Jinmeng Liu1, Fenghua Zhou2, Yanchun Chen3,4, Yingjun Guan3, Fandi Meng3, Zhenhan Zhao3, Xuemei Wang3, Xueshuai Gao3, Xin Jiang3, Haoyun Zhang4, Qing Wang4, Shuanhu Zhou5, Xin Wang6. 1. Laboratory of Biochemistry and Molecular Biology, Weifang Medical University Weifang 261053, Shandong, PR China. 2. Department of Pathology, Weifang Medical University Weifang 261053, Shandong, PR China. 3. Department of Histology and Embryology, Weifang Medical University Weifang 261053, Shandong, PR China. 4. Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University Weifang 261053, Shandong, PR China. 5. Department of Orthopaedic Surgery, Brigham and Women's Hospital, Harvard Medical School Boston 02115, MA, USA. 6. Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School Boston 02115, MA, USA.
Abstract
OBJECTIVES: We aimed to detect the expression profile of downstream signaling molecules of non-canonical Wnt pathway in SOD1G93A transgenic mice (ALS mice) and SOD1G93A mutant motor neuron-like hybrid (NSC-34) cells. Characterizing the molecular mechanism of the Wnt5a-mediated non-canonical Wnt/Ca2+ signaling pathway in motor neuron (MN) degeneration may provide a feasible approach to effective treatment of amyotrophic lateral sclerosis (ALS). METHODS: The expressions of CaMKII-α, CaMKII-β and TAK1 in the spinal cord of SOD1G93A ALS transgenic mice at different ages were determined using western blotting and immunofluorescence. The level of Ca2+ and cell apoptosis were assessed with flow cytometry and cell viability was evaluated using MTS assay. Cell proliferation was analyzed by the EdU cell proliferation assay. Neurite length was measured after treatment with retinoic acid. RESULTS: CaMKII-α, CaMKII-β, and TAK1 were down-regulated in the spinal cord of ALS mice. Ca2+ level and CaMKII-α, CaMKII-β, and TAK1 were down-regulated in SOD1G93A mutant NSC-34 cells. Expression of Ca2+, CaMKII-α, CaMKII-β, and TAK1 were up-regulated in SOD1G93A mutant NSC-34 cells after Wnt5a overexpression and down-regulated after Wnt5a knockdown. Overexpression of Wnt5a promoted cell viability and proliferation but inhibited cell apoptosis. Contrastingly, Wnt5a knockdown inhibited cell viability and proliferation but promoted cell apoptosis. CaMKII inhibitor KN-93 and CaMKII activator oleic acid reversed changes in cell viability, proliferation, apoptosis, and neurite outgrowth induced by Wnt5a overexpression and knockdown. CONCLUSIONS: This study demonstrates that Wnt5a protects MNs in ALS by regulating cell viability, proliferation, apoptosis, and neurite growth through the Wnt/Ca2+ signaling pathway. Our data indicate that the non-canonical Wnt/Ca2+ signaling pathway regulated by Wnt5a is involved in MN degeneration in ALS. AJTR
OBJECTIVES: We aimed to detect the expression profile of downstream signaling molecules of non-canonical Wnt pathway in SOD1G93A transgenic mice (ALS mice) and SOD1G93A mutant motor neuron-like hybrid (NSC-34) cells. Characterizing the molecular mechanism of the Wnt5a-mediated non-canonical Wnt/Ca2+ signaling pathway in motor neuron (MN) degeneration may provide a feasible approach to effective treatment of amyotrophic lateral sclerosis (ALS). METHODS: The expressions of CaMKII-α, CaMKII-β and TAK1 in the spinal cord of SOD1G93A ALS transgenic mice at different ages were determined using western blotting and immunofluorescence. The level of Ca2+ and cell apoptosis were assessed with flow cytometry and cell viability was evaluated using MTS assay. Cell proliferation was analyzed by the EdU cell proliferation assay. Neurite length was measured after treatment with retinoic acid. RESULTS: CaMKII-α, CaMKII-β, and TAK1 were down-regulated in the spinal cord of ALS mice. Ca2+ level and CaMKII-α, CaMKII-β, and TAK1 were down-regulated in SOD1G93A mutant NSC-34 cells. Expression of Ca2+, CaMKII-α, CaMKII-β, and TAK1 were up-regulated in SOD1G93A mutant NSC-34 cells after Wnt5a overexpression and down-regulated after Wnt5a knockdown. Overexpression of Wnt5a promoted cell viability and proliferation but inhibited cell apoptosis. Contrastingly, Wnt5a knockdown inhibited cell viability and proliferation but promoted cell apoptosis. CaMKII inhibitor KN-93 and CaMKII activator oleic acid reversed changes in cell viability, proliferation, apoptosis, and neurite outgrowth induced by Wnt5a overexpression and knockdown. CONCLUSIONS: This study demonstrates that Wnt5a protects MNs in ALS by regulating cell viability, proliferation, apoptosis, and neurite growth through the Wnt/Ca2+ signaling pathway. Our data indicate that the non-canonical Wnt/Ca2+ signaling pathway regulated by Wnt5a is involved in MN degeneration in ALS. AJTR
Authors: Zachary Osking; Jacob I Ayers; Ryan Hildebrandt; Kristen Skruber; Hilda Brown; Daniel Ryu; Amanda R Eukovich; Todd E Golde; David R Borchelt; Tracy-Ann Read; Eric A Vitriol Journal: iScience Date: 2019-08-16