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Abstract #108016 Published in IGR 23-4
Deficiency of the RNA-binding protein ELAVL1/HuR leads to the failure of endogenous and exogenous neuroprotection of retinal ganglion cells
Pacwa A; Machowicz J; Akhtar S; Rodak P; Liu X; Pietrucha-Dutczak M; Lewin-Kowalik J; Amadio M; Smedowski AFrontiers in cellular neuroscience 2023; 17: 1131356
INTRODUCTION: 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 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 -silenced RGC in the rat glaucoma model. METHODS: The study consisted of and approaches. , we used rat B-35 cells to investigate, whether AAV-shRNA-HuR delivery affects survival and oxidative stress markers under temperature and excitotoxic insults. 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. RESULTS: Silencing of induced apoptosis and increased oxidative stress markers in B-35 cells. Additionally, shRNA treatment impaired the cellular stress response to temperature and excitotoxic insults. , 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. CONCLUSIONS: 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.
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