Study IDs Function of CLN3 Protein, Defects of Which Cause Juvenile Batten
Scientists used genetic engineering to create mice strain to study lysosomes
The CLN3 protein, defects of which cause juvenile Batten disease, is needed for the breakdown and clearance of the fat molecules used to make cell membranes, a new study reveals.
The study, “CLN3 is required for the clearance of glycerophosphodiesters from lysosomes,” was 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 that can break complex cellular components into simple molecules that can be repurposed 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 has not been clear.
“Lysosomes are fascinating both fundamentally and clinically: they supply the rest of the cell with nutrients, but we don’t always know how and when they supply them, and they are the places where many diseases, especially those that affect the brain, start,” Monther Abu-Remaileh, PhD, the study’s lead author from Sanford University, said in a press release.
Small cellular compartments
One of the main difficulties in studying lysosomes is that, despite their importance, these cellular compartments are extremely small, usually only occupying 1%-3% of the total volume of a cell. Consequently, it can be hard to see changes in the lysosome if you’re looking at the whole cell.
“If something happens and a molecule grows in abundance 200-fold in the lysosome, you would see only a two-fold increase if you look at the whole cell,” said Nouf Laqtom, PhD, former postdoctoral researcher in Abu-Remaileh’s lab and first author of the study.
To address this problem, researchers used genetic engineering to create a strain of mice suited to lysosome studies, dubbed LysoTag mice.
Simplistically, the mice were engineered so that all their lysosomes would have a molecular tag on the outside — otherwise, they were normal mice. By carefully grinding up tissue from the mice and then using magnets attached to receptors for this tag, the researchers could then isolate the lysosomes from all the other cellular components. The purified lysosomes could then be used for further study.
This experimental setup “can be used to rapidly isolate intact and highly pure lysosomes from mouse organs and to study metabolite changes that are not detectable using traditional tissue-based metabolite profiling,” the researchers wrote.
After creating the LysoTag mice, the researchers crossed these mice with mice harboring mutations in the CLN3 gene, breeding LysoTag mice with CLN3 mutations. The scientists then assessed how the lysosomal contents changed in these mice with modeled juvenile Batten disease.
Results showed lysosomes from CLN3-mutant mice had marked accumulation of glycerophosphodiesters, or GPDs, which are small molecules generated when the fatty molecules used to make cell membranes are broken down. Additional experiments in cell models confirmed a lack of CLN3 protein led to GPD accumulation in lysosomes.
“In the mouse brain and in human cells in culture, the loss of CLN3 causes a large lysosomal accumulation of GPDs,” the researchers wrote.
Theoretically, the accumulation of GPD in CLN3-deficient cells could occur because the CLN3 protein breaks down GDPs in lysosomes — but prior research has suggested GPDs are not broken down any further, and indeed, experiments here showed the CLN3 protein had no effect on GPDs themselves.
Instead, the researchers determined CLN3 is needed to get GPDs out of the lysosomes after membrane fats are broken down. Without CLN3, the GPDs cannot efficiently leave the lysosome, so they build up to toxic levels and ultimately cause disease.
“We show that CLN3, the loss of which causes a severe neurodegenerative disease in children, is required for the efflux of GPDs from the lysosome,” the researchers concluded, adding that these findings provide “a framework for future studies into how the loss of CLN3 might affect” cellular processes.
“You can’t develop new ways to diagnose or treat diseases if you don’t know what is changing in the lysosomes,” said Laqtom. “This method helps you make sure you’re looking in the right direction. It points you down the right path and keeps you from getting lost.”
More broadly, the team hope their LysoTag model may be a useful tool for other studies of the lysosome.
“These mice are freely available for anyone in the research community to use, and people are already starting to use them,” said Abu-Remaileh. “We hope that this will become the gold standard.”
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