Nanoparticles of alkylglyceryl-dextran-graft-poly(lactic acid) for drug delivery to the brain: Preparation and in vitro investigation.

Poly(lactic acid), which has an inherent tendency to form colloidal systems of low polydispersity, and alkylglyceryl-modified dextran - a material designed to combine the non-immunogenic and stabilising properties of dextran with the demonstrated permeation enhancing ability of alkylglycerols - have been combined for the development of nanoparticulate, blood-brain barrier-permeating, non-viral vectors. To this end, dextran, that had been functionalised via treatment with epoxide precursors of alkylglycerol, was covalently linked to poly(lactic acid) using a carbodiimide cross-linker to form alkylglyceryl-modified dextran-graft-poly(lactic acid). Solvent displacement and electrospray methods allowed the formulation of these materials into nanoparticles having a unimodal size distribution profile of about 100-200nm and good stability at physiologically relevant pH (7.4). The nanoparticles were characterised in terms of hydrodynamic size (by Dynamic Light Scattering and Nanoparticle Tracking Analysis), morphology (by Scanning Electron Microscopy and Atomic Force Microscopy) and zeta potential, and their toxicity was evaluated using MTT and PrestoBlue assays. Cellular uptake was evidenced by confocal microscopy employing nanoparticles that had been loaded with the easy-to-detect Rhodamine B fluorescent marker. Transwell-model experiments employing mouse (bEnd3) and human (hCMEC/D3) brain endothelial cells revealed enhanced permeation (statistically significant for hCMEC/D3) of the fluorescent markers in the presence of the nanoparticles. Results of studies using Electric Cell Substrate Impedance Sensing suggested a transient decrease of the barrier function in an in vitro blood-brain barrier model following incubation with these nanoformulations. An in ovo study using 3-day chicken embryos indicated the absence of whole-organism acute toxicity effects. The collective in vitro data suggest that these alkylglyceryl-modified dextran-graft-poly(lactic acid) nanoparticles are promising candidates for in vivo evaluations that would test their capability to transport therapeutic actives to the brain.

Absence of glial α-dystrobrevin causes abnormalities of the blood-brain barrier and progressive brain edema.

The blood-brain barrier (BBB) plays a key role in maintaining brain functionality. Although mammalian BBB is formed by endothelial cells, its function requires interactions between endotheliocytes and glia. To understand the molecular mechanisms involved in these interactions is currently a major challenge. We show here that α-dystrobrevin (α-DB), a protein contributing to dystrophin-associated protein scaffolds in astrocytic endfeet, is essential for the formation and functioning of BBB. The absence of α-DB in null brains resulted in abnormal brain capillary permeability, progressively escalating brain edema, and damage of the neurovascular unit. Analyses in situ and in two-dimensional and three-dimensional in vitro models of BBB containing α-DB-null astrocytes demonstrated these abnormalities to be associated with loss of aquaporin-4 water and Kir4.1 potassium channels from glial endfeet, formation of intracellular vacuoles in α-DB-null astrocytes, and defects of the astrocyte-endothelial interactions. These caused deregulation of tight junction proteins in the endothelia. Importantly, α-DB but not dystrophins showed continuous expression throughout development in BBB models. Thus, α-DB emerges as a central organizer of dystrophin-associated protein in glial endfeet and a rare example of a glial protein with a role in maintaining BBB function. Its abnormalities might therefore lead to BBB dysfunction.

In vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood-brain barrier.

A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood-brain-barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cell-substrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood-brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood-brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.

P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: relationship to pathology and potential target for treatment.

Duchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca(2+) homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca(2+) and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca(2+) influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration-regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease.

Knockdown of MLC1 in primary astrocytes causes cell vacuolation: a MLC disease cell model.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contains other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton; it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Re-examination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease.

Toward drug delivery into the brain: synthesis, characterization, and preliminary in vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles.

A series of O-substituted alkylglyceryl chitosans with systematically varied degrees of grafting was prepared through synthetic steps that involved the protection of amino moieties via phthaloylation and employed for the formulation of aqueous nanoparticulate systems that may be capable of delivering drugs to the brain. Dynamic light scattering studies have shown that nanoparticles with physiologically relevant aqueous stabilities may be prepared following the partial quaternization of these alkylglyceryl-modified chitosans. Preliminary in vitro tests using a mouse-brain endothelial cell model have indicated the efficient cellular uptake of these nanoparticles and identified butylglyceryl chitosan and butylglyceryl N,N,N-trimethyl chitosan as promising materials for the formulation of colloidal systems that could act as drug carriers into the brain.

Synaptic alpha-dystrobrevin: localization of a short alpha-dystrobrevin isoform in melanin-concentrating hormone neurons of the hypothalamus.

The expression of the two members of the dystrobrevin (DB) family in the adult brain was thought to be highly specific for the two main cell types: alpha-dystrobrevin (alpha-DB) and beta-dystrobrevin (beta-DB) has been identified as glial and neuronal proteins, respectively. In the present work we show that a subset of neurons in the hypothalamus contains alpha-DB. Comparative immunohistochemical studies with two alpha-DB antibodies of different specificity indicate that the neurons contain short alpha-DB isoform(s) alpha-DB-2 and/or alpha-DB-4. Immunoreactive multipolar or spindle-shaped neurons form clusters with bilateral symmetry, localized predominantly in the lateral hypothalamic area, with extensions into the zona incerta and the dorso-medial and ventro-medial hypothalamic region. alpha-DB immunoreactivity was localized in cell processes and at postsynaptic densities, furthermore in the endoplasmic reticulum within the perikarya. alpha-DB-positive neurons are beta-dystrobrevin immunoreactive, but alpha- and beta-DB do not co-localize with their usual molecular anchors like dystrophins or high molecular weight forms of utrophin. Colocalization with nNOS was also not observed. The pattern of alpha-DB immunoreactive neurons gave a perfect colocalization with melanin-concentrating hormone (MCH) neurons throughout the whole region studied. We propose that alpha-DB plays a role in a structure or regulation mechanism unique to MCH-expressing neurons.

Dystrobrevins in muscle and non-muscle tissues.

The alpha- and beta-dystrobrevins belong to the family of dystrophin-related and dystrophin-associated proteins. As constituents of the dystrophin-associated protein complex, alpha-dystrobrevin was believed to have a role predominantly in muscles and beta-dystrobrevin in non-muscle tissues. Recent reports described novel localisations and molecular characteristics of alpha-dystrobrevin isoforms in non-muscle tissues (developing and adult). While single and double knockout studies have revealed distinct functions of dystrobrevin in some tissues, these also suggested a strong compensatory mechanism, where dystrobrevins displaying overlapping tissue expression pattern and structure/function similarity can substitute each other. No human disease has been unequivocally associated within mutations of dystrobrevin genes. However, some significant exceptions to these overlapping expression patterns, mainly in the brain, suggest that dystrobrevin mutations might underlie some specific motor, behavioural or cognitive defects. Dystrobrevin binding partner DTNBP1 (dysbindin) is a probable susceptibility gene for schizophrenia and bipolar affective disorder in some populations. As dysbindin abnormality is linked to Hermansky-Pudlak syndrome, dystrobrevins and/or their binding partners may also be required for proper function of other non-muscle tissues.

Expression of alpha-dystrobrevin in blood-tissue barriers: sub-cellular localisation and molecular characterisation in normal and dystrophic mice.

The alpha- and beta-dystrobrevins (DBs) belong to a family of dystrophin-related and dystrophin-associated proteins that are members of the dystrophin-associated protein complex (DAPC). This complex provides a link between the cytoskeleton and the extracellular matrix or other cells. However, specific functions of the two dystrobrevins remain largely unknown, with alpha-DB being believed to have a role mainly in skeletal muscle. Here, we describe previously unknown expression patterns and the localisation and molecular characteristics of alpha-DB isoforms in non-muscle mouse tissues. We demonstrate a highly specific sub-cellular distribution of alpha-DB in organs forming blood-tissue barriers. We show alpha-DB expression and localisation in testicular Sertoli cells, stomach and respiratory epithelia and provide electron-microscopic evidence for its immunolocalisation in these cells and in the central nervous system. Moreover, we present the molecular characterisation of alpha-DB transcript in these tissues and provide evidence for a distinct heterogeneity of associations between alpha-DB and dystrophins and utrophin in normal and dystrophic non-muscle tissues. Together, our results indicate that alpha-DB, in addition to its role in skeletal muscle, may also be required for the proper function of specific non-muscle tissues and that disruption of DAPC might lead to tissue-blood barrier abnormalities.

Increased susceptibility to ATP via alteration of P2X receptor function in dystrophic mdx mouse muscle cells.

Pathological cellular hallmarks of Duchenne muscular dystrophy (DMD) include, among others, abnormal calcium homeostasis. Changes in the expression of specific receptors for extracellular ATP in dystrophic muscle have been recently documented: here, we demonstrate that at the earliest, myoblast stage of developing dystrophic muscle a purinergic dystrophic phenotype arises. In myoblasts of a dystrophin-negative muscle cell line established from the mdx mouse model of DMD but not in normal myoblasts, exposure to extracellular ATP triggered a strong increase in cytoplasmic Ca2+ concentrations. Influx of extracellular Ca2+ was stimulated by ATP and BzATP and inhibited by zinc, Coomassie Brilliant Blue-G, and KN-62, demonstrating activation of P2X7 receptors. Significant expression of P2X4 and P2X7 proteins was immunodetected in dystrophic myoblasts. Therefore, full-length dystrophin appears, surprisingly, to play an important role in myoblasts in controlling responses to ATP. Our results suggest that altered function of P2X receptors may be an important contributor to pathogenic Ca2+ entry in dystrophic mouse muscle and may have implications for the pathogenesis of muscular dystrophies. Treatments aiming at inhibition of specific ATP receptors could be of a potential therapeutic benefit.

Localized expression of specific P2X receptors in dystrophin-deficient DMD and mdx muscle.

Using a combination of molecular and immunohistochemical methods, we have obtained evidence for a distinctive change in the expression patterns of ATP-gated (P2X) receptor subunits in dystrophic muscle from both Duchenne muscular dystrophy (DMD) patients and the mdx mouse model. In control myofibres there was no staining for any P2X subtype studied here, although P2X1 stained the smooth muscle of the blood vessels and P2X6 nerves and the tunica intima in small arteries. In contrast, P2X1 and P2X6 were co-expressed strongly in small regenerating muscle fibres in the dystrophic muscles, whereas this expression decreased in fully regenerated fibres. Moreover, immunoreactivity for the P2X2 receptor re-appeared in dystrophic muscle, where it co-localised with the Type 1 fibres. There is, thus, a burst of production of several P2X receptor subtypes in regenerating dystrophic muscle, which may have implications for drug targets for this muscle pathology.

Aberrant expression, processing and degradation of dystroglycan in squamous cell carcinomas.

The alpha- and beta- dystroglycan (DG) proteins are involved in epithelial cell development, formation of the basement membrane and maintenance of tissue integrity. Recently, specific changes in the expression patterns of DGs have been described in some cancers. We studied the expression and localisation of alpha- and beta-DG using Western blotting, immunohistochemistry and reverse transcriptase-polymerase chain reaction analyses in samples of normal oral mucosa, oral squamous cell carcinoma (SCC) and cancer cell lines. The alpha- and beta-DG were localised in the basal layers of normal oral mucosa.However, beta-DG expression in cancer tissues showed evidence of aberrant expression, processing and degradation. alpha-DG was altered in all oral cancer samples and cell lines, despite the persistent presence of DG mRNA in cancer cells. Using matrix metalloproteinase (MMP) inhibitors, we determined that beta-DG degradation in carcinoma cell lines can be mediated by MMPs but this process is highly variable, even in cells from the same cancer type. Considering the multifaceted role of DG in epithelial development, it appears that the role of DG degradation in cancer growth and spread, although currently poorly understood, may be important.

Differential spatio-temporal expression of alpha-dystrobrevin-1 during mouse development.

Dystrobrevins are a family of dystrophin-related and dystrophin-associated proteins. alpha-dystrobrevin-1 knockout mice suffer from skeletal and cardiac myopathies. It has been suggested that the pathology is caused by the loss of signalling functions but the exact role of dystrobrevins is largely unknown. We have analysed the spatial and temporal expression of alpha-dystrobrevin-1 during mouse embryogenesis and found striking developmental regulation and distribution patterns. During development this protein was expressed not only in muscle but also in the CNS, sensory organs, epithelia and skeleton. Particularly interesting was the correlation of alpha-dystrobrevin-1 expression with the induction of various differentiation processes in the developing eye, inner ear, pituitary, blood-brain barrier, stomach epithelium and areas of the brain, dorsal root ganglia and spinal cord. In contrast, this specific expression at the induction phase decreased/disappeared at later stages of development.