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New method allows scientists to determine all molecules present in mouse lysosomes

A cartoon depiction of the new method, which allows scientists to isolate the lysosomes (left) of each cell in a mouse to use mass spectrometry (right) to analyze and identify all the molecules in them. Credit: Cindy Lin

Small but powerful, lysosomes play a surprisingly important role in cells, despite their small size. Making up only 1-3% of the cell by volume, these tiny sacs are the cell’s recycling centers, home to enzymes that break down unnecessary molecules into small pieces that can then reassemble to form new ones. Lysosomal dysfunction can lead to a variety of neurodegenerative or other diseases, but without ways to better study the inner contents of lysosomes, the exact molecules involved in diseases — and thus new drugs to address them — remain elusive.

A new method, reported in Nature on Sept. 21, scientists will be able to determine all the molecules present in the lysosomes of any cell in mice. By studying the contents of these molecular recycling centers, researchers can learn how the improper breakdown of cellular materials leads to certain diseases. Led by Stanford University’s Monther Abu-Remaileh, an institute scientist at Sarafan ChEM-H, the study team also learned more about the cause of a currently untreatable neurodegenerative disease known as Batten’s disease, information that could lead to new therapies.

“Lysosomes are both fundamentally and clinically fascinating: they supply the rest of the cell with nutrients, but we don’t always know how and when they deliver them, and they are the places where many diseases, especially those that affect the brain, start,” said Abu-Remaileh, an assistant professor of chemical engineering and genetics.

Some proteins commonly found in lysosomes have been linked to a number of diseases. Mutations in the genetic instructions for making those proteins lead to these “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 or treat these diseases.

If scientists want to study the role a particular protein plays in the cell, they can block or stimulate its function and see whether certain molecules appear or disappear in response. But studying the contents of lysosomes is a scaling problem. “If something happens and a molecule grows 200-fold in abundance in the lysosome, you would only see a two-fold increase if you look at the whole cell,” said Nouf Laqtom, lead author of the study. The revealing results are buried in the noise.

To muffle the sound, the researchers would have to separate lysosomes from everything else in the cell. They had previously developed a method to do just that in cells grown in labs, but they wanted to develop a way to do the same in mice.

Fishing on magnets

The first step in their quest to isolate lysosomes was to make a small change in the mice’s genes to install a small molecular tag on the surface of each lysosome throughout the animal. Anytime they want to stop and check the molecules in the mouse’s lysosomes, such as after fasting or feeding a specific food, they put the tag in the cells they want to study and then remove the tissue and grind it carefully. to break open the cells without disrupting the lysosomes inside.

To fish lysosomes out of the cellular sludge, the team relies on magnets. To their slurry, they add small magnetic beads, each decorated with molecular clamps that grip the lysosomal tag they had previously installed. They can selectively collect all the lysosomes using a second magnet and then pull the lysosomes apart to reveal the molecules that were safely tucked inside. Mass spectrometry, a set of tools that determines the weight of different molecules in a mixture, then helps the researchers identify the individuals in their lysosomal molecular potpourri. Those that grow or decline point scientists to certain pathways or features.

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

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

Know where to look

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

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

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

In healthy cells, the GPDs do not accumulate in the lysosome; they are exported to another part of the cell, where they are then broken down into smaller pieces. The researchers now think the CLN3 protein plays an important role in that export, either by bringing the molecules out directly or by helping another protein do that job. They found GPD molecules in the cerebrospinal fluid of patients with CLN3 disease, suggesting that clinicians might be able to monitor GPD levels to measure the success of future treatments. The team is now determining which of the GPD molecules could be toxic and how to target the proteins involved in making and exporting GPDs with new drugs. They are also applying their method to look at other diseases involving mutations in lysosomal genes, such as Parkinson’s disease.

“You can’t develop new ways to diagnose or treat disease if you don’t know what’s changing in the lysosomes,” said Laqtom, a former postdoctoral scientist in the Abu-Remaileh lab. “This method will help you make sure you’re looking in the right direction. It points you in the right direction and ensures you don’t get lost.”


Molecular tags reveal how damaged lysosomes are selected and marked for clearance


More information:
Nouf N. Laqtom et al, CLN3 is required for the clearance of glycerophosphodiesters from lysosomes, Nature (2022). DOI: 10.1038/s41586-022-05221-y

Provided by Stanford University

Quote: New method allows scientists to determine all molecules present in mouse lysosomes (2022, September 21) retrieved September 21, 2022 from https://phys.org/news/2022-09-method-scientists- molecules-lysosomes-mice .html

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