CLN7 Is a Chloride Channel, Opens Door to New Therapies, Study Finds
The CLN7 protein that is faulty in a form of late-infantile Batten disease is a chloride channel that regulates the function of lysosomes, the recycling compartment of the cell, a new study discovered.
Developing treatments to restore CLN7 function or modulate other lysosomal chloride channels may be a promising therapeutic strategy for treating CLN7-related diseases, the scientists suggested.
Details of the discovery were published in the journal Science Advances, in a study titled “CLN7 is an organellar chloride channel regulating lysosomal function.”
Mutations in the MFSD8 gene cause a form of late-infantile Batten disease, with an age of onset between 2 and 7 years old. The gene provides instructions for the CLN7 protein found embedded in the membrane of lysosomes, an acidic compartment within cells that degrades and recycles waste.
These gene mutations lead to the toxic buildup of waste — composed of fat and proteins called lipofuscins — eventually killing the cells. Nerve cells are susceptible to lipofuscin buildup, resulting in motor and mental impairments, seizures, speaking difficulties, and vision loss, all hallmarks of this disease type.
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 an 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 traffics and sorts material within the cell before reaching the lysosomes for degradation.
Overproducing the CLN7 protein promoted the release of calcium ions from lysosomes, which activated a protein called calmodulin, leading to the fusion of endosomes to lysosomes. This caused them to become enlarged, 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 following the electrical current. In cells with excess CLN7 protein, there was a significant increase in the outward flowing current, suggesting positively charged ions moved into the lysosome or negatively charged ions flowed out.
The team then replaced all the positively charged ions — such as calcium, potassium, sodium, and magnesium — with NMDG, a large positively charged molecule unable to pass through membrane channels. Unexpectedly, the outward current remained unchanged.
Because the only negative ion present was chloride, the team discovered that reducing the concentration of chloride ions eliminated the outward flowing current. This suggested that CLN7 mediated the flow of chloride ions out of lysosomes, the researchers noted.
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. Then, exposing cells to three chemicals known to block chloride channels reduced the flow of outward current in a dose-dependent manner.
Furthermore, altering the CLN7 protein to remove positively charged amino acids (the building blocks of proteins) predicted to interact with negative chloride ions significantly reduced the current compared with normal CLN7. Lastly, deleting the MFSD8 gene largely stopped chloride currents, while adding mouse CLN7 rescued these currents.
“These electrophysiological and pharmacological properties suggest that CLN7 acts as a chloride channel,” the researchers wrote.
Experiments also confirmed that CLN7 plays an indirect role in modulating the levels of calcium within lysosomes, as well as regulating lysosomal pH and establishing a membrane potential. That is a voltage difference between the inside and outside of the lysosome.
Next, the team investigated the impact of four MFSD8 gene mutations commonly found in late-infantile Batten disease patients. While all four mutations reduced chloride currents compared with normal CLN7, two of the mutations that lead to more severe symptoms had more serious defects in chloride channel function.
“These findings 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 bred to lack CLN7 was larger than normal cells due to the buildup of lipofuscins, as seen in CLN7 patients. Adding CLN7 back into the cells significantly reduced this buildup.
In mouse cells lacking CLN7, there also was a decrease in the lysosome’s ability to degrade proteins and an increase in the levels of fat-like lipid oxidation, “which may be an important factor leading to cell death in CLN7 disease,” the scientist wrote.
The mice themselves showed lipofuscins buildup in their brains and, at 4 to 6 months of age, had retinal degeneration, consistent with the loss of vision seen in patients.
“Together, our findings suggest that development of specific drugs to restore the function of mutant CLN7 or to regulate other similar lysosomal chloride ion channels/transporters … is expected to be a promising therapeutic strategy for mitigating CLN7-related diseases,” the scientists wrote.
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