Dharamdajal Kalicharan

Surface characteristics of nanoparticles determine their intracellular fate in and processing by human blood-brain barrier endothelial cells in vitro.

A polarized layer of endothelial cells that comprises the blood-brain barrier (BBB) precludes access of systemically administered medicines to brain tissue. Consequently, there is a need for drug delivery vehicles that mediate transendothelial transport of such medicines. Endothelial cells use a variety of endocytotic pathways for the internalization of exogenous materials, including clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis. The different modes of endocytosis result in the delivery of endocytosed material to distinctive intracellular compartments and therewith correlated differential processing. To obtain insight into the properties of drug delivery vehicles that direct their intracellular processing in brain endothelial cells, we investigated the intracellular processing of fixed-size nanoparticles in an in vitro BBB model as a function of distinct nanoparticle surface modifications. Caveolar endocytosis, adsorptive-mediated endocytosis, and receptor-mediated endocytosis were promoted by the use of uncoated 500-nm particles, attachment of the cationic polymer polyethyleneimine (PEI), and attachment of prion proteins, respectively. We demonstrate that surface modifications of nanoparticles, including charge and protein ligands, affect their mode of internalization by brain endothelial cells and thereby their subcellular fate and transcytotic potential.

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells

BackgroundCigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.MethodsWe studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.ResultsWe observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.ConclusionThe observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

Corticotropin-releasing factor and urocortin regulate spine and synapse formation: structural basis for stress-induced neuronal remodeling and pathology

Dendritic spines are important sites of excitatory neurotransmission in the brain with their function determined by their structure and molecular content. Alterations in spine number, morphology and receptor content are a hallmark of many psychiatric disorders, most notably those because of stress. We investigated the role of corticotropin-releasing factor (CRF) stress peptides on the plasticity of spines in the cerebellum, a structure implicated in a host of mental illnesses, particularly of a developmental origin. We used organotypic slice cultures of the cerebellum and restraint stress in behaving animals to determine whether CRF in vitro and stress in vivo affects Purkinje cell (PC) spine density. Application of CRF and urocortin (UCN) to cerebellar slice cultures increased the density of spines on PC signaling via CRF receptors (CRF-Rs) 1 and 2 and RhoA downregulation, although the structural phenotypes of the induced spines varied, suggesting that CRF-Rs differentially induce the outgrowth of functionally distinct populations of spines. Furthermore, CRF and UCN exert a trophic effect on the surface contact between synaptic elements by increasing active zones and postsynaptic densities and facilitating the alignment of pre- and post-synaptic membranes of synapses on PCs. In addition, 1 h of restraint stress significantly increased PC spine density compared with those animals that were only handled. This study provides unprecedented resolution of CRF pathways that regulate the structural machinery essential for synaptic transmission and provides a basis for understanding stress-induced mental illnesses.

Formation of E-cadherin/beta-catenin-based adherens junctions in hepatocytes requires serine-10 in p27(Kip1)

The adhesion between epithelial cells at adherens junctions is regulated by signaling pathways that mediate the intracellular trafficking and assembly of its core components. Insight into the molecular mechanisms of this is necessary to understand how adherens junctions contribute to the functional organization of epithelial tissues. Here, we demonstrate that in human hepatic HepG2 cells, oncostatin M-p42/44 mitogen-activated protein kinase signaling stimulates the phosphorylation of p27(Kip1) on Ser-10 and promotes cell-cell adhesion. The overexpression of wild-type p27 or a phospho-mimetic p27S10D mutant in HepG2 cells induces a hyper-adhesive phenotype. In contrast, the overexpression of a nonphosphorylatable p27S10A mutant prevents the mobilization of E-cadherin and beta-catenin at the cell surface, reduces basal cell-cell adhesion strength, and prevents the stimulatory effect of oncostatin M on cell-cell adhesion. As part of the underlying molecular mechanism, it is shown that in p27S10A-expressing cells beta-catenin interacts with p27 and is prevented from interacting with E-cadherin. The intracellular retention of E-cadherin and beta-catenin is also observed in hepatocytes from p27S10A knockin mice that express the p27S10A mutant instead of wild-type p27. Together, these data suggest that the formation of adherens junctions in hepatocytes requires Ser-10 in p27.

The dynamic developmental localization of the full-length corticotropin-releasing factor receptor type 2 in rat cerebellum.

Corticotropin releasing factor receptor 2 (CRF‐R2) is strongly expressed in the cerebellum and plays an important role in the development of the cerebellar circuitry, particularly in the development of the dendritic trees and afferent input to Purkinje cells. However, the mechanisms responsible for the distribution and stabilization of CRF‐R2 in the cerebellum are not well understood. Here, we provide the first detailed analysis of the cellular localization of the full‐length form of CRF‐R2 in rat cerebellum during early postnatal development. We document unique and developmentally regulated subcellular distributions of CRF‐R2 in cerebellar cell types, e.g. granule cells after postnatal day 15. The presence of one or both receptor isoforms in the same cell may provide a molecular basis for distinct developmental processes. The full‐length form of CRF‐R2 may be involved in the regulation of the first stage of dendritic growth and at later stages in the controlling of the structural arrangement of immature cerebellar circuits and in the autoregulatory pathway of the cerebellum.

Mozaic microscopy of pancreatic islets

The interplay between molecules that regulate (sub)cellular processes and between cells and tissues that regulate organisms is highly fascinating. Precise knowledge of the contribution of these molecules and cells helps us to understand the basis of biology under (patho)-physiological conditions. Microscopic techniques have aided in visualization of molecules, cells and tissues for many years. However, choosing a certain microscopic technique often limits our ability to look into detail at different scales. This is especially important when studying complex, heterogeneous tissue where cells of interest are sparsely distributed. We study beta-cells in health and disease (diabetes). Beta-cells are the insulin-producing cells located in the Islets of Langerhans in the pancreas. Other cell-types are present in these islets, including hormone-secreting alpha-cells and delta cells and Islets are highly vascularized and innervated. Islets are heterogeneous, and can consist of a few up to several thousands of cells. Islets are scattered through the exocrine (acinar) pancreas, and contribute only a few percent to the total pancreas, making quantitative imaging challenging.