Research of The Role of CLN7 in The Regulation of Lysosomal Function

Introduction

Lysosomes play a vital role in eliminating worn-out proteins, damaged organelles, and other unnecessary materials in eukaryotic cells. Dysfunction of lysosomes often leads to lysosomal storage diseases (LSDs), which are characterized by abnormal accumulation of unmetabolized substrates within lysosomes. Neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive lysosomal storage diseases that are associated with progressive symptoms including dementia, visual loss, cerebral atrophy, epilepsy, and early death. Each of the NCL subtypes is caused by mutations in one of the CLN genes. Although NCLs share common pathological and cellular characteristics, the functions of the CLN genes are diverse. One variant form of late-infantile NCL (vLINCL) is caused by mutations of a lysosomal membrane protein CLN7, the function of which has remained unknown. There is currently no effective treatment for vLINCL, and elucidating the function of CLN7 may facilitate the development of promising therapeutic targets.

Methods

All cells were cultured at 37°C in a humidified incubator supplied with 5% CO₂. HeLa and HEK293T cells were maintained in Dulbecco's modified eagle medium. The location of CLN7 in the lysosome was studied by subcellular localization and confocal observation. To further reveal the regulatory role of CLN7 on lysosomal function, cDNA constructs and transfections, knockdown of CLN7 in HEK293T cells using CRISPR-Cas9 technology, Western blots, electrophysiology experiments, calcium imaging, lysosomal pH imaging, iodide imaging, flow cytometry, retinal immunostaining, and visual water maze test were performed.

Results

The results of subcellular localization indicated that CLN7 was mainly localized to lysosomes and endosomes. Further results showed that overexpression of CLN7 increases endolysosomal chloride currents and enlarges endolysosomes through a Ca²⁺/calmodulin-dependent way.

Fig. 1 CLN7 localizes to lysosomes and endosomes and enlarges these organelles through CaM and Ca²⁺.

The results of whole-endolysosomal patch-clamp recordings and the effect of CLN7 on ion flux across the endolysosomal membrane indicate that human CLN7 and its yeast homolog exhibit characteristics of chloride channels and are sensitive to chloride channel blockers. Moreover, CLN7 regulates lysosomal chloride conductance, luminal pH, and lysosomal membrane potential and promotes the release of lysosomal Ca²⁺ through transient receptor potential mucolipin 1 (TRPML1).

Fig. 2 hCLN7 exhibits properties of chloride channels.

The pathogenic mutations in CLN7 lead to a decrease in chloride permeability, suggesting that reconstitution of lysosomal Cl⁻ homeostasis may be an effective strategy for the treatment of vLINCL.

Fig. 3 CLN7-KO mice recapitulate pathological features of human vLINCL.

In conclusion, the results of this study suggest that the development of specific drugs to restore the function of mutant CLN7 or to regulate other similar lysosomal chloride ion channels/transporters to compensate for the lack of CLN7-mediated chloride transport is expected to be a promising therapeutic strategy for mitigating CLN7-related diseases.

Summary

This study identifies CLN7 as a novel endolysosomal chloride channel and reveals its regulatory effect on lysosomal function. The pathogenic mutations in CLN7 lead to a decrease in chloride permeability, suggesting that reconstitution of lysosomal Cl⁻ homeostasis may be an effective therapeutic strategy for vLINCL.

Reference:

Wang, Y.; et al. CLN7 is an organellar chloride channel regulating lysosomal function. Science Advances, 2021, 7(51): eabj9608.

* For research use only. Not intended for any clinical use.

Quick Inquiry

Research of The Role of CLN7 in The Regulation of Lysosomal Function

Introduction

Methods

Results

Summary

Related Products and Services at Creative Biogene