Ling-Ping Cen

PI3K/akt, JAK/STAT and MEK/ERK pathway inhibition protects retinal ganglion cells via different mechanisms after optic nerve injury

Recently we unexpectedly found that PI3K/akt, JAK/STAT and MEK/ERK pathway inhibitors enhanced retinal ganglion cell (RGC) survival after optic nerve (ON) axotomy in adult rat, a phenomenon contradictory to conventional belief that these pathways are pro‐survival. In this study we showed that: (i) the RGC protection was pathway inhibition‐dependent; (ii) inhibition of PI3K/akt and JAK/STAT, but not MEK/ERK, activated macrophages in the eye, (iii) macrophage removal from the eye using clodronate liposomes significantly impeded PI3K/akt and JAK/STAT inhibition‐induced RGC survival and axon regeneration whereas it only slightly affected MEK/ERK inhibition‐dependent protection; (iv) in the absence of recruited macrophages in the eye, inhibition of PI3K/akt or JAK/STAT did not influence RGC survival; and (v) strong PI3K/akt, JAK/STAT and MEK/ERK pathway activities were located in RGCs but not macrophages after ON injury. In retinal explants, in which supply of blood‐derived macrophages is absent, MEK/ERK inhibition promoted RGC survival whereas PI3K/akt or JAK/STAT inhibition had no effect on RGC viability. However, MEK/ERK inhibition exerted opposite effects on the viability of purified adult RGCs at different concentrations in vitro, suggesting that this pathway may be bifunctional depending on the level of pathway activity. Our data thus demonstrate that inhibition of the PI3K/akt or JAK/STAT pathway activated macrophages to facilitate RGC protection after ON injury whereas the two pathways per se did not modulate RGC viability under the injury conditions (in the absence of the pathway activators). In contrast, the MEK/ERK pathway inhibition protected RGCs via macrophage‐independent mechanism(s).

Differential roles of phosphatidylinositol 3-kinase/akt pathway in retinal ganglion cell survival in rats with or without acute ocular hypertension

Intraocular pressure (IOP) elevation has often been used as an experimental model to study mechanisms underlying retinal ganglion cell (RGC) death associated with ocular ischemic injury and glaucoma. The aim of the present study, using both in vitro and in vivo approaches, was to investigate the role of phosphatidylinositol 3-kinase (PI3K)/akt pathway in RGC viability in normal rats and rats following transient IOP elevation. For in vivo studies, pathway inhibitors were administered intravitreally on days 3, 9, and 15 post-2-h IOP elevation at 110 mm Hg. Toward the end of the 3-week examination period, the fluorescent dye Fluorogold was used to retrogradely label surviving RGCs. In order to examine the role of macrophages that were recruited into the eye following the pathway inhibition, clodronate liposomes were used to deplete phagocytic cells in the eye. PI3K/akt pathway activity and location in the retina were examined using Western blot and immunohistochemistry, respectively. Here we showed that PI3K/akt inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and KY12420 at low concentrations (2 microM or 20 microM) did not influence RGC survival but caused RGC loss at high concentration (200 muM) in retinal explants derived from intact rats. In contrast, both LY294002 and KY12420 at 20 microM led to RGC loss in retinal explants derived from IOP-elevated eyes. A detrimental action of phagocytic cells on RGC survival was also seen in these retinas. In vivo results confirmed the detrimental actions of PI3K/akt inhibition and macrophages on RGC survival in IOP-elevated, but not intact eyes even with high concentration of LY294002. Low level of PI3K/akt activity was detected in the ganglion cell layer (GCL) in intact retina. Acute IOP elevation activated PI3K/akt pathway in the inner nuclear layer and GCL including RGCs. This study thus demonstrates that PI3K/akt pathway mediates RGC survival after IOP elevation but not under normal condition.

Bilateral retinal microglial response to unilateral optic nerve transection in rats

When retinal ganglion cells undergo apoptosis after optic nerve (ON) injury, microglial cells proliferate and promptly clear the degenerated debris in the ipsilateral retina. However, microglial changes in the contralateral retina have not been fully elucidated. This study characterized the long-term bilateral retinal microglial responses after unilateral ON transection. We analyzed the time course of proliferation and morphology changes of microglial cells, between 3 days and 12 weeks post ON transection, of undisturbed and reactive microglia in bilateral retinas of adult Fischer rats with unilateral ON transection. Microglia in retinas without ON transection were distributed homogeneously and possessed a highly ramified morphology, as judged by immunohistochemistry for ionized calcium-binding adapter molecule 1 (Iba1). After ON transection, microglia density in the ipsilateral retina increased gradually from 3 days to 2 weeks, and decreased from 3 weeks to 12 weeks, along with dramatic inverted alteration of process branch points of microglia in the ganglion cell layer (GCL). Transformation of ramified microglia into ameboid-like macrophages with few branching processes was observed in the ipsilateral retina from 1 week to 3 weeks. Though an increase in microglial density was weak in the contralateral retina and could only be statistically detected in the central retina, the morphological alteration over time was obvious and similar to that of the ipsilateral retina. In the inner plexiform layer (IPL), cell density and morphological changes of microglia in both the ipsilateral and contralateral retina were not prominent. These findings indicates that, though proliferation of microglial cells is weak in the contralateral retina after unilateral ON transection, conspicuous alterations in microglial morphology occur bilaterally. These suggest that using the contralateral retina as a control in studies of retinal degeneration should be considered with caution.

Influence of macrophages and lymphocytes on the survival and axon regeneration of injured retinal ganglion cells in rats from different autoimmune backgrounds

The immune response after neural injury influences the survival and regenerative capacity of neurons. In the primary visual pathway, previous studies have described beneficial effects of macrophages and T‐cells in promoting neural survival and axonal regeneration in some rat strains. However, the contributions of specific cell populations to these responses have been unclear. In adult Fischer (F344) rats, we confirm prior reports that intravitreal macrophage activation promotes the survival of retinal ganglion cells (RGCs) and greatly enhances axonal regeneration through a peripheral nerve graft. Neonatal thymectomy that results in elimination of T‐cell production enhanced RGC survival after axotomy, but diminished the effect of intravitreal macrophage activation on axon regeneration. Thus, in F344 rats, lymphocytes appear to suppress RGC survival but augment the pro‐regenerative effects of macrophages. The cytotoxic effect of lymphocytes on RGCs was confirmed in in vitro studies; coculture of retinal explants with lymphocytes led to a 60% reduction in viable RGCs. Similar in vivo results were obtained in Sprague Dawley rats. By comparison, in adult Lewis rats, neither RGC survival nor axonal regeneration was increased after intravitreal macrophage activation. Neonatal thymectomy had only a small beneficial effect on RGC survival, and although Lewis lymphocytes reduced RGC viability in culture, they did so to a lesser extent. Thus, in addition to a complex role of lymphocytes, particularly T‐cells, after central nervous system injury, the present results demonstrate that the impact of macrophages is also influenced by genetic background.

Long-term survival and axonal regeneration of retinal ganglion cells after optic nerve transection and a peripheral nerve graft.

To investigate the effect of autologous peripheral nerve grafting on retinal ganglion cell survival and axonal regeneration after an injury, the optic nerve of adult Sprague–Dawley rats was transected and grafted with an autologous peripheral nerve from the peroneal branch of the left sciatic nerve. The numbers of both surviving and axon-regenerating retinal ganglion cells were determined at different times after surgery. The majority of retinal ganglion cells were rapidly lost within 3 weeks, followed by a slow and protracted phase of cell loss until the end of the 6-month study. FluoroGold-labelled axon-regenerating retinal ganglion cells were first detected by 2 weeks, followed by a period of high axonal regeneration that peaked at 8 weeks and accounted for over 35% of the total surviving retinal ganglion cells. However, retinal ganglion cells with regenerated axons eventually died. Our data thus indicate that axonal regeneration in the autologous peripheral nerve graft is insufficient to sustain the long-term survival of axotomized retinal ganglion cells.

Mutations of RagA GTPase in mTORC1 Pathway Are Associated with Autosomal Dominant Cataracts.

Cataracts are a significant public health problem with no proven methods for prevention. Discovery of novel disease mechanisms to delineate new therapeutic targets is of importance in cataract prevention and therapy. Herein, we report that mutations in the RagA GTPase (RRAGA), a key regulator of the mechanistic rapamycin complex 1 (mTORC1), are associated with autosomal dominant cataracts. We performed whole exome sequencing in a family with autosomal dominant juvenile-onset cataracts, and identified a novel p.Leu60Arg mutation in RRAGA that co-segregated with the disease, after filtering against the dbSNP database, and at least 123,000 control chromosomes from public and in-house exome databases. In a follow-up direct screening of RRAGA in another 22 families and 142 unrelated patients with congenital or juvenile-onset cataracts, RRAGA was found to be mutated in two unrelated patients (p.Leu60Arg and c.-16G>A respectively). Functional studies in human lens epithelial cells revealed that the RRAGA mutations exerted deleterious effects on mTORC1 signaling, including increased relocation of RRAGA to the lysosomes, up-regulated mTORC1 phosphorylation, down-regulated autophagy, altered cell growth or compromised promoter activity. These data indicate that the RRAGA mutations, associated with autosomal dominant cataracts, play a role in the disease by acting through disruption of mTORC1 signaling.

Stem cell therapy for retinal ganglion cell degeneration

The prospects of stem cell therapy for retinal ganglion cell (RGC) degeneration in human: RGC degeneration is a common pathologic cause of glaucoma and optic neuropathies, which are the leading cause of irreversible blindness and visual impairment in developed countries, currently affecting more than 100 million people worldwide. Intraocular pressure lowering can slow down glaucoma progression in a proportion of patients. Also, there is still no effective therapy for optic neuropathies. Besides, the degenerated RGCs in glaucoma cannot be repaired, and human retina has limited regenerative potential. Therefore, the development of new therapeutic treatments against RGC degeneration is needed. Cell replacement and neuroprotection are the principle strategies for glaucoma and optic neuropathy treatment. Replacing the diseased or degenerated cells by stem cell-derived RGCs should provide effective therapeutic treatment. However, complex circuitry in the retina makes cell replacement challenging and difficult for functional repair. Alternatively, neuroprotection is more realistic and applicable to preserve the patients’ vision. Numerous neuroprotection strategies have been investigated, including peripheral nerve grafting, electrical stimulation, application of neurotrophic factors (brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), direct intrinsic regeneration stimulation, RNA interference and human adult stem cells. Our group recently reported that the intravitreal transplantation of human periodontal ligament-derived stem cells (PDLSCs) ameliorates RGC degeneration after optic nerve injury in rats and promotes neural repair by enhancing axon regeneration through cell-cell interaction and neurotrophic factor secretion from PDLSCs (Cen et al., 2018). At present, there are 9 clinical trials on human adult stem cells for glaucoma and optic nerve diseases (www. clinicaltrials.gov/; Table 1). An emerging role of human adult stem cell therapy for glaucoma and optic neuropathy treatments is foreseeable in the near future.

Casein kinase-II inhibition promotes retinal ganglion cell survival and axonal regeneration

&NA; Neuron survival is critical for the maintenance of central nervous system physiology upon diseases or injury. We previously demonstrated that the blockage of phosphatidylinositol 3‐kinase/Akt and Janus kinase/STAT3 pathways promotes retinal ganglion cell (RGC) survival and axonal regeneration via macrophage activation; yet, the complexity of the inflammatory regulation for neural repair indicates the involvement of additional unresolved signaling pathways. Here we report the effects and underlying mechanism of casein kinase‐II (CK2) inhibition on RGC survival and axonal regeneration in rats after optic nerve (ON) injury. Adult rats received intravitreal injection of CK2 inhibitors, TBB (4,5,6,7‐Tetrabromo‐2‐azabenzimidazole) and DMAT (2‐Dimethylamino‐4,5,6,7‐tetrabromo‐1H‐benzimidazole), after ON transection and peripheral nerve (PN) grafting. Intravitreal application of TBB and DAMT effectively suppressed the CK2 phosphorylation activity in the retina, and enhanced RGC survival and axonal regeneration in vivo. Meanwhile, the numbers of infiltrating macrophages were increased. Removal of macrophages by clodronate liposomes significantly abolished the CK2 inhibition‐induced RGC survival and axonal regeneration. Clodronate liposomes also weakened the RGC protective effects by TBB and DMAT in vitro. In summary, this study revealed that inhibition of CK2 enhances RGC survival and axonal regeneration via macrophage activation in rats. CK2 could be a therapeutic target for RGC protection after ON injury.