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Cited by 414CRISPR and Genetic EngineeringFerroptosis and cancer prognosisAutophagy in Disease and Therapy

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

Open access
Tian, Ruilin, Abarientos, Anthony, Hong, Jason, Hashemi, Sayed Hadi, Yan, Rui, Dräger, Nina, Leng, Kun, Nalls, Mike A. +4 more·2021·Nature Neuroscience
DOI 10.1038/s41593-021-00862-0PMC PMC8254803

Published version of a preprint

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Abstract

Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type-specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here we present the first genome-wide CRISPR interference and CRISPR activation screens in human neurons. We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. 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. We also determine transcriptomic changes in neurons after perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain. Tian et al. conducted a genome-wide CRISPRi/CRISPRa screen in human neurons and uncovered a neuron-specific link among prosaposin, lipofuscin and ferroptosis. The CRISPRbrain data commons enables comparison of gene function across human cell types.

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Claims the paper makes

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  • Findingnone given

    The authors established a data commons named CRISPRbrain to enable systematic comparison of gene function across human cell types.

    “To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.”
    Quote in the paperNot a substantial claim
  • Findingown data

    PSAP knockdown triggers the formation of lipofuscin.

    “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.”
    Quote in the paperSubstantial claim
  • Findingnone given

    These are the first genome-wide CRISPR screens in human neurons.

    “To our knowledge, these are the first genome-wide CRISPR screens in human neurons.”
    Quote in the paperSubstantial claim
  • Findingown data

    Knockdown of the lysosomal protein prosaposin strongly sensitizes neurons to oxidative stress.

    “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.”
    Quote in the paperSubstantial claim
  • Findingown data

    The authors determined transcriptomic changes in neurons after perturbation of genes linked to neurodegenerative diseases.

    “We also determine transcriptomic changes in neurons following perturbation of genes linked to neurodegenerative diseases.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 58
  • Classified as probably not a substantial claim. 64

  • May combine two claims.

    Findingnone given

    PSAP deficiency caused these phenotypes only in neurons, not in iPSCs or HEK293 cells.

    “Intriguingly, PSAP deficiency caused these dramatic phenotypes only in neurons, but not in iPSCs or HEK293 cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The authors established a CRISPRa platform for overexpression of endogenous genes and genome-wide gain-of-function screens in human iPSC-derived neurons.

    “Here, we expand our toolbox by establishing a CRISPRa platform for overexpression of endogenous genes and genome-wide gain-of-function screens in human iPSC-derived neurons.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The authors conducted the first genome-wide CRISPRi and CRISPRa screens in human neurons based on a panel of cellular phenotypes, and CROP-seq screens to uncover transcriptional fingerprints of genes associated with neurodegenerative diseases.

    “We conduct the first genome-wide CRISPRi and CRISPRa screens in human neurons based on a panel of cellular phenotypes, and CROP-seq screens to uncover transcriptional fingerprints of genes associated with neurodegenerative diseases.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The screens uncovered an unexpected role for PSAP, whose knockdown caused lipofuscin formation, iron trapping, ROS generation, and ferroptosis.

    “These screens uncovered an unexpected role for the lysosomal protein prosaposin (PSAP), knockdown of which caused the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    FBXO7, a Parkinson's disease-associated gene whose deficiency causes complex I respiratory impairment and ROS production, increased ROS levels when knocked down in the screen.

    “FBXO7, a gene associated with Parkinson’s disease, whose deficiency was found to cause complex I respiratory impairment and ROS production37, also increased ROS levels when knocked down in our screen (Fig. 2d).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] Brunk, U. T., Jones, C. B. & Sohal, R. S. A novel hypothesis of lipofuscinogenesis and cellular aging based on interactions between oxidative stress and autophagocytosis. Mutat. Res. 275, 395–403 (1992). source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    ACSL4 inhibition prevents ferroptosis, consistent with a reduction of peroxidized lipids upon ACSL4 knockdown in the screen.

    “ACLS4 inhibition has been shown to prevent ferroptosis44, consistent with a reduction of peroxidized lipids upon knockdown in our screen (Fig. 2d).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Cui, Y. et al. CRISP-view: a database of functional genetic screens spanning multiple phenotypes. Nucleic Acids Res. 49, D848–D854 (2021). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    GO term enrichment analysis of lipid peroxidation hits revealed a strong enrichment of peroxisomal genes, knockdown of which increased lipid peroxidation.

    “Moreover, GO term enrichment analysis of lipid peroxidation hits revealed a strong enrichment of peroxisomal genes, knockdown of which increased lipid peroxidation, consistent with the important roles of peroxisomes in redox regulation and degradation of (poly-)unsaturated fatty acids.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Genes expressed at low or undetectable levels were strongly depleted from CRISPRi hits, whereas CRISPRa hits were not restricted by endogenous expression levels.

    “Indeed, genes expressed at low or undetectable levels were strongly depleted from CRISPRi hits (Fig. 1g, left), whereas CRISPRa hits were not restricted by endogenous expression levels (Fig. 1g, right).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Hits from CRISPRa screens showed high correlation (R = 0.82) between oxidative stress and unstressed conditions, suggesting no strong stress-specific phenotypes for overexpressed genes.

    “Hits from CRISPRa screens showed high correlation (R = 0.82) between oxidative stress and unstressed conditions, suggesting no strong stress-specific phenotypes for overexpressed genes (Extended Data Fig. 2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The likelihood of detecting hit genes was not related to the length of the gene or its coding sequence.

    “The likelihood of detecting hit genes was not related to the length of the gene or its coding sequence (Extended Data Fig. 1f,g).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Among overlapping genes, the authors identified known regulators of apoptosis (TP53, IKBIP), factors of neuronal survival (XIAP, GDPGP1, MAP3K1), and genes not previously implicated in neuronal survival (IGF2BP1, IGF2BP3).

    “Among these genes, we identified known regulators of apoptosis, such as TP53, IKBIP, and factors specifically of neuronal survival, such as XIAP17–20, GDPGP121 and MAP3K123,22–24, as well as genes not previously implicated in neuronal survival, such as IGF2BP1 and IGF2BP3.”
    Quote in the paperNot a substantial claim0 of 8 sources checked
    • cites [17] Dalfó, E. et al. Evidence of oxidative stress in the neocortex in incidental Lewy body disease. J. Neuropathol. Exp. Neurol. 64, 816–830 (2005). source

      Not checked in the free run.

    • cites [18] Subbarao, K. V., Richardson, J. S. & Ang, L. C. Autopsy samples of Alzheimer’s cortex show increased peroxidation in vitro. J. Neurochem. 55, 342–345 (1990). source

      Not checked in the free run.

    • cites [19] Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014). source

      Not checked in the free run.

    • cites [20] Wang, C. et al. Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide. J. Cell Biol. 212, 545–560 (2016). source

      Not checked in the free run.

    • cites [21] Taguchi, K. et al. Keap1 degradation by autophagy for the maintenance of redox homeostasis. Proc. Natl Acad. Sci. USA 109, 13561–13566 (2012). source

      Not checked in the free run.

    • cites [22] Reho, J. J., Guo, D.-F. & Rahmouni, K. Mechanistic target of rapamycin complex 1 signaling modulates vascular endothelial function through reactive oxygen species. J. Am. Heart Assoc. 8, e010662 (2019). source

      Not checked in the free run.

    • cites [23] Delgado-Camprubi, M., Esteras, N., Soutar, M. P., Plun-Favreau, H. & Abramov, A. Y. Deficiency of Parkinson’s disease-related gene Fbxo7 is associated with impaired mitochondrial metabolism by PARP activation. Cell Death Differ. 24, 120–131 (2017). source

      Not checked in the free run.

    • cites [24] Doll, S. et al. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat. Chem. Biol. 13, 91–98 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Disruption of mTORC1 pathway genes (MTOR, RPTOR, MLST8, RHEB) reduced ROS and/or lipid peroxidation levels, whereas knockdown of NPRL3 induced ROS and lipid peroxidation levels.

    “Disruption of genes involved in the mTORC1 pathway, including components of the mTORC1 complex (MTOR, RPTOR, and MLST8) or its activator RHEB reduced ROS and/or lipid peroxidation levels, whereas knockdown of NPRL3, a mTORC1 inhibitor, induced ROS and lipid peroxidation levels in neurons (Fig. 2d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    While some CRISPRi and CRISPRa hits were also survival-relevant in other cell types, a substantial fraction of hits was neuron-specific.

    “While some CRISPRi and CRISPRa hits were also survival-relevant in other cell types based on previously reported screens, a substantial fraction of hits was neuron-specific (Extended Data Fig. 1h–j).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    These lysosomal phenotypes were not observed in HEK293 cells, iPSCs, astrocytes, or microglia, and only to a small extent in NPCs.

    “Again, these lysosomal phenotype were not observed in HEK293 cells, iPSCs and derived astrocytes and microglia, and only to a small extent in iPSC-derived neuronal progenitor cells (Fig. 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In medium lacking antioxidants, PSAP KO caused a dramatic decrease in survival around Day 11, with complete death of all neurons by Day 14.

    “Strikingly, however, when we cultured these neurons in medium lacking antioxidants (–AO), PSAP KO caused a dramatic decrease in survival around Day 11, resulting in complete death of all neurons by Day 14 (Fig. 3h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PSAP KO neurons showed a substantial increase in ROS and peroxidized lipid levels, confirming the screen results.

    “Indeed, we observed a substantial increase in ROS and peroxidized lipid levels in PSAP KO neurons, confirming our screen results (Fig. 3e).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    The authors conducted focused secondary screens with an sgRNA library targeting 730 hit genes.

    “To further investigate hit genes from the genome-wide ROS and lipid peroxidation screens in high-throughput, we conducted focused secondary screens with an sgRNA library targeting 730 hit genes (Fig. 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Knockdown of PSAP altered lysosomal status and strongly induced ROS, lipid peroxidation, and iron levels.

    “Surprisingly, we identified that knockdown of PSAP altered lysosomal status and strongly induced ROS, lipid peroxidation, and iron levels when depleted (Fig. 2d,f,g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both CTSD and GM2A were hits in at least one redox screen, showing similar knockdown phenotypes as PSAP.

    “Intriguingly, both CTSD and GM2A were also hits in at least one of the redox screens, showing similar knockdown phenotypes as PSAP (Fig. 3b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The screens for survival under oxidative stress and levels of ROS and peroxidized lipids uncovered many categories of known redox regulators, validating the sensitivity of the approach.

    “In summary, our screens for survival of oxidative stress and levels of ROS and peroxidized lipids uncovered many categories of known redox regulators, validating the sensitivity of our approach and supporting the notion that core mechanisms of redox regulation are conserved across different cell types.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Knockdown of CD44 increased lipid peroxidation in the screen, as expected because CD44v stabilizes xCT and increases cysteine uptake for GSH synthesis, inhibiting ferroptosis.

    “By contrast, knockdown of CD44, whose splicing variant CD44v stabilizes the cystine/glutamate antiporter xCT at the plasma membrane and increases cysteine uptake for GSH synthesis, thereby inhibiting ferroptosis46,47, increased lipid peroxidation in our screen as expected (Fig. 2d).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [46] Topol, A., Tran, N. N. & Brennand, K. J. A guide to generating and using hiPSC derived NPCs for the study of neurological diseases. J. Vis. Exp. e52495 (2015). source

      Not checked in the free run.

    • cites [47] Cheng, C., Fass, D. M., Folz-Donahue, K., MacDonald, M. E. & Haggarty, S. J. Highly expandable human iPS cell-derived neural progenitor cells (NPC) and neurons for central nervous system disease modeling and high-throughput screening. Curr. Protoc. Hum. Genet 92, 21.8.1–21.8.21 (2017).

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    ROS induction in PSAP KO neurons could be rescued by overexpression of PSAP cDNA, confirming that phenotypes were not driven by off-target genome editing.

    “Moreover, ROS induction in PSAP KO neurons could be rescued by the overexpression of PSAP cDNA (Fig. 3e), confirming that phenotypes were not driven by off-target genome editing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    WDR45 was found among genes affecting lysosomal status and iron levels; it is involved in autophagosome formation and lysosomal degradation and is associated with NBIA.

    “Among these genes, we found WDR45, which is involved in autophagosome formation and lysosomal degradation and is also associated with Neurodegeneration with Brain Iron Accumulation (NBIA) in line with previous studies55,56.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [55] Wojcik, M., Hauser, M., Li, W., Moon, S. & Xu, K. Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells. Nat. Commun. 6, 7384 (2015). source

      Not checked in the free run.

    • cites [56] Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The viability of PSAP KO neurons under -AO condition was not rescued by Z-VAD-FMK but was fully rescued by ferroptosis inhibitors DFO and ferrostatin-1.

    “Intriguingly, the viability of PSAP KO neurons under the -AO condition was not rescued by Z-VAD-FMK, a pan-caspase inhibitor that blocks apoptosis, but was fully rescued by ferroptosis inhibitors, including the iron chelator deferoxamine (DFO) and the lipid peroxidation inhibitor ferrostatin-1 (Fig. 3i,j), suggesting that mild oxidative stress triggers ferroptosis in PSAP KO neurons.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dramatically enlarged lysosomes were observed in PSAP KO neurons, reflected in increased lysotracker signal by flow cytometry.

    “Strikingly, we observed dramatically enlarged lysosomes in PSAP KO neurons (Fig. 4c), which were also reflected in an increased lysotracker signal by flow cytometry (Fig. 5a), as expected based on our screen results (Fig. 2f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PSAP deficiency caused a substantial increase of iron levels in neurons, but not in other cell types.

    “Indeed, we observed that PSAP deficiency caused a substantial increase of iron levels in neurons (Fig. 5f), but not other cell types (Fig. 5g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overexpression of INSR, ATP5F1C, SOX5, GSX2, and GBX2 downregulated a neurogenesis-related gene module (M1) and upregulated a cell cycle-related gene module (M5).

    “overexpression of which downregulated a gene module related to neurogenesis (M1) and upregulated a gene module related to the cell cycle (M5) (Fig. 6g, bottom)”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    CRISPRbrain is complementary to the Human Cell Atlas and serves as an open-access platform for sharing and exploring functional genomics screening data in differentiated human cell types across labs.

    “Our CRISPRbrain Data Commons is complementary to the Human Cell Atlas and serves as an open-access platform for sharing and exploring functional genomics screening data in differentiated human cell types across labs.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    CRISPRbrain features screens with simple readouts and complex readouts, and allows browsing and searching by parameters of interest.

    “CRISPRbrain currently features screens with “simple” readouts (such as survival and fluorescent reporter levels), as well as screens with complex readouts (such as transcriptomes) Screens can be browsed and searched based on parameters of interest”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingreasoning

    CRISPRbrain is the first database focused on screens in non-cancerous human cell types for a broad range of phenotypes, including high-dimensional transcriptomic phenotypes.

    “CRISPRbrain is the first database focused on screens in non-cancerous human cell types for a broad range of phenotypes, including high-dimensional transcriptomic phenotypes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    Within populations of cells expressing sgRNAs for a specific target gene, target knockdown or overexpression levels were heterogeneous in some cases.

    “Within the population of cells expressing sgRNAs for a specific target gene, the levels of target knockdown or overexpression were heterogeneous in some cases.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Oxidative stress is one of the predominant stresses in aging and neurodegenerative diseases.

    “We apply our functional genomics platforms to systematically identify genetic modifiers of levels of reactive oxygen species and peroxidized lipids, and neuronal survival under oxidative stress, one of the predominant stresses in aging6 and neurodegenerative diseases7.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [6] Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5, e19760 (2016). source

      Not checked in the free run.

    • cites [7] Yilmaz, A., Peretz, M., Aharony, A., Sagi, I. & Benvenisty, N. Defining essential genes for human pluripotent stem cells by CRISPR–Cas9 screening in haploid cells. Nat. Cell Biol. 20, 610–619 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    A monoclonal CRISPRa-iPSC line was generated and had a normal karyotype.

    “A monoclonal line of these CRISPRa-iPSCs was generated and a normal karyotype was confirmed (Extended Data Fig. 1a).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Knockdown of certain genes can have remarkably different impacts on cell survival and gene expression in different isogenic human cell types, including stem cells and neurons.

    “Cell-type specific gene function is also supported by our recent finding that knockdown of certain genes can have remarkably different impacts on cell survival and gene expression in different isogenic human cell types, including stem cells and neurons3.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Kampmann, M. CRISPR-based functional genomics for neurological disease. Nat. Rev. Neurol. 16, 465–480 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mutations in ubiquitously expressed trafficking factors or aminoacyl-tRNA synthetases can cause neurodegenerative diseases such as Charcot-Marie-Tooth disease.

    “Striking examples are familial mutations causing neurodegenerative diseases, such as hereditary motor and sensory neuropathies (Charcot-Marie-Tooth disease), which can be caused by mutations in ubiquitously expressed trafficking factors (RAB7A, DNM2, DYNC1H1), or aminoacyl-tRNA synthetases (AARS, GARS, HARS, MARS)2.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The authors previously conducted a sub-genome scale CRISPRi screen targeting 2,325 druggable genome genes to reveal genes controlling neuronal survival.

    “We previously conducted a sub-genome scale CRISPRi screen to reveal genes controlling neuronal survival using an sgRNA library targeting 2,325 genes representing the “druggable genome”3.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Kampmann, M. CRISPR-based functional genomics for neurological disease. Nat. Rev. Neurol. 16, 465–480 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Gene function can be cell-type specific, as shown by mutations in broadly expressed genes causing cell-type specific defects and disease.

    “In addition to gene expression, gene function can also be cell type-specific, as evidenced by the fact that mutations in broadly expressed or housekeeping genes can lead to cell-type specific defects and disease states.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Lipofuscin traps iron, which generates reactive oxygen species and triggers 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.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Knockdown of a single component of a protein complex could abrogate the function of the entire complex and result in a phenotype, whereas overexpression of a single subunit by CRISPRa would generally be insufficient to increase complex function.

    “Second, many genes encode proteins that form complexes (such as the V-ATPase complex), for which knockdown of a single component could abrogate the function of the entire complex and result in a phenotype, whereas overexpression of a single subunit by CRISPRa would generally be insufficient to induce an increased function of the complex.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Paralogs tend to have less deleterious loss-of-function effects in CRISPR-Cas9 screens in different human cell lines.

    “Paralogs tend to have less deleterious loss-of-function effect in CRISPR-Cas9 screens in different human cell lines16.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Yao, Y. et al. Enhanced brain levels of 8,12-iso-iPF2α-VI differentiate AD from frontotemporal dementia. Neurology 61, 475–478 (2003). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ACSL4 enriches cellular membranes with long PUFAs and is required for ferroptosis.

    “ACSL4, encoding Acyl-CoA Synthetase Long Chain Family Member 4, which enriches cellular membranes with long PUFAs is required for ferroptosis44,45.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [44] Cui, Y. et al. CRISP-view: a database of functional genetic screens spanning multiple phenotypes. Nucleic Acids Res. 49, D848–D854 (2021). source

      Not checked in the free run.

    • cites [45] Guo, X. et al. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature 579, 427–432 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The V-ATPase complex mediates acidification of the endo-lysosomal compartment through ATP hydrolysis-coupled proton transport, and its dysfunction can cause neurodegenerative diseases.

    “The V-ATPase complex mediates acidification of the endo-lysosomal compartment through ATP hydrolysis-coupled proton transport, and its dysfunction can cause neurodegenerative diseases15.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Hensley, K. et al. Brain regional correspondence between Alzheimer’s disease histopathology and biomarkers of protein oxidation. J. Neurochem. 65, 2146–2156 (1995). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Increasing mTORC1 signaling induces ROS production, whereas inhibiting mTORC1 reduces ROS.

    “This is consistent with previous observations that increasing mTORC1 signaling induced ROS production34, whereas inhibiting mTORC1 reduced ROS34–36.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [34] Bartz, F. et al. Identification of cholesterol-regulating genes by targeted RNAi screening. Cell Metab. 10, 63–75 (2009). source

      Not checked in the free run.

    • cites [35] Puri, V. et al. Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage. J. Biol. Chem. 278, 20961–20970 (2003). source

      Not checked in the free run.

    • cites [36] Glaros, E. N. et al. Glycosphingolipid accumulation inhibits cholesterol efflux via the ABCA1/apolipoprotein A-I pathway: 1-phenyl-2-decanoylamino-3-morpholino-1-propanol is a novel cholesterol efflux accelerator. J. Biol. Chem. 280, 24515–24523 (2005). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    ROS are mainly generated by proton leak from the electron transport chain, and knockdown of electron transport chain components could increase proton leakage.

    “This was expected because ROS are mainly generated by proton leak from the electron transport chain, and knockdown of electron transport chain components could increase proton leakage.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    A gene not expressed in neurons will not have a CRISPRi knockdown phenotype but may have a CRISPRa overexpression phenotype.

    “First, a gene that is not expressed in neurons will not have a CRISPRi knockdown phenotype, but may have a CRISPRa overexpression phenotype.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Knockdown of a single gene may not lead to a phenotype due to redundancy, for example because gene paralogs can compensate for each other's loss-of-function.

    “Last, knockdown of a single gene may not lead to a phenotype due to redundancy. For example, gene paralogs can compensate for each other’s loss-of-function.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Neurons have a unique vulnerability to redox imbalance-induced oxidative stress, which is often found in the brain of patients with neurodegenerative diseases.

    “Given the unique vulnerability of neurons to redox imbalance-induced oxidative stress, which is often found in the brain of patients with neurodegenerative diseases25–29, we sought to apply our functional genomics toolkit to systematically identify factors that are important for redox homeostasis and oxidative stress response in human neurons.”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [25] Yuan, H., Li, X., Zhang, X., Kang, R. & Tang, D. Identification of ACSL4 as a biomarker and contributor of ferroptosis. Biochem. Biophys. Res. Commun. 478, 1338–1343 (2016). source

      Not checked in the free run.

    • cites [26] Ishimoto, T. et al. CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc− and thereby promotes tumor growth. Cancer Cell 19, 387–400 (2011). source

      Not checked in the free run.

    • cites [27] Weber, R. A. et al. Maintaining iron homeostasis is the key role of lysosomal acidity for cell proliferation. Mol. Cell 77, 645–655 (2020). source

      Not checked in the free run.

    • cites [28] Haack, T. B. et al. Exome sequencing reveals de novo WDR45 mutations causing a phenotypically distinct, X-linked dominant form of NBIA. Am. J. Hum. Genet. 91, 1144–1149 (2012). source

      Not checked in the free run.

    • cites [29] Diaz-Font, A. et al. A mutation within the saposin D domain in a Gaucher disease patient with normal glucocerebrosidase activity. Hum. Genet. 117, 275–277 (2005). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    GPX4 utilizes glutathione to reduce peroxidized lipids and thus prevents ferroptosis, a non-apoptotic cell death caused by iron-dependent lipid peroxidation.

    “GPX4 utilizes glutathione to reduce peroxidized lipids and thus prevents ferroptosis, which is a non-apoptotic type of cell death caused by iron-dependent lipid peroxidation30.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Radha Rama Devi, A. et al. Acute Gaucher disease-like condition in an Indian infant with a novel biallelic mutation in the prosaposin gene. J. Pediatr. Genet. 8, 81–85 (2019). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Intracellular labile ferrous iron can contribute to ROS generation and lipid peroxidation via Fenton reactions.

    “Since intracellular labile ferrous iron (Fe2+) can contribute to ROS generation and lipid peroxidation via Fenton reactions52, we asked whether some of the redox hits act through changing iron homeostasis.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [52] Komarov, P. G. et al. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285, 1733–1737 (1999). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Lipofuscin is an insoluble aggregate of peroxidized lipids, proteins, and metals in lysosomes of postmitotic cells, is a hallmark of aging, and is associated with several neurodegenerative diseases.

    “Lipofuscin is an insoluble aggregate of peroxidized lipids, proteins and metals in lysosomes of postmitotic cells, such as neurons, which is a hallmark of aging and also associated with several neurodegenerative diseases71.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [71]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Disruption of the electron transport chain may lead to mitochondrial damage, causing release of labile iron and reduced iron utilization, thus increasing labile iron levels.

    “Disruption of the electron transport chain may lead to mitochondrial damage which can cause the subsequent release of labile iron and reduced iron utilization, thus increasing labile iron levels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Loss of PSAP increases ROS levels and leads to increased lipid peroxidation, resulting in neuronal ferroptosis in response to mild oxidative stress.

    “Loss of PSAP increases ROS levels and leads to increased lipid peroxidation, resulting in neuronal ferroptosis in response to mild oxidative stress.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The four saposins, along with GM2A, function as activators for glycosphingolipid degradation by lysosomal hydrolases.

    “These four saposins, along with the lysosomal protein GM2A (GM2 Ganglioside Activator), function as activators for glycosphingolipid degradation by lysosomal hydrolases65 (Fig. 3a).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [65]

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Accumulation of ether lipids was recently characterized as a feature of hypoxia.

    “Interestingly, the accumulation of ether lipids was also recently characterized as a feature of hypoxia66.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [66]

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Lysotracker is a fluorescent acidotropic probe used to detect lysosome localization, quantify lysosome number and size, and monitor autophagy.

    “Lysotracker is a fluorescent acidotropic probe for labeling and tracking acidic organelles in live cells, which has been used to detect lysosome localization, quantify the number and sizes of lysosomes48,49 and to monitor autophagy50,51.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [48] Tcw, J. et al. An efficient platform for astrocyte differentiation from human induced pluripotent stem cells. Stem Cell Rep. 9, 600–614 (2017). source

      Not checked in the free run.

    • cites [49] Brownjohn, P. W. et al. Functional studies of missense TREM2 mutations in human stem cell-derived microglia. Stem Cell Rep. 10, 1294–1307 (2018). source

      Not checked in the free run.

    • cites [50] Piñero, J. et al. DisGeNET: a discovery platform for the dynamical exploration of human diseases and their genes. Database (Oxf.) 2015, bav028 (2015). source

      Not checked in the free run.

    • cites [51] Bersuker, K. et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575, 688–692 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PSAP is a strong genetic modifier of cholesterol levels, and accumulated glycosphingolipids can lead to lysosomal cholesterol accumulation by inhibiting cholesterol efflux.

    “This finding mirrored the results of previous studies that PSAP is a strong genetic modifier of cholesterol levels67 and that accumulated glycosphingolipids can lead to the accumulation of cholesterol in lysosomes by inhibiting cholesterol efflux68,69.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [67]

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    • cites [68]

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    • cites [69]

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PSAP encodes prosaposin, which is proteolytically processed by cathepsin D (CTSD) in the lysosome to generate saposins A, B, C, and D.

    “PSAP encodes prosaposin, which is a pro-protein that is proteolytically processed by cathepsin D (encoded by CTSD) in the lysosome to generate four cleavage products: saposins A, B, C, and D64.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [64] Liao, Y., Wang, J., Jaehnig, E. J., Shi, Z. & Zhang, B. WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs. Nucleic Acids Res. 47, W199–W205 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    There is currently no consensus on the molecular mechanisms driving lipofuscin formation.

    “There is currently no consensus on the molecular mechanisms driving lipofuscin formation71.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [71]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mitochondria are a major site of cellular iron storage and utilization in processes such as heme synthesis and iron-sulfur cluster biogenesis.

    “Mitochondria are a major site of cellular iron storage and utilization in processes such as heme synthesis and iron-sulfur cluster biogenesis59.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [59] Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Lipofuscin accumulates iron from lysosomal degradation of iron-rich proteins or organelles, and accumulated iron in lipofuscin has been proposed to generate ROS through the Fenton reaction.

    “Lipofuscin is known to accumulate iron from lysosomal degradation of iron-rich proteins or organelles (e.g. mitochondria), and accumulated iron in lipofuscin has been proposed to generate ROS through the Fenton reaction73,74.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [73]

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    • cites [74]

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  • May combine two claims.

    Premisecitation

    PSAP variants are associated with lysosomal storage disorders and Parkinson's Disease.

    “Given the screen phenotypes of PSAP, which were unexpected based on its known functions, and the association of PSAP variants not only with lysosomal storage disorders60–62 but also Parkinson’s Disease63, we decided to further investigate the underlying mechanisms of PSAP in neuronal redox regulation.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [60] Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 559 (2008). source

      Not checked in the free run.

    • cites [61] Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017). source

      Not checked in the free run.

    • cites [62] Soneson, C., Love, M. I. & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res. 4, 1521 (2015). source

      Not checked in the free run.

    • cites [63] Pang, X. P., Hershman, J. M., Chung, M. & Pekary, A. E. Characterization of tumor necrosis factor-α receptors in human and rat thyroid cells and regulation of the receptors by thyrotropin. Endocrinology 125, 1783–1788 (1989). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A previous study reported that overexpression of NQO1 stabilizes p53.

    “A previous study reported that overexpression of NQO1 stabilizes p5399.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [99]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CRISPR perturbation can be coupled to single-cell RNA sequencing using CROP-seq or Perturb-Seq to provide transcriptomic phenotypes.

    “CRISPR perturbation can be coupled to single-cell RNA sequencing, using CROP-seq or Perturb-Seq strategies78–80, to provide rich transcriptomic phenotypes.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [78]

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    • cites [79]

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    • cites [80]

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    SOX5, GSX2, and GBX2 are transcription factors maintaining neural progenitor cell fate and/or self-renewal.

    “SOX5, GSX2 and GBX2, which are transcription factors maintaining neural progenitor cell fate and/or their self-renewal89–91”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [89]

      Not checked in the free run.

    • cites [90]

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    • cites [91]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Lipofuscin formation in PSAP knockout neurons accumulates iron and generates ROS that peroxidize lipids.

    “which in turn accumulates iron and generates ROS that peroxidized lipids.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    EBF3 is an apoptosis inducer and is upregulated in the hippocampus of AD model mice.

    “EBF3, an apoptosis inducer85 that is also upregulated in the hippocampus of AD model mice86”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [85]

      Not checked in the free run.

    • cites [86]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Upregulation of NQO1 was proposed to be a neuroprotective mechanism against oxidative stress in neurodegenerative diseases.

    “The upregulation of NQO1 was proposed to be a neuroprotective mechanism against oxidative stress in neurodegenerative diseases.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Nrf2 canonically functions upstream of NQO1 by inducing antioxidant genes including NQO1 in response to oxidative stress.

    “Nrf2 canonically functions upstream of NQO1 by inducing the expression of many antioxidant genes, including NQO1, in response to oxidative stress100.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [100]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The Nrf2 pathway is induced in some neurodegenerative diseases, in patient brains and in patient iPSC-derived neurons.

    “the Nrf2 pathway is also induced in some neurodegenerative diseases, both in patient brains101 and in patient iPSC-derived neurons98.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [101]

      Not checked in the free run.

    • cites [98]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    NQO1 is thought to be a cytoprotective factor through its antioxidant functions.

    “NQO1, encoding the NAD(P)H:Quinone Oxidoreductase 1, is thought to be a cytoprotective factor through its antioxidant functions92.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [92]

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Elevated levels and activity of NQO1 have been found in the brains of patients with different neurodegenerative diseases and in patient iPSC-derived neurons.

    “Elevated levels and activity of NQO1 have been found in the brains of patients with different neurodegenerative diseases93–98, as well as in patient iPSC-derived neurons98.”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [93]

      Not checked in the free run.

    • cites [94]

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    • cites [95]

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    • cites [96]

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    • cites [97]

      Not checked in the free run.

    • cites [98]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    VPS54 and PON2 have been implicated in ALS, and PAXIP1 is associated with Alzheimer's disease.

    “VPS54 and PON2 have been implicated in Amyotrophic Lateral Sclerosis (ALS)82,83 whereas PAXIP1 is associated with Alzheimer’s disease (AD)84.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [82]

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    • cites [83]

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    • cites [84]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GWASs have uncovered hundreds of genes associated with human neurodegenerative diseases.

    “genome-wide association studies (GWASs) have uncovered hundreds of genes that are associated with human neurodegenerative diseases75,76.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [75]

      Not checked in the free run.

    • cites [76]

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisenone given

    GPX4 has a major role in reducing lipid peroxidation and suppressing ferroptosis.

    “Given the major role of GPX4 in reducing lipid peroxidation and suppressing ferroptosis”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lipofuscin accumulation has been characterized in Alzheimer's disease, Parkinson's disease, Huntington's disease, and GRN-associated frontotemporal dementia.

    “It has also been characterized in Alzheimer’s disease117,118, Parkinson’s disease119, Hungtinton’s disease120,121 and GRN-associated frontotemporal dementia (FTD)122.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [117]

      Not checked in the free run.

    • cites [118]

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    • cites [119]

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    • cites [120]

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    • cites [121]

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Accumulation of lipofuscin is a pathological hallmark of many degenerative diseases, including neuronal ceroid lipofuscinosis and inherited age-related macular degenerations.

    “The accumulation of lipofuscin is a pathological hallmark of many degenerative diseases, such as neuronal ceroid lipofuscinosis (NCL)114 and inherited age-related macular degenerations115,116.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [114]

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    • cites [115]

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    • cites [116]

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Abnormal lipid metabolism has been observed in neurodegenerative diseases.

    “Abnormal lipid metabolism has been observed in neurodegenerative diseases124–126.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [124]

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    • cites [125]

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    • cites [126]

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The Human Cell Atlas has obtained gene expression profiles for more than 30 human organs in more than 4.5 million cells.

    “gene expression profiles for >30 human organs in > 4.5M cells have been obtained so far (https://data.humancellatlas.org/).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Oxidative stress is one of the predominant types of stress in aging and neurodegenerative diseases.

    “One of the predominant types of stress in aging and neurodegenerative diseases is oxidative stress6,7”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [6] Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5, e19760 (2016). source

      Not checked in the free run.

    • cites [7] Yilmaz, A., Peretz, M., Aharony, A., Sagi, I. & Benvenisty, N. Defining essential genes for human pluripotent stem cells by CRISPR–Cas9 screening in haploid cells. Nat. Cell Biol. 20, 610–619 (2018). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    Lipofuscin has traditionally been considered a byproduct of aging and a consequence of defective cellular homeostasis.

    “lipofuscin has traditionally been considered a byproduct of aging and as a consequence of defective cellular homeostasis”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Lipofuscin formation leads to iron accumulation and strongly induces ROS production, oxidizing lipids and leading to neuronal ferroptosis under oxidative stress.

    “which leads to iron accumulation and strongly induces ROS production, oxidizing lipids and leading to neuronal ferroptosis under oxidative stress.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisereasoning

    The physiological and pathological functions of lipofuscin have not been well characterized.

    “its physiological and pathological functions have not been well characterized.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Premisenone given

    A comprehensive understanding of how neurons regulate redox homeostasis and maintain survival under oxidative stress is lacking.

    “yet a comprehensive understanding of how neurons regulate redox homeostasis and maintain survival under oxidative stress is lacking.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Studies with human iPSCs at UCSF were approved by the Human Gamete, Embryo, and Stem Cell Research Committee.

    “Studies with human iPSCs at UCSF were approved by the Human Gamete, Embryo, and Stem Cell Research (GESCR) Committee.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Accumulation of some glycosphingolipids, especially simple gangliosides, caused by inhibition of lysosome membrane recycling, contributes to neurodegeneration in cultured neurons and animal models.

    “Accumulation of some glycosphingolipids, especially simple gangliosides, caused by inhibition of lysosome membrane recycling, contributes to neurodegeneration both in cultured neurons and in animal models127.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [127]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The parental iPSC line used was WTC11 iPSCs carrying a single-copy doxycycline-inducible mouse NGN2 at the AAVS1 locus.

    “WTC11 iPSCs harboring a single-copy of doxycycline-inducible mouse NGN2 at the AAVS1 locus (Ngn2-iPSCs,9) were used as the parental iPSC line for further genetic engineering.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Colacurcio, D. J. & Nixon, R. A. Disorders of lysosomal acidification—the emerging role of v-ATPase in aging and neurodegenerative disease. Ageing Res. Rev. 32, 75–88 (2016). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Single sgRNAs were assigned to about 35,000 CRISPRi cells and about 21,000 CRISPRa cells, which were retained for downstream analysis.

    “Single sgRNAs could be assigned to about 35,000 CRISPRi cells and about 21,000 CRISPRa cells, which were retained for further analysis (Supplementary Table 5)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The CRISPRa iPSC line had CLYBL integration confirmed by PCR genotyping.

    “Monoclonal lines were isolated by limiting dilution and CLYBL integration was confirmed by PCR genotyping.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    An unsupervised outlier detection method based on local outlier factor was used to select cells in which functional perturbations occurred.

    “To select cells in which functional perturbations happened, we leveraged an unsupervised outlier detection method based on the local outlier factor (LOF) using the LocalOutlierFactor function in the Python package scikit-learn (version 0.23.0).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The genome-wide CRISPRi and CRISPRa libraries hCRISPRi-v2 and hCRISPRa-v2 each consist of seven sublibraries, H1 through H7.

    “The genome-wide CRISPRi and CRISPRa libraries hCRISPRi-v2 and hCRISPRa-v210, consisting of 7 sublibraries each (H1-H7), were packaged into lentivirus as previously described3.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Dandage, R. & Landry, C. R. Paralog dependency indirectly affects the robustness of human cells. Mol. Syst. Biol. 15, e8871 (2019). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Hit genes in the pooled CRISPR screens were determined using a Gene score cutoff corresponding to an empirical false discovery rate of 5%.

    “Hit genes were determined based on Gene score cutoff corresponding to an empirical false discovery rate (FDR) of 5%.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The PSAP KO iPSC line had a normal karyotype.

    “A normal karyotype was confirmed (Cell Line Genetics).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    In CROP-seq, the mean reads per cell were around 48,000 for CRISPRi and 36,000 for CRISPRa.

    “The mean reads per cell was around 48,000 for CRISPRi and 36,000 for CRISPRa.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The PSAP KO iPSC line had protein-level knockout confirmed by western blot and immunofluorescence.

    “Protein level KO was confirmed by western blot and immunofluorescence (Fig. 5c,d).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The PSAP KO iPSC line had frameshift indels confirmed by Sanger sequencing.

    “One clonal line was selected and frameshift indels were confirmed by Sanger sequencing.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The CRISPRi and CRISPRa iPSC infections achieved MOIs of 0.4-0.6 and approximately 1000x coverage per library element.

    “CRISPRi- and CRISPRa-iPSCs were infected by the sgRNA libraries at MOIs of 0.4–0.6 (as measured by the BFP fluorescence from the lentiviral vector) with approximately 1000x coverage per library element.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    In CROP-seq, approximately 58,000 CRISPRi neurons and 38,000 CRISPRa neurons were detected.

    “Approximately 58,000 CRISPRi neurons and 38,000 CRISPRa neurons were detected.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    In CROP-seq, the median number of genes detected per cell was around 4,341 for CRISPRi and 3,100 for CRISPRa.

    “The median number of genes detected per cell was around 4,341 for CRISPRi and 3,100 for CRISPRa.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Untargeted lipidomics compared wild-type and PSAP KO neurons in triplicate.

    “Triplicates of samples for WT and PSAP KO neurons were analyzed.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    CROP-seq cells were filtered to retain those with more than 2000 genes detected for CRISPRi and more than 1500 for CRISPRa, and with less than 0.15% mitochondrial transcript counts.

    “To ensure data quality, a stringent criterion was applied to filter cells based on the number of genes detected (> 2000 for CRISPRi and > 1500 for CRISPRa) and percentage of mitochondrial transcript counts (< 0.15%).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The CRISPRa iPSC line used for further experiments had a normal karyotype.

    “Karyotype testing (Cell Line Genetics) was normal for the clonal line used for further experiments in this study, which we termed CRISPRa iPSCs.”
    Quote in the paperNot a substantial claim
101 of 101

  • The paper relies on this without support.

    Neurodegenerative diseases often show selective vulnerability of specific neuronal subtypes even when the mutated gene is expressed broadly.

    “Moreover, neurodegenerative diseases are often characterized by the selective vulnerability of specific neuronal subtypes, even if the mutated gene is expressed throughout the brain or even throughout the whole body.”

  • The paper relies on this without support.

    The cell types used as comparisons (HEK293, iPSCs, NPCs, astrocytes, microglia) are adequate to establish neuron-specific effects.

    “Interestingly, PSAP KO or knockdown in five other cell types we tested (HEK293 cells, iPSCs, and iPSC-derived neuronal progenitor cells, astrocytes and microglia, Extended Data Fig. 3a) did not increase ROS levels (Fig. 3f), suggesting a neuron-specific role for PSAP in redox regulation.”

  • The paper relies on this without support.

    Peroxisomes have important roles in redox regulation and degradation of polyunsaturated fatty acids.

    “Moreover, GO term enrichment analysis of lipid peroxidation hits revealed a strong enrichment of peroxisomal genes, knockdown of which increased lipid peroxidation, consistent with the important roles of peroxisomes in redox regulation and degradation of (poly-)unsaturated fatty acids.”

  • The paper relies on this without support.

    The in vitro iPSC-derived neuron models and CRISPR screens reflect relevant aspects of human neuronal biology and disease.

    “supporting the notion that neurons have cell-type specific vulnerabilities, which may explain why defects in some generally expressed genes specifically cause neurological diseases.”

  • The paper relies on this without support.

    The -AO culture condition adequately models oxidative stress in neurodegenerative disease brains.

    “under oxidative stress conditions that are commonly found in the brains of patients with neurodegenerative diseases.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Neurons are vulnerable to toxic lipids, such as oxidized lipids, particularly when lysosomes are compromised.

    “neurons are vulnerable to toxic lipids, such as oxidized lipids65,66, particularly when lysosomes are compromised67.”

    From Decreased lipidated ApoE‐receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes (2025) · cites this paper as [67]

  • A distance measure between sets of cells describes difference or similarity between expression profiles of cells treated with distinct perturbations, which can infer unique or shared mechanisms or identify perturbation targets.

    “Such a distance measure describes the difference or similarity between the expression profiles of cells treated with distinct perturbations, thus inferring unique or shared mechanisms or identifying perturbation targets, which tend to produce similar shifts in molecular profiles20,21.”

    From scPerturb: harmonized single-cell perturbation data (2024) · cites this paper as [21]

  • More than 100 genes perturbed with CRISPRa in one dataset are perturbed with CRISPRi perturbations in another dataset of the same cell line, either in one publication or across multiple studies.

    “More than 100 genes perturbed with CRISPRa in one dataset are perturbed with CRISPRi perturbations in another dataset of the same cell line, either in one publication21 or across multiple studies20,49.”

    From scPerturb: harmonized single-cell perturbation data (2024) · cites this paper as [21]

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References 153 · Cited by 414

Authors
Tian, R., Abarientos, A., Hong, J., Hashemi, S. H., Yan, R., Dräger, N., Leng, K., Nalls, M. A., Singleton, A. B., Xu, K., Faghri, F., & Kampmann, M.
Affiliations
  • University of California, San Francisco (US)
  • University of Illinois Urbana-Champaign (US)
  • University of California, Berkeley (US)
  • National Institutes of Health (US)
Corresponding author
  • Tian, Ruilin
  • Kampmann, Martin
ORCID
Venue
Nature Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2021-05-24
Volume
24
Issue
7
Pages
1020-1034
ISSN
  • 1097-6256 (ISSN-L)
  • 1546-1726
DOI
10.1038/s41593-021-00862-0
PMC
PMC8254803
OpenAlex
W3164329676
Semantic Scholar
6338712759f5aa74801214fd04e63f9b75091ded
Cited by
414
References
153
Citations by year
2026: 71 · 2025: 93 · 2024: 100 · 2023: 65 · 2022: 54 · 2021: 27 · 2020: 4
FWCI
22.82
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Neuroscience (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute on Aging (F30 AG066418, ZIA AG000957-16, R01 AG062359, R56 AG057528)
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (DP2 GM132681, DP2 GM119139)
  • U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (U54 NS100717)
  • Paul G. Allen Family Foundation (Allen Distinguished Investigator Award)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Ferroptosis and cancer prognosis
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
Keywords
ferroptosis, lysosomal dysfunction, prosaposin, lipofuscin, oxidative stress, human neurons, neurodegenerative diseases, reactive oxygen species, iron accumulation, aging
MeSH terms
Ferroptosis, Humans, Lysosomes, Neurons, Oxidative Stress, Gene Expression Profiling, Saposins, Clustered Regularly Interspaced Short Palindromic Repeats

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