Nat Neurosci | Large-scale CRISPRi/a Screening Reveals New Mechanism of Neuronal Iron Death

Editor | As the terminally differentiated cells with the longest survival time in the human body, neurons are subject to many cell stresses during the aging process, and oxidative stress is one of the key ones.

Oxidative stress is caused by the excessive accumulation of reactive oxygen species (ROS) in cells.

Excessive reactive oxygen species can cause damage to DNA, protein and lipids and cause cell death.

The brain is particularly sensitive to reactive oxygen species, because the brain consumes a lot of oxygen, is rich in metals with high redox activity (such as iron and copper) and polyunsaturated fatty acids that are easily damaged by reactive oxygen species.
At the same time, the number of antioxidant factors is relatively limited.

Many studies have shown that oxidative stress is involved in the occurrence of neurodegenerative diseases.

Clinically, the level of reactive oxygen species in the brain of patients with neurodegenerative diseases increases, and the damage caused by reactive oxygen species also increases [1].

In vitro studies using induced pluripotent stem cell models have also confirmed that neurons carrying neurodegenerative disease gene mutations are more sensitive to oxidative stress [2].

On May 24, 2021, Martin Kampmann's team at the University of California, San Francisco published a research article Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis in Nature Neuroscience, which systematically explored how human neurons regulate reactive oxygen species and respond The mechanism of oxidative stress, and unexpectedly discovered a neuron-specific iron death pathway.

Tian Ruilin, an assistant professor at the School of Medicine of Southern University of Science and Technology, is the first author and co-corresponding author of this article.

First, in order to find the necessary pathways for neurons to respond to oxidative stress, the authors used the previously developed technology for CRISPRi screening in induced pluripotent stem cell (iPSC) differentiation of human neurons [3].
Genes necessary for survival under oxidative stress conditions were screened across the genome.

It is worth mentioning that, unlike other studies that often use strong ROS inducers (such as H2O2 or rotenone), the authors in this study created a gentle slow motion closer to physiological conditions by removing the antioxidant factors in the culture medium.
Oxidative stress stimulation.

The screening results showed that the genes on the iron death inhibitor GPX4 and the GPX4 synthesis pathway are necessary for neurons to survive under oxidative stress stimulation conditions, and knockdown of these genes under normal conditions has no phenotype, indicating that under oxidative stress conditions Lower neurons are prone to iron death.

Next, in order to find genes that directly regulate ROS homeostasis in neurons, the authors performed a genome-wide screening based on flow sorting using fluorescent indicators that can characterize intracellular ROS levels and lipid oxidation levels, respectively.

Interestingly, in addition to some known genes that can regulate ROS, the screening has also identified many new genes that affect ROS homeostasis, including PSAP.
PSAP encodes the protein prosaposin, which is located in the lysosome and participates in glycosphingology.
Degradation of lipids (glycosphingolipids).

How does a lysosomal protein participate in ROS regulation? The author has conducted an in-depth exploration of the mechanism behind it.

Through lipidomics analysis, neurons after PSAP knockout have a large amount of glycosphingolipid accumulation.

Surprisingly, when PSAP knocked-out neurons were cultured in a culture medium depleted of antioxidant factors, the cells all died after ten days, while normal neurons were still alive.

In addition, the death of PSAP knockout neurons cannot be rescued by apoptosis inhibitors, but can be completely rescued by iron death inhibitors, indicating that PSAP loss causes neuronal iron death (ferroptosis).

Furthermore, the authors found that PSAP knockout triggers lysosomal lipid metabolism disorders, leading to the formation of lipofuscin (lipofuscin).

A lot of active Fe2+ is accumulated in lipofuscin, which generates ROS through Fenton reaction, which leads to lipid oxidation and neuronal iron death.

It is very interesting that this pathway of PSAP knockout leading to iron death is neuron-specific, and the above phenotype was not found in HEK293, iPSC and iPSC differentiated neural stem cells and glial cells.

This study investigated the regulation of neuronal oxidative stress through a large-scale CRISPR screening system, and clarified a new mechanism of neuronal iron death caused by PSAP-involved lysosomal lipid metabolism disorder (below).

It is worth noting that clinical studies have found that mutations in the PSAP gene can cause Parkinson's disease, and a phenotype similar to this study was also found in patient brain samples.

At the same time, a large amount of iron accumulation is also found in the degenerated brain regions of many patients with neurodegenerative diseases, suggesting that iron death may be an important mechanism for neuron loss in neurodegenerative diseases, so it is possible to inhibit neuronal iron death Intervene in the progress of neurodegenerative diseases.

 In addition to the schematic diagram of the mechanism of neuronal iron death caused by PSAP loss, this study has other important contributions.

Technically, the author has developed a CRISPRa system that is inducible in iPSC differentiated neurons, which is complementary to the previous CRISPRi, and realizes genome-wide overexpression screening in human neurons.

In terms of data resources, this research has completed the genome-wide screening of multiple phenotypes in human neurons for the first time.
The author has constructed the CRISPRbrain database website (http://crisprbrain.
org/), which contains different cell types and cell phenotypes.
Screening results (based on survival, fluorescent signal, or single-cell transcriptome phenotype) facilitate researchers to explore the functions of genes of interest.

CRISPRbrain database website Nanke Datian Ruilin's research group is committed to developing new CRISPR screening technologies and applying them to the research of human nervous system diseases.

Research group website: https://sustech.
tianlab.
top/.

Now actively recruiting postdoctoral fellows, with superior scientific research conditions and generous remuneration.

Resume delivery (for those who are interested, please send your resume and other materials to): https://jinshuju.
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1038/s41593- 021-00862-0 Platemaker: Eleven References 1.
Barnham, KJ, Masters, CL & Bush, AI Neurodegenerative diseases and oxidative stress.
Nat.
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3, 205–214 (2004) 2.
Silva , MC et al.
Human iPSC-Derived Neuronal Model of Tau-A152T Frontotemporal Dementia Reveals Tau-Mediated Mechanisms of Neuronal Vulnerability.
Stem Cell Rep.
7, 325–340 (2016) 3.
Tian, ​​R.
et al.
CRISPR Interference -Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.
Neuron 104, 239-255.
e12 (2019).
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