As in other organ systems, gene and drug delivery to ocular tissues such as the retina and cornea is hampered by inefficient penetration of therapeutic molecules across the plasma membrane. We describe the use of a novel peptide for ocular delivery (POD) with protein transduction properties, for delivery of small and large molecules across the plasma membrane. POD enters cells within 5 minutes in a temperature dependent manner. POD can compact and deliver plasmid DNA, achieving transgene expression in >50% of human embryonic retinoblasts. Delivery of small interfering RNA (siRNA) duplexes to cells using POD, allowed for silencing of transgene expression by >50%. POD could also be used to deliver quantum dots in vitro and in vivo. Upon ocular delivery, POD rapidly entered neural retina and localized to retinal pigment epithelium (RPE), photoreceptor, and ganglion cells. Additionally, POD was able to enter corneal epithelium, sclera, choroid, and the dura of the optic nerve via topical application. POD also functions as a bacteriostatic, a useful property for a carrier of molecules to post mitotic neural ocular tissues.

Recently we described a novel cell penetrating peptide, POD (peptide for ocular delivery) that could deliver small molecules including fluorescent dyes into retinal cells. The objective of the current study was to examine whether biologically relevant macromolecules such as proteins, genetically fused with POD could also be delivered into retinal tissues in vivo. We generated a POD-GFP fusion protein and examined its cell and tissue penetrating properties. We found that endogenously expressed POD-GFP fusion protein localized to the nucleus, suggesting that POD acts as a nuclear localization signal. Adenovirus (Ad) vectors expressing POD-GFP fusion protein were constructed and the recombinant protein was purified from Ad-infected human embryonic retinoblasts (HER). Exogenously supplied POD-GFP fusion protein rapidly transduced A549 and HER cells and colocalized in part with markers of late endosomes, from which it could escape. Following subretinal delivery, POD-GFP localized to the retinal pigment epithelium and the photoreceptor cell bodies. When injected into the vitreous, POD-GFP localized to the ganglion cells and the inner nuclear layer of the retina as well as the lens capsule. Topical application of POD-GFP to ocular surfaces resulted in uptake by the corneal epithelium. POD-GFP also transduced non-ocular tissues, including the epidermis of the skin following topical application.

Follistatin is recognized to be an important regulator of cellular differentiation and secretion through its potent ability to bind and bioneutralize activin with which it is colocalized in many tissue systems. The 288-residue follistatin molecule is comprised of a 63-residue N-terminal segment followed by three repeating 10-cysteine "follistatin domains" also represented in several extracellular matrix proteins. We have used chemical modifications and mutational analyses to define structural requirements for follistatin bioactivity that previously have not been investigated systematically. Mutant follistatins were stably expressed from Chinese hamster ovary cell cultures and assayed for activin binding in a solid-phase competition assay. Biological activities were determined by inhibition of activinmediated transcriptional activity and by suppression of follicle-stimulating hormone secretion by cultured anterior pituitary cells. Deletion of the entire N-terminal domain, disruption of N-terminal disulfides, and deletion of the first two residues each reduced activin binding to <5 % of expressed wild-type follistatin and abolished the ability of the respective mutants to suppress activin-mediated responses in both bioassay systems. Hence, the three follistatin domains inherently lack the ability to bind or neutralize activin. Activin binding was impaired after oxidation of at least one tryptophan, at position 4, in FS-288. Mutation of Trp to Ala or Asp at either positions 4 or 36 eliminated activin binding and bioactivity. Mutation of a third hydrophobic residue, Phe-52, reduced binding to 20%, whereas substitutions for the individual Lys and Arg residues in the N-terminal region were tolerated. These results establish that hydrophobic residues within the N-terminal domain constitute essential activin-binding determinants in the follistatin molecule. The correlation among the effects of mutation on activin binding, activin transcriptional responses, and follicle-stimulating hormone secretion substantiates the concept that, at least in the pituitary, the biological activity of follistatin is attributable to its ability to bind and bioneutralize activin. Follistatin (FS)1 has gained recognition as an important mediator of cell secretion, development, and differentiation in a number of tissue and organ systems. Follistatin was first isolated from ovarian follicular fluid as a protein factor capable of suppressing FSH secretion by pituitary cells in culture in a manner similar to inhibin (reviewed in Refs. 1-4). Cloning and sequencing (5) showed it to be a protein of 288 amino acids (FS-288), unrelated to inhibin, with a C-terminal-extended form (FS-315) derived from alternative splicing. No "receptor" for follistatin has been found, but its mode of action in the pituitary became clear with the demonstration (6) that the protein binds the activin A homodimer with high affinity, approaching irreversibility because of its slow dissociation rate (7). Multiple lines of evidence have now shown that, rather than "presentin...

The Golgi complex plays an important role in cholesterol trafficking in cells, and amyloid ␤-peptides (A␤s) alter cholesterol trafficking. The hypothesis was tested that fresh and aged A␤-(1-42) would differentially modify Golgi cholesterol content in DINTC1 astrocytes and that the effects of A␤-(1-42) would be associated with the region of the Golgi complex. Two different methods were used to determine the effects of A␤-(1-42) on Golgi complex cholesterol. Confocal microscopy showed that fresh A␤-(1-42) significantly increased cholesterol and that aged A␤-(1-42) significantly reduced cholesterol content in the Golgi complex. Isolation of the Golgi complex into two fractions using density gradient centrifugation showed effects of aged A␤-(1-42) similar to those observed with confocal microscopy but revealed the novel finding that fresh A␤-(1-42) had opposite effects on the two Golgi fractions suggesting a specificity of A␤-(1-42) perturbation of the Golgi complex. Phosphatidylcholine-phospholipase D activity, cell membrane cholesterol, and apolipoprotein E levels were associated with effects of fresh A␤-(1-42) on cholesterol distribution but not with effects of aged A␤-(1-42), arguing against a common mechanism. Extracellular A␤-(1-42) targets the Golgi complex and disrupts cell cholesterol homeostasis, and this action of A␤-(1-42) could alter cell functions requiring optimal levels of cholesterol.