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Two different human diseases, X-linked myotubular myopathy and Charcot-Marie-Tooth disease, result from mutant MTM1 or MTMR2 lipid phosphatases. Although events involved in endosomal PI(3)P and PI(3,5)P 2 synthesis are well established and pivotal in receptor signaling and degradation, enzymes involved in phosphoinositide degradation and their roles in trafficking are incompletely characterized. Here, we dissect the functions of the MTM1 and MTMR2 myotubularins and establish how they contribute to endosomal PI(3)P homeostasis. By mimicking loss of function in disease through siRNA-mediated depletion of the myotubularins, excess PI(3)P accumulates on early (MTM1) and late (MTMR2) endosomes. Surprisingly, the increased PI(3)P blocks the egress of epidermal growth factor receptors from early or late endosomes, suggesting that the accumulation of signaling receptors in distinct endosomes may contribute to the unique disease etiologies when MTM1 or MTMR2 are mutant. We further demonstrate that direct myotubularin binding to the type III PI 3-kinase complex hVps34/hVps15 leads to phosphatase inactivation. The lipid kinase-phosphatase interaction also precludes interaction of the PI 3-kinase with Rab GTPase activators. Thus, unique molecular complexes control kinase and phosphatase activation and locally regulate PI(3)P on discrete endosome populations, thereby providing a molecular rationale for related human myo-and neuropathies. INTRODUCTIONHow phophoinositide synthesis and degradation are coordinately regulated remains a key question in endocytic membrane trafficking. The importance of phosphoinositides in endocytic trafficking has been largely studied by inhibiting lipid kinases or overexpressing lipid phosphatases to reduce lipid levels (Futter et al., 2001;Chaussade et al., 2003;Lu et al., 2003;Petiot et al., 2003;Tsujita et al., 2004;Johnson et al., 2006). The fact that inactivation of lipid phosphatases results in disease points to the importance of optimal phosphoinositide levels in homeostasis (Laporte et al., 1996;Bolino et al., 2000;Bitoun et al., 2005;Niemann et al., 2006;Spinosa et al., 2008).Endocytic membrane trafficking is critically dependent on the local synthesis of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-phosphate (PI(3,5)P 2 ) (Futter et al., 2001;Chaussade et al., 2003;Ikonomov et al., 2003;Lu et al., 2003;Petiot et al., 2003;Stein et al., 2003;Tsujita et al., 2004;Johnson et al., 2006;Shisheva, 2008). PI(3)P is generated on early endosomes as well as on late endosomes by the type III PI 3-kinase complex hVps34/hVps15 (Gillooly et al., 2000;Simonsen et al., 2001;Stein et al., 2003). PI(3)P is responsible for the temporal recruitment of proteins required for endosome fusion and membrane invagination. Endosomal synthesis of PI(3)P is initiated with the activation of the Rab5 and Rab7 GTPases on early and late endosomes, respectively (Christoforidis et al., 1999;Feng et al., 2001;Murray et al., 2002;Stein et al., 2003Stein et al., , 2005. The activated GTPases bind an...
The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.
During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.
Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P 2 binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P 2 are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM.
The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.
The problem of targeted delivery of antisense and siRNA oligonucleotides can be resolved into two distinct aspects. The first concerns devising ligand-oligonucleotide or ligand-carrier moieties that bind with high selectivity to receptors on the cell type of interest and that are efficiently internalized by endocytosis. The second concerns releasing oligonucleotides from pharmacologically inert endomembrane compartments so that they can access RNA in the cytosol or nucleus. In this review we will address both of these aspects. Thus we present information on three important receptor families, the integrins, the receptor tyrosine kinases, and the G protein-coupled receptors in terms of their suitability for targeted delivery of oligonucleotides. This includes discussion of receptor abundance, internalization and trafficking pathways, and the availability of suitable high affinity ligands. We also consider the process of oligonucleotide uptake and intracellular trafficking and discuss approaches to modulating these processes in a pharmacologically productive manner. Hopefully the basic information presented in this review will be of value to investigators involved in designing delivery approaches for oligonucleotides.
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