Metabolic Deregulation of the Blood-Outer Retinal Barrier in Retinitis Pigmentosa

Retinitis pigmentosa (RP) initiates with diminished rod photoreceptor function, causing peripheral and nighttime vision loss. However, subsequent loss of cone function and high-resolution daylight and color vision is most debilitating. Visual pigment-rich photoreceptor outer segments (OS) undergo phagocytosis by the retinal pigment epithelium (RPE), and the RPE also acts as a blood-outer retinal barrier transporting nutrients, including glucose, to photoreceptors. We provide evidence that contact between externalized phosphatidylserine (PS) on OS tips and apical RPE receptors activates Akt, linking phagocytosis with glucose transport to photoreceptors for new OS synthesis. As abundant mutant rod OS tips shorten in RP, Akt activation is lost, and onset of glucose metabolism in the RPE and diminished glucose transport combine to cause photoreceptor starvation and accompanying retinal metabolome changes. Subretinal injection of OS tip mimetics displaying PS restores Akt activation, glucose transport, and cone function in end-stage RP after rods are lost

Defective pituitary function and gamete interactions in beta-1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action. In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions

Systemic autoimmunity in TAM triple knockout mice causes inflammatory brain damage and cell death.

The Tyro3, Axl and Mertk (TAM) triply knockout (TKO) mice exhibit systemic autoimmune diseases, with characteristics of increased proinflammatory cytokine production, autoantibody deposition and autoreactive lymphocyte infiltration into a variety of tissues. Here we show that TKO mice produce high level of serum TNF-α and specific autoantibodies deposited onto brain blood vessels. The brain-blood barrier (BBB) in mutant brains exhibited increased permeability for Evans blue and fluorescent-dextran, suggesting a breakdown of the BBB in the mutant brains. Impaired BBB integrity facilitated autoreactive T cells infiltrating into all regions of the mutant brains. Brain autoimmune disorder caused accumulation of the ubiquitin-reactive aggregates in the mutant hippocampus, and early formation of autofluorescent lipofuscins in the neurons throughout the entire brains. Chronic neuroinflammation caused damage of the hippocampal mossy fibers and neuronal apoptotic death. This study shows that chronic systemic inflammation and autoimmune disorders in the TKO mice cause neuronal damage and death

TAM receptor deficiency affects adult hippocampal neurogenesis

182 hlm. : - ; 21 cm

T lymphocytes invade into TKO brain.

<p>(A, B) The WT (A) and TKO (B) mice at 7 weeks of age were prepared for brain sections, which were immunostained with anti-CD3 antibody to show T cell infiltration. (C) The brain section from the TKO mouse at age of 10 month old was stained with hematoxylin and eosin (H&E). Scale bars, 50 µm. (D) Brain diagram shows the regions of figures A–C. (E, F) Flow cytometric analysis of infiltrated TCRαβ-positive cells in TKO brains. Cell preparation and flow cytometry procedures were described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064812#s2" target="_blank">Methods and Materials</a>. There are increased TCRαβ-positive cells in the TKO brain (E, 5.6% of leukocytes) than the WT brain (F, 0.8% of leukocytes). This is one representative for each genotype, n = 3.</p

TKO mice produce increased proinflammatory cytokines.

<p>(A) TNF-α level in the WT and TKO mouse serum were measured by Ready-set-go ELISA kits (eBiosciences). Data are shown as means±SD, n = 9, p = 0.0002. (B) Thioglycollate-induced peritoneal macrophages (MФ) were treated with LPS for 2, 3, 4 and 5 hrs. TNF-α released into medium was measured as above. Data are shown as means ±SD for five wells per group in a single experiment and are representative of those in three experiments. N = 3, **P<0.001. (C) Real-time qPCR quantification of IL-1β and IL-6 mRNA in the lymph nodes. The total RNA was extracted by TRIzol and reversely transcribed using transcribed using qScript™ cDNA Supermix kit (Quanta Biosciences, MD). Real time qPCR was performed to measure the relative mRNA level of IL-1β and IL-6 genes in the WT (open) and TKO (solid) L.N. Data are shown as means ± SD, n = 3, *P<0.05, **P<0.002. Statistics was performed by the one-way ANOVA test using ProStat Ver 5.5.</p