Xiaoli Guo

Regulation of the severity of neuroinflammation and demyelination by TLR-ASK1-p38 pathway

Apoptosis signal‐regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen‐activated protein kinase (MAPK) kinase kinase which plays important roles in stress and immune responses. Here, we show that ASK1 deficiency attenuates neuroinflammation in experimental autoimmune encephalomyelitis (EAE), without affecting the proliferation capability of T cells. Moreover, we found that EAE upregulates expression of Toll‐like receptors (TLRs) in activated astrocytes and microglia, and that TLRs can synergize with ASK1‐p38 MAPK signalling in the release of key chemokines from astrocytes. Consequently, oral treatment with a specific small molecular weight inhibitor of ASK1 suppressed EAE‐induced autoimmune inflammation in both spinal cords and optic nerves. These results suggest that the TLR‐ASK1‐p38 pathway in glial cells may serve as a valid therapeutic target for autoimmune demyelinating disorders including multiple sclerosis.

Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration

Glia, the support cells of the central nervous system, have recently attracted considerable attention both as mediators of neural cell survival and as sources of neural regeneration. To further elucidate the role of glial and neural cells in neurodegeneration, we generated TrkBGFAP and TrkBc-kit knockout mice in which TrkB, a receptor for brain-derived neurotrophic factor (BDNF), is deleted in retinal glia or inner retinal neurons, respectively. Here, we show that the extent of glutamate-induced retinal degeneration was similar in these two mutant mice. Furthermore in TrkBGFAP knockout mice, BDNF did not prevent photoreceptor degeneration and failed to stimulate Müller glial cell proliferation and expression of neural markers in the degenerating retina. These results demonstrate that BDNF signalling in glia has important roles in neural protection and regeneration, particularly in conversion of Müller glia to photoreceptors. In addition, our genetic models provide a system in which glia- and neuron-specific gene functions can be tested in central nervous system tissues in vivo.

Dock3 Stimulates Axonal Outgrowth via GSK-3β-Mediated Microtubule Assembly

Dock3, a new member of the guanine nucleotide exchange factors, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes axonal outgrowth downstream of brain-derived neurotrophic factor (BDNF) signaling. We previously showed that Dock3 forms a complex with Fyn and WASP (Wiskott–Aldrich syndrome protein) family verprolin-homologous (WAVE) proteins at the plasma membrane, and subsequent Rac1 activation promotes actin polymerization. Here we show that Dock3 binds to and inactivates glycogen synthase kinase-3β (GSK-3β) at the plasma membrane, thereby increasing the nonphosphorylated active form of collapsin response mediator protein-2 (CRMP-2), which promotes axon branching and microtubule assembly. Exogenously applied BDNF induced the phosphorylation of GSK-3β and dephosphorylation of CRMP-2 in hippocampal neurons. Moreover, increased phosphorylation of GSK-3β was detected in the regenerating axons of transgenic mice overexpressing Dock3 after optic nerve injury. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the CNS, where it regulates cell polarity and promotes axonal outgrowth by stimulating dual pathways: actin polymerization and microtubule assembly.

Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced RGC apoptosis. In this study, we found that ONI-induced p38 activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor and inducible nitric oxide synthase in microglia were decreased when the ASK1-p38 pathway was blocked. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 pathways could be beneficial in the treatment of ONI.

Dock3 attenuates neural cell death due to NMDA neurotoxicity and oxidative stress in a mouse model of normal tension glaucoma

Dedicator of cytokinesis 3 (Dock3), a new member of the guanine nucleotide exchange factors for the small GTPase Rac1, promotes axon regeneration following optic nerve injury. In the present study, we found that Dock3 directly binds to the intracellular C-terminus domain of NR2B, an N-methyl-D-aspartate (NMDA) receptor subunit. In transgenic mice overexpressing Dock3 (Dock3 Tg), NR2B expression in the retina was significantly decreased and NMDA-induced retinal degeneration was ameliorated. In addition, overexpression of Dock3 protected retinal ganglion cells (RGCs) from oxidative stress. We previously reported that glutamate/aspartate transporter (GLAST) is a major glutamate transporter in the retina, and RGC degeneration due to glutamate neurotoxicity and oxidative stress is observed in GLAST-deficient (KO) mice. In GLAST KO mice, the NR2B phosphorylation rate in the retina was significantly higher compared with Dock3 Tg:GLAST KO mice. Consistently, glaucomatous retinal degeneration was significantly improved in GLAST KO:Dock3 Tg mice compared with GLAST KO mice. These results suggest that Dock3 overexpression prevents glaucomatous retinal degeneration by suppressing both NR2B-mediated glutamate neurotoxicity and oxidative stress, and identifies Dock3 signaling as a potential therapeutic target for both neuroprotection and axonal regeneration.

Neuroprotection, Growth Factors and BDNF-TrkB Signalling in Retinal Degeneration

Neurotrophic factors play key roles in the development and survival of neurons. The potent neuroprotective effects of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF) and nerve growth factor (NGF), suggest that they are good therapeutic candidates for neurodegenerative diseases. Glaucoma is a neurodegenerative disease of the eye that causes irreversible blindness. It is characterized by damage to the optic nerve, usually due to high intraocular pressure (IOP), and progressive degeneration of retinal neurons called retinal ganglion cells (RGCs). Current therapy for glaucoma focuses on reduction of IOP, but neuroprotection may also be beneficial. BDNF is a powerful neuroprotective agent especially for RGCs. Exogenous application of BDNF to the retina and increased BDNF expression in retinal neurons using viral vector systems are both effective in protecting RGCs from damage. Furthermore, induction of BDNF expression by agents such as valproic acid has also been beneficial in promoting RGC survival. In this review, we discuss the therapeutic potential of neurotrophic factors in retinal diseases and focus on the differential roles of glial and neuronal TrkB in neuroprotection. We also discuss the role of neurotrophic factors in neuroregeneration.

Valproic acid prevents retinal degeneration in a murine model of normal tension glaucoma

Valproic acid (VPA) is widely used for treatment of epilepsy, mood disorders, migraines and neuropathic pain. It exerts its therapeutic benefits through modulation of multiple mechanisms including regulation of gamma-aminobutyric acid and glutamate neurotransmissions, activation of pro-survival protein kinases and inhibition of histone deacetylase. The evidence for neuroprotective properties associated with VPA is emerging. Herein, we investigated the therapeutic potential of VPA in a mouse model of normal tension glaucoma (NTG). Mice with glutamate/aspartate transporter gene deletion (GLAST KO mice) demonstrate progressive retinal ganglion cell (RGC) loss and optic nerve degeneration without elevated intraocular pressure, and exhibit glaucomatous pathology including glutamate neurotoxicity and oxidative stress in the retina. VPA (300mg/kg) or vehicle (PBS) was administered via intraperitoneal injection in GLAST KO mice daily for 2 weeks from the age of 3 weeks, which coincides with the onset of glaucomatous retinal degeneration. Following completion of the treatment period, the vehicle-treated GLAST KO mouse retina showed significant RGC death. Meanwhile, VPA treatment prevented RGC death and thinning of the inner retinal layer in GLAST KO mice. In addition, in vivo electrophysiological analyses demonstrated that visual impairment observed in vehicle-treated GLAST KO mice was ameliorated with VPA treatment, clearly establishing that VPA beneficially affects both histological and functional aspects of the glaucomatous retina. We found that VPA reduces oxidative stress induced in the GLAST KO retina and stimulates the cell survival signalling pathway associated with extracellular-signal-regulated kinases (ERK). This is the first study to report the neuroprotective effects of VPA in an animal model of NTG. Our findings raise intriguing possibilities that the widely prescribed drug VPA may be a novel candidate for treatment of glaucoma.

Interleukin-1 attenuates normal tension glaucoma-like retinal degeneration in EAAC1-deficient mice.

Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of retinal ganglion cells (RGCs) and optic nerves. Although glaucoma is often associated with elevated intraocular pressure, recent studies have shown a relatively high prevalence of normal tension glaucoma (NTG) in glaucoma patient populations. In the mammalian retina, glutamate/aspartate transporter (GLAST) is localized to Müller glial cells, whereas excitatory amino acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs. Since the loss of GLAST or EAAC1 leads to retinal degeneration similar to that seen in NTG, we examined the effects of interleukin-1 (IL-1) on RGC death in GLAST- and EAAC1-deficient mice. IL-1 promoted increased glutamate uptake in Müller cells by suppressing intracellular Na(+) accumulation, which is necessary to counteract Na(+)-glutamate cotransport. The observed trends for the glutamate uptake increase in the wild-type (WT), GLAST- and EAAC1-deficient mice were similar; however, the baseline glutamate uptake and intracellular Na(+) concentration in the GLAST-deficient mice were significantly lower than those in the wild-type mice. Consistently, pretreatment with IL-1 exhibited no beneficial effects on glutamate-induced RGC degeneration in the GLAST-deficient mice. In contrast, IL-1 significantly increased glutamate uptake by Müller cells and the number of surviving RGCs in the wild-type and EAAC1-deficient mice. Our findings suggest that the use of IL-1 for enhancing the function of glutamate transporters may be useful for neuroprotection in retinal degenerative disorders including NTG.

Dock3 regulates BDNF-TrkB signaling for neurite outgrowth by forming a ternary complex with Elmo and RhoG

Dock3, a new member of the guanine nucleotide exchange factor family, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes neurite outgrowth through the formation of a protein complex with Fyn and WAVE downstream of brain‐derived neurotrophic factor (BDNF) signaling. Here, we report a novel Dock3‐mediated BDNF pathway for neurite outgrowth. We show that Dock3 forms a complex with Elmo and activated RhoG downstream of BDNF‐TrkB signaling and induces neurite outgrowth via Rac1 activation in PC12 cells. We also show the importance of Dock3 phosphorylation in Rac1 activation and show two key events that are necessary for efficient Dock3 phosphorylation: membrane recruitment of Dock3 and interaction of Dock3 with Elmo. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the central nervous system where it stimulates actin polymerization by multiple pathways.

Spermidine Ameliorates Neurodegeneration in a Mouse Model of Normal Tension Glaucoma.

PURPOSE To assess the therapeutic potential of spermidine in mice with excitatory amino acid carrier 1 (EAAC1) deletion (EAAC1 knockout [KO] mice), a mouse model of normal tension glaucoma. METHODS Spermidine, at 30 mM in drinking water, was administered to EAAC1 KO mice from 5 to 12 weeks old. Optical coherence tomography, multifocal electroretinograms, and the measurement of intraocular pressure (IOP) were performed at 5, 8, and 12 weeks old. Histopathology analyses were carried out at 8 and 12 weeks old, and immunoblot and immunohistochemical analyses of 4-hydroxy-2-nonenal (4-HNE) in the retina were performed at 8 weeks old. RESULTS Spermidine ameliorated retinal degeneration and improved visual function in EAAC1 KO mice at both 8 and 12 weeks old, without affecting IOP. A significant increase of 4-HNE was observed in vehicle-treated EAAC1 KO mice, but spermidine treatment reduced this increase, suggesting that spermidine alleviated the severity of the glaucoma-like phenotype by acting as an antioxidant. CONCLUSIONS The results from this study suggest that oral spermidine administration could be a useful treatment for retinal degenerative disorders including glaucoma.