Wnt5a Protects Motor Neuron in Amyotrophic Lateral Sclerosis by Regulating Wnt/Ca2+ Signaling Pathway

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that lead to the degeneration and death of motor neurons (MNs). Wnt signaling participates in multiple neurodegenerative processes. Wnt5a is a critical signaling molecule in Wnt signaling pathway. Intracellular Ca 2+ homeostasis is disrupted in a number of neurodegenerative diseases, and Ca 2+ /Calmodulin dependent protein kinase II (CaMKII) involved in various diseases associated with Ca 2+ signaling abnormalities. Here, we found that Wnt5a modulated MNs degeneration during ALS. CaMKII is a key effector of signals derived from Wnt/Ca 2+ signaling pathway. Its main subtypes CaMKII-α and CaMKII-β were down-regulated in the spinal cord of SOD1 G93A transgenic mice (ALS mice) and SOD1 mutant motor neuron like hybrid (NSC-34) cells. In addition, results showed that CaMKII-α and CaMKII-β were positively regulated by Wnt5a in vitro. Using specic CaMKII inhibitor and activator, we found that Wnt5a promoted NSC-34 cells viability and proliferation, inhibited cells apoptosis and promoted cells neurite outgrowth via the Wnt/Ca 2+ signaling pathway. These results indicate that Wnt5a confers neuroprotection by promoting neuronal proliferation, inhibiting cell apoptosis and promoting neurite growth through Wnt/Ca 2+ signaling pathway. Therefore, targeting Wnt5a can be an effective strategy to treat ALS. as well as intracellular Ca 2+ (level of SOD1 mutant NSC-34 cells) were detected at 48 h after over-expressing and knocking down Wnt5a. A correct intracellular Ca 2+ level is crucial for MNs function The ow cytometry results showed the percent of Ca 2+ in SOD1 mutant NSC-34 cells were increased by 6.68% after overexpressing Wnt5a and decreased by 12.97% after knocking down Wnt5a (p = 0.036, p = 0.031, Fig.3a-d). Western blot analysis showed that the expression levels of CaMKII-α and CaMKII-β proteins in SOD1 mutant NSC-34 cells increased 2.48 fold and 1.19-fold after overexpression of Wnt5a, while they decreased by 73.32% and 75.66% after knocking down Wnt5a, respectively (p = 0.0003, p = 0.0017, p = 0.015, p = 0.03, p = 0.0003, p = 0.006, Fig.3e-h). These results suggest that the level of intracellular Ca 2+ and the expression levels of CaMKII-α and CaMKII-β are manipulated by overexpression and knockdown of Wnt5a.

enhances improved symptoms and motor function of SOD1 mutants in ALS transgenic mice and increases the survival rate of MNs [11][12][13]. But there is still a lot of research to be done on targeted therapy for ALS. Wnt signaling is a conserved pathway in animal development and is highly associated with the occurrence and development of a variety of neurodegenerative diseases, including the pathogenic mechanisms of ALS. Presently, more than 19 genes associated with Wnt signaling have been pro led in both mice and humans. The Wnt genes encode Wnt proteins to activate the Wnt signaling pathway. The extra-cellular Wnt signal stimulates several intracellular signal transduction cascades, including the βcatenin-dependent pathway (Wnt/β-catenin pathway) for canonical and non canonical for β-cateninindependent pathway. It is the β-catenin-independent pathway that comprise the Wnt/Planar Cell Polarity (Wnt/PCP) and the Wnt/Ca 2+ pathways [14][15][16]. Knowledge of the Wnt pathway and the involved genes is a promising method to search for possible targets to counteract the harmful effects of neurodegenerative diseases. Wnt ligands are well known for activating the canonical β-catenin pathway, which regulates acetylcholine receptor (AChR) aggregation as well as the formation and maintenance of neuromuscular junctions (NMJ). The ndings suggested that β-catenin distribution could be an underlying factor affecting the onset of neurodegeneration in fALS [17]. Therefore, it may provide a feasible method for the treatment of ALS by targeting the Wnt/β-catenin pathway to maintain the stability of NMJ [18]. At present, the involvement of non-canonical Wnt signaling pathways in neuronal survival and death processes remains unclear [19].
Wnt5a is an important non-canonical Wnt signaling pathway ligand. It uses Ca 2+ as a second messenger to achieve its biological effect [20]. Wnt5a binds to the homologous frizzled (Fzd) receptor, which leads to an increase in the intracellular Ca 2+ concentration. The released Ca 2+ then activates CaMKII to regulate many biological processes such as cytoskeleton rearrangement, cell adhesion and migration [21,22]. CaMKII is mainly composed of α and β subtypes in neurons and plays a role in the regulation of longterm potentiation, axon growth and neurotransmitter release [23]. Some research suggests that Wnt5aactivated Ca 2+ signaling plays an important role in neurite outgrowth of various neuronal cell types [24]. It can also protect neurons in Alzheimer's disease (AD) by regulating the Wnt/Ca 2+ signaling pathway [25]. However, for ALS, there is no reliable evidence to indicate whether Wnt5a can affect the function of MNs through the Wnt/Ca 2+ signaling pathway.
Here we detect the expression of CaMKII-α and CaMKII-β in ALS mice and SOD1 mutant NSC-34 cells and explore the role of Wnt5a in degeneration of MNs. This study further gives insights into the molecular mechanism of ALS, which may provide new strategies for treating ALS.

Materials And Methods
Animals and tissue preparation Amyotrophic lateral sclerosis transgenic mice (ALS mice) with SOD1 G93A gene mutation and Wide-type mice (WT mice) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Male ALS mice aged 6-8 weeks were mated with adult female WT mice. The tail of newborn mice was cut after four weeks and the genomic DNA of the tail tissue was ampli ed using polymerase chain reaction (PCR) for genotyping. Adult ALS mice were randomly divided into three groups based on the early stage (95 days), middle stage (108 days) and late stage (122 days). Each group of ALS mice was matched with WT Littermate of the same age as the control group. The spinal cord tissues were directly isolated from some mice and stored in -80°C refrigerator to be used for extracting protein and RNA. Other mice were perfused with 4% paraformaldehyde, and their spinal cords were stripped out and frozen sections were prepared for immuno uorescence staining.
The secondary antibodies used include Alexa Fluor 488-conjugated anti-mouse IgG (1:400, Jackson ImmunoResearch) and Cy3 conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch). PBS was used instead of the rst antibody in the control group and there was no speci c staining identi ed. The images were observed and obtained using uorescence microscope (Olympus, Tokyo, Japan). The numbers of double positive cells were measured using the ImageJ software.

MTS viability assay
The cells viability was measured using Cell Titer 96 ® AQueous One Solution Cell Proliferation Assay.
Brie y, 1×10 4 transfected cells were plated into each well of a 96-well plate. The cells were cultured in CO 2 incubator after transfection. MTS reagent and 10% FBS medium were added into each well in the ratio of 1:4, and the plate was then incubated in CO 2 incubator for 40 min. The cell viability was detected at 0, 24, 48, 72 and 96 h after transfection. Absorbance (OD) was measured at 490 nm. Finally, the cells proliferation curve was drawn at various time points.

EdU cell proliferation assay
Cell proliferation was assessed using 5-Ethynyl-2-deoxyuridine (EdU) DNA proliferation assay at 48 h after transfection. The number of cells in the S phase was assessed according to the manual of Cell-Light TMEdU Apollo ® 567 In Vitro Kit (RiboBio). Brie y, 1×10 5 cells were cultured in each well of 96-well plates. The transfected cells were labeled with 0.1% reagent A. Then 4% paraformaldehyde was incubated for 15 min at room temperature to x the cells. 1×Apollo staining reaction solution was added to each well, and incubated in dark for 30 min. After being washed by PBS for 3 times, DNA staining was performed using Hoechst 33342 reaction solution for 15 min. The cells images were observed under a uorescence microscope (Olympus). The percentage of EdU-positive cells was calculated from ve random elds.

Cell apoptosis assay
The apoptosis of the NSC-34 cells were determined using FITC Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Brie y, the cells were plated in 6-well culture plates at a density of 1×10 6 /well, then cultured with 48 h. After incubation, cells were collected into 1.5 ml EP tubes, rinsed with PBS and re-suspended in 1×Annexin V binding buffer. Then, the cells were stained with 5 μl annexin V-FITC and 5 μl PI in darkness for 20 min at room temperature. After staining, apoptotic cell in each treatment (1×10 5 cells) was analyzed using a ow cytometry. All the experiments were performed in triplicate.

Measurement of neurite outgrowth
The NSC-34 cells were cultured on coverslips coated with poly-L-lysine, followed by treatment with 10 µmol/L retinoic acid (RA, Sigma-Aldrich) for 48 h after transfection to induce the neurite outgrowth. Then, cells were photographed using the microscope (Leica Microsystems CMS GmbH). The length of neurite was de ned as the distance from the soma to the tip of the longest branch [28]. Five random elds were used for image analysis in each group. The data on neurite length were obtained from three independent experiments.

Statistical Analysis
All data were statistically analyzed using SPSS software 22.0 and expressed as mean ± standard deviation (SD). The differences between groups were analyzed using two-tailed Student' t-test. The differences between groups were signi cant at p < 0.05. All graphs were generated using GraphPad Prism 7.0 software.

Results
The expression of CaMKII-α and CaMKII-β were down-regulated in the spinal cord of ALS mice CaMKII is the key molecule of Wnt/Ca 2+ signaling pathway. To indicate the distribution and localization of CaMKII-α and CaMKII-β in ALS, their expression was detected using double immuno uorescence staining technique. The results showed that CaMKII-α and CaMKII-β were expressed in the anterior horn of gray matter of spinal cord and co-expressed with neurons labeled with β-tubulin III. Meanwhile, the number of CaMKII-α/β-tubulin III and CaMKII-β/β-tubulin III double-positive cells in ALS middle stage were reduced by 49.45% and 58.87% compared to WT mice in the ventral horn of gray matter (p = 0.0037, p = 0.0001, Fig. 1a-c).
The level of Ca 2+ and the expression of CaMKII-α and CaMKII-β were down-regulated in SOD1 mutant

NSC-34 cells model
To further verify the effect of SOD1 gene mutation on the key signal molecules in Wnt/Ca 2+ signaling pathway in vitro, a NSC-34 cell model was constructed by transfecting the pcDNA3.1-G93A-SOD1 and pcDNA3.1-WT-SOD1 plasmids. The expression levels of CaMKII-α and CaMKII-β as well as intracellular Ca 2+ were detected at 48 h after transfection. The ow cytometry results demonstrated that the percent of Ca 2+ was decreased by 16.02% in NSC-34 cells transfected with the pcDNA3.1-G93A-SOD1 plasmid, compared with that transfected with the pcDNA3.1-WT-SOD1 plasmid (p = 0.048, Fig. 2a, b). Western blot analysis showed that the expression of CaMKII-α and CaMKII-β proteins were decreased by 62.77% and 74.54% in NSC-34 cells that carried the pcDNA3.1-G93A-SOD1 plasmid than those that carried the pcDNA3.1-WT-SOD1 plasmid (p = 0.022, p = 0.001, Fig. 2c, d). These results indicate that the changes of CaMKII-α and CaMKII-β expression were consistent with those of animal model.
Overexpression and knockdown of Wnt5a up-regulated and down-regulated Ca 2+ , CaMKII-α and CaMKII-β levels in SOD1 mutant NSC-34 cells To study the regulation of Wnt5a on Wnt/Ca 2+ signaling molecules in ALS, the expression levels of CaMKII-α and CaMKII-β as well as intracellular Ca 2+ (level of SOD1 mutant NSC-34 cells) were detected at 48 h after over-expressing and knocking down Wnt5a. A correct intracellular Ca 2+ level is crucial for MNs function [29]. The ow cytometry results showed the percent of Ca 2+ in SOD1 mutant NSC-34 cells were increased by 6.68% after overexpressing Wnt5a and decreased by 12.97% after knocking down Wnt5a (p = 0.036, p = 0.031, Fig.3a-d). Western blot analysis showed that the expression levels of CaMKII-α and CaMKII-β proteins in SOD1 mutant NSC-34 cells increased 2.48 fold and 1.19-fold after overexpression of Wnt5a, while they decreased by 73.32% and 75.66% after knocking down Wnt5a, respectively (p = 0.0003, p = 0.0017, p = 0.015, p = 0.03, p = 0.0003, p = 0.006, Fig.3e-h). These results suggest that the level of intracellular Ca 2+ and the expression levels of CaMKII-α and CaMKII-β are manipulated by overexpression and knockdown of Wnt5a.
All the above results supported that KN-93 can block the Wnt/Ca 2+ pathway, while OA can activate the Wnt/Ca 2+ pathway in ALS. Wnt5a may mitigate neurodegeneration and protect MNs through the Wnt/Ca 2+ pathway in ALS.

Discussion
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of MNs in the cortex, brainstem and spinal cord [5]. It was described by French neurologist Charcot in 1869 that, affects the upper and lower MNs [32]. Advanced stage of the disease may lead to the loss or death of MNs with the loss of motor function [33]. The ALS pathogenic genes include C9ORF72, SOD1, TARDBP, FUS, OPTN, PFN1, MATR3, TUBA4A, and TBK1 among others [34]. SOD1 gene was the rst gene associated with ALS, in 1993 [3,35]. Previous researches showed that massive MNs died in the ventral horn, as well as the loss of myelinated axons in the ventral motor roots of the spinal cord in SOD1 G93A animal model [36].
Wnt signaling pathway regulates multiple cell functions and controls many aspects of development, including cell proliferation, apoptosis, migration and cell polarity [21]. Wnt signaling pathway mediated by Wnt3a, Wnt5a and Wnt7a can antagonize neurotoxicity of AD β-amyloid protein, protect hippocampal neurons and improve cognitive function of patients. The activation of Wnt/β-catenin signaling pathway is related to the viability of Parkinson's Diseases (PD) dopaminergic neurons, which is conducive to maintain the integrity of dopaminergic neurons. Hence, targeting Wnt signaling molecules may confer therapeutic effects in neurodegenerative diseases [37][38][39].
Previously, the differential expression of canonical Wnt signal ligands (Wnt1, Wnt2, Wnt3a, and Wnt10a) and receptors (Fzd2, Fzd4, Fzd7 and LRP5) in the spinal cords of ALS mice were examined at different stages using transcriptional microarray analysis. Meanwhile, the expression of non-canonical Wnt ligand Wnt5a is also abnormal in ALS mice [40,41]. In the current study, it was con rmed that canonical Wnt signaling pathway can be activated in the pathogenesis of ALS [4]. Furthermore, it was found that Wnt1, a canonical Wnt signaling molecule, can promote the proliferation and viability of SOD1 mutant NSC-34 cells, inhibit cell apoptosis and protect MNs. In addition, silencing RNA helicase DDX3 was found to inhibit the proliferation of SOD1 mutant NSC-34 cells, promote cell apoptosis and affect the outgrowth of neurite through Wnt signaling molecule CK1ε [42]. These results indicate that Wnt signaling pathway regulates MNs degeneration in ALS.
Studies have found that canonical and non-canonical Wnt signaling pathways modulate the occurrence and development of various diseases [43,44]. Wnt5a is a key signaling molecule that participates in the non-canonical Wnt signaling pathway. In our previous study, Wnt5a abnormal expression was detected in the spinal cord of ALS mice [41]. Wnt5a mediated non-canonical signaling pathway plays an important role in neuronal damage [31,45]. In mammals, Wnt5a activates intracellular Ca 2+ release and further activates CaMKII to regulate neurite outgrowth and cell function mediated by Wnt/Ca 2+ pathway [46]. Spalloni et al. [47] found a decrease of the CaMKII-α autophosphorylation at threonine-286 in cortical M1 region of mice overexpressing the SOD1 G93A gene. In this study, it was found that the number of CaMKIIα/β-tubulin III and CaMKII-β/β-tubulin III double-positive cells in ALS middle stage were less than WT mice in the ventral horn of gray matter, which indicated that CaMKII-α, and CaMKII-β expression were related to the degeneration of MNs in ALS. The results of qRT-PCR and western blotting also showed that the expression of CaMKII-α and CaMKII-β were decreased in the onset period, which demonstrated that CaMKII-α and CaMKII-β were involved in the development of ALS. Intracellular Ca 2+ homeostasis is disrupted in many neurodegenerative diseases. High stimulation of glutamate receptor in MNs and subsequent excitotoxicity induced by increased calcium ion implantation may lead to MNs degeneration in ALS patients and SOD1 mutant mice model [48]. This study detected that the intracellular Ca 2+ level of NSC-34 cells carrying the pcDNA3.1-G93A-SOD1 plasmid was lower than those of NSC-34 cells transfecting the pcDNA3.1-WT-SOD1 plasmid at 48 h. CaMKII is a key molecule in the pathological cascade downstream of abnormal Ca 2+ signaling [49][50][51]. Our results showed that mRNA and protein levels of CaMKII-α and CaMKII-β at 48 h in NSC-34 cells carrying the pcDNA3.1-G93A-SOD1 plasmid were lower than in NSC-34 cells transfected with the pcDNA3.1-WT-SOD1 plasmid. These results suggested that the mutation of SOD1 reduced the expression of CaMKII-α and CaMKII-β, indicating that Wnt/Ca 2+ signaling pathway was involved in the pathogenesis of ALS.
To determine whether Wnt5a regulate Wnt/Ca 2+ signaling pathway in the pathogenesis of ALS, NSC-34 cells were respectively transfected with pcDNA3.1-G93A-SOD1 plasmid and pcDNA3.1-Wnt5a plasmid or siRNA-Wnt5a. We found that overexpression of Wnt5a up-regulated the intracellular Ca 2+ level and CaMKII-α and CaMKII-β expression, whereas knockdown of Wnt5a down-regulated them. These results suggested that Wnt5a positively regulated the intracellular Ca 2+ level and expression of CaMKII-α and CaMKII-β in ALS.
The loss of MNs was also linked to the pathogenesis of ALS. Martin [52] found that apoptosis was activated in ALS. SOD1 is an antioxidant enzyme, which can protect neurons from the damage of free superoxide radicals. The mutation of SOD1 can stimulate protein aggregation and lead to apoptosis [53].
Han et al. [54] found that the increase of Wnt5a promoted cell proliferation. On the contrary, knocking down Wnt5a reduced cell proliferation. This study supports that overexpression of Wnt5a promoted the cell proliferation of SOD1 mutant NSC-34 cells and knockdown of Wnt5a reduced the cell proliferation of SOD1 mutant NSC-34 cells. Meanwhile, over-expressing Wnt5a reduced the cell apoptosis of SOD1 mutant NSC-34 cells and knockdown of Wnt5a promoted the cell apoptosis of SOD1 mutant NSC-34 cells. These ndings imply that Wnt5a is important for cell growth and exerted neuroprotective effect through Wnt/Ca 2+ pathway on SOD1 mutant NSC-34 cells.
Neurons are made of three distinct sections: the soma, which contains the nucleus and the majority of the cellular organelles, a long axonal process to transmit information, and a complex dendritic arbor that receives information from neighboring neurons [55,56]. Mature neurites include axons and dendrites, which are supported by microtubule cytoskeleton composed of bundles of microtubules. Studies have shown that the degeneration of ALS MNs is related to the changes of cytoskeleton. SOD1 G93A mutation can affect the cytoskeleton state to regulate the neurite outgrowth of MNs [57]. CaMKII is a cytoskeletonrelated protein. Overexpression of CaMKII-α leads to neurite outgrowth [58]. Lund et al. [23] con rmed that the expression of CaMKII was changed after axonal injury in the sciatic MNs. To clarify whether Wnt5a regulates the neurite outgrowth to affect the function of ALS MNs, this study detected the neurite outgrowth of SOD1 mutant NSC-34 cells using RA. Overexpression of Wnt5a increased the length of SOD1 mutant NSC-34 cells and the percentages of cells with one or more neurites. Similarly, knocking down Wnt5a inhibited the neurite outgrowth of SOD1 mutant NSC-34 cells. It is suggested that Wnt5a may participate in neuronal degeneration by regulating the neurite outgrowth in ALS.
Finally, speci c CaMKII inhibitor KN-93 and activator OA were used to verify the role of Wnt/Ca 2+ pathway mediated by Wnt5a. The results showed that KN-93 reversed the NSC-34 cells viability, proliferation, apoptosis and neurite outgrowth after overexpression of Wnt5a and OA rescued the NSC-34 cells viability, proliferation, apoptosis and neurite outgrowth after knocking down Wnt5a. These results suggested that Wnt5a regulate the cells function to affect neuron motor degeneration through Wnt/Ca 2+ signaling pathway. In Arredondo's study, they determined that Wnt5a signals induce neurogenesis through CaMKII, and promotes dendritic development of newborn neurons through activating Wnt/JNK and Wnt/CaMKII signaling using speci c inhibitors [59]. These results are in line with those results of us. Wnt5a has certain signi cance and value for the continuous research, development and clinical application of ALS.
However, whether Wnt5a regulates canonical and non-canonical Wnt signaling pathways through binding different cell receptors in development of ALS is still unclear and needs further research. Availability of data and materials All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Medical Ethics Committee of Weifang medical university (protocol code2018-No.156 and date of approval 26 February 2018).

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.