MiR-125b-5p Regulates Remodeling Of Retinal Rod Bipolar Cells In RCS Rats Via BDNF

Background:Retinal degeneration is a kind of sight-threaten diseases due to genetic and environmental factors.With the degeneration of photoreceptors,second-order neurons will remodel owing to deafferentation including neuronal death,neuronal migration and neuronal circuit rewiring.It is important to study the remodeling of the retinal neurons for understanding the pathological mechanisms of retinal degeneration and seeking the windowing period of the possible therapeutic strategies.The Royal College of Surgeon(RCS)rat model is a good retinal degeneration model.In RCS rats,retinal pigment epithelial(RPE)cells fail to phagocytose shed photoreceptors outer segments due to a mutation in mertk gene,which lead to progressive photoreceptor degeneration.In the visual information transmission pathway,the optical signal is first transmitted to the retinal bipolar cells via the photoreceptor cells and finally to the ganglion cells.Thus,when photoreceptor cells are degenerated,the retinal remodeling of secondary neurons starts with bipolar cells.Revealing the remodeling response of retinal bipolar cells is important for understanding the retinal degeneration process.However,the significance of retinal bipolar cell remodeling and its molecular regulation mechanism is unclear.Studies suggest miRNAs are not only involved in the retinal degeneration,but also participate in the neuronal development and neuritogenesis.However,there are no reports about whether miRNAs are involved in the ectopic neuritogenesis of retinal rod bipolar cells during the retinal degeneration.Hypothesis: During the retinal degeneration of RCS rats,miRNAs might be involved with the regulation of ectopic neuritogenesis of rod bipolar cells.Object: To study the remodeling and its function of ectopic neuritogenesis of rod bipolar cellsduring the retinal degeneration of RCS rats,and to elucidate the molecular regulation mechanism of this remodeling reaction.Methods and Results: This study consists of three parts:Part One.The study of remodeling of ectopic neuritogenesis of rod bipolar cellsduring the retinal degeneration of RCS rats1.Identification of RCS rat animal model from morphology and function by immunohistochemistry and electroretinogram assays to prove that the death of photoreceptors and decreased electrophysiological function during the retinal degeneration.2.Study of the morphology changes of the axons in the rod bipolar cells of RCS rats and showed that there were no significant changes.3.Study of the morphology changes of the dendrites in the rod bipolar cells of RCS rats and showed that some dendrites retracted and then extended into the outer nuclear layer(which we termed ectopic dendrites).Moreover,the ectopic dendrites formed synaptic connection with remaining photoreceptor cells.It suggested that during retinal degeneration of RCS rats,the dendrites of rod bipolar cells were abnormally protruded,and this abnormal protrusions had information transfer ability.Part Two.Regulatory pathway of ectopic neuritogenesis of rod bipolar cellsduring retinal degeneration of RCS rats1.Study of the miRNAs expression profile by microRNA panel assay and showed that the expression of miR-125b-5p downregulated compared with the time-matched normal rats retinas.It suggested that mi R-125b-5p might be involved in the retinal degeneration of RCS rats.2.By bioinformatic prediction,the target of miR-125b-5p was BDNF which was further proved by dual luciferase reporter gene assay.3.Study of the location of miR-125b-5p and BDNF in the retinas of RCS rats by in situ hybridization immunohistochemistry and showed that the rod bipolar cells were miR-125b-5p and BDNF positive.It suggested that mi R-125b-5p might regulate the remodeling of ectopic neuritogenesis of rod bipolar cells via BDNF during retinal degeneration of RCS rats.Part Three.miR-125b-5p regulates ectopic synaptogenesis of rod bipolar cells in the retina of RCS rats via BDNF1.We studied the effect of the overexpression and downregulation of mi R-125b-5p on the ectopic neuritogenesis of retinal rod bipolar cells.It showed that overexpression of miR-125b-5p in the retinas of RCS rats diminished the ectopic dendrites of RBCs and b-wave of flash electroretinogram was compromised.On the contrary,down-regulation of miR-125b-5p increased the ectopic dendrites of the RBCs and the b-wave of flash ERG was improved.It suggested miR-125b-5p regulated ectopic synaptogenesis of rod bipolar cells during retinal degeneration of RCS rats.2.We studied the effect of the BDNF treatment on the ectopic neuritogenesis of retinal rod bipolar cells.It showed that BDNF treatment increased the ectopic dendrites of the RBCs and the b-wave of flash ERG was improved.It suggested BDNF regulated ectopic synaptogenesis of rod bipolar cells during retinal degeneration of RCS rats.3.We studied the expression of the downstream of BDNF during the retinal degeneration of RCS rats and found that rod bipolar cells were TrkB and CREB positive.Moreover,overexpression of miR-125b-5p down-regulated the expression of Trk B and CREB while BDNF treatment up-regulated the expression of Trk B and CREB.It suggested BDNF-TrkB-CREB regulated ectopic synaptogenesis of rod bipolar cells during retinal degeneration of RCS rats.Conclusions:1.During the retinal degeneration of RCS rats,some dendrites of rod bipolar cells extended into the outer nuclear layer and formed synaptic connection with remaining photoreceptor cells.2.During the retinal degeneration of RCS rats,miR-125b-5p regulated ectopic neuritogenesis in the rod bipolar cells through modulation of BDNF-TrkB-CREB pathway.3.Down-regulation of miR-125b-5p or exogenous BDNF treatment increased RBC ectopic dendritesand improved b-wave,which suggested that the synaptic connections between the ectopic dendrites of rod bipolar cells and remaining photoreceptor cells had information transfer ability.Besides,the the inner neuron information input were enhanced to save the function of degenerative retina.4.Exogenous BDNF treatment could be a potential therapy for retinal degeneration.