The Deletion Of ATP13A2 Activates The Mechanism Of The Canonical Wnt Signaling Pathway

Objective:Parkinson’s Disease(PD)is the second-most common neurodegenerative disorder in the world.Kufor–Rakeb syndrome(KRS)is an autosomal recessive form of the early-onset parkinsonism with pyramidal degeneration and dementia.ATP13A2(also known as PARK9)is the pathogenic gene of KRS.Researchers deem that the mutation of ATP13A2 destroys autophagosomes-lysosomes fusion,leading to accumulation of α-synuclein and damage mitochondrial function,results in the loss of dopaminergic neurons with enhancing cytotoxicity.In order to further understand the mechanism of ATP13A2 in PD and the other related diseases,the previous work of our laboratory used the proximity labeling with Turbo ID system to screen out the low-density lipoprotein receptor-related protein 6(LRP6)that interacts with ATP13A2.LRP6 is a single-pass membrane surface protein and an important receptor component of the classical Wnt signaling pathway.Studies have found that abnormal activation of the Wnt signaling pathway can induce apoptosis,and the reduction of β-catenin degradation will result in a decrease in the proportion of dopamine neurons in the proliferating neurons.Epigenetic studies have found that Wnt signaling pathway-related genes in the brains of PD patients are highly methylated,and the Wnt target gene’s protein expression in midbrain dopaminergic neurons is reduced,involved in the neurogenesis.Therefore,the Wnt signaling pathway is closely related to PD.Methods and Results:Firstly,we verify the interaction between ATP13A2 and LRP6 by the co-immunoprecipitation in HEK293 cells,at the same time,immunofluorescence experiment also observes the good co-localization between them.Because ATP13A2 plays an important role in maintaining the normal lysosomal function,we find that LRP6 and the phosphorylated LRP6 are relatively increased and the cytoplasmic β-catenin is also significantly increased in ATP13A2 KD HEK293 cells and ATP13A2 KO mouse embryonic fibroblasts(MEFs).These data all marks the activation of the canonical Wnt signaling pathway.Therefore,we confirm that the knockdown of ATP13A2 activates the canonical Wnt signaling pathway,while the overexpression of ATP13A2 represses the pathway by the Dualluciferase Reporter Assay(DLR).To explore the mechanism of LRP6 increase caused by the deletion of ATP13A2,we find that the half-life of LRP6 is not affected in the ATP13A2 KD HEK293 cells or ATP13A2 KO MEFs with the treatment of cycloheximide(CHX)that can inhibit the synthesis of new proteins,which suggests that ATP13A2 may affect the gene expression of LRP6.Then we confirm that the deletion of ATP13A2 increases LRP6 m RNA expression level by Real-time PCR.Further,we search the DNA sequence of the LRP6 promoter region in NCBI and constructed the recombinant plasmid with p GL4.22 as the vector.The DLR shows that the deletion of ATP13A2 increases the transcription activity of LRP6.Conclusions:Our research is the first to confirms that deletion of PD-related gene ATP13A2 enhances the transcriptional expression of LRP6,which in turn activates the canonical Wnt signaling pathway.This study reveals the new function of ATP13A2 in the Wnt signaling pathway,and provides new ideas and evidence for the pathogenesis of neurodegenerative diseases.