A new way to improve the study of cellular recycling centers

Small but powerful lysosomes play a surprisingly important role in cells despite their small size. These tiny sacs make up only 1-3% of a cell by volume, and are the cell’s recycling centers, home to enzymes that break apart unnecessary molecules into tiny pieces that can then be reassembled to form new ones. Lysosomal dysfunction can lead to a variety of neurodegenerative or other diseases, but without methods to better study the internal contents of lysosomes, disease-associated microparticles—and thus new drugs to target them—remain elusive.

A new method, mentioned in temper nature On September 21, scientists are allowed to identify all the molecules contained in the lysosomes of any cell in mice. Studying the contents of these molecular recycling centers can help researchers learn how improper decomposition of cellular materials leads to certain diseases. Led by Stanford University Munther Abu RumailaInstitute’s world in Sarafan Chem-HThe study team also learned more about the cause of the currently incurable neurodegenerative disease known as Batten disease, information that could lead to new treatments.

“Lysosomes are wonderful both fundamentally and clinically: they supply nutrients to the rest of the cell, but we don’t always know how and when to supply them, and they are where many diseases start, especially those that affect the brain,” said Abu Rumaila, associate professor of chemical engineering and genetics.

Certain proteins normally found in lysosomes are associated with a number of diseases. Mutations in the genetic instructions for making these proteins lead to “lysosomal storage disorders,” as they are collectively called, but the functions of some of these proteins have long puzzled scientists. Information about how these proteins work could help scientists develop better ways to diagnose, monitor, and treat these diseases.

If scientists want to study the role a particular protein plays in a cell, they can either block or stimulate its function and see if specific molecules appear or disappear in response. But the study of lysosome contents is a problem of scale. “If something happens and the molecule grows in abundance 200 times in the present particle, you will only see a twofold increase if you look at the whole cell,” said Nouf Laktoum, first author of the study. The revealing results are buried in the noise.

To quiet the noise, researchers have to separate the lysosomes from everything else in the cell. They had previously developed a way to do this in cells grown in labs, but wanted to develop a way to do the same in mice.

magnet fishing

The first step in their quest to isolate lysosomes was to make a small change in the mice’s genes to fix a small molecular marker on the surface of each lysosome in the whole animal. Anytime they want to stop and check for molecules in mouse lysosomes, such as after fasting or feeding them a certain food, they turn on the marker in the cells they want to examine, then remove the tissue and carefully grind it to break down the cells without disrupting the particles within.

To catch lysosomes from cellular sludge, the team relies on magnets. To their slurry they add tiny magnetic beads each adorned with molecular clips that capture the lysosomal tag they attached earlier. They can selectively collect all the lysosomes using a second magnet, then decompose the lysosomes to reveal the ones that have been safely placed inside. Mass spectrometry, a set of tools that determines the weights of different molecules in a mixture, then helps researchers identify individuals in their lysosomal molecular driers. Those that grow or decrease would direct scientists to specific pathways or jobs.

Except for the little extra tag on each lysosome, these “LysoTag” mice are normal lab mice. Now, almost any researcher can use these mice to study the role of lysosomes in various diseases.

“These mice are freely available for anyone in the research community to use, and people have already started using them,” Abu Rumaila said. “We hope this will become the gold standard.”

Knowing where to look

The team was eager to apply their method of studying lysosomes found in brain cells to better understand neurodegenerative lysosomal storage diseases, starting with CLN3 or juvenile Batten disease. “We really see this as one of the most pressing problems we can help solve,” Abu Rumaila said.

Caused by a mutation in the gene that codes for a protein called CLN3, Juvenile Batten disease is fatal and leads to vision loss, seizures, and progressive motor and mental decline in children and young adults. The CLN3 protein has been found on the lysosomal membrane, but no one has determined its function in the cell or how its dysfunction leads to the observed symptoms.

Using LysoTag mice, the researchers collaborated with experts at both Sarafan ChEM-H Metabolism Knowledge Center and the Whitehead Institute Metabolomics Core, and they found a significant increase in the amount of a type of molecule called a glycerophosphodiester, or GPD for short, in mice with the CLN3 mutation. GPDs are formed temporarily during the breakdown of fat molecules that make up the membranes of every cell in our bodies.

In healthy cells, GPD does not accumulate in the lysosome. It is exported to a different part of the cell, where it is then broken down into smaller pieces. The researchers now believe that the CLN3 protein plays an important role in this export, either by transporting molecules directly or by helping another protein do the job. They found GPD particles in the cerebrospinal fluid of patients with CLN3, suggesting that doctors can monitor GPD levels to gauge the success of future treatments. The team is now identifying which GPD molecules may be toxic and how the proteins involved in making and exporting GPD can be targeted with new drugs. They are also applying their method to research in other diseases that involve mutations in lysosomal genes, such as Parkinson’s disease.

“You can’t develop new ways to diagnose or treat diseases if you don’t know what changes in the lysosomes,” said Laktoum, a former postdoctoral researcher in the Abu Rumaila lab. “This method helps you make sure that you are looking in the right direction. It directs you to the right path and prevents you from getting lost.”

Reference: Laqtoum NN, Dong W, Midoh-On, et al. CLN3 is required for the removal of glycerophosphodiesterase from lysosomes. temper nature. 2022: 1-7. dui: 10.1038 / s41586-022-05221-y

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