Hyun Yi

Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina.

BackgroundThe Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk) family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1) and Dickkopf 2 (Dkk2) are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3) protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death.ResultsDkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis.ConclusionThese data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina.

Expression of brain-derived neurotrophic factor is regulated by the Wnt signaling pathway

Brain-derived neurotrophic factor (BDNF) is a well characterized neurotrophin that mediates a wide variety of activities in the central nervous system, including neuronal differentiation, neuroprotection, and synaptic plasticity. The canonical Wnt signaling pathway is a critical regulator of embryonic development and homeostasis in adult tissues. Our group and others recently demonstrated that Wnt signaling induces BDNF expression in neurons and glia. However, the precise relationship between BDNF and Wnt signaling pathways is not understood. Here, we investigated Wnt signaling regulation of BDNF at the transcriptional level using a combination of bioinformatics and molecular analyses. Analysis of the BDNF gene promoter identified seven binding motifs for Wnt-dependent TCF/LEF transcription factors. Furthermore, specific BDNF promoters were induced by the Wnt3a ligand using chloramphenicol acetyl transferase reporter assays and a dominant-negative TCF4 gene reduced Wnt3a-mediated induction. Finally, Wnt3a induced expression of BDNF and other members of the BDNF signaling pathway in glia cells. Therefore, these data indicate that BDNF is a direct target of Wnt signaling, which provides a new insight into the interaction between two essential signaling pathways.

The Wnt/β-Catenin Pathway Cross-Talks with STAT3 Signaling to Regulate Survival of Retinal Pigment Epithelium Cells

Wnt/β-catenin signaling is an essential pathway that regulates numerous cellular processes, including cell survival. The molecular mechanisms contributing to pro-survival Wnt signaling are mostly unknown. Signal transducer and activator of transcription proteins (STATs) are a well-described family of transcription factors. STAT3 induces expression of anti-apoptotic genes in many tissues and is a downstream mediator of protective growth factors and cytokines. In this study, we investigated whether pro-survival Wnt signaling is mediated by STAT3. The Wnt3a ligand activated Wnt signaling in the retinal pigment epithelium ARPE-19 cell line and significantly increased the viability of cells exposed to oxidative stress. Furthermore, Wnt3a increased STAT3 activation and nuclear translocation, as measured by an antibody against phosphorylated STAT3. Reducing STAT3 levels with siRNA eliminated Wnt3a-dependent protection from oxidative stress. Together, these data demonstrate a previously unknown link between Wnt3a-mediated activation of STAT3 and cell survival, and indicate cross-talk between two important pro-survival signaling pathways.

Novel role for the innate immune receptor toll-like receptor 4 (TLR4) in the regulation of the wnt signaling pathway and photoreceptor apoptosis

Recent evidence has implicated innate immunity in regulating neuronal survival in the brain during stroke and other neurodegenerations. Photoreceptors are specialized light-detecting neurons in the retina that are essential for vision. In this study, we investigated the role of the innate immunity receptor TLR4 in photoreceptors. TLR4 activation by lipopolysaccharide (LPS) significantly reduced the survival of cultured mouse photoreceptors exposed to oxidative stress. With respect to mechanism, TLR4 suppressed Wnt signaling, decreased phosphorylation and activation of the Wnt receptor LRP6, and blocked the protective effect of the Wnt3a ligand. Paradoxically, TLR4 activation prior to oxidative injury protected photoreceptors, in a phenomenon known as preconditioning. Expression of TNFα and its receptors TNFR1 and TNFR2 decreased during preconditioning, and preconditioning was mimicked by TNFα antagonists, but was independent of Wnt signaling. Therefore, TLR4 is a novel regulator of photoreceptor survival that acts through the Wnt and TNFα pathways.

Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

BACKGROUND:Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection–related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of &ggr;-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. METHODS:The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBP&bgr; was tested using immunohistochemistry. RESULTS:In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time–effect curves in the HSV vector expressing GAD67 was increased compared with that in the control vectors (P = 0.0005). Intrathecal GABA-A/B agonists elevated mechanical threshold in the pain model. The HSV vectors expressing GAD67 reversed the lowered GABA immunoreactivity in the spinal dorsal horn in the neuropathic rats. HSV vectors expressing GAD67 in the neuropathic rats reversed the increased signals of mitochondrial superoxide in the spinal dorsal horn. The vectors expressing GAD67 reversed the upregulated immunoreactivity expression of pCREB and pC/EBP&bgr; in the spinal dorsal horn in rats exhibiting NP. CONCLUSIONS:Based on our results, we suggest that GAD67 mediated by HSV vectors acting through the suppression of mitochondrial reactive oxygen species and transcriptional factors in the spinal cord decreases pain in the HIV-related neuropathic pain model, providing preclinical evidence for gene therapy applications in patients with HIV-related pain states.

Mechanical allodynia induced by nucleoside reverse transcriptase inhibitor is suppressed by p55TNFSR mediated by herpes simplex virus vector through the SDF1 alpha/CXCR4 system in rats

BACKGROUND:In the human immunodeficiency virus (HIV)–associated sensory neuropathy, neuropathic pain associated with the use of nucleoside reverse transcriptase inhibitors (NRTIs) in patients with HIV/acquired immunodeficiency syndrome is clinically common. While evidence demonstrates that neuropathic pain is influenced by neuroinflammatory events that include the proinflammatory molecules, tumor necrosis factor-&agr; (TNF-&agr;), stromal cell–derived factor 1-&agr; (SDF1-&agr;), and C-X-C chemokine receptor type 4 (CXCR4), the detailed mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In this study, we investigated the role of these proinflammatory molecules in the dorsal root ganglion (DRG) and the spinal dorsal horn in NRTIs-mediated neuropathic pain state. METHODS:Neuropathic pain was induced by intraperitoneal administration of 2′,3′-dideoxycytidine (ddC, one of the NRTIs). Mechanical threshold was tested using von Frey filament fibers. Nonreplicating herpes simplex virus (HSV) vectors expressing p55 TNF soluble receptor (p55TNFSR) were inoculated into hindpaw of rats. The expression of TNF-&agr;, SDF1-&agr;, and CXCR4 in both the lumbar spinal cord and the L4/5 DRG was examined using Western blots. Intrathecal CXCR4 antagonist was administered. RESULTS:The present study demonstrated that (1) systemic ddC induced upregulation of TNF-&agr;, SDF1-&agr;, and CXCR4 in both the lumbar spinal cord and the L4/5 DRG; (2) p55TNFSR mediated by a nonreplicating HSV vector reversed mechanical allodynia induced by systemic ddC; (3) intrathecal administration of the CXCR4 antagonist AMD3100 increased mechanical threshold; and (4) HSV vector expressing p55TNFSR reversed upregulation of TNF-&agr;, SDF1-&agr;, and CXCR4 induced by ddC in the lumbar spinal dorsal horn and the DRG. CONCLUSIONS:Our studies demonstrate that TNF-&agr; through the SDF1/CXCR4 system is involved in the NRTIs-related neuropathic pain state and that blocking the signaling of these proinflammatory molecules is able to reduce NRTIs-related neuropathic pain. These results provide a novel mechanism-based approach (gene therapy) to treating HIV-associated neuropathic pain.

Spinal CPEB-mtROS-CBP signaling pathway contributes to perineural HIV gp120 with ddC-related neuropathic pain in rats.

Human immunodeficiency virus (HIV) patients treated with nucleoside reverse transcriptase inhibitors (NRTIs), have been known to develop neuropathic pain. While there has been a major shift away from some neurotoxic NRTIs in current antiretroviral therapy, a large number of HIV patients alive today have previously received them, and many have developed painful peripheral neuropathy. The exact mechanisms by which HIV with NRTIs contribute to the development of neuropathic pain are not known. Previous studies suggest that cytoplasmic polyadenylation element-binding protein (CPEB), reactive oxygen species (ROS), and cAMP-response element-binding protein (CREB)-binding protein (CBP), are involved in the neuroimmunological diseases including inflammatory/neuropathic pain. In this study, we investigated the role of CPEB, mitochondrial ROS (mtROS), or CBP in neuropathic pain induced by HIV envelope protein gp120 combined with antiretroviral drug. The application of recombinant gp120 into the sciatic nerve plus systemic ddC (one of NRTIs) induced mechanical allodynia. Knockdown of CPEB or CBP using intrathecal antisense oligodeoxynucleotide (AS-ODN) reduced mechanical allodynia. Intrathecal mitochondrial superoxide scavenger mito-tempol (Mito-T) increased mechanical withdrawal threshold. Knockdown of CPEB using intrathecal AS-ODN, reduced the up-regulated mitochondrial superoxide in the spinal dorsal horn in rats with gp120 combined with ddC. Intrathecal Mito-T lowered the increased expression of CBP in the spinal dorsal horn. Immunostaining studies showed that neuronal CPEB positive cells were co-localized with MitoSox positive profiles, and that MitoSox positive profiles were co-localized with neuronal CBP. Our studies suggest that neuronal CPEB-mtROS-CBP pathway in the spinal dorsal horn, plays an important role in the gp120/ddC-induced neuropathic pain in rats.

Inhibition of Mitochondrial Fission Protein Reduced Mechanical Allodynia and Suppressed Spinal Mitochondrial Superoxide Induced by Perineural Human Immunodeficiency Virus gp120 in Rats.

BACKGROUND:Mitochondria play an important role in many cellular and physiologic functions. Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis. The most common complaint of human immunodeficiency virus (HIV)-sensory neuropathy is pain on the soles in patients with HIV, but the exact molecular mechanisms of HIV neuropathic pain are not clear. In the present study, we investigated the role of mitochondrial dynamin-related protein 1 (Drp1, a GTPase that mediates mitochondrial fission) in the perineural HIV coat glycoprotein gp120-induced neuropathic pain state. METHODS:Neuropathic pain was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. Mechanical threshold was tested using von Frey filaments. The mechanical threshold response was assessed over time using the area under curves. Intrathecal administration of antisense oligodeoxynucleotide (ODN) against Drp1, mitochondrial division inhibitor-1 (mdivi-1), or phenyl-N-tert-butylnitrone (a reactive oxygen species scavenger) was given. The expression of spinal Drp1 was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. RESULTS:Intrathecal administration of either antisense ODN against Drp1 or mdivi-1 decreased mechanical allodynia (a sensation of pain evoked by nonpainful stimuli) in the gp120 model. Intrathecal ODN or mdivi-1 did not change basic mechanical threshold in sham surgery rats. Intrathecal Drp1 antisense ODN decreased the spinal expression of increased Drp1 protein induced by peripheral gp120 application. Intrathecal phenyl-N-tert-butylnitrone reduced mechanical allodynia. Furthermore, both intrathecal Drp1 antisense ODN and mdivi-1 reversed the upregulation of mitochondrial superoxide in the spinal dorsal horn in the gp120 neuropathic pain state. CONCLUSIONS:These data suggest that mitochondrial division plays a substantial role in the HIV gp120-related neuropathic pain state through mitochondrial reactive oxygen species and provides evidence for a novel approach to treating chronic pain in patients with HIV.

HSV vector-mediated GAD67 suppresses neuropathic pain induced by perineural HIV gp120 in rats through inhibition of ROS and Wnt5a

Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite the extensive research, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. Loss of GABAergic tone may have an important role in the neuropathic pain state. Glutamic acid decarboxylase 67 (GAD67) is one of the isoforms that catalyze GABA synthesis. Here, we used recombinant herpes simplex virus (HSV-1) vectors that encode gad1 gene to evaluate the therapeutic potential of GAD67 in peripheral HIV gp120-induced neuropathic pain in rats. We found that (1) subcutaneous inoculation of the HSV vectors expressing GAD67 attenuated mechanical allodynia in the model of HIV gp120-induced neuropathic pain, (2) the anti-allodynic effect of GAD67 was reduced by GABA-A and-B receptors antagonists, (3) HSV vectors expressing GAD67 reversed the lowered GABA-IR expression and (4) the HSV vectors expressing GAD67 suppressed the upregulated mitochondrial superoxide and Wnt5a in the spinal dorsal horn. Taken together, our studies support the concept that recovering GABAergic tone by the HSV vectors may reverse HIV-associated neuropathic pain through suppressing mitochondrial superoxide and Wnt5a. Our studies provide validation of HSV-mediated GAD67 gene therapy in the treatment of HIV-related neuropathic pain.

MnSOD mediated by HSV vectors in the periaqueductal gray suppresses morphine withdrawal in rats

Morphine appears to be the most active metabolite of heroin; therefore, the effects of morphine are important in understanding the ramifications of heroin abuse. Opioid physical dependence (withdrawal response) may have very long-lasting effects on the motivation for reward, including the incubation of cue-induced drug-seeking behavior. However, the exact mechanisms of morphine withdrawal (MW) are not clear yet, and its treatment remains elusive. Periaqueductal gray (PAG) is one of the important sites in the pathogenesis of MW. Here, we used recombinant herpes simplex virus (HSV) vectors that encode the sod2 gene expressing manganese superoxide dismutase (MnSOD) to evaluate its therapeutic potential in MW. Microinjection of HSV vectors expressing MnSOD into the PAG reduced the MW syndrome. MnSOD vectors suppressed the upregulated mitochondrial superoxide, and endoplasmic reticulum stress markers (glucose-related protein 78 (GRP78) and activating transcription factor 6 alpha (ATF6α)) in the PAG induced by MW. Immunostaining showed that mitochondrial superoxide, GRP78 and ATF6α were colocalized with neuronal nuclei (a neuronal-specific marker), suggesting that they are located in the neurons in the PAG. These results suggest that overexpression of MnSOD by HSV vectors may relieve opioid dependence. This study may provide a novel therapeutic approach to morphine physical withdrawal response.