Xiaohong Zhou

Involvement of Insulin/Phosphoinositide 3-Kinase/Akt Signal Pathway in 17β-Estradiol-mediated Neuroprotection

In the present study, we tested the hypothesis that 17β-estradiol (βE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H2O2)-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both βE2 and 17α-estradiol (αE2) significantly protected against H2O2-induced retinal neuron degeneration; however, progesterone had no effect. βE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [32γATP] were used as substrate. Phospho-Akt levels were also transiently increased by βE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of βE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of βE2, suggesting that βE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that βE2 and αE2, but not progesterone, identified phosphorylated insulin receptor β-subunit (IRβ) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRβ activation of PI3K. Systemic administration of βE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of βE2 activated retinal IRβ, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.

Nanoceria Inhibit the Development and Promote the Regression of Pathologic Retinal Neovascularization in the Vldlr Knockout Mouse

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR.

Nanoceria extend photoreceptor cell lifespan in tubby mice by modulation of apoptosis/survival signaling pathways

Cerium oxide nanoparticles, nanoceria, are inorganic antioxidants that have catalytic activities which mimic those of the neuroprotective enzymes superoxide dismutase and catalase. We have previously shown that nanoceria preserve retinal morphology and prevent loss of retinal function in a rat light damage model. In this study, the homozygous tubby mutant mouse, which exhibits inherited early progressive cochlear and retinal degeneration, was used as a model to test the ability of nanoceria to slow the progression of retinal degeneration. Tubby mice were injected systemically, intracardially, with 20 μl of 1mM nanoceria in saline, at postnatal day 10 and subsequently at P20 and P30 whereas saline injected and uninjected wild type (or heterozygous tubby) served as injected and uninjected controls, respectively. Assays for retinal function, morphology and signaling pathway gene expression were performed on P34 mice. Our data demonstrate that nanoceria protect the retina by decreasing Reactive Oxygen Species (ROS), up-regulating the expression of neuroprotection-associated genes; down-regulating apoptosis signaling pathways and/or up-regulating survival signaling pathways to slow photoreceptor degeneration. These data suggest that nanoceria have significant potential as global agents for therapeutic treatment of inherited retinal degeneration and most types of ocular diseases.

Delay of photoreceptor degeneration in tubby mouse by sulforaphane

In this study, the homozygous tubby (tub/tub) mutant mouse, with an early progressive hearing loss and photoreceptor degeneration, was used as a model system to examine the effects of systemic administration of a naturally occurring isothiocyanate, sulforaphane (SF), on photoreceptor degeneration. Several novel observations have been made: (i) the mRNA and protein expression of thioredoxin (Trx), thioredoxin reductase (TrxR) and NF‐E2‐related factor‐2 (Nrf2) were significantly reduced even prior to photoreceptor cell degeneration in the retinas of tub/tub mice, suggesting that retinal expression of the Trx system is impaired and that Trx regulation is involved in the pathogenesis of retinal degeneration in this model, (ii) intraperitoneal injection with SF significantly up‐regulated retinal levels of Trx, TrxR, and Nrf2, and effectively protected photoreceptor cells in tub/tub mice as evaluated functionally by electroretinography and morphologically by quantitative histology, and (iii) treatment with PD98059, an inhibitor of extracellular signal‐regulated kinases (ERKs), blocked SF‐mediated ERKs activation and up‐regulation of Trx/TrxR/Nrf2 in the retinas of tub/tub mice. This suggests that ERKs and Nrf2 are involved in the mechanism of SF‐mediated up‐regulation of the Trx system to protect photoreceptor cells in this model. These novel findings are significant and could provide important information for the development of a unique strategy to prevent sensorineural deafness/retinal dystrophic syndromes and also other forms of inherited neurological disorders.

Analysis of Human Adenovirus Type 19 Associated with Epidemic Keratoconjunctivitis and its Reclassification as Adenovirus Type 64

PURPOSE Human adenovirus species D type 19 (HAdV-D19) has been associated with epidemic keratoconjunctivitis (EKC), a highly inflammatory infection of the ocular surface. Confusion exists regarding the origins of HAdV-D19. The prototype virus (HAdV-D19p) does not cause EKC, while a virus identified later with the identical serologic determinant is a significant ocular pathogen. METHODS High throughput genome sequencing and bioinformatics analysis were performed on HAdV-D19p and three HAdV-D19 EKC strains, and compared to the previously sequenced clinical isolate, HAdV-D19 (C) and HAdV-D37. Corneas of C57BL/6J mice were injected with HAdV-D19p, HAdV-D19 (C), or virus-free buffer, and inflammation assessed by clinical examination, flow cytometry, and cytokine ELISA. Confocal microscopy and real-time PCR of infected corneal cell cultures were used to test viral entry. RESULTS HAdV-D19 (C) and the other clinical EKC isolates showed nearly 100% sequence identity. EKC strains diverged from HAdV-D19p in the penton base, E3, and fiber transcription units. Simplot analysis showed recombination between EKC-associated HAdV-D19 with HAdV-D37, HAdV-D22, and HAdV-D19p, the latter contributing only the hexon gene, the principal serum neutralization determinant. HAdV-D19p induced stromal keratitis in the C57BL/6J mouse, but failed to infect productively human corneal epithelial cells. These data led to retyping of the clinical EKC isolates with a HAdV-D19 hexon gene as HAdV-D64. CONCLUSIONS HAdV-D19 associated with EKC (HAdV-D64) originated from a recombination between HAdV-D19p, HAdV-D37, and HAdV-D22, and was mischaracterized because of a shared hexon gene. HAdV-D19p is not infectious for corneal epithelial cells, thus explaining the lack of any association with keratitis.

Viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis.

Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.

Predicting the Next Eye Pathogen: Analysis of a Novel Adenovirus

ABSTRACT For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses. IMPORTANCE The ongoing dance between a virus and its host distinctly shapes how the virus evolves. While human adenoviruses typically cause mild infections, recent reports have described newly characterized adenoviruses that cause severe, sometimes fatal human infections. Here, we report a systems biology approach to show how evolution has affected the disease potential of a recently identified novel human adenovirus. A comprehensive understanding of viral evolution and pathogenicity is essential to our capacity to foretell the potential impact on human disease for new and emerging viruses. The ongoing dance between a virus and its host distinctly shapes how the virus evolves. While human adenoviruses typically cause mild infections, recent reports have described newly characterized adenoviruses that cause severe, sometimes fatal human infections. Here, we report a systems biology approach to show how evolution has affected the disease potential of a recently identified novel human adenovirus. A comprehensive understanding of viral evolution and pathogenicity is essential to our capacity to foretell the potential impact on human disease for new and emerging viruses.

Neuroprotective effect of overexpression of thioredoxin on photoreceptor degeneration in Tubby mice.

The Tubby mouse is a phenotypic model for sensorineural deafness and retinal dystrophy including Usher syndrome type 1. Thioredoxin is a small 13kDa protein which, when ubiquitously expressed as a transgene in the mouse, provides protection against multiple disease states including light-induced and oxidative stress-induced neurodegeneration and is down-regulated in the Tubby retina. We tested if overexpression of human thioredoxin in the Tubby mouse inhibits retinal degeneration and loss of visual function. Electroretinography, immunocytochemistry, quantitative histology, RT-PCR and Western blots were used to obtain data which showed that thioredoxin overexpression prevented loss of photoreceptors and retinal function. Analysis of signal pathways showed that thioredoxin up-regulated neurotrophic factors BDNF and GDNF and activated survival signaling pathways Akt, Ras/Raf1/ and the ERKs while inhibiting the ASK1/JNK apoptosis pathway. Relationships between the Tubby gene, its pathological phenotype and regulation of the thioredoxin system remain to be established.

17β‐estradiol (βE2) protects human retinal Müller cell against oxidative stress in vitro: Evaluation of its effects on gene expression by cDNA microarray

17β‐estradiol (βE2) is an effective neuroprotectant against hydrogen peroxide (H2O2)‐induced retinal neuronal cell death and light‐induced photoreceptor degeneration. Müller cells are the principal macroglia responsible for supporting retinal neuronal survival, information processing and removing metabolic waste. However, the role of βE2 on human Müller cells is unclear. In this study, the effects of βE2 on human Müller cell survival and gene expression were examined. Our data revealed that βE2 is able to increase human Müller cell viability after exposure to H2O2 through inhibition of apoptosis. Microarray analysis revealed significant changes in the expression of 69 genes (total of 21,324 genes screened) in cultured human Müller cells 6 h after βE2 treatment. Four of the βE2‐responsive genes [thrombospondin 1 (TSP1), mitogen‐activated protein kinase kinase kinase 3 (MAP3K3), large conductance calcium‐activated potassium channel β2 subunit (KCNMB2), and SRY (sex‐determining region Y)‐box 11 (SOX11)] were validated by both real‐time qRT‐PCR and semi‐quantitative RT‐PCR. Interestingly, exposure of human Müller cells to βE2 increased pigment epithelium‐derived factor (PEDF) gene expression as measured by both RT‐PCR and real time qRT‐PCR. Our data demonstrate, for the first time, that βE2 protects cultured human Müller cells against H2O2‐induced cell death through the inhibition of apoptosis. This protective effect may operate through regulation of genes, such as TSP1, MAP3K3, SOX11, TSP1, and PEDF, and may in turn exert an important role in protecting retinal neurons.

Molecular mechanisms underlying cochlear degeneration in the tubby mouse and the therapeutic effect of sulforaphane

As with Usher syndrome observed in humans, the two main phenotypes of the tubby mouse are progressive hearing loss and retinal degeneration. Yet, the mechanism underlying the tub-related cochlear degeneration is still unclear. The reduction/oxidation (redox) imbalance in the cell is related to many kinds of diseases. This study examined expressions of thioredoxin (Trx) and Trx reductase (TrxR), an important redox system in the cell, and the related upstream and downstream proteins of the Trx/TrxR in the tubby mouse cochlea. This report also examined the therapeutic effect of sulforaphane (SF) on the cochlear degeneration, which showed a protective effect on the tub-related retinal degeneration in our previous report. The results showed that the tub-mutation resulted in a significant suppression of Trx and TrxR expressions. Expression level of Nrf2 (NFE2 related factor 2), a transcription factor that regulates expression of Trx and TrxR and others, was also suppressed in the tubby mouse cochlea. Furthermore, a lowered level of activated extracellular signal-regulated kinase (p-ERK) was observed in the tubby mouse cochlea. In contrast, caspase-3 expression and activity were enhanced in the tubby mouse, suggesting apoptotic cell death. The tub-related molecular alterations in the cochlea were prevented by chronic treatment with SF. As a result, the SF-treatment significantly delayed the tub-related cochlear degeneration. Other unknown proteins may contribute to tubby-related degeneration because Nrf2 regulates many other antioxidants besides Trx/TrxR and sulforaphane did not prevent cochlear degeneration completely although it completely prevented alterations of Nrf2 and Trx/TrxR.