Mechanisms Of The Impairment Of Motor Neurons And The Changes Of Autophagy In Rats Induced By BMAA

Backgroud and Objective : Amyotrophic lateral sclerosis(ALS) is the most common motor neuron disease. At present the pathogenesis of ALS is still unknown. Gene mutation, oxidative stress, protein misfolding and abnormal accumulation, mitochondrial dysfunction, toxic effect of excitatory amino acids, axonal transport defects, intracellular abnormality of proteolytic pathway and immune inflammatory lesions were involved. Studies have shown that abnormal folding and deposition of mutant proteins are the key factors leading to the onset of ALS. Autophagy-lysosome system plays an important role in the process of degradating abnormal aggregation of proteins in cells. Dysfunction of autophagy would accelerate the progression of ALS. In this study, we established the rat model of damaged motor neuron disease induced by the toxicity of BMAA, and explored the mechanisms of motor neuron impairment and related changes of autophagy.Methods: The newborn wistar rats were randomly divided into the experimental group and the control group. The experimental group was given subcutaneous injection of BMAA 300mg/kg(freshly dissolved in PBS solution, 20ul/g) for 2 days on PND 9-10. The control group was given subcutaneous injection of PBS. From the beginning of the 4th week, weight test and neurological function assessment were performed every week on the two groups. Resting potentials, light contraction potentials and strong contraction potentials were measured in the gastrocnemius muscles after the animal models were prepared for 24 weeks. Then the rats in the experimental group and the control group were sacrificed respectively to collect the cerebral hemispheres, spinal cords and gastrocnemius muscles. The morphological changes of the gastrocnemius muscles of rats were observed by HE staining. The anterior horn motor neurons of lumbar spinal cords were observed with cresyl violet Nissl staining. LC3 b, P62, GFAP and LC3 b in lumbar spinal cords and cerebral motor cortexes were observed by immunohistochemistry and immunofluorescence. The expressions of TNF-α, i NOS, GLT-1, GFAP, CD11 b, LC3 b, P62 in the spinal cords were analyzed by western blot. And the m RNA level of TNF-α, GLT-1, LC3 II and P62 were measured by q RT-PCR.RESULTS: Compared with the control group, the rats in the experimental group had no significant changes in the body weight and neurological function score from start to the 24 th week(P all ﹥0.05). From the 22 nd to 24 th week, the motor function scores were 0.8±0.84, 1.0±0.71, 1.0±0.71 respectively, and significant difference was detected at 23 rd and 24 th week as compared to the control group. The denervation of the gastrocnemius muscles of the rats in the experimental group was shown with electromyography in the 24 th week. Resting potentials showed as fibrillation potentials. Light contraction presented showed as increased amplitude and extended time limit. Strong contraction showed as typical denervation. By HE staining, gastrocnemius muscles of the experimental group showed that part of muscle fibers were atrophic, and some were triangular or polygonal which were manifestations of typical nerval damage. The loss of motor neurons in the spinal cord anterior horns was observed with the method of Nissl’s staining(23.07±1.63 vs 49.10±2.25, P﹤0.05). The expression of Iba-1, P62, GFAP and LC3 b in the cerebral motor cortexes and lumbar spinal cords in the experimental group were increased by immunohistochemistry and immunofluorescence. And the expressions of TNF-α, i NOS, GFAP, CD11 b, LC3 b, P62 in the experimental group were significantly increased by western blot(P all ﹤0.05). q RT-PCR showed that the m RNA levels of tnf-α, LC3 II and P62 were also increased(P all ﹤0.05). The expression of GLT-1 was decreased by western blot and q RT-PCR(P﹤0.05).Conclusion:1.The rat model of motor neuron disease induced by BMAA in rats was successfully established.2.The increase of immune inflammatory reaction, the activation of glial cells, and the toxic effect of excitatory amino acids may be the mechanisms of impairment of motor neurons induced by BMAA.3.In the experimental group, the markers of autophagy bodies and autophagy substrates were all increased. This may indicate that although the level of autophagy induced by BMAA was increased in the rats treated with BMAA, it could not meet the requirement of the effective elimination of abnormal aggregation of proteins in cells. Consequently the increasing accumulation of abnormal proteins may impair the motor neurons through a variety of ways.