| [Background]Amyotrophic lateral sclerosis (ALS) is an adult-onset chronic neurodegenerative disease, characterized by the progressive and fatal loss of motor neurons in the brainstem and spinal cord, leading to atrophy and paralysis of skeletal muscle and ultimately death. Approximately20%of familial ALS cases possess one or more mutations in the gene that encodes copper-zinc superoxide dismutase1(SOD1). Although more than125different mutations in SOD1have been identified in patients with familial ALS, most of the symptoms caused by these mutations are similar, suggesting that the progressive degeneration of motor neurons in ALS is caused by one or several factors. The SOD1A transgenic mice can recapitulate the progression of human ALS symptoms, while the wild-type mice expressing identical copies do not occur. The SOD1G93A transgenic mice are considered a reliable model for researching the pathogenesis of ALS. The putative mechanisms include growth factor deficiency, mitochondrial abnormalities, axonal disorganization, oxidative stress, inflammation, excitotoxicity, and protein aggregation, among others. Despite intense effort, few therapeutic options have emerged for slowing down disease progression. The exact mechanisms responsible for motor neuron degeneration in ALS, however, are not completely understood.Canonical Wnt/β-catenin signaling pathway is a complex network composed of a variety of proteins with interaction and plays an important role in a variety of cellular events, including cell proliferation, differentiation, migration and morphogenesis. Extensive studies have shown abnormal Wnt signaling is relevant in a variety of neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and so on, however, the role of the Wnt signaling pathway in the pathogenesis of ALS is still unclear.Based on the above analysis, we designed the experiment. In the present study, the expression changes of the key signaling molecules in the Wnt/β-catenin signaling pathway were detected in the SOD1G93A transgenic mice, primary cultured astrocytes model from SOD1G93A transgenic mice and N2a neural cell model with SOD1mutation using the methods of morphology and molecular biology, such as immunofluorescence techniques, laser scanning confocal microscopy techniques, RT-PCR, Western blot, cell culture and so on. The expression changes of Wnt molecules were detected by the combination of a set of studies in vitro and in vivo and in different expression levels in order to explore the relationship between the Wnt signaling pathway and the pathogenesis of ALS, and reveal the role and mechanism of Wnt/p-catenin signaling pathway to find new targets for the treatment of ALS, Which may provide scientific basis for the prevention and treatment of ALS.[Methods]1.60ALS transgenic mice were selected and were randomly divided into three groups. Mice were killed at95d (early stage),108d (middle stage) and122d (end stage). Spinal cords of some mice were removed, and then total RNA was extracted and reverse transcribed to cDNA. The mRNA expression changes of the key signaling molecules in the Wnt/(3-catenin signaling pathway, such as Wnt ligands (Wnt2, Wnt3a and Wnt7a), GSK-3β, β-catenin and Cyclin D1, were detected in the spinal cord of ALS transgenic mice by RT-PCR.Total and nuclear protein from the spinal cord of some mice were extracted separartly, and the protein expression changes of the signaling molecules were detected by Western blot. Some mice received daily intraperitoneal injection of50mg/kg BrdU for5days starting at the age of90days. Animals were perfused intracardially with4%paraformaldehyde at1,14,28days after the last injection and frozen sections were prepared. The expression pattern and the relationship between the Wnt signaling molecules and cell proliferation and differentiation in the spinal cord of ALS mice were detected using immunohistochemistry, double and multiple immunofluorescence labeling techniques. Wild-type littermates were selected as the control in each group.2. The spinal cords of three day-old newborn ALS mice were removed under sterile conditions and then digested by trypsin. Primary astrocytes were cultured and purified in vitro. Primary astrocytes of wild-type littermates were considered as the control group. The expression changes of Wnt3a、β-catenin and Cyclin D1were detected in the primary cultured astrocytes model using Western blot and double immunofluorescence techniques.3. Mouse neuroblastoma cell line N2a was transfected with pEGFP-WT-SOD1and pEGFP-G93A-SOD1plasmids to prepare the wild-type and SOD1mutant neural cell model. The protein were extracted from the cells24h and48h after transfection. Cell implantation on slides were prepared from the cells24h and48h after transfection. The expression changes of Wnt3a、β-catenin and Cyclin D1were detected in the ALS cell model using Western blot and immunofluorescence techniques.[Results]1. The expression changes of the key signaling molecules in the Wnt/(3-catenin signaling pathway in the spinal cord of ALS transgenic mice(1)The expression changes of Wnt ligands in the spinal cord of ALS transgenic mice At the age of95d,108d, and122d, the levels of Wnt2,Wnt3a and Wnt7a mRNA and protein in the spinal cord of ALS were all increased compared to those in wild-type mice. The observed differences in expression were statistically significant (p<0.05, p<0.01). The majority of the immunopositive cells of the three kinds of Wnt ligands were all located in the ventral horns of the gray matter, the locus of neurodegeneration. At95d,108d and122d, the immunoreactivities of Wnt3a, Wnt2and Wnt7a in the ALS mice were all stronger than those in wild-type mice at the same time points. Double immunofluorescence staining showed that at95d,108d and122d, Wnt3a+/GFAP+, Wnt2+/GFAP+, and Wnt7a+/GFAP+double-positive cells in the ALS mice were all increased with neurodegeneration, especially at122d(p<0.05, p<0.01). Wnt3a+/β-tubulinⅢ+double-positive cells were decreased in the ALS mice at108d and122d (p<0.05). BrdU and Wnt3a double immunofluorescence staining was used to demonstrate the localization of Wnt3a as the representative of the three kinds of Wnt ligands in proliferating cells, however, no BrdU+/Wnt3a+double-positive cells were detected in both ALS mice and wild-type mice.(2) The expression changes of GSK-3β in the spinal cord of ALS transgenic mice At95d,108d and122d, no significant changes were observed in GSK-3β mRNA and total protein in the spinal cord of ALS and wild-type mice. However, the level of phospho-GSK-3β (Ser9) protein in the spinal cord in ALS mice was upregulated significantly compared with wild-type mice (p<0.05, p<0.01). Most of the phospho-GSK-3β-immunopositive cells were located in the ventral horns of the gray matter. At95d,108d and122d, the immunoreactivities of phospho-GSK-3p (Ser9) of ALS mice were all stronger than those in wild-type mice at the same time points. In order to determine whether GSK-3β was colocalized with β-tubulinⅢ, GFAP or BrdU, double immunofluorescence staining was performed. The result revealed that GSK-3β was expressed in neurons but not in mature astrocytes. No BrdU+/GSK-3β+double-positive cells was detected in both ALS mice and wild-type mice.(3) The expression changes of β-catenin in the spinal cord of ALS trans genic mice At the age of95d,108d, and122d, there were no changes seen in β-catenin mRNA and total protein levels in the spinal cord of ALS and wild-type mice. At the age of95d, there were no significant changes observed in nuclear β-catenin protein levels in the spinal cord of ALS and wild-type mice. At the age of108d and122d, nuclear β-catenin protein levels in the spinal cord of ALS were increased. Compared with wild-type mice, there was statistically significant in ALS mice(p<0.05). The results obtained from our immunofluorescence labeling showed that in ALS mice, the number of P-catenin-positive cells localized to the nuclei was increased; however, the number of P-catenin-positive cells localized to the cell membrane was clearly decreased at the age of108d and122d, compared with wild-type mice. Double immunofluorescence staining revealed that in ALS mice, the number of β-catenin+/GFAP+double-positive cells was increased(p<0.05, p<0.01), but the number of β-catenin+/β-tubulinⅢ+double-positive cells in the ventral horn was decreased at the age of108d, and122d(p<0.05). No BrdU+/β-catenin+double-positive cells were detected in ALS mice and wild-type mice at the age of95d,108d, and122d.(4) The expression changes of Cyclin D1in the spinal cord of ALS transgenic mice Cyclin D1mRNA and protein were upregulated in the ALS spinal cord compared with the wild-type spinal cord at95d,108d, and122d(p<0.05, p<0.01). The results of immunofluorescence labeling showed that the number of Cyclin D1-positive cells was distinctly increased in the gray and white matter as well as in the central canal of spinal cord in ALS mice at95d,108d, and122d. Double immunofluorescence staining showed that the number of BrdU and Cyclin D1double-positive cells in the spinal cord of ALS mice (95-day-old) was significantly increased than that in wild-type mice (p<0.05). Cyclin D1+/GFAP+and Cyclin D1+/β-tubulinⅢ+double-positive cells were mainly located in the ventral horns of the gray matter. In ALS mice, the number of Cyclin D1+/GFAP+and Cyclin D1+/β-tubulinⅢ+double-positive cells was both increased at108d and122d (p<0.05). In particular, we found a robust increase in Cyclin D1+/GFAP+double-positive cells, especially at122d (p<0.01). To identify the differentiation of proliferating cells, triple labeling of BrdU/Cyclin D1/β-tubulinⅢ and BrdU/Cyclin D1/GFAP was performed. The results showed that BrdU+/Cyclin D1+/GFAP+triple-positive cells were detected in ALS mice, however, BrdU+/Cyclin D1+/β-tubulinⅢ+triple-positive cells were not detected, implying that proliferating cells differentiated mainly into glial cells2. The expression changes of the key signaling molecules in the Wnt/β-catenin signaling pathway in the primary cultured astrocytes from the ALS mice(1) The expression changes of Wnt3a in the primary cultured astrocytes from the newborn ALS mice The results obtained from Western blot showed that the expression of Wnt3a protein was upregulated in the primary cultured astrocytes from the newborn ALS mice, compared with those from the wild-type newborn mice, and the difference was statistically significant (p<0.05). Double labeling revealed that the expression of Wnt3a was increased in the primary cultured astrocytes from the newborn ALS mice.(2) The expression changes of P-catenin in the primary cultured astrocytes from the newborn ALS mice Western blot analysis was performed to examine possible changes in β-catenin protein expression. The results showed that β-catenin protein level in the primary cultured astrocytes from the newborn ALS mice was increased, compared to those from the wild-type newborn mice. There was statistically significant difference (p<0.05). Double labeling revealed that in the primary cultured astrocytes from the newborn ALS mice, the expression of β-catenin was increased.(3) The expression changes of Cyclin D1in the primary cultured astrocytes from the newborn ALS mice The expression of Cyclin D1protein was upregulated in the primary cultured astrocytes from the newborn ALS mice, compared with those from the wild-type newborn mice, and there was statistically significant difference (p<0.05). Double immunofluorescence staining showed that the expression of Cyclin D1was increased in the primary cultured astrocytes from the newborn ALS mice.3. The expression changes of the key signaling molecules in the Wnt/β-catenin signaling pathway in the ALS cell model(1) The expression changes of Wnt3a in the ALS cell model Compared with N2a cells transfected with pEGFP-WT-SOD1, the expression of Wnt3a protein of the N2a cells transfected with pEGFP-G93A-SOD1was downregulated at24h and48h, and there was statistically significant difference (p<0.05). The immunofluorescence staining showed that the expression of Wnt3a was decreased in the N2a cells transfected with pEGFP-G93A-SOD1at24h and48h.(2) The expression changes of β-catenin in the ALS cell model Compared with N2a cells transfected with pEGFP-WT-SOD1, the expression of β-catenin protein of the N2a cells transfected with pEGFP-G93A-SOD1were all downregulated at24h and48h, and there was statistically significant difference (p<0.05). The immunofluorescence staining showed that the expression of β-catenin was decreased in the N2a cells transfected with pEGFP-G93A-SOD1at24h and48h.(3) The expression changes of Cyclin D1in the ALS cell model The expression level of Cyclin D1mRNA and protein of the N2a cells transfected with pEGFP-G93A-SOD1were all increased at24h and48h compared with N2a cells transfected with pEGFP-WT-SOD1, and the difference was statistically significant (p<0.05). The immunofluorescence staining showed that the expression of Cyclin Dl was increased in the N2a cells transfected with pEGFP-G93A-SOD1at24h and48h.[Conclusions]1. Upregulation of Wnt3a, Wnt2and Wnt7a activates the Wnt/β-catenin signaling pathway and upregulates the expression of the target gene Cyclin D1, which is associated with the degeneration of the motor neurons and glial proliferation in the spinal cord of ALS transgenic mice.2. The expression of Wnt3a,β-catenin and Cyclin Dl, three key signaling molecules, are all upregulated in the primary cultured astrocytes from the newborn ALS mice.3. The mutation of SOD1downregulates the expression of Wnt3a, β-catenin and upregulates the expression of the target gene Cyclin D1in the N2a neural cells.After comprehensive analysis of our study, the results show that the Wnt/β-catenin signaling pathway plays a significant role in the pathogenesis of ALS. Abnormalities of Wnt/β-catenin signaling pathway are closely related to the occurrence and progress of ALS. |
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