Intervention Study On Rilmenidine In NSC34Cells Transfected With ALS-related Proteins

Objective:Amyotrophic lateral sclerosis(ALS) is a neurodegenerative disordercharacterized by selectively involved upper and lower motor neurons, whichis adult-onset, ultimately dying in fatal respiratory failure after3to5years，and do not having available treatment now. The exactly pathogenicmechanism of ALS is still unknown, the mutant of SOD1gene and WTTDP-43gene is the hot topic, which leading to the misfolding and prone toaggregating of intra-celluar proteins make motor neuron degeneration,resulting in the generation and development of ALS. We all known that theautophagosome-lysosome pathway is the most important degradationmachinery to clear the misfolding and aggregated proteins.In order to find safer ways of inducing autophagy for clinical purposes,Claudia rose and his colleagues screened United States Food and DrugAdministration-approved drugs, founding that rilmenidine, centrally actinganti-hypertensive drug and the analogue of clonidine,, could induce autophagyin a HD mouse model and reduced levels of the mutant huntingtin fragment.Moreover, rilmenidine administration attenuated the signs of disease in a HDmouse model. It is has been confirmed that Huntington’s disease (HD) iscaused by mutant huntingin aggregations leading to the death of motor neuron.Accordingly, we assessed whether autophagy activated by rilmenidine is ableto accelerate ALS related proteins degradation in NSC34cells. NSC34cellwere treated with rilmenidine, levels of endogenous LC3-Ⅱ and P62weredetected by immunoblotting. To verify that whether the inducing autophagyeffect of rilmenidine is real. Fllowingly, NSC34cells transient-transfectedwith WT SOD1，G93A SOD1，WT TDP-43, TDP-25, TDP-35bulid ALS cellmodel, comparing the levels of transient-transfected proteins between the medication group and the control group by immunoblotting. lastly, to furtherprove that rilmenidine degrade protein aggregations by autophagy pathway,we used the specific autophagy inhibitor3-MA. In conclusion, our dateindicate the ALS related pathogenic proteins degradation effect of rilmenidinedepends on the autophaty pathway possibly to protect against neurondegeneration. It is possible that,at some level, what we founded supplytheoretical basis and experimental data in the research of rilmenidine and ALSrelated and so on animal model, even in the clinical trials.Methods:1Cell culture and generation of NSC34cell lines stably transfected withWT TDP-43and SOD1NSC34is a hybrid cell line that expresses many properties of motorneurons. NSC34was routinely maintained in the high glucose formulation ofDMEM supplemented with10%FBS and antibiotics at37and5%CO2incubator.NSC34cell transient-transfected with EGFP-WT SOD1、EGFP-G93A SOD1、EGFP-TDP-25、EGFP-TDP-35、MYC-WT TDP-43bulidALS cell model, which were maintained in selective medium(0.5%mg/ml ofG-418)containing10%PBS for passages.2Therapeutic agent and administration protocolNSC34cells were treated with rilmenidine, with concentrations varyingfrom0to20uM. Levels of endogenous LC3-Ⅱ, P62were detected byimmunoblotting, to verify that whether the inducing autophagy effect ofrilmenidine is existing. Furthermore,which concentration of rilmenidine weused is most valid to induce autophagy. NSC34cells transient-transfectedwith WT SOD1, G93A S0D1, WT TDP-43,TDO25,TDP-35were differentlydivided into the medical intervention group and the blank control group, levelsof the ALS related proteins were detected by immunoblotting to observingthe protein degradation effect of rilmenidine. Meanwhile we used3-MA andrilmenidine interposed in the NSC34cell transient-transfected TDP-25,levels of LC3-Ⅱand transient-transfected proteins were detected byimmunoblotting, to verify that whether the protein degradation effect of rilmenidine depends on the autophagy pathway.3ImmunoblottingCells were collected and total prtein were extracted. Protein etracts werequantified using BCA protein measure method. Prtein samples were separatedin10%or12%SDS-PAGE gels, transferred onto PVDF membranes. Themembranes was then incubated overnight at4℃with the relatively primaryantibody. Followingly, the membrane was then incubated with afluorescence-conjugated secondary antibody. The bands of interest on themembranes were detected using an Odyssey Infrared Imaging System. Theoriginal green or red color of a band was converted to black and white colorsfor data presentation.Results:1the endogenous protein level of LC3-Ⅱ and P62in the NSC34celllines after given rilmenidine intervention.The expression of LC3-Ⅱ and P62was measured by westernblottingtingting. The result were that the endogenous protein levels of LC3-Ⅱwere apparently up-regulated in the NSC34cell lines after given10uMrilmenidine, compared to vehicle treatment group.(P <0.05), on this condition,the levels of P62were distinctly down-regulated (P <0.05).2The ALS related protein levels after given rilmenidine interventionThe expression of WT TPD43was measured with an anti-myc antibody.The rest of the ALS related protein levels was measured with an anti-EGFPantibody. Our date founded that rilmenidine significantly accelerated theclearance of G93A S0D1, TDP-25, TDP-35, compared to vehicletreatmentgroup(P <0.05), but rilmenidine had no effect on WT TDP-43andWT SOD1degradation(P>0.05).3The effects of degrading proteins of rilmenidine was attenuated by3-MA.The protein levels of TDP-25in NSC34cell lines clearly up-regulatedafter given3-MA and rilmenidine intervention, compared to rilmenidine alonetreatment group (P <0.05). Conclusion:1we found that LC3-Ⅱacting as the marker of autophagic vacuolewere increased in the NSC34cell lines after rilmenidine intervention,compared to vehicle treatment group,on the contrary, the protein levelsof P62were decreased,P62act as a marker of autophagic flux and as abridge between degraded subsrate and autophagsome. We confirmed thatrilmenidine has an obvious effect on inducing autophagy.2We all known that ALS related mutant gene result in proteinaggregations leading to ALS. NSC34cells transient-transfected withG93A S0D1,WT SOD1,WT TDP-43,TDP-35,TPD25were treated with10uM rilmenidine, we observed that rilmenidine prominently promotedthe clearance of G93A S0D1,TDP-25,TDP-35,but had no effect on WTSOD1and WT TPD43degradation. We demonstrate that rilmenidinecould accerlerate the degradation of G93A S0D1as well as TDP-25andTDP-35(two truncated fragment of WT TDP-43).but there was no effecton the degradation of WT SOD1and WT TDP-43.3It has been found that autophagy-lysosome pathway(ALP) andubiquitin-proteasome system(UPS) are the two major systems fordegradation of protein aggregations, to further verify rilmenidinedegrades protein aggregations by autophagy-Lysosome pathway beyondubiquitin-proteasome,3-MA，which blocks autophagsome generation,was used. We found that3-MA could attenuate the effection of degradedprotein aggregations of rilmenidine, compared to rilmenidine aloneintervention.4In a word, rilmenidine could accerlerate the degradation of ALSrelated protein aggregations,which depends on autophagy-lysosomepathway.