Whole genome CRISPR screens identify a LRRK2-regulated pathway for extracellular tau uptake by human neurons

  1. Lewis D. Evans
  2. Alessio Strano
  3. Eleanor Tuck
  4. Ashley Campbell
  5. James Smith
  6. Christy Hung
  7. Tiana S. Behr
  8. Bernardino Ghetti
  9. Benjamin Ryskeldi-Falcon
  10. Emre Karakoc
  11. Francesco Iorio
  12. Alastair Reith
  13. Andrew R. Bassett
  14. Frederick J. Livesey

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Abstract

Extracellular release and cellular uptake of pathogenic forms of the microtubule-associated protein tau contribute to the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease. Defining the cellular mechanisms and pathways for tau entry to human neurons is essential to understanding tauopathy pathogenesis and the rational design of disease-modifying therapeutics. Whole genome CRISPR loss-of-function screens in human iPSC-derived excitatory neurons, the major neuronal cell type affected in these diseases, enabled the delineation of the different cellular pathways for uptake of extracellular monomeric and fibrillar tau. Monomeric and fibrillar tau are both taken up by human neurons by receptor-mediated endocytosis, but involve different routes of entry at the neuronal surface: the low-density lipoprotein LRP1 is the primary receptor for monomeric tau, but contributes less to fibrillar tau entry. Similarly, endocytosis of monomeric tau is dependent on the familial Parkinson’s disease gene LRRK2, but not required for endocytosis of fibrillar tau. These findings implicate LRP1 and LRRK2 in the pathogenesis of tauopathies and Parkinson’s disease and identify LRRK2 as a potential therapeutic target for altering progression of these diseases.

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  1. Review Commons

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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    This study describes genome-wide, FACS-based, pooled CRISPR knock-out screens carried out in human cortical neurons, to determine the cellular factors that are required for endocytosis of monomeric and fibrillar tau protein. The screens combined fluorescent tau species uptake with labelled transferrin endocytosis (which is predominantly clathrin-dependent). This allowed identification of genes that had specific effects on tau endocytosis versus general endocytosis.

    The study identified a plethora of genes/proteins that are required for tau endocytosis. Bioinformatics analysis convincingly demonstrated that the genes required for uptake of both forms of tau are enriched for various endocytic machineries; there was a partial overlap, as well as some important differences, in the classes of machinery involved for monomeric versus fibrillar tau. Reassuringly, the screen for monomeric tau identified LPR1 as important for its endocytosis, consistent with the previous literature, and individual validation results for several other genes confirmed their effect.
    Importantly, the study also identified LRRK2 as being important for the uptake of monomeric tau. Further experiments were carried out with gene edited neurons lacking LRRK2, or expressing mutated LRRK2, to characterise this finding in more detail. These identified morphological abnormalities in the endolysosomal system, and also validated that LRRK2 regulates neuronal endocytosis of other key molecules that have been linked to neurodegenerative diseases, such as alpha-synuclein and Abeta. The precise mechanism of this effect of LRRK2 is not clear, and I'm sure will be a fruitful topic for additional studies; it is beyond the scope of the present study.

    Overall, I think this is a well-conducted study that is nicely written with well-presented data. The data are largely convincing. The strengths of this study include that:

    • the studies are carried out in human neurons, important target cells of tauopathies.
    • the screen is nicely designed and the QC presented is thorough.
    • it defines the landscape of cellular processes that are involved in tau endocytosis, a process that is highly likely to be of pathological relevance to major neurological disorders.
    • an important mechanistic link between LRRK2 mutations and tau uptake is identified and further characterised.
    • in virtually all cases (apart from a few experiments, e.g. Figure 6f), the studies are carried out with sufficient replicates and the statistical analysis is, as far as I can tell, appropriate (I do not have detailed experience in the statistical analysis of functional genomics datasets).

    My criticisms of this study are all minor:

    • in the initial QC of the screen, it would be interesting to see immunofluorescence microscopy assays with labelled tau species to further validate the FACS-based uptake assay is behaving as expected. At the time-point examined by FACS, is most tau in an endosomal compartment (as would be expected)? Furthermore, as an optional point for the authors to consider, in general I think the paper would be enhanced by inclusion of representative immunofluorescence images (as extended information) to supplement the FACS data in some of the subsequent figures, for example those in Figure 4a-d and Figure 6; although I think the conclusions of the paper are supported without such images, they would provide a nice visual representation of the effects. -in Figure 2f there is validation of a selection of screen hits by targeted CRISRP knock-out of the genes involved and FACS-based assays. Was this done with different CRISPR guides to those used in the initial screen, to provide further reassurance that there are no off-target effects? In addition, depletion of the mRNA/protein of interest is not confirmed in these validation experiments and this should be shown.
    • in figure 4, the LAMP1 labelling is poorly resolved and it is difficult to see how the surface area of individual pucta could have been accurately measured. In addition, LAMP1 labelling is used as a proxy for the lysosomal compartment and I'm sure the authors appreciate that LAMP1 also labels late endosomal and autophagic compartments. I would suggest additional labelling for a lysosomal enzyme (e.g. cathepsin B or D) to provide additional specificity. This also tends to allow better delineation of individual vesicles than LAMP1, allowing easier measurement of lysosomal size.
    • on page 12, regarding the vacuolar ATPase hits from the screen, referring to Figures 4c,d, it is stated that the results indicate "both forms of tau protein are trafficked via intracellular acid compartments of neurons". However, the function of the vacuolar ATPase has also been linked to effects on clathrin-mediated endocytosis (see PMID: 23263279) and this could provide a more direct explanation for the effect seen. This possibility should be mentioned. In addition, I think the authors overstate the case that the Brefeldin experiments "confirm" the dependency of tau uptake on ER-Golgi transport. Brefeldin was used for 24 hours and so there could be many knock-on effects of this treatment. The authors should either soften this statement or provide additional evidence (e.g. through other methods of blocking ER-Golgi and Golgi traffic such as depletion of individual key proteins involved in the process - which could be selected from the screen hits ) to support it.
    • in certain figures bar graphs are shown, and these would be improved if they also showed the individual replicate data points.

    Referees cross-commenting

    Re Reviewer 1's comments:

    1. Since all results rely on isogenic iPSC lines from only one donor, authors need to confirm their finding using iPSC lines form another donor.
      • Although the authors could consider this, I don't think this is strictly necessary. To my mind one of the key strengths of the study is that the lines used are isogenic, meaning that genetic background effects are controlled for. Perhaps the authors could deal with this by recognising this limitation of the study in the text.
    2. There are no sufficient attempts to assess the effects on synaptic functions and neurotoxicity.
      • I think that this is beyond the scope of the current study.
    3. It is unclear how many technical replicates and how many independent experiments are performed in each experiment.
      • This is a fair point. It can sometimes be a little moot as to what constitutes a replicate for a biological repeat in such cell biology experiments, and the authors should clarify more clearly what they have done, and whether they consider it a replicate or biological repeat.
    4. Since FACS may detect tau uptake in only soma, the effects of tau uptake should be evaluated by imaging entire neurons including axon and dendrites.
      • I made a similar point in my review.
    5. In addition to RAP and LRP1 domain 4, it should be considered validating the results using LRP1 KO models or knockdown approaches.
      • The authors could consider this. My opinion was that two orthogonal approaches was sufficient.
    6. Detailed descriptions in the Methods section for the neuronal differentiation, reagent catalog numbers, reagent concentrations, experimental procedures, and analytical methods should be provided.
      • Agreed
    7. The concentrations and catalog numbers of RAP chaperone and LRP1 domain 4 is unclear
      • Agreed
    8. Individual data should be included as dots in all bar graphs.
      • Agreed

    Significance

    In conclusion, I feel that this is an important study that provides a conceptual advance to the field, especially in delineating the landscape of cellular functions involved in tau endocytosis and in providing a mechanistic linkage between LRRK2 function and tau endocytosis, as well as the endocytosis of other key neurodegeneration-associated molecules. I think that it will be of interest to a broad readership, including basic and translational scientists in the fields of Alzheimer's and Parkinson diseases and other prevalent neurodegenerative disorders. I anticipate that this paper will provide information that stimulates many subsequent studies.

  3. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The authors investigated the cellular uptake of tau in neurodegenerative diseases. Using a genome-wide CRISPR loss-of-function screening in human iPSC-derived excitatory neurons, they identified distinct cellular pathways involved in the uptake of extracellular monomeric and fibrillar tau. The screening results revealed that LRRK2, along with the previously recognized LRP1, plays a role in the uptake of monomeric tau. While LRP1 was critical for the uptake of monomeric tau, it did not contribute to the uptake of fibrillar tau. Similarly, the endocytosis of monomeric tau was dependent on the familial Parkinson's disease gene LRRK2, but LRRK2 was not required for the endocytosis of fibrillar tau. These findings suggest that LRP1 and LRRK2 are involved in the pathogenesis of tauopathies and Parkinson's disease, highlighting LRRK2 as a potential therapeutic target for these diseases.

    1. Since all results rely on isogenic iPSC lines from only one donor, authors need to confirm their finding using iPSC lines form another donor.
    2. There are no sufficient attempts to assess the effects on synaptic functions and neurotoxicity.
    3. It is unclear how many technical replicates and how many independent experiments are performed in each experiment.
    4. Since FACS may detect tau uptake in only soma, the effects of tau uptake should be evaluated by imaging entire neurons including axon and dendrites.
    5. In addition to RAP and LRP1 domain 4, it should be considered validating the results using LRP1 KO models or knockdown approaches.
    6. Detailed descriptions in the Methods section for the neuronal differentiation, reagent catalog numbers, reagent concentrations, experimental procedures, and analytical methods should be provided.
    7. The concentrations and catalog numbers of RAP chaperone and LRP1 domain 4 is unclear
    8. Individual data should be included as dots in all bar graphs.

    Significance

    While their findings are interesting, there are several concerns which should be further addressed.

  4. Version published to 10.1101/2020.08.11.246363v2 on bioRxiv
  5. Version published to 10.1101/2020.08.11.246363v1 on bioRxiv