The isolation and culturing of rodent retinal ganglion cells (RGC) is a key step in studying the function and cellular response of this crucial cell type. Typical methods used for isolation of RGCs include immunopanning or magnetic bead separation with antibodies targeting RGC specific protein markers. However, in developmental research, many of the most common markers, such as Thy-1, are not expressed in early stages of development. To help study these crucial early stage RGCs, we have developed a novel method that utilizes a transgenic mouse with a GFP tag on the protein BRN3 and a low-pressure fluorescence-activated cell sorter (FACS) system.

UNASSIGNED: ELAVL1/HuR is a keystone regulator of gene expression at the posttranscriptional level, including stress response and homeostasis maintenance. The aim of this study was to evaluate the impact of hur silencing on the age-related degeneration of retinal ganglion cells (RGC), which potentially describes the efficiency of endogenous neuroprotection mechanisms, as well as to assess the exogenous neuroprotection capacity of hur-silenced RGC in the rat glaucoma model.
UNASSIGNED: The study consisted of in vitro and in vivo approaches. In vitro, we used rat B-35 cells to investigate, whether AAV-shRNA-HuR delivery affects survival and oxidative stress markers under temperature and excitotoxic insults. In vivo approach consisted of two different settings. In first one, 35 eight-week-old rats received intravitreal injection of AAV-shRNA-HuR or AAV-shRNA scramble control. Animals underwent electroretinography tests and were sacrificed 2, 4 or 6 months after injection. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. For the second approach, animals received similar gene constructs. To induce chronic glaucoma, 8 weeks after AAV injection, unilateral episcleral vein cauterization was performed. Animals from each group received intravitreal injection of metallothionein II. Animals underwent electroretinography tests and were sacrificed 8 weeks later. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology.
UNASSIGNED: Silencing of hur induced apoptosis and increased oxidative stress markers in B-35 cells. Additionally, shRNA treatment impaired the cellular stress response to temperature and excitotoxic insults. In vivo, RGC count was decreased by 39% in shRNA-HuR group 6 months after injection, when compared to shRNA scramble control group. In neuroprotection study, the average loss of RGCs was 35% in animals with glaucoma treated with metallothionein and shRNA-HuR and 11.4% in animals with glaucoma treated with metallothionein and the scramble control shRNA. An alteration in HuR cellular content resulted in diminished photopic negative responses in the electroretinogram.
UNASSIGNED: Based on our findings, we conclude that HuR is essential for the survival and efficient neuroprotection of RGC and that the induced alteration in HuR content accelerates both the age-related and glaucoma-induced decline in RGC number and function, further confirming HuR\'s key role in maintaining cell homeostasis and its possible involvement in the pathogenesis of glaucoma.

暂无摘要。

暂无摘要。

Amyloid-beta (Aβ)-related alterations, similar to those found in the brains of patients with Alzheimer\'s disease, have been observed in the retina of patients with glaucoma. Decreased levels of brain-derived neurotrophic factor (BDNF) are believed to be associated with the neurotoxic effects of Aβ peptide. To investigate the mechanism underlying the neuroprotective effects of BDNF on Aβ1-40-induced retinal injury in Sprague-Dawley rats, we treated rats by intravitreal administration of phosphate-buffered saline (control), Aβ1-40 (5 nM), or Aβ1-40 (5 nM) combined with BDNF (1 µg/mL). We found that intravitreal administration of Aβ1-40 induced retinal ganglion cell apoptosis. Fluoro-Gold staining showed a significantly lower number of retinal ganglion cells in the Aβ1-40 group than in the control and BDNF groups. In the Aβ1-40 group, low number of RGCs was associated with increased caspase-3 expression and reduced TrkB and ERK1/2 expression. BDNF abolished Aβ1-40-induced increase in the expression of caspase-3 at the gene and protein levels in the retina and upregulated TrkB and ERK1/2 expression. These findings suggest that treatment with BDNF prevents RGC apoptosis induced by Aβ1-40 by activating the BDNF-TrkB signaling pathway in rats.

Optic atrophy (OA) with autosomal inheritance is a form of optic neuropathy characterized by the progressive and irreversible loss of vision. In some cases, this is accompanied by additional, typically neurological, extra-ocular symptoms. Underlying the loss of vision is the specific degeneration of the retinal ganglion cells (RGCs) which form the optic nerve. Whilst autosomal OA is genetically heterogenous, all currently identified causative genes appear to be associated with mitochondrial organization and function. However, it is unclear why RGCs are particularly vulnerable to mitochondrial aberration. Despite the relatively high prevalence of this disorder, there are currently no approved treatments. Combined with the lack of knowledge concerning the mechanisms through which aberrant mitochondrial function leads to RGC death, there remains a clear need for further research to identify the underlying mechanisms and develop treatments for this condition. This review summarizes the genes known to be causative of autosomal OA and the mitochondrial dysfunction caused by pathogenic mutations. Furthermore, we discuss the suitability of available in vivo models for autosomal OA with regards to both treatment development and furthering the understanding of autosomal OA pathology.

More than 30 years after discovering Leber\'s hereditary optic neuropathy (LHON) as the first maternally inherited disease associated with homoplasmic mtDNA mutations, we still struggle to achieve effective therapies. LHON is characterized by selective degeneration of retinal ganglion cells (RGCs) and is the most frequent mitochondrial disease, which leads young people to blindness, in particular males. Despite that causative mutations are present in all tissues, only a specific cell type is affected. Our deep understanding of the pathogenic mechanisms in LHON is hampered by the lack of appropriate models since investigations have been traditionally performed in non-neuronal cells. Effective in-vitro models of LHON are now emerging, casting promise to speed our understanding of pathophysiology and test therapeutic strategies to accelerate translation into clinic. We here review the potentials of these new models and their impact on the future of LHON patients.

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β-catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co-cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt-dependent target proteins that may mediate the axonal growth.

For years it has been known that unilateral optic nerve lesions induce a bilateral response that causes an inflammatory and microglial response in the contralateral un-injured retinas. Whether this contralateral response involves retinal ganglion cell (RGC) loss is still unknown. We have analyzed the population of RGCs and the expression of several genes in both retinas of pigmented mice after a unilateral axotomy performed close to the optic nerve head (0.5 mm), or the furthest away that the optic nerve can be accessed intraorbitally in mice (2 mm). In both retinas, RGC-specific genes were down-regulated, whereas caspase 3 was up-regulated. In the contralateral retinas, there was a significant loss of 15% of RGCs that did not progress further and that occurred earlier when the axotomy was performed at 2 mm, that is, closer to the contralateral retina. Finally, the systemic treatment with minocycline, a tetracycline antibiotic that selectively inhibits microglial cells, or with meloxicam, a non-steroidal anti-inflammatory drug, rescued RGCs in the contralateral but not in the injured retina. In conclusion, a unilateral optic nerve axotomy triggers a bilateral response that kills RGCs in the un-injured retina, a death that is controlled by anti-inflammatory and anti-microglial treatments. Thus, contralateral retinas should not be used as controls.

Glaucoma is a common cause of visual impairment and blindness, characterized by retinal ganglion cell (RGC) death. The mechanisms that trigger the development of glaucoma remain unknown and have gained significant relevance in the study of this neurodegenerative disease. P2X7 purinergic receptors (P2X7R) could be involved in the regulation of the synaptic transmission and neuronal death in the retina through different pathways. The aim of this study was to characterize the molecular signals underlying glaucomatous retinal injury. The time-course of functional, morphological, and molecular changes in the glaucomatous retina of the DBA/2J mice were investigated. The expression and localization of P2X7R was analysed in relation with retinal markers. Caspase-3, JNK, and p38 were evaluated in control and glaucomatous mice by immunohistochemical and western-blot analysis. Furthermore, electroretinogram recordings (ERG) were performed to assess inner retina dysfunction. Glaucomatous mice exhibited changes in P2X7R expression as long as the pathology progressed. There was P2X7R overexpression in RGCs, the primary injured neurons, which correlated with the loss of function through ERG measurements. All analyzed MAPK and caspase-3 proteins were upregulated in the DBA/2J retinas suggesting a pro-apoptotic cell death. The increase in P2X7Rs presence may contribute, together with other factors, to the changes in retinal functionality and the concomitant death of RGCs. These findings provide evidence of possible intracellular pathways responsible for apoptosis regulation during glaucomatous degeneration.