Mutations in CLCN6 as a Novel Genetic Cause of Neuronal Ceroid Lipofuscinosis in Patients and a Murine Model

Objective: The aim of this study was to explore the pathogenesis of CLCN6-related disease and to assess whether its Cl^-/H⁺-exchange activity is crucial for the biological role of ClC-6.

Methods: We performed whole-exome sequencing on a girl with development delay, intractable epilepsy, behavioral abnormities, retinal dysfunction, progressive brain atrophy, suggestive of neuronal ceroid lipofuscinoses (NCLs). We generated and analyzed the first knock-in mouse model of a patient variant (p.E200A) and compared it with a Clcn6^-/- mouse model. Additional functional tests were performed with heterologous expression of mutant ClC-6.

Results: We identified a de novo heterozygous p.E200A variant in the proband. Expression of disease-causing ClC-6^E200A or ClC-6^Y553C mutants blocked autophagic flux and activated transcription factors EB (TFEB) and E3 (TFE3), leading to autophagic vesicle and cholesterol accumulation. Such alterations were absent with a transport-deficient ClC-6^E267A mutant. Clcn6^E200A/+ mice developed severe neurodegeneration with typical features of NCLs. Mutant ClC-6^E200A, but not loss of ClC-6 in Clcn6^-/- mice, increased lysosomal biogenesis by suppressing mTORC1-TFEB signaling, blocked autophagic flux through impairing lysosomal function, and increased apoptosis. Carbohydrate and lipid deposits accumulated in Clcn6^E200A/+ brain, while only lipid storage was found in Clcn6^-/- brain. Lysosome dysfunction, autophagy defects, and gliosis were early pathogenic events preceding neuron loss.

Interpretation: CLCN6 is a novel genetic cause of NCLs, highlighting the importance of considering CLCN6 mutations in the diagnostic workup for molecularly undefined forms of NCLs. Uncoupling of Cl^- transport from H⁺ countertransport in the E200A mutant has a dominant effect on the autophagic/lysosomal pathway. ANN NEUROL 2024;96:608-624.