By integrating CRISPR-based functional genomics and stem cell technology, researchers based at the University of California, San Francisco (UCSF), have uncovered pathways that control the neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. The researchers, led by Martin Kampmann, PhD, associate professor at UCSF, determined how individual genes in human-stem-cell-generated neurons could, upon inactivation or activation, affect the ability of the neurons to cope with toxic, oxygen-containing molecules.

To their surprise, the researchers found that neurons became more vulnerable to oxidative stress if a gene encoding a lysosomal protein was disabled. This discovery was reported May 24 in the journal Nature Neuroscience, in an article titled, “Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis.”

“Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis,” the article’s authors wrote. “We also determine transcriptomic changes in neurons after perturbation of genes linked to neurodegenerative diseases.”

The article described how the UCSF researchers turned individual genes off and on by using genetic screens that incorporated CRISPR inactivation (CRISPRi) and CRISPR activation (CRISPRa) machinery. The article also asserted that the UCSF researchers are the first to present the results of genome-wide CRISPRi and CRISPRa screens in human neurons.

The article added that the CRISPRi/CRISPRa approach could be applied with many different human cell types, not just neurons. To help realize this possibility, the UCSF researchers have established a data commons named CRISPRbrain.

“This is the key next step in uncovering the mechanisms behind disease genes,” said Kampmann. “There are lots of human genetics studies linking specific genes to specific diseases. The work we’re doing can provide insight into how changes in these genes lead to disease and allow us to target them with treatments.”

To identify genes that might be involved in neurodegenerative diseases such as Alzheimer’s and related forms of dementia, Kampmann and colleagues evaluated stem-cell-generated human neurons after individual genes had been turned on and off. The researchers were looking specifically for downstream changes in gene expression that would produce oxidative stress in the cell. Such stress is thought to contribute to neurodegeneration.

Among the most interesting of the team’s findings was that switching off the gene for a protein called prosaposin, which normally assists with the cell’s recycling of waste products, greatly increased the levels of oxidative stress. In neurons, prosaposin is associated with a part of the cell called the lysosome, where biological molecules and toxins are sorted through and dealt with in a variety of ways.

“At first glance, prosaposin should have nothing to do with oxidative molecules,” Kampmann noted. “It caught our attention because this gene had recently been linked to Parkinson’s disease. What was really exciting was that now, with the results from this CRISPR screen, we had a cell-based model to help us understand what’s behind that linkage.”

The team then embarked on what Kampmann called a “detective story” to find out how the lack of prosaposin is linked to neurodegeneration. The researchers found that suppression of the gene led to buildup of a substance called age pigment, which has been seen in aging cells whose lysosomes no longer degrade material as efficiently. The researchers discovered that age pigment trapped iron, generating reactive oxygen molecules that triggered ferroptosis, an iron-dependent process that leads to cell death.

“By simply inactivating a single gene,” Kampmann emphasized, “in only days we could generate a hallmark of aging that would normally take decades to develop in the human body.”

The cascade of changes Kampmann and colleagues observed are specific to the function of neurons and are related to just one set of conditions. He said the results make a case for using CRISPRi/CRISPRa to perform similar screens looking for changes that prompt other kinds of disease-related environments in neurons and other types of differentiated cells.

To that end, the team created CRISPRbrain, an open-access database that is designed to let scientists share and study large-scale data sets like the ones generated in the current study. Applying advanced computational technology such as machine learning can then detect patterns in this sea of data.

“By becoming the data commons for screens of many different cell types from many different labs and in different disease contexts, we can achieve a critical mass of information,” Kampmann said. “There’s enormous power in aggregating and cross-analyzing all of this.”

The UCSF team’s next step is to perform similar screens on neurons made from stem cells derived from patients with mutations known to contribute to neurodegeneration, as well as look at other cells such as astrocytes and microglia that play roles in brain disease.

Kampmann’s hope is that the technology and database are widely adopted: “Now that we can do this in a systematic way, we can really interpret the underlying processes of how genes contribute to disease and find pathways to treat those conditions.”

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