Yoram Altschuler

Surface Charge of Nanoparticles Determines Their Endocytic and Transcytotic Pathway in Polarized MDCK Cells

A major challenge in drug delivery is the internalization through the apical plasma membrane of the polarized epithelial cells lining organs facing the external environment, e.g., lungs and the gastrointestinal tract. The reduced permeation of drugs entering through this pathway is in part due to the mucosal barrier and low rate of endocytosis at these membranes. We investigated the possible role of nanoparticle surface charge on its entry through the apical plasma membrane and its intracellular pathway. We found that both cationic and anionic nanoparticles are targeted mainly to the clathrin endocytic machinery. A fraction of both nanoparticle formulations is suspected to internalize through a macropinocytosis-dependent pathway. A significant amount of nanoparticles transcytose and accumulate at the basolateral membrane. Some anionic but not cationic nanoparticles transited through the degradative lysosomal pathway. Taken together, these observations indicate that cationic nanoparticles, in addition to their potential for drug delivery to epithelia, may be promising carriers for transcytosing drugs to the blood stream.

The activity of antiepileptic drugs as histone deacetylase inhibitors.

Summary:u2002 Purpose: Valproic acid (VPA), one of the widely used antiepileptic drugs (AEDs), was recently found to inhibit histone deacetylases (HDACs). HDAC inhibitors of a wide range of structures, such as hydroxamic acids, carboxylic acids, and cyclic tetrapeptides, have various effects on transformed and nontransformed cells, including neuromodulation and neuroprotection. The aim of this study was to assess comparatively the activity of traditional and newer AEDs as HDAC inhibitors.

α–Synuclein and PolyUnsaturated Fatty Acids Promote Clathrin Mediated Endocytosis and Synaptic Vesicle Recycling

α‐Synuclein (αS) is an abundant neuronal cytoplasmic protein implicated in Parkinson’s disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, αS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in αS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that αS acts with PUFAs to enhance the internalization of the membrane‐binding dye, FM 1‐43. Specifically, αS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin‐mediated endocytosis in neuronal and non‐neuronal cultured cells. Moreover, αS expression and PUFA‐enhanced basal and ‐evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted αS mouse brains. We suggest that αS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis.

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.

The mammalian Nek1 kinase is involved in primary cilium formation

Recent studies implicate primary cilium (PC) proteins in the etiologies of various polycystic kidney diseases (PKD). NIMA‐related kinases (NRKs) are conserved serine/threonine kinases, which are usually defined as ‘mitotic kinases’. Murine mutants for the NRKs, nek1 (kat mice) suffer from PKD, suggesting that it may be involved in cilium control. We demonstrated herein that Nek1 is localized to basal body region and that Nek1 overexpression inhibits ciliogenesis in Madin–Darby canine kidney epithelial cells. The number of primary cilia is dramatically reduced in kat2J mouse embryonic fibroblasts culture. It is thus hypothesized that Nek1 links cell cycle progression and the PC cycle.

The apical compartment: trafficking pathways, regulators and scaffolding proteins

Defects in the trafficking of apical membrane proteins in polarized epithelial cells are often associated with diseases, including cystic fibrosis, Liddles syndrome, nephrogenic diabetes insipidus and Dubin-Johnson syndrome. In recent years, we have learned much about the specialized apical trafficking pathways in polarized cells. Many laboratories have identified signals that direct proteins within these pathways and have defined protein interactions that mediate specific steps in the sorting and stabilization of these proteins. In addition, many cytosolic proteins, including lipid kinases, GTPases, ATPases and scaffolding/adaptor proteins that lack enzymatic activity, regulate the trafficking of proteins through these pathways. Recent advances in the field include the role of small GTPases, unconventional myosins and lipid kinases in apical endocytosis and transcytosis, and the identification of PDZ proteins that regulate apical membrane trafficking of receptors, transporters and ion channels.

A safety and tolerability study of differently-charged nanoparticles for local pulmonary drug delivery

Nanoparticle (NP) based drug delivery systems provide promising opportunities in the treatment of lung diseases. Here we examined the safety and tolerability of pulmonary delivered NPs consisting of PEG-PLA as a function of particle surface charge. The rationale for such a comparison should be attributed to the differential pulmonary toxicity of positively and negatively charged PEG-PLA NP. Thus, the local and systemic effects of pulmonary administered NPs were investigated following 5days of daily endotracheal instillation to BALB/c mice that were euthanized on the eighth or nineteenth day of the experiment. We collected bronchoalveolar lavages and studied hematological as well as histochemistry parameters. Notably, the cationic stearylamine based PEG-PLA NPs elicited increased local and systemic toxic effects both on the eighth and nineteenth day. In contrast, anionic NPs of similar size were much better tolerated with local inflammatory effects observed only on the eighth experimental day after pulmonary instillation. No systemic toxicity effect was observed although a moderate change was noted in the platelet count that was not considered to be of clinical significance. No pathological observations were detected in the internal organs following instillation of anionic NPs. Overall these observations suggest that anionic PEG-PLA NPs are useful pulmonary drug carriers that should be considered as a promising therapeutic drug delivery system.

Toxicity Mechanisms of Amphotericin B and Its Neutralization by Conjugation with Arabinogalactan

ABSTRACT Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells.

β-tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure

In epithelial cells, the apical junctional complex (AJC), composed of tight junctions (TJs) and adherens junctions (AJs), maintains cell‐surface polarity by forming a fence that prevents lateral movement and diffusion of proteins and lipids between the apical and basolateral PM and holds the epithelial monolayer intact through cell‐cell contacts. Disassembly of this complex is a prime event in development and cell transformation. Maintenance of the AJC has been shown to involve mainly the actin cytoskeleton. Recent findings also point to the involvement of the microtubule (MT) system. Here we show the first evidence that in polarized epithelial MDCK cells, ARF‐like protein 2 (ARL2) and β‐tubulin cofactor D, known to be involved in MT dynamics, have a role in disassembly of the AJC followed by cell dissociation from the epithelial mono‐layer, which is not dependent on MT depolymerization. In addition, we show that β‐tubulin cofactor D is partially localized to the lateral PM through its 15 C‐terminal amino acids and intact MTs. ARL2 inhibited β ‐tubulin cofactor D‐dependent cell dissociation from the monolayer and AJC disassembly. To our knowledge, this is the first evidence that β‐tubulin cofactor D plays a role in cells independent of its presumed role in folding tubulin heterodimers. We conclude that ARL2 and β ‐tubulin cofactor D participate in AJC disassembly and epithelial depolarization.— Shultz, T., Shmuel, M., Hyman, T., and Altschuler, Y. β‐tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure. FASEB J. 22, 168–182 (2008)

pH-specific sequestration of phosphoglucose isomerase/autocrine motility factor by fibronectin and heparan sulphate

Phosphoglucose isomerase (PGI) is a glycolytic enzyme that moonlights as a cytokine under the aliases autocrine motility factor (AMF), neuroleukin and maturation factor. The cytokine function of PGI/AMF targets multiple cell types however mechanisms that regulate and sequester this ubiquitous, circulating cytokine remain largely unidentified. PGI/AMF is shown here to exhibit fibronectin (FN)-dependent cell surface association at both neutral and acid pH. Direct PGI/AMF binding to FN and fluorescence resonance energy transfer (FRET) between PGI/AMF and FN were detected only at pH 5. At neutral pH, the interaction of PGI/AMF with FN is receptor-mediated requiring prior clathrin-dependent endocytosis. PGI/AMF and FN do not co-internalize and PGI/AMF undergoes a second round of endocytosis upon recycling to the plasma membrane indicating that recycling PGI/AMF receptor complexes associate with FN fibrils. Heparan sulphate does not affect cell association of PGI/AMF at neutral pH but enhances the FN-independent cell surface association of PGI/AMF at acid pH identifying two distinct mechanisms for PGI/AMF sequestration under acidic conditions. However, only PGI/AMF sequestration by FN at acid pH was able to stimulate cell motility upon pH neutralization identifying FN as a pH-dependent cytokine trap for PGI/AMF. The multiple ways of cellular association of PGI/AMF may represent acquired mechanisms to regulate and harness the cytokine function of PGI/AMF.