Pathobiologic Mechanisms of Neurodegeneration in Osteopetrosis Derived From Structural and Functional Analysis of 14 ClC-7 Mutants

Abstract

ClC‐7 is a chloride‐proton antiporter of the CLC protein family. In complex with its accessory protein Ostm‐1, ClC‐7 localizes to lysosomes and to the osteoclasts' ruffled border, where it plays a critical role in acidifying the resorption lacuna during bone resorption. Gene inactivation in mice causes severe osteopetrosis, neurodegeneration, and lysosomal storage disease. Mutations in the human CLCN7 gene are associated with diverse forms of osteopetrosis. The functional evaluation of ClC‐7 variants might be informative with respect to their pathogenicity, but the cellular localization of the protein hampers this analysis. Here we investigated the functional effects of 13 CLCN7 mutations identified in 13 new patients with severe or mild osteopetrosis and a known ADO2 mutation. We mapped the mutated amino acid residues in the homology model of ClC‐7 protein, assessed the lysosomal colocalization of ClC‐7 mutants and Ostm1 through confocal microscopy, and performed patch‐clamp recordings on plasma‐membrane‐targeted mutant ClC‐7. Finally, we analyzed these results together with the patients' clinical features and suggested a correlation between the lack of ClC‐7/Ostm1 in lysosomes and severe neurodegeneration. © 2020 American Society for Bone and Mineral Research (ASBMR).

Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

“…Perhaps upon binding PI(3,5)P2 the 5phosphate revises the H-bond network and induces a gating-controlling conformational change to inhibit the transporter. This model is consistent with a large body of evidence for a role of the cytoplasmic domain in slow gating of ClC-7, including a report that the ClC-7 C-terminal domain is necessary for voltage-dependent gating that corroborates the importance of this interface (Di Zanni et al, 2021;Ludwig et al, 2013;Sartelet et al, 2014).…”

The Role of the Lysosomal Cl−/H+ Antiporter ClC-7 in Osteopetrosis and Neurodegeneration

“…In relation to the genotype-phenotype correlation of ARO, Di Zanni et al observed that some ARO mutations associated with neurodegeneration (R126H, A299V/E, P582H and G780R) reduced lysosomal localization and produced no or little current when expressed at the plasma membrane. In contrast to this, ARO mutations without neurodegeneration (L90P, P376L, A511T, G780W/R, A590T and R791C) preserved ion transport activity [102]. While these are interesting observations, it should be kept in mind that the protein localization is often altered by overexpression in heterologous systems.…”

“…Several functional analyses of disease causing mutations have been conducted [7,11,78,102,103], and in combination with the very recent structures of the ClC-7/Ostm1 complex [5,6], this provides the opportunity to test if there is any correlation between alterations of ClC-7 functional properties and the phenotype of the disease. The first observation that emerges from the mapping of ARO and ADO II mutations onto the structure of ClC-7 is that there is no single hot-spot, and mutations are distributed throughout the whole protein.…”

Autosomal recessive osteopetrosis: mechanisms and treatments

“…In terms of genetic characterization, biallelic mutations of the CLCN7 gene are reported to cause severe forms of osteopetrosis, showing bone damage, hematological failure and primary neurodegeneration, with symptoms resembling lysosomal storage defects ( Kornak et al, 2001 ; Sobacchi et al, 2007 ). Neurodegeneration is probably due to the defective localization of ClC-7 in lysosomal membranes, because patients bearing mutations that maintain correct membrane targeting do not present CNS involvement ( Di Zanni et al, 2021 ). Additionally, CLCN7 mutations could account for the milder IAO, when patients suffer from mild symptoms and reach adulthood.…”