Miriam S. Domowicz

Selecting Improved Peptidyl Motifs for Cytosolic Delivery of Disparate Protein and Nanoparticle Materials

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptides ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

Proteoglycans in brain development

Proteoglycans, as part of the extracellular or cell-surface milieu of most tissues and organ systems, play important roles in morphogenesis by modulating cell-matrix or cell-cell interactions, cell adhesiveness, or by binding and presenting growth and differentiation factors. Chondroitin sulfate proteoglycans which constitute the major population of proteoglycans in the central nervous system may influence formation of neuronal nuclei, establishment of boundaries for axonal growth and act as modulators of neuronal outgrowth during brain development, as well as during regeneration after injury. There is a paucity of information on the role of chondroitin sulfate proteoglycans in central nervous system organogenesis. In the chick embryo, aggrecan has a regionally specific and developmentally regulated expression profile during brain development. By Northern and Western blot analysis, aggrecan expression is first detected in chick brain on embryonic day 7 (E7), increases from E7 to E13, declines markedly after E16, and is not evident in hatchling brains. The time course and pattern of aggrecan expression observed in ventricular zone cells suggested that it might play a role in gliogenesis. We have analyzed the role of aggrecan during brain development using a aggrecan-deficient model, nanomelia. In nanomelic chicks, expression and levels of neurocan and brevican is not affected, indicating a non-redundant role for these members of the aggrecan gene family. Our analysis of the aggrecan-deficient model found a severely altered phenotype which affects cell behavior in a neuronal culture paradigm and expression of astrocytic markers in vivo. Taken together our results suggest a function for aggrecan in the specification of a sub-set of glia precursors that might give rise to astrocytes in vivo. Published in 2004.

Domain Organization, Genomic Structure, Evolution, and Regulation of Expression of the Aggrecan Gene Family

Proteoglycans are complex macromolecules, consisting of a polypeptide backbone to which are covalently attached one or more glycosaminoglycan chains. Molecular cloning has allowed identification of the genes encoding the core proteins of various proteoglycans, leading to a better understanding of the diversity of proteoglycan structure and function, as well as to the evolution of a classification of proteoglycans on the basis of emerging gene families that encode the different core proteins. One such family includes several proteoglycans that have been grouped with aggrecan, the large aggregating chondroitin sulfate proteoglycan of cartilage, based on a high number of sequence similarities within the N- and C-terminal domains. Thus far these proteoglycans include versican, neurocan, and brevican. It is now apparent that these proteins, as a group, are truly a gene family with shared structural motifs on the protein and nucleotide (mRNA) levels, and with nearly identical genomic organizations. Clearly a common ancestral origin is indicated for the members of the aggrecan family of proteoglycans. However, differing patterns of amplification and divergence have also occurred within certain exons across species and family members, leading to the class-characteristic protein motifs in the central carbohydrate-rich region exclusively. Thus the overall domain organization strongly suggests that sequence conservation in the terminal globular domains underlies common functions, whereas differences in the central portions of the genes account for functional specialization among the members of this gene family.

AGGRECAN IS EXPRESSED BY EMBRYONIC BRAIN GLIA AND REGULATES ASTROCYTE DEVELOPMENT

Determination of the molecules that regulate astrocyte development has been hindered by the paucity of markers that identify astrocytic precursors in vivo. Here we report that the chondroitin sulfate proteoglycan aggrecan both regulates astrocyte development and is expressed by embryonic glial precursors. During chick brain development, the onset of aggrecan expression precedes that of the astrocytic marker GFAP and is concomitant with detection of the early glial markers GLAST and glutamine synthetase. In co-expression studies, we established that aggrecan-rich cells contain the radial glial markers nestin, BLBP and GLAST and later in embryogenesis, the astroglial marker GFAP. Parallel in vitro studies showed that ventricular zone cultures, enriched in aggrecan-expressing cells, could be directed to a GFAP-positive fate in G5-supplemented differentiation media. Analysis of the chick aggrecan mutant nanomelia revealed marked increases in the expression of the astrocyte differentiation genes GFAP, GLAST and GS in the absence of extracellular aggrecan. These increases in astrocytic marker gene expression could not be accounted for by changes in precursor proliferation or cell death, suggesting that aggrecan regulates the rate of astrocyte differentiation. Taken together, these results indicate a major role for aggrecan in the control of glial cell maturation during brain development.

Viral and cellular requirements for entry of herpes simplex virus type 1 into primary neuronal cells.

Herpes simplex virus (HSV) causes many disease states including mucosal lesions, encephalitis or disseminated infection in the immunocompromised host. These diverse clinical manifestations reflect the capacity of the virus to infect both epithelial and neuronal cell types. Determining the requirements for virus entry into both cell types may provide insights into the pathogenesis of HSV. Previous studies have focused on identifying viral and cellular requirements for entry using epithelial cells. However, little is known about the requirements for binding and entry into neuronal cells. The purpose of the studies reported here was to identify viral and cellular components involved in entry of HSV-1 into primary neuronal cells. Heparan sulfate glycosaminoglycans were found to serve as a receptor for entry of HSV-1 into primary neuronal cells. Evidence to support this includes the findings that heparin (an analogue of heparan sulfate) competitively inhibited virus binding and expression of immediate early virus gene products. In addition, heparitinase removed viral receptors and inhibited virus entry. In epithelial cells, deletion of HSV-1 glycoprotein C (gC) results in virions that have reduced specific binding activity (virus particles bound per cell) and specific infectivity. However, in neuronal cells, it was found that deletion of gC resulted in no loss in specific binding activity, but did result in significant impairment of virus entry as measured by expression of immediate early viral gene product. Taken together, these findings suggest cell-type differences in virus binding and entry and a different role for gC in neuronal cell infection.

Delivery and Tracking of Quantum Dot Peptide Bioconjugates in an Intact Developing Avian Brain

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-β-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

Cold pre‐conditioning neuroprotection depends on TNF‐α and is enhanced by blockade of interleukin‐11

J. Neurochem. (2011) 117, 187–196.

Aggrecan modulation of growth plate morphogenesis.

Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder.

S103L reactive chondroitin sulfate proteoglycan (aggrecan) mRNA expressed in developing chick brain and cartilage is encoded by a single gene

A large chondroitin sulfate proteoglycan (CSPG) identified in embryonic chick brain, and synthesized exclusively by neurons in a developmentally expressed pattern that coincides with migration and establishment of neuronal nuclei, reacts with a monoclonal antibody (S103L) developed against the cartilage-specific CSPG, aggrecan. The relationship of the brain and cartilage S103L CSPGs was established by chemical, biosynthetic and molecular analyses. Significant posttranslational differences (absence of keratan sulfate (KS), less CS, and different sulfation patterns) distinguish the brain S103L species from the cartilage S103L species. However, quantitative and qualitative Northern analysis, cassette RT-PCR and direct cloning and sequencing of the entire brain-specific S103L CSPG coding sequence, all indicate that the brain and cartilage core proteins are identical. Thus, although the S103L CSPG synthesized by chick brain and cartilage are the product of a single gene, they are clearly biochemically distinct and differentially expressed proteoglycan products, suggesting tissue specific roles for these proteoglycan homologs.

Role of the C-terminal G3 Domain in Sorting and Secretion of Aggrecan Core Protein and Ubiquitin-mediated Degradation of Accumulated Mutant Precursors

Aggrecan is a complex multidomain macromolecule that undergoes extensive processing and post-translational modification. A thorough understanding of the events and signals that promote translocation of aggrecan through the secretory pathway is lacking. To investigate which features of the C-terminal G3 region are necessary for successful translocation of the core protein, a number of deletion constructs based on the chick aggrecan cDNA sequence were prepared and transiently expressed in COS-1 cells and the natural host, embryonic chick chondrocytes; stable cell lines were established as well. The present results clearly establish a precise requirement for that portion of the G3 C-lectin domain encoded by exon 15 for: (i) translocation from the endoplasmic reticulum (ER) to the Golgi, (ii) secretion from the cell, (iii) galactosylation of chondroitin sulfate (CS) chains, (iv) generation of Ca+2-dependent galactose binding ability. Furthermore, in the absence of this subdomain there is excess accumulation in the ER of expression products leading to a stress-related response involving the chaperones Grp78 and protein disulfide isomerase, followed by degradation via a ubiquitin-proteosome pathway. All of these events in the model system faithfully mimic the naturally occurring nanomelic mutant, which also elicits a ubiquitin-mediated degradation response due to the accumulation of the truncated core protein precursor. This study represents the first report of the mode of degradation of overexpressed or misfolded proteoglycans and suggests that, although proteoglycans follow different glycosylation pathways from other glycoproteins, they are monitored by an ER surveillance system similar to that which detects other misfolded proteins.