Protein delivery into live cells by incubation with an endosomolytic agent.

We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTAT and a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

RIP1 kinase mediates arachidonic acid-induced oxidative death of oligodendrocyte precursors.

Oxidative damage is implicated in many neurological disorders including ischemic cerebral white matter injury. Oligodendrocyte precursors (preOLs) are intrinsically highly susceptible to various forms of oxidative stress. Here we report the identification of RIP1 kinase as a signaling molecule that mediates arachidonic acid- and glu-tathione depletion-induced oxidative death of preOLs. Blockade of RIP1 kinase activity with the specific allosteric inhibitor, necrostatin-1, rescued preOLs from arachidonic acid, cystine deprivation, and buthionine sulfoximine, but not hydrogen peroxide, induced necrosis. Arachidonic acid triggered robust production of reactive oxygen species (ROS) and sustained activation of the JNK pathway in preOLs, whereas inhibition of JNK significantly prevented cell death. Treatment of cells with necrostatin-1 efficiently abolished arachidonic acid-induced ROS production and JNK activation, indicating that RIP1 kinase activation is an upstream event. This study provides the first evidence that RIP1 kinase may play an active role in arachidonic acid- and glutathione depletion-mediated oxidative damage and suggests the therapeutic potential of necrostatin-1 in protecting undifferentiated OLs against oxidative injury.