The CLN7 protein that is defective in a form of late-infant Batten disease is a chloride channel that regulates the function of lysosomes, the cell’s recycling compartment, a new study finds.
Developing treatments to restore CLN7 function or modulate other lysosomal chloride channels could be a promising therapeutic strategy for treating CLN7-related diseases, the scientists suggested.
Details of the discovery were published in the journal Scientists progressin a study titledCLN7 is an organellar chloride channel regulating lysosomal function.”
Mutations in the MFSD8 gene causes a form of late-infantile Batten disease, with an age of onset between 2 and 7 years. The gene provides instructions for the CLN7 protein found in the membrane of lysosomes, an acidic compartment inside cells that degrades and recycles waste.
These genetic mutations lead to the toxic buildup of waste products — made up of fats and proteins called lipofuscins — that eventually kill cells. Nerve cells are sensitive to the accumulation of lipofuscin, leading to motor and mental impairments, seizures, slurred speech and loss of vision, all hallmarks of this type of disease.
Because the exact function of CLN7 is unknown, researchers at the University of Science and Technology of China designed a study to determine its role, with the aim of supporting the identification of therapeutic targets and the development of new therapies.
The team first confirmed that the CLN7 protein was located in the membrane of lysosomes but also in endosomes, a different type of compartment that transports and sorts material within the cell before reaching the lysosomes for degradation.
Overproduction of the CLN7 protein promoted the release of calcium ions from lysosomes, which activated a protein called calmodulin, leading to fusion of endosomes to lysosomes. This caused them to grow larger, indicating that CLN7 may play a role in transporting ions across membranes.
Based on these findings, the flow of ions, which carry an electric charge, was measured by tracking electric current. In cells with excess CLN7 protein, there was a significant increase in outgoing current, suggesting that positively charged ions moved into the lysosome or negatively charged ions moved out.
The team then replaced all positively charged ions – such as calcium, potassium, sodium and magnesium – with NMDG, a large positively charged molecule unable to cross membrane channels. Unexpectedly, the outgoing current remained unchanged.
Since the only negative ion present was chloride, the team found that reducing the concentration of chloride ions eliminated the outgoing current. This suggests that CLN7 mediated the flow of chloride ions out of lysosomes, the researchers noted.
The CLN7 protein further displayed properties seen in other chloride channels, such as allowing the flow of other negatively charged ions such as iodide (iodine ion) and fluoride. Next, exposing the cells to three chemicals known to block chloride channels reduced outward current flow in a dose-dependent manner.
Additionally, modifying the CLN7 protein to remove positively charged amino acids (the building blocks of proteins) believed to interact with negative chloride ions significantly reduced current compared to normal CLN7. Finally, delete the MFSD8 The gene largely stopped chloride currents, while the addition of mouse CLN7 rescued these currents.
“These electrophysiological and pharmacological properties suggest that CLN7 acts as a chloride channel,” the researchers wrote.
Experiments have also confirmed that CLN7 plays an indirect role in modulating calcium levels in lysosomes, as well as regulating lysosomal pH and establishing membrane potential. It is a voltage difference between the inside and the outside of the lysosome.
Next, the team studied the impact of four MFSD8 genetic mutations commonly seen in patients with late-infantile Batten disease. While all four mutations reduced chloride currents compared to normal CLN7, two of the mutations that led to more severe symptoms had more severe defects in chloride channel function.
“These results may provide a partial mechanistic explanation for the differential degrees of clinical symptoms among these four CLN7 mutations,” the scientists noted.
Finally, the size of lysosomes in cultured embryonic cells isolated from mice raised without CLN7 was larger than normal cells due to the accumulation of lipofuscins, as seen in CLN7 patients. Adding CLN7 to the cells dramatically reduced this accumulation.
In mouse cells lacking CLN7, there was also a decrease in the ability of the lysosome to degrade proteins and an increase in levels of oxidation of fat-like lipids, “which may be an important factor leading to cell death. in CLN7 disease,” the scientist wrote. .
The mice themselves showed an accumulation of lipofuscins in their brains and, at the age of 4 to 6 months, showed retinal degeneration, consistent with the vision loss observed in the patients.
“Together, our findings suggest that the development of specific drugs to restore the function of mutant CLN7 or to regulate other similar lysosomal chloride ion channels/transporters…should be a promising therapeutic strategy to ameliorate CLN7-related diseases. “, wrote the scientists.