Study Of TBN On Motor Dysfunction And Its Mechanism In SOD1^G93A Mouse Model Of Amyotrophic Lateral Sclerosis

Research objectives:Amyotrophic lateral sclerosis（ALS）is a neuromuscular disease caused by the degeneration of motor neurons in the brain and spinal cord.Currently,only two drugs,i.e.,riluzole and edaravone,were approved by the FDA for the treatment of ALS.There is an urgent need for drugs that can suppress the progression of ALS.TBN,（2-[（1,1-dimethylethyl）-oxidoimino]-methyl]-3,5,6-trimethylpyrazine）,a novel tetramethylpyrazine nitrone,was developed in our laboratory for the treatment for neurological diseases including ALS.Previous studies had shown that TBN can scavenge different types of free radicals（such as O₂·-,·OH and ONOO-）and had demonstrated good therapeutic effects in animal models of neurodegenerative diseases such as ioschemic stroke,Alzheimer’s disease and Parkinson’s disease.Hence,the current project performed researches on the efficacy of TBN in the ALS model of SOD1^G93A transgenic mice and explore its preliminary mechanism of action.This research provides scientific evidence supporting the development of TBN for the treatment of ALS.Research methods:The ALS model of human SOD1^G93A（h SOD1）transgenic mice were used in this experiment.The onset time of mice was determined by observing the phenotypic characteristics and behavioral experiments of SOD1^G93A transgenic mice.The motor ability and limb muscle strength of mice were evaluated by the pole-climbing test,suspension test and limb grasp test before（13 weeks old）,during（every 2 weeks）and at the end of administration of drugs（19 weeks old）,respectively.At the end of the experiment,the gastrocnemius muscles on both sides of mice legs were weighed to analyze the degree of gastrocnemius atrophy.The muscle morphology of gastrocnemius was observed by H&E and Masson staining to evaluate the degree of atrophy and fibrosis.Nissl staining was used to assess the survival motor neurons of the anterior horn.The contents of malondialdehyde（MDA）,reactive oxygen species（ROS）,8-hydroxydeoxyguanosine（8-OHDG）,glutathione（GSH）,human mutant SOD1（h SOD1）and total antioxidant capacity（T-AOC）in the serum of each group were determined by the Elisa kit.Immunohistochemistry,immunofluorescence and Western blot were used to explore the effects of TBN on h SOD1 level in the spinal cord of ALS mice,as well as the anti-inflammatory effect,antioxidant capacity and effects of TBN on autophagy in this model.Experimental results:TBN had no significant effect on the weight change of SOD1^G93A mice.Different doses of TBN could significantly improve the motor disorder of SOD1^G93A mice,and enhance the limb coordination ability,muscle endurance and muscle strength of SOD1^G93A mice.Statistical results of gastrocnemius muscle weight showed that TBN significantly reduced the degree of muscle fiber atrophy of gastrocnemius muscle and also significantly lowered the fibrosis of gastrocnemius muscle in SOD1^G93A mice.The statistical results of the number of motor neurons in the anterior horn of the spinal cord showed that different doses of TBN（10 mg/kg,30 mg/kg,60 mg/kg）could significantly improve the survival rate of spinal motor neurons in SOD1^G93A mice.Preliminary mechanism of action study show that:1)TBN can significantly reduce the content of lipid peroxide MDA and oxygen-reactive cluster ROS in serum of SOD1^G93A mice;2)TBN had no effect on the levels of carbonyl peroxide 8-OHDG,glutathione GSH and total antioxidant capacity T-AOC in serum of SOD1^G93A mice;3)TBN not only significantly reduced the content of h SOD1 in serum and spinal cord of SOD1^G93A mice（P<0.05）,but also obviously depressed the expression and accumulation of h SOD1 in the spinal cord of SOD1^G93A mice.4)TBN had no significant effect on the expression of m TOR autophagy pathway related proteins in the spinal cord of SOD1^G93A mice.5)TBN can significantly inhibit the excessive activation of glial cells in the spinal cord of SOD1^G93A mice;6)TBN can significantly increase the expression of PGC-1αpathway proteins（PGC-1α,Nrf2 and HO-1）in the spinal cord of SOD1^G93A mice.Experimental conclusion:TBN demonstrated significant efficacy in the SOD1^G93A transgenic mouse model of ALS.The therapeutic effect of TBN may be related to activating the antioxidant pathway of PGC-1α,reducing the accumulation of h SOD1 protein,and inhibiting neuroinflammation in the SOD1^G93Amice.TBN is a promising clinical candidate for the treatment of ALS.