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Purpose: Glaucoma is a distinct neuropathy characterized by the chronic and progressive death of retinal ganglion cells (RGCs). The etiology of RGC death remains unknown. Risk factors for glaucomatous RGC death are elevated intraocular pressure and glial production of tumor necrosis factor-alpha (TNF-α). Previously, the authors showed that glaucoma causes a rapid upregulation of a neurotrophin receptor truncated isoform lacking the kinase domain, TrkC.T1, in retina. Here they examined the biological role of TrkC.T1 during glaucoma progression., Methods: Rat and mouse models of chronic ocular hypertension were used. Immunofluorescence Western blot analysis and in situ mRNA hybridization were used to identify cells upregulating TrkC.T1. A genetic model of engineered mice lacking TrkC.T1 (TrkC.T1(-/-)) was used to validate a role for this receptor in glaucoma. Pharmacologic studies were conducted to evaluate intravitreal delivery of agonists or antagonists of TrkC.T1, compared with controls, during glaucoma. Surviving RGCs were quantified by retrograde-labeling techniques. Production of neurotoxic TNF-α and α2 macroglobulin were quantified., Results: TrkC.T1 was upregulated in retinal glia, with a pattern similar to that of TNF-α. TrkC.T1(-/-) mice had normal retinas. However, during experimental glaucoma, TrkC.T1(-/-) mice had lower rates of RGC death and produced less TNF-α than wild-type littermates. In rats with glaucoma, the pharmacologic use of TrkC antagonists delayed RGC death and reduced the production of retinal TNF-α., Conclusions: TrkC.T1 is implicated in glaucomatous RGC death through the control of glial TNF-α production. Overall, the data point to a paracrine mechanism whereby elevated intraocular pressure upregulated glial TrkC.T1 expression in glia; TrkC.T1 controlled glial TNF-α production, and TNF-α caused RGC death.

The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

[structure: see text] An efficient total stepwise solid-phase synthesis of oligonucleotide-(3'-->N)-peptide conjugates is described that makes use of either a controlled pore glass support or macroporous polystyrene beads. Extending our previous homoserine linker approach, we prepared a range of conjugates containing one of four different cell or nuclear penetration peptides together with oligonucleotides containing 2'-deoxynucleoside or 2'-O-methylribonucleoside phosphodiesters, or gapmers containing 2'-deoxyphosphorothioates. The route also allows incorporation of a fluorescent label within the conjugate for cell uptake studies.