The Role And Mechanism Of JIP1 In The Development And Injury Of RGCs

BackgroundVisual information is transmitted to the visual cortex of the brain by the optic nerve formed by retinal ganglion cells(RGCs)and their axons,which is an important step in the formation of vision.Thus,the structural and functional integrity of RGCs is crucial to vision.RGC apoptosis and its axonal mutation are critical in the development of eye diseases,including glaucoma,diabetic retinopathy,and Leber's Hereditary Optic Neuropathy(LHON),and even responsible for vision impairment and loss.However,RGCs are a kind of non-renewable cells,for which number and function losses cannot be reversed after apoptosis.Currently,no specific therapy is available for RGC number and function losses.Measures to protect RGCs during disease and to regulate their apoptosis to delay the progression of disease and reduce the severity of symptoms have become a research focus.C-jun N-terminal kinase(JNK)is a recognized signaling pathway related to apoptosis.A number of studies have shown that the inhibitors of JNK can repress the apoptosis of nerve cells in pathological state to some extent,but bring obvious side effects due to blocking the normal physiological functions of JNK.Endogenous mitogen-activated protein kinase 8 interaction protein 1(Mapk8ip1 or JIP1)is a scaffold protein in the JNK pathway.It can phosphorylate JNK and activate the downstream pathway by binding to JNK,MKK7,MLK,etc.,and thus change the activation state and action mode of JNK and the downstream molecules.The current study aimed to answer the following questions: Does JIP1 play an important role in the normal development of RGCs and their apoptosis triggered by external stimulation? Can regulation of JIP1 affect the above processes?PurposeTo investigate the role of JIP1 protein in the normal development of RGCs and the process of stress-related acute injury,and to explore the underlying regulatory mechanism,so as to provide a theoretical basis for the treatment of RGC injury.Methods and resultsThis study can be divided into the following two parts:Part 1 The role of JIP1 in the development of RGCs and the underlying mechanism1.Primary RGCs were isolated from C57 wild-type mice and cultured in vitro.On the 6th day of culture,a JIP1-specific probe and antibody were used for in situ hybridization and immunohistochemical(IHC)staining.We found that JIP1 mRNA was mainly distributed in the cytoplasm of RGCs,and the protein distributed in the cell body and neurites,with relatively high expression of protein in the head of axons.2.The JIP1 gene knockout mouse strain was introduced into the experiment,and the JIP1 expression was identified by PCR amplification of mouse genomic DNA and agar-gel electrophoresis.The mRNA and protein of mouse retina were extracted and detected by RT-PCR and WB,respectively.JIP1 mRNA and protein expressions could be detected in the retina of wild-type mice,but not be found in JIP1 knockout homozygous mice.3.The number of RGCs was counted through TUJ1 IHC staining of mouse retina,the thickness of GCC determined through SD-OCT,and the PhNR amplitude measured through visual electrophysiology testing.At the age of 8 to 10 weeks,no significant difference in the number of RGCs,thickness of GCC and RGC function was found between JIP1 knockout mice and wild-type mice.4.By IHC staining and western blot(WB)detection,we found that at the age of 8-10 weeks at the resting state,JIP1 gene knockout mice showed no significant difference in the expression site and level for the proteins including TUJ1,NF200,PSD95 and SYN in the retina from wild-type mice.5.IHC staining of the retinal sections of mice aged 8-10 weeks showed that at the resting state,the expression site of GFAP was same in the retina between wild-type mice and JIP1 knockout mice.WB detection further revealed that the expression level of GFAP protein was significantly higher in JIP1 knockout mice than in wild-type mice.6.By extracting retinal proteins from mice of different ages for WB detection,we discovered that JIP1 protein was highly expressed at P3 and P7 after birth in wild-type mice,and the expression decreased with the increasing age.Meanwhile,the expression of JIP3 protein gradually increased after birth,reaching a peak at P30.However,the expression of JIP3 protein peaked obviously in advance in the retina of JIP1 knockout mice,reaching a high level at P7 and P14.Part 2 The role of JIP1 in RGC damage and the underlying mechanism1.Rotenone(31.18 mM,0.5 ?L)was intravitreally injected into wide-type mice aged 8-10 weeks to induce an injured model.Fluorescence staining of retinal sections and visual electrophysiological testing confirmed that 1 hour after injection,the RGC number and PhNR amplitude were remarkably reduced,and 24 hours after injection,these two variables were further reduced.No significant difference was observed between 3 days and 24 hours after injection.2.At 24 hours after intravitreal injection of rotenone or vehicle,fluorescence staining,SD-OCT and visual electrophysiological testing were performed to determine the RGC number,GCC thickness and PhNR amplitude,respectively.These three parameters were significantly lower in the rotenone group than in the vehicle group.Within the vehicle group,no significant difference was found between wild-type mice and JIP1 knockout mice.Within the rotenone group,the above parameters were significantly higher in JIP1 knockout mice than in wild-type mice.3.Before and 24 hours,3 days,7 days after intravitreal injection of rotenone,TUJ1 IHC staining and visual electrophysiological testing were used to determine the RGC number and PhNR amplitude,respectively.The number of RGCs and the amplitude of PhNR were significantly reduced compared with those before rotenone injection.At the three time points after injection,the RGC number and PhNR amplitude were both higher in JIP1 knockout mice than in wild-type mice.4.Wild-type mice and JIP1 gene knockout mice were divided into three groups: mice intravitreally injected with 0.5 ?L of rotenone(31.18 mM)to induce an injured model;mice intravitreally injected with 0.5 ?L of vehicle to rule out the effect of the vehicle;and mice without treatment used as a negative control to observe the changes induced by JIP1 KO.By TUNEL staining,we found that the apoptotic index of RGC layer(the ratio of TUNEL-positive cells to DAPI-positive cells in RGC layer)showed no significant difference between the vehicle group and the non-injection group.In contrast,the apoptotic index of RGC layer was significantly lower in JIP1 knockout mice than in wild-type mice in the rotenone group.5.After treatment as described above,pJNK was detected by IHC staining in retinal slices.In the non-injection group and the vehicle group,a small amount of pJNK protein was distributed in RGC layer and outer plexiform layer.After intravitreal injection of rotenone,the pJNK protein was located in the same layers,but the fluorescence intensity increased significantly,with a greater increase in wild-type mice than in JIP1 knockout mice.WB detection of total retinal protein in mice revealed that the retinal JNK phosphorylation ratio(pJNK/JNK)was not significantly different between the vehicle group and the non-injection group,while significantly increased in the rotenone group,but with a lesser increase in JIP1 knockout mice than in wild-type mice.6.After treatment as described above,the total retinal protein of mice was detected by WB.We found that the retinal activation caspase-3 ratio(cleaved caspase-3 / caspase-3)was not significantly different between the vehicle group and the non-injection group,whereas significantly increased in the rotenone group,but with a lesser increase in JIP1 knockout mice than in wild-type mice.7.The primary RGCs from wild-type mice were cultured in vitro for 6 days.After 12-hour treatment with rotenone,IHC staining was performed and revealed that JIP1 protein tended to accumulate towards the cell body from the tip of the cell.8.Experiments using primary RGCs from wild-type mice and JIP1 knockout mice were divided into three groups: cells treated with rotenone for 12 hours,cells treated with solvent of same amount for 12 hours,and cells receiving no treatment.IHC and WB detection demonstrated that the apoptosis index,the phosphorylation ratio of JNK and the ratio of activated caspase 3 were not significantly different between the solvent group and the non-treatment group.These parameters were significantly increased in the rotenone group,but the increase was lesser in degree in primary RGCs from JIP1 knockout mice than in those from wild-type mice.Conclusions:This study confirmed that RGCs express JIP1 and the expression location and function of JIP1 protein are different at the resting state and stress state.The number,function and synaptic protein expression of RGCs after JIP1 knockout were not significantly affected at the resting state,possibly attributed to astrocyte activation and JIP3 compensation.Intravitreal injection of rotenone could successfully establish a mouse model of acute retinal injury,and the RGC number and function damage reached the peak 24 hours after injection.JIP1 gene knockout can inhibit the damaging effect caused by intravitreal injection of rotenone,and has a protective effect on the number and function of RGCs.The cell damage caused by rotenone involved the jip1-jnk-caspase3-apoptosis signaling axis,and the knockout of JIP1 could inhibit apoptosis-related signal transduction,thus exerting a protective effect on RGCs.