Zhuo Yh

An agonistic TrkB mAb causes sustained TrkB activation, delays RGC death, and protects the retinal structure in optic nerve axotomy and in glaucoma.

PURPOSE Brain-derived neurotrophic factor (BDNF) receptors TrkB and p75(NTR) are expressed in the retina. However, exogenous BDNF does not provide retinal ganglion cells (RGCs) with long-lasting neuroprotection in vivo during optic nerve axotomy or in glaucoma rat models of neurodegeneration. The authors set out to answer the hypothesis that a selective TrkB agonist might afford more efficient neuroprotection. METHODS Animal models of acute neurodegeneration (complete optic nerve axotomy) and chronic neurodegeneration (ocular hypertension, glaucoma) were used. After intravitreal delivery of test agents or controls, surviving RGCs were quantified. Transient or sustained activation of TrkB receptors in vivo was quantified by Western blot analysis retinal samples for TrkB-phosphotyrosine. Time-dependent changes to the neuronal retinal layers were quantified longitudinally by Fourier domain-optical coherence tomography. RESULTS The authors show that a selective TrkB agonist caused long-lived TrkB activation and significantly delayed RGC death in these models of acute and chronic retinal injury in vivo. Importantly, using noninvasive retinal imaging, they also show that a selective TrkB agonist caused preservation of the retinal structure in both animal models, with maintenance of the layers comprising neurons and neuronal fibers. CONCLUSIONS In animal models of acute and chronic neurodegeneration, a TrkB agonist affords long-lasting neuroprotection by causing sustained TrkB activation. The use of structural end points could have prognostic value to evaluate neuroprotection. This work contributes to the understanding of neurotrophic mechanisms underlying RGC death in glaucoma and optic nerve axotomy.

In Glaucoma the Upregulated Truncated TrkC.T1 Receptor Isoform in Glia Causes Increased TNF-α Production, Leading to Retinal Ganglion Cell Death

PURPOSE Glaucoma is a distinct neuropathy characterized by the chronic and progressive death of retinal ganglion cells (RGCs). The etiology of RGC death remains unknown. Risk factors for glaucomatous RGC death are elevated intraocular pressure and glial production of tumor necrosis factor-alpha (TNF-α). Previously, the authors showed that glaucoma causes a rapid upregulation of a neurotrophin receptor truncated isoform lacking the kinase domain, TrkC.T1, in retina. Here they examined the biological role of TrkC.T1 during glaucoma progression. METHODS Rat and mouse models of chronic ocular hypertension were used. Immunofluorescence Western blot analysis and in situ mRNA hybridization were used to identify cells upregulating TrkC.T1. A genetic model of engineered mice lacking TrkC.T1 (TrkC.T1(-/-)) was used to validate a role for this receptor in glaucoma. Pharmacologic studies were conducted to evaluate intravitreal delivery of agonists or antagonists of TrkC.T1, compared with controls, during glaucoma. Surviving RGCs were quantified by retrograde-labeling techniques. Production of neurotoxic TNF-α and α2 macroglobulin were quantified. RESULTS TrkC.T1 was upregulated in retinal glia, with a pattern similar to that of TNF-α. TrkC.T1(-/-) mice had normal retinas. However, during experimental glaucoma, TrkC.T1(-/-) mice had lower rates of RGC death and produced less TNF-α than wild-type littermates. In rats with glaucoma, the pharmacologic use of TrkC antagonists delayed RGC death and reduced the production of retinal TNF-α. CONCLUSIONS TrkC.T1 is implicated in glaucomatous RGC death through the control of glial TNF-α production. Overall, the data point to a paracrine mechanism whereby elevated intraocular pressure upregulated glial TrkC.T1 expression in glia; TrkC.T1 controlled glial TNF-α production, and TNF-α caused RGC death.

Mitochondria-Targeted Peptide MTP-131 Alleviates Mitochondrial Dysfunction and Oxidative Damage in Human Trabecular Meshwork Cells

PURPOSE To investigate the antioxidative ability of a novel mitochondria-targeted peptide MTP-131 in immortalized human trabecular meshwork (iHTM) and glaucomatous human trabecular meshwork (GTM(3)) cell lines. METHODS Cultured iHTM and GTM(3) cells were pretreated with MTP-131 for 1 hour, and sustained oxidative stress was induced by subjecting TM cells to 200 μM hydrogen peroxide (H(2)O(2)) for 24 hours. Untreated cells and cells incubated with H(2)O(2) alone were used as controls. Lactate dehydrogenase (LDH) assay was used to determine cell viability. Changes of mitochondrial membrane potential (ΔΨm) and generation of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry and confocal microscopy. Activation of caspase 3 was quantified by Western blotting, and apoptosis was measured by flow cytometry. Release of cytochrome c and changes in cytoskeleton were analyzed by confocal microscopy. Data were analyzed with commercial data analysis software and P < 0.05 was considered to be statistically significant. RESULTS In both iHTM and GTM(3) cells, decrease of ΔΨm and elevation of intracellular ROS were detected after sustained oxidative stress induced by H(2)O(2). When cells were pretreated with MTP-131, the H(2)O(2)-induced mitochondrial depolarization was prevented; intracellular ROS, LDH release, and apoptosis were significantly decreased; release of cytochrome c from mitochondria to cytoplasm and activation of caspase 3 were inhibited. In addition, cytoskeleton changes caused by H(2)O(2) were also alleviated by MTP-131. CONCLUSIONS Mitochondria-targeted peptide MTP-131 could prevent both iHTM and GTM(3) cells from sustained oxidative stress induced by H(2)O(2).

Using neurogenin to Reprogram Chick RPE to Produce Photoreceptor-like Neurons

PURPOSE One potential therapy for vision loss from photoreceptor degeneration is cell replacement, but this approach presents a need for photoreceptor cells. This study explores whether the retinal pigment epithelium (RPE) could be a convenient source of developing photoreceptors. METHODS The RPE of chick embryos was subjected to reprogramming by proneural genes neurogenin (ngn)1 and ngn3. The genes were introduced into the RPE through retrovirus RCAS-mediated transduction, with the virus microinjected into the eye or added to retinal pigment epithelial explant culture. The retinal pigment epithelia were then analyzed for photoreceptor traits. RESULTS In chick embryos infected with retrovirus RCAS-expressing ngn3 (RCAS-ngn3), the photoreceptor gene visinin (the equivalent of mammalian recoverin) was expressed in cells of the retinal pigment epithelial layer. When isolated and cultured as explants, retinal pigment epithelial tissues from embryos infected with RCAS-ngn3 or RCAS-ngn1 gave rise to layers of visinin-positive cells. These reprogrammed cells expressed genes of phototransduction and synapses, such as red opsin, the alpha-subunit of cone transducin, SNAP-25, and PSD-95. Reprogramming occurred with retinal pigment epithelial explants derived from virally infected embryos and with retinal pigment epithelial explants derived from normal embryos, with the recombinant viruses added at the onset of the explant culture. In addition, reprogramming took place in retinal pigment epithelial explants from both young and old embryos, from embryonic day (E)6 to E18, when the visual system becomes functional in the chick. CONCLUSIONS The results support the prospect of exploring the RPE as a convenient source of developing photoreceptors for in situ cell replacement.

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimers disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48h after 5% O(2) hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.