Kathryn E. Bollinger

Quantitative Proteomics: TGFβ2 Signaling in Trabecular Meshwork Cells

PURPOSE Transforming growth factor beta 2 (TGFβ₂) is often elevated in the aqueous humor (AH) and trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) and appears to contribute to POAG pathogenesis. To better understand TGFβ₂ signaling in the eye, TGFβ₂-induced proteomic changes were identified in cells cultured from the TM, a tissue involved in intraocular pressure (IOP) elevation in glaucoma. METHODS Primary cultures of human TM cells from four donors were treated with or without TGFβ₂ (5 ng/mL) for 48 hours; then cellular protein was analyzed by liquid chromatography-mass spectrometry iTRAQ (isobaric tags for relative and absolute quantitation) technology. RESULTS A total of 853 proteins were quantified. TGFβ₂ treatment significantly altered the abundance of 47 proteins, 40 of which have not previously been associated with TGFβ₂ signaling in the eye. More than half the 30 elevated proteins support growing evidence that TGFβ₂ induces extracellular matrix remodeling and abnormal cytoskeletal interactions in the TM. The levels of 17 proteins were reduced, including four cytoskeletal and six regulatory proteins. Both elevated and decreased regulatory proteins implicate TGFβ₂-altered processes involving transcription, translation, and the glutamate/glutamine cycle. Altered levels of eight mitochondrial proteins support TGFβ₂-induced mitochondrial dysfunction in the TM that in POAG could contribute to oxidative damage in the AH outflow pathway, TM senescence, and elevated IOP. CONCLUSIONS The results expand the repertoire of proteins known to participate in TGFβ₂ signaling, provide new molecular insight into POAG, and establish a quantitative proteomics database for the TM that includes candidate glaucoma biomarkers for future validation studies.

Late-onset inner retinal dysfunction in mice lacking sigma receptor 1 (σR1).

PURPOSE Sigma receptor 1 (σR1) is expressed abundantly in the eye, and several reports suggest that this putative molecular chaperone plays a role in lens cell survival, control of intraocular pressure (IOP), and retinal neuroprotection. The present study examined the consequence of the absence of σR1 on ocular development, structure, and function. METHODS Wild-type (σR1⁺/⁺), heterozygous (σR1⁺/⁻), and homozygous (σR1⁻/⁻, knockout) mice aged 5 to 59 weeks were subjected to comprehensive electrophysiological testing and IOP measurement. The eyes were examined by light and electron microscopy and subjected to morphometric examination and detection of apoptosis. RESULTS Cornea and lens of σR1⁻/⁻ mice were similar to wild-type mice in morphologic appearance at all ages examined, and IOP was within normal limits. Comprehensive ERG and morphometric analyses initially yielded normal findings in the σR1⁻/⁻ mice compared with those in the wild-type. By 12 months, however, significantly decreased ERG b-wave amplitudes and diminished negative scotopic threshold responses, consistent with inner retinal dysfunction, were detected in σR1⁻/⁻ mice. Concomitant with these late-onset changes were increased TUNEL- and active caspase 3-positive cells in the inner retina and significant loss of cells in the ganglion cell layer, particularly in the central retina. Before these functional and structural abnormalities, there was ultrastructural evidence of axonal disruption in the optic nerve head of σR1⁻/⁻ mice as early as 6 months of age, although there were no alterations observed in retinal vascularization in σR1⁻/⁻ mice. CONCLUSIONS These data suggest that lack of σR1 leads to development of late-onset retinal dysfunction with similarities to optic neuropathy.

Sigma Receptor Ligand, (+)-Pentazocine, Suppresses Inflammatory Responses of Retinal Microglia

PURPOSE To evaluate the effects of the σ 1 receptor (σR1) agonist, (+)-pentazocine, on lipopolysaccharide (LPS)-induced inflammatory changes in retinal microglia cells. METHODS Retinal microglia cells were isolated from Sprague-Dawley rat pups. Cells were treated with LPS with or without (+)-pentazocine and with or without the σR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor α (TNF-α), interleukin 10, (IL-10), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO) were determined. Reactive oxygen species (ROS) formation was assayed, and levels of mitogen-activated protein kinases (MAPKs) were analyzed by using Western blot. RESULTS The σR1 protein was expressed in retinal microglia. Incubation with LPS and/or (+)-pentazocine did not alter cell viability or σR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-α, IL-10, MCP-1, and NO. Pretreatment with (+)-pentazocine inhibited the LPS-induced morphologic changes. Release of TNF-α, IL-10, MCP-1, and NO was reduced with (+)-pentazocine. Intracellular ROS formation was suppressed with (+)-pentazocine. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (+)-pentazocine. The σR1 antagonist BD1063 blocked the (+)-pentazocine-mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (+)-pentazocine-mediated suppression of LPS-induced TNF-α, IL-10, MCP-1, NO, and intracellular ROS release. CONCLUSIONS Treatment with (+)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (+)-pentazocine may exert neuroprotective effects through manipulation of microglia.

Prevalence and management of elevated intraocular pressure after placement of an intravitreal sustained-release steroid implant.

Purpose of review The fluocinolone acetonide intravitreal implant was approved by the United States Food and Drug Administration in April, 2005 for treatment of noninfectious posterior segment uveitis (NIPU). This therapy is effective with respect to prevention of uveitis recurrence. However, corticosteroid-associated increased intraocular pressure frequently occurs in implanted eyes and must be managed appropriately to prevent glaucomatous vision loss. Recent findings Pooled results from three recent prospective randomized trials show that 75% of eyes receiving the fluocinolone acetonide implant required intraocular pressure (IOP) lowering therapy at some point within the 3-year study course. Eyes requiring surgical intervention had a high rate of hypotony but also showed stable postoperative visual acuity. Summary Fluocinolone acetonide intravitreal implants are an effective therapy for NIPU. However, patients receiving this treatment are at high risk for development of vision-threatening increased IOP. Therefore, we recommend that patients treated with fluocinolone acetonide implants receive frequent IOP monitoring and referral to a glaucoma specialist if pressure control is not achieved.

Intraocular Pressure Outcome of Patients with Fluocinolone Acetonide Intravitreal Implant for Noninfectious Uveitis

PURPOSE To evaluate the intraocular pressure (IOP) outcomes of patients with noninfectious posterior uveitis treated with a fluocinolone acetonide (FA) intravitreal implant over an 8-year period. DESIGN Retrospective clinical case series. PARTICIPANTS Forty-seven eyes of 35 patients. METHODS Retrospective review of medical records of all patients receiving FA implants between June 2001 and March 2009 was performed. Data were pooled to record visual acuity (VA) and to characterize the incidence and outcome of glaucoma surgical intervention. MAIN OUTCOME MEASURES Incidence of glaucoma surgery and IOP before and after glaucoma surgical intervention. Visual acuity before and after FA implant placement. RESULTS Nineteen of 42 eyes (45%) receiving FA implants over the course of the study period required glaucoma surgical intervention. Success of IOP-lowering surgery was achieved in 94%, 94%, and 92% of eyes at 6, 12, and 24 months postoperatively. No patient lost more than 1 line of VA. There was an average 2-line gain of acuity at 3 years after FA implant placement for those patients who underwent IOP-lowering surgery. CONCLUSIONS Patients receiving FA implants have a significant risk of increased IOP that frequently necessitates glaucoma surgery. Glaucoma surgery, when necessary, offers an effective method to decrease IOP. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosure may be found after the references.

Diabetes and Overexpression of proNGF Cause Retinal Neurodegeneration via Activation of RhoA Pathway

Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotosin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75NTR, cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75NTR and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75NTR expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.

Immunocytochemical evidence of Tulp1-dependent outer segment protein transport pathways in photoreceptor cells

Tulp1 is a protein of unknown function exclusive to rod and cone photoreceptor cells. Mutations in the gene cause autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In tulp1-/- mice, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment, indicating that Tulp1 is involved in protein transport from the inner segment to the outer segment. To investigate this further, we sought to define which outer segment transport pathways are Tulp1-dependent. We used immunohistochemistry to examine the localization of outer segment proteins in tulp1-/- photoreceptors, prior to retinal degeneration. We also surveyed the condition of inner segment organelles and rhodopsin transport machinery proteins. Herein, we show that guanylate cyclase 1 and guanylate cyclase activating proteins 1 and 2 are mislocalized in the absence of Tulp1. Furthermore, arrestin does not translocate to the outer segment in response to light stimulation. Additionally, data from the tulp1-/- retina adds to the understanding of peripheral membrane protein transport, indicating that rhodopsin kinase and transducin do not co-transport in rhodopsin carrier vesicles and phosphodiesterase does not co-transport in guanylate cyclase carrier vesicles. These data implicate Tulp1 in the transport of selective integral membrane outer segment proteins and their associated proteins, specifically, the opsin and guanylate cyclase carrier pathways. The exact role of Tulp1 in outer segment protein transport remains elusive. However, without Tulp1, two rhodopsin transport machinery proteins exhibit abnormal distribution, Rab8 and Rab11, suggesting a role for Tulp1 in vesicular docking and fusion at the plasma membrane near the connecting cilium.

Deletion of thioredoxin‐interacting protein preserves retinal neuronal function by preventing inflammation and vascular injury

Retinal neurodegeneration is an early and critical event in several diseases associated with blindness. Clinically, therapies that target neurodegeneration fail. We aimed to elucidate the multiple roles by which thioredoxin‐interacting protein (TXNIP) contributes to initial and sustained retinal neurodegeneration.

(+)-Pentazocine Reduces NMDA-Induced Murine Retinal Ganglion Cell Death Through a σR1-Dependent Mechanism

Purpose To evaluate, in vivo, the effects of the sigma-1 receptor (σR1) agonist, (+)-pentazocine, on N-methyl-D-aspartate (NMDA)-mediated retinal excitotoxicity. Methods Intravitreal NMDA injections were performed in C57BL/6J mice (wild type [WT]) and σR1−/− (σR1 knockout [KO]) mice. Fellow eyes were injected with phosphate-buffered saline (PBS). An experimental cohort of WT and σR1 KO mice was administered (+)-pentazocine by intraperitoneal injection, and untreated animals served as controls. Retinas derived from mice were flat-mounted and labeled for retinal ganglion cells (RGCs). The number of RGCs was compared between NMDA and PBS-injected eyes for all groups. Apoptosis was assessed using TUNEL assay. Levels of extracellular-signal–regulated kinases (ERK1/2) were analyzed by Western blot. Results N-methyl-D-aspartate induced a significant increase in TUNEL-positive nuclei and a dose-dependent loss of RGCs. Mice deficient in σR1 showed greater RGC loss (≈80%) than WT animals (≈50%). (+)-Pentazocine treatment promoted neuronal survival, and this effect was prevented by deletion of σR1. (+)-Pentazocine treatment resulted in enhanced activation of ERK at the 6-hour time point following NMDA injection. The (+)-pentazocine–induced ERK activation was diminished in σR1 KO mice. Conclusions Targeting σR1 activation prevented RGC death while enhancing activation of the mitogen-activated protein kinase (MAPK), ERK1/2. Sigma-1 receptor is a promising therapeutic target for retinal neurodegenerative diseases.

Efficient Endocytic Uptake and Maturation in Drosophila Oocytes Requires Dynamitin/p50

Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. In this study, we examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of Yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. We therefore propose that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor colocalized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation.