Differential Responses of Retinal Neurons and Glia Revealed via Proteomic Analysis on Primary and Secondary Retinal Ganglion Cell Degeneration.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATHTM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage.

Visual Function and Survival of Injured Retinal Ganglion Cells in Aged Rbfox1 Knockout Animals.

Rbfox1 is a multifunctional RNA binding protein that regulates various aspects of RNA metabolism important for neuronal differentiation and normal physiology. Rbfox1 has been associated with neurodevelopmental and neurological conditions as well as age-related neurodegenerative diseases such as Alzheimer's and Parkinson's. We have shown that in mammalian retinas Rbfox1 is expressed in retinal ganglion cells (RGCs) and in amacrine cells (ACs). This study investigates the effect of advanced age (22-month-old mice) on visual function, retinal morphology and survival of injured retinal ganglion cells (RGC) in Rbfox1 knockout (KO) animals. A visual cliff test, which was used to evaluate visual function, showed that 22-month old Rbfox1 KO mice have profound depth perception deficiency. Retinal gross morphology in these animals appeared to be normal. Optic nerve crush (ONC) induced axonal injury resulted in approximately 50% of RGC loss in both Rbfox1 KO and age-matched control animals: the average RGC densities in uninjured control and Rbfox1 KO animals were 6274 ± 1673 cells/mm2 and 6004 ± 1531 cells/mm2, respectively, whereas 1 week after ONC, RGC numbers in the retinas of control and Rbfox1 KO mice were reduced to 2998 ± 858 cells/mm2 and 3036 ± 857 cells/mm2, respectively (Rbfox1 KO vs. Rbfox1 KO + ONC, p < 0.0001 and control vs. control + ONC, p < 0.0001). No significant difference between RGC numbers in Rbfox1 KO + ONC and age-matched control + ONC animals was observed, suggesting that Rbfox1 has no effect on the survival of injured RGCs. Interestingly, however, contrary to a commonly accepted view that the number of RGCs in old (18 month of age) compared to young animals is reduced by approximately 40%, the RGC densities in 22-month-old mice in this study were similar to those of 4-month-old counterparts.

Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics.

To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.

Comparative lipid profiling dataset of the inflammation-induced optic nerve regeneration.

In adult mammals, retinal ganglion cells (RGCs) fail to regenerate following damage. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; this can lead to permanent vision loss and, eventually, blindness. Lipids are crucial for the development and maintenance of cell membranes, myelin sheaths, and cellular signaling pathways, however, little is known about their role in axon injury and repair. Studies examining changes to the lipidome during optic nerve (ON) regeneration could greatly inform treatment strategies, yet these are largely lacking. Experimental animal models of ON regeneration have facilitated the exploration of the molecular determinants that affect RGC axon regeneration. Here, we analyzed lipid profiles of the ON and retina in an ON crush rat model using liquid chromatography-mass spectrometry. Furthermore, we investigated lipidome changes after ON crush followed by intravitreal treatment with Zymosan, a yeast cell wall derivative known to enhance RGC regeneration. This data is available at the NIH Common Fund's Metabolomics Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, the Metabolomics Workbench, http://www.metabolomicsworkbench.org, where it has been assigned Project ID: PR000661. The data can be accessed directly via it's Project DOI: doi: 10.21,228/M87D53.

The effect of celastrol on the ocular hypertension-induced degeneration of retinal ganglion cells.

Celastrol, a quinine methide triterpene extracted from the perennial vine Tripterygium wilfordii, has been identified as a neuroprotective agent in various models of neurodegenerative disorders. We have reported earlier that systemic and intravitreal administration of celastrol stimulate the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC) and that mechanisms underlying celastrol׳s RGC protection may be associated with inhibition of TNF-alpha-mediated cell death. The present study evaluates the effect of celastrol on the survival of RGCs injured by ocular hypertension. Intraocular pressure (IOP) elevation resulted in approximately 23% of RGCs loss. Reduction in RGC numbers was observed in all four retinal quadrants: 30% in superior, 17% in inferior, 11% in nasal and 35% in temporal regions. Celastrol (1 mg/kg) or vehicle (DMSO) was administered three times per week by intraperitoneal injection, starting on the day of laser photocoagulation of the TM and continued for the entire duration of the experiment (5 weeks). Celastrol treatment stimulated RGC survival by an average of 24% in the entire retina compared to the vehicle-treated group. RGC numbers were increased in all four quadrants: approximately 40%, 17%, 15% and 30% more RGCs were counted in the superior, inferior, nasal and temporal regions, respectively. The average RGC numbers for the entire retinas of the celastrol/IOP group were only ∼5% and 10% lower than that in vehicle- or celastrol-injected animals with normal IOP, respectively. Our data indicate a significant celastrol-mediated neuroprotection against elevated IOP-induced injury.

Optic Nerve Regeneration After Crush Remodels the Injury Site: Molecular Insights From Imaging Mass Spectrometry.

Purpose: Mammalian central nervous system axons fail to regenerate after injury. Contributing factors include limited intrinsic growth capacity and an inhibitory glial environment. Inflammation-induced optic nerve regeneration (IIR) is thought to boost retinal ganglion cell (RGC) intrinsic growth capacity through progrowth gene expression, but effects on the inhibitory glial environment of the optic nerve are unexplored. To investigate progrowth molecular changes associated with reactive gliosis during IIR, we developed an imaging mass spectrometry (IMS)-based approach that identifies discriminant molecular signals in and around optic nerve crush (ONC) sites. Methods: ONC was performed in rats, and IIR was established by intravitreal injection of a yeast cell wall preparation. Optic nerves were collected at various postcrush intervals, and longitudinal sections were analyzed with matrix-assisted laser desorption/ionization (MALDI) IMS and data mining. Immunohistochemistry and confocal microscopy were used to compare discriminant molecular features with cellular features of reactive gliosis. Results: IIR increased the area of the crush site that was occupied by a dense cellular infiltrate and mass spectral features consistent with lysosome-specific lipids. IIR also increased immunohistochemical labeling for microglia and macrophages. IIR enhanced clearance of lipid sulfatide myelin-associated inhibitors of axon growth and accumulation of simple GM3 gangliosides in a spatial distribution consistent with degradation of plasma membrane from degenerated axons. Conclusions: IIR promotes a robust phagocytic response that improves clearance of myelin and axon debris. This growth-permissive molecular remodeling of the crush injury site extends our current understanding of IIR to include mechanisms extrinsic to the RGC.

Heat shock proteins in the retina: Focus on HSP70 and alpha crystallins in ganglion cell survival.

Heat shock proteins (HSPs) belong to a superfamily of stress proteins that are critical constituents of a complex defense mechanism that enhances cell survival under adverse environmental conditions. Cell protective roles of HSPs are related to their chaperone functions, antiapoptotic and antinecrotic effects. HSPs' anti-apoptotic and cytoprotective characteristics, their ability to protect cells from a variety of stressful stimuli, and the possibility of their pharmacological induction in cells under pathological stress make these proteins an attractive therapeutic target for various neurodegenerative diseases; these include Alzheimer's, Parkinson's, Huntington's, prion disease, and others. This review discusses the possible roles of HSPs, particularly HSP70 and small HSPs (alpha A and alpha B crystallins) in enhancing the survival of retinal ganglion cells (RGCs) in optic neuropathies such as glaucoma, which is characterized by progressive loss of vision caused by degeneration of RGCs and their axons in the optic nerve. Studies in animal models of RGC degeneration induced by ocular hypertension, optic nerve crush and axotomy show that upregulation of HSP70 expression by hyperthermia, zinc, geranyl-geranyl acetone, 17-AAG (a HSP90 inhibitor), or through transfection of retinal cells with AAV2-HSP70 effectively supports the survival of injured RGCs. RGCs survival was also stimulated by overexpression of alpha A and alpha B crystallins. These findings provide support for translating the HSP70- and alpha crystallin-based cell survival strategy into therapy to protect and rescue injured RGCs from degeneration associated with glaucomatous and other optic neuropathies.

Programmed cell death-1 is expressed in large retinal ganglion cells and is upregulated after optic nerve crush.

Programmed cell death-1 (PD-1) is a key negative receptor inducibly expressed on T cells, B cells and dendritic cells. It was discovered on T cells undergoing classical programmed cell death. Studies showed that PD-1 ligation promotes retinal ganglion cell (RGC) death during retinal development. The purpose of this present study is to characterize PD-1 regulation in the retina after optic nerve crush (ONC). C57BL/6 mice were subjected to ONC and RGC loss was monitored by immunolabelling with RNA-binding protein with multiple splicing (Rbpms). Time course of PD-1 mRNA expression was determined by real-time PCR. PD-1 expression was detected with anti-PD-1 antibody on whole mount retinas. PD-1 staining intensity was quantitated. Colocalization of PD-1 and cleaved-caspase-3 after ONC was analyzed. Real-time PCR results demonstrated that PD-1 gene expression was significantly upregulated at day 1, 3, 7, 10 and 14 after ONC. Immunofluorescent staining revealed a dramatic increase of PD-1 expression following ONC. In both control and injured retinas, PD-1 tended to be up-expressed in a subtype of RGCs, whose somata size were significantly larger than others. Compared to control, PD-1 intensity in large RGCs was increased by 82% in the injured retina. None of the large RGCs expressed cleaved-caspase-3 at day 5 after ONC. Our work presents the first evidence of PD-1 induction in RGCs after ONC. This observation supports further investigation into the role of PD-1 expression during RGC death or survival following injury.

Axonal Transport in the Rat Optic Nerve Following Short-Term Reduction in Cerebrospinal Fluid Pressure or Elevation in Intraocular Pressure.

PURPOSE: To examine the influence of short-term reduction in cerebrospinal fluid pressure (CSFP) as compared with short-term elevation in IOP on axonal transport. METHODS: The study included 111 adult Sprague-Dawley rats. For 6 hours, IOP was unilaterally elevated to 40 mm Hg (IOP40-group; n = 27), IOP was unilaterally increased to a value of 25 mm Hg below the mean blood pressure (PP25-group; n = 27), or CSFP was reduced by continuous aspiration of cerebrospinal fluid (Low-CSFP-group; n = 27). A sham control group (with a trocar in cisterna magna without cerebrospinal fluid release) included 24 rats. The left eyes of the IOP40 study group and PP25 study group served as additional contralateral control group. Orthograde axonal transport was examined by intravitreally injected rhodamine-ß-isothiocyanate; retrograde axoplasmic flow was assessed by fluorogold injected into the superior colliculi. RESULTS: At 24 hours after baseline, rhodamine-ß-isothiocyanate (RITC) staining intensity of the optic nerve was lower (P < 0.05) in the IOP40-group, PP25-group, and Low-CSFP-group than in the control groups. At 6 hours after the fluorogold injection, fluorogold fluorescence was significantly lower in the IOP40-group, the PP25-group, and the Low-CSFP-group than in the control groups. At 5 days after baseline, the fluorogold fluorescence no longer differed significantly between the IOP40-group or the Low-CSFP-group and the control groups. At 1 week after baseline, retinal ganglion cell density was markedly reduced only in the PP25-group. CONCLUSIONS: Both short-term lowering of CSFP and short-term rise in IOP were associated with a disturbance of both the orthograde and retrograde axonal transport. The findings support the notion of an association between abnormally low CSFP and optic nerve damage.

Celastrol supports survival of retinal ganglion cells injured by optic nerve crush.

The present study evaluates the effect of celastrol on the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC). Celastrol, a quinine methide triterpene extracted from the perennial vine Tripterygium wilfordii (Celastraceae), has been identified as a potential neuroprotective candidate in a comprehensive drug screen against various neurodegenerative diseases. Two weeks after ONC, the average density of remaining RGCs in retinas of animals treated with daily intraperitoneal (i.p.) injections of celastrol (1mg/kg) was approximately 1332 cells/mm(2), or 40.8% of the Celastrol/Control group. In retinas of the Vehicle/ONC group about 381 RGCs/mm(2) were counted, which is 9.6% of the total number of RGCs in the DMSO/Control group. This corresponds to approximately a 250% increase in RGC survival mediated by celastrol treatment compared to Vehicle/ONC group. Furthermore, the average RGC number in retinas of ONC animals treated with a single intravitreal injection of 1mg/kg or 5mg/kg of celastrol was increased by approximately 80% (760 RGCs/mm(2)) and 78% (753 RGCs/mm(2)), respectively, compared to Vehicle/ONC controls (422 cells/mm(2)). Injection of 0.2mg/kg of celastrol had no significant effect on cell survival, with the average number of RGCs being 514 cells/mm(2) in celastrol-treated animals versus 422 cells/mm(2) in controls. The expression levels of Hsp70, Hsf1, Hsf2, HO-1 and TNF-alpha in the retina were analyzed to evaluate the roles of these proteins in the celastrol-mediated protection of injured RGCs. No statistically significant change in HO-1, Hsf1 and Hsp70 levels was seen in animals with ONC. An approximately 2 fold increase in Hsf2 level was observed in celastrol-treated animals with or without injury. Hsf2 level was also increased 1.8 fold in DMSO-treated animals with ONC injury compared to DMSO-treated animals with no injury suggesting that Hsf2 induction has an injury-induced component. Expression of TNF-alpha in retinas of celastrol-treated uninjured and ONC animals was reduced by approximately 2 and 1.5 fold compared to vehicle treated animals, respectively. The observed results suggest that mechanisms underlying celastrol׳s RGC protective effect are associated with inhibition of TNF-alpha-mediated cell death.

Bis(zinc-dipicolylamine), Zn-DPA, a new marker for apoptosis.

PURPOSE: To characterize the labeling of apoptotic cells with a molecular probe of bis(zinc(II)-dipicolylamine) (Zn-DPA) conjugated with a fluorescent reporter in a rat model of retinal ganglion cell (RGC) degeneration induced by N-methyl-D-aspartate (NMDA). METHODS: Adult Wistar rats were given unilateral intravitreal injections of 3 µL 40 mM neutralized NMDA and euthanized at 1, 2, 4, 24, and 48 hours. One hour before euthanasia, 3 µL Zn-DPA conjugated with fluorescein (Zn-DPA 480) was intravitreally injected. Prelabeling of RGC with retrograde fluorogold (FG), TUNEL, and immunohistochemistry with III ß-tubulin and vimentin were performed. RESULTS: Fluorescence labeling of Zn-DPA 480 was observed in the retinas from 1 hour up to 24 hours after NMDA injection, whereas the labeling was reduced at 48 hours postinjection. At both 4 and 24 hours postinjection, most Zn-DPA 480-positive cells in the RGC layer were labeled by FG and III ß-tubulin. The number of TUNEL-positive cells increased from 4 to 24 hours. At 24 hours, 95.7% of Zn-DPA 480-positive cells were TUNEL positive, whereas 95.1% of TUNEL-positive cells were Zn-DPA 480 positive. The numbers of Zn-DPA 480-positive cells at 1 and 2 hours after NMDA injection were significantly higher than TUNEL. CONCLUSIONS: Our findings demonstrate that intravitreal injection of fluorescent Zn-DPA 480 labels retinal neurons undergoing apoptosis and that recognition of exposed phosphatidylserine appears earlier than detection of DNA fragmentation, indicating the potential of Zn-DPA as an imaging probe for tracking degenerating retinal neurons.

Crystallins in retinal ganglion cell survival and regeneration.

Crystallins are heterogeneous proteins classified into alpha, beta, and gamma families. Although crystallins were first identified as the major structural components of the ocular lens with a principal function to maintain lens transparency, further studies have demonstrated the expression of these proteins in a wide variety of tissues and cell types. Alpha crystallins (alpha A and alpha B) share significant homology with small heat shock proteins and have chaperone-like properties, including the ability to bind and prevent the precipitation of denatured proteins and to increase cellular resistance to stress-induced apoptosis. Stress-induced upregulation of crystallin expression is a commonly observed phenomenon and viewed as a cellular response mechanism against environmental and metabolic insults. However, several studies reported downregulation of crystallin gene expression in various models of glaucomatous nerodegeneration suggesting that that the decreased levels of crystallins may affect the survival properties of retinal ganglion cells (RGCs) and thus, be associated with their degeneration. This hypothesis was corroborated by increased survival of axotomized RGCs in retinas overexpressing alpha A or alpha B crystallins. In addition to RGC protective functions of alpha crystallins, beta and gamma crystallins were implicated in RGC axonal regeneration. These findings demonstrate the importance of crystallin genes in RGC survival and regeneration and further in-depth studies are necessary to better understand the mechanisms underlying the functions of these proteins in healthy RGCs as well as during glaucomatous neurodegeneration, which in turn could help in designing new therapeutic strategies to preserve or regenerate these cells.

The dark phase intraocular pressure elevation and retinal ganglion cell degeneration in a rat model of experimental glaucoma.

Intraocular pressure (IOP) elevation is considered as a major risk factor causing the progression of vision deterioration in glaucoma. Although it is known that the IOP level changes widely throughout the day and night, how the dark or light phase IOP elevation contributes to retinal ganglion cell (RGC) degeneration is still largely unclear. To examine the profile of IOP, modified laser photocoagulation was applied to the trabecular meshwork of Brown Norway rats and both light and dark phase IOPs were monitored approximately 1-2 times a week. The relationship between IOP elevation and RGC degeneration was investigated while RGC body loss was analyzed with Rbpms immunolabeling on retinal wholemount and axonal injury in the optic nerve was semi-quantified. The baseline awake dark and light IOPs were 30.4 ± 2.7 and 20.2 ± 2.1 mmHg respectively. The average dark IOP was increased to 38.2 ± 3.2 mmHg for five weeks after the laser treatment on 270° trabecular meshwork. However, there was no significant loss of RGC body and axonal injury. After laser treatment on 330° trabecular meshwork, the dark and light IOPs were significantly increased to 43.8 ± 4.6 and 23 ± 3.7 mmHg respectively for 5 weeks. The cumulative dark and light IOP elevations were 277 ± 86 and 113 ± 50 mmHg days respectively while the cumulative total (light and dark) IOP elevation was 213 ± 114 mmHg days. After 5 weeks, regional RGC body loss of 29.5 ± 15.5% and moderate axonal injury were observed. Axonal injury and loss of RGC body had a high correlation with the cumulative total IOP elevation (R(2) = 0.60 and 0.65 respectively). There was an association between the cumulative dark IOP elevation and RGC body loss (R(2) = 0.37) and axonal injury (R(2) = 0.51) whereas the associations between neuronal damages and the cumulative light IOP elevation were weak (for RGC body loss, R(2) = 0.01; for axonal injury, R(2) = 0.26). Simple linear regression model analysis showed statistical significance for the relationships between the total cumulative IOP elevation and RGC body loss (P = 0.009), and axonal injury (P = 0.016). To examine the role of light and dark IOP elevation in RGC body loss and axonal injury, analyses for the association between different light/dark IOP factors and percentage of RGC body loss/axonal injury grading were performed and only the association between the cumulative dark IOP elevation and axonal injury showed statistical significance (P = 0.033). The findings demonstrated that the cumulative total (light and dark) IOP elevation is a risk factor to RGC degeneration in a rat model of experimental glaucoma using modified partial laser photocoagulation at 330° trabecular meshwork. Further investigations are required to understand the role of longer term light and dark phase IOP elevation contributing to the progression of degeneration in different compartments of RGCs.

Neuronal programmed cell death-1 ligand expression regulates retinal ganglion cell number in neonatal and adult mice.

OBJECTIVES: During mouse retina maturation, the final number of retinal ganglion cells (RGCs) is determined by highly regulated programmed cell death. Previous studies demonstrated that the immunoregulatory receptor programmed cell death-1 (PD-1) promotes developmental RGC death. To identify the functional signaling partner(s) for PD-1, we identified retinal expression of PD-1 ligands and examined the effect of PD-1 ligand expression on RGC number. We also explored the hypothesis that PD-1 signaling promotes the development of functional visual circuitry. METHODS: Characterization of retinal and brain programmed cell death-1 ligand 1 (PD-L1) expression were examined by immunofluorescence on tissue sections. The contribution of PD-ligands, PD-L1, and programmed cell death-1 ligand 2 (PD-L2) to RGC number was examined in PD-ligand knockout mice lacking 1 or both ligands. Retinal architecture was assessed by spectral-domain optical coherence tomography, and retinal function was analyzed by electroretinography in wild-type and PD-L1/L2 double-deficient mice. RESULTS: PD-L1 expression is found throughout the neonatal retina and persists in adult RGCs, bipolar interneurons, and Müller glia. In the absence of both PD-ligands, there is a significant numerical increase in RGCs (34% at postnatal day 2 [P2] and 18% in adult), as compared to wild type, and PD-ligands have redundant function in this process. Despite the increased RGC number, adult PD-L1/L2 double-knockout mice have normal retinal architecture and outer retina function. CONCLUSION: This study demonstrates that PD-L1 and PD-L2 together impact the final number of RGCs in adult mice and supports a novel role for active promotion of neuronal cell death through PD-1 receptor-ligand engagement.

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury.

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).

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 ß-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 ß-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.

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.

Thioredoxins 1 and 2 protect retinal ganglion cells from pharmacologically induced oxidative stress, optic nerve transection and ocular hypertension.

Oxidative damage has been implicated in retinal ganglion cell (RGC) death after optic nerve transection (ONT) and during glaucomatous neuropathy. Here, we analyzed the expression and cell protective role of thioredoxins (TRX), key regulators of the cellular redox state, in RGCs damaged by pharmacologically induced oxidative stress, ONT and elevated intraocular pressure (IOP). The endogenous level of thioredoxin-1 (TRX1) and thioredoxin-2 (TRX2) in RGCs after axotomy and in RGC-5 cells after glutamate/buthionine sulfoximine (BSO) treatment showed upregulation of TRX2, whereas no significant change was observed in TRX1 expression. The increased level TRX-interacting protein (TXNIP) in the retinas was observed 2 and 5 weeks after IOP elevation. TRX1 level was decreased at 2 weeks and more prominently at 5 weeks after IOP increase. No change in TRX2 levels in response to IOP change was observed. Overexpression of TRX1 and TRX2 in RGC-5 treated with glutamate/BSO increased the cell survival by 2- and 3-fold 24 and 48 h after treatment, respectively. Overexpression of these proteins in the retina increased the survival of RGCs by 35 and 135% 7 and 14 days after ONT, respectively. In hypertensive eyes, RGC loss was approximately 27% 5 weeks after IOP elevation compared to control. TRX1 and TRX2 overexpression preserved approximately 45 and 37% of RGCs, respectively, that were destined to die due to IOP increase.