| Research background and purpose:Lanthionine Synthetase C-Like 2(LanCL2),a member of the LanCL protein family,is widely expressed in humans and mice.Human LanCL2 have the highest expression in the brain.LanCL2 is highly similar in sequence and crystal structure to LanCL1,which protects neurons from damage caused by oxidative stress.In addition,LanCL2 is a binding receptor for abscisic acid(ABA).The highest content of ABA is found in the brain.Exogenous ABA can cross the blood-brain barrier.By injecting exogenous ABA into the brain of mice or directly giving mice ABA-rich diets,can improve the cognitive impairment of mice and protect the brain from neuroinflammation and oxidative stress.These studies suggest that LanCL2 may play a pivotal role in the central nervous system(CNS).At present,there are few reports on the specific role and mechanism of LanCL2 in the CNS.In this study,we propose to explore the potential physiological functions of LanCL2 in the CNS by specifically knocking out it.Materials and Methods:1.The 2-month-old C57BL/6 mice were sacrificed by neck removal,and the cerebellum,spinal cord,hippocampus,cerebral cortex,testis,heart,liver and spleen of the mice were separated.The histamin of the mice was extracted,and the protein level of LanCL2 was detected by Western blot.2.Primary cortical neuron cells of C57BL/6 mice were cultured.and the expression level of LanCL2 in primary neurons detected by cell immunofluorescence staining and Western blot.Neurotrophic factors(IGF-1,EGF,PDGF and BDNF)were added into the medium of primary neuron cells,and the expression level of LanCL2 in primary neurons induced by different neurotrophic factors was detected by Western blot.3.By using Crispr/Cas9 technology,the 2 exon of LanCL2 was conditionally knocked out in the central nervous system of mice to obtain the LanCL2 conditional knockout mice,which were mated and bred with Nestin-Cre mice to obtain the control mice and homozygous mice required for the experiment.The mouse DNA was extracted by tail clipping for gene identification.Mouse cerebral cortex proteins were isolated and extracted for Western blot and q PCR verification.4.The LanCL2 CNS-specific knockout mice and their same sex control mice were5.selected,and the body shapes of the two mice were measured and compared.From the first week after birth,the weight changes of both were continuously weighed and recorded for 40 weeks.LanCL2 CNS-specific knockout mice and their same sex control mice were killed by neck removal,and the whole brain of the mice was taken.The brain volume and weight of the two mice were measured and weighed.6.4-month-old LanCL2 CNS-specific knockout mice and same litter,same sex control mice were selected to compare the emotional responses and motor coordination ability through open-field experiments and gait analysis.10-month-old LanCL2CNS-specific knockout mice and same litter,same sex control mice were selected to compare the athletic endurance and cognitive ability through rotarod test and novel object recognition.7.4-month-old LanCL2 CNS-specific knockout mice and same litter,same sex control mice were sacrificed by neck removal.The whole brain and spinal cord of the two kinds of mice were separated,and histamin and RNA were extracted.The protein levels of LanCL1 and GFAP in cerebral cortex and MBP in spinal cord were detected by Western blot,and the m RNA levels of LanCL1 in cerebral cortex and CNP,MBP,MOG,MOBP,PLP and Tmem10 in spinal cord were detected by q PCR.8.The LanCL2 CNS-specific knockout mice and their same sex control mice were selected,perfusion them after chloral hydrate anesthesia.Then the brains,cervical enlargement and lumbar enlargement of both mice were removed and immersed in paraformaldehyde for 24-48 h.Then,perform frozen sections after sucrose gradient dehydration.Immunofluorescence staining was used to detect the expression of neuron marker Nue N in cerebral cortex,hippocampus and spinal cord respectively.The distribution and activation of astrocyte and microglia in the frontal cortex,hippocampus,corpus callosum and striatum were detected by immunofluorescence staining,respectively.Expression of MBP in spinal cord and corpus callosum were also detected by immunofluorescence staining.9.The LanCL2 CNS-specific knockout mice and their same sex control mice were selected at the age of 10 months.After anesthesia with chloral hydrate,the hippocampal part of the mice was perfused,fixed,and the cell structure of the hippocampal neurons of the two mice was observed and analyzed by electron microscopy.10.HEK293 T cells were resuscitated and cultured,and the cells were stimulated with LPS(50 ng/m L,100 ng/m L,500 ng/m L,1 μg/m L,5 μg/m L)for 24 h to extract cell proteins.The protein level of LanCL2 in cells detects by Western blot.Result:1.LanCL2 is highly expressed in the CNS and testis,but less expressed in the heart,liver and spleen.2.LanCL2 is expressed in the cell body and synapse of neurons,and the expression level of LanCL2 protein gradually increases with time.3.The expression of LanCL2 protein increased under the induction of neurotrophic factors.4.The mouse model of LanCL2 gene specific knockout in CNS was successfully established.5.There was no significant difference in the m RNA and protein levels of LanCL1 in the cerebral cortex of mice with LanCL2 gene specific knockout in CNS compared with the control group.6.The mice with LanCL2 gene-specific knockout in CNS lost weight and brain size.7.Compared with the control mice,LanCL2 CNS-specific knockout mice have no difference in activity in the central area of the open field,and there is no difference in staying time on the rotarod.The average step length and hind limb step width are smaller than those of the control mice,and the cognitive index is lower than that of the control mice.8.The specific knockout of LanCL2 in CNS makes the cortical cells arranged more closely and the cortex more thinner.Compared with control mice,hippocampal neurons are sparse,mitochondrial structure in hippocampal neurons is destroyed and ribosomes are reduced,and there is no significant difference between spinal neurons and control mice.9.The specific knockout of LanCL2 in CNS causes astrocytes and microglia activation in the hippocampus,striatum,corpus callosum,and spinal cord.10.The expression of LanCL2 protein was increased under the stimulation of high concentration LPS of 5 μg/mol.11.Decreased expression of MBP in spinal cord and corpus callosum of LanCL2CNS-specific knockout mice.And there is a downward trend on the m RNA levels of CNP,MBP,PLP and Tmem10,while the m RNA levels of MOG and MOBP decreased significantly.Conclusion:In this study,the expression distribution of lanthionine synthetase c-like 2 protein(LanCL2)in various organs and neurons was detected,and it was found that LanCL2 was highly expressed in the central nervous system and testis,but less expressed in the heart,liver and spleen.In addition,the expression level of LanCL2 protein increased gradually with the maturation and differentiation of neurons,and the expression level increased after induction by neurotrophic factor.This suggests that LanCL2 plays a role in the development of CNS.In order to further study the functional role of LanCL2 in the CNS,we constructed the LanCL2 gene specific knockout mouse model in CNS,which was the first time.It was found that there was no physiological compensation of LanCL1 in LanCL2 CNS-specific knockout mice.By observing and comparing the morphological differences between LanCL2 CNS-specific knockout mice and control mice,it was shown that the body weight and brain volume of LanCL2 CNS-specific knockout mice were obviously reduced,and this difference was not affected by gender.The results of behavioral analysis show that the specific knockout of LanCL2 gene in the CNS will not lead to significant anxiety and exercise endurance disorders in mice,but will lead to a certain degree of motor coordination and cognitive impairment in mice.However,no neuron death has been found after LanCL2 specific knockout.The experimental results show that the specific knockout of LanCL2 gene in the central nervous system will make the neurons in the cerebral cortex of mice more closely arranged and thinner,and lead to the sparse distribution of neurons in the hippocampus of mice,and the mitochondrial structure in neurons will be destroyed and ribosomes will be reduced.And there is no obvious effect on spinal cord neurons of LanCL2CNS-specific knockout mice.In addition,the specific knockout of LanCL2 in CNS causes microglia and astrocyte activation in the mouse brain and spinal cord,but this activation dose not cause polar imbalance between astrocytes and microglia.Neuroinflammation is often characterized by microglial and astrocyte activation.However,the specific mechanism of inflammatory response in the brain and spinal cord of LanCl2-specific knockout mice needs to be further explored.The expression of MBP is reduced in the spinal cord of LanCL2-specific knockout mice,and there is a decreasing trend in the m RNA levels of CNP,PLP and Tmem10,and the m RNA levels of MOG and MOBP are significantly down-regulated,indicating that specific knockout the LanCL2 gene in the CNS may affect myelination. |
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