin-eosin staining of eye sections derived from 4 dpf micro-injected larvae (CTRL =  control injections; S-MPO =  splicing morpholino; T-MPO =  translation morpholino; RESCUED =  translation morpholino plus 200 pg/egg of mutant CD mRNA). Images at high magnification clearly show the palisade of microvilli of RPE cells (which contain melanin granules, MG) that interdigitate in the layer of photoreceptor cells (PRC) in CTRL, S-MPO and RESCUED zebrafish, while this is completely absent in T-MPO CD KD zebrafish (note the RPE cells containing melanine granules close to the PRC layer). Scale bar is 20 µm. Images representative of five (three for Rescued) independent experiments.</p

Phenotype of zebrafish cathepsin D following down-regulation by two different morpholinos at 4 dpf.

<p>Representative images of 4 dpf larvae resulting from control injection (CTRL), injection with S-MPO or with T-MPO. <b>A</b>) Larvae grown in the presence of PTU (+PTU). The main phenotypic alterations produced by T-MPO CD KD were: 1. microphtalmia (empty arrow); 2. absence of inflated swim-bladder (asterisk); 3. reduced yolk adsorption (arrow); 4. reduced body length (standard length, SL); 5. reduced oro-anal tract length (snout-vent length, SVL). <b>B</b>) Eyes magnification of 4 dpf larvae grown in the presence of PTU; the eye area is enclosed by the dotted circle. <b>C</b>) Body length, oro-anal tract length and eye area ratios calculated between CTRL and S-MPO or CTRL and T-MPO larvae. Measurements were done with the ImageJ software. Ten image sets obtained from 5 different experiments were analysed. Data are given as mean ± S.D. Differences in SL, SVL and eye area data were statistically highly significant (**, p ≤0,01) according to Student's <i>t</i> test. <b>D</b>) Larvae grown in the absence of PTU (-PTU). Following T-MPO CD knock-down the larvae showed skin hyper-pigmentation (arrows). <b>E</b>) Eyes magnification of 4 dpf larvae grown in the absence of PTU: dark-field images show normal iridophore reflections. Scale bar in <b>A</b> and <b>D</b> is 200 µm. Data presented in this figure have been reproduced in five independent experiments.</p

Immunofluorescence staining of CD in microvilli of RPE cells in zebrafish following cathepsin D knock-down and rescue.

<p>Immunofluorescence staining of CD and α-actin in eye sections derived from 4 dpf micro-injected larvae (CTRL =  control injections; S-MPO =  splicing morpholino; T-MPO =  translation morpholino; RESCUED =  translation morpholino plus 200 pg/egg of mutant CD mRNA). Nuclei are stained with DAPI (blue). As negative control, CTRL larvae sections have been incubated only with secondary antibodies (neg). Note the intense staining for CD in the RPE layer (identifiable by the actin-positive microvilli) in CTRL, S-MPO and RESCUED zebrafish. This experiment confirms the efficient KD of CD by T-MPO in agreement with western blotting data. These images are representative of three independent experiments. Scale bar is 10 µm.</p

Zebrafish cathepsin D protein expression.

<p><b>A</b>) Western blotting of CD in homogenates of un-fertilized eggs (UFE), embryos (at 30% epiboly, 1 dpf and 2 dpf) and larvae (at 3 dpf and 4 dpf). The filter was first probed with a polyclonal antibody against rat CD, stripped and re-probed with a monoclonal antibody against β-actin. The arrow points to the single-chain CD (41 kDa). Asterisks point to aspecific bands. The position of standard molecular weight proteins is indicated. One representative gel out of five independent experiment is shown. <b>B</b>) Colloidal Coomassie G-250 stained gel (after blotting) showing whole homogenate proteins of each sample loaded. <b>C</b>) Western blotting validation of the polyclonal anti-CD antibody against homogenates of 4 dpf larvae, PAC2 cells and SH-SY5Y cells (transfected with either the empty vector or the vector carrying the cDNA for human CD or for zebrafish CD). The position of the CD molecular forms recognized by the antibody is indicated on the right. The position of standard molecular weight is indicated on the left. Essentially a similar pattern of CD expression was obtained in two other independent experiments.</p

Rescue of T-MPO phenotypes by mutated zebrafish CD mRNA.

<p><b>A</b>) T-MPO target sequence and corresponding mutated sequence of the <i>in vitro</i> synthesized CD mRNA. <b>B</b>) Western blotting of CD in homogenates of 2 dpf embryos and 4 dpf larvae deriving from control injection (CTRL), injection with T-MPO alone (T-MPO) and injection with T-MPO plus 200 pg/egg of mutant CD mRNA (RESCUED). The filter was incubated with polyclonal antibody against rat CD, stripped and re-probed with monoclonal antibody against β-actin. The densitometry ratio of CD <i>vs</i> actin expression is shown. (a.u. =  arbitrary units). Data reproduced in two to three independent experiments. The empty arrow points to CD expressed by the exogenous mRNA. <b>C</b>) Representative image of 4 dpf larvae obtained from control injection (CTRL), injection with T-MPO alone or with T-MPO plus 200 pg/egg of mutant zebrafish CD mRNA (RESCUED). Larvae body length (SL) and oro-anal tract length (SVL) are indicated. The experiment shows that synthesis of protein CD driven by exogenous CD mRNA in T-MPO injected zebrafish was sufficient to rescue a normal phenotype. Scale bar in <b>C</b> is 200 µm. Data presented in this figure have been reproduced in three independent experiments.</p