Jacky M. K. Kwong

RNA binding protein with multiple splicing: a new marker for retinal ganglion cells.

PURPOSE To characterize expression of the RNA binding protein (RBPMS) in the retina as a specific marker for retinal ganglion cells (RGCs). METHODS Optic nerve transection (ONT) was performed on adult male Wistar rats. Retrograde RGC labeling was performed with FluoroGold (FG) applied to the cut surface of the optic nerve. RBPMS mRNA and protein expression in the retina was analyzed by in situ hybridization and immunohistochemistry, respectively. The expression of RBPMS in various rat tissues was analyzed with semiquantitative RT-PCR. RESULTS RBPMS mRNA and protein expression was localized primarily to irregularly shaped cells in the ganglion cell layer of the retina. Quantitative analysis showed that almost 100% of RGCs labeled by FG were also RBPMS-positive, irrespective of their location relative to the optic nerve head. Approximately 94% to 97% of RBPMS-positive cells were also positive for Thy-1, neurofilament H, and III beta-tubulin. In 2-week ONT retinas, the remaining few RGCs were weakly stained with RBPMS compared with intact RGCs in control retinas. Outside the retina, expression of RBPMS was observed in the heart, kidney, liver, and lungs. No expression was detected in any neuronal tissues except the retina. CONCLUSIONS The data indicate that in the retina RBPMS is selectively expressed in RGCs and therefore could serve as a marker for RGC quantification in normal retinas and for estimation of RGC loss in ocular neuropathies.

Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma.

We investigated the neuroprotective effect of thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2) which play critical roles in the regulation of oxidative stress on retinal ganglion cells (RGCs) in a rat glaucoma model. Expression of Trx1 and Trx2 and Trx-interacting protein (Txnip) was observed in the RGC layer (GCL), nerve fiber layer and inner nuclear layer. Txnip-, Trx1- and Trx2-expressing cells in the GCL were primarily colocalized with RGCs. The increased Txnip protein level was observed 2 and 5 weeks after glaucoma induction. Trx1 level decreased 2 weeks after glaucoma induction and more prominently after 5 weeks. No change in Trx2 levels was detected. The effects of Trx1 and Trx2 overexpression on RGC survival were evaluated 5 weeks after glaucoma induction. In nontransfected and EGFP-transfected (used as a negative control) retinas, RGC loss was approximately 27% compared with control. The loss of RGCs in Trx1- and Trx2- transfected retinas was approximately 15 and 17%, respectively. Thus, Trx1 and Trx2 preserved 45 and 37% of cells, respectively that were destined to die in glaucomatous retinas. The results of this study provide evidence for the involvement of oxidative stress in RGC degeneration in experimental glaucoma and point to potential strategies to reduce its impact.

Calpain and N-methyl-d-aspartate (NMDA)-induced excitotoxicity in rat retinas.

Calpain-mediated proteolysis has been implicated as a major process in neuronal cell death in both acute insults and the chronic neurodegenerative disorders in the central nerves system. However, activation of calpain also plays a protective function in the early phase of excitotoxic neuronal death. The exact role of calpains in neuronal death and recovery after exposure to N-methyl-D-aspartate (NMDA) is not clearly known. The purpose of present study was to examine the involvement of mu- and m-calpain in NMDA-induced excitotoxicity in the adult rat retina. Increased immunoreactivity of mu-calpain was noted in RGC layer cells and in the inner nuclear layer with maximal expression at 12 h after NMDA injection. This was further confirmed with Western blotting. TdT-mediated biotin-dUTP nick end labeling (TUNEL) positive cells in the inner retina co-localized with moderate or intense mu-calpain immunoreactivity. In contrast, there was no remarkable change in m-calpain immunoreactivity at any time point after NMDA injection. Simultaneous injection of 2 nmol of a calpain inhibitor (calpain inhibitor II) significantly reduced the number of TUNEL-positive cells in the inner retina at 18 h after NMDA injection and preserved RGC-like cells counted at 7 days after injection. The results of this study showed that mu-calpain may be involved in mediating NMDA-induced excitotoxicity in the rat retina and calpain inhibitors may play a therapeutic role in NMDA related disease.

Modulation of alpha and beta crystallin expression in rat retinas with ocular hypertension-induced ganglion cell degeneration

The expression of alpha (alphaA and alphaB) and beta (betaA1/A3, betaA2, betaA4, and betaB2) crystallin genes were analyzed at the mRNA and protein levels in rat retinas with ocular hypertension-induced ganglion cell death. An animal model with progressive loss of retinal ganglion cells (RGC) was generated by elevation of intraocular pressure (IOP). The estimated RGC loss was approximately 8% and 20% at 2 and 5 weeks post IOP elevation, respectively. mRNA and protein quantification showed that alpha and beta crystallin genes were downregulated at both transcriptional (alphaA, alphaB, betaA1/A3, betaA4, and betaB2 approximately 50% and betaA2~40%) and protein (alphaA~50%, alphaB~63%, betaA1/A3~70%, and betaB2~38%) levels 2 weeks after IOP elevation. In experimental retinas 5 weeks after IOP elevation, the levels of crystallin mRNAs were higher than at 2 weeks and were comparable to that of control retinas. However, the levels of the corresponding proteins were still lower (alphaA, alphaB, and betaB2 approximately 37% and betaA1/A3~70%) than in control retinas. Furthermore, we found that the expression of these genes in the retina is predominantly localized to the cells in the GCL and to a lesser degree in the INL and ONL. Colocalization of the crystallin-positive and Fluorogold retrogradely labeled cells indicated that the cells expressing alpha and beta crystallins in the GCL are RGCs. In summary, we showed that alpha and beta crystallins are expressed in the retina predominantly by RGCs and that their expression is affected by ocular hypertension.

Activation of autophagy in retinal ganglion cells

Autophagy has been shown to be activated in neuronal cells in response to injury and suggested to have a cell‐protective role in neurodegenerative diseases. In this study, we investigated the activation of autophagy in retinal ganglion cells (RGCs) following optic nerve transection (ONT) and evaluated its effect on RGC survival. Expression of several autophagy‐related genes, including Atg5, Atg7, and Atg12, and autophagy markers microtubule‐associated protein 1 light chain 3–II (LC3‐II) and beclin‐1 were analyzed at the transcriptional or protein level 1, 3, and 7 days after ONT. Transcription of the Atg5, Atg7, and Atg12 genes was up‐regulated 1.5‐ to 1.8‐fold in the retina 3 days after ONT compared with that in the controls. Expression of Atg12 mRNA was increased 1.6‐fold 1 day after ONT. Seven days after ONT, expression of Atg5, Atg7, and Atg12 mRNA was comparable to that in the untreated retinas. Western blot analysis of proteins isolated from RGCs showed 1.6‐, 2.7‐, and 1.7‐fold increases in LC3‐II level 1, 3, and 7 days after ONT, respectively, compared with those in the controls. Expression of beclin‐1 was 1.7‐fold higher 1 day after RGCs were axotomized, but 3 and 7 days after ONT it was comparable to that of the control. Inhibition of autophagy with bafilomycin A1, 3‐methyladenine, and Wortmannin in RGC‐5 cells under serum‐deprived conditions decreased cell viability by approximately 40%. These results suggest possible activation of autophagy in RGCs after optic nerve transection and demonstrate its protective role in RGC‐5 cells maintained under conditions of serum deprivation.

Protective effect of thioredoxins 1 and 2 in retinal ganglion cells after optic nerve transection and oxidative stress.

PURPOSE Oxidative stress has been implicated in retinal ganglion cell (RGC) death pathways after optic nerve transection (ONT) and during glaucomatous neuropathy. The authors investigated the expression and cell-protective roles of thioredoxins (cytosolic Trx1 and mitochondrial Trx2), important regulators of the cellular redox state, on RGCs after ONT and pharmacologic oxidative stress induction. METHODS ONT was performed on adult Wistar rats. Trx1 and Trx2 quantitative and spatial expression were examined with Western blot and immunohistochemistry, respectively. Electroporation and calcium phosphate-mediated procedures were used to deliver Trx1 and Trx2 expression constructs to RGCs in vivo and to cultured RGC-5 cells, respectively. Cell-protective effects of Trx1 and Trx2 overexpression on RGCs after ONT and on RGC-5 cells treated with glutamate/buthionine sulfoximine (BSO) were determined by RGC density analysis and cell viability assay, respectively. RESULTS Upregulation of Trx1 and Trx2 was observed in RGCs at different times after ONT and in RGC-5 cells after glutamate/BSO treatment. Trx1 and Trx2 overexpression in RGC-5 cells increased their survival rate by approximately twofold and threefold 24 and 48 hours after glutamate/BSO treatment, respectively. A neuroprotective effect of Trx1 and Trx2 overexpression on RGCs was also observed in vivo; the survival rate of RGCs was increased by 35% and 135%, respectively, 1 and 2 weeks after ONT. CONCLUSIONS These findings provide evidence for in vitro and in vivo cell-protective effects of Trx1 and Trx2 on RGCs against oxidative stress-induced neurodegeneration.

The Role of αA- and αB-Crystallins in the Survival of Retinal Ganglion Cells after Optic Nerve Axotomy

PURPOSE Stress-induced crystallin expression is commonly viewed as activation of the cell survival mechanism. The authors analyzed the expression of alphaA- and alphaB-crystallins in a rat optic nerve transection (ONT) model characterized by specific retinal ganglion cell (RGC) degeneration and determined their role in RGC survival. METHODS ONT was performed on adult Wistar rats. Quantitative and spatial expression were examined with Western blot analysis and immunohistochemistry, respectively. Electroporation was used to deliver alphaA and alphaB expression constructs to RGCs. Cell-protective effects of alphaA and alphaB overexpression after ONT were determined by RGC density analysis. RESULTS Expression of alphaA and alphaB in the retina was observed predominantly in the ganglion cell layer, where most crystallin-positive cells were colocalized with RGCs. Levels of alphaA and alphaB proteins after ONT were decreased 1.6-fold. The effect of alphaA and alphaB overexpression on RGC survival was evaluated 7 and 14 days after axotomy. At day 7 after ONT, 1426 +/- 70 and 1418 +/- 81 RGCs/mm(2) were present in retinas electroporated with alphaA and alphaB expression constructs, respectively, compared with 1010 +/- 121 RGCs/mm(2) in sham-transfected or 1016 +/- 88 RGCs/mm(2) in nontransfected retinas. Numbers of surviving RGCs at 14 days were 389 +/- 57 and 353.57 +/- 60 cells/mm(2) after alphaA and alphaB transfection, respectively, compared with 198 +/- 29 cells/mm(2) after transfection with the vector alone or 206 +/- 60 cells/mm(2) in nontransfected retinas. CONCLUSIONS Increases of approximately 95% and 75% in RGC survival mediated by alphaA and alphaB overexpression, respectively, were observed 14 days after ONT. At day 7, the RGC protective effect of alphaA and alphaB overexpression was approximately 40%.

Hyperthermic pre-conditioning protects retinal neurons from N-methyl-d-aspartate (NMDA)-induced apoptosis in rat

Glutamate-induced excitotoxicity is associated with a selective loss of retinal neurons after retinal ischemia and possibly in glaucoma. Since heat shock protein (HSP) 70 is known to play a protective role against ischemic neuronal injury, which is also linked to excitotoxicity, we studied the expression of inducible (HSP72) and constitutive (HSC70) forms of HSP70 in apoptosis of retinal ganglion cells (RGCs) after intravitreal injection of 8 nmoles N-methyl-D-aspartate (NMDA), a glutamate receptor agonist. Approximately 18 h after NMDA injection, there were increased numbers of TUNEL-positive cells and cells with elevated HSP72 immunoreactivity in the retinal ganglion cell layer (RGCL), but there were no noticeable changes in HSC70 immunoreactivity. These HSPs positive cells were also Thy-1 positive, a marker for RGCs. Hyperthermic pre-conditioning, which is known to induce HSPs, given 6 or 12 h prior to NMDA injection ameliorated neuronal loss in the RGCL as counted 7 days after NMDA injection but pre-conditioning at 18 h prior to NMDA injection did not have any ameliorative effect. Quercetin, an inhibitor of HSP synthesis, abolished the ameliorative effect of hyperthermic pre-conditioning. Pre-conditioning elevated HSP72 but not HSC70 immunoreactivity and reduced the number of TUNEL-positive cells in the RGCL at 18 h. Our results suggest that intravitreal injection of NMDA induces an up-regulation of HSP72 in a time-dependent manner but not HSC70 in RGCs, indicating a stress response of HSP72 in RGCs and other inner retinal neurons after exposure to NMDA. Hyperthermic pre-conditioning given within a therapeutic window is neuroprotective to the retina against NMDA-induced excitotoxicity, likely by inhibiting apoptosis through the modulation of HSP72 expression.

Severe Neurologic Impairment in Mice with Targeted Disruption of the Electrogenic Sodium Bicarbonate Cotransporter NBCe2 (Slc4a5 Gene)

The choroid plexus lining the four ventricles in the brain is where the majority of cerebrospinal fluid (CSF) is produced. The secretory function of the choroid plexus is mediated by specific transport systems that allow the directional flux of nutrients and ions into the CSF and the removal of toxins. Normal CSF dynamics and chemistry ensure that the environment for neural function is optimal. Here, we report that targeted disruption of the Slc4a5 gene encoding the electrogenic sodium bicarbonate cotransporter NBCe2 results in significant remodeling of choroid plexus epithelial cells, including abnormal mitochondrial distribution, cytoskeletal protein expression, and ion transporter polarity. These changes are accompanied by very significant abnormalities in intracerebral ventricle volume, intracranial pressure, and CSF electrolyte levels. The Slc4a5−/− mice are significantly more resistant to induction of seizure behavior than wild-type controls. In the retina of Slc4a5−/− mice, loss of photoreceptors, ganglion cells, and retinal detachment results in visual impairment assessed by abnormal electroretinogram waveforms. Our findings are the first demonstration of the fundamental importance of NBCe2 in the biology of the nervous system.

Quantitative Analysis of Retinal Ganglion Cell Survival with Rbpms Immunolabeling in Animal Models of Optic Neuropathies

PURPOSE To investigate whether a recently described retinal ganglion cell (RGC) marker Rbpms (RNA binding protein with multiple splicing) could be used for RGC quantification in various models of RGC degeneration. METHODS Optic nerve crush, excitotoxicity, and elevated intraocular pressure (IOP) rat models were used. Topographic analysis of Rbpms immunolabeling was performed on retinal wholemounts. Retrograde labelings with Fluorogold (FG) and III β-tubulin immunohistochemistry were compared. RESULTS In the optic nerve crush model, 37%, 87%, and 93% of Rbpms-positive cells were lost 1, 2, and 4 weeks, respectively. Significant loss of Rbpms-positive cells was noted 1 week after intravitreal injection of 12, 30, and 120 nmol N-methyl-d-aspartate (NMDA), whereas coinjection of 120 nmol of NMDA along with MK-801 increased the cell number from 10% to 59%. Over 95% of Rbpms-positive cells were FG- and III β-tubulin-positive after injury caused by optic nerve crush and NMDA injection. In rats with elevated IOP, induced by trabecular laser photocoagulation, there was a significant loss of Rbpms-positive cells compared with that of contralateral controls (P = 0.0004), and cumulative IOP elevation showed a strong linear relationship with the quantification of RGCs by Rbpms immunolabeling and retrograde labeling with FG. More than 99% of the remaining Rbpms-positive cells were double-labeled with FG. CONCLUSIONS Rbpms can reliably be used as an RGC marker for quantitative evaluation in rat models of RGC degeneration, regardless of the nature and the location of the primary site of the injury and the extent of neurodegeneration.