Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence.

Purpose: To undertake the first ultrastructural characterization of human retinal pigment epithelial (RPE) differentiation from fetal development to adolescence. Methods: Ten fetal eyes and three eyes aged six, nine, and 17 years were examined in the temporal retina adjacent to the optic nerve head by transmission electron microscopy. The area, number, and distribution of RPE organelles were quantified and interpreted within the context of adjacent photoreceptors, Bruch's membrane, and choriocapillaris maturation. Results: Between eight to 12 weeks' gestation (WG), pseudostratified columnar epithelia with apical tight junctions differentiate to a simple cuboidal epithelium with random distribution of melanosomes and mitochondria. Between 12 to 26 WG, cells enlarge and show long apical microvilli and apicolateral junctional complexes. Coinciding with eye opening at 26 WG, melanosomes migrate apically whereas mitochondria distribute to perinuclear regions, with the first appearance of phagosomes, complex granules, and basolateral extracellular space (BES) formation. Significantly, autophagy and heterophagy, as evidenced by organelle recycling, and the gold standard of ultrastructural evidence for autophagy of double-membrane autophagosomes and mitophagosomes were evident from 32 WG, followed by basal infoldings of RPE cell membrane at 36 WG. Lipofuscin formation and deposition into the BES evident at six years increased at 17 years. Conclusions: We provide compelling ultrastructural evidence that heterophagy and autophagy begins in the third trimester of human fetal development and that deposition of cellular byproducts into the extracellular space of RPE takes place via exocytosis. Transplanted RPE cells must also demonstrate the capacity to subserve autophagic and heterophagic functions for effective disease mitigation.

Glial, Neuronal, Vascular, Retinal Pigment Epithelium, and Inflammatory Cell Damage in a New Western Diet-Induced Primate Model of Diabetic Retinopathy.

This study investigated retinal changes in a Western diet (WD)-induced nonhuman primate model of type 2 diabetes. Rhesus nonhuman primates, aged 15 to 17 years, were fed a high-fat diet (n = 7) for >5 years reflective of the traditional WD. Age-matched controls (n = 6) were fed a standard laboratory primate diet. Retinal fundus photography, optical coherence tomography, autofluorescence imaging, and fluorescein angiography were performed before euthanasia. To assess diabetic retinopathy (DR), eyes were examined using trypsin digests, lipofuscin autofluorescence, and multimarker immunofluorescence on cross-sections and whole mounts. Retinal imaging showed venous engorgement and tortuosity, aneurysms, macular exudates, dot and blot hemorrhages, and a marked increase in fundus autofluorescence. Post-mortem changes included the following: decreased CD31 blood vessel density (P < 0.05); increased acellular capillaries (P < 0.05); increased density of ionized calcium-binding adaptor molecule expressing amoeboid microglia/macrophage; loss of regular distribution in stratum and spacing typical of ramified microglia; and increased immunoreactivity of aquaporin 4 and glial fibrillary acidic protein (P < 0.05). However, rhodopsin immunoreactivity (P < 0.05) in rods and neuronal nuclei antibody-positive neuronal density of 50% (P < 0.05) were decreased. This is the first report of a primate model of DR solely induced by a WD that replicates key features of human DR.

Novel morphometric analysis of higher order structure of human radial peri-papillary capillaries: relevance to retinal perfusion efficiency and age.

We apply novel analyses to images of superficial capillaries that are located near and around the optic disc of the human retina: the radial peri-papillary capillaries (RPCs). Due to their unique perfusion of the nerve fibre layer the RPCs are particularly significant for optic-neuropathies. The inputs to the analysis were z-stacks from 3D confocal fluorescence microscopy from 62 human retinas aged 9 to 84 years. Our aim was to find morphometric correlates of age. The retinas had no ophthalmic history. The analysis was undertaken in two stages: (1) converting the z-stacks to 3D tubular networks of vessels, and (2) characterizing the tubular networks using features derived from the Minkowski functionals (MFs). The MFs measure: the capillary volume, surface area, mean breadth, and Euler number. The mean breadth is related to tortuosity, wall shear stress and resistance to flow, and the Euler number is related to the density of loops (collaterals). Features derived from the surface area, mean breadth and Euler number were most related to age (all p ≤ 0.006). The results indicate the importance of pressure-equalizing loops and tortuosity as quantitative measures related to perfusion efficiency. The novel morphometric analysis could quantify disease-related accelerated aging and vessel malformation.

P2X7 receptor signaling during adult hippocampal neurogenesis.

Neurogenesis is a persistent and essential feature of the adult mammalian hippocampus. Granular neurons generated from resident pools of stem or progenitor cells provide a mechanism for the formation and consolidation of new memories. Regulation of hippocampal neurogenesis is complex and multifaceted, and numerous signaling pathways converge to modulate cell proliferation, apoptosis, and clearance of cellular debris, as well as synaptic integration of newborn immature neurons. The expression of functional P2X7 receptors in the central nervous system has attracted much interest and the regulatory role of this purinergic receptor during adult neurogenesis has only recently begun to be explored. P2X7 receptors are exceptionally versatile: in their canonical role they act as adenosine triphosphate-gated calcium channels and facilitate calcium-signaling cascades exerting control over the cell via calcium-encoded sensory proteins and transcription factor activation. P2X7 also mediates transmembrane pore formation to regulate cytokine release and facilitate extracellular communication, and when persistently stimulated by high extracellular adenosine triphosphate levels large P2X7 pores form, which induce apoptotic cell death through cytosolic ion dysregulation. Lastly, as a scavenger receptor P2X7 directly facilitates phagocytosis of the cellular debris that arises during neurogenesis, as well as during some disease states. Understanding how P2X7 receptors regulate the physiology of stem and progenitor cells in the adult hippocampus is an important step towards developing useful therapeutic models for regenerative medicine. This review considers the relevant aspects of adult hippocampal neurogenesis and explores how P2X7 receptor activity may influence the molecular physiology of the hippocampus, and neural stem and progenitor cells.

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models.

AIMS/HYPOTHESIS: A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy. METHODS: CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-ß) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line. RESULTS: CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B). CONCLUSIONS/INTERPRETATION: CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.

P2X7 Receptors Regulate Phagocytosis and Proliferation in Adult Hippocampal and SVZ Neural Progenitor Cells: Implications for Inflammation in Neurogenesis.

Identifying the signaling mechanisms that regulate adult neurogenesis is essential to understanding how the brain may respond to neuro-inflammatory events. P2X7 receptors can regulate pro-inflammatory responses, and in addition to their role as cation channels they can trigger cell death and mediate phagocytosis. How P2X7 receptors may regulate adult neurogenesis is currently unclear. Here, neural progenitor cells (NPCs) derived from adult murine hippocampal subgranular (SGZ) and cerebral subventricular (SVZ) zones were utilized to characterize the roles of P2X7 in adult neurogenesis, and assess the effects of high extracellular ATP, characteristic of inflammation, on NPCs. Immunocytochemistry found NPCs in vivo and in vitro expressed P2X7, and the activity of P2X7 in culture was demonstrated using calcium influx and pore formation assays. Live cell and confocal microscopy, in conjunction with flow cytometry, revealed P2X7+ NPCs were able to phagocytose fluorescent beads, and this was inhibited by ATP, indicative of P2X7 involvement. Furthermore, P2X7 receptors were activated with ATP or BzATP, and 5-ethynyl-2'-deoxyuridine (EdU) used to observe a dose-dependent decrease in NPC proliferation. A role for P2X7 in decreased NPC proliferation was confirmed using chemical inhibition and NPCs from P2X7-/- mice. Together, these data present three distinct roles for P2X7 during adult neurogenesis, depending on extracellular ATP concentrations: (a) P2X7 receptors can form transmembrane pores leading to cell death, (b) P2X7 receptors can regulate rates of proliferation, likely via calcium signaling, and (c) P2X7 can function as scavenger receptors in the absence of ATP, allowing NPCs to phagocytose apoptotic NPCs during neurogenesis. Stem Cells 2018;36:1764-1777.

Pathophysiology, screening and treatment of ROP: A multi-disciplinary perspective.

The population of infants at risk for retinopathy of prematurity (ROP) varies by world region; in countries with well developed neonatal intensive care services, the highest risk infants are those born at less than 28 weeks gestational age (GA) and less than 1 kg at birth, while, in regions where many aspects of neonatal intensive and ophthalmological care are not routinely available, more mature infants up to 2000 g at birth and 37 weeks GA are also at risk for severe ROP. Treatment options for both groups of patients include standard retinal laser photocoagulation or, more recently, intravitreal anti-VEGF drugs. In addition to detection and treatment of ROP, this review highlights new opportunities created by telemedicine, where screening and diagnosis of ROP in remote locations can be undertaken by non-ophthalmologists using digital fundus cameras. The ophthalmological care of the ROP infant is undertaken in the wider context of neonatal care and general wellbeing of the infant. Because of this context, this review takes a multi-disciplinary perspective with contributions from retinal vascular biologists, pediatric ophthalmologists, an epidemiologist and a neonatologist. This review highlights the latest insights regarding cellular and molecular mechanisms in the formation of the retinal vasculature in the human infant, pathogenesis of ROP, detection and treatment of severe ROP, the risks and benefits of anti-VEGF therapy, the identification of new therapies over the horizon, and the optimal neonatal care regimen for best ROP outcomes, and the benefits and pitfalls of telemedicine in the remote screening and diagnosis of ROP, all of which have the potential to improve ROP outcomes.

Increased Indoleamine 2,3-Dioxygenase and Quinolinic Acid Expression in Microglia and Müller Cells of Diabetic Human and Rodent Retina.

Purpose: We investigated the relationship between inflammation, neuronal loss, and expression of indoleamine 2, 3-dioxygenase (IDO) and quinolinic acid (QUIN) in the retina of subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) and in the retina of rats with T1D. Methods: Retinas from T1D (n = 7), T2D (n = 13), and 20 age-matched nondiabetic human donors and from T1D (n = 3) and control rats (n = 3) were examined using immunohistochemistry for IDO, QUIN, cluster of differentiation 39 (CD39), ionized calcium-binding adaptor molecule (Iba-1, for macrophages and microglia), Vimentin (VIM; for Müller cells), neuronal nuclei (NeuN; for neurons), and UEA1 lectin (for blood vessels). Results: Based on morphologic criteria, CD39+/ionized calcium binding adaptor molecule 1(Iba-1+) resident microglia and CD39-/Iba-1+ bone marrow-derived macrophages were present at higher density in T1D (13% increase) and T2D (26% increase) human retinas when compared with controls. The density and brightness of IDO+ microglia were increased in both T1D and T2D human retinas. The intensity of QUIN+ expression on CD39+ microglia and VIM+ Müller cells was greatly increased in both human T1D and T2D retinas. T1D retinas showed a 63% loss of NeuN+ neurons and T2D retinas lost approximately 43% when compared with nondiabetic human retinas. Few QUIN+ microglia-like cells were seen in nondiabetic retinas, but the numbers increased 18-fold in T1D and 7-fold in T2D in the central retina. In T1D rat retinas, the density of IDO+ microglia increased 2.8-fold and brightness increased 2.1-fold when compared with controls. Conclusions: Our findings suggest that IDO and QUIN expression in the retinas of diabetic rats and humans could contribute to the neuronal degeneration that is characteristic of diabetic retinopathy.

Insulin-like growth factor binding protein-3 links obesity and breast cancer progression.

Obesity is associated epidemiologically with poor breast cancer prognosis, but the mechanisms remain unclear. Since IGF binding protein-3 (IGFBP-3) influences both breast cancer growth and adipocyte maturation, it may impact on how obesity promotes breast oncogenesis. This study investigated the role of endogenous IGFBP-3 on the development of obesity and subsequently on breast tumor growth. Wild-type (WT) C57BL/6 or IGFBP-3-null (BP3KO) mice were fed a high-fat diet (HFD) or control chow-diet for 15 weeks before orthotopic injection with syngeneic EO771 murine breast cancer cells. When the largest tumor reached 1000 mm3, tissues and tumors were excised for analysis. Compared to WT, BP3KO mice showed significantly reduced weight gain and mammary fat pad mass (contralateral to tumor) in response to HFD, despite similar food intake. EO771 tumor weight and volume were increased by HFD and decreased by BP3KO. Despite differences in tumor size, tumors in BP3KO mice showed no differences from WT in the number of mitotically active (Ki67+) and apoptotic (cleaved caspase-3+) cells, but had greater infiltration of CD3+ T-cells. These data suggest that endogenous (circulating and/or stromal) IGFBP-3 is stimulatory to adipose tissue expansion and enhances mammary tumor growth in immune-competent mice, potentially by suppressing T-cell infiltration into tumors.

The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome.

Copy number variations to chromosome 21 (HSA21) cause intellectual disability and Down Syndrome, but our understanding of the HSA21 genetic factors which contribute to fetal brain development remains incomplete. Here, we focussed on the neurodevelopmental functions for EURL (also known as C21ORF91, Refseq Gene ID:54149), a protein-coding gene at the centromeric boundary of the Down Syndrome Critical Region (DSCR) of HSA21. We report that EURL is expressed during human and mouse cerebral cortex development, and we report that alterations to EURL mRNA levels within the human brain underlie Down Syndrome. Our gene perturbation studies in mice demonstrate that disruptions to Eurl impair progenitor proliferation and neuronal differentiation. Also, we find that disruptions to Eurl impair the long-term positioning and dendritic spine densities of cortical projection neurons. We provide evidence that EURL interacts with the coiled-coil domain-containing protein CCDC85B so as to modulate ß-catenin levels in cells. Further, we utilised a fluorescent reporter (8xTOPFLASHd2EGFP) to demonstrate that disruptions to Eurl alter ß-catenin signalling in vitro as well as in vivo. Together, these studies highlight EURL as an important new player in neuronal development that is likely to impact on the neuropathogenesis of HSA21-related disorders including Down Syndrome.

Differential expression of sirtuins in the aging rat brain.

Although there are seven mammalian sirtuins (SIRT1-7), little is known about their expression in the aging brain. To characterize the change(s) in mRNA and protein expression of SIRT1-7 and their associated proteins in the brain of "physiologically" aged Wistar rats. We tested mRNA and protein expression levels of rat SIRT1-7, and the levels of associated proteins in the brain using RT-PCR and western blotting. Our data shows that SIRT1 expression increases with age, concurrently with increased acetylated p53 levels in all brain regions investigated. SIRT2 and FOXO3a protein levels increased only in the occipital lobe. SIRT3-5 expression declined significantly in the hippocampus and frontal lobe, associated with increases in superoxide and fatty acid oxidation levels, and acetylated CPS-1 protein expression, and a reduction in MnSOD level. While SIRT6 expression declines significantly with age acetylated H3K9 protein expression is increased throughout the brain. SIRT7 and Pol I protein expression increased in the frontal lobe. This study identifies previously unknown roles for sirtuins in regulating cellular homeostasis and healthy aging.

Loss of survival factors and activation of inflammatory cascades in brain sympathetic centers in type 1 diabetic mice.

Neuroinflammation and neurodegeneration have been observed in the brain in type 1 diabetes (T1D). However, little is known about the mediators of these effects. In T1D mice with 12- and 35-wk duration of diabetes we examined two mechanisms of neurodegeneration, loss of the neuroprotective factors insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) and changes in indoleamine 2,3-dioxygenase (IDO) expression in the brain, and compared the response to age-matched controls. Furthermore, levels of matrix metalloproteinase-2 (MMP-2), nucleoside triphosphate diphosphohydrolase-1 (CD39), and ionized calcium-binding adaptor molecule 1 (Iba-1) were utilized to assess inflammatory changes in astrocytes, microglia, and blood vessels. In the diabetic hypothalamus (HYPO), we observed 20% reduction in neuronal soma diameter (P<0.05) and reduced neuronal expression of IGFBP-3 (-32%, P<0.05) and IGF-I (-15%, P<0.05) compared with controls at 35 wk. In diabetic HYPO, MMP-2 expression was increased in astrocytes (46%, P<0.01), and IDO⁺ cell density rose by (62%, P<0.05). CD39 expression dropped by 30% (P<0.05) in microglia and blood vessels. With 10 wk of systemic treatment using minocycline, an anti-inflammatory agent that crosses the blood-brain barrier, MMP-2, IDO, and CD39 levels normalized (P<0.05). Our results suggest that increased IDO and early loss of CD39⁺ protective cells lead to activation of inflammation in sympathetic centers of the CNS. As a downstream effect, the loss of the neuronal survival factors IGFBP-3 and IGF-I and the neurotoxic products of the kynurenine pathway contribute to the loss of neuronal density observed in the HYPO in T1D.

Evidence for lymphatics in the developing and adult human choroid.

PURPOSE: Lymphatics subserve many important functions in the human body including maintenance of fluid homeostasis, immune surveillance, and tumor metastasis. Our aim was to provide structural and phenotypic evidence of lymphatic-like structures in the human choroid, including details of its development. METHODS: Using multiple-marker immunohistochemistry (IHC), choroids from human fetal eyes (8-26 weeks gestation) and adults (17-74 years) were examined with lymphatic- and vascular-specific markers: prospero homeobox-1 (PROX-1), lymphatic vascular endothelium receptor-1 (LYVE-1), podoplanin, D2-40, endomucin, VEGF-C, vascular endothelial growth factor receptor-3 (VEGFR-3 or Flt4), UEA lectin, platelet endothelial cell adhesion molecule-1 (PECAM-1), CD34, and CD39. Transmission electron microscopy (TEM) was used to establish evidence for choroidal lymphatics, and to provide details of stratification and relative frequency of lymphatics compared to choroidal blood vessels. RESULTS: Immunohistochemistry and TEM indicated a central-to-peripheral topography of lymphatic formation, with numerous blind-ended lymph sacs just external to the choriocapillaris, as well as the presence of infrequent precollector and collector lymphatic channels. Characteristic ultrastructural features of lymphatics in adult human choroid included anchoring filaments, luminal flocculent protein but absence of erythrocytes, fragmented and/or absent basal lamina, absence of intracellular Weibel-Palade bodies, infrequent pericyte ensheathment, and lack of fenestrae. CONCLUSIONS: The system of blind-ended initial lymphatic segments seen just external to the fenestrated vessels of the choriocapillaris is ideally placed for recirculating extracellular fluid and strategically placed for immune surveillance. The presence of a system of lymphatic-like channels in the human choroid provides an anatomical basis for antigen presentation in the posterior eye, with a possible route from the eye to the sentinel lymph nodes, similar to that already described for anterior eye lymphatics.

P2X7 receptors mediate innate phagocytosis by human neural precursor cells and neuroblasts.

During early human neurogenesis there is overproduction of neuroblasts and neurons accompanied by widespread programmed cell death (PCD). While it is understood that CD68(+) microglia and astrocytes mediate phagocytosis during target-dependent PCD, little is known of the cell identity or the scavenger molecules used to remove apoptotic corpses during the earliest stages of human neurogenesis. Using a combination of multiple-marker immunohistochemical staining, functional blocking antibodies and antagonists, we showed that human neural precursor cells (hNPCs) and neuroblasts express functional P2X7 receptors. Furthermore, using live-cell imaging, flow cytometry, phagocytic assays, and siRNA knockdown, we showed that in a serum-free environment, doublecortin(+) (DCX) neuroblasts and hNPCs can clear apoptotic cells by innate phagocytosis mediated via P2X7. We found that both P2X7(high) DCX(low) hNPCs and P2X7(high) DCX(high) neuroblasts, derived from primary cultures of human fetal telencephalon, phagocytosed targets including latex beads, apoptotic ReNcells, and apoptotic hNPC/neuroblasts. Pretreatment of neuroblasts and hNPCs with 1 mM adenosine triphosphate (ATP), 100 µM OxATP (P2X7 antagonist), or siRNA knockdown of P2X7 inhibited phagocytosis of these targets. Our results show that P2X7 functions as a scavenger receptor under serum-free conditions resembling those in early neurogenesis. This is the first demonstration that hNPCs and neuroblasts may participate in clearance of apoptotic corpses during pre target-dependent neurogenesis and mediate phagocytosis using P2X7 as a scavenger receptor.

Ultrasmall superparamagnetic iron oxide nanoparticle prelabelling of human neural precursor cells.

Stem cells prelabelled with iron oxide nanoparticles can be visualised using magnetic resonance imaging (MRI). This technique allows for noninvasive long-term monitoring of migration, integration and stem cell fate following transplantation into living animals. In order to determine biocompatibility, the present study investigated the biological impact of introducing ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) into primary human fetal neural precursor cells (hNPCs) in vitro. USPIOs with a mean diameter of 10-15 nm maghemite iron oxide core were sterically stabilised by 95% methoxy-poly(ethylene glycol) (MPEG) and either 5% cationic (NH2) end-functionalised, or 5% Rhodamine B end-functionalised, polyacrylamide. The stabilising polymer diblocks were synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation. Upon loading, cellular viability, total iron capacity, differentiation, average distance of migration and changes in intracellular calcium ion concentration were measured to determine optimal loading conditions. Taken together we demonstrate that prelabelling of hNPCs with USPIOs has no significant detrimental effect on cell biology and that USPIOs, when utilised at an optimised dosage, are an effective means of noninvasively tracking prelabelled hNPCs.

Mapping NAD(+) metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence.

Over the last decade, the importance of NAD(+) has expanded beyond its role as an essential cofactor for energy metabolism. NAD(+) has emerged as a major signalling molecule that serves as the sole substrate for several enzymatic reactions including the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), NAD-dependent protein deacetylases or CD38, and transcriptional factors by a new class of histone deacetylases known as sirtuins. NAD(+) levels are regulated by the metabolic status and cellular stress caused by oxidative stress and DNA damage. Since a detailed study of NAD(+) metabolism in the healthy ageing mammalian brain is nascent, we examined the effect of ageing on intracellular NAD(+) metabolism in different brain regions in female Wistar rats in young (3 months), middle aged (12 months) and older adults (24 months). Our results are the first to show a significant decline in intracellular NAD(+) levels and NAD:NADH ratio with ageing in the CNS, occurring in parallel to an increase in lipid peroxidation and protein oxidation (o- and m-tyrosine) and a decline in total antioxidant capacity. Hyperphosphorylation of H2AX levels was also observed together with increased PARP-1 and PARP-2 expression, and CD38 activity, concomitantly with reduced NAD(+) and ATP levels and SIRT1 function in the cortex, brainstem, hippocampus and cerebellum. Reduced activity of mitochondrial complex I-IV and impaired maximum mitochondrial respiration rate were also observed in the ageing rat brain. Among the multiple physiological pathways associated with NAD(+) catabolism, our discovery of CD38 as the major regulator of cellular NAD(+) levels in rat neurons indicates that CD38 is a promising therapeutic target for the treatment of age-related neurodegenerative diseases.

CNS inflammation and bone marrow neuropathy in type 1 diabetes.

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.

Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.