Regulatory Function Of Zebrafish Slc30a10 In Manganese Homeostasis

Manganese?Mn?,an essential element,is required for the catalytic activity of numerous cellular enzymes in many organs and tissues.It is widely involved in not only the metabolic processes of amino acids,lipids,carbohydrates,but also immune functions,the growth of bones and connectives tissues,blood coagulation and nerve conduction.Either manganese deficiency or accumulation could directly lead to malfunction of a cell.Therefore,the maintenance of manganese homeostasis is essential for a living organism.It is known that cellular manganese is tightly regulated by metal transporters,such as SLC30A10,which has been proposed as one of the manganese exporters.Autosomal recessive mutations in the human SLC30A10 gene have been identified in HMDPC?hypermanganesemia with dystonia,polycythemia,and cirrhosis,OMIM#613280?.Currently,standard treatments for HMDPC are chelation therapies using intravenous disodium calcium edetate?EDTA-Ca Na₂?or oral iron?ferrous fumarate?supplementation.Since the non-specific feature of EDTA-Ca Na₂might cause many adverse effects,it is urgently needed to have a faithful animal model to screen for safer and more efficacious drugs for treating HMDPC patients.To our knowledge,there are no reliable animal models that closely recapitulate symptoms of human HMDPC patients.This study aims to generate a novel HMDPC disease model for better understanding the regulatory mechanism of Mn in the development of HMDPC by using genetic advantages of zebrafish.Zebrafish serves as a suitable model for studying manganese metabolism.Firstly,human SLC30A10 has high sequence homology compared to zebrafish Slc30a10.Secondly,similar tissue sensitive pattern to Mn,such as brain and liver,has been observed between human and zebrafish.Thirdly,the zebrafish model presents similar phenotype of the locomotion deficits to those patients with hypermanganesemia.Here,we generated two Slc30a10 mutant zebrafish models using CRISPR/Cas9.We measured the levels of Mn in mutant animals.Mn levels were lower in 1-week-old mutant embryos compared to wild-type embryos.While in 3-week-old larvae,we found higher Mn levels in the mutants compared to wild-type animals.Finally,in 4-month-old adults,systemic Mn levels were significantly higher in both male and female mutant animals compared to wild-type controls.No locomotion defects were observed in the mutant embryos during the hatching stage.However,when exposure to relatively low concentrations of Mn²⁺,mutant embryos-but not wildtype embryos-had impaired locomotion and other defects,including tremor,postural instability,disorientation,and impaired balance.Under natural growth condition,our Slc30a10 mutant zebrafish begin to develop neurological defects at approximately 4 months of age.Increased extracellular GABA and impaired dopaminergic transmission might be attributed to the locomotion defects of mutant zebrafish upon Mn exposure.Additionally,mutant animals developed steatosis,liver fibrosis,and polycythemia accompanied by increased epo expression.These phenotypes could be partially rescued by EDTA-Ca Na₂chelation therapy and iron supplementation.Taken together,we have successfully generated a HMDPC disease model,which faithful recapitulates symptoms presented in human patients.Mechanistically,we found that ATP2C1?a member of the P-type family of ATPases?could partially compensate the regulatory role of SLC30A10 in Mn metabolism.ATP2C1 pumps Ca²⁺and Mn²⁺ions from the cytosol into the Golgi apparatus.In the absence of SLC30A10,cells under Mn stress could utilize ATP2C1 to pump Mn from the cytoplasm via Golgi vesicular trafficking.When the expression levels of SLC30A10 are sufficient,cells mainly utilize SLC30A10 as the primary Mn exporter under Mn stress without initiation of the ATP2C1-mediated mobilization of Mn.Notably,when cells express a mutant form of SLC30A10,Mn accumulates in the cytoplasm,causing Mn-induced toxicity.In conclusion,we developed and functionally characterized two independent lines of Slc30a10 mutant zebrafish.We demonstrated that these zebrafish disease models faithfully recapitulate HMDPC patients'symptoms,which provides a novel invaluable model for further studying both molecular mechanisms and screening potential drugs for the disease.Mechanistically,we found that ATP2C1 plays a compensatory role in the context of SLC30A10 dysfunction,thereby maintaining Mn metabolism.These findings not only shed light on our understanding of the role that the Mn exporter SLC30A10 plays in the pathophysiology of HMDPC,but also provide experimental evidence for targeting ATP2C1 in managing HMDPC and related disorders.