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The CLN3 protein, whose defects cause juvenile Batten disease, is necessary for the breakdown and elimination of fat molecules used to make cell membranes, a new study reveals.

The study, “CLN3 is required for the clearance of glycerophosphodiesters from lysosomeswas published in Nature.

Batten disease is a type of lysosomal storage disorder – a group of genetic conditions that impair the function of lysosomes. Lysosomes are the recycling centers of the cell; they house a number of specialized enzymes capable of breaking down complex cellular components into simple molecules that can be reused by the cell.

Juvenile Batten disease is caused by mutations in the gene CLN3, which provides instructions for making a protein of the same name. While the CLN3 protein is known to play a role in lysosomes, its exact function is unclear.

“Lysosomes are both fundamentally and clinically fascinating: they provide nutrients to the rest of the cell, but we don’t always know how and when they provide them, and they are the place where many diseases, especially those affecting the brain, begin,” said Monther Abu-Remaileh, PhD, lead author of the Sanford University study, in a press release.

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Small cell compartments

One of the main difficulties in studying lysosomes is that, despite their importance, these cellular compartments are extremely small, typically occupying only 1-3% of a cell’s total volume. Therefore, it can be difficult to see changes in the lysosome if you look at the whole cell.

“If something happens and a molecule grows in abundance 200 times in the lysosome, you would only see a two-fold increase if you look at the whole cell,” said Nouf Laqtom, PhD, a former postdoctoral researcher in the lab. of Abu-Remaileh and first author of the study.

To solve this problem, the researchers used genetic engineering to create a mouse strain suitable for lysosome studies, called LysoTag mice.

Simplistically, the mice were engineered so that all of their lysosomes had a molecular tag on the outside – otherwise, they were normal mice. By carefully grinding up the tissues of the mice, then using magnets attached to the receptors for this tag, the researchers were then able to isolate the lysosomes from all other cellular components. The purified lysosomes could then be used for further study.

This experimental setup “can be used to rapidly isolate intact, highly pure lysosomes from mouse organs and to study metabolite changes that are not detectable using traditional tissue-based metabolite profiling,” wrote Researchers.

After creating the LysoTag mice, the researchers crossed these mice with mice carrying mutations in the CLN3 gene, breeding LysoTag mice with CLN3 mutations. The scientists then assessed how the lysosomal content changed in these mice with modeled juvenile Batten disease.

The results showed lysosomes from CLN3-the mutant mice had a marked accumulation of glycerophosphodiesters, or GPDs, which are small molecules generated when fatty molecules used to make cell membranes are broken down. Additional experiments in cell models confirmed that a lack of CLN3 protein led to GPD accumulation in lysosomes.

“In mouse brain and cultured human cells, loss of CLN3 causes significant lysosomal accumulation of GPD,” the researchers wrote.

Theoretically, GPD accumulation in CLN3-deficient cells could occur because the CLN3 protein breaks down GDPs in lysosomes – but previous research has suggested that GPDs are no longer broken down, and indeed, experiments here have showed that the CLN3 protein had no effect on the GPDs themselves.

Instead, the researchers determined that CLN3 is required to extract GPDs from lysosomes after the breakdown of membrane fats. Without CLN3, GPDs cannot effectively leave the lysosome, so they accumulate to toxic levels and ultimately cause disease.

“We show that CLN3, the loss of which causes severe neurodegenerative disease in children, is required for the efflux of GPDs from the lysosome,” the researchers concluded, adding that these results provide “a framework for future studies on how whose loss of CLN3 could affect “cellular processes”.

“You can’t develop new ways to diagnose or treat disease if you don’t know what’s changing in lysosomes,” Laqtom said. “This method helps you make sure you are looking in the right direction. This shows you the right way and prevents you from getting lost.

More broadly, the team hopes that its LysoTag model can be a useful tool for further studies on the lysosome.

“These mice are freely available to anyone in the research community, and people are already starting to use them,” Abu-Remaileh said. “We hope it will become the gold standard.”