Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis

Ruilin Tian, Anthony Abarientos, Jason Hong, Sayed Hadi Hashemi, Rui Yan, Mike A. Nalls, Andrew B. Singleton, Ke Xu, Faraz Faghri, Martin Kampmann

Preprint posted on 22 July 2020 https://www.biorxiv.org/content/10.1101/2020.06.27.175679v2

Article now published in Nature Neuroscience at http://dx.doi.org/10.1038/s41593-021-00862-0

Fishing for fishies: comprehensive genome-wide genetic screens to identify genes important for neuronal survival in different stress conditions.

Selected by Berrak Ugur

Categories: cell biology, genetics, neuroscience

Background

As the sequencing technology gets better and better, the list of potential disease-causing genes is also getting bigger. However, having a pile of genes associated with disease does not elucidate their cellular or physiological function. Therefore, there is a need for systemic identification of the function of these “unknown” genes (Kampmann, 2020). These functions may also differ from cell type to cell type, making the understanding of their role in disease context even more challenging. For example, loss of genes associated with certain neurodegenerative disorders may cause specific defects in certain neuronal subtypes whereas other neurons remain unaffected by the loss of these genes. To systemically analyze genes required for neuronal survival at different physiological conditions, Kampmann Lab used human induced pluripotent stem cell (iPSC)-derived neurons to perform genome-wide knockdown and gene-activation screens. Their study provides a very compressive resource that will guide more research to assign functions to genes.

Key Findings:

Genes important for selenocysteine incorporation regulate redox balance in neurons

Previously, Kampmann lab developed a strategy to control specific knockdown of genes (CRISPRi) in human neurons (Tian et al., 2019), now they expand this toolkit to control specific gene activation (CRISPRa). In this preprint, they perform a genome-wide CRISPRi and CRIPRa screen to identify genes important for neuronal survival. As neurons are known to be sensitive to oxidative stress, they also perform the same screen in the presence and absence of antioxidants to isolate genes that are important for regulating oxidative stress in neurons. In addition to scoring for survival, the authors also asses how alterations in gene expression affect reactive oxygen species (ROS) and lipid oxidation. As a result of these screens they find out that genes involved in selenocycteine incorporation to proteins are important for neuronal survival under oxidative stress.

Lysosome and iron secondary screens identify prosaposin as a gene important for iron and lysosome homeostasis in neurons

Following the ROS and lipid peroxidation screen, the authors focus on 730 hit genes and perform a secondary screen to see how their loss alter lysosome and iron levels by using fluorescent indicators. The authors identified prosaposin (PSAP) as a gene whose knockdown resulted in increased lysosomal signal and high ROS, lipid perodixation and iron levels. PSAP is a proprotein that is cleaved by a lysosomal protein cathepsin D (Hiraiwa et al., 1997). This cleavage induces four saposins that are involved in glycosphingolipid degradation (Hiraiwa et al., 1997). Interestingly, these proteins are associated with various neurodegenerative diseases including Alzheimer’s and Parkinson’s (Mendsaikhan et al., 2019; Oji et al., 2020). To investigate how loss of PSAP affects neurons, the authors generated PSAP KO iPSCs and characterized its function in iPSC derived neurons. They observed that loss of PSAP did not affect stem cells but led to an increase in ROS in neurons, indicating that PSAP has neuron specific function in ROS homeostasis. In addition, loss of PSAP caused increased glycosphingolipid, cholesterol and iron levels. Moreover, inhibiting iron toxicity rescued the survival of PSAP KO neurons whereas inhibiting apoptosis did not have an effect. These results indicate that PSAP regulates ROS balance through ferroptosis that is an iron dependent cell death pathway. Looking in the ultrastructure of PSAP KO neurons, the authors revealed that these neurons accumulated highly pigmented lipid containing lysosomal residues called lipofuscin. Interestingly, lipofuscin-bound iron is known to be a source of oxidants (Höhn et al., 2010). However, the details of how PSAP loss regulates lipofuscin accumulation and ferroptosis pathway are not exactly known (Fig. 1).

Figure 1. Model of how prosaposin loss may lead to neuronal ferroptosis. https://doi.org/10.1101/2020.06.27.175679

Although not mentioned in this preLight, the authors perform single-cell RNA seq in some of the targets identified in their CRISPRi and CRISPRa screen. The aim of this characterization is to have the transcriptomic profile of neurodegeneration associated genes and I highly recommend checking the preprint for more info.

Take home messages:

Genes involved in selenocysteine pathway are indispensable for neurons to survive oxidative stress
Loss of prosaposin (PSASP) blocks glycosphingolipid in lysosome leading to formation lipofuscin
Loss of prosaposin leads to neuronal ferroptosis under oxidative stress
Checkout CRISPRbrain to navigate screen results and how alterations in certain neurodegenerative disease-causing genes affect expression of different genes

What I liked about this story:

This is truly an impressive report of screens to identify genes involved in neuronal survival at various conditions. I had a chance to explore the database generated based on this and other studies and it will be an inspiration for many scientists throughout the world.

Remaining Questions:

You observe that loss of PSAP leads to accumulation of cholesterol. Do you think that this accumulation is independent of lipofuscin accumulation? Do you think that there are any genes identified in your screen that caused cholesterol accumulation but not lipofuscin formation?

You show that knockdown of Acyl-CoA Synthetase Long Chain Family Member 4, leads to a decrease in peroxidized lipids. Do you think that knockdown of ACSL4 in PSAP KO neurons would rescue some of the phenotypes? In other words, do you think that your screen results can guide us in identifying a genetic pathway involved in neuronal ferroptosis?

References:

Hiraiwa, M., Martin, B.M., Kishimoto, Y., Conner, G.E., Tsuji, S., and O’Brien, J.S. (1997). Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): its mechanism and inhibition by ganglioside. Arch. Biochem. Biophys. 341, 17–24.

Höhn, A., Jung, T., Grimm, S., and Grune, T. (2010). Lipofuscin-bound iron is a major intracellular source of oxidants: role in senescent cells. Free Radic. Biol. Med. 48, 1100–1108.

Kampmann, M. (2020). CRISPR-based functional genomics for neurological disease. Nat. Rev. Neurol.

Mendsaikhan, A., Tooyama, I., Bellier, J.-P., Serrano, G.E., Sue, L.I., Lue, L.-F., Beach, T.G., and Walker, D.G. (2019). Characterization of lysosomal proteins Progranulin and Prosaposin and their interactions in Alzheimer’s disease and aged brains: increased levels correlate with neuropathology. Acta Neuropathol. Commun. 7, 215.

Oji, Y., Hatano, T., Ueno, S.-I., Funayama, M., Ishikawa, K.-I., Okuzumi, A., Noda, S., Sato, S., Satake, W., Toda, T., et al. (2020). Variants in saposin D domain of prosaposin gene linked to Parkinson’s disease. Brain J. Neurol. 143, 1190–1205.

Tian, R., Gachechiladze, M.A., Ludwig, C.H., Laurie, M.T., Hong, J.Y., Nathaniel, D., Prabhu, A.V., Fernandopulle, M.S., Patel, R., Abshari, M., et al. (2019). CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. Neuron 104, 239-255.e12.

Posted on: 24 August 2020

doi: https://doi.org/10.1242/prelights.24131

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Author's response

Ruilin Tian and Martin Kampmann shared

The accumulation of cholesterol is likely independent of lipofuscin formation, but might be dependent on the accumulation of glycosphingolipids in PSAP KO neurons. Cholesterol levels have been previously found to be tightly coupled to sphingolipid levels. It has been reported that glycosphingolipids accumulation could inhibit cholesterol efflux and lead to cholesterol accumulation (PMID: 12657626, PMID: 15890646). We did not check the cholesterol levels for other hit genes, but there might be genes knockdown of which causes cholesterol accumulation but not lipofuscin formation.
Good question. We have not checked if knockdown of ACSL4 in PSAP KO neurons could rescue some of the phenotypes. Modifier screens in the PSAP KO background could potentially identify other factors in the same pathway regulating neuronal ferroptosis.

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