Imaging rhodopsin degeneration in vivo in a new model of ocular ischemia in living mice.

Delivery of antibodies to monitor key biomarkers of retinopathy in vivo represents a significant challenge because living cells do not take up immunoglobulins to cellular antigens. We met this challenge by developing novel contrast agents for retinopathy, which we used with magnetic resonance imaging (MRI). Biotinylated rabbit polyclonal to chick IgY (rIgPxcIgY) and phosphorylthioate-modified oligoDNA (sODN) with random sequence (bio-sODN-Ran) were conjugated with NeutrAvidin-activated superparamagnetic iron oxide nanoparticles (SPION). The resulting Ran-SPION-rIgPxcIgY carries chick polyclonal to microtubule-associated protein 2 (MAP2) as Ran-SPION-rIgP/cIgY-MAP2, or to rhodopsin (Rho) as anti-Rho-SPION-Ran. We examined the uptake of Ran-SPION-rIgP/cIgY-MAP2 or SPION-rIgP/cIgY-MAP2 in normal C57black6 mice (n = 3 each, 40 µg/kg, i.c.v.); we found retention of Ran-SPION-rIgP/cIgY-MAP2 using molecular contrast-enhanced MRI in vivo and validated neuronal uptake using Cy5-goat IgPxcIgY ex vivo. Applying this novel method to monitor retinopathy in a bilateral carotid artery occlusion-induced ocular ischemia, we observed pericytes (at d 2, using Gd-nestin, by eyedrop solution), significant photoreceptor degeneration (at d 20, using anti-Rho-SPION-Ran, eyedrops, P = 0.03, Student's t test), and gliosis in Müller cells (at 6 mo, using SPION-glial fibrillary acidic protein administered by intraperitoneal injection) in surviving mice (n ≥ 5). Molecular contrast-enhanced MRI results were confirmed by optical and electron microscopy. We conclude that chimera and molecular contrast-enhanced MRI provide sufficient sensitivity for monitoring retinopathy and for theranostic applications.

Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration.

Although phosphatidylinositol 5-phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3-kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P-binding motifs, we identified the phosphoinositide 5-kinase PIKfyve and the phosphoinositide 3-phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P(2). The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P-producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.

A stress survival response in retinal cells mediated through inhibition of the serine/threonine phosphatase PP2A.

Cell survival signalling involving the PI3K/Akt survival pathway can be negatively regulated by several phosphatases including PP2A. When retinal-derived 661W cells were subjected to trophic factor deprivation this initiated a survival response through inhibition of the activity of PP2A and subsequent upregulation of the Erk and Akt survival pathways. We show this survival response via inhibition of PP2A activity was due in part to increased reactive oxygen species production when retinal cells were deprived of trophic factors. Inhibition of PP2A activity was mediated by a rapid and transient increase in phosphorylation at Tyr307, accompanied by an increase in demethylation and a decrease in the methylated form. Pre-treatment with N-acetyl-L-cysteine, which is involved in scavenging reactive oxygen species, prevented PP2A inhibition and subsequent upregulation of survival pathways. Pre-treatment with the Src family kinase inhibitor PP2 resulted in approximately 50% reduction in cellular levels of phospho-PP2A in trophic factor-deprived 661W cells, suggesting an Src tyrosine kinase had a role to play in this redox regulation of cell survival. We observed similar events in the rd10 mouse retina where there was an increased survival response prior to retinal cell death mediated through an increase in both phospho-PP2A and phospho-Gsk. Together, these results demonstrate that when retinal cells are stressed there is an initial struggle to survive, mediated through inhibition of PP2A and subsequent upregulation of survival pathways, and that these events occur simultaneously with production of reactive oxygen species, thus suggesting an important cell-signalling role for reactive oxygen species.

Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide.

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H(2)O(2), can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non-mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H(2)O(2). This study demonstrated that 661W cells, a retina-derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well-established regulators. The roles of Nox2 and Nox4 in producing pro-survival H(2)O(2) were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time-lapse imaging of dichlorofluorescein fluorescence (generated upon H(2)O(2) production) in retinal explants was established and it showed that explants also produce a burst of H(2)O(2). The increase in H(2)O(2) production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro-survival role of Nox2 and Nox4 in retina-derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.

IQGAP1 makes PI(3)K signalling as easy as PIP, PIP2, PIP3.

Despite being one of the most studied signalling pathways, precisely how phospholipid synthesis is regulated in the phosphoinositide signalling cascade remains unclear. The scaffold protein IQGAP1 is now shown to orchestrate the assembly of a multi-enzyme complex that streamlines PtdIns(3,4,5)P3 synthesis to facilitate Akt activation in response to extracellular stimuli.

PIP4kγ is a substrate for mTORC1 that maintains basal mTORC1 signaling during starvation.

Phosphatidylinositol-5-phosphate 4-kinases (PIP4ks) are a family of lipid kinases that specifically use phosphatidylinositol 5-monophosphate (PI-5-P) as a substrate to synthesize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Suppression of PIP4k function in Drosophila results in smaller cells and reduced target of rapamycin complex 1 (TORC1) signaling. We showed that the γ isoform of PIP4k stimulated signaling through mammalian TORC1 (mTORC1). Knockdown of PIP4kγ reduced cell mass in cells in which mTORC1 is constitutively activated by Tsc2 deficiency. In Tsc2 null cells, mTORC1 activation was partially independent of amino acids or glucose and glutamine. PIP4kγ knockdown inhibited the nutrient-independent activation of mTORC1 in Tsc2 knockdown cells and reduced basal mTORC1 signaling in wild-type cells. PIP4kγ was phosphorylated by mTORC1 and associated with the complex. Phosphorylated PIP4kγ was enriched in light microsomal vesicles, whereas the unphosphorylated form was enriched in heavy microsomal vesicles associated with the Golgi. Furthermore, basal mTORC1 signaling was enhanced by overexpression of unphosphorylated wild-type PIP4kγ or a phosphorylation-defective mutant and decreased by overexpression of a phosphorylation-mimetic mutant. Together, these results demonstrate that PIP4kγ and mTORC1 interact in a self-regulated feedback loop to maintain low and tightly regulated mTORC1 activation during starvation.

AKT facilitates EGFR trafficking and degradation by phosphorylating and activating PIKfyve.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that controls cell proliferation, growth, survival, metabolism, and migration by activating the PI3K (phosphatidylinositol 3-kinase)-AKT and ERK (extracellular signal-regulated kinase)-RSK (ribosomal S6 kinase) pathways. EGFR signaling to these pathways is temporally and spatially regulated. Endocytic trafficking controls the access of EGFR to these downstream effectors and also its degradation, which terminates EGFR signaling. We showed that AKT facilitated the endocytic trafficking of EGFR to promote its degradation. Interfering with AKT signaling reduced both EGFR recycling and the rate of EGFR degradation. In AKT-impaired cells, EGFRs were unable to reach the cell surface or the lysosomal compartment and accumulated in the early endosomes, resulting in prolonged signaling and increased activation of ERK and RSK. Upon EGF stimulation, AKT phosphorylated and activated the kinase PIKfyve [FYVE-containing phosphatidylinositol 3-phosphate 5-kinase], which promoted vesicle trafficking to lysosomes. PIKfyve activation promoted EGFR degradation. Similar regulation occurred with platelet-derived growth factor receptor (PDGFR), suggesting that AKT phosphorylation and activation of PIKfyve is likely to be a common feedback mechanism for terminating RTK signaling and reducing receptor abundance.