Disruption of ClC-3-mediated 2Cl(-)/H(+) exchange leads to behavioural deficits and thalamic atrophy.

CLCN3 encodes ClC-3, an endosomal 2Cl⁻/H⁺ exchanger, with pathogenic variants causing a neurodevelopmental condition marked by developmental delays, intellectual disability, seizures, hyperactivity, anxiety, and brain and retinal abnormalities. Clcn3(-/-) mice show hippocampal and retinal degeneration, recapitulating key symptoms observed in humans. ClC-3 forms homodimers (ClC-3/ClC-3) and heterodimers with ClC-4 (ClC-3/ClC-4), with overlapping brain expression. This suggests distinct functional roles for homo- and heterodimeric assemblies and raises the question of which brain regions specifically depend on ClC-3/ClC-3 rather than ClC-3/ClC-4 complexes. Using ex vivo PET tracer analyses, Clcn3(-/-) and Clcn3(td/td) mice, we found neurodegeneration in the hippocampus and thalamus of Clcn3(-/-), while Clcn3(td/td) mice showed thalamic degeneration and altered neuronal excitability, including changes in action potential threshold and after hyperpolarization. Clcn3(td/td) mice carrying a transport-deficient p.E281Q ClC-3 variant that still associates with ClC-4, thereby allowing ClC-4 to be sorted to endosomes as ClC-4/ClC-3 heterodimers, unlike in the Clcn3(-/-) model. Clcn3(td/td) mice also exhibited reduced weight, hyperactivity, and motor deficits, reflecting clinical features. Lower ClC-4 levels in thalamus predict a predominant thalamic expression of ClC-3/ClC-3 homodimers. Overall, our findings indicate a region-specific function of ClC-3/ClC-3 homodimeric complexes and highlight the importance of ClC-3 transport activity in thalamic neuron survival, with electrophysiological dysfunction likely contributing to neurodegeneration.