The CRISPRi / CRISPRa screen reveals neuron-specific pathways that can lead to dementia

By integrating CRISPR-based functional genomics with stem cell technology, researchers at the University of California, San Francisco have discovered pathways that control the neural response to chronic oxidative stress associated with neurodegenerative diseases. Researchers led by Dr. Martin Kampman show how individual genes in neurons produced in human stem cells are capable of coping with toxic oxygen-containing molecules when inactivated or activated. Decided whether to affect.

Surprisingly, researchers found that when the gene encoding the lysosomal protein was disabled, neurons became more vulnerable to oxidative stress.This finding was reported in the journal on May 24th. Nature Neuroscience, “Genome-wide CRISPRi / a screening of human neurons associates lysosomal disorders with ferroptosis.. “

“Unexpectedly, knockdown of the lysosomal protein prosaposin causes the formation of lipofuscin, which is characteristic of aging, traps iron, produces reactive oxygen species, and causes ferroptosis, thereby putting neurons into oxidative stress. It sensitizes strongly, but not other cell types, “the author of the article said. I have written. “It also determines the transcriptional changes in neurons after perturbation of genes associated with neurodegenerative diseases.”

In this article, UCSF researchers describe how to turn individual genes on and off using gene screening that incorporates the CRISPR inactivation (CRISPRi) and CRISPR activation (CRISPRa) mechanisms. .. The article also claims that UCSF researchers were the first to publish the results of genome-wide CRISPRi and CRISPRa screening in human neurons.

This article adds that the CRISPRi / CRISPRa approach can be applied to a wide variety of human cells, not just neurons. To realize this potential, USCF researchers have set up a data commons named CRISPR brain.

“This is an important next step in unraveling the mechanism behind the disease gene,” said Kampmann, an associate professor at UCSF. “There are many studies of human genetics that associate specific genes with specific diseases. Our research provides insights into how changes in these genes lead to disease and in treatment. It makes it possible to target them. “

To identify genes that may be involved in neurodegenerative diseases such as Alzheimer’s disease and related dementia, Kampmann et al. Worked on humans produced in stem cells after turning individual genes on and off. Evaluated neurons. Researchers were particularly looking for downstream changes in gene expression that cause oxidative stress in cells. Such stress is thought to contribute to neurodegeneration.

The most interesting part of the team was turning off a gene for a protein called prosaposin. This usually helped recycle cellular waste products and significantly increased the level of oxidative stress. In neurons, prosaposin is associated with parts of the cell called lysosomes, which classify biomolecules and toxins and process them in a variety of ways.

“At first glance, prosaposin should have nothing to do with the oxidative molecule,” Kanpan said. “It caught our attention because this gene has recently been associated with Parkinson’s disease. What was really exciting was the results of this CRISPR screen to understand what’s behind that linkage. We have a useful cell-based model. “

The team then embarked on what Kampmann called a “detective novel” to investigate how lack of prosaposin was associated with neurodegeneration. Researchers have found that gene suppression leads to the accumulation of a substance called an aging pigment. This was seen in senescent cells where lysosomes stopped degrading the substance efficiently. Researchers have discovered that aging pigments trap iron and produce reactive oxygen species that cause ferroptosis, an iron-dependent process that leads to cell death.

“By simply inactivating a single gene, in just a few days we were able to create the aging trait that normally takes decades to develop in the human body,” Kampmann emphasized.

The cascade of changes observed by Kampmann and colleagues is unique to neuronal function and is associated with only one set of conditions. He said the results indicate that similar screenings should be performed using CRISPRi / CRISPRa to look for changes that promote other types of disease-related environments in neurons and other types of differentiated cells. ..

To that end, the team created a CRISPR brain. It is an open access database designed to allow scientists to share and study large datasets such as those generated in current research. Advanced computational techniques such as machine learning can be applied to detect ocean patterns in this data.

“We can get a great deal of information from many different laboratories by becoming a data commons for screening of different cell types in different disease situations,” says Kampmann. “Aggregating all of this and cross-analyzing it is enormous.”

The next step in the UCSF team is to perform similar screening on neurons made from stem cells from patients with mutations known to contribute to neurodegeneration, and astrocytes that play a role in brain disease. And other cells such as microglia.

Kampmann’s hope is that technology and databases will be widely adopted. “Now that we can do this systematically, we can actually interpret the underlying processes of how genes contribute to disease and find pathways to treat those conditions. . “