A toxic brew of lysosomal lipids, reactive iron atoms, and oxidative stress can spell doom for human neurons. This is the upshot of the first-ever CRISPR screens at the genome-wide level in these cells. Researchers led by Martin Kampmann at the University of California, San Francisco, used the genome-editing tool to dial up or down expression of each protein-coding gene in the human neuronal genome. They uncovered a surprising connection between endolysosomal processing and the iron-dependent cell-death pathway called ferroptosis.
- First genome-wide CRISPR screens in human neurons tweaks gene expression.
- Endolyosomal function, oxidative stress, and iron homeostasis genes key to neuronal survival.
- Sans lysosomal prosaposin, neurons might die by ferroptosis.
- Data available on CRISPRbrain, a communal database.
Zeroing in on that pathway, the researchers found that in the absence of the lysosomal protein prosaposin, glycosphingolipids accumulate in the lysosomes, setting off oxidative stress. This results in a toxic mesh of ferrous ions and peroxidized lipids that can kill neurons. The findings connect pathways that have been implicated separately in neurodegenerative disease, and support the idea that iron-rich “aging pigments,” commonly spotted in older brains, might not be so benign after all.
The findings were posted in a manuscript on bioRxiv on February 5 and the data is the first entered into CRISPRbrain, a communal database in which researchers can share CRISPR screening data gathered from different cell types.
“This is a beautiful study using CRISPRi and CRISPRa to identify genes controlling neuronal response to oxidative stress,” commented Fenghua Hu of Cornell University in Ithaca, New York. “The cell-type-specific effect of gene perturbation is extremely interesting and explains why the neuron is the cell most vulnerable to oxidative stress during neurodegeneration.” CRISPRi and CRISPRa interfere with and activate genes, respectively.
Ellen Sidransky of the National Institutes of Health in Bethesda, Maryland, called the study groundbreaking, noting that it directs attention to the importance of lysosomal pathways in neurodegeneration. “This is an important step toward uncovering molecular mechanisms underlying various neurodegenerative diseases, and the results will likely yield new therapeutic targets,” she wrote.
In its original form, as discovered in bacteria, CRISPR is a way to snip specific regions in the genome with the Cas9 nuclease. Single guide RNAs, affixed with a Cas9 binding sequence, direct the nuclease to specific sequences for cutting. Researchers co-opted the mechanism to edit the genome at will, deleting or replacing nucleic acids of choice. In recent years, researchers have devised multiple variations on this theme, among them CRISPR interference and CRISPR activation. CRISPRi uses a catalytically “dead” form of Cas9 (dCas9) to block transcription of a targeted gene, rather than permanently editing its sequence. In CRISPRa, this dCas9 is fused to an activation domain of a transcription factor, thus revving up expression of targeted genes instead.
By tweaking expression levels, these forms of CRISPR influence the genome similarly to most common genetic variations identified in genome-wide association studies, Kampmann noted. Previously, he and his colleagues had deployed genome-wide CRISPRi and CRISPRa screens in human cancer cell lines (Gilbert et al., 2014). More recently, they used CRISPRi in human induced pluripotent stem cell (iPSC)-derived neurons, targeting a subset of more than 2,000 genes representing the “druggable genome” (Tian et al., 2019).
With this new study, first author Ruilin Tian, now at Southern University of Science and Technology, Shenzhen, China, and colleagues pulled out all the stops. They targeted the entire protein-coding genome in human neurons with CRISPRi and CRISPRa, in search of genes critical to neuron survival.
Screening for Survival. Human iPSCs were transfected to express dCas9 for CRISPRi, or dCas9 fused with a transcription factor for CRISPRa. Later, they were transduced with a library of single guide RNAs, including several for each protein-coding gene in the genome. Fourteen days later, sgRNA frequencies serve as proxies for effects on neuronal survival. [Courtesy of Tian et al., bioRxiv, 2021.]
For both types of screen, the researchers transduced iPSCs with the machinery for CRISPRi or CRISPRa. Later, they transduced the CRISPR-competent iPSCs en masse with a library of sgRNAs targeting each protein-coding gene in the genome. This was done such that only one type of guide RNA infected each cell. They then cultured the cells for two weeks. In that time, cells transduced with sgRNAs targeting essential genes were more likely to die, thus reducing the frequency of that sgRNA in the population of transduced cells. By measuring sgRNAs left on day 14, the researchers could gauge each sgRNA’s influence on survival.
From the CRISPRi screen, genes involved in oxidative stress leapt out as essential for neuronal survival. Superoxide dismutases SOD1 and SOD2 were among the top 10 genes that neurons could not live without. Endolysosomal genes, including subunits of the vacuolar ATPase, which acidifies the lysosome, were also important. Other must-haves included genes involved in cholesterol biosynthesis, iron homeostasis, protein folding, mRNA processing, and autophagy. In the CRISPRa screen, ramping up expression of apoptotic genes docked survival, as expected.
For some genes, including those involved in protein homeostasis, modulating expression in either direction dampened survival, suggesting that neurons narrowly tune their expression. A majority of the genes identified in these screens had not come up in previous CRISPR screens of other cell types, such as cancer cells, suggesting they were uniquely critical for neurons.
Given the prominence of oxidative stress genes that emerged from these survival screens, the researchers ran a series of secondary CRISPR screens to identify genes that influenced survival under conditions of mild oxidative stress, i.e., when the cells were grown in the absence of antioxidants typically added to the media. Under these conditions, they found that Glutathione Peroxidase 4 (GPX4)—an enzyme that uses glutathione to lower the oxidation state of peroxidized lipids—was essential. This enzyme also happens to be essential for putting the kibosh on ferroptosis (Yang et al., 2014).
In fact, oxidized phospholipids are the signature of ferroptosis. In this cell-death pathway, ferrous iron atoms provoke the production of ROS via a chemical process known as the Fenton reaction. This is when iron reacts with hydrogen peroxide formed by incomplete respiration to produce hydroxl radicals. The peroxidized lipids then cause damage to cellular membranes, ultimately rupturing the cells (Dixon et al., 2012).
Focusing on lipid oxidation, Kampmann's group next employed CRISPRi screens to hunt for genes that influenced levels of ROS or peroxidized lipids. Here, they found many expected genes, such as those involved in electron transport or autophagy—pathways that can cause and curtail oxidative stress, respectively. The screens also turned up key regulators of ferroptosis, including GPX4.
One shocker that emerged was prosaposin, a lysosomal protein. In the lysosome, Cathepsin D chops up prosaposin, releasing four activator proteins, saposin A, B, C, D, which assist in the degradation of glycosphingolipids by lysosomal hydrolase enzymes. Without full expression of prosaposin, levels of both ROS and lipid peroxides soared in neurons. Why? Further screens revealed that prosaposin was critical for lysosomal function, but also for keeping reactive iron levels in check inside of neurons.
How was prosaposin involved in all these pathways? To find out, the researchers knocked out the PSAP genes in human iPSCs, and differentiated them into neurons. Compared to their PSAP-replete counterparts, these KO neurons churned out more ROS and had high levels of peroxidized lipids. Interestingly, when the researchers knocked out PSAP from other cell types, including astrocytes and microglia, these toxic signs of stress did not appear, suggesting PSAP specifically kept the peace in neurons.
Bathed in a sea of antioxidants, the PSAP-less neurons survived normally for up to two weeks in culture. However, when the researchers cultured the cells sans antioxidants, they started to die off rapidly after 11 days, and by 14 days, all had perished. Kampmann described the neuronal demise as a messy affair, with neurons appearing to pop.
Apoptosis inhibitors did not save the cells from this fate. Rather, inhibitors of ferroptosis—the iron chelator deferoxamine (DFO) and the lipid peroxidation inhibitor ferrostatin-1—rescued them. This suggested that mild oxidative stress trips off ferroptosis, at least when PSAP is absent.
Lyso Lipids Stoke Ferroptosis. Without prosaposin, glycosphingolipids accumulate in the lysosome, which form lipofuscin granules. The iron in these granules produces reactive oxygen species, which oxidize lipids. Peroxidized lipids damage membranes and kill cells via ferroptosis. [Courtesy of Tian et al., bioRxiv, 2021.]
Lipofuscin Pile-Up. Without prosaposin, electron-dense granules of lipids and iron clogged lysosomes of human neurons. [Courtesy of Tian et al., bioRxiv, 2021.]
What is the connection between PSAP and ferroptosis? Examining the PSAP KO neurons via biochemistry, electron microscopy, and super-resolution microscopy, the researchers found that the lysosomes were dramatically enlarged, and chock-full of glycosphingolipids. Strikingly, they found that these lipid-logged organelles were also electron-dense, suggesting they were loaded with iron. In fact, these densities bore an uncanny resemblance to lipid-iron granules called lipofuscin, also known as aging pigment. Lipofuscin soaks up the metal ions from the detritus of iron-rich organelles such as mitochondria, and this iron is thought to provoke the production of ROS via the Fenton reaction.
Strikingly, none of these phenotypes—from lipid accumulation to lysosomal enlargement to lipofuscin or peroxidized lipids—occurred when the researchers knocked PSAP out of other cell types, including iPSC neural progenitor cells, microglia, and astrocytes.
“The unexpected link between saposin-mediated glycosphingolipid degradation, iron metabolism, lipofuscin accumulation, and oxidative stress underscores an important role of lysosomes in regulating iron dynamics in neurons,” wrote Hu.
Could this cascade play out in the aging brain? All of the culprits are there, Kampmann said. For one, oxidative stress is known to rise in the brain with age, and lysosomal function also flags. Levels of not only lipofuscin, but reactive iron increase in aging brains and even more so in neurodegenerative disease (Zhang et al., 2021; Mar 2019 news).
Genetic risk variants for neurodegenerative diseases point to this cascade, as well. Many occur in or around endolysosomal genes. PSAP variants have been linked to lysosomal storage disorders, and, more recently, to Parkinson’s disease (Oji et al., 2020). Sidransky noted that SapC, one of prosaposin’s products, is an essential activator for the enzyme glucocerebrosidase, a lysosomal protein linked to parkinsonism (Jun 2011 news; Jun 2014 news; Oct 2019 news).
Why would these pathways only manifest in neurons? George Perry of the University of Texas, San Antonio, explained that neurons, given their long-lived, post-mitotic nature, have a higher priority to survive than other cells do. As such, they have evolved mechanisms to sequester and detoxify mounting detritus from defunct organelles, and to survive in stressful environs despite faltering function. Perry views lipofuscin as a necessary component of this survival regime. “Lipofuscin has always been relegated to the backwaters of aging research, and this study helps bring it to the forefront,” Perry said. Eventually, the system fails and events such as ferroptosis ensue, he added.
Ashley Bush of the University of Melbourne in Australia made a similar point, noting that despite its dismissal as a meddlesome pigment, lipofuscin is known to concentrate iron. If it was released from these granules in its reactive form, this could explain the inevitable rise in brain iron levels with aging in all mammalian species studied, he wrote (Kurz et al., 2011). The current paper provides evidence for how this could be sinister, rendering neurons vulnerable to ferroptosis under conditions of slight oxidative stress, he added.
The UCSF researchers are continuing to hone their CRISPR tools, and to wield them in different kinds of cells. Case in point: Another manuscript they posted on bioRxiv screened a cell line to look for genes involved in the aggregation of α-synuclein (See et al., 2021). Using a FRET-based sensor, these cells fluoresce when α-synuclein fibrils snap together inside the cell. First author Stephanie See and colleagues uncovered that endolysosomal transport genes had a surprisingly strong sway over α-synuclein aggregation. Notably, they found that a small-molecule inhibitor of PIK-fyve—an endosomal enzyme that mediates endolysosomal trafficking—blocked α-synuclein aggregation. They think it did so by preventing α-synuclein from reaching the lysosome, from where it busts out into the cytoplasm and begins to aggregate.
“Both manuscripts functionally validate strong hits and solidify the endolysosomal network as a hub for neuronal dysfunction in neurodegeneration,” commented Jessica Young of the University of Washington in Seattle.
Interestingly, PIK-fyve inhibitors also reportedly correct autophagy deficits caused by mutations in the C9ORF72 gene. Hexanucleotide repeat expansions in this gene, which cause amyotrophic lateral sclerosis and frontotemporal degeneration, not only derail its endogenous functions of promoting autophagy, but also produce toxic dipeptides that clog the floundering cellular digestive system (Feb 2018 news). In this scenario, PIK-fyve inhibitors promote autophagy.
Separately, PIK-fyve inhibitors have also emerged as a potential treatment for Ebolavirus and SARS-CoV2, presumably by halting the viruses' inexorable march through the endolysosomal system (Kang et al., 2020; clinicaltrials.gov).—Jessica Shugart
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- Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61. Epub 2014 Oct 9 PubMed.
- Tian R, Gachechiladze MA, Ludwig CH, Laurie MT, Hong JY, Nathaniel D, Prabhu AV, Fernandopulle MS, Patel R, Abshari M, Ward ME, Kampmann M. CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. Neuron. 2019 Oct 23;104(2):239-255.e12. Epub 2019 Aug 15 PubMed.
- Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, Cheah JH, Clemons PA, Shamji AF, Clish CB, Brown LM, Girotti AW, Cornish VW, Schreiber SL, Stockwell BR. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014 Jan 16;156(1-2):317-331. PubMed.
- Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012 May 25;149(5):1060-72. PubMed.
- Zhang G, Zhang Y, Shen Y, Wang Y, Zhao M, Sun L. The Potential Role of Ferroptosis in Alzheimer's Disease. J Alzheimers Dis. 2021;80(3):907-925. PubMed.
- Oji Y, Hatano T, Ueno SI, Funayama M, Ishikawa KI, Okuzumi A, Noda S, Sato S, Satake W, Toda T, Li Y, Hino-Takai T, Kakuta S, Tsunemi T, Yoshino H, Nishioka K, Hattori T, Mizutani Y, Mutoh T, Yokochi F, Ichinose Y, Koh K, Shindo K, Takiyama Y, Hamaguchi T, Yamada M, Farrer MJ, Uchiyama Y, Akamatsu W, Wu YR, Matsuda J, Hattori N. Variants in saposin D domain of prosaposin gene linked to Parkinson's disease. Brain. 2020 Apr 1;143(4):1190-1205. PubMed.
- Kurz T, Eaton JW, Brunk UT. The role of lysosomes in iron metabolism and recycling. Int J Biochem Cell Biol. 2011 Dec;43(12):1686-97. Epub 2011 Sep 3 PubMed.
- See SK, Chen M, Bax S, Tian R, Woerman A, Tse E, Johnson IE, Nowotny C, Muñoz EN, Sengstack J, Southworth DR, Gestwicki JE, Leonetti MD, Kampmann M. PIKfyve inhibition blocks endolysosomal escape of α-synuclein fibrils and spread of α-synuclein aggregation. bioRxiv. January 22, 2021
- Kang YL, Chou YY, Rothlauf PW, Liu Z, Soh TK, Cureton D, Case JB, Chen RE, Diamond MS, Whelan SP, Kirchhausen T. Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2. Proc Natl Acad Sci U S A. 2020 Aug 25;117(34):20803-20813. Epub 2020 Aug 6 PubMed.
- Kurz T, Eaton JW, Brunk UT. The role of lysosomes in iron metabolism and recycling. Int J Biochem Cell Biol. 2011 Dec;43(12):1686-97. Epub 2011 Sep 3 PubMed.
- Ashraf A, So PW. Spotlight on Ferroptosis: Iron-Dependent Cell Death in Alzheimer's Disease. Front Aging Neurosci. 2020;12:196. Epub 2020 Jul 14 PubMed.
- Tian R, Abarientos A, Hong J, Hashemi SH, Yan R, Dräger N, Leng K, Nalls MA, Singleton AB, Xu K, Faghri F, Kampmann M. Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis. Nat Neurosci. 2021 Jul;24(7):1020-1034. Epub 2021 May 24 PubMed.