Purpose Phosphatidylinositol-3-phosphate (PI(3)P), and Vps34, the type III phosphatidylinositol 3-kinase primarily responsible for its production, are important for function and survival of sensory neurons, where they have key roles in membrane processing events, such as autophagy, endosome processing, and fusion of membranes bearing ubiquitinated cargos with lysosomes. We examined their roles in the most abundant class of secondary neurons in the vertebrate retina, the ON-bipolar cells (ON-BCs). Methods A conditional Vps34 knockout mouse line was generated by crossing Vps34 floxed mice with transgenic mice expressing Cre recombinase in ON-BCs. Structural changes in the retina were determined by immunofluorescence and electron microscopy, and bipolar cell function was determined by electroretinography. Results Vps34 deletion led to selective death of ON-BCs, a thinning of the inner nuclear layer, and a progressive decline of electroretinogram b-wave amplitudes. There was no evidence for loss of other retinal neurons, or disruption of rod-horizontal cell contacts in the outer plexiform layer. Loss of Vps34 led to aberrant accumulation of membranes positive for autophagy markers LC3, p62, and ubiquitin, accumulation of endosomal membranes positive for Rab7, and accumulation of lysosomes. Similar effects were observed in Purkinje cells of the cerebellum, leading to severe and progressive ataxia. Conclusions These results support an essential role for PI(3)P in fusion of autophagosomes with lysosomes and in late endosome maturation. The cell death resulting from Vps34 knockout suggests that these processes are essential for the health of ON-BCs.

The P23H mutation in rhodopsin (Rho), the rod visual pigment, is the most common allele associated with autosomal dominant retinitis pigmentosa (adRP). The fate of misfolded mutant Rho in rod photoreceptors has not yet been elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized, but mutant P23H-Rho protein was mislocalized in the inner segments. Heterozygotes exhibited slowly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases. mRNA levels for both the mutant human rhodopsin allele and the WT mouse rhodopsin levels were reduced, but protein levels revealed selective degradation of the mutant protein. The results suggest the mutant rods undergo an adaptative process that prolongs survival despite unfolded protein accumulation in the ER. The P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.

Although protein kinases and phosphatases participate in integrin αIIbβ3 signaling, whether integrin functions are regulated by the catalytic subunit of protein phosphatase 1 (PP1c) isoforms are unclear. We show that siRNA mediated knockdown of all PP1c isoforms (α, β and γ1) in 293 αIIbβ3 cells decreased adhesion to immobilized fibrinogen and fibrin clot retraction. Selective knockdown of only PP1cγ1 did not alter adhesion or clot retraction, while depletion of PP1cβ decreased both functions. Unexpectedly, knockdown of PP1cα enhanced αIIbβ3 adhesion to fibrinogen and clot retraction. Protein interaction studies revealed that all PP1c isoforms can interact with the integrin αIIb subunit. Phosphoprofiling studies revealed an enhanced activation of mitogen-activated protein kinase (MAPK) p38 in the PP1cα depleted cells. Enhanced adhesive phenotype displayed by the PP1cα depleted 293 αIIbβ3 cells was blocked by pharmacological inhibition of p38. Conversely, the decreased adhesion of PP1cα overexpressing cells was rescued by the expression of constitutively active p38α or p38γ. Thus, PP1c isoforms have distinct contribution to the outside-in αIIbβ3 signaling-dependent functions in 293 αIIbβ3 cells. Moreover, PP1cα negatively regulates integrin function by suppressing the p38 pathway.

190 Signal transduction mediated by the kinases and phosphatases are critical for platelet activation at the site of vascular injury. Compared to the kinases, a role for phosphatases in platelet activation is less understood. Our previous studies have focused on the roles of serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) in regulating integrin αIIbβ3functions. However, platelets also express protein phosphatase 2B (PP2B) and its role in platelet function is unexplored. PP2B-Aα and PP2B-Aβ constitute two ubiquitous isoforms of the PP2B catalytic subunit. Due to the general concerns regarding the specificity of the PP2B inhibitors, we have utilized mice deficient in the β isoform of the catalytic subunit of PP2B (PP2B-Aβ) to explore the role of PP2B in platelet functions. Mice lacking PP2B-Aα are short lived and are not considered in this study. Loss of PP2B-Aβ did not cause any compensatory increase in the PP2B-Aα levels in platelets. Compared to the wild type (WT) platelets, PP2B-Aβ−/− platelets displayed increased aggregation in response to low doses of protease-activated receptor 4-activating peptide (PAR4-AP), ADP, collagen and collagen related peptide (CRP). Enhanced α granule secretion in response to the low doses of PAR4-AP and CRP was noticed in PP2B-Aβ−/− platelets, relative to the WT platelets. Functions regulated by the outside-in αIIbβ3 integrin signaling like adhesion to immobilized fibrinogen and fibrin clot retraction were enhanced in the PP2B-Aβ−/− platelets. These studies indicate that PP2B-Aβ negatively regulate platelet functions in vitro. Consistent with these observations, PP2B-Aβ−/− mice exhibited a shorter tail bleeding time compared to the WT mice. In a FeCl3 induced endothelial denudation injury model, PP2B-Aβ−/− mice showed decreased time to occlusion in the carotid artery, and reduced number of emboli compared to the WT mice. These studies indicate that PP2B-Aβ suppress multiple murine platelet functions that contribute to an occlusive thrombi. Unlike a positive thrombus promoting role for the PP1cγ that was noticed in our previous study, PP2B-Aβ suppressed murine platelet activation, suggesting that different subtypes of Ser/Thr phosphatases have distinct roles in murine platelet activation. Disclosures: No relevant conflicts of interest to declare.

The P23H mutation in rhodopsin (Rho), the visual pigment protein in rod photoreceptor neurons, is the most common genetic cause of autosomal dominant retinitis pigmentosa (adRP), a retinal disease that causes blindness. Despite multiple studies in animal models, the subcellular details of the fate of misfolded mutant Rho in rod photoreceptors have not been completely defined. We generated a new mouse model of adRP, in which the P23H-Rho mutant allele is fused to Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized there, but mutant P23H-Rho protein was specifically mislocalized in the inner segments of rods. Despite this cellular phenotype, the P23HhRhoRFP heterozygous mice exhibited only slowly progressing retinal degeneration; in ERG recordings, scotopic a-wave amplitudes were reduced by 24% and 26% at 30 days and 90 days respectively, and the corresponding scotopic b-waves by 18% and 24%. Outer nuclear layer thickness was still 80% of WT at 90 days, but at 364 days had declined to 40% of WT. Transmission electron microscopy revealed greatly expanded membrane lamellae in the inner segment, and by fluorescence imaging, we determined that the mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. TUNEL staining revealed a slow pace of cell death involving chromosomal endonucleolytic degradation. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases in levels of messages encoding the proteins BiP, CHOP, ATF6, XBP1, PERK, Eif2α and Derlin-1, but a decreased level of total Rhodopsin (mouse + human) mRNA levels. The decline in the rate of cell death after an initial burst suggests that P23HhRhoRFP mutant rods undergo an adaptative process that prolongs survival despite gross P23HhRhoRFP protein accumulation in the ER. Because of its slowly progressing nature, and easy visualization of the mutant protein, the P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.