MODULATION OF TRPML1/TFEB PATHWAY FOR THE TREATMENT OF WILSON DISEASE

Wilson disease (WD) is an autosomal recessive disorder characterized by toxic copper accumulation in several tissues, resulting in hepatic and neurological impairment. WD is caused by mutations in the P-type ATPase copper transporting B (ATP7B) gene, responsible for copper supply to cuproproteins and removal of excessive copper by biliary excretion. In ATP7B-knockout cells, copper overload triggers autophagy and lysosomal exocytosis via transcription factor EB (TFEB), stimulating copper clearance and reducing toxicity. Lysosomal Ca2+ release through transient receptor potential mucolipin 1 (TRPML1) activates calcineurin, which dephosphorylates TFEB, promoting its nuclear translocation. In this study, we investigated the therapeutic potential of TRPML1/TFEB axis for the treatment of WD. We first assessed the effects of TFEB and TRPML1 activation in vitro using the TRPML1 agonists ML-SA5 and ML-SA1. In copperchallenged ATP7B-KO cells, TRPML1 agonists improved cell viability and mitochondrial membrane polarization, indicating protection against copper-induced mitochondrial damage. We then moved to an in vivo proof-of-concept study using Atp7b-/- mice. We generated AAV8 vectors expressing TFEB, TRPML1, or GFP (control) with a hepatocytespecific promoter and administered them intravenously to 6-week-old Atp7b-/- mice. After twelve weeks, mice overexpressing TRPML1 – and, to a lesser extent, TFEB – showed decreased serum transaminase and alkaline phosphatase activities, reduced liver inflammation and fibrosis, and normalized copper levels in brain and urine. TFEB and TRPML1 overexpression also increased biliary copper excretion, suggesting enhanced lysosomal exocytosis at the canalicular domain of hepatocyte plasma membrane. Conversely, in vivo testing of ML-SA5 in Atp7b-/- mice did not produce measurable benefits, and TRPML1 activation with ML-SA1 or ML-SA5 did not improve WD organoid survival under copper stress. These results highlight the need for further studies to elucidate the mechanisms of action of these TRPML1 agonists. In conclusion, this work provides proof-of-concept for the efficacy of TRPML1 and TFEB overexpression in ameliorating WD and proposes TRPML1/TFEB axis as a novel target for the treatment of this severe genetic disease.