Renping Zhou

Loss of ephrin-A5 function disrupts lens fiber cell packing and leads to cataract

Cell–cell interactions organize lens fiber cells into highly ordered structures to maintain transparency. However, signals regulating such interactions have not been well characterized. We report here that ephrin-A5, a ligand of the Eph receptor tyrosine kinases, plays a key role in lens fiber cell shape and cell–cell interactions. Lens fiber cells in mice lacking ephrin-A5 function appear rounded and irregular in cross-section, in contrast to their normal hexagonal appearance in WT lenses. Cataracts eventually develop in 87% of ephrin-A5 KO mice. We further demonstrate that ephrin-A5 interacts with the EphA2 receptor to regulate the adherens junction complex by enhancing recruitment of β-catenin to N-cadherin. These results indicate that the Eph receptors and their ligands are critical regulators of lens development and maintenance.

Differentiation of the midbrain dopaminergic pathways during mouse development

Dopaminergic (DA) neurons in the substantia nigra (SN) and ventral tegmental area (VTA) of the midbrain project to the dorsolateral caudate/putamen and to the ventromedially located nucleus accumbens, respectively, establishing the mesostriatal and the mesolimbic pathways. Disruptions in this system have been implicated in Parkinsons disease, drug addiction, schizophrenia, and attention deficit hyperactivity disorder. However, progress in our understanding has been hindered by a lack of knowledge of how these pathways develop. In this study, different retrograde tracers, placed into the dorsolateral caudate/putamen and the nucleus accumbens, were used to analyze the development of the dopaminergic pathways. In embryonic day 15 mouse embryos, both SN and VTA neurons, as well as their fibers, were doubly labeled by striatal injections into the dorsolateral and ventromedial striatum. However, by birth, the SN DA neurons were labeled exclusively by DiA placed in the dorsolateral striatum, and the VTA DA neurons were labeled only by DiI injected into the ventromedial striatum. These data suggest that initial projections from midbrain DA neurons target nonspecifically to both the dorsolateral striatum and the nucleus accumbens. Later during development, the separate mesostriatal and mesolimbic pathways differentiate through the selective elimination of mistargeted collaterals. J. Comp. Neurol. 476:301–311, 2004.

Mistargeting hippocampal axons by expression of a truncated Eph receptor

Topographic mapping of axon terminals is a general principle of neural architecture that underlies the interconnections among many neural structures. The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been implicated in the formation of topographic projection maps. We show that multiple Eph receptors and ligands are expressed in the hippocampus and its major subcortical projection target, the lateral septum, and that expression of a truncated Eph receptor in the mouse brain results in a pronounced alteration of the hippocamposeptal topographic map. Our observations provide strong support for a critical role of Eph family guidance factors in regulating ontogeny of hippocampal projections.

Mitogen-activated protein kinase pathway mediates effects of brain-derived neurotrophic factor on differentiation of basal forebrain oligodendrocytes

Previous studies indicate that brain‐derived neurotrophic factor (BDNF), through the mediation of the trkB receptor, modulates the expression of differentiated traits in basal forebrain (BF) oligodendrocytes (OLGs). Specifically, BDNF up‐regulates the expression of myelin basic protein (MBP), proteolipid protein (PLP), and myelin associated glycoprotein (MAG; Du et al. [ 2006 ] Mol. Cell. Neurosci. 31:366–375). However, the signaling cascades mediating the effects of BDNF have not been defined. The current study employs biochemical and molecular biological approaches to examine the involvements of the mitogen‐activated protein kinase (MAPK) pathway, the phosphatidylinositol‐3 kinase (PI3K) pathway, and the phospholipase C‐γ (PLC‐γ) pathway. Our results indicate that, in BF OLGs, BDNF activates the MAPK pathway and the PLC‐γ pathway but not the PI3K‐Akt signaling cascade. By using specific inhibitors and mutated dominant negative or constitutively active forms of MAPK kinase, we demonstrate that the MAPK pathway is mediating the effects of BDNF on expression of differentiated traits in BF OLGs.

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development

Background Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases. Methodology/Principal Findings Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca2+ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents. Conclusions/Significance These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.

Ephrins stimulate or inhibit neurite outgrowth and survival as a function of neuronal cell type

The Eph family of tyrosine kinase receptors and ligands play key roles in cell segregation and axon targeting in the developing nervous system. Interactions between the ligands and receptors cause repulsion or degeneration of receptor‐positive axons from several brain regions including the retina, hippocampus, thalamus, and midbrain dopaminergic system. We extend these previous observations by showing that three A‐ephrins also negatively regulate the growth of neurites from striatal and olfactory neurons. In addition to negative effects, however, we also report a trophic activity of the A‐ephrins: Ephrin‐A2 and A5 promote survival and neurite outgrowth of sympathetic neurons. These observations provide support to the notion that ephrins may function as either negative or positive signals in the developing nervous system. J. Neurosci. Res. 60:427–436, 2000

Increased neuroglobin levels in the cerebral cortex and serum after ischemia-reperfusion insults

Neuroglobin (NGB) is a newly discovered protein localized in neurons of the central and peripheral nervous systems in vertebrates. It functions to bind, store, and facilitate the utilization of oxygen in neuronal cells. Recent studies suggest that it may modulate hypoxic and ischemic injury. The major goal of the present study is to characterize the dynamic changes of NGB protein in the brain and serum in a global forebrain ischemia-reperfusion model using gerbils. The sensitivity and validity of serum NGB as a potential biomarker for brain injury were further evaluated. Global cerebral ischemia-reperfusion models were induced by bilateral carotid occlusion for 20 min followed with 2-, 8-, 16-, 24-, 48-, or 72-h reperfusion in forty-six Mongolian gerbils. Sham-operated and operated animals were sacrificed at the designated time after reperfusion. Brains were fixed for immunocytochemical study to evaluate the time-dependent expression of NGB, and the concentration of NGB in serum was measured by enzyme-linked immunosorbent assay. Our results showed that the expression of NGB was upregulated in the cerebral cortex but significantly downregulated in the hippocampus from 2 to 72 h of reperfusion after 20 min of bilateral common carotid arteries occlusion. The concentration of NGB in serum was significantly increased at 8 h and reached a peak at 48 h of reperfusion. There is a significant correlation between NGB levels in the serum and severity of neuronal damage in the gerbil brain. In summary, the upregulation of NGB in cerebral cortex and downregulation in hippocampus after reperfusion insults in the gerbil brain are consistent with the fact that cerebral cortex is more tolerant to hypoxic or ischemic injury than the hippocampus. Moreover, the changes of NGB levels in serum may be used to monitor the extent of brain damage in ischemic brain diseases.

Kinetic analysis of the binding of monomeric and dimeric ephrins to Eph receptors: Correlation to function in a growth cone collapse assay

Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide‐linked Fc fragments, is best described using a bivalent analyte model as a two‐step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30‐ to 6000‐fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A‐class ephrin/Eph interactions than in the B‐class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions.

Distribution of EphA5 receptor protein in the developing and adult mouse nervous system

The EphA5 receptor tyrosine kinase plays key roles in axon guidance during development. However, the presence of EphA5 protein in the nervous system has not been fully characterized. To examine EphA5 localization better, mutant mice, in which the EphA5 cytoplasmic domain was replaced with β‐galactosidase, were analyzed for both temporal and regional changes in the distribution of EphA5 protein in the developing and adult nervous system. During embryonic development, high levels of EphA5 protein were found in the retina, olfactory bulb, cerebral neocortex, hippocampus, pretectum, tectum, cranial nerve nuclei, and spinal cord. Variations in intensity were observed as development proceeded. Staining of pretectal nuclei, tectal nuclei, and other areas of the mesencephalon became more diffuse after maturity, whereas the cerebral neocortex gained more robust intensity. In the adult, receptor protein continued to be detected in many areas including the olfactory nuclei, neocortex, piriform cortex, induseum griseum, hippocampus, thalamus, amygdala, hypothalamus, and septum. In addition, EphA5 protein was found in the claustrum, stria terminalis, barrel cortex, and striatal patches, and along discrete axon tracts within the corpus callosum of the adult. We conclude that EphA5 function is not limited to the developing mouse brain and may play a role in synaptic plasticity in the adult. J. Comp. Neurol. 514:310–328, 2009.

Characterization of the receptors for axon guidance factor netrin-4 and identification of the binding domains

Netrins are a family of secreted protein related to laminin and act as tropic cues directing axon growth and cell migration during neural development. Netrin-4 is a novel member of netrin family recently identified in the vertebrate with neuritis elongation promoting activity; however, the receptors for netrin-4 are still unknown. To better understand the function and signal transduction pathway of netrin-4, the potential receptors for netrin-4 were studied in this paper. The netrin-4 protein was prepared by introducing a eukaryotic expression vector with a secretable alkaline phosphatase tag (AP4) into COS7 cells to allow the expression of AP4-netrin4 fusion protein. Axon guidance activity of netrin-4 was confirmed by using the cortical explants. After incubation with cultured primary cortical neurons, the neurons were distinctly labeled by the AP4-coupled netrin-4 ligands. In contrast, the binding activity of AP4-netrin4 to neurons could be completely competed by the exogenously expressed netrin-4 protein without AP4 tag, indicating specificity of netrin-4 binding to the potential receptors. Moreover, netrin-4 could also bind to CHO cells transfected with the plasmids expressing two known receptors for netrin-1, Deleted in Colorectal Cancer (DCC) and UNC5 homolog 1 (UNC5H1) respectively. As there are three domains in netrin-4, we further tried to narrow down the region containing binding sites with the receptors. Interestingly, only the N-terminal domain (LNT) could bind to DCC and UNC5H1. A further ligand-receptor binding analysis showed that both the N- and the C-terminal domain (NCT) but not the EGF-like (EGFL) domain of netrin-4 could bind to the surface of cultured primary neurons, indicating the existence of novel receptors for netrin-4. After competed by netrin-4, we confirmed that the binding of AP tagged netrin-4 domains to neurons were also netrin-4 dependent. The binding activity of the N-terminal domain of netrin-4 is about 3-fold higher than that for the C-terminal domain. In summary, our data here indicated that the two known receptors for netrin-1, DCC and UNC5H1, are also receptors for netrin-4, while only LNT but not EGFL and NCT is the key domain for specific binding. In addition, there are novel receptors for netrin-4, where both LNT and NCT but not EGFL are key domains for binding.