Sifting through cellular recycling centers
A cartoon representation of the new method, which allows scientists to isolate the lysosomes (left) of any cell in a mouse to analyze and identify using mass spectrometry (right) all the molecules inside them. Credit: Cindy Lin

lysosomes are small but important in cells. These small sacs are the cell's recycling centers, where the cell'sidases break down unneeded molecule into small pieces that can be reassembled to form new ones. Without ways to better study the inner contents of lysosomes, the exact molecules involved in diseases remain elusive.

The new method allows scientists to determine all the molecule present in the lysosomes of any cell. The improper degradation of cellular materials can lead to diseases. The study's team was led by Monther Abu-Remaileh, an institute scholar at Sarafan CHEM-H.

"Lysosomes are fascinating both fundamentally and clinically, but we don't always know how and when they supply them, and they are the places where many diseases, including those that affect the brain, start," said Abu-Remaileh.

There are a number of diseases associated with the lysosomes. Some of the functions of some of the lysosomal storage disorders have puzzled scientists. Scientists can use information about how these proteins work to develop better ways to diagnose and treat diseases.

If scientists want to study the function of a particular molecule in the cell, they can either block it or see if it appears or disappears. It is difficult to study the contents of lysosomes. "If 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. The results aren't revealed in the noise.

The researchers had to separate the lysosomes from the rest of the cell. They wanted to develop a way to do the same thing in mice as they had done in cells grown in labs.

It is possible to fish for magnets.

The first step in isolating lysosomes was to change the genes of the mice in order to put a tag on them. When they want to check on the molecules in the mouse lysosomes, they turn on the tag in the cells they want to examine, and then remove the tissue and grind it up.

The team uses magnets to fish lysosomes. They add tiny magnetic beads that are decorated with a molecule that grab a hold of the lysosomal tag. They can use a second magnet to collect all the lysosomes and then break them apart. The weights of different molecule in a mixture can be determined with the help of mass spectrometry. Scientists would point to certain pathways or functions if they grew or decreased.

The "LysoTag" mice are normal laboratory mice. These mice can be used to study the role of lysosomes.

Abu-Remaileh said that people are already starting to use the mice. This will be the gold standard.

Knowing where to go.

The team wanted to apply their method to study the lysosomes found in brain cells to better understand the diseases. Abu-Remaileh said this is one of the most urgent problems they can help solve.

Children and young adults with juvenile Batten disease are at risk of vision loss, seizures, and progressive motor and mental decline. The lysosome is home to the CLN3 protein, but no one has figured out how it works in the cell or how it leads to symptoms.

The increase in the amount of a molecule called a glycerophosphodiester, or GP, was found by the researchers using their LysoTag mice. The GPDs are formed during the process of degradation of the fat in the cells.

GPDs are exported to a different part of the cell, where they are degraded into smaller pieces. The researchers believe that the CLN3Protein plays an important role in that export, either by helping anotherprotein do that job or directly shuttling out the molecule. The discovery of GPD in the cerebral spinal fluid of patients with CLN3 disease suggests that clinicians could possibly monitor GPD levels to measure the success of future treatments. The team is trying to figure out which GPD molecule is toxic and how the proteins involved in making and exporting GPDs could be targeted. Parkinson's disease is one of the diseases that they're looking at using their method.

If you don't know what is happening in the lysosomes, you can't develop new ways to diagnose or treat diseases. You can use this method to make sure you are looking in the right place. It tells you where to go and keeps you out of trouble.

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 Journal information: Nature