. Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis. bioRxiv. February 5, 2021

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  1. The paper by Tian et al. is truly groundbreaking, because, for the first time, the investigators successfully performed CRISPR-based screens in neurons derived from human induced pluripotent stem cell models, opening many new avenues of research that may lead to an enhanced understanding of the specific functions of human genes in different types of neurons. This is an important step toward uncovering molecular mechanisms underlying various neurodegenerative diseases, and the results will likely yield new therapeutic targets.

    Among the strong “hits” were genes influencing reactive oxygen species (ROS), lipid peroxidation, and levels of labile iron. One of these hits, PSAP, encoding the lysosomal protein prosaposin, modifies lysosomal function. Knockdown of PSAP strongly induced ROS, lipid peroxidation, and iron levels, which were aspects not previously associated with this pre-protein.

    In the lysosome, prosaposin is cleaved by cathepsin into four saposins that are known cofactors for different lysosomal enzymes. In fact, one of the saposins, Sap C, is an essential activator for the enzyme glucocerebrosidase, a known protein linked to parkinsonism. This finding once again directs attention to lysosomal pathways in neurodegeneration.

    Overall, the genome-wide CRISPR screen described opens up multiple new paths to pursue, and undoubtedly will lead to many new stories like that of the evolving implications of prosaposin deficiency. The data will be available so that all investigators can interact with this enormous dataset through the new data commons described—CRISPRbrain.

    View all comments by Ellen Sidransky
  2. Two new studies from the Kampmann lab use CRISPR technology to uncover regulators of the endolysosomal pathway that impact neurodegeneration. Using CRISPR screens either in hiPSC-derived neurons (Tian et al. 2021) or HEK293 cells (See et al., 2021),  they identified modifiers of neurodegenerative phenotypes, including endolysosomal dysfunction, endolysosomal trafficking, and oxidative stress.

    This work is exciting because it combines unbiased genome-wide screening with relevant phenotypic readouts implicated in neurodegeneration, moving beyond assays of just cell survival. Both manuscripts functionally validate strong hits and solidify the endolysosomal network as a hub for neuronal dysfunction in neurodegeneration.

    See et al. implicate inhibition of PIKfyve, a phosphatidylinositol kinase, in mediating a-synuclein aggregation by altering trafficking from early endosomes to lysosomes. Tian et al. show that depletion of PSAP, a lysosomal prosapsosin, increased reactive oxygen species and lysosomal cholesterol, enlarged lysosomes, and increased lipofuscin and iron deposits in neurons.

    Many of these phenotypes were not observed non-neuronal cells, including hiPSC-derived microglia, similar to what we have seen for SORL1 depletion in our group (Knupp et al., 2020). This highlights not only the need for cell-type specific assays to understand how disruption of these pathways uniquely affect the diverse cells of the central nervous system, but also to take unique cellular responses into account when designing therapeutic strategies.

    Finally, Tian et al., describe CRISPRbrain, a database which focuses on screens done in non-cancerous cells lines. This will be an extremely valuable resource for collating phenotypic data that impact neurodegeneration.

    References:

    . PIKfyve inhibition blocks endolysosomal escape of α-synuclein fibrils and spread of α-synuclein aggregation. bioRxiv. January 22, 2021

    . Depletion of the AD Risk Gene SORL1 Selectively Impairs Neuronal Endosomal Traffic Independent of Amyloidogenic APP Processing. Cell Rep. 2020 Jun 2;31(9):107719. PubMed.

    View all comments by Jessica Young
  3. This is a beautiful study using CRISPRi and CRISPRa to identify genes controlling neuronal response to oxidative stress. The cell type-specific effect of gene perturbation is extremely interesting and explains why the neuron is the most vulnerable cell type to oxidative stress during neurodegeneration.

    The unexpected link between saposin-mediated glycosphingolipid degradation, iron metabolism, lipofuscin accumulation, and oxidative stress underscores an important role of lysosome in regulating iron dynamics in neurons.

    Finally, CRISPRbrain will be a very useful platform for researchers in the neuroscience field to explore functional genomics screening data.

    View all comments by William Hu
  4. The See et al. manuscript builds off of previous work by the Kampmann lab to identify genetic modifiers of neurodegeneration through CRISPR interference (CRISPRi) screens, including the Tian et al. paper. The authors generated a cell line that stably expresses a FRET-based reporter of α-synuclein aggregation, and then, through careful validation experiments, demonstrated that these cells can faithfully report aggregation triggered by the addition of preformed α-synuclein fibrils via a FACS based assay. A genome-wide CRISPRi screen using these cells revealed that phosphatidylinositol kinases are key regulators of α-synuclein aggregation seeded by the extracellularly derived fibrils. Accordingly, both genetic and pharmacologic inhibition of PIKfyve was found to suppress α-synuclein aggregation following addition of recombinant α-synuclein fibrils or fibrils derived from multiple system atrophy (MSA) patient brains.

    In their discussion, the authors mention that the PIKfyve inhibitor apilimod has been shown to block SARS-CoV-2 infection in vitro and is being evaluated in a COVID19 clinical trial. Recently, a genome-wide CRISPR KO screen to identify SARS-CoV-2 host factors found that several components of the phosphatidylinositol biosynthetic pathway are required for infection with SARS-CoV-2, as well as the coronaviruses HCoV-OC43 and HCoV-229E (Wang et al., 2021). Together these findings raise the intriguing possibility that diverse pathogenic agents that enter cells through the endolysosomal pathway require overlapping mechanisms for exit into the cytosol and propagation to other cells. This suggests that some therapies found to suppress coronavirus infection may hold therapeutic potential for the treatment of synucleinopathies and vice versa. This may be great news for the neurodegeneration field considering the current intense focus on developing antiviral drugs for the treatment of COVID19.

    While this study represents a considerable technical achievement, the requirement to use lipofectamine to promote uptake of fibrils into cells limits the ability of the screen to identify genes that regulate α-synuclein uptake. Future studies can overcome this limitation by assessing aggregation in neurons, which should more readily uptake extracellular α-synuclein (Gerdes et al., 2020). As the Tian et al. paper demonstrated the ability to perform genome-wide CRISPRi screens in iPSC-derived neurons, they are well suited to follow up on this exciting work.

    References:

    . Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses. Cell. 2021 Jan 7;184(1):106-119.e14. Epub 2020 Dec 9 PubMed.

    . A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol. Nat Commun. 2020 Jun 1;11(1):2729. PubMed.

    View all comments by Nathaniel Safren
  5. This elegant screen by Kampmann and colleagues suggests that PIKFYVE inhibition may be able to block the pathological spread of misfolded proteins that cause neurodegeneration (See et al., 2021). 

    This is an exciting finding because blocking the spread of pathology could greatly modify the course of disease if done early enough. Together with our results showing that PIKFYVE inhibition potently reduces neurodegeneration and pre-existing pathology in C9ORF72 ALS/FTD induced neurons and mice, I think this provides a strong rationale for investigating PIKFYVE inhibition as a therapeutic target for neurodegeneration (Feb 2018 news). 

    References:

    . PIKfyve inhibition blocks endolysosomal escape of α-synuclein fibrils and spread of α-synuclein aggregation. bioRxiv. January 22, 2021

    View all comments by Justin Ichida
  6. This very interesting study adds considerable detail to the emerging picture that lysosomal acidification is intimately linked to cellular iron homeostasis, mitochondrial function, and oxidative stress. As Yambire et al. showed in 2019, reduced lysosomal acidification leads to apparent accumulation of ferric iron in lysosomes and a deficiency of ferrous iron, causing problems in mitochondrial biogenesis and inflammatory responses. (I previously commented on this paper in January 2020).

    Tian et al. also describe how correct lysosomal acidification is necessary for cellular iron homeostasis (Weber et al., 2020). It is likely significant that work from Nixon and colleagues has shown that fAD mutations of PSEN1 (Lee et al., 2010) and increased levels of the APP fragment C99 (Jiang et al., 2019)—such as are predicted from many fAD mutations of APP (see Checler et al., 2021)—both reduce lysosomal acidification. So, fAD mutations of PSEN1 and APP may be unified in their effects on iron homeostasis leading to mitochondrial dysfunction, oxidative stress (inducing Aβ production that would also accumulate due to lysosomal dysfunction), and inflammation.

    The important protective function of Prosaposin in neurons observed by Tian et al. is consistent with results from our earliest transcriptome analyses of knockdown of presenilin 1 or presenilin 2 function in zebrafish embryos using morpholino antisense oligonucleotides. Both treatments caused increased levels of prosaposin transcripts, which can be viewed as a homeostatic response (Newman et al., 2009). 

    References:

    . Is γ-secretase a beneficial inactivating enzyme of the toxic APP C-terminal fragment C99?. J Biol Chem. 2021 Mar 1;:100489. PubMed.

    . Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99). J Neurosci. 2019 Jul 3;39(27):5255-5268. Epub 2019 May 1 PubMed.

    . Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010 Jun 25;141(7):1146-58. PubMed.

    . Altering presenilin gene activity in zebrafish embryos causes changes in expression of genes with potential involvement in Alzheimer's disease pathogenesis. J Alzheimers Dis. 2009;16(1):133-47. PubMed.

    . Maintaining Iron Homeostasis Is the Key Role of Lysosomal Acidity for Cell Proliferation. Mol Cell. 2020 Feb 6;77(3):645-655.e7. Epub 2020 Jan 23 PubMed.

    . Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo. Elife. 2019 Dec 3;8 PubMed.

    View all comments by Michael Lardelli

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  1. Dysfunctional Lysosomes Cause Ferroptosis in Neurons