A research team led by Prof. WANG Xiaochen from the Institute of Biophysics of the Chinese Academy of Sciences identify the lysosomal Cl^-/H⁺ antiporter CLH-6/ClC-7 as an important factor for protecting lysosome integrity. The study was published online in Journal of Cell Biology.

Lysosomes are acidic membrane-bound organelles responsible for degrading extra- and intra-cellular macromolecules, recycling catabolites, and acting as signaling hubs to maintain cell and tissue homeostasis. Dysfunction of lysosomes is associated with metabolic disorders, neurodegenerative diseases, and cancer. Lysosomal membrane integrity is vital for cell homeostasis, but the underlying mechanisms are poorly understood.

Prof. WANG's lab utilizes the multicellular organism Caenorhabditis elegans to investigate how lysosome membrane integrity is maintained and how it contributes to animal development and longevity. They perform large scale genetic screens to identify genes important for the maintenance of lysosome integrity. In this study, the researchers demonstrate that CLH-6, the C. elegans ortholog of the lysosomal Cl^-/H⁺ antiporter ClC7, protects lysosome membrane integrity via cathepsin activation.

Wang lab identified clh-6 from a forward genetic screen, whose loss-of-function causes accumulation of the membrane damage reporter GFP::Gal3. Consistent with this, transmission electron microscopy analyses revealed severe membrane damage of lysosomes in clh-6(lf) mutants. In addition to lysosomal damage, the structural integrity of endomembrane compartments such as endosomes, Golgi and ER are also affected in clh-6 mutants, probably due to leakage of lysosomal hydrolases upon lysosomal membrane damage. Expression of ClC-7 efficiently rescues the lysosome integrity defect in clh-6 mutants, which suggests that CLH-6 and ClC-7 play a conserved role in preserving lysosomal membrane stability. clh-6 mutants have a shortened lifespan and significantly reduced brood size, which suggests that CLH-6 function is important for worm fertility and longevity.

The researchers investigated how lysosome membrane integrity is affected in clh-6 mutants. They found that loss of clh-6 function causes cargo accumulation in lysosomes, and reducing cargo delivery or increasing CPL-1/cathepsin L or CPR-2/cathepsin B expression suppresses lysosomal defects in clh-6 mutants. Moreover, inactivation of CPL-1 or CPR-2, like CLH-6 inactivation, affected cargo digestion and causes lysosomal membrane rupture. These data suggest that loss of CLH-6 impairs cargo degradation, leading to membrane damage of lysosomes.

In clh-6 mutants, lysosomes are acidified as in wild type but contain lower chloride levels, and cathepsin B and L activities are significantly reduced. MST assay revealed that Cl^- binds to CPL-1 and CPR-2 in vitro, and Cl^- supplementation increases lysosomal cathepsin B and L activities in a concentration-dependent manner. These results collectively suggest that CLH-6 maintains the luminal chloride levels required for cathepsin activity, thus facilitating substrate digestion to protect lysosomal membrane integrity.

This study demonstrates that lysosomal Cl^-/H⁺ antiporter CLH-6/ClC-7 plays an essential role in the maintenance of lysosome membrane integrity and reveals the role of luminal Cl^- in modulating cathepsin activity in lysosomes. Loss of the ClC-7 chloride channel leads to osteopetrosis and causes neurodegeneration. The finding that CLH-6/ClC-7 maintains lysosome integrity via cathepsin activation provides new insights into the pathogenesis of CLCN7 deficiency-related human diseases.

Article link: https://doi.org/10.1083/jcb.202210063

Contact: WANG Xiaochen

Institute of Biophysics, Chinese Academy of Sciences

Beijing 100101, China

Email: wangxiaochen@ibp.ac.cn

（Reported by Dr. WANG Xiaochen's group）